diff --git a/CONTRIBUTORS.yaml b/CONTRIBUTORS.yaml index 70b63265371eae..9688d89182ee16 100644 --- a/CONTRIBUTORS.yaml +++ b/CONTRIBUTORS.yaml @@ -195,6 +195,11 @@ astrovsky01: orcid: 0000-0002-7901-7109 joined: 2019-06 +adamtaranto: + name: Adam Taranto + email: adam.p.taranto@gmail.com + joined: 2023-01 + aurelienmoumbock: name: Aurélien F. A. Moumbock email: amaf.aurelien@yahoo.com @@ -362,7 +367,9 @@ burkemlou: name: Melissa Burke email: melissa@biocommons.org.au joined: 2023-12 - + affiliations: + - AustralianBioCommons + B0r1sD: name: Boris Depoortere joined: 2024-04 @@ -1364,6 +1371,13 @@ mccalluc: name: Chuck McCallum joined: 2018-06 +mcharleston: + name: Michael Charleston + email: michael.charleston@utas.edu.au + joined: 2023-01 + affiliations: + - UTas + mcmaniou: name: Maria Christina Maniou joined: 2021-09 @@ -1635,6 +1649,12 @@ RareSeas: name: Adelaide Rhodes joined: 2021-05 +PatCapon39: + name: Patrick Capon + joined: 2024-02 + affiliations: + - AustralianBioCommons + patrick-austin: name: Patrick Austin email: patrick.austin@stfc.ac.uk diff --git a/ORGANISATIONS.yaml b/ORGANISATIONS.yaml index 02f8ce7de0909a..9c43f2761dd8ac 100644 --- a/ORGANISATIONS.yaml +++ b/ORGANISATIONS.yaml @@ -73,6 +73,17 @@ ifb: avatar: "/training-material/shared/images/ifb.png" github: false +AustralianBioCommons: + name: Australian BioCommons + url: https://www.biocommons.org.au/ + avatar: "/training-material/shared/images/Australian-Biocommons-Favicon-RGB.png" + +UTas: + name: University of Tasmania + url: https://www.utas.edu.au/ + avatar: "/training-material/shared/images/UTas_logo.png" + github: false + pndb: name: Pôle National de Données de Biodiversité url: https://www.pndb.fr/ @@ -88,4 +99,4 @@ vib: qiime2: name: QIIME2 url: https://qiime2.org/ - avatar: https://avatars.githubusercontent.com/u/18176583?s=200&v=4 + avatar: https://avatars.githubusercontent.com/u/18176583?s=200&v=4 \ No newline at end of file diff --git a/_config.yml b/_config.yml index 0c2e57ba4c3efe..a4b777d6b1b73d 100644 --- a/_config.yml +++ b/_config.yml @@ -108,7 +108,11 @@ icon-tag: feedback: far fa-comments galaxy-advanced-search: fas fa-angle-double-down galaxy-show-active: fa fa-map-marker - galaxy-barchart: fas fa-chart-bar + galaxy-barchart: fas fa-chart-column + galaxy-visualise: fas fa-chart-column + galaxy-visualize: fas fa-chart-column + galaxy-vis-config: fas fa-angle-double-left + galaxy-viz-config: fas fa-angle-double-left galaxy-bug: fas fa-bug galaxy-chart-select-data: fas fa-database galaxy-clear: fas fa-times-circle diff --git a/_includes/contributors-line.html b/_includes/contributors-line.html index 2d55177862475e..c7d10311a6e6d9 100644 --- a/_includes/contributors-line.html +++ b/_includes/contributors-line.html @@ -1,7 +1,7 @@
{% if include.page.contributors %}
- {{ locale['authors'] | default: "Authors" }}: {% include _includes/contributor-list.html contributors=include.page.contributors badge=true newcontributors=include.newcontributors %} + {% unless include.authorsonly %}{{ locale['authors'] | default: "Authors" }}: {% endunless %}{% include _includes/contributor-list.html contributors=include.page.contributors badge=true newcontributors=include.newcontributors %}
{% elsif include.page.contributions %}
diff --git a/_includes/slides-youtube-selector.html b/_includes/slides-youtube-selector.html new file mode 100644 index 00000000000000..83f18ef00ee20c --- /dev/null +++ b/_includes/slides-youtube-selector.html @@ -0,0 +1,54 @@ +
+
{{ include.title }}
+

This content is available in multiple, equivalent formats. Choose your preferred format below.

+ +
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+ Open in new tab
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diff --git a/_includes/youtube.html b/_includes/youtube.html new file mode 100644 index 00000000000000..80b48053a31498 --- /dev/null +++ b/_includes/youtube.html @@ -0,0 +1,14 @@ + diff --git a/_layouts/base_slides.html b/_layouts/base_slides.html index 9782df5130175a..04e7eae7f6f7dd 100644 --- a/_layouts/base_slides.html +++ b/_layouts/base_slides.html @@ -95,9 +95,13 @@ {% endif %} {% endif %} +
+
# {{ page.title | default: topic.title }} +
+
{% include _includes/contributors-line.html page=page authorsonly=true %} @@ -211,8 +215,16 @@ } {{ 'main' | load_bundle }} - {% if page.mathjax %} - + {% if page.js_requirements.mathjax %} + + {% endif %} diff --git a/_layouts/base_slides_ai4life.html b/_layouts/base_slides_ai4life.html index 8d0498f85a8e89..ae13002f29e245 100644 --- a/_layouts/base_slides_ai4life.html +++ b/_layouts/base_slides_ai4life.html @@ -205,7 +205,7 @@ } {{ 'main' | load_bundle }} - {% if page.mathjax %} + {% if page.js_requirements.mathjax %} {% endif %} diff --git a/_plugins/jekyll-mathjax.rb b/_plugins/jekyll-mathjax.rb index acbe2f78026b70..748e348f1ddebe 100644 --- a/_plugins/jekyll-mathjax.rb +++ b/_plugins/jekyll-mathjax.rb @@ -1,6 +1,15 @@ + Jekyll::Hooks.register :pages, :post_init do |page| page.data['js_requirements'] = { - 'mathjax' => page.content =~ /\$\$/, + 'mathjax' => page.content =~ /\$\$/ || page.content =~ /\\\(/, 'mermaid' => page.content =~ /```mermaid/ || page.content =~ /pre class="mermaid"/ || page.data['layout'] == 'workflow-list', } + + # some fixes for mathjax: escape underscores + # both in inline mode \\( .. \\) and block mode $$ ..$$ + if page.content + page.content = page.content.gsub(/\$\$(.*?)\$\$/) { |m| m.gsub('_', '\\\\_') } + page.content = page.content.gsub(/\\\\\((.*?)\\\\\)/) { |m| m.gsub('_', '\\\\_') } + + end end diff --git a/assets/css/slides.scss b/assets/css/slides.scss index f2c92657142d3b..2dbfb9e9077700 100644 --- a/assets/css/slides.scss +++ b/assets/css/slides.scss @@ -377,6 +377,11 @@ body.remark-container { text-align: left !important; } + .pull-bottom { + position: fixed; + bottom: 3em; + } + .requirements li { list-style-type: none; text-align: left; @@ -447,6 +452,13 @@ body.remark-container { margin-right: 0.5em; } + .center { + // Tables aren't centered by text-align: center + * { + margin: auto; + } + } + .btn { display: inline-block; @@ -532,6 +544,6 @@ body.remark-container { text-align: center; font-size: 150%; padding: 0.3em; - + } } diff --git a/bin/schema-slides.yaml b/bin/schema-slides.yaml index 936915589a8a76..fe541278fff4ea 100644 --- a/bin/schema-slides.yaml +++ b/bin/schema-slides.yaml @@ -204,6 +204,7 @@ mapping: - /assets/images/BioNT_Logo.png - topics/ai4life/images/AI4Life-logo_giraffe-nodes.png - assets/images/genomics_intro.png + - shared/images/biocommons-utas.png required: true description: | A logo identifier (e.g. GTN) should be used by default, but may be swapped out for special logos from the assets folder. diff --git a/shared/images/Australian-Biocommons-Favicon-RGB.png b/shared/images/Australian-Biocommons-Favicon-RGB.png new file mode 100644 index 00000000000000..2da9f770436266 Binary files /dev/null and b/shared/images/Australian-Biocommons-Favicon-RGB.png differ diff --git a/shared/images/Australian-Biocommons-Logo-Horizontal-RGB.png b/shared/images/Australian-Biocommons-Logo-Horizontal-RGB.png new file mode 100644 index 00000000000000..666a1daeb0886b Binary files /dev/null and b/shared/images/Australian-Biocommons-Logo-Horizontal-RGB.png differ diff --git a/shared/images/UTas_logo.png b/shared/images/UTas_logo.png new file mode 100644 index 00000000000000..98953ac75fa1ce Binary files /dev/null and b/shared/images/UTas_logo.png differ diff --git a/shared/images/biocommons-utas.png b/shared/images/biocommons-utas.png new file mode 100644 index 00000000000000..3580a90e7413e1 Binary files /dev/null and b/shared/images/biocommons-utas.png differ diff --git a/topics/contributing/tutorials/create-new-tutorial-content/tutorial.md b/topics/contributing/tutorials/create-new-tutorial-content/tutorial.md index de4b5ce1ea948a..81a71762e24d93 100644 --- a/topics/contributing/tutorials/create-new-tutorial-content/tutorial.md +++ b/topics/contributing/tutorials/create-new-tutorial-content/tutorial.md @@ -349,6 +349,8 @@ Surround your math expression with two `$` signs on each side (like in LaTeX mat $$ 5 + 5 $$ +- Note: if inline mode is not working correctly, you can force it by using the following delimiters instead of dollar signs: `\\( 5 +5 \\)` + Dollar signs are therefore *reserved characters* for instructing the templating system to open/close LaTeX math blocks. If you want to use a `$` within your expression, you will need to *escape* it: `$$ a + 3\$ = 5\$ $$` will be rendered as: $$ a + 3\$ = 5\$ $$ diff --git a/topics/evolution/metadata.yaml b/topics/evolution/metadata.yaml index 36a80c11bd40a0..34ac9b2260bb50 100644 --- a/topics/evolution/metadata.yaml +++ b/topics/evolution/metadata.yaml @@ -8,7 +8,14 @@ requirements: - type: internal topic_name: introduction docker_image: '' + +subtopics: + - id: phylogenetics + title: "Phylogenetics" + description: "Learn about evolution through Phylogenetics, the study of evolutionary relationships among groups of organisms." + editorial_board: - cstritt - pvanheus + edam_ontology: ["topic_3299"] diff --git a/topics/evolution/tutorials/abc_intro_phylo-intro/images b/topics/evolution/tutorials/abc_intro_phylo-intro/images new file mode 120000 index 00000000000000..77c95679a02665 --- /dev/null +++ b/topics/evolution/tutorials/abc_intro_phylo-intro/images @@ -0,0 +1 @@ +../abc_intro_phylo/images \ No newline at end of file diff --git a/topics/evolution/tutorials/abc_intro_phylo-intro/slides.html b/topics/evolution/tutorials/abc_intro_phylo-intro/slides.html new file mode 100644 index 00000000000000..1dabcbd41ed842 --- /dev/null +++ b/topics/evolution/tutorials/abc_intro_phylo-intro/slides.html @@ -0,0 +1,56 @@ +--- +layout: tutorial_slides +logo: shared/images/biocommons-utas.png +title: "Phylogenetics - Back to Basics - Introduction" +zenodo_link: "" +subtopic: phylogenetics +priority: 1 +contributors: + - mcharleston +--- + + + +![Hand drawn phylogenetic tree from Charles Darwin’s notebook with clades A, B, C, D branching from a common ancestor. Prefaced with handwriting that reads ‘I think’ and annotated with other illegible notes.](images/Darwin_tree.png ) + +Charles Darwin, 1837 Notebeook entry +--- +![Model of the external structure of the SARS-CoV-2 virion](images/SARS-CoV-2-www.ncbi.nlm.nih.png) +.right[https://en.wikipedia.org/wiki/Coronavirus; CC BY-SA 4.0] +--- + +![Two-part figure showing a phylogenetic tree of SARS-CoV-2 strains on the left and genomic epidemiology of SARS-CoV-2 with subsampling focused globally over the 6 months leading up to November 2023 on the right](images/CovidNextStrain20231102.png) +.right[https://nextstrain.org/ncov/gisaid/global/6m] +--- +# Covid-19, latest tree from NextStrain + +![Phylogenetic tree for SARS-CoV-2 strains coloured by strain grouping](images/CovidTree20231102NextStrain.png) +--- +# Terminology + +![Schematic of a phylogenetic tree where features such as nodes/taxa, edges/branches are annotated and colour coded. The root of the tree is at the top of the image and the tree branches into two clades as you move towards the bottom of the image. The clades are formed of hypothetical common ancestors and five extant taxa which are labelled as the ‘in group’. Two additional taxa are appended to the right hand side of the tree and are labelled as the outgroup.](images/TreeAnatomy.png) +--- +# Phylogenetic tree of hexapods + +![Circular phylogenetic tree of hexapods (insects). Clades are colour coded and labelled with common names e.g. ‘Fleas’. Silhouettes of representative species are shown around the outside of the tree.](images/Hexapoda_phylogenetic_tree.png) +.left[https://doi.org/10.1371/journal.pone.0109085; CCBY 4.0 DEED license] +--- +# Sequence alignment + +![Screenshot of sequence visualisation output from Galaxy. Fifteen Anolis DNA sequences are arranged in rows. The nucleotides are colour coded and arranged in columns: A(blue), T (green), C (pink), G (orange). The top half of the image shows approximately 50 bases of each sequence. The lower half of the image shows a zoomed out heatmap-like image of a larger portion of the sequences.](images/UnalignedAnolis.png) +--- +# Building trees from distances + +![Flow chart illustrating how sequence alignment data or dis/similarity measures are used to calculate and calculate phylogenetic distances. Colours and shapes are used to differentiate different sections of the flowchart, guiding the viewer through each step from left to right. The flowchart begins with Sequence Alignment or Dis/Similarity Measures. These are used to form a distance matrix (D) which is used to select two nodes (x and y) forming a new node z. The distance matrix is updated with the new node z until no further nodes can be formed.](images/TreeConstruction.drawio.png) +--- +# Searching for trees + +![Screenshot of a phylogenetic tree of Anolis species. The root of the tree is on the left and the species are listed vertically on the right. The tree consists of multiple branching events and clades and includes bootstrap values.](images/PhyloVisTree.png) +--- +# Phylogenetic Networks + +![Screenshot of a phylogenetic network of Anolis species. The root of the network is at the centre of the image and clades radiate outwards forming a circular network.](images/ST4-HKY85.png) +--- +# Thank you! + +Let's begin diff --git a/topics/evolution/tutorials/abc_intro_phylo-mle-trees/images b/topics/evolution/tutorials/abc_intro_phylo-mle-trees/images new file mode 120000 index 00000000000000..77c95679a02665 --- /dev/null +++ b/topics/evolution/tutorials/abc_intro_phylo-mle-trees/images @@ -0,0 +1 @@ +../abc_intro_phylo/images \ No newline at end of file diff --git a/topics/evolution/tutorials/abc_intro_phylo-mle-trees/slides.html b/topics/evolution/tutorials/abc_intro_phylo-mle-trees/slides.html new file mode 100644 index 00000000000000..09b6d6fe71a24a --- /dev/null +++ b/topics/evolution/tutorials/abc_intro_phylo-mle-trees/slides.html @@ -0,0 +1,795 @@ +--- +layout: tutorial_slides +logo: shared/images/biocommons-utas.png +title: "Phylogenetics - Back to Basics - Estimating trees from alignments" +zenodo_link: "" +subtopic: phylogenetics +priority: 5 +contributors: + - mcharleston + +--- + +# Likelihood + +*A way to select models, given data* + +--- +# Why Likelihood? +
+
+* _Statistical rigour!_ Likelihood requires a statistical model and gains scientific rigour to defend hypotheses made. +* _Comparing hypotheses!_ We can compare models of evolution and determine which is better supported by the data. +* _Accuracy!_ Likelihood is generally the most accurate method to estimate phylogenies. + + + +--- +# Likelihood Definition +
+
+The *likelihood* of a model is a quantity that is *proportional* to the probability that the model gave you the data. +
+
+It is easy(ish) to calculate the probability that a given model yields a particular outcome -- the data set. +
+
+Maximum Likelihood (ML) seeks to find the model for which this probability is the *highest*. + + +--- +# Dice example +
+
+ +Imagine I have a set of very strange 10-sided dice, where the sides of my .blue[blue] die are labelled (1,1,2,3,4,5,5,6,6,6), and the sides of the .red[red] die are labelled (1,1,2,2,3,3,4,4,5,6). +
+
+Both dice have the same range of possible outcomes, 1 through 6, but the relative probabilities are different. + +| score | P.red[red] | P.blue[blue]| +|:--:|:--:|:--:| +| 1 | 0.2 | 0.2 | +| 2 | 0.1 | 0.2 | +| 3 | 0.1 | 0.2 | +| 4 | 0.1 | 0.2 | +| 5 | 0.2 | 0.1 | +| 6 | 0.3 | 0.1 | +| Total | 1.0 | 1.0 | + + +--- +# Dice example +
+
+ +Suppose I choose a die and roll it three times, getting values 3, 6, and 2. +
+
+Which die is most likely to have been used? +
+
+If it were the .blue[blue] die then the probability of this outcome is \\(0.1\times 0.3 \times 0.1 = 0.003\\). +
+
+If it were the .red[red] die then this probability is \\(0.2\times 0.1 \times 0.2 = 0.004\\). +
+
+Neither of these probabilities is high! But the larger of the two is for the .red[red] die: thus, given the data, we would say the maximum likelihood estimate of the "model" (here, which die was used) is the .red[red] die. + + +--- +# Slightly formal, with Bayes +
+We could write this example more formally as + +* Data \\(D\\) is the outcome \\((3,6,2)\\); + +* \\(P(\color{blue}{B})\\) and \\(P(\color{red}{R})\\) are the probabilities of choosing the blue and red dice (say 0.5 each); + +* \\(P(D|\color{blue}{B})\\) and \\(P(D|\color{red}{R})\\) are the probabilities of the data given the model "blue die" and "red die"; + +* \\(P(\color{blue}{B}|D)\\) is the probability of the *model* given the *data*, and it can be written + +\\( + P(\color{blue}{B}|D) = \frac{P(D|\color{blue}{B})P(\color{blue}{B})}{P(D)} = \frac{P(D|\color{blue}{B})P(\color{blue}{B})}{P(D|\color{blue}{B})P(\color{blue}{B})+P(D|\color{red}{R})P(\color{red}{R})} +\\) +
+
+-- this is *Bayes' formula*. +
+
+* Similarly we can write \\(P(\color{red}{R}|D)\\) as + +\\( + P(\color{red}{R}|D) = \frac{P(D|\color{red}{R})P(\color{red}{R})}{P(D)} = \frac{P(D|\color{red}{R})P(\color{red}{R})}{P(D|\color{blue}{B})P(\color{blue}{B})+P(D|\color{red}{R})P(\color{red}{R})} +\\) +
+
+* These conditional probabilities can be thought of as the relative likelihoods of the model, given the data. + + +--- +# Important Concepts learned +
+
+The dice example was very simple but it showed us important concepts: + +1. We could turn around the probability of the data given a model to the probability of a model, given the data; +
+2. Of the models considered, it was possible to choose one that was more likely; +
+3. These likelihoods can be really small, and don't have to sum to 1 (so they're not probabilities really). + + +--- +# Likelihood values in practice +
+
+In phylogenetics the probability of an alignment given a tree and its probabilities become really tiny so in order to avoid underflow numerical errors we use the natural log of the probabilities, and call it the *log-likelihood*. + + + +--- +# Probability of a substitution +
+
+Now suppose we have a probability matrix whose entries are the probabilities of nucleotide substitution in some fixed time, say 1 million years: +
+
+.image-50[ ![Formula where the probability of nucleotide substitutions P is expressed in a matrix with the nucleotides AGCT on both the x and y axis. The probability of changing from any nucleotide to another is 0.01. Probability of not changing is 0.97. Long description provided in the video recording at 11:20.](images/05-mle-02-probmatrix-1.png) ] +
+
+We could then calculate the probability of going from one nucleotide to another, say *A* to *G*, in 1M years by reading it from the graph: + +\\( + Pr(A\to G) = P_{A,G} = 0.01. +\\) + + +--- + +# Probability of sequences changing +
+
+To calculate the probability of multiple nucleotides changing we multiply the individual probabilities together -- which is making direct use of the assumption that each nucleotide site evolves independently of the others. +
+
+We would then calculate the probability of going from sequence **AAGT** to **AAGA** as + +
+ +$$ +\begin{align} +prob & = P(A\to A)\times P(A\to G) \times P(G\to G) \times P(T\to A) \\\\ + & = 0.97 \times 0.01 \times 0.97 \times 0.01 \\\\ + & \approx 9.4\times 10^{-6} +\end{align} +$$ + +--- +# Changing the time +
+
+To calculate the probability of sequences changing in our toy example for say *two* million years, we would *square* the probability matrix \\(P\\): +
+
+.image-50[ ![Formula where the probability of sequences changing over two million years P2 is expressed in a matrix with the nucleotides AGCT on both the x and y axis. The probability of changing from any nucleotide to another is 0.0196. Probability of not changing is 0.9412. +Long description provided in the video recording at 13:12.](images/05-mle-03-probmatrixsquared-1.png)] +
+
+Notice that the probability of going from A to G is not quite twice what it was before, since now there is a small chance that A will change to G and then back again. +
+
+So if we have a probability matrix for a fixed time period we can convert it to a probability matrix for multiples of that time. + +--- +# Probabilities to Rates +
+
+This doesn't generalise to arbitrary time periods, so instead we use a *rate* matrix, like this: +
+
+.image-50[ ![Formula where the rate of nucleic acid subtitutions Q is expressed as a matrix with the nucleotides AGCT on both the x and y axis. The rate of change from any nucleotide to another is 0.01. The rate of not changing is -0.03. Long description provided in the video recording at 15:56](images/05-mle-04-ratematrix-1.png) ] +
+
+Converting a rate matrix to a probability requires a matrix *exponentiation*, giving formulae like this: + +\\( + P(t) = e^{Qt}. +\\) + +We won't worry here about technical details, but note that some classes of rate matrix are easier to work with than others, the easiest being the JC69 (one parameter) matrix. + + +--- +# Likelihood uses rate matrices +
+
+* Many models of sequence evolution available, including different base (nucleotide) frequencies, and different sites being allowed different rates. + +* Likelihoods will be expressed as a (negative) log-likelihood, and these are used to compare models. + +* Typically these will be quite large (e.g., -37000, -80000), but it is the *relative likelihoods* that matter, so the *difference* between log likelihoods. + + +.blue[A model is selected to give the best chance of getting the phylogeny right.] + +--- +# A huge range of models +
+
+* More parameter-rich models fit better; we need to avoid over-fitting. + +* The (log)-likelihood value can be adjusted to penalise the model, based on the number of parameters. + +* Common penalties are the Akaike Information Criterion (AIC). + +* There is also a "corrected" AIC (AICc), and a "Bayesian Information Criterion" penalty (BIC). + +--- +# Penalised likelihood +
+
+For a model with \\(k\\) free parameters and a log likelihood \\(L\\), here are the penalty functions: +
+
+AIC is the original information-theoretic correction: +\\( AIC = 2k-2\ln(L)\\) +
+
+AICc is a correction for small sample sizes and is equal to +\\( AICc = AIC + \frac{2k^{2}+2k}{n-k-1} \\) +
+
+BIC is defined by +\\( BIC = k\ln(n) - 2\ln(L)\\) +(where \\(n\\) is a measure of sample size). +
+
+All of these are in common use but the commonest is AIC, and that is the method we use in the tutorial. + +--- +# Phylogenetic likelihood is mostly directionless +
+
+The likelihood calculations in this tutorial are based on *reversible* models: that means that there is no difference between the likelihood of going from sequence A to sequence B of from B to A. +
+
+This means that the output trees are *unrooted*. +
+
+(There are some asymmetric models included in IQTree but they are well beyond the scope of this tutorial and are not in common use.) + +--- +# Likelihood of a tree and model +
+
+Now we have the huge task: + +```{code} +ML Search: + for every tree $T$ in tree space { + for each parameter in the substitution rate matrix { + for each branch length { + Calculate the (log-)likelihood of the alignment + } + } + } +``` + +... and find the best combination! +
+
+All this means that *Maximum Likelihood is slow*. + +--- +# Likelihood under JC69 +
+
+.image-50[ ![Formula where rate of nucleic acid substitutions according to Jukes-Cantor 69 model QJC69 are expressed as a matrix with the nucleotides AGCT on both the x and y axis. The rate of change from any nucleotide to another is represented by the character alpha. The rate of not changing is represented by an asterisk. Long description provided in the video recording at 26:47](images/05-mle-05-JC69-1.png) ] + +.reduce-75[ where the asterisk is a short-hand to make the row-sums equal 0. ] +
+
+* This model has only one parameter. +* Note: we cannot separate this from the overall rate (just as you can't tell people how "far" somewhere is by assuming how fast they'll travel). +* Thus tree search is "only" over all trees and all branch lengths. + + +--- +# Likelihood under HKY85 +
+
+.image-50[ ![Formula where rate of nucleic acid substitutions according to the HKY85 model QHKY85 is expressed as a matrix with the nucleotides AGCT on both the x and y axis. The rate of change from any nucleotide to another is a factor of variation in nucleotide frequencies πA; πG; πC; πT and transition/transversion rates kappa. The rate of not changing is represented by an asterisk. Long description provided in the video recording at 28:06](images/05-mle-06-HKY85-1.png) ] +
+
+* The HKY85 model has *four* parameters: \\(\pi_{A}\\), \\(\pi_{G}\\), \\(\pi_{C}\\) (and this fixes \\(\pi_{T}\\)), and a transition/ transversion rate ratio \\(\kappa\\). + +* Can fix \\(\pi\\) or estimate it from the observed frequencies, or even estimate it by searching for the best values (which means it takes longer of course). + + +--- +# GTR +
+
+The General Time Reversible model has *nine* parameters: three from the base frequencies and six more from the parameters in the substitution rate matrix: +
+
+.image-50[ ![Formula where rate of nucleic acid substitutions according to the GTR model QGTR is expressed as a matrix with the nucleotides AGCT on both the x and y axis. The rate of change from any nucleotide to another incorporates nine parameters represented by Greek letters. The rate of not changing is represented by an asterisk. Long description provided in the video recording at 30:00](images/05-mle-07-GTR-1.png) ] +
+
+(The Greek letters are not standardised: they just show the structure.) + +--- +# Rates across sites +
+
+* Some sites are non-coding: they evolve *faster* than others. + +* Third codon position sites are largely redundant: for most amino acids, the third nucleotide is *free to vary*. + +* Some are under *strong selection* and evolve slowly. + +* Some are so important that they must stay in the same state else a protein will not fold properly: these are *fixed*. +
+
+This means that even if the same rate matrix might be applicable at different sites, the overall substitution rate might be hundreds of times faster at one site than another. +
+
+Models that accommodate this are called "Rates Across Sites" or RAS models. +
+
+.blue[By default, IQTree includes RAS models.] + + +--- +# Rates across sites +
+
+![Schematic representing differences in the rate of substitutions at different sites. A line with two dots on it represents two positions within a gene. Blue arrows connect each dot to separate rate matrices. The matrices are identical but the matrix on the right is annotated 5x indicating that there are 5 times more substitutions at this site. Described at 33:00](images/05-mle-08-RAS-1.png) + +While the proportions of substitution types might be the same, the overall *rate* at these two sites can differ. + +--- + +# IQTree +
+
+.image-25[ + ![IQTree logo](images/05-mle-09-IQTreeLogo-1.png) +] + +IQTree is at the forefront of maximum likelihood phylogenetic tools. It is by far the most advanced such tool and is under active development, adding new models and features. + +It is available from [IQtree.org](http://www.iqtree.org/). + +_To cite IQTree2, please use_ + +*B.Q. Minh, H.A. Schmidt, O. Chernomor, D. Schrempf, M.D. Woodhams, A. von Haeseler, R. Lanfear* (2020) IQ-TREE 2: New models and efficient methods for phylogenetic inference in the genomic era. _Mol. Biol. Evol._, 37:1530-1534. [https://doi.org/10.1093/molbev/msaa015](https://doi.org/10.1093/molbev/msaa015) + + +--- +# Tree Space +
+
+_A way to navigate among trees_ +--- +# What is "tree space"? +
+
+.left[ A *network*: ] + * whose nodes are *trees*, and + * whose edges are *adjacencies between trees*, where + * each node has some *score* or weight: here, it will be *likelihood*. +
+
+.left[ As we cannot check every tree we must wander through the set of trees in the hope of finding "good" ones, under the assumption that the best (most likely) trees will have a lot in common with each other.] +
+
+.left[ Fortunately this turns out to be the case! The best trees tend to clump together in tree space.] + +--- +# Tree perturbations 1: NNI + +*N*earest *N*eighbour *I*nterchange is a way to transform one tree into another -- so we can search for the best tree. + +An NNI move consists of effectively collapsing an internal branch of the tree and re-expanding it out in either of two possible ways. + +For example, given the original tree + +.image-25[ ![Schematic of an unrooted phylogenetic tree. There are three connected internal nodes x,y,z and five tips A, B, C, D, E. A and B are connected to x; C is connected to Y; E and D are connected to z.](images/05-mle-10-initialtree-1.png) ] +
+
+there are two internal edges: \\(x-y\\) and \\(y-z\\). We can do an NNI move on each of them. + + +--- +# Tree perturbations 1: NNI +
+
+![Schematic of an unrooted phylogenetic tree. There are three connected internal nodes x,y,z and five tips A, B, C, D, E. A and B are connected to x; C is connected to Y; E and D are connected to z. The edge connecting x and y is coloured red.](images/05-mle-11-select-1.png) + +Select edge \\(xy\\) to collapse. + +--- +# Tree perturbations 1: NNI +
+
+![Schematic of a non-binary phylogenetic tree. There are two connected internal nodes xy and z and five tips A, B, C, D, E. A, B and C are connected to xy; D and E are connected to z. The internal node xy coloured red.](images/05-mle-12-collapse-1.png) + +\\(xy\\) is collapsed. + +--- +# Tree perturbations 1: NNI +
+
+![Schematic of two phylogenetic trees. The tree on the left has three connected internal nodes x,y,z and five tips A, B, C, D, E. A and C are connected to x; B is connected to Y; E and D are connected to z. The edge connecting x and y is red. The tree on the right has three connected internal nodes x,y,z and five tips A, B, C, D, E. B and C are connected to x; A is connected to Y; E and D are connected to z. The edge connecting x and y is red. +](images/05-mle-13-expand-1.png) + +Expand \\(xy\\) in either of two ways. +
+
+For an unrooted binary tree with \\(n\\) leaves, there are \\(2(n-3)\\) neighbouring trees under this move. + +--- +# Simple example: five taxa +
+
+![Fifteen different unrooted binary phylogenetic trees containing five taxa are arranged in a star shaped configuration. Blue lines connect the trees and represent NNI moves. Long description provided at 40:00 in the video recording.](images/05-mle-14-treespace-1.png) + + +--- +# Tree perturbations 2: SPR +
+
+*S*ubtree *P*runing and *R*egrafting works by choosing an edge, cutting the tree at that edge, and then reconnecting the smaller part anywhere else in the larger part. + +![Schematic of an unrooted phylogenetic tree. The tree has four connected internal nodes w,x,y,z and six tips A, B, C, D, E, F. A and B are connected to w; C is connected to x; D is connected to y; E and F are connected to z. The edge connecting w and z is red.](images/05-mle-15-SPR-select-1.png) +--- +# Tree perturbations 2: SPR +
+
+![Schematic of an unrooted phylogenetic tree. The tree has four connected internal nodes w,x,y,z and six tips A, B, C, D, E, F. A and B are connected to w; C is connected to x; D is connected to y; E and F are connected to z. There is a black dot on the edge connecting w and z.](images/05-mle-16-SPR-trim-1.png) + +Trim the root of the subtree... +--- +# Tree perturbations 2: SPR +
+
+![Schematic of an unrooted phylogenetic tree. The tree has four connected internal nodes w,x,y,z and six tips A, B, C, D, E, F. A and B are connected to w; C is connected to x; D is connected to y; E and F are connected to z. There is a black dot on the edge connecting w and z. A red arrow points from this dot to the edge connecting y and D.](images/05-mle-17-SPR-graft-yD-1.png) + +Regraft to \\(y-D\\), +--- +# Tree perturbations 2: SPR +
+
+![Schematic of an unrooted phylogenetic tree. The tree has four connected internal nodes w,x,y,z and six tips A, B, C, D, E, F. A and B are connected to w; C is connected to x; D is connected to y; E and F are connected to z. There is a black dot on the edge connecting w and z. Red arrows point from this dot to the edge connecting y and D and the edge connecting y and z.](images/05-mle-18-SPR-graft-yz-1.png) + +... or \\(y-z\\), +--- +# Tree perturbations 2: SPR +
+
+![Schematic of an unrooted phylogenetic tree. The tree has four connected internal nodes w,x,y,z and six tips A, B, C, D, E, F. A and B are connected to w; C is connected to x; D is connected to y; E and F are connected to z. There is a black dot on the edge connecting w and z. Red arrows point from this dot to the edges connecting y and D; y and z; and z and E.](images/05-mle-19-SPR-graft-zE-1.png) + +... or \\(z-E\\), +--- +# Tree perturbations 2: SPR +
+
+![Schematic of an unrooted phylogenetic tree. The tree has four connected internal nodes w,x,y,z and six tips A, B, C, D, E, F. A and B are connected to w; C is connected to x; D is connected to y; E and F are connected to z. There is a black dot on the edge connecting w and z. Red arrows point from this dot to the edges connecting y and D; y and z; z and E; and z and F.](images/05-mle-20-SPR-graft-zF-1.png) + +... or \\(z-F\\). + +Under this move there are more neighbours: \\(4(n-3)(n-2)\\) possible moves. + +--- +# Tree perturbations 3: TBR +
+
+*T*ree *B*isection and *R*econnection works by cutting the tree at any internal edge, then re-rooting each of the parts just formed, and reconnecting the roots: + +![Schematic of an unrooted phylogenetic tree. The tree has four connected internal nodes w,x,y,z and six tips A, B, C, D, E, F. A and B are connected to w; C is connected to x; D is connected to y; E and F are connected to z. The edge connecting x and y is red.](images/05-mle-21-TBR-select-1.png) +--- +# Tree perturbations 3: TBR +
+
+![Schematic of two phylogenetic trees. In the tree on the left node w is connected to nodes A, B and C. In the tree on the right node z is connected to nodes D, E and F. ](images/05-mle-22-TBR-trim-1.png) +--- +# Tree perturbations 3: TBR +
+
+
+![Schematic of two phylogenetic trees. In the tree on the left node w is connected to nodes A, B and C. In the tree on the right node z is connected to nodes D, E and F. Black dots are shown on the edges connecting A-w and D-z.](images/05-mle-23-TBR-selectroots-1.png) + +Selecting edges as roots of the new subtrees +--- +# Tree perturbations 3: TBR +
+
+
+![Schematic of two phylogenetic trees. In the tree on the left node w is connected to nodes A, B and C. In the tree on the right node z is connected to nodes D, E and F. Black dots are shown on the edges connecting A-w and D-z. A dashed line connects the trees through these dots.](images/05-mle-24-TBR-regraft-1.png) + +And reconnect! +
+
+*Note*: There is no neat form for the number of neighbours of a given tree under the SPR moves, but it's more than TBR. + +--- +
+
+ +![Photograph of desert landscape with multiple hills. From https://en.wikipedia.org/wiki/File:Painted_Hills_2009.08.13.11.08.52.jpg](images/Painted_Hills_2009.08.13.11.08.52.jpg) + +Painted Hills near Mitchell, Oregon. + +This file is licensed under the Creative Commons Attribution-Share Alike 3.0 Unported license. + + +--- +# Hill Climbing +
+
+Tree Search algorithms work essentially by +
+
+1. Starting with a reasonably good estimate, e.g., using Neighbour-Joining; + +2. Checking all the neighbouring trees under whichever perturbation is being used for an acceptable new tree; + +3. If an acceptable new tree is available, move to it! If not, then stop. + - Most simply, "acceptable" means "better than this one". +
+
+This kind of heuristic search is called *hill climbing*, from the obvious analogy. + +--- +
+
+ +![Two networks containing 15 phylogenetic trees are shown. Lines connect the trees in the networks forming a star shaped pattern. The network on the right is additionally annotated with lines that trace over selected connections between trees multiple times. Long description in video recording at 47:17 Image from: https://doi.org/10.1101/746362](images/CaoEtAl2019Fig3cropped.jpg) + +This figure from Cao _et al._ (2019) illustrates moving around in tree space. + +Source: [https://doi.org/10.1101/746362](https://doi.org/10.1101/746362) + +--- + +# _Anolis_ phylogeny example +
+
+.pull-left[ +![Photograph of a green lizard](images/Anole.jpg) +] +.pull-right[ +_Anolis_ is a highly speciose genus of lizards -- more than 425 species (unless as some would have it, many should be moved to another genus) -- native to the Americas. +
+
+.reduce50[ Graphic: "Picture of a lizard (Anolis carolinensis) I (DanielCD) took in Atascocita, Texas (USA) on 4/22/05. Atascocita is in southeast Texas just north of Houston." + +This file is licensed under the Creative Commons Attribution-Share Alike 3.0 Unported license. ] +] +--- + +layout: true + +# `iqtree` output + +--- + +.reduce70[ +``` +mac@mac-pc-443 ~/N/p/t/2/AusBioCommons-Phylogenetics> iqtree2 -s anolis-aligned.fasta +IQ-TREE multicore version 2.1.3 COVID-edition for Linux 64-bit built Apr 21 2021 +Developed by Bui Quang Minh, James Barbetti, Nguyen Lam Tung, +Olga Chernomor, Heiko Schmidt, Dominik Schrempf, Michael Woodhams. + +Host: mac-pc-443 (AVX2, FMA3, 31 GB RAM) +Command: iqtree2 -s anolis-trimmed-aligned-clustal.fasta -redo +Seed: 225340 (Using SPRNG - Scalable Parallel Random Number Generator) +Time: Thu Dec 21 13:26:51 2023 +Kernel: AVX+FMA - 1 threads (8 CPU cores detected) + +HINT: Use -nt option to specify number of threads because your CPU has 8 cores! +HINT: -nt AUTO will automatically determine the best number of threads to use. + +Reading alignment file anolis-trimmed-aligned-clustal.fasta ... Fasta format detected +Alignment most likely contains DNA/RNA sequences +Alignment has 55 sequences with 1462 columns, 1138 distinct patterns +913 parsimony-informative, 172 singleton sites, 377 constant sites + Gap/Ambiguity Composition p-value + 1 Anolis.acutus 3.56% passed 50.37% + 2 A.aeneus 5.54% failed 2.25% + 3 A.agassizi 3.01% failed 1.82% + 4 A.ahli 3.15% passed 81.11% + 5 A.aliniger 2.74% passed 43.47% + 6 A.alutaceous 2.94% passed 9.34% + 7 A.angusticeps 2.80% passed 57.88% + 8 A.bahorucoensis 2.53% passed 21.76% + 9 A.barahonae 3.90% failed 1.72% + 10 A.bartschi 3.76% passed 30.22% + 11 A.bimaculatus 4.65% passed 32.77% +``` +] +--- + + +.reduce70[ +``` +Create initial parsimony tree by phylogenetic likelihood library (PLL)... 0.010 seconds +Perform fast likelihood tree search using GTR+I+G model... +Estimate model parameters (epsilon = 5.000) +Perform nearest neighbor interchange... +Estimate model parameters (epsilon = 1.000) +1. Initial log-likelihood: -37100.382 +2. Current log-likelihood: -37098.639 +Optimal log-likelihood: -37097.515 +Rate parameters: A-C: 1.41395 A-G: 4.11730 A-T: 1.55773 C-G: 0.69506 C-T: 7.28507 G-T: 1.00000 +Base frequencies: A: 0.339 C: 0.261 G: 0.118 T: 0.282 +Proportion of invariable sites: 0.186 +Gamma shape alpha: 0.832 +Parameters optimization took 2 rounds (0.144 sec) +Time for fast ML tree search: 0.993 seconds +``` +] +--- + + +.reduce70[ +``` +ModelFinder will test up to 286 DNA models (sample size: 1462) ... + No. Model -LnL df AIC AICc BIC + 1 GTR+F 41407.688 115 83045.375 83065.197 83653.445 + 2 GTR+F+I 39252.694 116 78737.387 78757.569 79350.744 + 3 GTR+F+G4 37164.452 116 74560.904 74581.085 75174.261 + 4 GTR+F+I+G4 37096.959 117 74427.917 74448.462 75046.562 + 5 GTR+F+R2 37597.901 117 75429.802 75450.347 76048.447 + 6 GTR+F+R3 37179.249 119 74596.499 74617.780 75225.718 + 7 GTR+F+R4 37074.898 121 74391.795 74413.828 75031.590 + 8 GTR+F+R5 37037.210 123 74320.421 74343.219 74970.791 + 9 GTR+F+R6 37031.233 125 74312.465 74336.043 74973.410 + 21 SYM+R5 37262.481 120 74764.963 74786.618 75399.470 + 22 SYM+R6 37261.627 122 74767.254 74789.668 75412.336 + 34 TVM+F+R5 37097.645 122 74439.290 74461.704 75084.373 + 35 TVM+F+R6 37095.642 124 74439.285 74462.471 75094.942 + 47 TVMe+R5 37745.606 119 75729.213 75750.494 76358.432 + 48 TVMe+R6 37742.081 121 75726.162 75748.195 76365.957 + 60 TIM3+F+R5 37076.842 121 74395.685 74417.718 75035.480 + 61 TIM3+F+R6 37070.459 123 74386.918 74409.717 75037.288 + 73 TIM3e+R5 37747.554 118 75731.107 75752.019 76355.039 + 74 TIM3e+R6 37740.393 120 75720.786 75742.442 76355.293 + 86 TIM2+F+R5 37042.461 121 74326.921 74348.954 74966.716 + 87 TIM2+F+R6 37036.463 123 74318.927 74341.725 74969.297 + 99 TIM2e+R5 37274.306 118 74784.612 74805.524 75408.544 +100 TIM2e+R6 37273.302 120 74786.604 74808.259 75421.111 +112 TIM+F+R5 37081.068 121 74404.136 74426.169 75043.931 +113 TIM+F+R6 37074.732 123 74395.464 74418.262 75045.834 +125 TIMe+R5 37757.456 118 75750.913 75771.824 76374.845 +126 TIMe+R6 37750.820 120 75741.641 75763.296 76376.148 +138 TPM3u+F+R5 37129.051 120 74498.103 74519.758 75132.610 +``` +] +--- + +.reduce70[ +``` +Estimate model parameters (epsilon = 0.100) +1. Initial log-likelihood: -37042.461 +Optimal log-likelihood: -37042.422 +Rate parameters: A-C: 1.77793 A-G: 5.08796 A-T: 1.77793 C-G: 1.00000 C-T: 8.61510 G-T: 1.00000 +Base frequencies: A: 0.339 C: 0.261 G: 0.118 T: 0.282 +Site proportion and rates: (0.321,0.030) (0.225,0.316) (0.232,1.114) (0.196,2.482) (0.026,6.784) +Parameters optimization took 1 rounds (0.163 sec) +Computing ML distances based on estimated model parameters... +Computing ML distances took 0.054833 sec (of wall-clock time) 0.054830 sec(of CPU time) +Computing RapidNJ tree took 0.000283 sec (of wall-clock time) 0.000282 sec (of CPU time) +Log-likelihood of RapidNJ tree: -37150.315 +``` +] +--- + +.reduce70[ +``` +------------------------------------------------------------------- +| INITIALIZING CANDIDATE TREE SET | +-------------------------------------------------------------------- +Generating 98 parsimony trees... 0.879 second +Computing log-likelihood of 98 initial trees ... 1.283 seconds +Current best score: -37042.422 + +Do NNI search on 20 best initial trees +Estimate model parameters (epsilon = 0.100) +BETTER TREE FOUND at iteration 1: -37042.003 +Estimate model parameters (epsilon = 0.100) +BETTER TREE FOUND at iteration 2: -37036.294 +Iteration 10 / LogL: -37042.212 / Time: 0h:0m:5s +Iteration 20 / LogL: -37058.987 / Time: 0h:0m:6s +Finish initializing candidate tree set (10) +Current best tree score: -37036.294 / CPU time: 6.458 +Number of iterations: 20 +``` +] + +--- + +.reduce70[ +``` +-------------------------------------------------------------------- +| OPTIMIZING CANDIDATE TREE SET | +-------------------------------------------------------------------- +Estimate model parameters (epsilon = 0.100) +BETTER TREE FOUND at iteration 27: -37036.066 +Iteration 30 / LogL: -37044.404 / Time: 0h:0m:9s (0h:0m:31s left) +Iteration 40 / LogL: -37036.365 / Time: 0h:0m:11s (0h:0m:26s left) +Iteration 50 / LogL: -37036.142 / Time: 0h:0m:14s (0h:0m:22s left) +Iteration 60 / LogL: -37038.691 / Time: 0h:0m:16s (0h:0m:19s left) +Iteration 70 / LogL: -37039.589 / Time: 0h:0m:19s (0h:0m:15s left) +Iteration 80 / LogL: -37049.077 / Time: 0h:0m:21s (0h:0m:12s left) +Iteration 90 / LogL: -37036.399 / Time: 0h:0m:23s (0h:0m:9s left) +Iteration 100 / LogL: -37036.108 / Time: 0h:0m:26s (0h:0m:7s left) +Iteration 110 / LogL: -37036.508 / Time: 0h:0m:28s (0h:0m:4s left) +Iteration 120 / LogL: -37036.242 / Time: 0h:0m:31s (0h:0m:1s left) +TREE SEARCH COMPLETED AFTER 128 ITERATIONS / Time: 0h:0m:33s +``` +] + +--- + +.reduce70[ +``` +-------------------------------------------------------------------- +| FINALIZING TREE SEARCH | +-------------------------------------------------------------------- +Performs final model parameters optimization +Estimate model parameters (epsilon = 0.010) +1. Initial log-likelihood: -37036.066 +Optimal log-likelihood: -37036.065 +Rate parameters: A-C: 1.78140 A-G: 5.12638 A-T: 1.78140 C-G: 1.00000 C-T: 8.65177 G-T: 1.00000 +Base frequencies: A: 0.339 C: 0.261 G: 0.118 T: 0.282 +Site proportion and rates: (0.320,0.029) (0.222,0.311) (0.230,1.085) (0.200,2.454) (0.027,6.674) +Parameters optimization took 1 rounds (0.074 sec) +BEST SCORE FOUND : -37036.065 +Total tree length: 10.598 + +Total number of iterations: 128 +CPU time used for tree search: 32.804 sec (0h:0m:32s) +Wall-clock time used for tree search: 33.018 sec (0h:0m:33s) +Total CPU time used: 33.400 sec (0h:0m:33s) +Total wall-clock time used: 33.500 sec (0h:0m:33s) + +Analysis results written to: + IQ-TREE report: anolis-aligned.fasta.iqtree + Maximum-likelihood tree: anolis-aligned.fasta.treefile + Likelihood distances: anolis-aligned.fasta.mldist + Screen log file: anolis-aligned.fasta.log + +Date and Time: Thu Dec 21 13:28:52 2023 +``` +] + +--- + +.image-100[ + +![Screenshot of a phylogenetic tree of Anolis species. The root of the tree is at the left of the image and is connected to Anolis species on the right by multiple branches.](images/AnolisIQTree.png) + +] + + +--- +layout: false + +# Beyond Maximum Likelihood Trees +
+
+* Bayesian analyses with BEAST and MrBayes +* Networks -- next video! + +--- +layout: false + +# Thank you! + +.center[_Next - phylogenetic networks!_] + diff --git a/topics/evolution/tutorials/abc_intro_phylo-msa/images b/topics/evolution/tutorials/abc_intro_phylo-msa/images new file mode 120000 index 00000000000000..77c95679a02665 --- /dev/null +++ b/topics/evolution/tutorials/abc_intro_phylo-msa/images @@ -0,0 +1 @@ +../abc_intro_phylo/images \ No newline at end of file diff --git a/topics/evolution/tutorials/abc_intro_phylo-msa/slides.html b/topics/evolution/tutorials/abc_intro_phylo-msa/slides.html new file mode 100644 index 00000000000000..be5182d1e5cd48 --- /dev/null +++ b/topics/evolution/tutorials/abc_intro_phylo-msa/slides.html @@ -0,0 +1,561 @@ +--- +layout: tutorial_slides +logo: shared/images/biocommons-utas.png +title: "Phylogenetics - Back to Basics - Multiple Sequence Alignment" +zenodo_link: "" +subtopic: phylogenetics +priority: 3 +contributors: + - mcharleston + +--- + + +# Motivation +
+
+We use sequence alignment to: +
+
+- identify complex relationships among multiple species - more than just pairwise comparisons; +- find homologous parts (sites / loci) in sequences that may be under different selection dynamics; +- and to build phylogenetic trees! + +
+
+Multiple sequence alignment (MSA) is a required step in molecular phylogenetics +--- + +# Sequence Alignment +
+
+.left[One of the best understood and best solved bioinformatics problem is *how to align two sequences*.] +
+.left[In order to do this we need to know:] +
+1. what an "alignment" really means; +
+2. how to judge how good an alignment is; +
+3. an algorithm to do the alignment. +--- + +# What is an alignment? +
+Given two sequences like **GGGCTGAA** and **GGGACTG** "an alignment" is a _mapping_ of their positions (a.k.a. "sites") to a common ordering, by inserting gaps in one sequence or another: +
+
+![Alignment of two DNA sequences GGGACTG and GGCTGAA. In the alignment gaps, indicated by -, are added to the sequences to form GGGACTG-- and -GG-CTGAA so that homologous sites are aligned at positions 2, 3, 5, 6, 7. ](images/03-msa-01-what-is-an-alignment.pdf-1.png)

+--- +# Alignments represent homology +
+
+ +

Our goal with sequence alignment is to identify which regions, down to individual positions in a molecular sequence, are homologous: that is, their shared evolutionary history is the same as that of the taxa of interest.

+
+ +

It's like tracing the origin of a set of transcribed documents through all their copies back to the original, where each was only copied (with maybe some mistakes) from one predecessor.

+
+

Once we have identified homologous sites then we can analyse their differences and similarities under an evolutionary model - a crucial centrepiece of phylogenetic analysis, and in fact of all comparative analysis of molecular sequences.

+--- + +# Sequences evolve on a tree +
+
+![Schematic example of a phylogenetic tree where species are represented by short DNA sequences to demonstrate how sequences evolve on a tree. The tree branches multiple times from a common ancestor to five extant taxa.](images/03-msa-02-a-tree-only.pdf-1.png) +--- + +# Sequences evolve on a tree +
+
+![A schematic of a phylogenetic tree showing the evolution of different DNA sequences from a common ancestral sequence. An A to G substitution is highlighted in the first branching event.](images/03-msa-02-b.pdf-1.png) +--- +# Sequences evolve on a tree +
+
+![A schematic of a phylogenetic tree showing the evolution of different DNA sequences from a common ancestral sequence. A deletion of a T is highlighted in the first branching event.](images/03-msa-02-c-deleteT.pdf-1.png) +--- +# Sequences evolve on a tree +
+
+![A schematic of a phylogenetic tree showing the evolution of different DNA sequences from a common ancestral sequence. An insertion of TT is highlighted in one of the branches.](images/03-msa-02-d-insertTT.pdf-1.png) +--- +# Sequences evolve on a tree +
+
+![A schematic of a phylogenetic tree showing the evolution of different DNA sequences from a common ancestral sequence. An A to G substitution is highlighted in one of the branches.](images/03-msa-02-e-AtoG.pdf-1.png) +--- +# Sequences evolve on a tree +
+
+![A schematic of a phylogenetic tree showing the evolution of different DNA sequences from a common ancestral sequence. An insertion of a T is highlighted in one of the branches.](images/03-msa-02-f-insertT.pdf-1.png) +--- +# Sequences evolve on a tree +
+
+![A schematic of a phylogenetic tree showing the evolution of different DNA sequences from a common ancestral sequence. Two T to G substitutions have occurred on one branch.](images/03-msa-02-g-2xTtoG.pdf-1.png) +--- +# Sequences evolve on a tree +
+
+![A schematic of a phylogenetic tree showing the evolution of different DNA sequences from a common ancestral sequence. A C to A substitution has occurred on one branch.](images/03-msa-02-h-CtoA.pdf-1.png) +--- +# Sequences evolve on a tree +
+
+![A schematic of a phylogenetic tree showing the evolution of different DNA sequences from a common ancestral sequence. A deletion of a T has occurred on one branch.](images/03-msa-02-i-deleteT.pdf-1.png) +--- +# Sequences evolve on a tree +
+
+![A schematic of a phylogenetic tree showing the evolution of different DNA sequences from a common ancestral sequence. An A to C substitution has occurred on one branch.](images/03-msa-02-j-AtoC.pdf-1.png) +--- +# Let's align these +
+
+Gaps don't remain in the history so we only have +
+
+.image-50[ ![Five unaligned DNA sequences are arranged vertically. Nucleotides are colour coded G = orange, C = blue, A = green, T = pink](images/03-msa-03-a-unaligned.pdf-1.png) ] +--- +# A good alignment +
+
+This alignment reflects truth +
+
+.image-50[ ![A possible alignment of five DNA sequences arranged vertically. Gaps have been introduced to align homologous sites. Nucleotides are colour coded G = orange, C = blue, A = green, T = pink](images/03-msa-03-b-truth.pdf-1.png) ] +--- +# Although... +
+
+ +But this alignment also looks "good" (?) +
+
+.image-50[ ![A possible alignment of five DNA sequences arranged vertically. Gaps have been introduced to align homologous sites. Nucleotides are colour coded G = orange, C = blue, A = green, T = pink](images/03-msa-03-c-good1.pdf-1.png) ] + +--- + +# Although... +
+
+ +And so does this +
+
+.image-50[ ![A possible alignment of five DNA sequences arranged vertically. Gaps have been introduced to align homologous sites. Nucleotides are colour coded G = orange, C = blue, A = green, T = pink](images/03-msa-03-d-good2.pdf-1.png) ] + +--- + +# This is nonsensical +
+Only matches and indels! Win? +
+
+.image-50[ ![A possible alignment of five DNA sequences arranged vertically. Gaps have been introduced to align homologous sites. Nucleotides are colour coded G = orange, C = blue, A = green, T = pink](images/03-msa-03-e-nonsense.pdf-1.png) ] +
+
+Here, the indels do not make sense as having come from the same phylogenetic history. + +--- + +# Fixing errors +
+
+![A possible alignment of six DNA sequences arranged vertically drawing attention to a position, 1, where gaps have been introduced in nearly all sequences and, 2, gaps are not aligned but could be.](images/03-msa-04-a-fixingerrors-badly.pdf-1.png) + +At 1 a gap has been inserted in EVERY sequence. + +--- + +# Fixing errors +
+
+![A possible alignment of six DNA sequences arranged vertically drawing attention to a position, 1, where gaps have been introduced in nearly all sequences and, 2, gaps are not aligned but could be.](images/03-msa-04-a-fixingerrors-badly.pdf-1.png) + +At 2 the gaps don't line up, but they *could*. + +--- + +# Fixing errors +
+
+![A possible alignment of six DNA sequences arranged vertically drawing attention to a position 1 where gaps have been removed to improve alignment at position 2.](images/03-msa-04-b-fixingerrors-well.pdf-1.png) + + +This fixes both problems. + +--- + +# Rating alignments +
+This can be a bit of an art. +
+.left[You need to:] + +- balance the number of gaps with number of mismatches + +- notice when there are insertions / deletions that don't make sense, where there are alternative arrangements that do + +--- + +# Pair-wise Alignment +
+
+.center[*Dynamic Programming Approach*] + +--- + +# Sequence Similarity +
+
+To do any kind of comparison we need a distance or similarity measure. +
+Without it we can't say whether, e.g., *these* two species are more similar than *those* two. +
+- **Sequence Dissimilarity** + - Two main methods first: Hamming and *p*-distances, counting the number or proportion of differences between sequences +- **Edit cost** + - a measure of the amount of evolutionary "work" that has to be done to change one character state into another one +- **Likelihood** + - under a specific model of evolution, what is the probability that we would observe these sequences? + +--- + +# Hamming Distance +
+The *Hamming* distance between two sequences is just the number of differences between them. +
+It makes no distinction between substitutions or insertions/deletions, and not between transitions and transversions. +
+
+.image-40[ ![An alignment of two DNA sequences. Positions that are not homologous are indicated by an asterisk and assigned the value 1.](images/03-msa-05-hamming-distance.pdf-1.png) ] + +Sequence length: 20 +

Number of differences: 5

+ +

Hamming Distance = 5

+

P-distance = 5/20 = 0.25 or 25%

+--- + +# Edit Cost +
+
+We use a matrix of costs to describe how much evolutionary "work" must be done to convert one character to another. +
+
+.pull-left[ + +This is the *Edit cost matrix*. + +|-|A|C|G|T|-| +|------|--------------------| +|**A**|0|1|2|1|5| +|**C**|1|0|1|2|5| +|**G**|2|1|0|1|5| +|**T**|1|2|1|0|5| +|**-**|5|5|5|5|n/a| +] +.pull-right[ + + +

.image-75[ + ![An alignment of two DNA sequences. Positions that are not homologous are indicated by an asterisk and assigned the values 1, 2 or 5.](images/03-msa-06-edit-distance.pdf-1.png) +]

+

Complete edit-cost = 1 + 1 + 2 + 1 + 5 = 10

+] +
+ + + +
+Using edit costs we can describe better the relationships between sequences: for example it's less common for an *A* to change to a *G* in the above than for an *A* to change to a *T*, in turn less common than for an *A* to remain as an *A*. +-- + +Also note we have introduced a bigger cost for aligning any nucleotide with a gap, reflecting our believe that insertion/deletion events are less common than substitutions. + +--- +# What price a gap? +
+
+There are two basic methods for assigning a cost *c* to a gap of length *g* in a sequence. +
+
+**Linear cost**: \\(c = -dg\\) + where \\(d\\) is the _gap open penalty_; +
+
+**Affine cost**: \\(c = -d - (g-1)e\\) + where \\(e\\) is the _gap extension penalty_. +
+
+Typical values are \\(d = 10, e = 0.1\\). +
+
+The affine gap cost method is the most complex method we can use in order to solve the alignment of two sequences quickly. +
+
+More complex models prohibit the use of dynamic programming to solve the alignment (and must use _heuristics_). + +--- +# Numbers of alignments + +For two sequences of length \\(x\\) and \\(y\\), there are \\(\frac{(x+y)!}{x!\ y!}\\) possible alignments. +
+For three sequences, of length \\(x\\), \\(y\\) and \\(z\\) say, there are \\(\frac{(x+y+z)!}{x!\ y!\ z!}\\) alignments. +
+For \\(n\\) sequences of length 10, this increases rapidly: + +| n | Hash alignments | +|-----|------------------| +| 2 | 184756 | +| 3 | 5.55 × 10^12 | +| 4 | 4.71 × 10^21 | +| 5 | 4.83 × 10^31 | +| 6 | 3.64 × 10^42 | +| 7 | 1.45 × 10^54 | +| 8 | 2.38 × 10^66 | +| 9 | 4.94 × 10^85 | +| 10 | 2.35 × 10^92 | + + +
+In general it is not possible - even with really fast computers - to guarantee optimal multiple alignments, even with simple costing schemes. +--- +# Dynamic Programming +
+
+.left[Dynamic Programming (DP) is a common method to solve many types of problems, including pairwise sequence alignment. ] +
+- Solves local problems optimally +- Amalgamates these into globally optimal complete solutions +- "Fast" +--- +# DP overview +
+
+Dynamic Programming solves problems by breaking them down recursively into (slightly) smaller problems. + +![Schematic representation of dynamic programming showing how the process solves a problem by breaking it down and finding optimal partial solutions that can be used to infer the full solution.](images/DPOverviewDiagram.png) + +In terms of sequence alignment, this comes down to basing alignment of two sequences up to positions *i* and *j* in terms of the best alignments yet found for the two sequences, up to positions \\(i-1,j-10\\), \\(i,j-1\\), and \\(i-1,j\\). +--- +#DP overview (cont.) +
+
+![Schematic representation of how alignment of two DNA sequences progresses. Three scenarios are shown: 1. both sequences are identical 2. the sequences differ and a gap is introduced in the top sequence 3. the sequences differ and a gap is introduced in the bottom sequence.](images/03-msa-10-advancing-seqs.pdf-1.png) + +--- +# Dynamic Programming alignment + +1. Array two sequences along the top and left sides of a cost matrix. +2. Fill in the cells of the matrix from top-left to bottom right. + At each stage find the minimum cost sub-alignment and add to it: +

2.1 Find the maximum score from the previous cells including gap costs;

+

2.2 Put the result into this new cell;

+

2.3 Note which cell we chose with a pointer or reference.

+
+ +.image-25[ ![Schematic of the dynamic programming alignment described on the slide](images/03-DPcell.png) ] + + +We add the cost of the best solution to the previous cells (above, to the left and above-left) to the best possible score for this cell. + +--- + +#Alignment example +
+
+We will align two amino acid sequences next: +
+
+Input sequences: +
+
+.center[ +| x | HEAGAWGHEE | +|---|---| +| **y** | **PAWHEAE** | +] + +--- +# Filling in the cost matrix +
+
+Edit costs for these two sequences: +
+ +| | H | E | A | G | A | W | G | H | E | E | +|------|----|----|----|----|----|----|----|----|----|----| +| **P** | -2 | -1 | -1 | -2 | -1 | -4 | -2 | -2 | -1 | -1 | +| **A** | -2 | -1 | 5 | 0 | 5 | -3 | 0 | -2 | -1 | -1 | +| **W** | -3 | -3 | -3 | -3 | -3 | 15 | -3 | -3 | -3 | -3 | +| **H** | 10 | 0 | -2 | -2 | -2 | -3 | -2 | 10 | 0 | 0 | +| **E** | 0 | 6 | -1 | -3 | -1 | -3 | -3 | 0 | 6 | 6 | +| **A** | -2 | -1 | 5 | 0 | 5 | -3 | 0 | -2 | -1 | -1 | +| **E** | 0 | -6 | -1 | -3 | -1 | -3 | -3 | 0 | 6 | 6 | + +--- +# Dynamic Programming: fill in +
+
+![Cost matrix of possible alignments of the amino acid sequences HEAGAWGHEE on the x-axis and PAWHEAE on the y axis. Arrows indicate the direction taken to reach each score.](images/03-msa-08-a-DP-forward.pdf-1.png) +--- + +# Dynamic Programming: backtrack +
+
+![Cost matrix of possible alignments of the amino acid sequences HEAGAWGHEE on the x-axis and PAWHEAE on the y axis. The optimal alignment is indicated in red. Arrows indicate the direction taken to reach each score.](images/03-msa-08-b-DP-backtrack.pdf-1.png) + +--- +# Alignments are paths in the table +
+
+![Cost matrix of possible alignments of the amino acid sequences HEAGAWGHEE on the x-axis and PAWHEAE on the y axis. Arrows indicate the direction taken to reach this alignment.](images/03-msa-09-alignments-are-paths.pdf-1.png) +--- +# Conclusion of simple alignment +
+
+Aligned sequences: +
+
+ +| | | | | | | | | | | | | +|---------|-----|-----|-----|-----|-----|-----|-----|-----|-----|-----|-----| +| $$s_{1}$$ | H | E | A | G | A | W | G | H | E | - | E | +| $$s_{2}$$ | - | - | P | - | A | W | - | H | E | A | E | +| | -5 | -5 | -1 | -5 | +5 | +15 | -5 | +10 | +6 | -5 | +6 | + +
+
+Total cost = 16 + +--- +# Generalising this approach +
+
+Without too much effort it is possible to generalise this approach: +
+
+**Needleman-Wunsch** is used for _local_ alignment; +
+
+**Smith-Waterman** is used for _global_ alignment; +
+
+**longest match** can be found by setting high match costs and large negative mis-match and gap costs; +
+
+**BLAST** uses this system once good patching pairs have been found; +
+
+**Affine gap scores** as mentioned, accounting for gap opening and gap extension penalties to differ. + +--- +#Properties of DP Pairwise Alignment +
+
+- Pairwise Sequence Alignment takes an amount of *time* that is proportional to the number of cells in the table, which is roughly the product of the lengths of the two sequences. + + That means it is \\(O(nm)\\) if the lengths of the sequences are \\(n\\) and \\(m\\); usually as these are about the same we can write \\(O(n^{2})\\): it's *quadratic in sequence length*. + +- The amount of space required is also quadratic in \\(n\\). + There is a linear-space version of the DP method (which is rarely used as it takes longer). + +- This solution is globally optimal. It will always produce an optimal alignment, though there may be more than one. + +- With more sequences, the size of the table increase: + For \\(k\\) sequences the algorithm is \\(O(n^{k})\\): not practical. + +--- + +# Multiple Sequence Alignment: Aligning groups of sequences using heuristics +
+
+.center[*Aligning multiple sequences*] +--- +#Aligning multiple sequences +
+
+We cannot check all possible alignments (there are simply too many), so we must use make a compromise. +
+
+We will use pairwise alignment (which is easy) and build up a multiple sequence alignment from pairs of sequences. +
+
+These *heuristic* methods are used in Clustal, GCG and others. +--- +# Progressive Alignment +
+
+.left[The process is quite simple:] + +1. Align all pairs of sequences using DP (dynamic programming). +2. Create a distance matrix based on the alignments. +3. Form a *guide tree* from the distance matrix. This is not the same as a phylogenetic tree, nor should it be interpreted as one! +4. Progressively align the pairs of sequences with DP, creating summary (consensus) sequences as we go. +--- +# MAFFT, Muscle, T-COFFEE, k-align +
+
+- Clustal is not that great to be honest +- Other very good automatic alignment methods exist, such as Muscle, T-Coffee, k-align, di-align. +- My current favourites are Muscle and MAFFT. + +--- +# MAFFT +
+
+ +"MAFFT offers various multiple alignment strategies. They are classified into three types, (a) the progressive method, (b) the iterative refinement method with the WSP score, and (c) the iterative refinement method using both the WSP and consistency scores. In general, there is a tradeoff between speed and accuracy. The order of speed is a > b > c, whereas the order of accuracy is a < b < c. The results of benchmarks can be seen here. The following are the detailed procedures for the major options of MAFFT." +
+
+
+
+
+Source: MAFFT is available at https://mafft.cbrc.jp/; first paper 10.1093/nar/gkf436 + +--- +# MAFFT algorithms overview +
+
+![Flow chart giving an overview of algorithms used by the program MAFFT to convert distance matrices into alignments. The flow chart is described in the video recording at 37:52.](images/03-msa-11-MAFFT-1.png) +--- + +# *Anolis* example +
+
+![Screenshot from the program SeeView showing a multiple sequence alignment of Anolis species. DNA sequences are aligned vertically and nucleotides are colour coded. Aligned sites can be identified by solid lines of colour that run from top to bottom of the image. Full description included in the video recording at 40:06.](images/AnolisPartialAlignment2.png) + +Around site 982-990 there is a 2 bp gap in all sequences. + +--- +# *Anolis* example +
+
+![Screenshot from the program SeeView showing a multiple sequence alignment of Anolis species. DNA sequences are aligned vertically and nucleotides are colour coded. Aligned sites can be identified by solid lines of colour that run from top to bottom of the image. Full description included in the video recording at 40:06.](images/AnolisPartialAlignmentGapsAligned.png) + +Here I have lined up these gaps: more substitutions? but makes sense. +--- + +# *Anolis* example +
+
+![Screenshot from the program SeaView showing a multiple sequence alignment of Anolis species. DNA sequences are aligned vertically and nucleotides are colour coded. Aligned sites can be identified by solid lines of colour that run from top to bottom of the image. Full description included in the video recording at 40:06.](images/AnolisPartialAlignmentGapsAlignedTidied.png) + + +We can remove the gap-only sites now. There's more to do! + +--- +# What's Next? +
+
+Once your sequences are properly aligned, they can be used for _phylogenetic analysis_. +--- +# Thank you! +
+
+.center[*Next: building trees from distances!*] + +--- diff --git a/topics/evolution/tutorials/abc_intro_phylo-networks/images b/topics/evolution/tutorials/abc_intro_phylo-networks/images new file mode 120000 index 00000000000000..77c95679a02665 --- /dev/null +++ b/topics/evolution/tutorials/abc_intro_phylo-networks/images @@ -0,0 +1 @@ +../abc_intro_phylo/images \ No newline at end of file diff --git a/topics/evolution/tutorials/abc_intro_phylo-networks/slides.html b/topics/evolution/tutorials/abc_intro_phylo-networks/slides.html new file mode 100644 index 00000000000000..6762006707acf2 --- /dev/null +++ b/topics/evolution/tutorials/abc_intro_phylo-networks/slides.html @@ -0,0 +1,73 @@ +--- +layout: tutorial_slides +logo: shared/images/biocommons-utas.png +title: "Phylogenetics - Back to Basics - Phylogenetic Networks" +zenodo_link: "" +subtopic: phylogenetics +priority: 6 +contributors: + - mcharleston +--- + +#About these slides +
+
+The following phylogenetic networks of _Anolis_ species were generated in SplitsTree. They represent selected screenshots of networks explored as a live demo in the video associated with these slides. +--- +#Uncorrected P model +
+Phylogenetic network of _Anolis_ species created in SplitsTree using the uncorrected P model. +![Circular phylogenetic network with many branches splitting outwards from a central point to multiple Anolis species at the periphery. The network is oriented so that the species Diplolaemus darwinii and Phenacosaurus acutirostrus appear on the right hand side.](images/06-networks-uncorrectedP.png) +--- +#Exploring splits +
+Click on the branches of the network to explore different splits. Here _A. ahli_ appears in two different splits highlighted in yellow. + +.pull-left[ ![Screen shot of a circular phylogenetic network with multiple branches splitting out from a central point. At the bottom left of the image a split containing A. ahli, A. equestris2 and several other species is highlighted in yellow](images/06-networks-explore_split_1.png) ] + +.pull-right[ ![Screen shot of a circular phylogenetic network with multiple branches splitting out from a central point. At the top left of the image a split containing A. ahli, A. bimaculatus and several other species is highlighted in yellow](images/06-networks-explore_split_2.png) ] + +--- +#Let's follow a split +
+This is the network made using the uncorrected p model. We will follow the split containing _A. lineatopus_ and _A. limifrons_ through different models. + +![Screen shot of a circular phylogenetic network with multiple branches splitting out from a central point. At the bottom left of the image a split containing A. lineatopus and A. limifrons is highlighted in yellow](images/06-networks-unP-follow-split.png) + +--- +#Jukes Cantor Model +
+These networks were created using the Jukes Cantor model. Note that the species _A. limifrons_ appears in two alternate splits. + +.pull-left[ ![Screen shot of a circular phylogenetic network with multiple branches splitting out from a central point. At the bottom left of the image a split containing A. limifrons and A. humilis is highlighted in yellow](images/06-networks-JC_split1.png) ] + +.pull-right[ ![Screen shot of a circular phylogenetic network with multiple branches splitting out from a central point. At the bottom left of the image a split containing A. lineatopus and A. limifrons is highlighted in yellow](images/06-networks-JC_split2.png) ] +--- +#HKY85 Model +
+This network was created with the HKY85 model. Note that _A. limfrons_ is paired with _A. humilis_ in this split. + +![Screen shot of a circular phylogenetic network with multiple branches splitting out from a central point. At the top left of the image a split containing A. humilis and A. limifrons is highlighted in yellow](images/06-networks-HKY85_split.png) +--- +#Bootstrapped network +
+![Screen shot of a circular phylogenetic network with multiple branches splitting out from a central point. Each branch is annotated with a value between 0-100](images/06-networks-bootstrap.jpg) +--- +#Bootstrapped network - a closer look +
+Let's explore our two splits again + +.pull-left[ ![Close up screen shot of a section of a circular phylogenetic network with multiple branches splitting out from a central point. The split containing A. humilis and A. limifrons is highlighted in yellow and is annotated with the value 99.99](images/06-networks-bootstrap-split1.jpg) ] + +.pull-right[ ![Close up screen shot of a section of a circular phylogenetic network with multiple branches splitting out from a central point. The split containing A. lineatopus and A. limifrons is highlighted in yellow and is annotated with the value 38.3](images/06-networks-bootstrap-split2.jpg) ] +--- +#Bootstrapped network - a closer look +
+This the centre of our bootstrapped network. Note that the values are smaller. +![Close up screen shot of a section of a circular phylogenetic network with multiple branches splitting out from a central point. The splits form a star like shape and are annotated with numbers](images/06-networks-bootstrap-centre.jpg) + + +--- +# Thank you! + +--- \ No newline at end of file diff --git a/topics/evolution/tutorials/abc_intro_phylo-terminology/images b/topics/evolution/tutorials/abc_intro_phylo-terminology/images new file mode 120000 index 00000000000000..77c95679a02665 --- /dev/null +++ b/topics/evolution/tutorials/abc_intro_phylo-terminology/images @@ -0,0 +1 @@ +../abc_intro_phylo/images \ No newline at end of file diff --git a/topics/evolution/tutorials/abc_intro_phylo-terminology/slides.html b/topics/evolution/tutorials/abc_intro_phylo-terminology/slides.html new file mode 100644 index 00000000000000..8f57d4198b07e9 --- /dev/null +++ b/topics/evolution/tutorials/abc_intro_phylo-terminology/slides.html @@ -0,0 +1,73 @@ +--- +layout: tutorial_slides +logo: shared/images/biocommons-utas.png +title: "Phylogenetics - Back to Basics - Terminology" +zenodo_link: "" +subtopic: phylogenetics +priority: 2 +contributors: + - mcharleston +--- + +# Phylogenetic tree of hexapods + +![Circular phylogenetic tree of hexapods (insects). Clades are colour coded and labelled with common names e.g. ‘Fleas’. Silhouettes of representative species are shown around the outside of the tree.](images/Hexapoda_phylogenetic_tree.png) +.right[https://doi.org/10.1371/journal.pone.0109085; CCBY 4.0 DEED license] + +--- +# Beginning with the leaves of the tree only + +![The start of a phylogenetic tree schematic. Five rectangles labelled as Extant taxa 1-5 are evenly distributed horizontally.](images/01-leaves-only.png) + +--- + + +![Schematic of a phylogenetic tree composed of five extant taxa (white boxes) and their hypothetical ancestors (purple boxes). Coloured lines represent relationships between taxa. The root of the tree is a hypothetical common ancestor at the top of the image and extant taxa are at the bottom. The tree branches from this common ancestor twice on the left to form two clades containing extant taxa 1 and 2; or extant taxa 3. The tree branches once to the right to form a clade containing extant taxa 4 and 5.](images/02-rooted-tree.png) + +--- + + +![Schematic of a phylogenetic tree where internal nodes are represented by purple circles and extant taxa by white rectangles. Coloured lines represent the relationship between nodes and taxa. The root of the tree is at the top of the image and indicated by an arrow. The tree branches multiple times from this ancestor through additional ancestors forming three clades composed of five extant taxa at the bottom of the image.](images/03-this-is-the-root-only.png) + +--- + +![Schematic of a phylogenetic tree where internal nodes are represented by purple circles and extant taxa by white rectangles. Coloured lines represent the relationship between nodes and taxa. The internal nodes are indicated by an arrow and labelled with “Internal nodes represent hypothetical ancestors”. The root of the tree is at the top of the image. The tree branches multiple times from this node through additional internal nodes forming three clades composed of five extant taxa at the bottom of the image.](images/04-hypothetical-ancestors-only.png) + +--- + +![Schematic of a phylogenetic tree where internal nodes are represented by purple circles and extant taxa by white rectangles. Coloured lines represent the relationship between nodes and taxa. An arrow and text box indicates that yellow lines are “Internal edges/branches show the relationships between all taxa”. The root of the tree is at the top of the image. The tree branches multiple times from this node through additional internal nodes forming three clades composed of five extant taxa at the bottom of the image.](images/05-internal-branches-only.png) + +--- + +![Schematic of a phylogenetic tree where internal nodes are represented by purple circles and extant taxa by white rectangles. Coloured lines represent the relationship between nodes and taxa. An arrow and text box indicates that green lines connecting extant taxa to their nearest common ancestor are “Pendant edges/branches are always in every tree where the taxa differ so are not interesting”. The root of the tree is at the top of the image. The tree branches multiple times from this node through additional internal nodes forming three clades composed of five extant taxa at the bottom of the image.](images/06-pendant-branches-only.png) + +--- + +![Schematic of a phylogenetic tree where internal nodes are represented by purple circles and extant taxa by white rectangles. Coloured lines represent the relationship between nodes and taxa. The root of the tree is at the top of the image. The tree branches multiple times from this node through additional internal nodes forming three clades composed of five extant taxa at the bottom of the image. Red dotted lines are drawn alongside the branches that form a path between extant taxa 2 and 3, and the root and extant taxa 4. Boxed text and red arrows indicate that the dotted lines are “Evolutionary distance is path length”.](images/07-path-lengths-only.png) + +--- + +![Schematic of a phylogenetic tree where internal nodes are represented by purple circles and extant taxa by white rectangles. Coloured lines represent the relationship between nodes and taxa. The root of the tree is at the top of the image. The tree branches multiple times from the root through additional internal nodes forming three clades composed of five extant taxa at the bottom of the image. A yellow dotted line circles a branch containing three extant taxa and two internal nodes on the left. Text states “The leaves and internal nodes all descendant from a given internal node form a subtree or clade’.](images/08-clades-only.png) + +--- + +![Schematic of a phylogenetic tree where internal nodes are represented by purple circles and extant taxa by white rectangles. Coloured lines represent the relationship between nodes and taxa. The root of the tree is at the top of the image. The tree branches multiple times from the root through additional internal nodes forming three clades composed of five extant taxa at the bottom of the image. These five extant taxa are highlighted by a blue box and labelled as the ingroup.](images/09-ingroup-only.png) + +--- + +![Schematic of a phylogenetic tree where internal nodes are represented by purple circles and extant taxa by white rectangles. Coloured lines represent the relationship between nodes and taxa. The root of the tree is at the top of the image. The tree branches multiple times from the root through additional internal nodes forming three clades composed of five extant taxa at the bottom of the image. Two additional taxa appear to the right of the tree and are labelled as the outgroup.](images/10-outgroup-only.png) + +--- + +![Schematic of a phylogenetic tree where internal nodes are represented by purple circles and extant taxa by white rectangles. Coloured lines represent the relationship between nodes and taxa. The root of the tree is at the top of the image. The tree branches multiple times from the root through additional internal nodes forming three clades composed of five extant taxa at the bottom of the image. Two additional taxa appear to the right of the tree and are labelled as the outgroup. This outgroup is joined to the tree by blue lines pass through “A confident common ancestor to the ingroup and outgroup”.](images/11-rooted-only.png) + +--- + +![Schematic of a phylogenetic tree where features such as nodes/taxa, edges/branches are annotated and colour coded. The root of the tree is at the top of the image and the tree branches into two clades as you move towards the bottom of the image. The clades are formed of hypothetical common ancestors and five extant taxa which are labelled as the ‘in group’. Two additional taxa are appended to the right hand side of the tree and are labelled as the outgroup.](images/11-rooted.png) + +--- +# Thank you! + +Next - alignment! + +--- diff --git a/topics/evolution/tutorials/abc_intro_phylo-trees/graphics b/topics/evolution/tutorials/abc_intro_phylo-trees/graphics new file mode 120000 index 00000000000000..77c95679a02665 --- /dev/null +++ b/topics/evolution/tutorials/abc_intro_phylo-trees/graphics @@ -0,0 +1 @@ +../abc_intro_phylo/images \ No newline at end of file diff --git a/topics/evolution/tutorials/abc_intro_phylo-trees/slides.html b/topics/evolution/tutorials/abc_intro_phylo-trees/slides.html new file mode 100644 index 00000000000000..ace59d9db98215 --- /dev/null +++ b/topics/evolution/tutorials/abc_intro_phylo-trees/slides.html @@ -0,0 +1,533 @@ +--- +layout: tutorial_slides +logo: shared/images/biocommons-utas.png +title: "Phylogenetics - Back to Basics - Building Trees" +zenodo_link: "" +subtopic: phylogenetics +priority: 4 +contributors: + - mcharleston +--- + +# Why _build_ trees? +
+The main reason we end up building a tree is that searching tree space for an optimal one takes too long when the number of taxa gets large. +
+.reduce70[ +| _n_ | # trees | yes, but how much is that _really_? +| --- |---|:--| +| 3 | 3 | enumerable by hand | +| 4 | 15 | enumerable by hand | +| 5 | 105 | enumerable by hand on a rainy day | +| 6 | 945 | enumerable by computer | +| 7 | 10395 | still searchable very quickly on computer | +| 8 | 135135 | a bit more than the number of hairs on your head | +| 9 | 2027025 | population of Sydney living west of Parramatta | +| 10 | 34459425 | \\(\approx\\) upper limit for exhaustive searching; about the number of possible combinations of numbers in the National Lottery | +| 20 | $$8.2\times 10^{21}$$ | \\( \approx \\) upper limit for branch-and-bound searching | +| 48 | $$ 3.21 \times 10^{70} $$ | \\(\approx\\) number of particles in the universe | +| 136 | $$ 2.11 \times 10^{267} $$ | number of trees to choose from in the ``Out of Africa'' data¹ | +] + +.footnote[ +¹ Vigilant et al., 1991 +] + +--- +# Tree space is worse than "space" space +
+![Hubble Space Telescope image of two colliding galaxies](graphics/NGC2207+IC2163.jpg) +
+NGC 2207 and IC 2163 galaxies colliding. +
+.footnote[Source: https://commons.wikimedia.org/wiki/Commons:Featured_pictures/Astronomy] +--- +# Why should building work then? +
+
+Some parts of a phylogeny can be confidently accepted: when there are two species much more similar to each other than they are to any other species, we can confidently say that they are likely to be each other's closest relatives, in the set of species of interest. +
+
+If molecular sequences evolve at a nice steady rate - the "molecular clock" hypothesis - and if there's neither too little nor too much change, this can be good enough. + +--- +# Distances from trees +
+
+Before we talk about building a tree from distances, we need to think about how distances are reflected by trees. +
+
+The most natural way to infer distances from trees is by adding the lengths of branches between each pair of nodes. +
+
+Such distances are called *patristic* distances (I don't know why). +--- +# Patristic Distances +
+
+![Schematic of a phylogenetic tree containing internal nodes a, b, c. Nodes d, e, f, g and h form the tips. The branches of the tree are annotated with numbers that represent the evolutionary distance between species. Described at 3:05 in the video recording.](graphics/04-NonUltrametricTree1-1.png) + +Rooted binary tree with branch lengths +--- +# Patristic Distances +
+
+![Schematic of a phylogenetic tree containing internal nodes a, b, c. Nodes d, e, f, g and h form the tips. The distance between nodes f and g via the internal node c is highlighted with a red dotted line and annotated with the value 0.045. Described at 3:50 in the video recording.](graphics/04-NonUltrametricTree2-1.png) +
+Patristic distance between tips f and g is 0.02 + 0.025 = 0.045 +--- +# Patristic Distances +
+
+![Schematic of a phylogenetic tree containing internal nodes a, b, c. Nodes d, e, f, g and h form the tips. The distance between nodes g and h via the internal nodes c and b is highlighted with a red dotted line and annotated with the value 0.07. Described at 4:03 in the video recording.](graphics/04-NonUltrametricTree3-1.png) +
+Patristic distance between tips g and h is 0.025 + 0.01 + 0.035 = 0.07 +--- +# Patristic Distances +
+
+![Schematic of a phylogenetic tree containing internal nodes a, b, c. Nodes d, e, f, g and h form the tips. The distance between nodes e and f the internal node a, the root and internal nodes b and c is highlighted with a red dotted line and annotated with the value 0.085.Described at 4:10 in the video recording.](graphics/04-NonUltrametricTree4-1.png) +
+Patristic distance between tips e and f is 0.02 + 0.015 + 0.02 + 0.01 + 0.02 = 0.085 + +--- +# Tree-like distances are easy +
+
+A set of distances between the tips (taxa) that match the patristic distances of some tree is called **tree-like**. +
+
+Given tree-like distances, most tree construction methods will work. +
+
+Yes, _most_ - not all! + +--- + +# Clock-like +
+
+The easiest possible distance data to work with are those where the distance from the root to every tip is the same. +
+
+Such trees (and the distances derived from them) are called _ultrametric_ (that is, they have the same root-to-tip distance for every tip). + +--- + +# Clock-like +
+
+For example, suppose this is the _true_ tree relating some species of interest, with actual branch lengths as labelled: +
+
+![Schematic of a phylogenetic tree containing internal nodes a, b, c. Nodes d, e, f, g and h form the tips. The branches of the tree are annotated with numbers that represent the evolutionary distance between species. The distance between the root and tip on all branches is the same. Described at 4:44 in the video recording.](graphics/04-UltrametricTreeScaled-1.png) + +--- + +# These distances are _ultrametric_ +
+.pull-left[ +.image-90[ ![Schematic of a phylogenetic tree containing internal nodes a, b, c. Nodes d, e, f, g and h form the tips. The branches of the tree are annotated with numbers that represent the evolutionary distance between species. The distance between the root and tip on all branches is the same. Described at 4:44 in the video recording.](graphics/04-UltrametricTree-1.png) ] +] +.pull.right[ +* The maximum root-to-tip distance is called the _height_ of the tree. +* Here, the root-to-tip distances are all the same. A set of distances with this property is called _ultrametric_. +* If the distances represent evolutionary time accurately and all the tips are in the present, then we should expect this property. +* Reconstructing trees from ultrametric distances is _super easy_. +] +--- + +# These distances are _ultrametric_ +
+
+.left-column40[ + ![Schematic of a phylogenetic tree containing internal nodes a, b, c. Nodes d, e, f, g and h form the tips. The branches of the tree are annotated with numbers that represent the evolutionary distance between species. The distance between the root and tip on all branches is the same. Described at 4:44 in the video recording.](graphics/04-UltrametricTree-1.png) +] +.right-column60[ +Distance matrix: +.center[ +| | d | e | f | g | h | +|:---:|:---:|:---:|:---:|:---:|:---:| +|**d** | 0 | 0.07 | 0.1\* | 0.1 | 0.1 | +|**e** | 0.07 | 0 | 0.1 | 0.1 | 0.1 | +|**f** | 0.1 | 0.1 | 0 | 0.04 | 0.06 | +|**g** | 0.1 | 0.1 | 0.04 | 0 | 0.06 | +|**h** | 0.1 | 0.1 | 0.06 | 0.06 | 0 | +] +.reduce70[\* Note; this is twice the root-to-tip distance.] +] + + +.pull-bottom[ +Any ultrametric tree satisfies the three-point condition (for rooted trees): for any three tips x, y, z, the larger two pairwise distances of D(x, y), D(x,z), D(y,z) will be equal. +] + +--- + +# These distances are _ultrametric_ +
+
+.left-column40[ + ![Schematic of a phylogenetic tree containing internal nodes a, b, c. Nodes d, e, f, g and h form the tips. The branches of the tree are annotated with numbers that represent the evolutionary distance between species. The distance between the root and tip on all branches is the same. Described at 4:44 in the video recording.](graphics/04-UltrametricTree-1.png) +] +.right-column60[ +Distance matrix: +.center[ +| | d | e | f | g | h | +|:---:|:---:|:---:|:---:|:---:|:---:| +|**d** | 0 | 0.07 | 0.1 | 0.1 | 0.1 | +|**e** | - | 0 | 0.1 | 0.1 | 0.1 | +|**f** | - | - | 0 | 0.04 | 0.06 | +|**g** | - | - | - | 0 | 0.06 | +|**h** | - | - | - | - | 0 | +] +
+Since this is a symmetric matrix we usually just show half of it... +] + +--- + +# These distances are _ultrametric_ +
+
+.left-column40[ + ![Schematic of a phylogenetic tree containing internal nodes a, b, c. Nodes d, e, f, g and h form the tips. The branches of the tree are annotated with numbers that represent the evolutionary distance between species. The distance between the root and tip on all branches is the same. Described at 4:44 in the video recording.](graphics/04-UltrametricTree-1.png) +] +.right-column60[ +Distance matrix: +.center[ +| | e | f | g | h | +|:---:|:---:|:---:|:---:|:---:|:---:| +|**d** | 0.07 | 0.1 | 0.1 | 0.1 | +|**e** | - | 0.1 | 0.1 | 0.1 | +|**f** | - | - | 0.04 | 0.06 | +|**g** | - | - | - | 0.06 | +] +
+... and only the non-zero entries. +] + +--- +# The general approach +
+
+![Flow chart illustrating how sequence alignment data or dis/similarity measures are used to calculate and calculate phylogenetic distances. Colours and shapes are used to differentiate different sections of the flowchart, guiding the viewer through each step from left to right. The flowchart begins with Sequence Alignment or Dis/Similarity Measures. These are used to form a distance matrix (D) which is used to select two nodes (x and y) forming a new node z. The distance matrix is updated with the new node z until no further nodes can be formed. Described at 8:20 in the video recording.](graphics/TreeConstruction.drawio.png) + +--- + +# Our distances +
+
+* We get distances from molecular sequences once they have been _aligned_. +* There are various ways to compare aligned sequences to obtain distances: + - uncorrected "p-distance" -- the proportion of sites that differ between the two sequences; + - the Jukes-Cantor (JC69) correction, which takes into account of multiple character state changes through time; + - The Hasegawa-Kishino-Yano (HKY85) model, which also allows for variation in nucleotide frequencies \\(\pi_{A}, \pi_{C}, \pi_{G}, \pi_{T}\\) as well as different transition/transversion rates; + - and more. + +--- + +# Jukes-Cantor / JC69 +
+This model has one single parameter, assuming that the base frequencies are each 25%: \\(\pi_{A}=\pi_{G}=\pi_{C}=\pi_{T}=0.25\\) +
+
+The rate matrix looks like this: +
+.image-40[ ![Rate matrix representing the Jukes Cantor model. The matrix has the nucleotides AGCT on both the x and y axis. The substitution rates are identical and represented by the character alpha. Asterisks are used as shorthand for values that make the row sums equal 0. Described at 11:31 in the video recording.](graphics/04-QJC69-1.png) ] +
+.reduce50[where the asterisk is a short-hand to make the row-sums equal 0.] +
+
+Under this model the expected number of substitutions between two sequences with a p-distance of \\(p\\) is +$$\hat{d} = \frac{-3}{4}\ln\left(1-\frac{4}{3}p\right)$$ + +--- +# Hasegawa-Kishino-Yano / HKY85 +
+This model allows for variation in nucleotide frequencies \\(\pi_{A}, \pi_{G}, \pi_{C}, \pi_{T}\\) as well as different transition/transversion rates using parameter \\(\kappa\\): +
+
+.image-40[ ![Rate matrix representing the HKY85 model. The matrix has the nucleotides AGCT on both the x and y axis. Variation in nucleotide frequencies πA; πG; πC; πT as well as different transition/transversion rates using parameter kappa. Described at 14:08 in the video recording.](graphics/04-QHKY85-1.png) ] +
+
+This model also allows for a correction to turn relative observed numbers of substitutions between the different bases into expected total number of substitutions between two sequences, but it is much more complex. + +--- + +# From alignment to distances +
+
+.left-column50[ + ![Screenshot from the program SeaView showing a multiple sequence alignment of Anolis species. DNA sequences are aligned vertically and nucleotides are colour coded. Aligned sites can be identified by solid lines of colour that run from top to bottom of the image. Full description included in the video recording at 40:06.](graphics/AnolisPartialAlignmentGapsAlignedTidied.png) +] +.right-column50[ + ![Figure representing a distance matrix (D) comparing sequences from A. acutus and A. aeneus.](graphics/04-AnolisDistanceMatrixCorner-1.png) +
+
+Once the alignment is complete, each pair of sequences is compared to give an estimated distance between them: this forms the distance matrix for tree building. +] +--- +# Example +
+
+_Ultrametric distances_ + +--- +# The true tree +
+
+![Schematic of an ultrametric phylogenetic tree containing internal nodes a, b, c. Nodes d, e, f, g and h form the tips. The branches of the tree are annotated with numbers that represent the evolutionary distance between species. The distance between the root and tip on all branches is the same.](graphics/04-TrueTreeUltrametric-1.png) + + +--- +# Original Distance Matrix +
+
+![Distance matrix representing the evolutionary distance between nodes d, e, f, g and h on a phylogenetic tree.](graphics/04-InitialDistancesUltrametric-1.png) +--- +# Original Distance Matrix +
+
+![Distance matrix representing the evolutionary distance between nodes d, e, f, g and h on a phylogenetic tree. The distance between g and f is highlighted in red and has a value of 0.04.](graphics/04-InitialDistancesUltrametric-2.png) +--- +
+
+
+
+
+![The letters d, e, f, g and h are equally distributed horizontally. They represent the tips of a phylogenetic tree. Described at 16:08 in the video recording.](graphics/04-TreeBuildStep1-1.png) +--- + + +![The letters d, e, f, g and h are equally distributed horizontally at the bottom of the image. They represent the tips of a phylogenetic tree. An internal node c sits above the tips and is connected to nodes f and g. Described at 16:08 in the video recording.](graphics/04-TreeBuildStep2-1.png) +--- +
+
+
+
+
+![The letters d, e, f, g and h are equally distributed horizontally at the bottom of the image. They represent the tips of a phylogenetic tree. An internal node c sits above the tips and is connected to nodes f and g. The distance from f to g via c is highlighted with a red dotted line and annotated with the value 0.04.Described at 16:08 in the video recording.](graphics/04-TreeBuildStep3-1.png) +
+
+The distance between f and g *D(f,g)* is the sum of the branch lengths on the path between them. +--- +
+
+
+
+
+ +![The letters d, e, f, g and h are equally distributed horizontally at the bottom of the image. They represent the tips of a phylogenetic tree. An internal node c sits above the tips and is connected to nodes f and g. The distance from f to g via c is highlighted with a red dotted line and annotated with the value 0.04. A blue dotted line represents the distance between f and h with the value 0.06. A green dotted line represents the distance between g and h with the value 0.06. Described at 16:08 in the video recording.](graphics/04-TreeBuildStep4-1.png) +
+
+Similarly the distances *D(f,h)* and *D(g,h)* are the sum of branch lengths. +--- +
+
+
+
+ +![The letters d, e, f, g and h are equally distributed horizontally at the bottom of the image. They represent the tips of a phylogenetic tree. An internal node c sits above the tips and is connected to nodes f and g. The distance from f to g via c is highlighted with a red dotted line and annotated with the value 0.04. A blue dotted line represents the distance between f and h with the value 0.06. A green dotted line represents the distance between g and h with the value 0.06. A brown line represents the path between c and h with the value 0.08. Described at 16:08 in the video recording.](graphics/04-TreeBuildStep5-1.png) +
+
+Therefore: +
+$$\mathbf{D(c,h)} = \frac{D(f,h)+D(g,h)-D(f,g)}{2}$$ + +--- +# Forming the next Distance Matrix +
+
+We need to remove columns and rows f and g, and add new column and row c. + +.image-75[ ![Distance matrix (D0) representing the evolutionary distance between nodes d, e, f, g on the y axis and e,f, g and h the x axis. Explanation provided at 19:00 in the video recording.](graphics/04-DistanceMatrixMod1-1.png) ] + +--- +# Forming the next Distance Matrix +
+
+The entries for *D(f,d), D(g,d)* etc get dropped. + +.image-75[ ![Distance matrix D1 representing the evolutionary distance between nodes d, e, f, g and c on the y-axis and nodes e, f, g, c and h on the x-axis. Some values have been struck out to illustrate how a matrix is sequentially updated during the tree building process. Explanation provided at 19:00 in the video recording.](graphics/04-DistanceMatrixMod2-1.png) ] +--- +# Forming the next Distance Matrix +
+
+We fill in the new entries using the formulae below: + +.image-75[ ![Distance matrix D1 representing the evolutionary distance between nodes d, e, f, g and c on the y-axis and nodes e, f, g, c and h on the x-axis. Some values have been struck out or replaced with a formula to illustrate how a matrix is sequentially updated during the tree building process. Explanation provided at 19:00 in the video recording.](graphics/04-DistanceMatrixMod3-1.png) ] +
+
+$$D(c,d) = \frac{1}{2}(D(f,d)+D(g,d)-D(f,g)) = \frac{1}{2}(0.1+0.1-0.04) = \mathbf{0.08}$$ +--- +# Forming the next Distance Matrix +
+
+We fill in the new entries using the formulae below: + +.image-75[ ![Distance matrix D1 representing the evolutionary distance between nodes d, e, f, g and c on the y-axis and nodes e, f, g, c and h on the x-axis. Some values have been struck out to illustrate how a matrix is sequentially updated during the tree building process. The distance from d to c is highlighted and is 0.08. Explanation provided at 19:00 in the video recording.](graphics/04-DistanceMatrixMod4-1.png) ] +
+
+$$D(c,e) = \frac{1}{2}(D(f,e)+D(g,e)-D(f,g)) = \frac{1}{2}(0.1+0.1-0.04) = \mathbf{0.08}$$ +--- +# Forming the next Distance Matrix +
+
+We fill in the new entries using the formulae below: + +.image-75[ ![Distance matrix D1 representing the evolutionary distance between nodes d, e, f, g and c on the y-axis and nodes e, f, g, c and h on the x-axis. Some values have been struck out to illustrate how a matrix is sequentially updated during the tree building. The distances from d to c and e to c are highlighted and are 0.08. Explanation provided at 19:00 in the video recording.](graphics/04-DistanceMatrixMod5-1.png) ] +
+
+$$D(c,h) = \frac{1}{2}(D(f,h)+D(g,h)-D(f,g)) = \frac{1}{2}(0.06+0.06-0.04) = \mathbf{0.04}$$ +--- +# Forming the next Distance Matrix +
+
+.image-75[ ![Distance matrix D1 representing the evolutionary distance between nodes d, e, f, g and c on the y-axis and nodes e, f, g, c and h on the x-axis. Some values have been struck out to illustrate how a matrix is sequentially updated during the tree building. The distances from d to c and e to c are highlighted and are 0.08. The distance between c and h is highlighted and is 0.04. Explanation provided at 19:00 in the video recording.](graphics/04-DistanceMatrixMod6-1.png) ] +--- +# Forming the next Distance Matrix +
+
+Completed new distance matrix: + +.image-50[ ![Distance matrix D1 representing the evolutionary distance between nodes d, e, c, and e, c and h on a phylogenetic tree.](graphics/04-NewDistanceMatrix1-1.png) ] + + + +--- + + +![Schematic of an ultrametric phylogenetic tree containing internal nodes a, b, c. Nodes d, e, f, g and h form the tips. The branches of the tree are annotated with numbers that represent the evolutionary distance between species. The distance between the root and tip on all branches is the same. Node c is circled in red and annotated with the words ‘Found this!’.](graphics/04-FoundC1-1.png) + + +--- + + +![Schematic of a phylogenetic tree containing internal nodes a and b. Nodes c, d, e, and h form the tips. The branches of the tree are annotated with numbers that represent the evolutionary distance between species. ](graphics/04-FoundC2-1.png) + + +Now we have reduced the problem by one taxon / sequence: we repeat until we have just two more taxa to join and that will give us the root! + +--- +# Non-clocklike trees +
+
+*Losing the ultrametric property* +--- +# Not perfectly clock-like +
+
+Now suppose this is the _true_ tree relating some species of interest, with actual branch lengths as labelled: +
+
+![Schematic of a rooted non-clocklike phylogenetic tree containing internal nodes a, b, c. Nodes d, e, f, g and h form the tips. The branches of the tree are annotated with numbers that represent the evolutionary distance between species. The distance between the root and tip on all branches is not the same. Description from 21:08 in the video recording.](graphics/04-NonclocklikeTree-1.png) +--- +
+
+.left-column40[ + ![Schematic of a rooted non-clocklike phylogenetic tree containing internal nodes a, b, c. Nodes d, e, f, g and h form the tips. The branches of the tree are annotated with numbers that represent the evolutionary distance between species. The distance between the root and tip on all branches is not the same. Description from 21:08 in the video recording.](graphics/04-NonclocklikeTree-1.png) +] +.right-column60[ +.image-75[ ![Distance matrix D from a non-clocklike phylogenetic tree comparing evolutionary distances between nodes d, e,f g on the y-axis and e, f, g and h on the x-axis. The smallest distance between e and h is highlighted and is 0.020. Description from 21:08 in the video recording.](graphics/04-NonclocklikeTreeDistances-1.png) ] +
+The smallest distance doesn't match a true pair of siblings. +] + +--- +# Neighbo[u]r-Joining +
+
+Neighbo[u]r-Joining solves this problem by accounting for the *net divergence* of node from the rest, so if distances are tree-like, even if they're not ultrametric, it *will* get the tree right. +
+
+The formula for net divergence, with *n* taxa (i.e., an *n* *x* *n* distance matrix) is +
+
+$$r_{i} = \frac{1}{n-2}\sum_{j\neq i}D(i,j)$$ +
+
+And the adjusted distance becomes +
+
+$$D^{\ast}(i,j) = D(i,j) - r(i) - r(j)$$ + +--- +# Adjusted distance matrix +
+Net divergences: +.center[ +| $$i$$ | $$r(i)$$ | +|:---:|:------:| +| d | 0.075666 | +| e | 0.050666 | +| f | 0.065666 | +| g | 0.072666 | +| h | 0.047333 | +] +
+Adjusted Distances: +
+.image-50[ ![Adjusted Distance matrix D* from a non-clocklike phylogenetic tree comparing evolutionary distances between nodes d, e,f g on the y-axis and e, f, g and h on the x-axis. The values have been adjusted using the Neighbor-Joining method. Description from 24:03 in the video recording.](graphics/04-NJAdjustedDistances-1.png) ] + + +--- + + +![Schematic of a rooted non-clocklike phylogenetic tree containing internal nodes a, b, c. Nodes d, e, f, g and h form the tips. The branches of the tree are annotated with numbers that represent the evolutionary distance between species. The distance between the root and tip on all branches is not the same. The paths from d to e via a and f to g via c are highlighted in purple. Description from 24:03 in the video recording](graphics/04-NJTriumphs-1.png) + +--- +#Realistic data +
+
+*Losing tree-like distances* +--- +* Real data are not tree-like in general. +* We should adjust the final branch lengths that are found. +* One criterion is _Minimum Evolution_; this minimises the sum of squared differences ("ordinary least squares"; OLS) between the patristic distances, say _G_, from the tree and the original distances _D_: +
+$$OLS = \sum_{i,j}(D(i,j) - G(i,j))^{2}$$ +
+This assumes that the estimates are independent of each other, which clearly isn't the case as the distances between tips in the tree often share parts of the paths between them. +
+
+.reduce70[ Further reading: Denis and Gascuel: doi.org/10.1016/S0166-218X(02)00285-8. ] + +--- +# _Anolis_ tree with uncorrected p-distances +
+
+![Screenshot of a phylogenetic tree of Anolis species created in SplitsTree using uncorrected p-distances. The root of the tree is at the centre of the image and branches outward. There are a large number of very short branches near the centre of the tree.The branches are labelled with Anolis species names. Described from 28:44 in the video recording.](graphics/04-AnolisTreeUncorrectedPDistances-1.png) + +--- +# _Anolis_ tree with JC69 distances +
+
+![Screenshot of a phylogenetic tree of Anolis species created in SplitsTree using the Jukes Cantor model. The root of the tree is at the centre of the image and branches outward. The branches are labelled with Anolis species names. Described from 29:21 in the video recording](graphics/04-AnolisTreeJC69Distances-1.png) + +--- +# _Anolis_ tree with HKY85 distances +
+
+![Screenshot of a phylogenetic tree of Anolis species created in SplitsTree using the HKY85 model. The root of the tree is at the centre of the image and branches outward. The branches are labelled with Anolis species names. Described from 29:55 in the video recording](graphics/04-AnolisTreeHKY85Distances-1.png) + + +--- +# Limitations +
+
+* A tree-building method will _always give you a tree_, even if the data didn't come from one. +* There is no information on "next best" trees. +* There is no measure of "goodness" for the most part (though you could always quote the least squares error). +* Lastly, there is by default no going back on bad decisions: because NJ is a greedy heuristic, once the tree is finished, we _stop_. Luckily, we have programs like FastTree to do some adjustment! + +--- + +#Thank you! +
+
+_Next - estimating trees from alignments_ +--- + + + diff --git a/topics/evolution/tutorials/abc_intro_phylo/data/anolis-raw.fst b/topics/evolution/tutorials/abc_intro_phylo/data/anolis-raw.fst new file mode 100644 index 00000000000000..f838b60b7299f3 --- /dev/null +++ b/topics/evolution/tutorials/abc_intro_phylo/data/anolis-raw.fst @@ -0,0 +1,111 @@ + +>Anolis.acutus +ATGAGTCCCACAATCTATACAATTATTATATCAAGCCTAGCAACAGGCACAATTATTACCATAACCAGCTACCACTGACTTATAGCTTGAATAGGCCTAGAACTTAATACACTAGCAATTATTCCAATCATTTCATCACAACACCACCCCCGATCTACAGAAGCTGCAACAAAATATTTTCTGACACAAGCAGCAGCATCCGCCATAATTTTATTCTCCAGCATATCCAATGCCTGANTTACTGGTACATGGGACATCACTCAAATATCTTACACCCCCTCTAATATTTTTTTAACCATAGCACTAGCTATAAAATTAGGTTTGGCACCAGTACACTTCTGATTTCCAGAAGTATTACAAGGATCAACCTTATTTACTGCTTTTATTATTTCAACATGACAAAAGTTAGCCCCAATATCACTAAGTTTTCTGACTATCAACAATCTNCCACCAGTAACTTTATTAGCACTAGGACTTTTTTCCTCTGTCATTGGAGGGTGGGGCGGTCTCAATCAAACACAAACCCGAAAAATTATAGCATATTCATCAATTGCTCACTTAGGTTGAATGGCTGTAATTTCCTCAATTGCAACAAACATTATAATTATAAACCTACTAATTTATCTAATAATAACAACAGCCCTATTTTCTTCCTTAATTTTAACCAAATCAAAAACAATTCAAGACACAACAAANACTTGAACAATATCACCCACTTTAACTATTACTATGATACTTCTGCTACTTTCACTTGGTGGCCTGCCGCCCCTTACAGGATTTTTACCAAAATGATTGATTTTAGAAGAATTAACAGCCCAAAACCTTCTACCCCTGGCAACAATTATATCAACTANTGCGCTTCTCAGCCTATTCTTCTACTTACGCCTAGCTTACACTACAACTTTAACACTTTCTCCTAACACCCTACAAGTAAAATTCAAATGACGATTTAAACCACACACACCAACTTATTTAATAACATTTTCTACCTTTGCCATTTTTCTCTTACCATTAGCACCACTAATTTTACTATAAAAACTTAGGATAAAATAATAAACCAAGAGCCTTCAAAGTCCAAAATAAGGGCGCAATCCCCTTAGTTTTTGTAAGACTTGTAAAATACTAATTTACATCTCATGAATGCAACTCAAGCGCTTTTATTAAGCCAAAGCCTCACTGAATAGGCGGGCCTTGATCCCACGACAAGTTAATTAACAGCTAACTGCCCTAACCAGCGGGCTTCTATTCGCTTCTCCCGTACGGGAGAAGCCCCGGAGCCCCTTAGGGCTCTTCTTCAAACTTGCATTTTGACGTGAAAACACTACAGAGCTTCTAGTAAAGAAAGGGATTAAACCAATGTAAATAAGTTTACAGCTTACCGCCTAACACTCGGCCACTTTACCTGTGTTTATTA +>A.aeneus +ATGAGCCCTGCAATATATACCATTATTTTATCTAGCCTTGCAACAGGCACTATTATTACAATATCCAGCTATCACTGATTAATAGCCTGAATTGGCCTAGAAATTAATACACTAGCAATCATTCCAATTATTTCCATACAACACCACCCACGATCAACGGAGGCCGCTACAAAGTACTTCTTAATTCAAGCAGCAGCTTCAGCTATAATTTTATTTGCTAGTACAACAAATGCCTGATACACAGGAACATGAAGTATTATCCAAATATCTACCTTACCTTCACATATTATATTAACCCTAGCATTATTAATAAAACTAGGACTAGCCCCAATACATTTTTGATTACCAGAAGTCCTTCAAGGATCAACTNTATCTACTGCTCTTATTATTACTACATGACAAAAACTTGCCCCAATATCCTTAATTTTAATAACTATAAATAACCTAAACCCATTAATTTTACTTCTACTTGGACTAACTTCCTCAATAATAGGGGGCTGAGGCGGACTAAATCAAACACAAATACGAAAAATTATAGCATTTTCATCAATCGCCCATCTAGGGTGAATAATAATAATAGCATCAATCATAACAAATATTTTCATCTTAAATCTTATAATTTACCTAATTATAACCTCCAATATATTCTACTCCATAATTATATTAAAAACTAAAACTATTCAAGACTCATCAACTTCATGAACAACCTCCCCAATACTTACCATTATTATAATACTTACCCTTTTATCACTAGGAGGTCTACCTCCTCTTACCGGATTTATACCAAAATGACTTATTTTAGAAGAACTAGCAACACAAAGCCTAATCATACTAGCAACTATAATAGCACTAGCATCACTGCTAAGCCTATTTTTTTACTTACGACTAAGTTATACTACTACTCTTACACTGTCCCCAAATACAACTCAAACAAAACATAAATGACGATTTAAACCATCAAAAACTACATCAATATTAATACTTTTAACTCCTCTAACTATATTTCTATTACCGATAACACCAATAGTACTATTATAGAAACTTAGGATTAAATAAACCAAGAGCCTTCAAAGCCCTAAATATGAGTCTAACTCTCATAGTTTCTGTAAGACTTGTGAAATATTAATCCACATCCTCTGAATGCAACTCAACCACTTTAATTAAGCTAAAATCTTTCTAAGTAGGCGGGCTTTGATCCCACGACAAATTAATTAACAGCTAACTACCCAAACCAGCGGGCTTCTACTCGCTTCTCCCGTACGGGAGAAGCCCCGGAGCCTTTTAGGGCTCGTCTTTAAATTTGCATTTTAATGTGGAAACACTTCAGGACTGTGATAAAGAAAGGAATTAAACCTCTATAAATAGGACTACAGCCTACTGCC +>A.agassizi +ATGAGCCCTATAATATACATAATCATTATCTCCAGCCTCACAACAGGCACCATCATTACCATGTCTAGTCATCACTGACTACTAGCTTGAATTGGCCTAGAAATCAACACATTAGCAATCATTCCAATCATCTCAATACAACACCACCCACGATCCACAGAAGCCGCCACAAAATATTTCCTAACACAAGCAGCAGCTTCCGCTATAATCCTATTCTCAAGTTCTACCAACGCCTGATACACAGGAACATGAGACATTACTCAAATAACTACCACTCACTCCAACATCCTACTAACCCTAGCACTATCAATAAAATTAGGACTGGCCCCCCTACACTTTTGACTACCCGAAGTATTACAAGGCTCCACCATAACCACAGCACTAATTATTACAACCTGACAAAAACTAGCCCCAATGTCATTAATTTATCTAACTATAAATAACCTCTTCCCAACAATCTTACTCTCACTAGGACTTTTATCTTCAATAGTTGGAGGATGGGGAGGCCTAAACCAAACACAAACACGAAAAATCATAGCATACTCATCAATTGCCCACCTAGGATGAATAGCAACAGTATCATCAATCATAACAAACATTTTAGTATTAAATCTACTAATCTACCTAACAATAACAACAACCATATTTTTCTCACTAATTCTATCAAAATCTAAAACAATCCAAGATACAACAACCTCTTGAACAACATCCCCAATACTAACGATTATTATGATACTAACCCTCCTATCACTAGGGGGCTTACCCCCTCTTACAGGCAACATACCAAAATGACTAATCCTAGAAGAACTAACAATGCAAAACCTAACCCTACTAGCACTTATTATAGCAATAGCATCACTACTAAGCCTATTCTTCTACTTACGACTAACCTACACAACAACACTCACACTTTCCCCCAACATCACACAAACAAAACACAAGTGACGATTCAAACCATCAACCAACATACTCCCACTAACTATTATAACCCCAACAACCCTATTACTCCTACCAATAACCCCCTTAATCATCCATTAGAAACTTAGGATAAAATTAAACCAAGAGCCTTCAAAGCTTAAAATATGAGTTAACCCTCATAGTTTCTGCAAGACTTGTGAAATATTATTACACATCTTCTGAATGCAACTCAAACACTTTAATTAAGCTAAAGCCTCTCTGAATAGGCGGGCCTTGATCCCACGATATATTAATTAACAGCTAACTGCCCAAACCAGCGGGCTTCTATTCGCTTCTCCCGTACGGGAGAAGCCCCGGAGCCCTTCTGGGCTCGTCTTCAAATTTGCATTTTGACGTGAAACACCTCGGGGCTGTGATAAAGAGAGGAATCAACCCCCCTTAGATAGGACTACAGCCTACTGCCTAACATTCGGCCACTTTACCTGTGTTTATCAATCGAT +>A.ahli +ATGAGCCCAATAATATACACAATTATACTATCAAGCCTAGCAACAGGCACTATCGTTACCATAACGAGCTACCACTGACTCCTAGCCTGAATCGGACTAGAAATAAACACTTTATCAATTATTCCAATTATTTCTACCATACACCACCCACGATCAACAGAGGCCGCCACAAAATACTTTTTAACACAAGCAGCGGCTTCTGCCATAATCTTGTTTTCAAGCATAATTAACGCCTGACAAACCGGATCATGGGATATTACTCAATTATCATCTACACCATCACACATTCTATTAACCATGGCATTAGCAATAAAACTGGGCCTAGTCCCAGTACATTTTTGACTACCAGAAGTTATTCAGGGTTCAACAATAACCACGGCACTCATTATTACCACATGACAAAAACTTGCCCCAATTTCCCTAATTTATTTATCTATTAATAACCTCTCAACAGTGGTCCTAATAACCATGGGCCTACTATCTTCAATAGTCGGAGGCTGAGGCGGACTAAACCAAACACAGACACGAAAAATTATGGCATATTCATCAATCGCACATCTAGGATGAATAACCACTGTTTCCTCAATTATACCAAACATTATAGTTTTAAACTTAATAATTTACCTAATTACAACCGCAGCCACATTTTTCACACTAATACTGTTTAAATCAAAAACTATTCAAGATACAACAATAAACTGAACCTTATCCCCAATAACAACCGTCATAATAATAATTCTATTACTCTCACTTGGAGGCCTCCCACCATTAACTGGATTTATACCAAAATGATTAATTCTTGAAACACTAACAACCCAAAACTTTATTATAGCAGCAGTAGCTATAGCCATTTCAGCACTACTAAGCCTATTTTTTTACCTTCGACTTACCTACACTACAACCCTCACACTTGCACCAAATACTACACATACAAAACTAAAATGACGATTTAAACCAAACCTGCCAACCTTTCTAATACTTTCTTTTTCTACCACTTCAATATTTGCCCTACCACTTATGCCTCTCATAACACAATAAAAACTTAGGAATAACTAAACCAAAGGCCTTCAAAGCCCTAAATAAGGGTGCAATCCCCTTAGTTTTTGTAAGACTTGTGGGAGCTAACCCACATCTCCTGAATGCAACTCAAATGCTTTTATTAAACTAAAGCCTTCCANACAGGTGGGCCTTGATCCCACAAAAACTAGTTAACAGCTAGTTACCCAAGCCAGCGGGCTTCTATTCGCTTCTCCCGTACGGGAGAAGCCCCGGAGCCCTTTTGGGCTCATCTTCAAACTTGCATTTTGATGTGGGTCACTTCGGGACTTTGATAAAGAAAGGAATTAAACCAATGTAAGTAGGACTACAGCCTATCGCCTAACACTCGGCCAATTTACCTGTGTCAACTAATCGTTGA +>A.aliniger +ATGAGCCCTACAGTTTATTCAATTATTTTGTCAAGCCTACCAACAGGCACAGTTATTACTATAACCAGCTACCATTGATTAATAGCCTGAGTCGGGCTAGAAATTAACACACTCGCAATTATTCCTGTTGTTTCAATACAACATCACCCACGGTCCACAGAAGCCGCCACAAAATATTTTCTAACACAAGCAGCAGCCTCCGCCTTAATTCTATTTGCTAGCACAACTAACGCCTGATCAACGGGCACATGAGATATTACCCAAATATCCTCTACCCCCTCTCATATCCTTTTAACTATCGCGCTTGCTATAAAACTAGGCCTAGCACCCACCCACTTCTGATTACCAGAAGTAGTTCAAGGTTCAACTATAACAACAGCGTTTATTATTATAACATGGCAAAAACTAGCCCCAATATCATTAATTTATTTAACCATAAATAATCTTTCCCCAACAATTCTTCTTCTATTAGGAATAGCCTCATCTATGCTTGGTGGATGAGCAGGACTAAATCAAACACAGACCCGAAAAATTATAGCATTTTCATCAATTGCCCACCTTGGATGAATAGCTGTAATTGCATCTATTATGACAAATATTCTTACTCTTAACCTTATACTTTACTTAATTATGACAACAACCGCGTTCTTTACCCTAATTTTATCAAAATCAAAAACAATTCAAGATACAACAGCAACCTGAACTCTCTCCCCAACCCTAACTATTATTATAATGTTATCACTTCTTTCACTAGGCGGACTTCCACCATTAACCGGATTTATACCAAAATGATTAATTTTAGAAGAATTAACTACCCAAAAGTTAGCCCCCCTAGCTACTATTATAGCAGCATCCGCCTTACTCAGCTTATTTTTTTATCTCCGATTAGCCTACACTACAACACTCACATTTTCCCCAAACACTACACAAACAAAATTCAAGTGACGATTTAAACTACCCAACCTAGCTCCCCCACTAACAATTACCACCCCCCTAACTATTTTTCTTCTCCCCCTTACACCATTAATTTTAGCATAAGAAATTTAGGATAATATTAAACCAAGGACCTTCAAAGTCCAAGACATGGGCGAAACCCCCATAATTTCTGCAAGACTTGTAAAACATTAATTTACATCTCCTGAATGCAACTCAAGCACTTTTATTAAGCTAAAGCCTCTCTGAATAAGCGGGCCTCGATCCCGCGACAAATTAGTTAACAGCTAATAACCCAAACCAGCGGGCTTCTATTCGCTTCTCCCGTACGGGAGAAGCCCCGGAGCCCTTTAGGGCTCTTCTTCAAACTTGCATTTTGACGTGGAACACCCCGGGACTTTGATAAAGAGGGGAATTAAACCCCTATTAATGGGTCTACAGCCCACCGCCTAACATTCGGCCACCCTACCTGTGTCTATTAATCGTTGA +>A.alutaceous +ATGAACCCAACAATTATTATAATTACCCTAACCAGCCTGGCAACTGGTACAGTTATTACCATACATAGCTTCCATTGATTAATGGCCTGANTCGGATTAGANATCAATACACTATCAATTATTCCAATAATTTCAACATTACACCACCCACGATCAACTGAAGCTGCTACAAAATATTTCCTCACCCAAGCAGCTGCTTCANCTTTAATCCTTTTTTCAAGCACAATTAATGCCTGACAAACAGGATCATGAGACATTACCCAACTATCATCAACCCCCTCACACATTTTATTAACTATGGCTTTGGCCATAAAACTAGGACTCGCCCCACTTCACTTTTGATTACCAGAAGTAATTCAAGGGTCTTCCATAAACACAGCCTTAATTATTGTNACTTGACAAAAACTAGCCCCAATATCCCTAATCTTTTTAACCATAAATAATTTATCAACAACTATTTTAATGATAATAGGACTTGTATCTTCACTTGTCGGAGGATGAGCAGGACTTAATCAAACACAAACTCGAAAAATTATAGCCTACTCATCTATTGCTCACCTAGGGTGAATAGCAGCCATTTCCTCAATTATAACAAACATTTTAATCCTCAACCTATTAATTTACCTAATTATAACTACCTCAGTATTTTGCTTTATTATTATTACAAAATCAAAAACAATCCAAAACACAGCTACTATTTGGACAATATCACCAACACTAACAATTACCATAATACTTTCTTTACTTTCACTAGGAGGACTACCGCCACTAACAGGATTTATACCAAAATGAATAATTATAGAAGAACTTATTAATCAAAACTTTACTACAATTACTACTATGATAGCACTATCGTCCTTATTAAGCCTATTTTTTTATATTCGACTAACATATACAACTTCACTTACACTATCCCCAAACACAACCCAAACGAAATTTAAATGACGATTTTACCCAAACATAAAAACCACAGTAATTATAGTCTCTACAACAGTCACAATTTTTATATTACCGATACTTCCAATAATTTTGATATAAAAACTTAGGATAAACTAAACCAAGAGCCTTCAAAGCTCAAAAAAGGGGTGTAACCCCCCTAGTTTTTGTAAGACTTGTAAAATACTAATTTACATCTCTTGAATGCAACTTAAACACTTTAATTAAGCTAAAACCTTACTGAATAGGCGGGCTTTTAACCCACGAAAAATTAATTAACAGCTAATTTCCCAAACCAACGGGCTTCTATTCGCTTCCCCCGTACGGGAGAAGCCCCGGAGCCTTTAAGGGCTCTTCTTCAAACTTGCAATTTGACGTAAATCACTTACTTCGGGGCTTGATAAAGAAAGGAATTAAACCAATGTGAATAGGACTACAGCCTACCGCCTACCATTCGGCCACTTTACCTGTGTTTATTAATCGTTGA +>A.angusticeps +ATGAGCCCCCCCATTTTTACAATTATCATCTCAAGTCTAGCAACAGGTACAATTATTACCATAACCAGCTACCATTGACTCATAGCCTGAGTTGGTCTAGAAATAAATACACTAGCAATTATTCCTATTATTTCAACAACACATCACCCACGAGCCACAGAAGCTTCCACAAAATATTTTCTTACACAAGCTGCAGCCTCTGCTCTAATTTTATTTTCTAGTATAATTAACGCATGACACACAGGATCTTGAGACATTACTCAAATATTATCTACCCAATCAAATACTATATTGACTATAGCCCTAGCTATAAAACTAGGGCTGGCCCCACTTCACTTTTGACTACCAGAAGTAATCCAAGGATCAACAATACTAACCGCCTTTATTATTACTACATGACAAAAACTAGCCCCAATATCTCTAATTTTTTTAACTATAAATAATATATCCACAACAATCCTCCTACTAATAGGAATATTATCCTCACTTGTTGGCGGTTGAGGGGGGCTAAACCAAACACAAACCCGAAAGATCATAGCATACTCATCAATTGCTCACCTTGGATGAATAGCCACAATTTCATCTATTATAACCAACATCCTAATCTTAAACCTAGTTCTTTACCTAATTATGACTACATCAATGTTTGTCTCTTTAATTATTTCTAAGTCAAAAACAATCCAAAATACAGCAGAAACCTGAACACTCTCACCTACTCTAACAGTTTTTATGTTACTTTCACTCCTATCTTTAGGTGGACTACCACCCCTAACTGGGTTTATTCCAAAATGACTAGTTATAGAAGAACTAATTTTACAAAATTTTAATTTTCCAGCTATTTTAATAGCCATGTCCTCCCTACTAAGTCTATTTTTTTACCTTCGAATTACCTACACAACCACACTATCGCTATCACCTAATACAACTCAAATAAAATTTAAATGACGTTTTTATCCAAAAACCACAACCACCTTATCCTCTTCCACCGCAACAATAACAACCCTCCTTCTTCCACTTACACCACTAATTTTGTTATAAAAACTTAGGATAAAATTAAACCAAGGGCCTTCAAAGCCCAAGACATGGGTTAAACTCCCATAGTTTTTGTAAGACTTGTGAAACATTATTACACATCTCCTGAATGCAACTCAAGCACTTTTATTAAGCCAAAGCCTCACTGAATAGGCGGGCCTTGATCCCACGACAAATTAATTAACAGCTAAATACCCAAACCAGCGGGCTTCTATTCGCTTCTCCCGTACGGGAGAAGCCNCCGAGCCTTTAGGGCTCCTCTTCAGACTTGCATTTTGACGTGAAAACACTTCGGGACTTTTGATAAAGAAAGGAATTAAACCTGTGTAAGTAGGTCTACAGCCTACTGCCTAACACTCGGCCACTTTACCTGTGTTTATTAACCGTTGA +>A.bahorucoensis +ATGAGCCCCATAATTTACTCAATTGTATTCTCAAGCCTAGCNACAGGTACTATTATTACTATAACCAGCTACCACTGATTTATGGCCTGAATCGGACTAGAAATTAATACACTAGCAGTAATCCCCATTATTTCAACACTACACCACCCACGATCTACAGAAGCTGCTACAAAATACTTCTTAACACAAGCAGCAGCCTCCGCCACAATCCTATTTTCAAGTATAATTAATGCCTGACAAACAGGCACATGAGATATTACCCAATTATCCACAACCCCCGCCCACATCCTACTGACTATAGCACTTGCCATAAAACTAGGCCTAGTCCCAGCCCATTTCTGACTACCAGAAGTCCTCCAAGGTTCAACCATTACAACTGCCCTCATTATTACTACATGACAAAAACTCGCTCCAATATCACTAATTTTCTTAACCATTAACAACCTGTCCTCAACAGCCCTTCTATTATTAGGCTTAATGTCCTCATTATTAGGCGGCTGAGCCGGACTAAACCAAACACAAACCCGAAAAATTATAGCATTTTCATCCATTGCTCACCTTGGATGAATAGCCACTATTTCATCAATTATAACCAATATCCTTATCATAAACTTACTACTTTATTTAATTATGACAACAACTATATTTTACTCCCTTATTTTATCAAAATCTAAAACTATTCAAGACACAATAACCACCTGAACACTCTCGCCAGCCCTAACTATTATAATAATGTTGTTTCTCCTCTCCCTAGGGGGGCTACCTCCACTAACTGGCTTTATCCCAAAATGACTTATCCTAGAAGAACTTACTACCCAAAACCTTGCCCCACTAGCTATTATTATAGCAATATCAGCCCTTCTTAGCCTATTTTTTTATTTGCGTCTAGCCTATACTACAACACTAACACTTTCTCCTAATACAACCCAAATAAAACCTAATTGACGATTTAAACCACTAGCCCAAACCACCCCCCTAACAGCTATGACCCCCCTATCTATTTTTTTACTCCCACTTATGCCTTTAATTTTATACTAGAAACTTAGGATAACCTCAAAACCAAGAGCCTTCAAAGCTCAAAATATGGGTGAAACCCCCGTAGTTTCTGCCAAAACTTGTGAAACTTTAATTCACATCTGCTGAATGCAACTCAAACACTTTTATTAAGCTAAAGCCTCACTGAATAGGCGGGCTTTGATCCCACAACAAATTAGTTAACAGCTAAACACCCAATCCAGCGGGCTTCTATTCGCTTCTCCCGTACGGGAGAAGCCCCGGAGCCTTTTTAGGGCTCCTCTTCAAACTTGCATTTTGACGTGAAACACCCCGGGACTGCATGATAAGGAAGGGAATTGAGCCCCTATAAATAGGACTACAGCCTACCACCTAGCATTCGGTCACCTTACCTGTGTCCATTAATCGTTGA +>A.barahonae +ATGAGCCCGCTAATTTATATGATTATTTTATCAAGCTTAGCAACAGGCACAATTATTACAATAACGAGTTTTCATTGAATTATAGCTTGAATTGGGTTAGAAATCAACACCTTAGCAATTATCCCAATTATTTCTATATTACACCACCCACGTTCTACTGAAGCAGCCACAAAATATTTTCTTACACAAGCAGCAGCATCCGCTATAATCCTATTTTCAAGTATAATTAATGCCTGACAAACAGGAACATGAGATATCACTCAAATAACTAATATCCAATCCAACATTCTATTAACAATAGCACTTNCCATAAAATTAGGTCTAGCACCACTACATTTCTGACTTCCAGAAGTATTACAAGGCTCAACCTTAAATACCGCCCTTATTATAATTACATGACAAAAGCTAGCCCCAATATCATTAATTTTTATAACCATTAATAACTTATCGACAACAATACTATTATTACTAGGATTAGTATCAACAATGCTAGGAGGATGAGGAGGATTAAACCAAACACAAATACGAAAAATAATAGCATATTCATCAATTGCACATTTAGGCTGAATAGCAACAATTTCTTCTATTATAACAAATATTCTTATTATAAACTTCTTAATCTATATAATTTTAACCTTATCTATATTTTTATTACTTATTTTCTCAAATTCTAAAACAATTCAAGACACCACAACAGCCTGAACCCTATCTCCAACCATTACTATTATAATAATACTTACACTCCTGTCATTAGGGGGATTACCCCCACTCACCGGATTTTTACCAAAATGACTAATTCTAGAAGAATTAACATCCCAAAACCTAACAACCCTGGCTATAATCATAGCAATATCCGCCCTGTTAAGTCTGTTTTTTTACCTTCGACTAACCTACACAACATCACTCACACTTTCCCCAAACACAGTATTTACAAAATTTAAATGACGATATAAGCCCATGATACCAACTCAAATAATAGTAACTTTTACTATTATATCTATCTTTTTACTTCCACTCGCCCCACTAATATTATTATAAAGACTTAGGATAACTAAACCAAGAGCCTTCAAAGCTCCAAATAGGAATGAGAACCTCCTAGTTTTTGTAAGACTTGTAAAACATTAATTTACATCTCTTGAATGCAAATCAAGCACTTTAATTAAGCTAAAGCCTTCCTGAATAGGCGGGCTTTGATCCCGCGACAAATTAATTAACAGCTAACTACCCAAACCAGCGGGCTTCTATTCGCTTCTCCCGTACGGGAGAAGCCCCGGAGCCCTTTTGGGCTCTTTTTCAAATTTGCATTTTGATGTGGAACACTTCGAGACTTTGATAAATAAAGGAATTAAACCAATGTAAGTAGGACTACAGCCTACTGCCTAACATTCGGCCACTTTACCTGTGTC +>A.bartschi +ATGAGCCCAATAATTTATTCTATCGACCTATCAAGCCTAGCAACAGGAACTATTATCACAATAACTAGCTATCATTGACTAATAGCCTGAATTGGACTAGAAATCAACACACTAGCAATTATTCCAATTATCTCAATTAAACACCACCCACGATCCACAGAAGCAGCTACAAAATATTTTCTCACACAAGCTGCAGCCTCCGCTACAATCCTATTTTCTAGCACAATCAATGCCTGACTAACAGGCACATGAGATATTACCCAAATCTCCACCACCCCTTCCCACATCTTACTAACCACAGCACTAGCCATAAAACTCGGCCTAGCCCCAATACACTTTTGACTTCCAGAAGTATTGCAAGGCTCAACCATAAAAACAGCTTTTATTATCACTACATGACAAAAACTTGCCCCCATATCTCTTATCTTTTTAACCATAAACAACCTTTCTACAACAATTTTAATCTTTCTAGGATTACTATCATCCGCAGTAGGCGGATGAGNNGGATTAAACCAAACACAAATACGAAAAATTATAGCATACTCATCAATCGCTCATCTAGGATGAATAGCCACAATCTCATCAATTATAACCAACCTTCTAATTATGAATCTCCTACTATATCTAATCATTACAACAGCCATATTTTGGGCCATAATCCTGTCAAACTCAAAGACAATTCAAGACACAACAACCACCTGAACCCTCTCACCAACCCTTACTATTATTATAATACTTTCCCTCCTCTCACTCGGAGGTCTACCCCCATTAACCGGATTTACACCAAAATGGCTGATCTTAGAAGAACTAGTATTTCAANNNNNNNNNNNNCTAGCCACCATTATAGCACTATCGGCTTTACTCAGCTTATTTTTTTACCTACGATTAACCTACACCACAACCCTAACAATATCCCCAAACACCATAAAAACAAAATTCAAATGACGGTTTAAATCCCTAACAACAACATTACCACTAGCAATATTTACCCCTATAACCATCTTTTTATTACCCATAACACCACTAATATTAATATAAGAAACTTAGGATAAACTTAAACCAAGAGCCTTCAAAGCTCAAAATGTGGGTGAAACCCCCATAGTCTCTGCAAGACTTGTGAAATATTAATCCACATCTCATGAATGCAACTCAAACACTTTAATTAAGCTAAAGCCTCCCTGAACAGGCGGGCCTCGATCCCACGATAAATTAATTAACAGCTAATTACCCAAACCAGCGGGCTTCTATTCGCTTCTCCCGTACGGGAGAAGCCCCGAGCCCCTTAGGGCTCTTCTTCAAACTTGCATTTTGACGTGAAACACCTCGAGGCTGTGATAAGAAAGGGAATTAAACCCCTGTAAACAGGACTACAGCCTGACACCTAGACTTCGGCCACCTTACCTGTGTCCATTAATCGTTGA +>A.bimaculatus +ATGAGCCCAACAATCAATATAATTATTTTATCAAGCCTAGCAACAGGCACCATCATTACCATAAGCAGCTTCCACTGATTAATAGCCTGAATCGGCCTAGAAATTAATACACTAGCAATTATTCCAATCCTCTCAACACCACACCACCCACGATCAACAGAAGCTGCTATAAAATACTTTTTAACACAAGCCGCAGCCTCGTCCATAATTTTATTCTCTAGCATGACTAATGCCTGATATTTAGGCACATGAGATATTACTCAATTAGCCTATACCCCCTCAACCATTATATTGACCGCAGCCCTTTCCATAAAATTAGGACTAGCCCCCCTACACTACTGACTTCCAGAAGTACTTCAAGGATCAACCCTCTCCACAGCCTTCATTATTGTTACATGACAAAAACTAGCCCCCATATCTCTAATTTATTTAACCATTAATAATCTTAATACCGAAACCCTCCTAACTTTAGGACTTCTATCAACCATTATTGGAGGTTGAAGCGGACTAAACCAAACACAAACCCGTAAAATTATAGCATATTCATCAATCGCACATCTAGGGTGAATGGCCTCCATTTCCTCAATTATAACAAACATTATAATCATAAACTTAGCAATTTATTTGATCATAACAACAGCCCTATTCTTCTCCCTTATTTTATCTAAATCAAAAACAATTCAAGACACAGCAACCACCTGAACAACCTCCCCAATTCTGGTTATAATAATAATATTATCACTACTGTCACTAGGAGGACTACCACCCCTAACAGGATTTGCACCAAAATGACTAATTATAGAAGAACTAATTGTCCAAAACCTAGCCCCAATAGCCACCGTTATAGCCCTCTCCGCCCTACTAAGCCTATTCTTCTACCTACGCCTAACCTATACAACAACACTTACACTTTCACCAAACACAACCCAAGCAAAATTTAAATGGCGACTAAAAGCACAGCAATGCCCACCCCGATCACAACAGTCCCAACAACAGTTACAGTACTTTTATTACCCCTTATACCCCATTCTGTCATAAANACTTAGGATAACCAAACCGAGAACCTTCAAAGTTCAAAATACGGGTGAAACCCCCATAGTTTTTGTTAAGACCTGTAAAACTCTAATTTACATCTTCTGAATGCAACTCAAATACCTTAATTAAGCTAAGGCCTCTCTGAATAGGCGGGCCTTGATCCCACAACAAATTAATTAACAGCTAATTACCCAAACCAGCGGGCTTCTATTCGCTTCTCCCGTACGGGAGAAGCCCCGGAGCCCCTTAGGGCTCTTTCTCAAACTTGCATTTTGACGTGANNNNCTACGTGGCTGTGATAAAGAAAGGAATTAAANNNNNNNNAATAGGTCTACAGCCTACCGCCTAACACTCGGCCACTTTACCTGTGTC +>A.brevirostrus +ATGAGCCCACTAATCCACACAATTATACTCTCAAGTCTAGCAACAGGCACTATTATTACTATATCTAGCCACCACTGACTAATAGCCTGAATTGGATTAGAAATTAACACACTAGCAATTATCCCCATCATTTCAACATCCCACCACCCACGATCAACAGAAGCTGCCACAAAATATTTCCTTACACAAGCAGCAGCCTCTGCCACCGTACTATTTTCTAGTATAATTAATGCCTGACAAACCGGAACATGAGACATCACTCAAATATCTTATGCACCATCCAACATACTTTTAACTATAGCACTCTCCATAAAGCTCGGACTAGCCCCGCTACACTTCTGACTTCCAGAGGTTCTTCAAGGCTCAACCCTACCCACCGCCCTCATTATTACCACATGGCAAAAACTAGCCCCTATAGCACTAATCTGCCTAACAATTAATAATCTTAACCCAGCAATCCTACTAATCTTAGGACTTTTATCCTCAACTATTGGGGGTTGGGCCGGCCTTAACCAAACACAAACCCGAAAAATTATAGCATATTCATCAATTGCCCACCTAGGATGAATAGCCGCAATCGCCTCAATCATAACAAACATCATAATTATAAACCTCCTAATTTACTTAATGATAACAACCGCCCTATTCTGCTCCCTAATCTTTTCAAAGTCAAAAACTATTCAAGATACAACAACCACCTGAGCCACATCCCCAATAATAACTATTACTACTGCGCTCTCATTATTATCATTAGGCGGACTTCCACCCCTAACAGGATTTGCACCAAAATGACTAATTCTAGAAGAGCTCACTACCCAAAACTTAATCCCCATAGCCGCTGTTATAGCCCTATTCTCACTTCTTAGCTTATTTTTTTACACTCGCCTAGTTTATACAACAACACTTACACTCCCGCCTAGCACACTTCAAACAAAATTTAAATGACGATTTAAACAGGCCCCACCAACCTCACCAATAACAATTTCATCAACAGCAGCCATCTTTCTTCTGCCTTTAGTACCTCTAATGTTGATATAAAAACTTAGGATAATAAAACCGAGAGCCTTCAAAGCTTTAAAAAAGGGTGTAAACCCCTTAGTTTTTGTAAGACTTGTGAGAGACTAACCCACATCTTCTGAATGCAACCCAAACACTTTAATTAAGCTAAAGCCTTCATGAATAGGCGGGCCTTGATCCCACGAAAATTTAATTAACAGCTAACTGCCCTAGCCAGCGGGCTTCTATTCGCTTCTCCCGTACGGGAGAAGCCCCGGAGCCTATTGGGGCTCTTCTTCAAACTTGCATTTTGACGTAAAACACCTCGAGGCTATGATAAAGAAAGGACTTGAACCAATATGGGTAGGTTTACAGCCTACCACCTAAACCTCGGTCACTTTACCTGTGTCTATTAATCGTTGA +>A.carolinensis +ATGAGCCCAACTATTCACATAATTATTTTATCAAGCCTAGCAACCGGCACCATTATTACCATAACCAGCTACCACTGACTTATAGCCTGAGTGGGCCTAGAAATAAATACCTTAGCAATTATTCCAATCATCTCAACAATACACCACCCGCGATCTACAGAAGCAGCCACAAAGTACTTCTTAACCCAAGCTGCAGCCTCCGCACTAATCTTATNTTCAAGCATAACAAATGCTTGAAACCTAGGCTCATGAGACATTACTCAAACATTAGCCCCCCCCTCACATATTCTATTAACCATAGCACTAGCCATAAAACTTGGCCTAGCCCCTCTACACTTCTGACTACCAGAAGTCATCCAAGGATCAACCATAACCACAGCCAACATTATTACCACATGACAAAAACTTGCCCCAATATCCCTTATTTTCCTAACCATAAACAACCTATCCACCACAATTTTATTATTAATAGGACTACTATCTTCACTTATCGGGGGATGGGGGGGATTGAACCAAACACAAACCCGAAAAATCATAGCATACTCATCAATTGCACACCTAGGATGAATAGCAACAATCTCTTCAANCATAACAAATATCNTTAAACATAAACTACAAATTTACCTTATCATAACAACTTCAATATTTCTNTCTCTTATTATTTCNAAATCAAAAACAATCCAAAACGCAACATCCACCTGAACCCTGTCCCCAGCACTTACAATCATTATAATACTCTCACTCCTTTCCTTAGGAGGACTACCCCCACTTACAGGACTTATACCGAAATGACTTATTATAGAAGAACTAATCTTACAAGACTTCAACTTACTAGCCACCATAATAGCTTTATCCTCCCTACTTAGCCTATTTTTTTACTTACGACTAGCCTATATAACCACACTTACACTATCTCCAAATACAACCCAAACAAAATTTAAATGACGATTTTACCCAAACACACCAACCGTCTTTCTATCTATCCCTGCTACAATTTCTATTTTCTTACTACCCCTAACCCCTCTAATCTTACTATAAAGACTTAGGATAAACTAAACCAAGAGCCTTCAAAGCTCAAAATAGGAGTTAAAACCCCCTAGTTTTTGCAAGACTTGTGAAACATTATTACACATCTCCTGAATGCAACTCAAGCACTTTAATTAAGCTAAAGCCTCTCTGAATAGGCGGGCCTTGATCCCACAACAAATTAATTAACAGCTAATTACCCAAACCAACGGGCTTCTATTCGCTTCTCCCGNACGGGAGAANNCAACGAGCCTTTAGGGCTCCTCTTCAAACTTGCATTTTGACGTGAAACACTTCGGGGCTTGATAAAGAAAGGAATTAAACCAATGTAAATAGGACTACAGCCTACTGCCTAACACTCGGCCACTTTACCAGTGTTTTTTAGTCGTTAA +>A.christophei +ATGAGCCCCTTAATTTTTACAATTATCCTGTCAAGCCTAGCAACAGGCACAGTTATTACTATAACCAGCTTCCATTGACTATTAGCTTGAATTGGGTTAGAAATCAATACACTAGCAATTATTCCAATTATTTCTACAATTCATCACCCACGCTCAACAGAAGCAGCCACAAAGTACTTCCTTACACAAGCAGCAGCATCAGCTATAGTCTTATTTTCAAGCATAATTAACGCCTGACAAACAGGAACATGAGACATTACTCAAATAACTACTGATCAATCTAACATTATATTAACAATAGCACTTGCTATAAAACTTGGATTAGCCCCANTACACTTCTGACTTCCAGAAGTATTACAAGGTTCAACCTTAAATACTGCTCTTATTGTAACCACATGACAAAAACTTGCCCCAATATCACTAATTTACCTAACTATAAACAACCTATCAACAACAACTTTGCTGCTACTAGGAGTAGTATCCTCAATATTAGGAGGATGGGGAGGACTTAATCAAACACAGACACGTAAAATTATAGCATACTCATCAATTGCACACCTAGGATGAATGGCCACAATTTCTTCTATTATAACAAACATTCTAATTATAAATTTATTAATTTATTTAATTTTAACAGTATCTATATTTCTTCTACTTATTTTTTTAAACTCCAAAACCATTCAAGATACTATAATAACCTGAACCCTCTCTCCAACCATAACCATTATAACTATACTTACACTTTTATCATTAGGTGGCCTACCACCACTAACCGGGTTTATACCAAAATGATTGATCTTAGAAGAACTAACAACTCAAAACTTAACCTCACTAGCTATGATTATGGCAATATCTGCTCTACTAAGTCTATTTTTTTATTTACGAGTTACATACACAACATCACTCACATTATCCCCTAATACAGTCCAAACAAAATTTAAATGACGATTTAAACCATTAATACCAACTCATCTAATTTTAATTTCAACTACAACAGCTATTTTTTTACTTCCCCTCACCCCGCTAATATTTCTATAAAGACTTAGGATAACATAAACCAAGAGCCTTCAAAGCTCAAAATATGGGAGAAACCCCCCTAGTTTTTGTAAGACTTGTAAAATATTAATTTACATCTCCTGAATGCAANTCAAGCACTTTTATTAAGCTAAAACCTTTCTGAATAGGTGGGCCTTGATCCCACGANAAATCAATTAACAGCTAATTACCCTAACCAGCGGGCTTCTATTCGCTTCTCCCGTACGGGAGAAGNCCGGAGCCCTTTTATGGGCTCTTTCTCANACTTGCATTTTGATGTGAAACACTTCGGGACTTTGATAAAGAAAGGAATTAAANNAATGTAAGTAGGACTACAGCCTACTGCCTATCATTCGGNNACTTTACCTGTGTCTATTAAGCGTTGA +>A.coelestinus +ATGAGCCCACTAATTTTTTCAATCGTCCTGTCAAGCCTAGCAACAGGCACTATTATTACCATAACCAGCTATCACTGATTAATAGCTTGAATTGGTCTAGAAATAAACACACTTGCTATTATTCCAATTATCTCAATACAACATCACCCTCGATCTACAGAAGCCGCTACAAAATATTTCCTTACACAAGCAGCAGCCTCCGCTATGATTTTATTCGCCAGCACAACAAATGCTTGATACACAGGCACATGAGACATCACTCAAATTTCCGCCACCCCATCCCACATCCTCCTAACTGCCGCATTAGCCATAAAACTAGGCCTAGCCCCTATACACTTCTGATTACCAGAAGTCCTTCAAGGGTCCACCTTAAAAACAAGCTTTATTATTGTCACATGACAAAAACTAGCCCCTATATCATTAATTTATTTAACTATAAATAATCTTTCCTCAACAATTCTTCTCTCACTAGGACTAGTTTCATCTATGGTAGGAGGATGGGGCGGACTAAACCAAACACAAACCCGAAAGATCATAGCATACTCATCAATTGCACATCTCGGATGAATGGCCTCAATCTCCTCCATTATAACTAATATTCTTGTTATAAATTTAGTACTTTACCTAATTATAACAACATCTGTATTCTACGCTTTGATCTTAACAAAGTCTAAAACAATTCAAGATACAACAACCACCTGAACACTCTCCCCAACTTTAACAATTATTATAATACTCTCACTTCTCTCCCTTGGAGGCTTACCCCCCTTAACTGGATTCACACCAAAATGACTTATCTTAGAAGAATTAACCACCCAAAATCTACTCCCCCTAGCCACTACTATAGCGATATCTACCCTACTCAGCCTGTTTTTTTACTTACGTTTAACCTATACCACAACCCTTACATTCTCCCCAAACACTACACATACAAAATTTAAATGACGATTCAAGTCAACTAATTTAACAATACCACTAACACTTACAGCACCCCTGAGCGTCCTCTTACTTCCTTTAGTCCCGCTAATTATAGTATAAGAAATTTAGGATAATATTAAACCAAGAGCCTTCAAAGCTCAAAATACGGGTGAAACCCCCATAATTTCTGTAAGACTTGTGAAATACTAATCCACATCTCATGAATGCAACTCAAGCACTTTTATTAAGCTAAAGCCTTTCTGAATAGGCGGGCCTCGAGCCCACGACAAATTAGTTAACAGCTAATTACCCAAACCAGCGGGCTTCTATTCGCTTCTCCCGTACGGGAGAAGCCCCGGAGCCCTTTAGGGCTCTTCTTCAAACTTGCATTTTGATGTGGAACACCCCAGGACTTGATGAGGAAGGGAATTAAACCCCTGTTAATGAGTCTACAGCTCACCACCTAACACTCGGCCACCTTACCTGTGTCTA +>A.cristatellus +ATGAGCNNNACAATCTACACAATTATTTTGTNNNNNCTAGCAACAGGCACTATCATCACTATAACTAGCTTCCACTGACTAATGGCCTGAATCGGACTAGAGCTTAATACGCTAGCAATTATCCCGATTATTTCAACATTACACCACCCACGATCAACAGAAGCCGCAACAAAATACTTCTTAACACAAGCAGCAGCCTCTGCAATAATTATGTTTTCTAGCATAATTAATGCCTGAAACATAGGAACATGGGACATTACTCAAATATCTTATACCCCATCAAATGTTCTTCTTTTACTAGCACTGGCCCTAAAACTAGGACTAGCCCCAGCACACTTCTGACTCCCAGAGGTATTACAGGGCTCAACCTTATTTATTGCCCTCATTATTACTACGTGGCAAAAACTGGCCCCAATGTCACTTATCTACATATGTACAAATAATCTTCCATCAGTAATTTTATTAATACTAGGCATTATCTCCTCCACTGTCGGGGGGTGGTCTGGATTAAACCAAACACAAACTCGAAAAATCATAGCATACTCATCAATTGCACACCTTGGATGAATGGCTATAATCGCCTCAATTATAACAAACATCATAATTATAAACCTGGTGATTTATTTAATAATAACAACCACCTTGTTTACCACCCTAATTTGCTCTAAATCAAAAACAATTCAAGACACAGCAACCACTTGAACAACATCACCAACCCTAACTATCATTACTTTACTTTCTCTTCTGTCACTTGGGGGACTCCCACCTCTTACAGGATTTTTACCAAAATGACTAGTTTTAGAAGAAATAATTGTACAAAACTTAACCCCACTCGCCACAACCATAGCAATTTCCGCCCTTCTTAGTCTATTCTTCTATCTACGCCTAACCTACACCACAACACTAACATTAGCTCCAAACACCCTACAAATAAACTTTAAATGACGGTTTAAACCAACCACATCATTTATTCTAATAACCCTTTCAACTATGACCATTTTTTTACTACCTCTCATACCACTAACCCTTATATAAAAACTTAGGATAACATAAACCAAGAACCTTCAAAGTTCAGAATAAGGGTGTAAACCCCTTAGTTTTTGCAAGACTTGTAANANATTAATTTACATCTCATGAATGCAACTCAAGCGCTTTTATTAAGCTAAAGCCTCTCTGAATAGGCGGGCCTTGATCCCACGATAAATTAATTAACAGCTAATTACCCAAACCAGCGGGCTTCTATTCGCTTCTCCCGTACGGGAGAAGCCCCGGAGCCAATANGGGCTNGCTTTCAAANTTGCATTTTGACGTGAAATACTACAGGACTCGTAGTAAAGAAAGGGTTTAAANCAATATAAANAGATTTACANGCTACCGCCTAANAGTCAGCCACTTTACCTGTGTCCATTAAGCGTTAA +>A.cuvieri +ATGAGCCCAACAATTCTCTCAATCATTTTATCAAGCCTAGCAGCAGGAACAATTATTACAATAACAAGCTTTCATTGATTAATAGCCTGAATTGGACTAGAAATTAATACACTAGCAATTATTCCAATTATCTCAATAATACATCACCCACGATCTACAGAAGCAGCCACAAAATATTTTCTCACACAAGCAGCAGCATCAGCTATAATCCTGTTCTCAAGCATAATTAATGCTTGACAAACAGGGACATGAGATATTACCCAAATATATACTCCTCAATCTAACATTTTATTAACTATAGCCCTTGCCATAAAAATAGGATTAGCCCCAATACACTTCTGACTACCAGAAGTACTTCAAGGTTCAACCTTAAACGCTGCTCTTATTATTACCACATGACAAAAAATCGCCCCAATGTCATTAATTTATTTAACCATTAATAATTTATCAACAACAACCTTATTAACACTAGGATTAATTTCTTCAATATGAGGAGGATGAGCAGGCCTAAACCAAACACAAACCCGAAAAATCACAGCATACTCATCTATTGCTCACATTGGATGAATGGCTACAATTTCTTCAATTATAACAAATATTCTTATTATAAACCTATTAATTTATTTAATTATAACAATCTCTATATTTAACTCACTAATTTTATCCAAATCTAAAACTATTCAAGACACGTCAATGACATGAACATTATCTCCAATATTAACTATTATTACTATACTTACACTCTTATCATTAGGGGGATTACCACCACTAACCGGATTTATACCAAAATGACTAATTCTAGAAGAATTGACAACCCAAAACTTAACTTCACTAGCCGTAATCATAGCAATAACTGCCCTATTAAGCCTTTTTTTTTACTTACGACTAACATACACAACAACACTTACATTATCACCAAACACAACACAAACAAAATTTAAATGACGATTTAAACCAAATTTACCTACATATTTACTAATAATCTCCTCTACAACAACCATCTTACTTCTTCCACTAACCCCCTTAATAATTTACTAAAAACTTAGGTTAACATAAACCGAGAGCCTTCAAAGCTCAAAACAGGAGTCAAAAACTCTTAGTTTTTGTAAGACTTGTAAAATACTAATTTACATCTTCTGAATGCAACTCAAACACTTTAATTAAGCTAAAGCCTACCTGAATAGGCGGGCCTTGATCCCACGACAAATTAATTAACAGCTAATTACCCAAACCAGCGGGCTTCTATTCGCTTCTCCCGTACGGGAGAAGGCCCGGAGCCCTTTTGGGCTCTTCTTCAAATTTGCATTTTGACGTGAAACACTTCAGGACTTTGATAAAGAAGGGAATTAAACCAATGTAAGTAGGACTACAGCCTACCGCCTAACATTCGGCCACTTTACCTGTGTTTATTAATCGTTGA +>A.distichus +ATGAGCCCGCCAATCTACGCAATTATACTATCAAGCTTAGCAACAGGCACCATTATCACTATAACCAGTTACCATTGACTAATGGCCTGAATTGGACTAGAAATTAATACACTAGCAATTCTTCCAATTATTGCAACATCACACAACCCACGATCCACAGAAGCTGCCACAAAATACTTTTTAACACAATCAGCAGCTTCCGCCACTATCTTATTTTCTAGCATACTTAACGCCTGACAAACCGGAACATGAGACATTACTCAAATATCTTATGCGCCATCTAACCTCCTTCTCTCCATAGCACTAGCCATAAAACTAGGACTAGCCCCCCTACACTTTTGACTCCCAGAAGTACTTCAAGGCTCAACTTTATTTTCTGCCCTCATTATTGTTACATGACAAAAATTAGCCCCAATATCACTGATTTATTTAACTATTAGTAACCTTAACCCAACTATTCTACTAATCTTAGGCCTCCTATCCTCAACTATTGGGGGATGGGGTGGGTTAAATCAAACACAAACCCGAAAAATCATAGCATATTCATCAATTGCTCACCTAGGATGAATGGCCATTATTGCCACCATTATAACAAACCTCATAGTCATGAACCTCTTAATTTATCTAATAATAACAACAGCCCTATTCTCCCTCCTAATTTTTTCCAAATCAAAAACCATTCAAGACACAACAACAACCTGAGCACTCTCCCCCACAATAACTATTATAATGACATTTCTATTATTATCATTAGGGGGGCTTCCCCCAATAACAGGATTTGCACCAAAATGACTAATTCTAGAAGAACTCACAACCCAAAATCTAATTCCTCTAGCTGTTTTAATAGCAGTATTTTCCCTTCTTAGCCTGTTTTTTTATACTCGACTAGCCTACACAACAACACTTACACTATCGCCCAATACACTACANANAAAATTTAAATGACGATTCAAACAAACCCTATCAACTTCACTAATAGTAACTTTATCAACAATAGCTATTTTTCTTCTACCACTTACACCCTTAATGTTAATATAAAAACTTAGGATATTAAAACCAAGGGCCTTCAAAGCCCTAAAAAAGGGTGCAAACCCCTTAGTTTTTGTAAGACTTGTGAAACACTAATCCACATCTTCTGAATGCAACCCAAACGCTTTCATTAAGCTAAAGCCTTTCTGAATAGGCGGGCCTTGATCCCGCGATAAATTAATTAACAGCTAATTACCCTAACCAGCGGGCTTCTATTCGCTTCTCCCGTACGGGAGAAGCCCCGGAGCCCTTTTGGGCTCTTCTTCAAACTTGCATTTTGACGTAAAACACCTCGAGGCTTTGATAAAGAAAGGGCTTCAACCAATATAAATAAGTTTACAACTTACCGCCTAAATTTCGGCCACTTTACCTGTGTTCATTAATCGTTGA +>A.equestris +ATGAGCCCAACAATTTATTCAATTATCCTATCAAGCCTTGCAANNNNNACAATTATTACTATAACCAGCCACCATTGACTAATAGCCTGANTCGGATTAGAAATTAACACATTAGCAATTATCCCAATTATTTCAACATTACACCACCCACGATCCACAGAAGCCGCCACAAAATATTTCCTAACACAAGCAGCTGCTTCTGCTATAATTTTATTTTCTAGCATAACAAATGCTTGATACACAGGTACATGAGACATTACCCAAATATCAGCCAACCCCTCCCATATTATATTAACTATGGCACTTGCCATAAAACTAGGCCTAGCACCTCTACACTTCTGACTACCAGAAGTACTCCAAGGCTCAACCATAAAAACCGCATTCATCATTACAACATGACAAAAACTTGCCCCAATATCATTAATCTACCTCATTATTAATAACTTATCCCCCACACTTCTCCTCCTATTAGGACTAATATCATCTACTCTGGGAGGCTGAGGAGGACTAAACCAAACCCAAACCCGAAAAATCATAGCCTATTCATCAATCGCCCACCTAGGTTGAATAGCTACAATCTCTTCAATCATAACCAATATTCTTGTTATAAACCTATTACTTTATATAACTATGACAACATCAATATTTTTTACCCTTATTTTATCAAAATCTAAAACAATTCAAGATACAACTACCTCATGAACACTATCTCCATCCCTAACCATCATTATAATATTATCACTTCTCTCATTAGGCGGACTACCCCCCCTAAGTGGATTTATACCAAAATGATTAATCCTAGAAGAACTNNNNNNCCAAAATCTTCCCCCACTAGCCACTATTATAGCAATATCTGCCCTACTTAGCTTATTCTTTTATCTACGACTTACCTACACTACTACCCTAACAATCTCCCCTAATACCTTACAAACTAAATTTAAATGACGATTCAAACCTACGACCTCAACCCTACCAATAATAATCTTTACCCCATTAACTATTTTTATGTTACCACTAACACCAATAATCATCATATAGAAACTTAGGTTTAAATAAACCAAGAGCCTTCAAAGCTCAAAATATGGATGAAAACCCCATAGTTTCTGCTAAGACTTGTAAAATACTAATTTACATCTCCTGAATGCAACTCAAATGCTTTTATTAAGCTAAAGCCTTCCTGAATAGGAGGGCCTTGATCCCACAATAAATTAATTAACAGCTAATTACCCAAACCAGCGGGCTTCTATTCGCTTCTCCCGTACGGGAGAAGCCCCGGAGCCCTATTGGGGCTCTTTTTCAAACTTGCATTTTGACGTGAAACACTTCAGGGCTTTGATAAGAAAAGGAATTAAACCTATNNNNNNAGGACTACAGCCTACCGCCTAACACTCAGCCATCTTACCTGTGTCTATTAACCGTTA +>A.etheridgei +ATGAGCCCTGCCATCTATACCATTATTTTATCAAGCCTAGCAACAGGCACACTCCTAACTATAACCAGCTTTCACTGATTAATAGCATGAATCGGATTAGAAATTAATACACTAGCAATAATTCCAATTATCTCAACACCTCACCAACCACGATCTACAGAAGCTGCTACAAAATATTTCCTCACACAAGCAGCAGCCTCCGCTATAATTTTATTTTCAAGCATAATTAATGCCTGACAAACAGGATCATGAGACATTACCCAAACATCTACTTTCCCCTCTCATATTCTTTTAACCATGGCCTTAGCAATAAAATTAGGCCTTGCCCCCTTACATTTTTGACTACCAGAAGTTCTTCAAGGCTCAACCCTAAACACAGCCCTAATTATTACCACATGACAAAAGCTAGCTCCAATGTCACTAATTTATTTAACAGTAGATAACCTATCCCCCTCAATTCTTCTCCTACTTGGTCTTCTATCTTCTCTAGTTGGCGGCTGAGGTGGACTAAATCAGACACAAACCCGAAAAATTATAGCATACTCATCAATTGCACATCTAGGATGAATAGCCACCGTAACTTCAATTATAACAAATATTCTTCTCATAAACCTTATAATTTATTTAATTATAACAACAACCATATTCTTCTCTTTAATTTTATCCAAATCCAAAACAATTCAAGACTCAACTATTACCTGAACACTCTCACCAACTCTAACTATTATTACAACACTTACACTTCTTTCACTCGGAGGATTACCCCCCCTAACCGGATTTATGCCAAAATGACTAATCTTAGAAGAATTAACAACTCAAAACTTAACCCCCATAGCTATTACCATAGCAATATCCACCCTACTTAGCTTATTTTTTTACTTGCGACTAACCTACACAACAGCTCTTACTTTATCCCCAAACACCATACAAACAAAGCTTAAATGACGATTTAAACTAAACCAACCAACTCAGCTAATAATGACTACTTCTACAATAACAATTTTTTTACTTCCTCTTACACCCTTACTCACATTATAAAAACTTAAGTTTAACTAAACTAAGAGCCTTCAAAGCCCAAAATATGGGTAAAACCCCCATAGTTTTTGTAAGACTTGTGAAATACTATTTCACATCTCTTGAATGCAACTCAAGCACTTTTATTAAGCTAAAGCCTTCCTGAATAGGCGGGCCTTGATCCCGCGATAAATTAATTAACAGCTAACTACCCTAGCCAGCGGGCCTCTATTCGCTTCTCCCGTACGGGAGAAGCCCCGGAGCCCTTTGTGGGCTCTTTTTCAAACTTGCATTTTGATGTGAAACACTTCGGGACTTTAATAAAGAAAGGAATTAAACCAATGTAAATAGGACTACAGCCTACCGCCTAACACTCGGCCACTTTACCTGTGTTTATCAAGCGTTGA +>A.garmani +ATGAGCCCAACCATCCTTATAATTATTATCTCAAGCCTGGCAACAGGTACCATTATTACCATAACAAGCCACCACTGACTCATAGCCTGAATCGGACTAGAAATAAATACCTTAGCTATTATTCCAATCATTACTACTATACACAACCCACGATCAACAGAAGCCGCCACAAAATACTTCTTAACACAAGCAGCAGCCTCTGCCATAATCTTATTCTCAAGCATAATTAATGCCTGACAAATAGGATCATGAAATATTATTAACTTAACATACGAACCCGCGCACATCCTATTAATTATGGCCCTAGCAATAAAACTTGGCCTCGCACCCATACACTTTTGACTGCCAGAAGTTCTTCAAGGCTCAACCTTAACCACTGCACTAATTATTACCACTTGACAAAAACTCGCCCCAATATCTTTAATTTATATAACCCTAAACAATCTATCGACCACAATTTTAATTTTACTCGGACTTCTATCCTCAATTATTGGAGGATGGGGGGGACTCAATCAGACCCAAACTCGAAAAATCATAGCATTTTCATCAATNCGACACCTGGGGTGGATAATTACTGTCTCATCAATTATGCCGAACATTATAATTTTAAACCTAATTATTTATTTAGTCATAACAACCGCCCTATTTTACTCACTAATCTTATCNAAATCAAATGGAATCCAAGACACAACAACAACTTGAACACTTTCACCCACAATAACTATTATAATGATAACTTTACTCCTCTCCCTAGGGGGCCTCCCACCACTAACTGGCTTTATACCAAAATGANTTATTCTAGAAGAACTAGTTGCCCAAAATCTTATCTCAACCGCCACTATTATAGCCATATCAGCACTACTAAGCCTATTTTTTTATCTACGACTAACCTATACAACAACACTAACCCTATCCCCGAACACTANACAAACAAAGTTTAAATGACGATTTAAACCAAACCGTACAACACCCGTTATTGGGGCCTCAGCTACTATCTCAATTTTCCTACTCCCTATAACCCCATTAATACTTCTATAAAGACTTAGGATAATTAAACCAAGGGCCTTCAAAGCCCTAAATAAGGGCCATACTCCCTTAGTTTTTGTAAGACTTATGAGGGACTATCCCATATCTCCTGAATGCAACTCAAGCACTTTTATTAAGCTAAAGCCTTATCTAAATAGGCGGGCCTTGATCCCACAAAAACTAGTTAACAGCTAATTACCCAATCCAGCGGGCTTCTCTTTAGCTTCTCCCGTACGGGAGAAGCCCCNGGGCCCTTTAGGGCTCTTTTTCAAACTTGCATTTTGATGTGAATCACTTCAGAGCTGTGATAAAGAAAGGAATTAAACCAATGTAAANNGGTCTACAGCCTACCGCCTAACATTCGGCCACTTTACCTGTGTCTATTAATCGTT +>A.grahami +ATGAGCCCATCAATCCTTATAATTATTATTTCAAGCCTGGCAACAGGCACTATTATTACTATAACAAGCCACCACTGACTTATAGCCTGAGTCGGACTAGAAATAAATACTTTGGCAATTATCCCAATTATTTCTACTACACACAGCCCGCGATCCACAGAAGCCGCTACAAAATATTTTTTAACACAAGCAGCTGCCTCTACCATAATCTTATTTTCAAGCATAACCAACGCCTGACAAACAGGCACATGAGACATTATTAACTTAACATATGAGCCCGCGCACATCCTGCTAACCCTGGCCCTAGCAATAAAACTAGGCCTTGCACCTATACACTTCTGANTACCAGAAGTATTACAAGGCTCAACCTTAACCACTGCACTAATTATTACCACTTGACAAAAACTAGCCCCAATATCCTTAATTTATATGACCCTAAACAACCTGTCAACCACAACTTTAATCTTACTAGGACTTCTATCTTCAATTATTGGGGGGTGGGGCGGACTAAACCAAACCCAAACCCGAAAAATTATAGCGTTTTCATCAATCGCACATCTTGGGTGAATAATTACTATTTCATCAATTATACCCAATATTATAGTTTTAAACCTGACAATCTACCTAATCATAACAATTGCCCTATTTTATTCACTAATTCTATCTAAATCAAAAACAATCCAAGATACAACANCAACTTGGACACTCTCACCCGCACTGACTGCCATAATAATAACACTACTTCTTTCCCTGGGAGGGCTACCACCACTAACGGGCTTTATGCCAAAATGACTTATCTTAGAAGAACTAGTAGCCCAAAACCTCACCCCAGCCGCCACTGCTATAGCCATATCAGCGCTACTAAGCCTGTTCTTCTACNTGCGGCTAACCTATACAACAACACTAACCCTGTCCCCAAATACCACTCAAACAAAACTTAAATGACGATTTAAGCCAAACCACACAACACTTCTCATAGGGGCCTCCTCCACAATCTCAATCTTTCTACTCCCCATGACCCCCTTAATACTTCTATAAAAACTTAGGATAATTAAACCAACGGCCTTCAAAGCCCTAAATAAGGCCACCATCCCCTTAGTTTTNGTAAGACTNATGGGTAACTACCCCATATCTCCTGAATGNAACTCAAACACTTTTATTAAGCTAAAGCCTTATCTAAANNNNNNNNNNNNNNNNNNNNAAAAATTAGTTAACAGCTAATTACCCAATCCACGGGNCTTCTATTTAGCTTCTCCCGTACGGGAAAGNCCCGGAGCCCTTTAGGGCTCTTTTTCAAACTTGCATTTTGATGTGGATCACTTCAGGGCTGTGATAAAGAAAGGAATTAAACCAATGTAAATAGGACTACAGCCTACCGCCTACATTCGGCCACTTTACCTGTGTTCATTAATCGTT +>A.humilis +ATGAGCCCCACTGTTCTTATAGTAATTTTATCAAGCCTAGCCACTGGGACTATCATCACCATAACAAGCCACCATTGACTATTTGCCTGACTCGGACTAGAAATAAATACCCTAGCAATTATTCCCACCATCACCACCCTCCACAACCCACGATCAACAGAAGCAGCCACAAAATATTTTCTAACACAAGCAGCAGCCTCTGCCATAATTTTATTTTCAAGCATAACTAATGCTTGACAAACCGGATCATGAGATATTACTAGCACGATATCTACACCAGCCCATATCCTACTAACTTTAGCACTAACAATAAAGCTAGGACTTGCCCCAATACACTTCTGACTACCCGAAGTTCTACAAGGCTCAACCCTAACTACAGCACTCATTATTACAACCTGACAAAAACTTGCACCTCTAACCCTAATTTACTTGACTATCTCTAATTTATCAACAATAATTTTAATTACCATAGGACTTTCATCAACTATCATTGGAGGGTGAGGCGGACTCAACCAAACACAAATCCGAAAAATCATAGCATACTCATCTATTGCCCACCTAGGATGAATACTGTCTATCTCATCTATTATAACAAACATTATAATTTTAAACTTGATAATTTACTTAATTGTAACAACAGCCCTATTTAGCTTCTTAATTTTTACTCGATCCAAAACAATTCAAGATACAATAATAACCTGAACACTCTCACCCATAACAACTTCTATAATAATATTACTATTTTTATCCCTTGGAGGACTACCCCCATTAACTGGATTTATTCCAAAATGACTTATTTTAGAAGAACTAATTACCCAAAATCTTGATTCAATAGCTATTATTATAGCCCTATCTACACTACTTAATTTATACTTTTACTTACGATTAACCTACACAACAGCCATTACACTATCCCCAAACATATTACAAACAGAATTTAAATGACGACTAAAACCGAAATTTACACACACTGTTATTAGTAACCTCATCCTCAATATCTATTTTTTTTCTTCCCTAATACCACTAATTTTATTATAAAAACTTAGGTTATTTAAACCAAGGGCCTTCAAAGCCCTAAATAAGGACACCAGCCCTTAGTTTTTGATAAGACTTATGGGAAACTAACCCACATCTTTTGAATGCAACCCAAACACTTTTATTAAGCTAAAGCCTCTCGGAGCAAGTGGGCCTTGATCCCACAAAAATTAGTTAACAGCTAATTACCCTAACCAGCGGGCTTCTACTCGNNNNNNNCCGTACGGGAGAANNCCCCGAGCCTTTTAGGGGCTCTTCTTCAAATTTGCATTTTGATGTAAGTCACTGCGGAGGCTCTGATAAAGAAAGGAGTTAAACCAATGTGTGCAGGACTACTGCCTGCCGCCTAACACTCGGCCACTTTACCTGNGTTTATTAGCCGTTAT +>A.insolitus +ATGAACCCAACTATTCTTACATTAATTTTATCAAGCTTAGCAACAGGTACAATCCTTACAATAATCAGCTTTCACTGACTACTCGCATGAATTGGTCTAGAGATTAATACCCTAGCAATTATTCCTATTATCTCAGCACCTCACCACCCCCGACCCACAGAAGCCTCCACAAAATATTTCCTTACACAAGCAGCTGCTTCTGCTACAATCTTATTTTCAAGCATAATTAATGCTTGACTAACAGGCACATGAAACATTACCCAAATATCCTCTACTCCCGCTAATATTCTTCTAACCCTAGCACTAGCAATAAAACTAGGACTTGCCCCCACACACTTCTGACTACCAGAAGTGCTTCAAGGCTCAACTATAAACACAGCCCTCATTATTCATACATGACAAAAACTTGCCCCAATGTCACTGATTTACTTAACAGTAAATAACTTATCCCCTACAATTCTATTATCCCTGGGCCTCTTATCTTCCCTAATTGGAGGCTGAGCCGGACTAAATCAAACACAAACTCGAAAAATTATAGCATACTCATCAATCGCACACCTAGGATGAATGGCCACAGTAGCCTCAATTATAACAAATATTCTTCTAATAAACCTCATAATCTACTTGTTAATAACAACAGCCATATTTTTCTCTCTTATTTTATCCAAAACTAAAACAATTCAAGACTCAGCAATAACCTGAGCACTTTCACCAGCCTTAACTACTATAACAATACTCACACTTCTCTCATTAGGCGGACTACCCCCACTTACTGGATTTATGCCCAAATGACTAATCCTAGAACAATTAGCCAGCCAAGACTTAGTGCCCCTAGCCACTATTATAACTATATCTGCCTTACTTAGCCTATTTTTTTATCTACGACTAACCTACACAACAACACTTACACTATCCCCTAACACTACACAAACAAAATTTAAATGACGATTTAAACTAAACCAACCTACCCAACTAACAACAACCACTATTACAATAGCAACCTTATTACTACCCATACTACCAATGCTCACACTATAAAAACTTAAGTTAAACCAAACTAAGAGCCTTCAAAGCTCAAAATAAGGGTAGAACCCCCTTAGTTTTTGTAAGACTTGTGAAACACTAACTCACATCTCCTGAATGCAACTCAGACACTTTTATTAAGCTAAAACCTTCATGAATAAGTGGGCCTTGATCCCACAACAAATTAATTAACAGCTAACTACCCTAACCAGCGGGCTTTTATTCGCTTCTCCCGTACGGGAGAANNCNCGGAGCCTTTTTGGGCTCCTTTTCAAACTTGCATTTTGACGTGAAGCACTTCGGGACTTTGATAAAGAAAGGAATTAAACCAATGTAAATAGGACTACAGCCTACCGCCTAGCACTCGGCCACTTTACCTGTGTTTATT +>A.krugi +ATGAGCCCTGCAATTTACACAATTATATTATCAAGCTTGGCAACAGGCACTATCATCACTATAACAAGCTTCCACTGACTAATAGCCTGAGTTGGACTAGAACTTAATACATTAGCAATTATCCCAATTATTTCAACATTACACCACCCCCGGGCCACAGAAGCCTCAACAAAGTATTTCCTCACTCAAGCAGCAGCCTCTGCCATAATTTTATTTTCTAGCATAATTAATGCCTGACACACGGGAACATGGGACATTACTCAAATATCTTACACCCCCTCCAACATCATTTTAACAATAGCACTAGCCATAAAGCTAGGACTTGCCCCAATGCACTTTTGACTTCCAGAAGTCCTACAAAACTCAACCCTTATTACTGCTTTTATTATTTCTACATGACAAAAACTTGCCCCAATATCACTAGTCTACTTGACTATTAATAACCTTCCACCAATAATTCTGCTTACCCTAGGAGTTATTTCCTCTACTATGGGCGGATGAGCAGGACTAAACCAAACACAAACTCGAAAAATTATAGCATACTCATCAATTGCACACCTGGGCTGAATGGCCTCTATTGCCTCAATCATAACAAACATTATAATCATGAATCTAATGATTTATTTAATTATAACAGCGGCCCTATTCTTCTCATTAATTTATTCTAAGTCAAAAACAATTAAAGACACAACATCTGCCTGAACAATATCCCCCACCCTAACTATTACAATAATGTTAACCCTTTTATCACTTGGAGGATTACCTCCCCTAACAGGATTTTTACCAAAATGACTAATCCTAGAAGAANNNNNNNNNNNNNNNCTTGCCCCATTAGCCACCACCNTAGCAATATCCGCTCTTCTAAGCCTATTCTTTTACCTACGCCTGGCCTATACAACAACACTAACACTTTCACCAAATACACTTCAAATAAAATTTAAATGACGATTTAAACCAACCACTACACCACCACTAATAATCTTTTCATCTATAGCCGTTTTTTTACTACCACTTACACCTTTAATCTTATTGTAAAAACTTAGGATAAGATAATAAACCAAGAACCTTCAAAGTTCAAAATAAGGGTGAAAGTCCCTTAGTTTTTGTAAGACTTGTGAAACATTAATTCACATCTCATGAATGCAACTCAAGCACTTTTATTAAGCTAAAGCCTCCCTGAATAGGCGGGCTTTGATCCCACGAAAAATTAATTAACAGCTAACTACCCATTCCAGCGGGCTTCTATTCGCTTCTCCCGTACGGGAGAAGCCCCGGAGCCCCTTAGGGCTCTTTTTCAAACTTGCATTTTGACGTGAAAGCCACTTCAAGGCTGTGACAAAGAAAGGATTCAAACCAATATAAGTAAGTTTACAGCCTACCGCCTAATATTCGGCCACTTTACCTGTGTCTATTAATC +>A.limifrons +ATGAGCCCATCAATTTTTATAATTATTTTATCAAGTCTAGCCACAGGGACAATTATTATTATAACAAGCCACCATTGACTTTTTGCCTGACTTGGCTTAGAAATAAACACTTTAGCAATTATCCCAACTATTTCTACTACCCATAACCCACGATCAACAGAAGCAGCTACAAAGTATTTTTTAACACAAGCAGCAGCCTCCGCCTTAATTTTATTTTCAAGTACAATTAATGCTTGACAAACAGGAACCTGAGATATTACTAACACACTATCTACACCCGCACACATTTTATTGACCCTAGCACTAGCAATAAAACTAGGACTTGCCCCAATACACTTCTGACTGCCAGAGGTATTACAAGGCGTTACATTAGTTACTGCACTTATTATTTCTACCTGACAAAAACTCGCACCACTAACCCTAATTTACTTAACAGTTAATAATCTATCAACAACAATTCTATTAGTTATGGGACTATTATCTGCCATTGTAGGCGGATGGGGCGGACTTAATCAAACACAAACTCGTAAAATCATAGCATATTCATCAATTGCTCACCTAGGCTGAATACTCTCTATTTCATCCATCATAGTTAATATTATACTTTTAAACTTANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAAAACAATCCAAGACACAATAATGACATGAACACTCTCACCCATACTAACCTCTATAATAATAACTTTACTATTGTCATTAGGTGGTTTACCACCTTTAACCGGATTTATACCAAAATGGCTAATCTTACAAGGACTCGTTGCCCAAAACCTTACCTCAGTAGCCATTATATTNNCAATCGCCACCTTACTTAGCTTATTTTTTTACTTACGATTAGCTTATACAACAGCCACTACACTTTCCCCAAAGCNATTACAAACAAAACTCAAATGACGATTAAAACCAATATTACCAACATTACTTGTGGCAGCCTCATCTATAATANGCCACCTTTTTTCTTCCTATACCCCCCTATTTTTACTATAAAAATTTAGGTTAACTAAACCAAGGGCCTTTAAAACCCTAAATAAGGACACTCACCCCTTAATTTTTGATAAGACCTATAAATGACTTATTTATATCTCCTGAATGCAANNNAACAACTTTCATTAAGCTAAAGCCTCTCTGAGAAACGGNNCCTTGANNNNNNAAAAATTAGTTAACACCTAATTACCCTAACCCACGGGCTTCTACTCGCTTCTCCCGTACGGAAGAACCCCNNGACCCCTTATGGGCTCTCCTNCAAACTTGCATTTTGACGTAATTCACTTCGANGCTTTGATAAAGAAAGGAATTAAACCAATGTNTACAGGACTACAGCCTGCCGCCTTACACTCGGCCACTTTACCTGTGTTTATTAATCGTTGA +>A.lineatopus +ATGAGCCCTGCTATCTTTTCAATTGTCATATCCAGCCTAGCAACAGGCACTATTATTACCATAACAAGCCACCACTGACTATTAGCCTGAATAGGGTTAGAAATAAATACCCTAGCAATTATTCCAATTATTTCTATTACCCACAACCCCCGAGCCACAGAAGCCGCCACAAAGTACTTCTTAACACAAGCAGCAGCCTCAGCCATAGTTTTATTTGCAAGCATAACTAATGCTTGACAAACAGGAACATGAGATATTACTAGCCTATCATATATGCCCTCACATATTTTATTAACTATAGCATTAGCTATAAAACTAGGGCTTGCCCCAATGCACTTCTGACTACCAGAAGTGCTACAAGGAACAGCCCTACCAACTTCACTTATTATTACTACTTGACAAAAACTTGCCCCAATGTCCCTAATTTATATAACTATTAATAGTTTATCAACCATAATTCTAATNTCACTAGGCCTACTATCCTCAATTATTGGAGGATGGGGAGGACTAAACCAAACACAAACTCGAAAAATTATAGCATTTTCATCAATCGCCCACCTAGGATGAATAATTACTATTTTTTCACTCATAACAAATATTATAATCTTAAACCTAACAATTTACTTAATCATAACAACAGCAATATTTTGCTCACTAATCTTATCAAAATCAAAAACAATACAAGATATTATGATAACTTGAACAATCTCACCCACCCTTACTACCCTAATAATAATTTTACTCCTCTCCCTGGGCGGCCTACCACCACTAACCGGATTTATACCAAAATGATTAATTCTAGAAGAATTGGTTGCCCAAAACCTTATCCCAACAGCTACTATTATAGCAATGTCGGCACTCCTCAGCCTATTTTTTTATTTACGACTTACCTATGTAACTGNACTTACACTCTCCCCAAACACAGCACAAACAAAATTCAAATGACGACTTAAACCAAACTCACCAAAACTGTTCATAATAGCTTCCTCCAGCAATATCAATATTTTTACTTCCCTAACCCCCCTTATATTACTATAAAAACTTAGGATAATTAAACCAAGGGCCTTCAAAGCCCTAAACAAGGGCACTACCCCCTTAGTTTTTGTAAGACTTATGGACAACTACACCATATCTTCTGAATGCAACTCAAACACTTTAATTAAGCTAAAGCCTCTTCCAAATAGGGGGGCCTTGATCCCACAATAAACTAGTTAACAGCTAATCGCCCAAGCCAGCGGGCTTCTATTTGCTTCTCCCGTACGGGAGAAGCCCCGGAGCCTTTAAGGGCTCCTTTTCAAACTTGCATTTTGATGTGAATCACTTCGGGGCTGTGATAAAGAAAGGAATTAAACCAATGTAAATAGGACTACAGCCTACCGCCTACCATTCGGCCACTTTACCTGTGTTTATTAATCGTTGA +>A.lineatus +ATGAGTCCAACAATTTTTATAATTATAGTGATAAGCCTAACAACCGGGACTATTATTACCTTAACAAGCCACCACTGACTTATGGCCTGAATAGGCCTGGAAATAAACACCTTAGCAATTATTCCAATCATTTCCTACACCCATAACCCACGATCCACCGAAGCTGCCACAAAATATTTCCTAACCCAAGCAGCAGCCTCCGCTACAATTTTATTTTCAAGTATAACTAATGCCTGACAAACAGGCACATGAGANNNNACCAATATATCTTACGAACCATCATGCATTCTATTAACAACAGCACTAGCAATAAAACTAGGACTCGCCCCCATACACTTCTGACTACCAGAAGTCGTTCAAGGAACAACACTAACTAATGCATTAATCATCACCACCTGACAAAAACTTGCACCAATAGCCCTAATTATGCTTACCATTAACAACCTATCAACAACAATTTTACTACTAATAGGACTACTTTCCGCAATCATTGGTGGATGGGGAGGACTAAACCAGACACAGACCCGAAAAATCATGGCCTACTCATCAATCGCACACCTCGGATGAATAATTACCATTTCATCAATCATAACAAACATCATACTAATTAACCTACTTATTTACTTAATTATAACCACAGCAATGTTCTGCCTACTAATTTCATCCAAATCAAAAACAATCCAAGATACAACAACAACATGAACACTATCACCAACACTAACTTCAATAGCATCAATTTTACTTTTATCCCTAGGAGGACTGCCACCATTAACCGGGTTTGTTCCAAAATGATTAATCCTAGAAGAATTAGTAACCCAAAACCTAATCCCAACAGCTATTATTATAGTAATATCATCGTTACTTAGCTTATTCTTCTACCTACGACTAACATATACAATAACCATAACACTGTCCCCTAACACAACACAAACAACATTTAAATGACGATCTAACCCTAACTTTAACTCTTCATTAATTATAATTTCATCAACAATATCGATCTTTTTACTTCCAATAACCCCATTAATACTACTATAAAAACTTAGGTTAACACAAACCAAGGGCCTTCAAAGCCCTAAATAAGGATGACCTCCCCTTAGTTTTTGTAAGACTTATGAAAACTACTTCATATCTTCTGATTGCAACTCAAACACTTTTATTAAGCTAAAGCCTTCACCTGAACAGGAGGGCCTCGATCCCACAAAAATTAGTTAACAGCTAATTACCCAAACCAGCGGGCTTCTATTCGCTTCTCCCGTACGGGAGAAGCCCCGGAGCCCCTTAAGGGCTCATTTTCAAACTTGCATTTTGACGTGAATCACTTCGGGACTTTGATAAAGAAAGGAATTAAACCAATGTGAATAGGTCTACAGCCTACCGCCTCTCATTCGGCCACTTTACCTGTGTCTATTAATCGTTA +>A.loysiana +ATGAACCCAGTCGTTATTNNNATTCTTCTATCAAGCCTAGCAACTGGTACTATTATTACTATAACTAGCTATCACTGATTAATAGCCTGAGTTGGACTAGAAATAAACACATTAGCAATTATTCCAATCATTTCAACAACTCACCACCCACGAGCCACAGAAGCAGCCACAAAATACTTCTTAACCCAAGCTGCAGCCTCCGCCCTAATCTTATTTTCAAGTACAATTAATGCTTGATACTCAGGCTCATGAAATATTACCCAAATACTAACCTTCCCATCACACATTCTCTTAGCCATAGCACTGGCCATAAAATTAGGCCTAGCCCCACTTCACTTCTGACTACCAGAAGTTATTCAAGGATCAACCATAATTACTGCATTTNTTATTACCACATGACAAAAACTAGCCCCTATGTCTCTTATTTTCATAACCTCAAACAATATGCCCACAACAATCTTTCTGCTTATAGGACTTCTTTCCTCATTCACCGGAGGGTGGGGCGGACTAAACCAAACACAGACCCGAAAAATTATAGCCTACTCATCAATCGCCCATCTAGGATGAATCGCCTCCATTTCCTCAATTATAACAAACATTATAATTATTAACCTACTAATCTACCTAATTATAACGACCTCCATATTTTTAACCCTTATTTTTTCTAACTCAAAAACAATCCAAAACACAGCAATTAATTGAACACTTTCTCCAACACTTACAACTATTATTCTACTTTCACTCCTATCACTAGGAGGACTACCACCCCTAACAGGATTTATACCAAAGTGACTAGTTATAGAAGAACTAATTTTACAAAACTTCTTTTTATTAGCCAGTATAATAGCCCTATCATCTCTACTCAGCCTATTTTTTTACCTCCGACTAACCTACACTACCACACTAACACTTTCCCCAAACACAACACAAACAAAATTTAAATGACGATTTTACCCTAACACAAANACAACCCCAATAATAATCTCAGCTACAATCTCTATTTTCCTTCTACCACTAACCCCACTCATTTTATCATAAANACTTAGGATAAACAAAACCAAGAGCCTTCAAAGCTCAAAATAGGAGTGCAATCCCCCTAGTTTTTGTAAGACCTGTGAAATACTATTACACATCTTCTGAATGCAACTCAAACACTTTTATTAAGCTAAAGCCTCCCTGAATAGGCGGGCCTTGATCCCACGACAAATTAATTAACAGCTAATTACCCTAACCAGCGGGCTTCTATTCGCTTCTCCCGTACGGGAGAAGCCCCGGAGCCTTTTATGGCCCTTCTTCAAACTTGCATTTTGACGTGAAAACACTACGAGACTCTGATAAAGAAAGGAATCAAACCAGTGTAAGTAGGTCTACAGCCTACCGCCTAACATTCGGCCACTTTACCTGTGTTTATTTACCGTTGA +>A.equestris2 +ATAACAAGTTATCATTGACTTATGGCCTGAATTGGACTAGAAATAAACACATTAGCAATTATTCCAATTANATTTACTATACACAACCCACGAGCCACAGAAGCTGCCACAAAGTACTTCCTAACACAAGCATCAGCATCAGCCATAATTTTATTTTCAAGCACAATTAACGCCTGACAAACAGGGACCTGAGACATTACTAACCTCTCATCCGAACCAGCACACATTTTATTAACCATTGCACTAGCAATAAAACTAGGCCTAGCCCCAATACACTTTTGACTACCAGAAGTACTACAAGGGTCAACCATAACCACAGCACTAGTTATCTCTACTTGACAAAAACTGGCCCCAATAGCCCTAATTTATCTGAACATTAATAATCTATCAACAGCTGTCCTAATCTTACTAGGACTCTTATCATCAATTATTGGGGGCTGAGGGGGACTTAATCAAACACAAACCCGAAAAATTATGGCATTTTCATCAATTGCACACCTAGGGTGAATAATTACTGTTTCATCTATTATAACTAATATCATGATTATAAACTTAATAATCTACTTAATTATAACTACAGCCCTATTTTACTCATTAATTTTATCCAAATCAAAAACTATTCAAGATACAACAACAGCCTGAANACTATCACCCACCCTCACCACTATAATAATAATTTTACTTCTGTCCTTAGGCGGACTACCACCATTAACAGGATTCATGCCTAAATGACTTATTTTAGAAGAACTAATTATTCAAAACCTTATCCCAACAGCCACTACTTTAGCAGTATCCACACTACTAAGCCTATTTTTTTACNTACGGCTTGCCTACACAACAACACTCACCCTATCCCCAAACACTATTCAGACAAAATTTAAATGACGATTTAAATTAAAATTTACAACATTTTTTATAATGCCACTTTCCACAATTTCAATTCTTCTCCTTCCTCTTACCCCGCTAATACTATTATAAAAACTTAGGATNNNNNNNAAACCAAGGGCCTTCAAAGCCCTAAATAAGGGNCCCACCCCCTTAGTTTTTGTAAGACTTATGGGTGTCTACTCCATATCTCCTGAATGCAAATCAAACACTTTTATTAAGCTAAAGCCTTGACCAAATAGGTGGGCCTTGATCCCNNAAAAATTAGTTAACAGCTAATTACCCAAACCAGAGGGCTTCCATTTGCTTCTCCCGTACGGGAGAACCCCNGGAGCCTTTCAGGGCTCATTTTCAAACTTGCATTTTGATGTGAATCACTTCGGGGCTGATAAAGAAAGGAATTAAACCAATGTAAATAGGACTACAGCCTACCGCCTACCATTCGGCCACTTT +>A.lucius +ATGAGCCCAACAATTTATATAATCACTTTATCAAGCCTAGCAACAGGGACTATTATCACCATAACAAGCTACCACTGACTAATAGCCTGAGTGGGGTTAGAAATCAACACACTATCAATTATTCCAATCATTTCAATAATACATCACCCACGATCCACAGAAGCTGCCACAAAATATTTCTTAACCCAAGCAGCAGCCTCAGCTCTAATTTTATTTTCTAGTACAACACACGCATGATATTCAGGCACATGAGATATTACCCAAACATCAAATATTACCTCCAACATTCTTCTAACAATAGCACTTACAATAAAATTGGGCCTAGCCCCATTACACTTTTGATTACCAGAAGTAGTTCAAGGATCGACTATAAATACAGCCTTTATCATTATCACATGACAAAAACTAGCCCCAATATCACTTATTTACATAACTATAAACAACTTATCCACAACAATTTTATTAATCATAGGATTAATATCATCAATAATTGGAGGCTGAGGCGGCCTAAACCAAACACAAGTCCGAAAAATTATAGCCTACTCATCAATTGCACATCTTGGATGAATAGCAACAATTTCAACAATTATAACTAATATTTTAGTTTTAAACCTATTAATTTACCTAATTATAACAACATCTATATTTCTTTTATTAATTTTAACCAATTCTAAAACAATTCAAGACATATCAACTACCTGAACTCTTTCACCAACCCTACTAATTATAATAATACTAACACTATTATCACTAGGCGGCCTACCCCCTCTAACAGGATTTATACCAAAATGATTAATCTTAGAAGAGCTAACAACCCAAAACTTAACCCCACTAGCTACCCTTATAGCACTATCAGCCCTATTAAGCTTATTCTTTTACCTACGATTAGCTTACACCACAGCCCTTACACTATCTCCAAACACACTACAAACAAAATTTAAATGACGATTTACCCAAAATATAGCAACACCAACAACAATTATTACCACTACAATAACAATTTTTTTGCTCCCTTTAACACCTCTTATACTCTCATAAAAACTTAGGATAATTCAAACCAAGGGCCTTCAAAGCCCAAAATAAGGGCTAATCCCCTTAGTTTTTGCAAGACTCATGTAATATTAATACACATCTCCTGAATGCAACTCAAACACTTTAATTAAGCTAGAGCCTCTCTGAATAGGCGGGCCTTGATCCCGCGACAAATCAATTAACAGCTAATTACCCAAACCAGCGGGCTTCTATTCTTCTCCCGTACGGGAGAAGCCCCGGAGCCTTTTAGGGCTCTTCTTCAAACTTGCAATTTGATGTAAGTCACTTCGGAGCTGTGATAAAGAAAGGAGTTGAACCAATGTAAATAGGACTACAGCCTACCGCCTATCAGTCGGCCACTTTACCTGTGTTTATTAATCGTTGA +>A.marcanoi +ATGAGCCCAACAATTTTTTCAATTATGCTATCGAGTCTAGCAACAGGCACCATTATTACTATAACAAGCTTTCACTGACTAATGGCCTGAGTCGGCTTAGAAATTAATACGCTAGCTGCTATTCCAATTATTTCAATACAACACCACCCTCGATCAACAGAAGCAGCCACAAAATACTTTTTAACACAAGCAACTGCATCCTCCTTAATTTTATTTTCAAGTATGATTAATGCTTGGCATACAGGAACATGAGACATTACTCAAATTTCAACTACCCAATCATGCACCCTTCTTACAATAGCAATCTCAATAAAGCTAGGCTTAGCCCCACTACACTTCTGACTACCAGAGGTTCTCCAAGGTTCAACAATATTTACTGCTTTAATTATTATAACATGACAGAAACTAGCCCCCATATCCCTACTTTTCCTCACTATAAACAACCTATCCACATCAATACTCTTAACAATTGGACTTGTATCAACAATAGTTGGCGGATGGGCAGGATTGAATCAGACACAAACACGAAAAATTATAGCATACTCATCAATTGCGCATCTTGGCTGAATAGCCCCTATTGCCTCAATTATAACAAACATTCTTATTATAAATTTACTTATTTACTTACTTCTTACAACGGCCCTATTTTCCTCTCTAACACTATCTAAATCAAAGACAATTCAAGATACATCAACAACCTGAACAATATCACCATATTTAACTATTATAACCATACTTATACTTCTTTCATTAGGCGGACTACCGCCACTTACAGGATTCATCCCAAAATGATTAGTATTAGAAGAACTAGTTACTCAAAACCTACTCCCACTAGCTACTATTATAGCAATAGCAGCTCTCCTTAGTCTTTTCTTTTATTTACGCCTAACCTATGCCACAGCACTAACACTTTCACCGAATACAATACAAACTAAATTTAAATGACGATTTAAACCTAATCACCCATCCGCAACAACAGCACTAACTTGTACAATCGCCATTTTTTTACTCCCAATAACCCCTCTTATTTTGCTATAAAAACTTAGGATAAACAAACCAAGGGCCTTCAAAGCCCGAAATAAGGGTGCAAGTCCCCTAGTTTTTGTAAAACTTGTACTATACTATAGCACATCTCTTGAATGCAACTCAAGCACTTTTATTAAGCTAAAGCCTCCCTGAACAGACGGGCCTCGATCCCGCGACAAATTAATTAACAGCTAACTGCCCAAACCAGCGGGCTTCAATTCGCTTCTCCCGTACGGGAGAAGCCCCGGAGCCTTTTAGGGCTCATCTTTAAATTTGCATTTTAACGTATAACACCTCGGGACTTTGATAAAGAAGGGGTTTAGCCCTCATGGGTGGAGCTACAAGCCACCGCCTATTATTCGGCCACTTTACCTGTGTTTTTTACTCGTTGA +>A.maynardi +ATGAGCCCACCATCNTATANAATTATTTTATCAAGCCTAGCAACCGGCACCATTATTACCATAACCAGCTATCACTGACTTATAGCCTGAGTCGGCCTAGAAATAAACACCTTAGCAATCATCCCAATCATCTCAACAATACATCACCCACGGTCTACAGAAGCAGCTACAAAATACTTCTTAACCCAAGCTGCAGCCTCAGCTCTAATTTTATTTTCAAGCATAACAAATGCTTGAAGCTCAGGCTCATGAGATATCACTCAAACATTAACCTCCCCCTCACATATTCTATTAACTATAGCACTAGCTATAAAGCTCGGACTAGCCCCAGTACACTTCTGACTACCAGAGGTAATCCAAGGCTCTTCCATAATTACAGCCTTCATTATCACCACATGACAAAAACTTGCCCCAATCTCACTTATCTTNATAACCATAAACAACCTATCTACTACAATCNNNTTATTAATAGGACTTCTATCTTCACTCATTGGAGGATGGGGAGGACTTAACCAAACACAAACCCGAAAAATCATAGCATACTCATCAATTGCACATCTAGGATGAATAGCAACAATTTCTTCAATCATAACAAATATTCTCATTATGAACTTATTAGTCTATCTTATTATAACAACCTCAATATTTCTCTCTCTTATCATCCCTAAATCAAAAACAATCCAAAACACAACATCCACCTGAACACTATCCCCAACACTTACAATTATTATGATACTCTCACTCCTCTCCTTAGGAGGACTACCCCCACTAACAGGGTTTGTACCAAAATGACTTATCATAGAAGAACTAATTTTGCAGGACTTCAACTTACTAGCCATCATAATAGCAATATCCTCCTTACTAAGCCTATTTTTTTACTTACGACTAACTTATGCAACCACACTTACACTATCTCCAAATACAACCCAAACAAAATTTAAATGACGATTTTACCCAAGCACACCAACCATCTTTCTACCTATTCCTGCTGCAATCTCCATCTTCCTACTCCCCCTAACCCCACTAATTTTGTTATAAAAACTTAGGATCAACTAAACCAAGAGCCTTCAAAGCTCAAAATAGGGATGAAAACCCCCTAGTTTTTGTAAGACTTGTGAAATATTATAACACATCTCCTGAATGCAACTCAAGCACTTTAATTAAGCTAAAGCCTCTCTGAACAGGCGGGCCTTGATCCCACAAAAAATTAATTAACAGCTAATTACCCAAACCAGCGGGCTTCTATTCGCTTCTCCCGTAACGGGAGAAGCCCCGGAGCCTTTAGGGCTCCTTNTCANACTTGCATTTTGACGTGAAACACTTCGGGGCTTGATAAAGAAGGGAATTANNCCCCTGTGAATAGGACTACAGCCTACTGCCTGACACTCGGCCACTTTACCTGTG +>A.microtus +ATGAGCCCGATAACTGACACAATCATTATTTCCAGCCTTACAACCGGCACNNNNNNNACTATAACCAGCTACCATTGATTGGTAGCTTGACTTGGACTAGAGATCAACACATTGGCAATCATTCCAATCATCTCAATACAACACCACCCCCGATCCACAGAAGCTGCCACAAAATACTTCCTCACACAAGCAGCAGCCTCTGCCATAGTCTTATTCGCAAGCACAACCAACGCTTGACATACAGGAACATGAGACATTACCCAAATAACCACCCCTCATTCCAACACTCTTCTTACCATGGCACTCTCAATAAAACTAGGATTAACCCCCGTACACTTCTGATTGCCAGAAGTACTACAAGGATCAACCATAACTACAGCACTCATTATTACAACATGACAAAAACTAGCTCCCATATCCCTAATTTATCTGACCATAAACAACCTCTCCCCAACAATCCTTATCACATTGGGGCTTCTATCTTCAATAGTAGGGGGGTGAGCGGGCCTAAACCAAACACAAACACGAAAAATTATAGCATACTCATCAATCGCTCACCTGGGATGAATAGCAGCAGTATCATCAATTATAACAAACATCCTAACACTAAATTTATTTATCTACTTAATAATAACAACAGCCTTATTTTATTCACTAATTTTATCAAAAACTAAAACAATCCAAGACACAACAACCTCTTGAGCAACCTCCCCCATACTAACAACTATCATAATGCTTACCCTTTTGTCATTAGGAGGCCTACCTCCACTTACAGGCTTTATACCAAAATGACTAATTCTAGAAGAACTAATGATACAAAACTTAGCCCCCCTAGCAATTGTTATAGCANNNACATCACTACTAAGCCTTTTTTTCTACCTACGACTAACCTACACAACCACACTTACTCTATCTCCAAATACAGTACAAACAAAACACAAATGACGATTTAAACCTTCAACTACCATACTTCCACTAACCATCATAACACCCGCAACCCTACTTCTATTACCAATAACACCCCTAGTTATTTGCTAGANACTTAGGATAAACCTAAACCAAGAGCCTTCAAAGCTCAANATATGAACTAGCCCTCATAGTTTCTGCAAGACTTGTGAAACATTAACACACATCTCTTGAATGCAACTCAAGCACTTTAATTAAGCTAAAGCCTCTCTGAATAGGCGGGCCTTGATCCCACGATATATTAATTAACAGCTAACTACCCAAACCAGCGGGCTTCTATTCGCTTCTCCCGTACGGGAGAAGCCCCGGAGCCTTTTTAGAGGCTCTTCTTCAAATTTGCATTCTGACGTGGAACACCCCAGGGCTGTGATAAAAAGAGGAATTAAACCCCTATTAATAGGACTACAGCCTACTGCCTTACATTCGGCCACTTTACCTGTGTTTATTAATCGCTGA +>A.occulatus +ATGAGCCCCAATAATCTACTTAATAGTTTAATTAGCTTATTTATANNNACAACACTAGTAACCACTAGCCACCACTGATTATTAGCGTGAGTTGGCTTGGAAATTAACACACTTGCAGCTATTCCACTTATCTCAACAAAACATCACCCCCGAGCTACAGAATCAGCCACAAAATACTTTTTAATTCAAGCAGCAGCCTCAGCTACAATCTTATTTTCAAGTACCATTAATGCTTGACACACAGGCTCATGAGACATTACCCAAATAACCACAAACCCATCCAATATTTTACTAACTATAGCCCTTGCCATAAAACTAGGCCTAGCCCCAACACACTTCTGACTCCCAGAAGTACTTCAAGGGTCAACTATAGACACTGCCCTAATTATTACAACATGACAAAAAATTGCCCCAATAACCCTAATTTACCTCACCATAAACAACCTATACCCAAGCATTCTAATAACAATAGGCCTATTATCTACAATAATCGGGGGTTGAGGAGGCCTAAACCAAACACAAACCCGAAAAATTATAGCATACTCATCAATTGCACACTTGGGTTGAATGGCCGTAATTTCCACCCTAATAACAAACATCTTTATTATAAACCTAATTATGTATCTAATTATAACAACAACCGCATTCTGGGCCCTAATCTGCTTAGAATCTAAAACTGTAAAAGACACAACAACTGCCTGAACAACCTCACCAACCATCACTTTAACACTTCTTATTTCCCTCTTATCCCTAGGAGGCCTTCCCCCTCTCTCTGGATTCTTACCAAAATGACTAATCCTAGAAGAACTTACAAACCAAAATCTACTACCATTAACCACCACTATAGCAGTCTCCTCCCTGCTAAGCCTGTTTTTTTACCTACGACTTACCTATAATACAACACTAACAATAGCCCCAAACACAATACAAACAAAATTCAAGTGACGATTTAAACCTAATAACCCACCCTTATTATTAACAGCTACTGCCCCACTAACCACTCTACTACTCCCGCTTACACCTCTTATATTAATATAGAAACTTAGATTTAAACAAACCAAGAGCCTTCAAAGCTCAAAATATGGGTGTAACCCCCATAGTTTCTGTAACACTTACAAAACTTTAATTTACATCTTTTGATTGCAACTCAAACACTTTTATTAAGCTAAAGCCTCCTTGAATAGTCGGGCTTCGATCCCACGACAAATTAATTAACAGCTAACTGCCCAAACCAGCGGGCTTCTACTCGCTTCTCCCGTACGGGAGAAGCCCCGGAGCCTTTTGGGGCTCCTTTTCAAACTTGCATTTTGACGTGAAAACACTTCGAGACTTTAATAAGNAAAGGAATCAAACCCCNGNAAATAGGACTACAGCCTACCGCCTAACACTCGGCCACCTTACCTGTGTCTATTAATCGTTA +>A.olssoni +ATGAACCCCACCATCTCCATAAATTATCTATCAAGCCTAGCAACAGGAACAATTATTACTATGACCAGCTTTCATTGATTAATAGCATGAATTGGATTAGAAGTCAACACACTAGCAATTATTCCAATCATCTCAGCCCCTCACCACCCACGATCAACAGAAGCTGCAACAAAATACTTTCTCACACAAGCAGCTGCCTCCGCTATAATTCTATTTGCCAGTATAATTAACGCCTGACAAACAGGCACATGAGACATTACTCAACTGTCAACCACCCCTGCCCACACCCTCCTAACTATAGCACTAGCCATAAAACTAGGACTTGCCCCACTTCACTTCTGATTACCAGAAGTTATTCAAGGTGCCACCCTTCCCACTGCTTTCATCATTGTAACATGGCAAAAACTTGCTCCTATATCACTTATTTTTTTAACCATAAACAACCTAAACCCAACAATTTTACTTCTCCTTGGACTTCTATCTTCTACAGTAGGAGGATGAGCCGGATTAAACCAAACACAAACCCGAAAAATTATAGCCTATTCATCAATTTCACACTTAGGCTGAATGGCCGCAATCTCCTCTATTATAACTAATATTCTTATTATAAACCTAACCCTTTATTTAATTATAACAACAACCATGTTTTATACCCTAATTTTAACTAAATCCAAAACAATCCAAGACTCAACCCTCACCTGATCACTTTCACCAGTACTAACAATTATTATAATACTAACACTCCTCTCACTAGGAGGACTACCTCCCCTAACCGGATTTTTACCAAAATGAATAATCCTAGAAGAACTTACTACCCAAAACCTAACCCCACTGGCCACCATAATAGCCATTTCAGCTTTACTAAGCCTATTTTTTTACCTACGCCTAACTTATACAACAACCTTAACACTCTCCCCAAACACAATACAAACAAAACTCAAATGGCGATTTAAATTAAATAAACCAACAACACTTATACTAATAACTTCTACAATTACCACCTTCTTACTCCCCCTATCACCACTCATTTTAATATAAAAACTTAGGTTTAACCAAACCAAGAGCCTTCAAAGCTCAAAATAAGGGTCAAACACCCTTAGTTTTTGTAAGACTTGTGAAATACTAATACACATCTTCTGAATGCAACTCAAACACTTTAATTAAGCTAAAGCCTCTCTGAATAGGCGGGCCTCGATCCCGCGACAAATTAATTAACAGCTAACTACCCAAACCAGCGGGCTTCTACTCGCTTCTCCCGTACGGGAGAAGCCCCGGAGCCGTTTGGGGCTCTTCTTCAAACTTGCATTTTGATGTGAAACACTTCGGGACTTTGATAAAGAAAGGAATTAAACCAATGTTAATAGGACTACAGCCTACTGCCTATCATTCGGCCACTTTACCTGTGTTCATTAAT +>A.ophiolepis +ATGAGCCCAACAATCTTTATAATTATTTTATCAAGTCTTGCAACTGGTACAATTATTACTATAACTAGTTATCACTGACTATTAGCCTGAATCGGCCTAGAAATTAATACCTTATCAATTATCCCACTTATTTCAACAACCCACCATCCACGAGCCACAGAAGCCGCTACCAAGTATTTTCTTACACAAGCAGCAGCTTCGGCCATAATTTTGTTTTCTAGTATAACTAATGCATGAGAGACAGGCACATGAGATATTACCCAGCTATCATCAACACCATCACACATCTTTCTTACAATAGCATTAGCAATAAAACTAGGTCTAGTCCCAGTACACTTTTGATTACCAGAAGTCCTACAAGGGTCAACCCTAACAACTGCACTTATTATTACTACATGACAAAAACTTGCACCAATATCTTTAGTCTACTTAACCATTAATTCCCTTTCAACAACAGTTTTATTATTAATGGGACTCATATCCACTGTTATTGCTGGATGAGCAGGACTTAACCAAACACAAACACGAAAAATTATAGCATACTCATCAATTGCTCACCTTGGATGAATAGTCGCTGTTTCTTCTATTATAATAAACCTCATAGTAATAAATCTAATTATTTATTTAATCATGACCTCTGCCCTATTTCAAGCACTAATTGTCTCTCAATCAAAAACAATTCAAGATACAACAATGACATGAGCACTATCACCAACCATTTCCATTTTAACCATAGCACTTCTACTATCACTTGGAGGCTTACCACCACTAACAGGATTTATCCCTAAATGATTAATTCTAGAAGAACTAGTAACCCAAAACCTCATTCCAACAGCAACAATAATAGCCCTATCTGCCTTACTAAGCCTTTTCTTTTACTTACGCCTAACTTATACCACAACACTAACACTATCACCAAACACAACTACTACAAAATTTAAATGACGACTTAAACCAAATCAACCTACACAACCCATACTTATTGCTACCACAACCTCTATCCTTCTACTTCCACTAGCACCACTCATGCTTATATAAAATAAAAACTTAGGATAACCTAAACCAAGGGCCTTCAAAGCCCTAAATAAGGGATAGACCCCCTTAGTTTTTGTAAGACTTGTGAAATATTAATTCACATCTCATGAATGCAACTCAAGCACTTTTATTAAGCTAAAGCCTTTAGGAATGGGCGGGCCTTGATCCCGCAAAAAATTAATTAACAGCTAATTACCCAAACCAGCGGGCTTCCATTCGCTTCTCCCGTACGGGAGAAGCCCCGGAGCCTTTTAGGGCTCCTCTTCAAACTTGCATTTTGATGTGGGTCACTTCGGGGCTTTGATAAAGAAAGGAATTGAACCAATGTTAGTAGGACTACAGCCTACCGCCTATTACTCAGCCACTTTACCTGTGTCTATTAATCGTTGA +>A.paternus +ATGAGCCCATTTATTCTTACAATTATTTTTTCAAGCTTAGCAACAGGCACAATTATTACTATAACCAGCTACCACTGACTTATGGCCTGAATTGGATTAGAGATAAACACACTAGCAATTATTCCCATTATCTCAACAACACATCACCCACGAGCCACAGAAGCTTCCACAAAATATTTTCTTACACAAGCTGCAGCCTCTGCCTTAATTTTATTTTCTAGTATAACCAATGCATGACATACGGGATCTTGAGACATTACTCAAATATTATTTTCTCCATCAAACACCATGCTAACCATAGCCCTAGCCATAAAACTAGGGTTAGCTCCACTTCACTTCTGGCTACCAGAAGTAATTCAAGGATCAACAATACTAACTGCCTTTATTATTACTACATGACAAAAACTAGCCCCAATATCTCTAATTTTTTTAACTATAAATAGTATGTCCACAACAATTCTTCTACTAATGGGAGTCTTATCCTCACTTGTTGGTGGTTGAGGGGGGTTGAACCAAACACAGACCCGAAAAATCATAGCATACTCATCAATTGCCCACCTCGGATGAATAGCTACAATTTCATCTATTATAACAAACATCTTAATTTTAAACCTAACTATTTACCTAATTATAACAACTTCCATATTTTTTTCCCTAATTATTTCTAAGTCAAAAACAATCCAAAACACAGCGGAGACCTGAACATTTTCACCCATACTAACGATTATAATAATACTTTCACTTCTTTCACTAGGGGGACTACCACCACTAACTGGATTTGTACCAAAGTGGCTAGTTATAGAAGAATTAATTTTACAAAATTTTAATCTTCCAGCTATTTTAATAGCTATATCTTCCCTACTAAGCCTATTTTTTTACCTTCGAATTTCCTACACAGCCACACTCTCACTATCACCTAATACAACTCAAATAAAATTCAAATGACGATTTTATCCAAAAACTACAACCACCTTAACCTCTTCCACCACAACAATAACAATCTTCCTCCTACCACTTACACCCATAATTTTATTATAAAAACTTAGGATCAAATAAACCAAGGGCCTTCAAAGCCCAAAATATGGGTTAAACACCCATAGTTTTTGTAAGACTTGTGAAACACTATTACACATCTCCTGAATGCAACTCAAGCACTTTTATTAAGCTAAAGCCTCACTGAATAGGCGGGCTTTGATCCCACGACAAATTAATTAACAGCTAATTACCCAAACCAGCGGGCTTCTATTCGCTTCTCCCGTCGGTGAAGAAACGGGAGAAGCCCCGGAGCCTTTAGGGCTCTTCTTCAAACTTGCATTTTGATGTGGAACACTTCGGGACTTTGATAAAGAAAGGAATTAAACCAATGTAAATAGGACTACAGCCTACTGCCTAACACTCGGCTACTTTACCTGTGTTTATTAACCGTTGATTCTTCTCAACC +>A.luciae +CAGCCACCATTGATTAATAGCTTGAATTGGATTAGAAATCAACACACTAGCCATTATTCCTATTATCTCAATACAACATCACCCACGAACTACAGAAGCTGCCACAAAATACTTTTTAACCCAAGCAGCAGCATCTGCCATAATCCTTTTTGCAAGCACAACAAACGCTTGATTTACAGGAACATGAAATATTACCCAAATATCAACTACCCCCTCCCATATCTTACTAACTATAGCACTAGCAATAAAACTAGGGCTAGCCCCAACACACTTCTGACTCCCAGAAGTACTACAAGGCTCAACCATAACCACCGCCCTAATCATTACCACATGACAAAAACTTGCCCCCATATCCCTAATTTACATAACCATAAACAACCTATCACCACTAATACTACTTCTACTAGGACTTATATCCTCAATCATTGGAGGCTGGGGCGGATTAAATCAAACACAAACACGAAAAATCATAGCATATTCATCCATCGCCCACCTAGGCTGAATAGCAGCCATTACATCAATTATAACAAATATTTTAATCTTAAACTTACTAATTTACCTTGTCATAACAATAACAATATTTTATTCCCTTATTTTACTAAAATTAAAAACAATCCAAGATACATCCACCTCCTGATCAACCTCCCCCACATTAACCACTACTACAATACTAACCCTTCTCTCACTGGGGGGCCTTCCCCCACTCACTGGATTTATGCCAAAATGACTTATTTTAGAAGAGCTAACCATACAAAACCTAGTTATACTAGCAACTATAATAGCACTAGCATCCTTACTAAGCCTATTCTTCTACTTACGCCTAAGCTACACAACAACCCTTACATTATCCCCAAATACCATCCAAACAAAACATAAATGACGATTTAAATCACCAACCACCACACACCCACTAATTACACTAATCCCTATAACTACCCTTATACTACCACTAACACCAACAATACTATCATAGAAACTTAGGACTAACCAAACCAAGAGCCTTCAAAGCCCTAAACATGAGTCTAACCCTCATAGTTTCTGTAAGACTTGTGAGATACTAACCCACATCTAATGAATGCAACTCAAACACTTTAATTAAGCTAAAGCCTTCCTGAGTAAGCGGGCCTTGATCCCGCGATATATTAATTAACAGCTAACTACCCAAACCAGCGGGCTTCTACTCGCTTCTCCCGTACGGGAGAAGCCCCGGAGCCTTTTAGGGCTCTTCTTTAAACTTGCATTTTAATGTGTAACACCCCGGGACCTCATGGTGGTAAAGAGAGGAATTAAACCCCTTTAAATAGGACTACAGCCTACCACCTAGCATTC +>A.paternus2 +ATGAGCCCATTTATTTTTACAATTATTTTTTCAAGCTTAGCAACAGGCACAATTATTACTATAACCAGCTACCACTGACTTATAGCCTGAGTTGGATTAGAAATAAACACACTAGCAATTATTCCCATTATCTCAACAACACATCACCCACGAGCCACAGAAGCTTCCACAAAATATTTTCTTACACAAGCTGCAGCCTCTGCCTTAATTTTATTTTCTAGTATAACCAATGCATGACATACGGGATCTTGAGACATTACTCAAATATTATTTACTCCATCAAACACCATGCTAACCATAGCCCTAGCCATAAAACTAGGGTTAGCTCCACTTCACTTCTGGCTACCAGAAGTAATTCAAGGATCAACAACACTAACCGCCTTTATTATTACTACATGACAAAAACTAGCCCCAATATCTCTAATTTTTTTAACTATAAATAGTATGTCCACAACAATTCTTCTACTAATGGGAGTCTTATCCTCACTTGTTGGTGGTTGAGGGGGGTTAAACCAAACACAGACCCGAAAAATCATAGCATACTCATCAATTGCTCACCTCGGATGAATAGCTACAATTTCATCTATTATAACAAACATCTTAATTTTAAACCTAACTATTTACCTAATTATAACAACTTCAATATTTTTTTCCCTAATTATTTCTAAGTCAAAAACAATCCAAAACACAGCAGAGACCTGAACATTTTCACCCATACTAACGATTATAATGATACTTTCACTTCTTTCACTAGGGGGACTACCACCACTAACTGGATTTATACCAAAATGGCTAGTTATAGAAGAATTAATTTTACAAAATTTTAATCTTCCAGCTATTTTAATAGCTATATCTTCCCTACTAAGCCTATTTTTCTACCTTCGAATTACCTACACAGCCACACTCTCACTATCACCTAATACAACTCAAATAAAATTCAAATGACGATTTTACCCAAAAACTACAACCACCTTAACCTCTTCCACCACAACAATAACAATCTTCCTCCTACCACTTACACCCATAATTTTATTATAAAAACTTAGGATTAAATAAACCAAGGGCCTTCAAAGCCCAAAATATGGGTTAAACACCCATAGTTTTTGTAAGACTTGTGAAANACTATTACACATCTCCTGAATGCAACTCAAGCACTTTTATTAAGCTAAAGCCTCACTGAATAGGCGGGCTTTGATCCCACGACAAATTAATTAACAGCTAATTACCCAAACCAGCGGGCTTCTATTCGCTTCTCCCGTACGGGAGAAGCCCCGGAGCCTTTAGGGCTCTTCTTCAAACTTGCATTTTGATGTGGAACACTTCGGGACTTTGATAAATAAAGGAATTANACCAATGTAAATAGGACTACAGCCTACTGCCTAACACTCGGCTACTTTACCCGTGTTTATTAACCGTTAT +>A.pumilis +ATGAACAGCCCAACCATTTTTTTTCTACTATCAAGCCTTCAACCCGGCACCACTATTACCATAACCAGCCATCACTGACTTATGGCCTGAGTAGGGCTAGAAATAAACACACTAGCTATTATCCCAATTATTTCAACAATACACCACCCACGAGCCACAGAAGCATCTACAAAATACTTCTTAACCCAAGCTGCAGCTTCCGCACTAATCTTATTCTCAAGCATAATTAGTGCCTGATATACAGGCTCATGAAATATTACCCAAACATTAACCTATCCATCACACATCTTACTGACCATGGCACTAGCCATAAAACTTGGCCTCGCCCCACTACACTTCTGACTACCGGAAGTAATTCAAGGATCAACCATACCCACAGCCTTCATTATTACCACATGACAAAAACTAGCCCCTATGTCACTTATCTTTTTAACCACAAACAATATATCCACAACAATTTTTTTAGCCTTAGGACTTTTATCCTCATTAATTGGAGGATGAGGAGGCCTAAACCAAACACAAACCCGAAAAATTATAGCCTATTCATCAATTGCCCATTTAGGATGAATAGCCTGTATCTCTTCAATTATAACAAACATTTTAATTCTAAACCTATTAATTTACCTAATTATAACAAGTTCAATATTTATAACCCTTATTTTATCTAAATCTAAAACCATTAAAGATACAACAACCACCTGAACACTCTCCCCCACACTTACAGTAATTATAATACTCTCCCTCCTTTCATTAGGGGGCCTCCCACCACTAACAGGATTTTTACCTAAATGACTAATTTTAGAAGAATTAATTTTACAAGACTTTCTCTTATTAGCCACAATAATAGCCTTAACATCACTATTTAGCCTGTTCTTCTACTTACGACTAACTTATACAACCCTTCTTACACTCTCACCAAACACAACCCAAACAAAATTTAAATGACGATTTTTCCCTAATACAAAAACAACCCCCACCATAATTTTCCTAACAATCTCCACCCTCTTACTTCCCTTAACCCCCCTCATCTTACTTTAAANACTTAGGATAACTAAACCATGAACCTTCAAAGCTCAAAATAAGGGTGTAAACCCCTTAGTTTTTGTAAGACTTGTGAAATACTAATTCACATCTCCTGAATGCAACTCAAACACTTTTATTAAGCTAAAGCCTCCCTGAATAGGCGAGCCTTGATCTCACAACAAATTAATTAACAGCTAATTACCCAAACCAGCGGGCTTCTATTCTCTTCTCCCGTACGGGAGAAGCCCCGGAGCCTTTATGGCTCATCTTCAAACTTGCAATTTGACGTAAAATCACTACAGAGCTTTGATAAAGAAAGGAATTAAACCAATGTAAGTAGGACTACAGCCTACCACCTAGCACTCGGCCACTTTACCTGTGTTTATTAATCGTTGA +>A.richardi +ATGAGCCCTGCAATATACACAATCATCTTATCCAGCCTTGCAACAGGTACTATTATTACTATGTCAAGTTACCACTGATTAATAGCTTGANTTGGCTTAGAAATTAATACACTAGCAATTATTCCAATTATTTCTATACAACACCACCCACGGTCTACAGAAGCCGCTACAAAATACTTCTTAATCCAAGCAGCAGCTTCAGCCATAATTTTNTTTGCAAGCACAACAAATGCCTGACATACAGGCACATGAAATATTATTCANATATCTACTACCCCTTCACACATTATATTAACCCTAGCACTATCAATAAATTTGGGACTAGCTCCAATACTCTTTTGCGTTCCAGAAGTCTTACAAGGATCAACCATATCTNCTGCCTTTNNNNNNACAACATGACAAAAACTTGCCCCAATATCCTTAATTTTAATAACTGCAAACAACCTATCCCCATTAATTTTACTTCTACTTGGACTCACTTCCTCAATGATAGGAGGATGAGGAGGACTAAATCAAACACAAATACGAAAAATCATAGCATTTTCATCAATTGCACACCTAGGATGAATAATAACAGNAGCATCAATCACAACAAATATTATAANNNNAAANNNNCTAATTTATTTAATTATAACCTCCACTATATTTTTATCCCTAATTATACTAAAAATTAACACAATTCAAGACTCCTCAACCTCATGAACAACCTCTCCCATACTAACTATTATAATAATATTAACCCTATTATCACTAGGAGGGCTCCCTCCTCTTACTGGGTTTATACCAAAATGACTCATTCTAGAAGAACTGACAACACAAAACCTAACCGTATTAGCAACTATAATAGCACTAGCTTCACTACTAAGCCTATTCTTTTACTTACGACTTAGTTATACTACTANTCTTACTCTATCCCCAAATACAACTCANANAANACATAAATGACGGTTCCAACCATTAACAACTACATCCCCACTAATAATTTTAACCCCTATAACCATTCTATTACTACCAATAACACCAATAATATTATTATAGAAACTTAAGATTAATAAACTAAGGACCTTCAAAGCCCTAAATATGAGTTCAACTCTCATAGTTTCTGTAAGACTTGTGAAATATTAATTCACATCCTCTGAATGCAACTCAACTACTTTAATTAAGCTAAAGCCTTTCTAAGTAGGCGGGCTTTGATCCCACGACAAATTAATTAACAGCTAACTACCCAAACCAGCGGGCTTCTACTCGCTTCTCCCGTACGGGAGAAGCCCCGGAGCCTTTTAGGGCTCATCTTTAAATTTGCATTTTAATGTGGAACACTTCAGGACTATNGATAAAAAAAGGAATTGAACCTCTATAAATAGGACTACAGCCTACTGCCTAACACTCGGCCATTTTACCT +>A.sagrei +ATGAGCCCAACAATCTTTATAATTATCATACTAAGTCTTGCAACTGGTACAATTATTACTACTACTAGCCACCACTGACTATTAGCCTGAATCGGCCTAGAAATTAATACCCTCTCAATTATTCCAATTATTTCAATAACCCACCACCCACGATCCACAGAAGCCGCTACCAAGTACTTTCTGACACAAGCAGCAGCCTCCGCCCTAATTTTATTTTCCAGTATAACTAATGCATGAGAAACAGGTACATGAGACATTACACAACTCTCATCAACACCATCACACATCTTATTAACAGTAGCACTAGCAATAAAACTAGGCCTCGTACCAATACACTTTTGACTGCCAGAAGTACTACAAGGGTCAAGCCTAATAACTGCACTTATTATTACTACATGACAAAAAATCGCACCTATAACTTTAATTTATCTAACAATTAACTCCCTCTCAACAACAATTCTGTTATTTATGGGACTTACATCCTCTATCATTGCAGGATGAGCTGGACTTAATCAAACACAAACACGGAAAATCATGGCATACTCATCAATTGCTCACCTAGGATGAATAATTGCTATTTCCTCTATTATGACGCATCTTATAGTAATAAACCTAATAATTTACTTAATTATAACCTCAGCCCTATTCCAAGCACTAATTTGCTCTAAATCAAAAACAATCCAAGACACAACAACAACCTGAGCCCTATCACCAGTTATTGCTACCATAACCATAGCACTATTATTATCATTAGGAGGCCTTCCACCATTAACTGGTTTTATACCAAAATGACTAATTCTAGAAGAACTAGTAACCCAAAATCTCATTCCAATAGCAACAGTAATAGCCCTGTCCGCTCTACTAAGCNNNTTTTTTTATCTACGCCTAACATATACTACAACACTAACACTTTCCCCAAACACAACCGCTACAAAATTTAAATGGCGACTTAAACTAAACCAACCTACACCACTAATGCTAATTACTGCTACAACCTCTATTCTTTTACTTCCACTAACACCACTAATACTTCTATAAAAACTTAGGATAACCTAAACCAAGGGCCTTCAAAGCCCTAAATAAGCTAGACGCCCTTAGTTTTTGTAAGACTTGTGAAAAACTAATTCACATCTCATGAATGCAACTCAAGCACTTTTATTAAGCTAAAGCCTTATGAATGAGCGGGCCTCGATCCCACAAAAAATTAATTAACAGCTAATTACCCAAACCAGCGGGCTTCCATTCGCTTCTCCCGTACGGGAGAANNNNCGGAGCCTTTTAGGGCTCCTTTTCAAACTTGCATTTTGACGTGGGTCACTTCGGGGCTTTGATAAAGAAAGGAATTGAACCAATGTTAGTAGGACTACAGCCTACCGCCTATCACTCGGCCACTTTACCTGTGTCTATTAATCGTTGA +>A.sheplani +ATGAGCCCAACCATCTACACAATTATTTTATCAAGCCTAGCAACCGGCACTATTATTACAATATCTAGTTATCACTGACTAATGGCCTGAATCGGCCTAGAGATTAATACACTAGCAATAATCCCGATCATCTCAACAACACATCACCCACGATCTACAGAAGCAGCCACAAAATATTTTTTAATTCAAGCTGCAGCCTCCGCCCTAATTTTGTTCTCAAGTATAACTAACGCCTGATATACAGGCTCCTGGGACATTACCCAAATAGTAAACAAGCCCTCAAATATTGCACTAACAGTAGCACTGGCCATAAAACTAGGACTGGCCCCACTTCACTTTTGATTACCAGAGGTAGTTCAAGGATCAACCACAACCACAGCACTTATTATTATAACCTGACAAAAACTAGCCCCCATATCACTGATTTTTCTTACCATAAATAACTTATCTACAACAGCCCTTCTACTGATAGGACTTCTGTCTTCAATAATTGGGGGCTGAGGGGGCTTAAACCAAACACAAGTACGAAAAATTATAGCATACTCATCAATTGCCCACCTAGGGTGGATAGCCTCCGTTTCCTCAATTATAACAAACCTTTTAATCATAAACCTGGTAATTTATATTATTGTAACAACATCCACCTTCTTCCCCCTAATTACTTCCAAATCTAAGACAATTCAAAATATAACTTCAACTTGAACACTCTCCCCCACACTAATTGTTGTTATAATATTATCCCTCCTGTCCTTAGGGGGCCTGCCCCCTTTAACTGGGTTTATTCCAAAGTGACTTATTATGGAAGAACTAATTCTACAAAACTTTAACCTATTTACCACTATAATAGCCCTGTCATCTTTATTAGGCCTCTTCTTTTACCTACGAATAGTCTACTTAACTACACTTACACTTTCCCCAAACGTGGCCCAAACAAAATTCAAATGACGATTTTATCCAAATACGACAACCAACTTTTTCTCAGCATTTTTCTTAATTTCTACCCTTCTTCTTCCCCTCACACCACTAATTTTAATATAAAAACTTAGGATAAATAAACCAAGAGCCTTCAAAGCTCAAAACATGGGTAAAACCCCCATAGTTTTTGTAAGACCTGTACATACTATTATACATCTCCTGAATGCAACTCAAGCACTTTAATTAAGCTAAAGCCTCTCTAAGTAGGCGGGCTTTGATCCCGCGACAAATTAATTAACAGCTAACTACCCAAACCAGCGGGCTTCTACTTGCTTCTCCCGTACGGGAGAAGCCCCGGAGCCTTTATGGCTCTTTTTCAAACTTGCATTTTGACGAGAAACCACTACAGGGCTCTGATAAAGAAAGGAGTTAAACCAATGTTAGCAGGACTACAGCCTGCCGCCTTACACTCGGCCACTTTACCTGTGTTTATTACCCGTTGA +>A.strahmi +ATGAGCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNACTATAACAAGCTACCACTGACTCATAGCCTGAATTGGACTAGAAATTAACACCTTAGCCATTATTCCAATTATTTCTATACAACACAACCCACGATCAACGGAAGCCGCAACAAAATACTTCCTAACTCAAGCAGCTGCATCCTCCCTAATTTTATTCGCAAGCCTATTTAACGCCTGACAAGTAGGCACATGAGATATTACACAACTATCAACTACACAAGCACACATTCTTTTGACTATGGCCCTATCAATAAAACTAGGCCTAGCCCCACTACACTTTTGATTACCAGAAGTAATTCAAGGATCTACTATATTTACTGCCTTAATTATTACAACATGACAAAAACTAGCCCCAATATCTCTTATTTTCCTCACCGCAAACAACTTATCAACGACAATTCTTTTAACAATAGGACTTCTGTCTGCAATAATCGGCGGATGAGCAGGACTTAACCAAACACAAACCCGAAAAATTATAGCATACTCATCAATTGCTCACCTTGGATGAATATCCACAATTGCCTCAATCTATACAAACATTTTTATTATAAACTTATTTATTTACTTACTACTAACAACAGCCATATTTTACTCCCTAATTTTTTCAAAATCAAAAACAATTCAAGATACATCAGTCACTTGAACAACTTCCCCATATTTAATGATTTTAACAACATTAACATTACTTTCATTAGGTGGACTTCCTCCCCTAACAGGATTTATCCCAAAATGACTCGTACTAGAAGAACTAACAACTCAAAATCTAATCCCTTTAGCCACTGTCATAGTAATAGCCGCTCTCCTCAGCCTATTTTTTTACCTACGCTTAGCCTATACTACAANACTTACACTTTCCCCAAACACAANACAAACTAAATTTAAATGACGATTTAAACCTAACATTAAAACCCCCCTAATAACTGCAACCCTTGTGGTTTCAATATTTTTACTTCCAATATCCCCAATACTTCTACTTTAAAGACTTAGGATAATTAAAACCAAGAGCCTTCAAAGCTCAAAATAAGGGTGCAACCCCCTTAGTTTTTGTAAGACTTGCACCATACTATAATGCATCTCCTGAATGCAACTCAAGCGATTTAATTAAACTAAAGCTTTACTGAACAGGCGGGCCTCGATCCCACGATAAATTAATTAACAGCTAATTACCCAAACCAGCGGGCTTCAATTCGCTTCTCCCGTACGGGAGAAGCCCCGGAGCCTTTTAGGGCTCATCTTTAAACTTGCATTTTAATGTAGTAACACTTCGGGGCTTTGGTACAGAAAGGAATTAACCAATGTGAGTAGGACTACAGCCTACCGCCTGACATTCGGCCACTATACCTGTGTACCTTAATCGTTGA +>A.stratulus +ATGAGCCCTATAATTTACACAATCATTTTGTCAAGCCTAGCAACAGGGACAATTATTACCATAACCAGCTACCACTGATTAATAGCTTGAATAGGCCTAGAACTTAATACTCTAGCAATTATTCCATTATCTCATACAACACACAACCCACGATCTACAGAAGCCGCAACAAAATACTTCTTAACACAAGCAGCAGCATCTGCTATAATCTTGTTTTCCAGCATAACCAACGCCTGCTTTACGGGCATATGAGACATTACTCAAATATCTTATTTACCATCTAATATTCTTCTAACCATGGCACTAGCCATAAAACTCGGCCTAGCACCAGTGCACTTCTGATTTCCAGAAGTCTTACAAGGCTCAACCCTACTTACCGCTTTTATTATTTCAACATGACAAAAACTGGCCCCAATATCACTAATTTTTTTGACAATAAGCAATCTTCCACCTATAATTTTATTAACTTTAGGACTTGTTTCCTCTATTATTGGTGGGTGAGGCGGACTTAACCAAACACAAACCCGAAAAATCATAGCATACTCATCAATCGCACACCTAGGATGAATAGCTGTAATTTCCTCAATTATAACAAACATTATAATTATAAACCTTTTAGTTTATTTAATAATAACAACAGCCCTATTTTTAGCCCTAATTTTATCTAAATCAAAAACAATTCAAGATACAACAAACACTTGAACAATATCCCCCACTTTAACTATTATTATGATACTTCTACTCCTCTCACTTGGTGGCCTGCCCCCCTTAACAGGGTTTTTACCGAAATGATTAATTTTAGAAGAACTAATAACCCAAAACCTTATTCCACTAGCAACACTTATAGCACTTACAGCCCTTCTTAGCTTATTCTTTTATCTTCGCCTAGCTTACACAACAACCTTAACACTTTCTCCAAACACACTACAAATAAAGTTTAAATGACGATTTAAACCAGCCACCAAGGCCTACCTAATATTATTTTCAACCCTAGCTATTTTTCTTTTACCACTTACACCACTAATTTTACTATAAAAACTTAGGATAATCTTAAACCAAGAGCCTTCAAAGTTCAAAATAAGGGTGCGATTCCCTTAGTTTTTGTAAGACTTGTAAAATACTAATTTACATCTCATGAATGCAACTCAAGCGCTTTTATTAAACTAAAGCCTCCCTGAACAGGCGGGCCTTGATCCCACGACAAATTAATTAACAGCTAACTGCCCTAACCAGCGGGCTTCTATTCGCTTCTCCCGTACGGGAGAAGCCCCGGAGCCTTTAAGGCTCTTCTTCAAACTTGCATTTTGACGTGAAAACACTACAGGGCTTTTAGTAAAGAAAGGGATTAAACCAATGTAAGTAAGTTTACAGCTTACCGCCTAACACTCAGCCACTTTACCTGTGTTTATTAATCGTTGA +>A.vanidicus +ATGAGCCCAACAATTTATACAACTATTCTAACCAGTCTTGCCACCGGAACAATTATTACAATAACCAGCCACCACTGATTAATAGCCTGANTCGGATTAGAAATNAATACATTAGCAATAATCCCAACTATCTCAACAATGCACCACCCTCGATCAACTGAAGCTGCCACAAAATACTTCTTAACTCAGGCAGCTGCCTCAGCCTTAATTCTCTTTTCTAGTATAACTAACGCCTGACAAACAGGCTCCTGAGATATTACCCATCTATCATCAACCCCAGCACATATTTTATTAACCATGGCCCTAGCAATAAAATTAGGCCTGGCCCCATTTCACTTTTGATTACCAGAAGTCCTACAAGGATCTGCTATAAATACTGCACTAATCATTACTACTTGACAAAAGCTAGCTCCAATATCTCTAATTTTCTTGACCGTAAATAACCTATCTACAACAATTCTTTTAACAATGGGACTTATATCTTCACTAATCGGAGGGTGGGCCGGACTAAATCAAACACAAACCCGAAAAATTATAGCTTTCTCATCAATCGCCCACCTAGGATGAATAGCAGCTATCTCTTCTATTATAACAAACATTTTAATTCTAAACCTACTTATTTACCTAATTATAACAACCTGCATGTTTTGCCTACTTATCATTACAAAATCTAAAACAATTCAAAACTCAACCATTATTTGAACACTATCCCCCACAATTACAATTATCATAATACTTTCACTTCTTTCTCTGGGGGGTTTACCACCACTTACTGGGTTCCTACCAAAATGATTTATTGTAGAAGAACTCATTACTCAAAACTTAATNACAGTTATTACCATTATAGCACTATCATCTCTACTTAGCTTATTCTTCTATATCCGACTAGCCTACACTACCACACTAACACTTTCTCCTAATACAACCCAAACAAAGTTCAAGTGACGATTTTACCCTAACACAAAATCTACCACAATTATAATCCCTGCAATAATTACTATCTTCATACTACCCTTAACACCAACAATCTTAATATAAAGACTTAGGATAAACAAACCAAGAGCCTTCAAAGCTCCAAACAAGGGTGCAACCCCCTTAGTTTTTGTAAGACCTGTAAATACTAATTTACATCTCCTGAATGCAACTCAAGCGCTTTAATTAAGCCAAGACCTAACTGAATAGACGGGCTTCGATCCCGCGATAAGTTAATTAACAGCTAACCACCCAAACCAGCGGGCTTCAATTCTGCTTCTCCCGTACGGGAGAAGCCCCGGAGCCTTTAGGGGCTCTTCTTCAAACTTGCAATTTGACGTAAGTCACTTCGGAGCTGTGTGATAAAGAAAGGAATTAAACCAATATTAGTAGGACTACGGCCTACCGCCTATCATTCGGCCACTTTACCAGTGTTTATTAATCGTTGA +>A.vermiculatus +ATGAGCCCAACAATCTACTCAATTATTTTATCTAGCCTAGCAACAGGCACCATCATTACTATAACTAGCTACCATTGACTAATGGCCTGAATTGGGCTAGAAATCAACACACTAGCAATCATTCCAATTATTTCAACTAAATACCACCCACGATCCACAGAAGCTGCCACAAAATACTTCCTCACACAAGCAGCAGCCTCTGCTATAATTTTATTTTCAAGCACAACTAATGCTTGATTAACAGGCACATGAGACATCACCCAAATCTCAACTACCCCTTCTCACATTCTACTAACAGCAGCACTAGCTATAAAACTAGGACTAGCCCCTATACATTTTTGACTCCCAGAAGTTCTACAAGGATCAACTATAAAAACAGCTTTTATTATTACAACATGACAAAAACTTGCTCCAATATCATTAATTTATTTAACCGTAAACAACCTATCTACAACAATTCTTCTTTCACTAGGATTATTATCATCCATAGTAGGAGGATGGGGAGGTTTAAATCAAACACAAATACGAAAAATCATAGCATACTCATCAATTGCTCACCTAGGATGAATAGCCACAATTTCATCAATCATAACCAACCTTCTAATAATAAATTTAATAATCTATTTAATTATAACAACAACCATATTTTGAGCCCTAATCTTGTCAAAATCCAAGACCATCCAAGACACAACAACCACCTGAACATTTTCACCATCTTTAACCATCATTACAATACTAACACTCCTTTCACTTGGTGGCCTACCCCCACTAACTGGATTTATGCCAAAATGACTTATCCTAGAAGAATTAACAACCCAAAACTTCACTCCACTAGCAACTATTATAGCACTATCCGCTTTACTAAGCCTATTTTTTTATTTGCGACTTACCTACACTACAACTTTAACTATGTCCCCAAACACCACAAAAACCAAATTTAAATGACGATTTAAACCATCAATAATAACACCACTACTAACAATAACCACACCAATGACTATTTTTTTTCTTCCTCTAACACCCCTTATCTTAATATAGAAACTTAGGATTAATATTAAACCAAGGGCCTTCAAAGCCCAAAATATGGGTATAACACCCATAGTTTCTGCAAGACCTGTGAAATACTAATTCACATCTTATGAATGCAACTCAAACACTTTAATTAAGCTAAAGCCTTACTGAATAGGCGGGCCTTGATCCCACAACAAATTAATTAACAGCTAATTACCTAAACCAGCGGGCTTCTATTCGCTTCTCCCGTACGGGAGAAGCCCCNNGACCCTTTAGGGCTCTTCTTCAAACTTGCATTTTGACGTGAAACACCTCGAGACTTTGGTAAGAAAGGGAATTAAGCCCCTGTAAACAGGACTACAGCCTGACACCTATGCTCGGCCACCTTACCTGTGTCCATTAATCGTTGA +>A.wattsi +ATGAGCCCAGCAATTCACATAATTATCTCATCAAGCCTAGCAACAGGCACCATTATTACCATGAACAGCCACCACTGATTAATGGCCTGACTTGGGCTAGAAATTAACACACTAGCAATTATTCCAATAATCACATCACCACATCACCCACGGTCCACAGAAGCCGCCATAAAATACTTCTTAACACAAGCAGCAGCCTCGGCCATAATCTTATTTTCTAGCATAATTAACGCCTGATATCTGGGGACATGAGACATCACCCAACTATCTTACACCCCGTCAGTTATTCTTTTAACCATAGCATTAGCCATAAAACTAGGCCTAGCCCCCACACACTTCTGACTTCCAGAAGTACTTCAAGGCTCAACCCTACCAACAGCCTTTATCATTGTAACATGACAAAAACTCGCCCCAATATCCCTAATCTTCTTAACCATTAATAACCTTAACACCCCCACACTATTAGTATTAGGCATTTTATCCTCCATTATCGGAGGCTGAGGCGGTCTTAATCAAACATATATTCGAAAGCTCATGGCATACTCATCAATTGCACACCTTGGATGAATGGCTTCTATTTCCTCAATCATAACAAACATTATAATCATAAATTTACTAGTCTATCTAATTATAACAATGGCCCTATTTTGCTCCCTCATTGTGTCCAATTCAAAAACAATTCAGGATACAACAACTGCCTGAACAGCTTCACCAGTATTAACTGCCTCCATACTACTTTNNCTTCTATCACTAGGGGGCCTCCCACCTCTAACAGGGTTCGCACCAAAATGATTAATCCTAAATGAACTAGTAGCCCAAAACTTAGTTCCCCTAGCCACTGCAATGGCTATGTCCGCATTACTAAGTCTATTCTTTTATCTTCGTCTCACCTATACAACAACACTCACACTAGCACCAAACACAACACAAATAAAATTTAAATGACGACTTTATCCAGCAACATTTACACCAGTTTTAACAATTTCAACCACACTAGCCGCGCTTCTTCTACCAATAACCCCGCTAATCTTATTATAAGAACTTAGGATAACTAAACCAAGAGCCTTCAAAGCCCAAAATAGGGGTGAAACTCCCCCAGTTCTTGCTAAGACCTGTGAAATTTTAATTCACATCTTCTGAATGCAACTCAAACACCTTCATTAGGCTAAGACCTTACTGAATAGGCGGGCCTTGATCCCACAACAAATTAATTAACAGCTAATTACCCTAGCCAGCGGGCTTCTATTCGCTTCTCCCGTACGGGAGAAGCCCCGGAGCCCTTTTGGGCTCTTTCTCAAACTTGCATTTTGACGTGGATCACTGCGGAACTGTGATAAAGAAAGGAATCNAACCAATGTAAGTAGGACTACAGCCTACTGCCTAGCACTCGGCCACTTTACCTGTGTCTATTAATCGTTGA +>Chamaeleolis.barbouri +ATGAGCCCAATTATACATACAATTATTTTATCAAGCCTAGCAACAGGCACAATTATTACTATAACTAGCTTTCACTGACTAATAGCATGAGTCGGCCTAGAAATCAACACCCTAGCAATGATTCCCATTATTTCAGCACCCCATCACCCCCGCTCCACAGAAGCAGCTACAAAATATTACCTCACACAAGCAGCAGCCTCCTCTATAATTTTACTCTCTAGCACACTCAACGCATGACAAACAGGATCATGAGATATTACTCAACCATCCACAACCCCCTCCCACATCATCTTAACAATAGCCCTTGCTATAAAACTAGGCCTAGCCCCTTTCCATTTCTGACTGCCAGAAGTCCTACAAGGATCAACCACACCCACCGTTTTTATTATTACTACATGACAAAAATTAGCCCCAATAACCCTAATTTTTCTAACATTAAATAACCTATCATCAACAATTCTATTAACATTGGGCTTTTTATCTACAATAATTGGAGGTTGAGGGGGACTTAACCAAACACAAATTCGAAAACTCATAGCATACTCATCCATTGCACACCTCGGATGAATAGCCACAATTTCTCTAATCTTATCAAATATTCTTATTATAAACCTACTAATTTACTTAATTATAACAACAACCATATTTTGTCTACTAATTATAACTAAATCCAAGACAATCCAAGACATAATAAACACCAAACCACTCTCACCAACCTTATCTATTATAATAATATTAACGCTACTTTCACTAGGAGGGCTCCCACCATTAACAGGATTTTTACCAAAATGACTAATTCTAGAAGAACTTACAACTCAAAATCTAACCCCCCTAGCAATTATTATAGCAATATCAACACTACTCAACCTATTTTTTTACCTACGACTTGTATATACAACAACCCTTACACTAGCACCAAACACCACACAAGCAAAACTCAAATGACGATTTAAACCAAACTTTTCAACCCCTACAACTTTAATTTTAATTACATTAACAATCTTTTTACACCCCCTAACCCCACTAATATTACAATAAAAATTTAGGTTTAACTAAACCAGGGGCCTTCAAAGCCCACAAAAAGGGTGTAAACCCCTTAATTTTTGTAAGACTTGTGAAATACTAATACACATCTCATGAATGCAACTCAAGCACTTTTATTAAGCTAAAGCCTCTCTGAATAAGCGGGCCTCGATCCCACGACAAATTAATTAACAGCTAATTACCCAAACCAGCGGGCTTCTATTCGCTTCTCCCGTACGGGAGAANNCCCGGAGCCTTTTAGGGCTCTTCTTTAAACTTGCATTTTAATGTGAAACACTTCGGGACCCTGATAAAGAAAGGAGTTAAACCAATGTTAGTAGGACTACAGCCTACTGCCTNNCACTCGGCCACTTTACCTGAGTTTATTA +>C.guamuhaya +ATGAGTCCAACCATTTACACCATTATTTTATCAAGCTTAGCAACAGGTACAATTATTACCATATCTAGCTATCACTGATTAATAGCCTGAGTCGGACTAGAGATCAACACTTTATCTATTATTCCTATTGTATCTATACTACACCACCCACGATCCACAGAAGCAGCAACAAAATATTTTCTTACACAAGCAGCAGCCTCAGCTATAATTTTATTCTCAAGCATAATTAACGCCTGACAAACGGGCACATGAGATATTACTCAAATNACCACTATTCCATCCAACACCCTCTTAACCATAGCACTAGCCATAAAATTAGGCCTAGCCCCAATACACTTCTGACTACCAGAAGTACTACAAGGATCAACTATAAACACCGCCCTAATTATTACTACATGACAAAAGCTTGCTCCAATATCATTAATCTTTATGACCATTAACAACTTATCAACACCAACCCTACTTCTACTAGGAGTTATTTCCTCATTAATTGGAGGATGAGCCGGACTAAATCAAACGCAAACCCGAAAAATTATGGCATACTCATCAATTGCTCATCTGGGATGAATAGCTACAATTTCATCAATTATAACAAATATCCTACTTATAAATATTTTAATTTACCTAATTTTAACTACATCTATATTTTTAATATTTATTTTTTCTAAATCCAAAACAATTCAAGACACTGCTACCACTTGGCCCTCTTCACCAGCCCTGACCGCTATAATAATAATTACACTACTATCACTGGGCGGCTTGCCCCCACTTACCGGCTTTATACCAAAATGAATAATCTTAGAAGAATTAATTAACCAAAACCTACCTGTAGTATCATTAATTATGGTAGCATCTACTTTACTAAGCCTATTTTTTTATTTACGACTAACCTACACAACCTCCCTTACTCTGTCCCCCAATACAACCCAAACAAAATTTAAATGACGACTTAAACCAAACATACAAACCTCTTTAATAATAGCACTATTTTCAATAACAGTATTTATACTTCCACTAACCCCACTAATTTTACTATAAAAACTTAGGATAAATTAAACCAAGAGCCTTCAAAGCTCGAAATAGGAGTTTAAACCTCCTAGTTTTTGTAAGACTTGTAAAATTCTAATTTACATCTATTGAATGCAACTCAAACACTTTAATTAAGCTAAAGCCTTACTGAATAAGCGGGCCTTGATCCCGCGATAAATTAATTAACAGCTAATTGCCCAAACCAGCGGGCTTTTATTCGCTTCTCCGTACGGGAGAAGCCCCGGAGCTTTCAGGGCTCTTCTTCAAATTTGCACTTTGATGTGAAACACTTCAGGACTTTGATAAATAAAGGAATTAAACCAATGTAAGTAGGACTACAGCCTACTGCCTAACACTCGGCCAATTTACCTGTGTC +>C.chamaeleonides +ATTTATACCATTATTTTATCAAGCTTAGCAACAGGCACAATTATTACTATATCTAGCTATCACTGATTAATAGCCTGAGTCGGATTAGAAATCAACACTTTATCTATTATCCCTATTGTATCTATATNNCATCACCCACGATCCACAGAAGCAGCAACAAAATATTTTCTTACACAAGCGGCAGCCTCAGCTATAATTTTATTCTCAAGCATAATTAACGCCTGACAAACGGGCACATGAGATATTACTCAAATAACCACTATTCCATCCAACACCCTTTTAACCATAGCATTAGCCATAAAACTAGGTCTAGCCCCAATACACTTCTGATTACCAGAAGTACTACAAGGGTCAACTATAGACACTGCCCTAATTATTGCCACATGACAAAAACTTGCTCCAATATCACTAATCTTTATAACCATTAACAACTTATCAACACCAACCCTGCTTCTACTAGGAGTTATCTCCTCATTAATTGGGGGGTGAGCCGGACTAAATCAAACACAAACCCGAAAAATTATGGCATACTCATACATTGCTCACCTAGGGTGAATAGCTACAATTTCATCAATTATAACAAACATCCTACTTATAAATATTTTAATTTACCTAGTTTTAACTACATCTATATTTTCTATATTTATTTTTTCTAAGTCCAAAACAATTCAAGACACTACTACCACTTGACCCTCTTCACCAGCCCTAACCACTATAATAATAATTACACTATTATCACTGGGCGGCTTACCCCCATTTACCGGCTTTATACCAAAATGAATAATTTTAGAAGAATTAATTAACCAAAACCTACTTGTAGTATCATTAATCATAGTAGCATCTACTTTACTAAGCCTATTTTTTTACTTGCGACTAACCTACACAACCTCCCTTACTCTATCCCCCAATACAACCCAAACAAAATTTAAATGACGACTTAAACCAAACATACACACCTCTTTAATAATAATAATATTTTCAATAACAATATTTATACTTCCACTAACCCCACTAATTTTATTATAAANACTTAGGATAAATTAAACCAAGAGCCTTCAAAGCTCAAAATAGGAGTTTAAATCTCCTAGTTTTTGTAAGACTTGTAAAATTCTAATTTACATCTATTGAATGCAACTCAAACACTTTAATTAAGCTAAAGCCTTACTGAATAAGNGGGCCTTGATCCCGCGAGAAATTAATTAACAGCTAACTGCCCAAACCAGCGGGCTTTTATTCTCTTCTCCCGTACGGGAGAAACCCCGGAGCCTTTTAGGGCTCTTCTTCAAATTTGCACTTTGATGTGAAATACTTCAGGACTTTGGTAAAGAAAGGAATTAAACCAATGTAAATAGGACTACAGCCTACTGCCTAACACTCGGCCAATTTACCTATGTCTGTTAATCGTTA +>Diplolaemus.darwinii +ATGAGCCCAACTACAATAATAATTATTACATCTAGCCTAGCCACGGANACAATCATCACCGCATCAAGCTACCACTGACTACTGGCCTGAGTAGGCCTAGAACTAAATACACTAGCAATTCTTCCAATAATTTCAAAATATCACCACCCACGAGCAACAGAAGCTGCAACAAAATATTTCCTAACACAAGCAGCAGCCTCCGCCATAATCATATTTTCAAGCACACTAAACGCCTGACAAACAGGCACATGAAACATCACACAACCAAACAACACAACATCAAGCATCTTACTAACAGTCGCACTAGCTATAAAACTAGGACTTGTGCCAATACACTTCTGACTACCAGAAGTATTACAAGGCTCCTCCATAAAAACAGCCCTCATTATTAGTACATGACAAAACGTAGCCCCAATAGCCCTTATTTTCCTGACATCCAACACACTATCAACCACAATTCTATTATTGATAGCCACACTCTCCACCATCATTGGTGGCTGAGGCGGATTAAACCAAACACAGCTACGAAAAATTATAGCATTCTCATCAATCGCCCATATTGGCTGAATGGCGGCAATCTCAACAATTTCAGAAAACATTTTAACCCTAAATTTAATACTTTATCTATTAATAACTTCCTCAACCTTCTCAGCACTTATTTTATCTAAATCAAAAACCATCAAAGACACATCCACATCACTAACAACCTCCACAACACTAACCATCATAATAATACTTATGCTACTATCACTAGGAGGACTCCCACCCCTAACAGGCTTTTTACCAAAGTGATTAGTACTAAAAGAGCTAACAATACAAGGACTACTTATTATTTCCACAACCCTAGCCATGTCCTCCCTCCTAAGCCTATTTTTTTACCTACGACTTTCATATACAACAACACTTACACTTGCCCCAAATACACTACTAATAAAACACAAATGACGATTCAAACCAATATCAAAAACACTTTTAATATCTACATCAACACCACTAGCCCTAATATTACTACCAATAACCCCACTAATATTATAAGAGACTTAGGCTAACTAACCAAACCAAGGGCCTTCAAAGCCCAANATAGAGATGAAAACCCTCTAGTTTCTGATAAGACCTGTGTAATACTAAAACACATCTCCTGAATGCAACCCAGACACTTTAATTAAGCTAAGGCCTCCCTAGATAGGGGGGCTTTGATCCCACGAAACTTTAGTTAACAGCTAAACACCCAAACCAGCGGGCTTCTATCCGCTTCTCCCGCACGGGAGAANNCCCGGAGCCCTTTATGGGCTCTTCAAATTTGCATTTTGACGTGCTTACACCACAGGACTTTGATAAAAAGAGGAGTTAAACCCCTGTAAATAGGACTACAGCCTACCTCCTAAACGCTCGGCCATTTTACCTGTGTTTATTAA +>Phenacosaurus.acutirostrus +ATGAGCCCAGTAACAATAACAATCCTACTCTCCAGCATCGCAGCAGGAACAATTCTTACCATATCTAGCTACCACTGGCTCTTGGCTTGACTCGGATTAGAAATTAATACCCTGGCAATCATCCCGATTATTTCAAAACAGCATCATCCCCGAGCTACCGAAGCTACAACAAAATATTTCCTAACACAAGCAGCCGCATCCGCCCTAATCCTATTCTCCAGCACAATTAATGCCTGACAAACGGGGACCTGAGATATCACACAAATAACTAATCCAACACCAACCACACTATTAACCGCCGCCCTGGCCATAAAGCTAGGACTTGCACCCACACACTTCTGACTTCCAGAGGTCCTGCAAGGCACTACAATAAACACAGCCTTAATTATTACAACATGACAAAAACTAGCCCCAATATCACTTATTTACCTAACAATAAACAACCTACCCCCAACAATCCTACTACTTACAGGAATACTGTCCTCACTCCTAGGCGGCTGGGGAGGACTAAATCAAACACAAACCCGAAAACTAATGGCTTACTCATCAATCGCCCACCTGGGGTGAATGGCCGTAATTTCCACGATAATACCGAACATTCTAATTCTTACCCTAACAACATACATTCTAATAACTACTGCAATATTCCTAACACTAATCTCTACTAAATCAAAAACAATCCAAGATGCATCTACATCATGGACAACTTCTCCAACCATTACCACTATATCTCTCCTCATCCTCCTATCCCTTGGAGGCTCCCCCCCTTTAAAAGGCTTTATTCCAAAATGACTCATTCTAGAAGAACTCTTCACACAAAACCTAGTCCCAACAGCAACTATCATAGCAATATCAGCCCTGCTAAGCCTATTCTTTTATCTCCGACTCACCTACACAACAANACTCACAATAGCCCCAAACACAACTCCAACAAAATACAAGTGACGATTCAAAATCTCCGCACCAAAACTTCTAACAATAACTACCCCCATAGCGCTCCTTCTACTACCAATAACCCCCCTACTCACTTCATAGAACCTTAGGCTAATCAAACCTTGGACCTTCAAAGTCCAAAATAGGAGTGCAAACCCCCTAGTTTCTGCAAGACCTGCGAACCATTAAAACGCATCCTCTGAATGCAACTCAGTCACTTTAATTAAGCTAAGGCCTCCCTAGACAGGCGGGCCTTGATCCCGCAAAAATTAGTTAACAGCTAATTACCCAAACCAGCGGGCTTCCATCCGCTTCTCCCGTACGGGAGAAGCCCCGGAGCCCTTTAGGGGCTCTTCTCCGAATTTGCACTTCGGCGTGTTCTCACTACAGGGCTGTGGTAAGCAGAGGAATTAAACCCCTGTCTATGAGTCTACAGCCCACCGCCTAAACACTCGGCCACCTTACCTGTGTTCATCACACGTTAC +>P.nicefori +ATGAGCCCAACAATATATTCAATTATTATCTCTAGCCTTGCAACCGGCACTATCATTACTATAACCAGTCACCATTGACTGCTAGCCTGTCTCGGACTAGAAATCAACACACTAGCAATTGTTCCAACCATTTCAATAAACCACAACCCACGAGCCACAGAAGCCACTATAAAGTACTTTTTAACACAAACAGCAGCTTCGGCCATAATTTTATTTGCAAGCACAACAAATGCTTGACACACAGGAACATGAGATATTACCCAAATATCAACAACCACCTCTTACATTTTACTTACTCTTGCATTAGCAATAAAACTAGGACTAGCCCCCCTACACTTCTGACTCCCAGAAGTACTACAAGGAACTACCCTACCCACAGCCCTAATCATTGTTACATGACAAAAACTTGCCCCTATATCATTAATTTACATAACCATTAACAACCTTTCTACAACATTACTACTAACACTAGGATTATTTTCCTCAATGGTTGGCGGATGAGCTGGACTAAACCAAACACAAACACGAAAAATAATAGCCTTCTCATCAATCGCTCACTTAGGATGAATATCAACAGTAGCATCAATTATAACAAACATTCTAATCTTAAATCTAATTATTTATTTAACTATAACAATAACCATATTTTACTCACTTATTTTATCAAAAGCTAAAACAATTAAAGATACCACCACCTCATGAACACTATCCCCAACATTAACAATTATTATAATACTCACTCTTCTCTCACTAGGGGGGCTCCCCCCATTAACCGGGTTTATACCAAAATGACTTATTCTAGAAGAACTAACAATTCAAAACCTATGCCCTCTAGCCCTTTTGATAGCATTAACATCACTATTAAGTTTATTTTTTTACTTACGGCTAACCTACACAACAACACTTACACTTTCTCCAAACACCATTTTAACAAAACACAAATGACGATTTAAACCAATAACTATAACAACCCCCATAACAATTTTAATCCCACTAACAATACTTATACTACCAATAACACCAATAATAATCTCTTAGAAATTTAGGCTAACATTAAACCAAGAGCCTTCAAAGCTCAAAATATGGACTATACCCATAATTTCTGTAAGACTTGTAAAATATTAATTTACATCTCATGAATGCAACTCAAGCACTTTAATTAAGCTAAAATCTACCATAAGTAGGCGGGCTTTGATCCCACGACATATTAATTAACAGCTAACTACCCAAACCAGCGGGCTTCTACTCGCTTCTCCCGCACGGGAGAAGCCCCGGAGCCTTTTAGGGCTCTTCTTCAAATTTGCATTTTGATATGGAACACCTCAGGGCTTTGATAAATGAGGAATTTAACCCCTTGTGTAGGACTACAAACTACTGCCTTACGTTCGGCCATTTTACCTGTGTTTCTTAATCGAT \ No newline at end of file diff --git a/topics/evolution/tutorials/abc_intro_phylo/data/anolis-trimmed-aligned-clustal.fst b/topics/evolution/tutorials/abc_intro_phylo/data/anolis-trimmed-aligned-clustal.fst new file mode 100644 index 00000000000000..cd42fb39d6c0e7 --- /dev/null +++ b/topics/evolution/tutorials/abc_intro_phylo/data/anolis-trimmed-aligned-clustal.fst @@ -0,0 +1,1430 @@ +>Anolis.acutus +ATGAGTCCCACAATCTATACAATTATTATATCAAGCCTAGCAACAGGCACAATTATTACC +ATAACCAGCTACCACTGACTTATAGCTTGAATAGGCCTAGAACTTAATACACTAGCAATT +ATTCCAATCATTTCATCACAACACCACCCCCGATCTACAGAAGCTGCAACAAAATATTTT +CTGACACAAGCAGCAGCATCCGCCATAATTTTATTCTCCAGCATATCCAATGCCTGANTT +ACTGGTACATGGGACATCACTCAAATATCTTACACCCCCTCTAATATTTTTTTAACCATA +GCACTAGCTATAAAATTAGGTTTGGCACCAGTACACTTCTGATTTCCAGAAGTATTACAA +GGATCAACCTTATTTACTGCTTTTATTATTTCAACATGACAAAAGTTAGCCCCAATATCA +CTAAGTTTTCTGACTATCAACAATCTNCCACCAGTAACTTTATTAGCACTAGGACTTTTT +TCCTCTGTCATTGGAGGGTGGGGCGGTCTCAATCAAACACAAACCCGAAAAATTATAGCA +TATTCATCAATTGCTCACTTAGGTTGAATGGCTGTAATTTCCTCAATTGCAACAAACATT +ATAATTATAAACCTACTAATTTATCTAATAATAACAACAGCCCTATTTTCTTCCTTAATT +TTAACCAAATCAAAAACAATTCAAGACACAACAAANACTTGAACAATATCACCCACTTTA +ACTATTACTATGATACTTCTGCTACTTTCACTTGGTGGCCTGCCGCCCCTTACAGGATTT +TTACCAAAATGATTGATTTTAGAAGAATTAACAGCCCAAAACCTTCTACCCCTGGCAACA +ATTATATCAACTANTGCGCTTCTCAGCCTATTCTTCTACTTACGCCTAGCTTACACTACA +ACTTTAACACTTTCTCCTAACACCCTACAAGTAAAATTCAAATGACGATTTAAACCACAC +ACACCAACTTATTTAATAACAT--TTT---CTACCTTTGCCATTTTTCTCTTACCATTAG +CACCACTAATTTTACTATAAAA---ACTTAGGATAAAATAATAAACCAAGAGCCTTCAAA +GTCCAAAATAAGGGCGCAATCCCC-TTAGTTTTTGTAAGACTTGTAAAATACTAATTTAC +ATCTCATGAATGCAACTCAAGCGCTTTTATTAAGCCAAAGCCTC-ACTGAA-TAGGCGGG +CCTTGATCCCACGACAAGTTAATTAACAGCTAACTGCCCTAACCAGC-GGGCTTCTATTC +GCTTCTCCCGTACGGGAG---------AAGCCCCGGAGCCCCTTA--GGGCTCTTCTTCA +AACTTGCATTTTGACGTGAAAACACTACAG-AGCTT---CTAGTAAAGAAAGGGATTAAA +CCAATGTAAATAAGTTTACAGCTTACCGCCTAACACT-CGGCCACTTTACCTGTGTTTAT +TA-------------------- +>A.aeneus +ATGAGCCCTGCAATATATACCATTATTTTATCTAGCCTTGCAACAGGCACTATTATTACA +ATATCCAGCTATCACTGATTAATAGCCTGAATTGGCCTAGAAATTAATACACTAGCAATC +ATTCCAATTATTTCCATACAACACCACCCACGATCAACGGAGGCCGCTACAAAGTACTTC +TTAATTCAAGCAGCAGCTTCAGCTATAATTTTATTTGCTAGTACAACAAATGCCTGATAC +ACAGGAACATGAAGTATTATCCAAATATCTACCTTACCTTCACATATTATATTAACCCTA +GCATTATTAATAAAACTAGGACTAGCCCCAATACATTTTTGATTACCAGAAGTCCTTCAA +GGATCAACTNTATCTACTGCTCTTATTATTACTACATGACAAAAACTTGCCCCAATATCC +TTAATTTTAATAACTATAAATAACCTAAACCCATTAATTTTACTTCTACTTGGACTAACT +TCCTCAATAATAGGGGGCTGAGGCGGACTAAATCAAACACAAATACGAAAAATTATAGCA +TTTTCATCAATCGCCCATCTAGGGTGAATAATAATAATAGCATCAATCATAACAAATATT +TTCATCTTAAATCTTATAATTTACCTAATTATAACCTCCAATATATTCTACTCCATAATT +ATATTAAAAACTAAAACTATTCAAGACTCATCAACTTCATGAACAACCTCCCCAATACTT +ACCATTATTATAATACTTACCCTTTTATCACTAGGAGGTCTACCTCCTCTTACCGGATTT +ATACCAAAATGACTTATTTTAGAAGAACTAGCAACACAAAGCCTAATCATACTAGCAACT +ATAATAGCACTAGCATCACTGCTAAGCCTATTTTTTTACTTACGACTAAGTTATACTACT +ACTCTTACACTGTCCCCAAATACAACTCAAACAAAACATAAATGACGATTTAAACCATCA +AAAACTACATCAATATTAATAC--TTTTAACTCCTCTAACTATATTTCTATTACCGATAA +CACCAATAGTACTATTATAGAA---ACTTA--GGAT-TAAATAAACCAAGAGCCTTCAAA +GCCCTAAATATGAGTCTAACTCTCATAGTTTCTGT-AAGACTTGTGAAATATTAATCCAC +ATCCTCTGAATGCAACTCAACCACTTTAATTAAGCTAAAATCTT-TCTAAG-TAGGCGGG +CTTTGATCCCACGACAAATTAATTAACAGCTAACTACCCAAACCAGC-GGGCTTCTACTC +GCTTCTCCCGTACGGGAG---------AAGCCCCGGAGCCTTTTA--GGGCTCGTCTTTA +AATTTGCATTTTAATGTGGAAACACTTCAG-GACT----GTGATAAAGAAAGGAATTAAA +CCTCTATAAATAGGACTACAGCCTACTGCC------------------------------ +---------------------- +>A.agassizi +ATGAGCCCTATAATATACATAATCATTATCTCCAGCCTCACAACAGGCACCATCATTACC +ATGTCTAGTCATCACTGACTACTAGCTTGAATTGGCCTAGAAATCAACACATTAGCAATC +ATTCCAATCATCTCAATACAACACCACCCACGATCCACAGAAGCCGCCACAAAATATTTC +CTAACACAAGCAGCAGCTTCCGCTATAATCCTATTCTCAAGTTCTACCAACGCCTGATAC +ACAGGAACATGAGACATTACTCAAATAACTACCACTCACTCCAACATCCTACTAACCCTA +GCACTATCAATAAAATTAGGACTGGCCCCCCTACACTTTTGACTACCCGAAGTATTACAA +GGCTCCACCATAACCACAGCACTAATTATTACAACCTGACAAAAACTAGCCCCAATGTCA +TTAATTTATCTAACTATAAATAACCTCTTCCCAACAATCTTACTCTCACTAGGACTTTTA +TCTTCAATAGTTGGAGGATGGGGAGGCCTAAACCAAACACAAACACGAAAAATCATAGCA +TACTCATCAATTGCCCACCTAGGATGAATAGCAACAGTATCATCAATCATAACAAACATT +TTAGTATTAAATCTACTAATCTACCTAACAATAACAACAACCATATTTTTCTCACTAATT +CTATCAAAATCTAAAACAATCCAAGATACAACAACCTCTTGAACAACATCCCCAATACTA +ACGATTATTATGATACTAACCCTCCTATCACTAGGGGGCTTACCCCCTCTTACAGGCAAC +ATACCAAAATGACTAATCCTAGAAGAACTAACAATGCAAAACCTAACCCTACTAGCACTT +ATTATAGCAATAGCATCACTACTAAGCCTATTCTTCTACTTACGACTAACCTACACAACA +ACACTCACACTTTCCCCCAACATCACACAAACAAAACACAAGTGACGATTCAAACCATCA +ACCAACATACTCCCACTAACTA--TTATAACCCCAACAACCCTATTACTCCTACCAATAA +CCCCCTTAATCATCCATTAGAA---ACTTA--GGAT-AAAATTAAACCAAGAGCCTTCAA +AGCTTAAAATATGAGTTAACCCTCATAGTTTCTGC-AAGACTTGTGAAATATTATTACAC +ATCTTCTGAATGCAACTCAAACACTTTAATTAAGCTAAAGCCTC-TCTGAA-TAGGCGGG +CCTTGATCCCACGATATATTAATTAACAGCTAACTGCCCAAACCAGC-GGGCTTCTATTC +GCTTCTCCCGTACGGGAG---------AAGCCCCGGAGCCCTTCT--GGGCTCGTCTTCA +AATTTGCATTTTGACGTGAAACACCTCGGG-GCT-----GTGATAAAGAGAGGAATCAAC +CCCCCTTAGATAGGACTACAGCCTACTGCCTAACATT-CGGCCACTTTACCTGTGTTTAT +CAATCGAT-------------- +>A.ahli +ATGAGCCCAATAATATACACAATTATACTATCAAGCCTAGCAACAGGCACTATCGTTACC +ATAACGAGCTACCACTGACTCCTAGCCTGAATCGGACTAGAAATAAACACTTTATCAATT +ATTCCAATTATTTCTACCATACACCACCCACGATCAACAGAGGCCGCCACAAAATACTTT +TTAACACAAGCAGCGGCTTCTGCCATAATCTTGTTTTCAAGCATAATTAACGCCTGACAA +ACCGGATCATGGGATATTACTCAATTATCATCTACACCATCACACATTCTATTAACCATG +GCATTAGCAATAAAACTGGGCCTAGTCCCAGTACATTTTTGACTACCAGAAGTTATTCAG +GGTTCAACAATAACCACGGCACTCATTATTACCACATGACAAAAACTTGCCCCAATTTCC +CTAATTTATTTATCTATTAATAACCTCTCAACAGTGGTCCTAATAACCATGGGCCTACTA +TCTTCAATAGTCGGAGGCTGAGGCGGACTAAACCAAACACAGACACGAAAAATTATGGCA +TATTCATCAATCGCACATCTAGGATGAATAACCACTGTTTCCTCAATTATACCAAACATT +ATAGTTTTAAACTTAATAATTTACCTAATTACAACCGCAGCCACATTTTTCACACTAATA +CTGTTTAAATCAAAAACTATTCAAGATACAACAATAAACTGAACCTTATCCCCAATAACA +ACCGTCATAATAATAATTCTATTACTCTCACTTGGAGGCCTCCCACCATTAACTGGATTT +ATACCAAAATGATTAATTCTTGAAACACTAACAACCCAAAACTTTATTATAGCAGCAGTA +GCTATAGCCATTTCAGCACTACTAAGCCTATTTTTTTACCTTCGACTTACCTACACTACA +ACCCTCACACTTGCACCAAATACTACACATACAAAACTAAAATGACGATTTAAACCAAAC +CTGCCAACCTTTCTAATACTTTCTTT--TTCTACCACTTCAATATTTGCCCTACCACTTA +TGCCTCTCATAACACAATAAAA---ACTTAGGAA---TAACTAAACCAAAGGCCTTCAAA +GCCCTAAATAAGGGTGCAA-TCCCCTTAGTTTTTGTAAGACTTGTGG-GAGCTAACCCAC +ATCTCCTGAATGCAACTCAAATGCTTTTATTAAACTAAAGCCTTCCANA---CAGGTGGG +CCTTGATCCCACAA-AAACTAGTTAACAGCTAGTTACCCAAGCCAGC-GGGCTTCTATTC +GCTTCTCCCGTACGGGAG---------AA-GCCCCGGAGCCCTTT-TGGGCTCATCTTCA +AACTTGCATTTTGATGTGGGTCACTTCGGG-ACT-----TTGATAAAGAAAGGAATTAAA +CCAATGTAAGTAGGACTACAGCCTATCGCCTAACACT-CGGCCAATTTACCTGTGTCAAC +TAATCGTTGA------------ +>A.aliniger +ATGAGCCCTACAGTTTATTCAATTATTTTGTCAAGCCTACCAACAGGCACAGTTATTACT +ATAACCAGCTACCATTGATTAATAGCCTGAGTCGGGCTAGAAATTAACACACTCGCAATT +ATTCCTGTTGTTTCAATACAACATCACCCACGGTCCACAGAAGCCGCCACAAAATATTTT +CTAACACAAGCAGCAGCCTCCGCCTTAATTCTATTTGCTAGCACAACTAACGCCTGATCA +ACGGGCACATGAGATATTACCCAAATATCCTCTACCCCCTCTCATATCCTTTTAACTATC +GCGCTTGCTATAAAACTAGGCCTAGCACCCACCCACTTCTGATTACCAGAAGTAGTTCAA +GGTTCAACTATAACAACAGCGTTTATTATTATAACATGGCAAAAACTAGCCCCAATATCA +TTAATTTATTTAACCATAAATAATCTTTCCCCAACAATTCTTCTTCTATTAGGAATAGCC +TCATCTATGCTTGGTGGATGAGCAGGACTAAATCAAACACAGACCCGAAAAATTATAGCA +TTTTCATCAATTGCCCACCTTGGATGAATAGCTGTAATTGCATCTATTATGACAAATATT +CTTACTCTTAACCTTATACTTTACTTAATTATGACAACAACCGCGTTCTTTACCCTAATT +TTATCAAAATCAAAAACAATTCAAGATACAACAGCAACCTGAACTCTCTCCCCAACCCTA +ACTATTATTATAATGTTATCACTTCTTTCACTAGGCGGACTTCCACCATTAACCGGATTT +ATACCAAAATGATTAATTTTAGAAGAATTAACTACCCAAAAGTTAGCCCCCCTAGCTACT +ATTATAGCAGCATCCGCCTTACTCAGCTTATTTTTTTATCTCCGATTAGCCTACACTACA +ACACTCACATTTTCCCCAAACACTACACAAACAAAATTCAAGTGACGATTTAAACTACCC +AACCTAGCTCCCCCACTAACAA--TTACCACCCCCCTAACTATTTTTCTTCTCCCCCTTA +CACCATTAATTTTAGCATAAGA---AATTTAGGATA-ATATTAAACCAAGGACCTTCAAA +GTCCAAGACATGGGCGAAACCCCCATAATTTCTGC-AAGACTTGTAAAACATTAATTTAC +ATCTCCTGAATGCAACTCAAGCACTTTTATTAAGCTAAAGCCTC-TCTGAA-TAAGCGGG +CCTCGATCCCGCGACAAATTAGTTAACAGCTAATAACCCAAACCAGC-GGGCTTCTATTC +GCTTCTCCCGTACGGGAG---------AAGCCCCGGAGCCC-TTTA-GGGCTCTTCTTCA +AACTTGCATTTTGACGTGGAACACCCCGGG-ACT-----TTGATAAAGAGGGGAATTAAA +CCCCTATTAATGGGTCTACAGCCCACCGCCTAACATT-CGGCCACCCTACCTGTGTCTAT +TAATCGTTGA------------ +>A.alutaceous +ATGAACCCAACAATTATTATAATTACCCTAACCAGCCTGGCAACTGGTACAGTTATTACC +ATACATAGCTTCCATTGATTAATGGCCTGANTCGGATTAGANATCAATACACTATCAATT +ATTCCAATAATTTCAACATTACACCACCCACGATCAACTGAAGCTGCTACAAAATATTTC +CTCACCCAAGCAGCTGCTTCANCTTTAATCCTTTTTTCAAGCACAATTAATGCCTGACAA +ACAGGATCATGAGACATTACCCAACTATCATCAACCCCCTCACACATTTTATTAACTATG +GCTTTGGCCATAAAACTAGGACTCGCCCCACTTCACTTTTGATTACCAGAAGTAATTCAA +GGGTCTTCCATAAACACAGCCTTAATTATTGTNACTTGACAAAAACTAGCCCCAATATCC +CTAATCTTTTTAACCATAAATAATTTATCAACAACTATTTTAATGATAATAGGACTTGTA +TCTTCACTTGTCGGAGGATGAGCAGGACTTAATCAAACACAAACTCGAAAAATTATAGCC +TACTCATCTATTGCTCACCTAGGGTGAATAGCAGCCATTTCCTCAATTATAACAAACATT +TTAATCCTCAACCTATTAATTTACCTAATTATAACTACCTCAGTATTTTGCTTTATTATT +ATTACAAAATCAAAAACAATCCAAAACACAGCTACTATTTGGACAATATCACCAACACTA +ACAATTACCATAATACTTTCTTTACTTTCACTAGGAGGACTACCGCCACTAACAGGATTT +ATACCAAAATGAATAATTATAGAAGAACTTATTAATCAAAACTTTACTACAATTACTACT +ATGATAGCACTATCGTCCTTATTAAGCCTATTTTTTTATATTCGACTAACATATACAACT +TCACTTACACTATCCCCAAACACAACCCAAACGAAATTTAAATGACGATTTTACCCAAAC +ATAAAAACCACAGTAATTATAG--TCTCTACAACAGTCACAATTTTTATATTACCGATAC +TTCCAATAATTTTGATATAAAA---ACTTA--GGATAAACT-AAACCAAGAGCCTTCAAA +GCTCAAAAAAGGGGTGTAACCCCCCTAGTTTTTGT-AAGACTTGTAAAATACTAATTTAC +ATCTCTTGAATGCAACTTAAACACTTTAATTAAGCTAAAACCTT-ACTGAA-TAGGCGGG +CTTTTAACCCACGAAAAATTAATTAACAGCTAATTTCCCAAACCAAC-GGGCTTCTATT- +CGCTTCCCCCGTACGGGA--------GAAGCCCCGGAGCCTTTA--AGGGCTCTTCTTCA +AACTTGCAATTTGACGTAAATCACTTACTTCGGGGC---TTGATAAAGAAAGGAATTAAA +CCAATGTGAATAGGACTACAGCCTACCGCCTACCATT-CGGCCACTTTACCTGTGTTTAT +TAATCGTTGA------------ +>A.angusticeps +ATGAGCCCCCCCATTTTTACAATTATCATCTCAAGTCTAGCAACAGGTACAATTATTACC +ATAACCAGCTACCATTGACTCATAGCCTGAGTTGGTCTAGAAATAAATACACTAGCAATT +ATTCCTATTATTTCAACAACACATCACCCACGAGCCACAGAAGCTTCCACAAAATATTTT +CTTACACAAGCTGCAGCCTCTGCTCTAATTTTATTTTCTAGTATAATTAACGCATGACAC +ACAGGATCTTGAGACATTACTCAAATATTATCTACCCAATCAAATACTATATTGACTATA +GCCCTAGCTATAAAACTAGGGCTGGCCCCACTTCACTTTTGACTACCAGAAGTAATCCAA +GGATCAACAATACTAACCGCCTTTATTATTACTACATGACAAAAACTAGCCCCAATATCT +CTAATTTTTTTAACTATAAATAATATATCCACAACAATCCTCCTACTAATAGGAATATTA +TCCTCACTTGTTGGCGGTTGAGGGGGGCTAAACCAAACACAAACCCGAAAGATCATAGCA +TACTCATCAATTGCTCACCTTGGATGAATAGCCACAATTTCATCTATTATAACCAACATC +CTAATCTTAAACCTAGTTCTTTACCTAATTATGACTACATCAATGTTTGTCTCTTTAATT +ATTTCTAAGTCAAAAACAATCCAAAATACAGCAGAAACCTGAACACTCTCACCTACTCTA +ACAGTTTTTATGTTACTTTCACTCCTATCTTTAGGTGGACTACCACCCCTAACTGGGTTT +ATTCCAAAATGACTAGTTATAGAAGAACTAATTTTACAAAATTTTAATTTTCCAGCTATT +TTAATAGCCATGTCCTCCCTACTAAGTCTATTTTTTTACCTTCGAATTACCTACACAACC +ACACTATCGCTATCACCTAATACAACTCAAATAAAATTTAAATGACGTTTTTATCCAAAA +ACCACAACCACCTTATCCTCTT--CCACCGCAACAATAACAACCCTCCTTCTTCCACTTA +CACCACTAATTTTGTTATAAAA---ACTTA--GGATAAAATTAAACCAAGGGCCTTCAAA +GCCCAAGACATGGGTTAAACTCCCATAGTTTTTGT-AAGACTTGTGAAACATTATTACAC +ATCTCCTGAATGCAACTCAAGCACTTTTATTAAGCCAAAGCCTC-ACTGAA-TAGGCGGG +CCTTGATCCCACGACAAATTAATTAACAGCTAAATACCCAAACCAGC-GGGCTTCTATTC +GCTTCTCCCGTACGG----------GAGAAGCCNCCGAGCCTTT--AGGGCTCCTCTTCA +GACTTGCATTTTGACGTGAAAACACTTCGG-GACTT---TTGATAAAGAAAGGAATTAAA +CCTGTGTAAGTAGGTCTACAGCCTACTGCCTAACACT-CGGCCACTTTACCTGTGTTTAT +TAACCGTTGA------------ +>A.bahorucoensis +ATGAGCCCCATAATTTACTCAATTGTATTCTCAAGCCTAGCNACAGGTACTATTATTACT +ATAACCAGCTACCACTGATTTATGGCCTGAATCGGACTAGAAATTAATACACTAGCAGTA +ATCCCCATTATTTCAACACTACACCACCCACGATCTACAGAAGCTGCTACAAAATACTTC +TTAACACAAGCAGCAGCCTCCGCCACAATCCTATTTTCAAGTATAATTAATGCCTGACAA +ACAGGCACATGAGATATTACCCAATTATCCACAACCCCCGCCCACATCCTACTGACTATA +GCACTTGCCATAAAACTAGGCCTAGTCCCAGCCCATTTCTGACTACCAGAAGTCCTCCAA +GGTTCAACCATTACAACTGCCCTCATTATTACTACATGACAAAAACTCGCTCCAATATCA +CTAATTTTCTTAACCATTAACAACCTGTCCTCAACAGCCCTTCTATTATTAGGCTTAATG +TCCTCATTATTAGGCGGCTGAGCCGGACTAAACCAAACACAAACCCGAAAAATTATAGCA +TTTTCATCCATTGCTCACCTTGGATGAATAGCCACTATTTCATCAATTATAACCAATATC +CTTATCATAAACTTACTACTTTATTTAATTATGACAACAACTATATTTTACTCCCTTATT +TTATCAAAATCTAAAACTATTCAAGACACAATAACCACCTGAACACTCTCGCCAGCCCTA +ACTATTATAATAATGTTGTTTCTCCTCTCCCTAGGGGGGCTACCTCCACTAACTGGCTTT +ATCCCAAAATGACTTATCCTAGAAGAACTTACTACCCAAAACCTTGCCCCACTAGCTATT +ATTATAGCAATATCAGCCCTTCTTAGCCTATTTTTTTATTTGCGTCTAGCCTATACTACA +ACACTAACACTTTCTCCTAATACAACCCAAATAAAACCTAATTGACGATTTAAACCACTA +GCCCAAACCACCCCCCTAACAG--CTATGACCCCCCTATCTATTTTTTTACTCCCACTTA +TGCCTTTAATTTTATACTAGAA---ACTTAGGATAA-CCTCAAAACCAAGAGCCTTCAAA +GCTCAAAATATGGGTGAAACCCCCGTAGTTTCTGCCAAAACTTGTGAAACTTTAATTCAC +ATCTGCTGAATGCAACTCAAACACTTTTATTAAGCTAAAGCCTC-ACTGAA-TAGGCGGG +CTTTGATCCCACAACAAATTAGTTAACAGCTAAACACCCAATCCAGC-GGGCTTCTATTC +GCTTCTCCCGTACGGGAG---------AAGCCCCGGAGCCTTTTTA-GGGCTCCTCTTCA +AACTTGCATTTTGACGTGAAACACCCCGGG-ACTGC---ATGATAAGGAAGGGAATTGAG +CCCCTATAAATAGGACTACAGCCTACCACCTAGCATT-CGGTCACCTTACCTGTGTCCAT +TAATCGTTGA------------ +>A.barahonae +ATGAGCCCGCTAATTTATATGATTATTTTATCAAGCTTAGCAACAGGCACAATTATTACA +ATAACGAGTTTTCATTGAATTATAGCTTGAATTGGGTTAGAAATCAACACCTTAGCAATT +ATCCCAATTATTTCTATATTACACCACCCACGTTCTACTGAAGCAGCCACAAAATATTTT +CTTACACAAGCAGCAGCATCCGCTATAATCCTATTTTCAAGTATAATTAATGCCTGACAA +ACAGGAACATGAGATATCACTCAAATAACTAATATCCAATCCAACATTCTATTAACAATA +GCACTTNCCATAAAATTAGGTCTAGCACCACTACATTTCTGACTTCCAGAAGTATTACAA +GGCTCAACCTTAAATACCGCCCTTATTATAATTACATGACAAAAGCTAGCCCCAATATCA +TTAATTTTTATAACCATTAATAACTTATCGACAACAATACTATTATTACTAGGATTAGTA +TCAACAATGCTAGGAGGATGAGGAGGATTAAACCAAACACAAATACGAAAAATAATAGCA +TATTCATCAATTGCACATTTAGGCTGAATAGCAACAATTTCTTCTATTATAACAAATATT +CTTATTATAAACTTCTTAATCTATATAATTTTAACCTTATCTATATTTTTATTACTTATT +TTCTCAAATTCTAAAACAATTCAAGACACCACAACAGCCTGAACCCTATCTCCAACCATT +ACTATTATAATAATACTTACACTCCTGTCATTAGGGGGATTACCCCCACTCACCGGATTT +TTACCAAAATGACTAATTCTAGAAGAATTAACATCCCAAAACCTAACAACCCTGGCTATA +ATCATAGCAATATCCGCCCTGTTAAGTCTGTTTTTTTACCTTCGACTAACCTACACAACA +TCACTCACACTTTCCCCAAACACAGTATTTACAAAATTTAAATGACGATATAAGCCCATG +ATACCAACTCAAATAATAGTAA--CTTTTACTATTATATCTATCTTTTTACTTCCACTCG +CCCCACTAATATTATTATAAAG---ACTTA--GGAT-AAC-TAAACCAAGAGCCTTCAAA +GCTCCAAATAGGAATGAGAACCTCCTAGTTTTTGT-AAGACTTGTAAAACATTAATTTAC +ATCTCTTGAATGCAAATCAAGCACTTTAATTAAGCTAAAGCCTT-CCTGAA-TAGGCGGG +CTTTGATCCCGCGACAAATTAATTAACAGCTAACTACCCAAACCAGC-GGGCTTCTATTC +GCTTCTCCCGTACGGGAG---------AAGCCCCGGAGCC-CTTTT-GGGCTCTTTTTCA +AATTTGCATTTTGATGTGGAACACTTCGAG-ACT-----TTGATAAATAAAGGAATTAAA +CCAATGTAAGTAGGACTACAGCCTACTGCCTAACATT-CGGCCACTTTACCTGTGTC--- +---------------------- +>A.bartschi +ATGAGCCCAATAATTTATTCTATCGACCTATCAAGCCTAGCAACAGGAACTATTATCACA +ATAACTAGCTATCATTGACTAATAGCCTGAATTGGACTAGAAATCAACACACTAGCAATT +ATTCCAATTATCTCAATTAAACACCACCCACGATCCACAGAAGCAGCTACAAAATATTTT +CTCACACAAGCTGCAGCCTCCGCTACAATCCTATTTTCTAGCACAATCAATGCCTGACTA +ACAGGCACATGAGATATTACCCAAATCTCCACCACCCCTTCCCACATCTTACTAACCACA +GCACTAGCCATAAAACTCGGCCTAGCCCCAATACACTTTTGACTTCCAGAAGTATTGCAA +GGCTCAACCATAAAAACAGCTTTTATTATCACTACATGACAAAAACTTGCCCCCATATCT +CTTATCTTTTTAACCATAAACAACCTTTCTACAACAATTTTAATCTTTCTAGGATTACTA +TCATCCGCAGTAGGCGGATGAGNNGGATTAAACCAAACACAAATACGAAAAATTATAGCA +TACTCATCAATCGCTCATCTAGGATGAATAGCCACAATCTCATCAATTATAACCAACCTT +CTAATTATGAATCTCCTACTATATCTAATCATTACAACAGCCATATTTTGGGCCATAATC +CTGTCAAACTCAAAGACAATTCAAGACACAACAACCACCTGAACCCTCTCACCAACCCTT +ACTATTATTATAATACTTTCCCTCCTCTCACTCGGAGGTCTACCCCCATTAACCGGATTT +ACACCAAAATGGCTGATCTTAGAAGAACTAGTATTTCAANNNNNNNNNNNNCTAGCCACC +ATTATAGCACTATCGGCTTTACTCAGCTTATTTTTTTACCTACGATTAACCTACACCACA +ACCCTAACAATATCCCCAAACACCATAAAAACAAAATTCAAATGACGGTTTAAATCCCTA +ACAACAACATTACCACTAGCAA--TATTTACCCCTATAACCATCTTTTTATTACCCATAA +CACCACTAATATTAATATAAGA---AACTTAGGATA-AACTTAAACCAAGAGCCTTCAAA +GCTCAAAATGTGGGTGAAACCCCCATAGTCTCTGC-AAGACTTGTGAAATATTAATCCAC +ATCTCATGAATGCAACTCAAACACTTTAATTAAGCTAAAGCCTC-CCTGAA-CAGGCGGG +CCTCGATCCCACGATAAATTAATTAACAGCTAATTACCCAAACCAGC-GGGCTTCTATTC +GCTTCTCCCGTACGGGAG---------AAGCCCCGAGC-CC-CTTA-GGGCTCTTCTTCA +AACTTGCATTTTGACGTGAAACACCTCGAG-GCT-----GTGATAAGAAAGGGAATTAAA +CCCCTGTAAACAGGACTACAGCCTGACACCTAGACTT-CGGCCACCTTACCTGTGTCCAT +TAATCGTTGA------------ +>A.bimaculatus +ATGAGCCCAACAATCAATATAATTATTTTATCAAGCCTAGCAACAGGCACCATCATTACC +ATAAGCAGCTTCCACTGATTAATAGCCTGAATCGGCCTAGAAATTAATACACTAGCAATT +ATTCCAATCCTCTCAACACCACACCACCCACGATCAACAGAAGCTGCTATAAAATACTTT +TTAACACAAGCCGCAGCCTCGTCCATAATTTTATTCTCTAGCATGACTAATGCCTGATAT +TTAGGCACATGAGATATTACTCAATTAGCCTATACCCCCTCAACCATTATATTGACCGCA +GCCCTTTCCATAAAATTAGGACTAGCCCCCCTACACTACTGACTTCCAGAAGTACTTCAA +GGATCAACCCTCTCCACAGCCTTCATTATTGTTACATGACAAAAACTAGCCCCCATATCT +CTAATTTATTTAACCATTAATAATCTTAATACCGAAACCCTCCTAACTTTAGGACTTCTA +TCAACCATTATTGGAGGTTGAAGCGGACTAAACCAAACACAAACCCGTAAAATTATAGCA +TATTCATCAATCGCACATCTAGGGTGAATGGCCTCCATTTCCTCAATTATAACAAACATT +ATAATCATAAACTTAGCAATTTATTTGATCATAACAACAGCCCTATTCTTCTCCCTTATT +TTATCTAAATCAAAAACAATTCAAGACACAGCAACCACCTGAACAACCTCCCCAATTCTG +GTTATAATAATAATATTATCACTACTGTCACTAGGAGGACTACCACCCCTAACAGGATTT +GCACCAAAATGACTAATTATAGAAGAACTAATTGTCCAAAACCTAGCCCCAATAGCCACC +GTTATAGCCCTCTCCGCCCTACTAAGCCTATTCTTCTACCTACGCCTAACCTATACAACA +ACACTTACACTTTCACCAAACACAACCCAAGCAAAATTTAAATGGCGACTAAAAGCACAG +CAATGCCCACCCCGATCACAACAGTCCCAACAACAGTTACAGTACTTTTATTAC--CCCT +TATACCCCATTCTGTCATAAAN---ACTTAGGATAA----CCAAACCGAGAACCTTCAAA +GTTCAAAATACGGGTGAAACCCCCATAGTTTTTGTTAAGACCTGTAAAACTCTAATTTAC +ATCTTCTGAATGCAACTCAAATACCTTAATTAAGCTAAGGCCTC-TCTGAA-TAGGCGGG +CCTTGATCCCACAACAAATTAATTAACAGCTAATTACCCAAACCAGC-GGGCTTCTATTC +GCTTCTCCCGTACGGGAG---------AAGCCCCGGAGCCCCTTA--GGGCTCTTTCTCA +AACTTGCATTTTGACGTGANNNNCTAC--G-TGGCT---GTGATAAAGAAAGGAATTAAA +NNNNNNNNAATAGGTCTACAGCCTACCGCCTAACACT-CGGCCACTTTACCTGTGTC--- +---------------------- +>A.brevirostrus +ATGAGCCCACTAATCCACACAATTATACTCTCAAGTCTAGCAACAGGCACTATTATTACT +ATATCTAGCCACCACTGACTAATAGCCTGAATTGGATTAGAAATTAACACACTAGCAATT +ATCCCCATCATTTCAACATCCCACCACCCACGATCAACAGAAGCTGCCACAAAATATTTC +CTTACACAAGCAGCAGCCTCTGCCACCGTACTATTTTCTAGTATAATTAATGCCTGACAA +ACCGGAACATGAGACATCACTCAAATATCTTATGCACCATCCAACATACTTTTAACTATA +GCACTCTCCATAAAGCTCGGACTAGCCCCGCTACACTTCTGACTTCCAGAGGTTCTTCAA +GGCTCAACCCTACCCACCGCCCTCATTATTACCACATGGCAAAAACTAGCCCCTATAGCA +CTAATCTGCCTAACAATTAATAATCTTAACCCAGCAATCCTACTAATCTTAGGACTTTTA +TCCTCAACTATTGGGGGTTGGGCCGGCCTTAACCAAACACAAACCCGAAAAATTATAGCA +TATTCATCAATTGCCCACCTAGGATGAATAGCCGCAATCGCCTCAATCATAACAAACATC +ATAATTATAAACCTCCTAATTTACTTAATGATAACAACCGCCCTATTCTGCTCCCTAATC +TTTTCAAAGTCAAAAACTATTCAAGATACAACAACCACCTGAGCCACATCCCCAATAATA +ACTATTACTACTGCGCTCTCATTATTATCATTAGGCGGACTTCCACCCCTAACAGGATTT +GCACCAAAATGACTAATTCTAGAAGAGCTCACTACCCAAAACTTAATCCCCATAGCCGCT +GTTATAGCCCTATTCTCACTTCTTAGCTTATTTTTTTACACTCGCCTAGTTTATACAACA +ACACTTACACTCCCGCCTAGCACACTTCAAACAAAATTTAAATGACGATTTAAACAGGCC +CCACCAACCTCACCAATAACAA--TTTCATCAACAGCAGCCATCTTTCTTCTGCCTTTAG +TACCTCTAATGTTGATATAAAA---ACTTAGGATAA----TAAAACCGAGAGCCTTCAAA +GCTTTAAAAAAGGGTGTAAACCCC-TTAGTTTTTGTAAGACTTGTGAGAGACTAACCCAC +ATCTTCTGAATGCAACCCAAACACTTTAATTAAGCTAAAGCCTT-CATGAA-TAGGCGGG +CCTTGATCCCACGAAAATTTAATTAACAGCTAACTGCCCTAGCCAGC-GGGCTTCTATTC +GCTTCTCCCGTACGGGAG---------AAGCCCCGGAGCCTATTG--GGGCTCTTCTTCA +AACTTGCATTTTGACGTAAAACACCTC--G-AGGCT---ATGATAAAGAAAGGACTTGAA +CCAATATGGGTAGGTTTACAGCCTACCACCTAAACCT-CGGTCACTTTACCTGTGTCTAT +TAATCGTTGA------------ +>A.carolinensis +ATGAGCCCAACTATTCACATAATTATTTTATCAAGCCTAGCAACCGGCACCATTATTACC +ATAACCAGCTACCACTGACTTATAGCCTGAGTGGGCCTAGAAATAAATACCTTAGCAATT +ATTCCAATCATCTCAACAATACACCACCCGCGATCTACAGAAGCAGCCACAAAGTACTTC +TTAACCCAAGCTGCAGCCTCCGCACTAATCTTATNTTCAAGCATAACAAATGCTTGAAAC +CTAGGCTCATGAGACATTACTCAAACATTAGCCCCCCCCTCACATATTCTATTAACCATA +GCACTAGCCATAAAACTTGGCCTAGCCCCTCTACACTTCTGACTACCAGAAGTCATCCAA +GGATCAACCATAACCACAGCCAACATTATTACCACATGACAAAAACTTGCCCCAATATCC +CTTATTTTCCTAACCATAAACAACCTATCCACCACAATTTTATTATTAATAGGACTACTA +TCTTCACTTATCGGGGGATGGGGGGGATTGAACCAAACACAAACCCGAAAAATCATAGCA +TACTCATCAATTGCACACCTAGGATGAATAGCAACAATCTCTTCAANCATAACAAATATC +NTTAAACATAAACTACAAATTTACCTTATCATAACAACTTCAATATTTCTNTCTCTTATT +ATTTCNAAATCAAAAACAATCCAAAACGCAACATCCACCTGAACCCTGTCCCCAGCACTT +ACAATCATTATAATACTCTCACTCCTTTCCTTAGGAGGACTACCCCCACTTACAGGACTT +ATACCGAAATGACTTATTATAGAAGAACTAATCTTACAAGACTTCAACTTACTAGCCACC +ATAATAGCTTTATCCTCCCTACTTAGCCTATTTTTTTACTTACGACTAGCCTATATAACC +ACACTTACACTATCTCCAAATACAACCCAAACAAAATTTAAATGACGATTTTACCCAAAC +ACACCAACCGTCTTTCTATCTA--TCCCTGCTACAATTTCTATTTTCTTACTACCCCTAA +CCCCTCTAATCTTACTATAAAG---ACTTA--GGATAAACT-AAACCAAGAGCCTTCAAA +GCTCAAAATAGGAGTTAAAACCCCCTAGTTTTTGC-AAGACTTGTGAAACATTATTACAC +ATCTCCTGAATGCAACTCAAGCACTTTAATTAAGCTAAAGCCTC-TCTGAA-TAGGCGGG +CCTTGATCCCACAACAAATTAATTAACAGCTAATTACCCAAACCAAC-GGGCTTCTATTC +GCTTCTCCCGNACG-GGA---------GAANNCAACGAGCCTTT--AGGGCTCCTCTTCA +AACTTGCATTTTGACGTGAAACACT-TCG---GGGC---TTGATAAAGAAAGGAATTAAA +CCAATGTAAATAGGACTACAGCCTACTGCCTAACACT-CGGCCACTTTACCAGTGTTTTT +TAGTCGTTAA------------ +>A.christophei +ATGAGCCCCTTAATTTTTACAATTATCCTGTCAAGCCTAGCAACAGGCACAGTTATTACT +ATAACCAGCTTCCATTGACTATTAGCTTGAATTGGGTTAGAAATCAATACACTAGCAATT +ATTCCAATTATTTCTACAATTCATCACCCACGCTCAACAGAAGCAGCCACAAAGTACTTC +CTTACACAAGCAGCAGCATCAGCTATAGTCTTATTTTCAAGCATAATTAACGCCTGACAA +ACAGGAACATGAGACATTACTCAAATAACTACTGATCAATCTAACATTATATTAACAATA +GCACTTGCTATAAAACTTGGATTAGCCCCANTACACTTCTGACTTCCAGAAGTATTACAA +GGTTCAACCTTAAATACTGCTCTTATTGTAACCACATGACAAAAACTTGCCCCAATATCA +CTAATTTACCTAACTATAAACAACCTATCAACAACAACTTTGCTGCTACTAGGAGTAGTA +TCCTCAATATTAGGAGGATGGGGAGGACTTAATCAAACACAGACACGTAAAATTATAGCA +TACTCATCAATTGCACACCTAGGATGAATGGCCACAATTTCTTCTATTATAACAAACATT +CTAATTATAAATTTATTAATTTATTTAATTTTAACAGTATCTATATTTCTTCTACTTATT +TTTTTAAACTCCAAAACCATTCAAGATACTATAATAACCTGAACCCTCTCTCCAACCATA +ACCATTATAACTATACTTACACTTTTATCATTAGGTGGCCTACCACCACTAACCGGGTTT +ATACCAAAATGATTGATCTTAGAAGAACTAACAACTCAAAACTTAACCTCACTAGCTATG +ATTATGGCAATATCTGCTCTACTAAGTCTATTTTTTTATTTACGAGTTACATACACAACA +TCACTCACATTATCCCCTAATACAGTCCAAACAAAATTTAAATGACGATTTAAACCATTA +ATACCAACTCATCTAATTTTAA--TTTCAACTACAACAGCTATTTTTTTACTTCCCCTCA +CCCCGCTAATATTTCTATAAAG---ACTTA--GGAT-AACATAAACCAAGAGCCTTCAAA +GCTCAAAATATGGGAGAAACCCCCCTAGTTTTTGT-AAGACTTGTAAAATATTAATTTAC +ATCTCCTGAATGCAANTCAAGCACTTTTATTAAGCTAAAACCTT-TCTGAA-TAGGTGGG +CCTTGATCCCACGANAAATCAATTAACAGCTAATTACCCTAACCAGC-GGGCTTCTATTC +GCTTCTCCCGTACGGGAG---------AAGNCCGGAGCCCTTTTAT-GGGCTCTTTCTCA +NACTTGCATTTTGATGTGAAACACTTCGGG-ACT-----TTGATAAAGAAAGGAATTAAA +NNAATGTAAGTAGGACTACAGCCTACTGCCTATCATT-CGGNNACTTTACCTGTGTCTAT +TAAGCGTTGA------------ +>A.coelestinus +ATGAGCCCACTAATTTTTTCAATCGTCCTGTCAAGCCTAGCAACAGGCACTATTATTACC +ATAACCAGCTATCACTGATTAATAGCTTGAATTGGTCTAGAAATAAACACACTTGCTATT +ATTCCAATTATCTCAATACAACATCACCCTCGATCTACAGAAGCCGCTACAAAATATTTC +CTTACACAAGCAGCAGCCTCCGCTATGATTTTATTCGCCAGCACAACAAATGCTTGATAC +ACAGGCACATGAGACATCACTCAAATTTCCGCCACCCCATCCCACATCCTCCTAACTGCC +GCATTAGCCATAAAACTAGGCCTAGCCCCTATACACTTCTGATTACCAGAAGTCCTTCAA +GGGTCCACCTTAAAAACAAGCTTTATTATTGTCACATGACAAAAACTAGCCCCTATATCA +TTAATTTATTTAACTATAAATAATCTTTCCTCAACAATTCTTCTCTCACTAGGACTAGTT +TCATCTATGGTAGGAGGATGGGGCGGACTAAACCAAACACAAACCCGAAAGATCATAGCA +TACTCATCAATTGCACATCTCGGATGAATGGCCTCAATCTCCTCCATTATAACTAATATT +CTTGTTATAAATTTAGTACTTTACCTAATTATAACAACATCTGTATTCTACGCTTTGATC +TTAACAAAGTCTAAAACAATTCAAGATACAACAACCACCTGAACACTCTCCCCAACTTTA +ACAATTATTATAATACTCTCACTTCTCTCCCTTGGAGGCTTACCCCCCTTAACTGGATTC +ACACCAAAATGACTTATCTTAGAAGAATTAACCACCCAAAATCTACTCCCCCTAGCCACT +ACTATAGCGATATCTACCCTACTCAGCCTGTTTTTTTACTTACGTTTAACCTATACCACA +ACCCTTACATTCTCCCCAAACACTACACATACAAAATTTAAATGACGATTCAAGTCAACT +AATTTAACAATACCACTAACAC--TTACAGCACCCCTGAGCGTCCTCTTACTTCCTTTAG +TCCCGCTAATTATAGTATAAGA---AATTTAGGATA-ATATTAAACCAAGAGCCTTCAAA +GCTCAAAATACGGGTGAAACCCCCATAATTTCTGT-AAGACTTGTGAAATACTAATCCAC +ATCTCATGAATGCAACTCAAGCACTTTTATTAAGCTAAAGCCTT-TCTGAA-TAGGCGGG +CCTCGAGCCCACGACAAATTAGTTAACAGCTAATTACCCAAACCAGC-GGGCTTCTATTC +GCTTCTCCCGTACGGGAG---------AAGCCCCGGAGCCC-TTTA-GGGCTCTTCTTCA +AACTTGCATTTTGATGTGGAACACCCCAGG-ACT-----TG-ATGAGGAAGGGAATTAAA +CCCCTGTTAATGAGTCTACAGCTCACCACCTAACACT-CGGCCACCTTACCTGTGTCTA- +---------------------- +>A.cristatellus +ATGAGCNNNACAATCTACACAATTATTTTGTNNNNNCTAGCAACAGGCACTATCATCACT +ATAACTAGCTTCCACTGACTAATGGCCTGAATCGGACTAGAGCTTAATACGCTAGCAATT +ATCCCGATTATTTCAACATTACACCACCCACGATCAACAGAAGCCGCAACAAAATACTTC +TTAACACAAGCAGCAGCCTCTGCAATAATTATGTTTTCTAGCATAATTAATGCCTGAAAC +ATAGGAACATGGGACATTACTCAAATATCTTATACCCCATCAAATGTTCTTCTTTTACTA +GCACTGGCCCTAAAACTAGGACTAGCCCCAGCACACTTCTGACTCCCAGAGGTATTACAG +GGCTCAACCTTATTTATTGCCCTCATTATTACTACGTGGCAAAAACTGGCCCCAATGTCA +CTTATCTACATATGTACAAATAATCTTCCATCAGTAATTTTATTAATACTAGGCATTATC +TCCTCCACTGTCGGGGGGTGGTCTGGATTAAACCAAACACAAACTCGAAAAATCATAGCA +TACTCATCAATTGCACACCTTGGATGAATGGCTATAATCGCCTCAATTATAACAAACATC +ATAATTATAAACCTGGTGATTTATTTAATAATAACAACCACCTTGTTTACCACCCTAATT +TGCTCTAAATCAAAAACAATTCAAGACACAGCAACCACTTGAACAACATCACCAACCCTA +ACTATCATTACTTTACTTTCTCTTCTGTCACTTGGGGGACTCCCACCTCTTACAGGATTT +TTACCAAAATGACTAGTTTTAGAAGAAATAATTGTACAAAACTTAACCCCACTCGCCACA +ACCATAGCAATTTCCGCCCTTCTTAGTCTATTCTTCTATCTACGCCTAACCTACACCACA +ACACTAACATTAGCTCCAAACACCCTACAAATAAACTTTAAATGACGGTTTAAACCAACC +ACATCATTTATTCTAATAACCC--TTT---CAACTATGACCATTTTTTTACTACCTCTCA +TACCACTAACCCTTATATAAAA---ACTTAGGATAA---CATAAACCAAGAACCTTCAAA +GTTCAGAATAAGGGTGTAAACCCC-TTAGTTTTTGCAAGACTTGTAANANATTAATTTAC +ATCTCATGAATGCAACTCAAGCGCTTTTATTAAGCTAAAGCCTC-TCTGAA-TAGGCGGG +CCTTGATCCCACGATAAATTAATTAACAGCTAATTACCCAAACCAGC-GGGCTTCTATTC +GCTTCTCCCGTACGGGAG---------AAGCCCCGGAGCCAATAN--GGGCTNGCTTTCA +AANTTGCATTTTGACGTGAAATAC-TACAG-GACTC---GTAGTAAAGAAAGGGTTTAAA +NCAATATAAANAGATTTACANGCTACCGCCTAANAGT-CAGCCACTTTACCTGTGTCCAT +TAAGCGTTAA------------ +>A.cuvieri +ATGAGCCCAACAATTCTCTCAATCATTTTATCAAGCCTAGCAGCAGGAACAATTATTACA +ATAACAAGCTTTCATTGATTAATAGCCTGAATTGGACTAGAAATTAATACACTAGCAATT +ATTCCAATTATCTCAATAATACATCACCCACGATCTACAGAAGCAGCCACAAAATATTTT +CTCACACAAGCAGCAGCATCAGCTATAATCCTGTTCTCAAGCATAATTAATGCTTGACAA +ACAGGGACATGAGATATTACCCAAATATATACTCCTCAATCTAACATTTTATTAACTATA +GCCCTTGCCATAAAAATAGGATTAGCCCCAATACACTTCTGACTACCAGAAGTACTTCAA +GGTTCAACCTTAAACGCTGCTCTTATTATTACCACATGACAAAAAATCGCCCCAATGTCA +TTAATTTATTTAACCATTAATAATTTATCAACAACAACCTTATTAACACTAGGATTAATT +TCTTCAATATGAGGAGGATGAGCAGGCCTAAACCAAACACAAACCCGAAAAATCACAGCA +TACTCATCTATTGCTCACATTGGATGAATGGCTACAATTTCTTCAATTATAACAAATATT +CTTATTATAAACCTATTAATTTATTTAATTATAACAATCTCTATATTTAACTCACTAATT +TTATCCAAATCTAAAACTATTCAAGACACGTCAATGACATGAACATTATCTCCAATATTA +ACTATTATTACTATACTTACACTCTTATCATTAGGGGGATTACCACCACTAACCGGATTT +ATACCAAAATGACTAATTCTAGAAGAATTGACAACCCAAAACTTAACTTCACTAGCCGTA +ATCATAGCAATAACTGCCCTATTAAGCCTTTTTTTTTACTTACGACTAACATACACAACA +ACACTTACATTATCACCAAACACAACACAAACAAAATTTAAATGACGATTTAAACCAAAT +TTACCTACATATTTACTAATAA--TCTCCTCTACAACAACCATCTTACTTCTTCCACTAA +CCCCCTTAATAATTTACTAAAA---ACTTA--GGTT-AACATAAACCGAGAGCCTTCAAA +GCTCAAAACAGGAGTCAAAAACTCTTAGTTTTTGT-AAGACTTGTAAAATACTAATTTAC +ATCTTCTGAATGCAACTCAAACACTTTAATTAAGCTAAAGCCTA-CCTGAA-TAGGCGGG +CCTTGATCCCACGACAAATTAATTAACAGCTAATTACCCAAACCAGC-GGGCTTCTATTC +GCTTCTCCCGTACGGGAG---------AAGGCCCGGAGCC-CTTTT-GGGCTCTTCTTCA +AATTTGCATTTTGACGTGAAACACTTCAGG-ACT-----TTGATAAAGAAGGGAATTAAA +CCAATGTAAGTAGGACTACAGCCTACCGCCTAACATT-CGGCCACTTTACCTGTGTTTAT +TAATCGTTGA------------ +>A.distichus +ATGAGCCCGCCAATCTACGCAATTATACTATCAAGCTTAGCAACAGGCACCATTATCACT +ATAACCAGTTACCATTGACTAATGGCCTGAATTGGACTAGAAATTAATACACTAGCAATT +CTTCCAATTATTGCAACATCACACAACCCACGATCCACAGAAGCTGCCACAAAATACTTT +TTAACACAATCAGCAGCTTCCGCCACTATCTTATTTTCTAGCATACTTAACGCCTGACAA +ACCGGAACATGAGACATTACTCAAATATCTTATGCGCCATCTAACCTCCTTCTCTCCATA +GCACTAGCCATAAAACTAGGACTAGCCCCCCTACACTTTTGACTCCCAGAAGTACTTCAA +GGCTCAACTTTATTTTCTGCCCTCATTATTGTTACATGACAAAAATTAGCCCCAATATCA +CTGATTTATTTAACTATTAGTAACCTTAACCCAACTATTCTACTAATCTTAGGCCTCCTA +TCCTCAACTATTGGGGGATGGGGTGGGTTAAATCAAACACAAACCCGAAAAATCATAGCA +TATTCATCAATTGCTCACCTAGGATGAATGGCCATTATTGCCACCATTATAACAAACCTC +ATAGTCATGAACCTCTTAATTTATCTAATAATAACAACAGCCCTATTCTCCCTCCTAATT +TTTTCCAAATCAAAAACCATTCAAGACACAACAACAACCTGAGCACTCTCCCCCACAATA +ACTATTATAATGACATTTCTATTATTATCATTAGGGGGGCTTCCCCCAATAACAGGATTT +GCACCAAAATGACTAATTCTAGAAGAACTCACAACCCAAAATCTAATTCCTCTAGCTGTT +TTAATAGCAGTATTTTCCCTTCTTAGCCTGTTTTTTTATACTCGACTAGCCTACACAACA +ACACTTACACTATCGCCCAATACACTACANANAAAATTTAAATGACGATTCAAACAAACC +CTATCAACTTCACTAATAGTAA--CTTTATCAACAATAGCTATTTTTCTTCTACCACTTA +CACCCTTAATGTTAATATAAAA---ACTTAGGATAT----TAAAACCAAGGGCCTTCAAA +GCCCTAAAAAAGGGTGCAAACCCC-TTAGTTTTTGTAAGACTTGTGAAACACTAATCCAC +ATCTTCTGAATGCAACCCAAACGCTTTCATTAAGCTAAAGCCTT-TCTGAA-TAGGCGGG +CCTTGATCCCGCGATAAATTAATTAACAGCTAATTACCCTAACCAGC-GGGCTTCTATTC +GCTTCTCCCGTACGGGAG---------AAGCCCCGGAGCCCTTTT--GGGCTCTTCTTCA +AACTTGCATTTTGACGTAAAACACCTC--G-AGGCT---TTGATAAAGAAAGGGCTTCAA +CCAATATAAATAAGTTTACAACTTACCGCCTAAATTT-CGGCCACTTTACCTGTGTTCAT +TAATCGTTGA------------ +>A.equestris +ATGAGCCCAACAATTTATTCAATTATCCTATCAAGCCTTGCAANNNNNACAATTATTACT +ATAACCAGCCACCATTGACTAATAGCCTGANTCGGATTAGAAATTAACACATTAGCAATT +ATCCCAATTATTTCAACATTACACCACCCACGATCCACAGAAGCCGCCACAAAATATTTC +CTAACACAAGCAGCTGCTTCTGCTATAATTTTATTTTCTAGCATAACAAATGCTTGATAC +ACAGGTACATGAGACATTACCCAAATATCAGCCAACCCCTCCCATATTATATTAACTATG +GCACTTGCCATAAAACTAGGCCTAGCACCTCTACACTTCTGACTACCAGAAGTACTCCAA +GGCTCAACCATAAAAACCGCATTCATCATTACAACATGACAAAAACTTGCCCCAATATCA +TTAATCTACCTCATTATTAATAACTTATCCCCCACACTTCTCCTCCTATTAGGACTAATA +TCATCTACTCTGGGAGGCTGAGGAGGACTAAACCAAACCCAAACCCGAAAAATCATAGCC +TATTCATCAATCGCCCACCTAGGTTGAATAGCTACAATCTCTTCAATCATAACCAATATT +CTTGTTATAAACCTATTACTTTATATAACTATGACAACATCAATATTTTTTACCCTTATT +TTATCAAAATCTAAAACAATTCAAGATACAACTACCTCATGAACACTATCTCCATCCCTA +ACCATCATTATAATATTATCACTTCTCTCATTAGGCGGACTACCCCCCCTAAGTGGATTT +ATACCAAAATGATTAATCCTAGAAGAACTNNNNNNCCAAAATCTTCCCCCACTAGCCACT +ATTATAGCAATATCTGCCCTACTTAGCTTATTCTTTTATCTACGACTTACCTACACTACT +ACCCTAACAATCTCCCCTAATACCTTACAAACTAAATTTAAATGACGATTCAAACCTACG +ACCTCAACCCTACCAATAATAA--TCTTTACCCCATTAACTATTTTTATGTTACCACTAA +CACCAATAATCATCATATAGAA---ACTTA--GGTT-TAAATAAACCAAGAGCCTTCAAA +GCTCAAAATATGGATGAAAACCCCATAGTTTCTGCTAAGACTTGTAAAATACTAATTTAC +ATCTCCTGAATGCAACTCAAATGCTTTTATTAAGCTAAAGCCTT-CCTGAA-TAGGAGGG +CCTTGATCCCACAATAAATTAATTAACAGCTAATTACCCAAACCAGC-GGGCTTCTATTC +GCTTCTCCCGTACGGGAG---------AAGCCCCGGAGCCCTATTG-GGGCTCTTTTTCA +AACTTGCATTTTGACGTGAAACACTTCAGG-GCT-----TTGATAAGAAAAGGAATTAAA +CCTATNNNNNNAGGACTACAGCCTACCGCCTAACACT-CAGCCATCTTACCTGTGTCTAT +TAACCGTTA------------- +>A.etheridgei +ATGAGCCCTGCCATCTATACCATTATTTTATCAAGCCTAGCAACAGGCACACTCCTAACT +ATAACCAGCTTTCACTGATTAATAGCATGAATCGGATTAGAAATTAATACACTAGCAATA +ATTCCAATTATCTCAACACCTCACCAACCACGATCTACAGAAGCTGCTACAAAATATTTC +CTCACACAAGCAGCAGCCTCCGCTATAATTTTATTTTCAAGCATAATTAATGCCTGACAA +ACAGGATCATGAGACATTACCCAAACATCTACTTTCCCCTCTCATATTCTTTTAACCATG +GCCTTAGCAATAAAATTAGGCCTTGCCCCCTTACATTTTTGACTACCAGAAGTTCTTCAA +GGCTCAACCCTAAACACAGCCCTAATTATTACCACATGACAAAAGCTAGCTCCAATGTCA +CTAATTTATTTAACAGTAGATAACCTATCCCCCTCAATTCTTCTCCTACTTGGTCTTCTA +TCTTCTCTAGTTGGCGGCTGAGGTGGACTAAATCAGACACAAACCCGAAAAATTATAGCA +TACTCATCAATTGCACATCTAGGATGAATAGCCACCGTAACTTCAATTATAACAAATATT +CTTCTCATAAACCTTATAATTTATTTAATTATAACAACAACCATATTCTTCTCTTTAATT +TTATCCAAATCCAAAACAATTCAAGACTCAACTATTACCTGAACACTCTCACCAACTCTA +ACTATTATTACAACACTTACACTTCTTTCACTCGGAGGATTACCCCCCCTAACCGGATTT +ATGCCAAAATGACTAATCTTAGAAGAATTAACAACTCAAAACTTAACCCCCATAGCTATT +ACCATAGCAATATCCACCCTACTTAGCTTATTTTTTTACTTGCGACTAACCTACACAACA +GCTCTTACTTTATCCCCAAACACCATACAAACAAAGCTTAAATGACGATTTAAACTAAAC +CAACCAACTCAGCTAATAATGA--CTACTTCTACAATAACAATTTTTTTACTTCCTCTTA +CACCCTTACTCACATTATAAAA---ACTTA--AGTT-TAACTAAACTAAGAGCCTTCAAA +GCCCAAAATATGGGTAAAACCCCCATAGTTTTTGT-AAGACTTGTGAAATACTATTTCAC +ATCTCTTGAATGCAACTCAAGCACTTTTATTAAGCTAAAGCCTT-CCTGAA-TAGGCGGG +CCTTGATCCCGCGATAAATTAATTAACAGCTAACTACCCTAGCCAGC-GGGCCTCTATTC +GCTTCTCCCGTACGGGAG---------AAGCCCCGGAGCCCTTTGT-GGGCTCTTTTTCA +AACTTGCATTTTGATGTGAAACACTTCGGG-ACT-----TTAATAAAGAAAGGAATTAAA +CCAATGTAAATAGGACTACAGCCTACCGCCTAACACT-CGGCCACTTTACCTGTGTTTAT +CAAGCGTTGA------------ +>A.garmani +ATGAGCCCAACCATCCTTATAATTATTATCTCAAGCCTGGCAACAGGTACCATTATTACC +ATAACAAGCCACCACTGACTCATAGCCTGAATCGGACTAGAAATAAATACCTTAGCTATT +ATTCCAATCATTACTACTATACACAACCCACGATCAACAGAAGCCGCCACAAAATACTTC +TTAACACAAGCAGCAGCCTCTGCCATAATCTTATTCTCAAGCATAATTAATGCCTGACAA +ATAGGATCATGAAATATTATTAACTTAACATACGAACCCGCGCACATCCTATTAATTATG +GCCCTAGCAATAAAACTTGGCCTCGCACCCATACACTTTTGACTGCCAGAAGTTCTTCAA +GGCTCAACCTTAACCACTGCACTAATTATTACCACTTGACAAAAACTCGCCCCAATATCT +TTAATTTATATAACCCTAAACAATCTATCGACCACAATTTTAATTTTACTCGGACTTCTA +TCCTCAATTATTGGAGGATGGGGGGGACTCAATCAGACCCAAACTCGAAAAATCATAGCA +TTTTCATCAATNCGACACCTGGGGTGGATAATTACTGTCTCATCAATTATGCCGAACATT +ATAATTTTAAACCTAATTATTTATTTAGTCATAACAACCGCCCTATTTTACTCACTAATC +TTATCNAAATCAAATGGAATCCAAGACACAACAACAACTTGAACACTTTCACCCACAATA +ACTATTATAATGATAACTTTACTCCTCTCCCTAGGGGGCCTCCCACCACTAACTGGCTTT +ATACCAAAATGANTTATTCTAGAAGAACTAGTTGCCCAAAATCTTATCTCAACCGCCACT +ATTATAGCCATATCAGCACTACTAAGCCTATTTTTTTATCTACGACTAACCTATACAACA +ACACTAACCCTATCCCCGAACACTANACAAACAAAGTTTAAATGACGATTTAAACCAAAC +CGTACAACACCCGTTATTGGGGCCTC--AGCTACTATCTCAATTTTCCTACTCCCTATAA +CCCCATTAATACTTCTATAAAG---ACTTAGGAT----AATTAAACCAAGGGCCTTCAAA +GCCCTAAATAAGGGCCATA-CTCCCTTAGTTTTTGTAAGACTTATGAGGGACTATCCCAT +ATCTCCTGAATGCAACTCAAGCACTTTTATTAAGCTAAAGCCTTATCTAAA-TAGGCGGG +CCTTGATCCCACAA-AAACTAGTTAACAGCTAATTACCCAATCCAGCGGGCTTCTCTTTA +GCTTCTCCCGTACGGGAG---------AA-GCCCCNGGGCCCTTT-AGGGCTCTTTTTCA +AACTTGCATTTTGATGTGAATCACTTCAGA-GCT-----GTGATAAAGAAAGGAATTAAA +CCAATGTAAANNGGTCTACAGCCTACCGCCTAACATT-CGGCCACTTTACCTGTGTCTAT +TAATCGTT-------------- +>A.grahami +ATGAGCCCATCAATCCTTATAATTATTATTTCAAGCCTGGCAACAGGCACTATTATTACT +ATAACAAGCCACCACTGACTTATAGCCTGAGTCGGACTAGAAATAAATACTTTGGCAATT +ATCCCAATTATTTCTACTACACACAGCCCGCGATCCACAGAAGCCGCTACAAAATATTTT +TTAACACAAGCAGCTGCCTCTACCATAATCTTATTTTCAAGCATAACCAACGCCTGACAA +ACAGGCACATGAGACATTATTAACTTAACATATGAGCCCGCGCACATCCTGCTAACCCTG +GCCCTAGCAATAAAACTAGGCCTTGCACCTATACACTTCTGANTACCAGAAGTATTACAA +GGCTCAACCTTAACCACTGCACTAATTATTACCACTTGACAAAAACTAGCCCCAATATCC +TTAATTTATATGACCCTAAACAACCTGTCAACCACAACTTTAATCTTACTAGGACTTCTA +TCTTCAATTATTGGGGGGTGGGGCGGACTAAACCAAACCCAAACCCGAAAAATTATAGCG +TTTTCATCAATCGCACATCTTGGGTGAATAATTACTATTTCATCAATTATACCCAATATT +ATAGTTTTAAACCTGACAATCTACCTAATCATAACAATTGCCCTATTTTATTCACTAATT +CTATCTAAATCAAAAACAATCCAAGATACAACANCAACTTGGACACTCTCACCCGCACTG +ACTGCCATAATAATAACACTACTTCTTTCCCTGGGAGGGCTACCACCACTAACGGGCTTT +ATGCCAAAATGACTTATCTTAGAAGAACTAGTAGCCCAAAACCTCACCCCAGCCGCCACT +GCTATAGCCATATCAGCGCTACTAAGCCTGTTCTTCTACNTGCGGCTAACCTATACAACA +ACACTAACCCTGTCCCCAAATACCACTCAAACAAAACTTAAATGACGATTTAAGCCAAAC +CACACAACACTTCTCATAGGGGCCTC--CTCCACAATCTCAATCTTTCTACTCCCCATGA +CCCCCTTAATACTTCTATAAAA---ACTTAGGAT----AATTAAACCAACGGCCTTCAAA +GCCCTAAATAAGGCCACCA-TCCCCTTAGTTTTNGTAAGACTNATGGGTAACTACCCCAT +ATCTCCTGAATGNAACTCAAACACTTTTATTAAGCTAAAGCCTTATCTAAA-NNNNNNNN +NNNNNNNNNNNNAA-AAATTAGTTAACAGCTAATTACCCAATCCACGGGNCTTCTATTTA +GCTTCTCCCGTACGGGAA---------AG--NCCCGGAGCCCTTT-AGGGCTCTTTTTCA +AACTTGCATTTTGATGTGGATCACTTCAGG-GCT-----GTGATAAAGAAAGGAATTAAA +CCAATGTAAATAGGACTACAGCCTACCGCCTACATT--CGGCCACTTTACCTGTGTTCAT +TAATCGTT-------------- +>A.humilis +ATGAGCCCCACTGTTCTTATAGTAATTTTATCAAGCCTAGCCACTGGGACTATCATCACC +ATAACAAGCCACCATTGACTATTTGCCTGACTCGGACTAGAAATAAATACCCTAGCAATT +ATTCCCACCATCACCACCCTCCACAACCCACGATCAACAGAAGCAGCCACAAAATATTTT +CTAACACAAGCAGCAGCCTCTGCCATAATTTTATTTTCAAGCATAACTAATGCTTGACAA +ACCGGATCATGAGATATTACTAGCACGATATCTACACCAGCCCATATCCTACTAACTTTA +GCACTAACAATAAAGCTAGGACTTGCCCCAATACACTTCTGACTACCCGAAGTTCTACAA +GGCTCAACCCTAACTACAGCACTCATTATTACAACCTGACAAAAACTTGCACCTCTAACC +CTAATTTACTTGACTATCTCTAATTTATCAACAATAATTTTAATTACCATAGGACTTTCA +TCAACTATCATTGGAGGGTGAGGCGGACTCAACCAAACACAAATCCGAAAAATCATAGCA +TACTCATCTATTGCCCACCTAGGATGAATACTGTCTATCTCATCTATTATAACAAACATT +ATAATTTTAAACTTGATAATTTACTTAATTGTAACAACAGCCCTATTTAGCTTCTTAATT +TTTACTCGATCCAAAACAATTCAAGATACAATAATAACCTGAACACTCTCACCCATAACA +ACTTCTATAATAATATTACTATTTTTATCCCTTGGAGGACTACCCCCATTAACTGGATTT +ATTCCAAAATGACTTATTTTAGAAGAACTAATTACCCAAAATCTTGATTCAATAGCTATT +ATTATAGCCCTATCTACACTACTTAATTTATACTTTTACTTACGATTAACCTACACAACA +GCCATTACACTATCCCCAAACATATTACAAACAGAATTTAAATGACGACTAAAACCGAAA +TTTACACACACTGTTATTAGTAACCTC-ATCCTCAATATCTATTTT-TTTTCTTCCCTAA +TACCACTAATTTTATTATA------AAAACTTAGGT-TATTTAAACCAAGGGCCTTCAAA +GCCCTAAATAAGGACACCA-GCCCTTAGTTTTTGATAAGACTTATGGGAAACTAACCCAC +ATCTTTTGAATGCAACCCAAACACTTTTATTAAGCTAAAGCCTCTCGGAGC--AAGTGGG +CCTTGATCCCACAA-AAATTAGTTAACAGCTAATTACCCTAACCAGCGGGCTTCTACTCG +NNNNNNNCCGTACGGGAG---------AANNCCCCGAGCCTTTTA-GGGGCTCTTCTTCA +AATTTGCATTTTGATGTAAGTCACTGCGGAGGCT-----CTGATAAAGAAAGGAGTTAAA +CCAATGTGTGCAGGACTACTGCCTGCCGCCTAACACT-CGGCCACTTTACCTGNGTTTAT +TAGCCGTTAT------------ +>A.insolitus +ATGAACCCAACTATTCTTACATTAATTTTATCAAGCTTAGCAACAGGTACAATCCTTACA +ATAATCAGCTTTCACTGACTACTCGCATGAATTGGTCTAGAGATTAATACCCTAGCAATT +ATTCCTATTATCTCAGCACCTCACCACCCCCGACCCACAGAAGCCTCCACAAAATATTTC +CTTACACAAGCAGCTGCTTCTGCTACAATCTTATTTTCAAGCATAATTAATGCTTGACTA +ACAGGCACATGAAACATTACCCAAATATCCTCTACTCCCGCTAATATTCTTCTAACCCTA +GCACTAGCAATAAAACTAGGACTTGCCCCCACACACTTCTGACTACCAGAAGTGCTTCAA +GGCTCAACTATAAACACAGCCCTCATTATTCATACATGACAAAAACTTGCCCCAATGTCA +CTGATTTACTTAACAGTAAATAACTTATCCCCTACAATTCTATTATCCCTGGGCCTCTTA +TCTTCCCTAATTGGAGGCTGAGCCGGACTAAATCAAACACAAACTCGAAAAATTATAGCA +TACTCATCAATCGCACACCTAGGATGAATGGCCACAGTAGCCTCAATTATAACAAATATT +CTTCTAATAAACCTCATAATCTACTTGTTAATAACAACAGCCATATTTTTCTCTCTTATT +TTATCCAAAACTAAAACAATTCAAGACTCAGCAATAACCTGAGCACTTTCACCAGCCTTA +ACTACTATAACAATACTCACACTTCTCTCATTAGGCGGACTACCCCCACTTACTGGATTT +ATGCCCAAATGACTAATCCTAGAACAATTAGCCAGCCAAGACTTAGTGCCCCTAGCCACT +ATTATAACTATATCTGCCTTACTTAGCCTATTTTTTTATCTACGACTAACCTACACAACA +ACACTTACACTATCCCCTAACACTACACAAACAAAATTTAAATGACGATTTAAACTAAAC +CAACCTACCCAACTAACAACAA--CCACTATTACAATAGCAACCTTATTACTACCCATAC +TACCAATGCTCACACTATAAAA---ACTTA--AGTT-AAACCAAACTAAGAGCCTTCAAA +GCTCAAAATAAGGGTAGAACCCCCTTAGTTTTTGT-AAGACTTGTGAAACACTAACTCAC +ATCTCCTGAATGCAACTCAGACACTTTTATTAAGCTAAAACCTT-CATGAA-TAAGTGGG +CCTTGATCCCACAACAAATTAATTAACAGCTAACTACCCTAACCAGC-GGGCTTTTATTC +GCTTCTCCCGTACGGGAG---------AANNCNCGGAGCCT-TTTT-GGGCTCCTTTTCA +AACTTGCATTTTGACGTGAAGCACTTCGGG-ACT-----TTGATAAAGAAAGGAATTAAA +CCAATGTAAATAGGACTACAGCCTACCGCCTAGCACT-CGGCCACTTTACCTGTGTTTAT +T--------------------- +>A.krugi +ATGAGCCCTGCAATTTACACAATTATATTATCAAGCTTGGCAACAGGCACTATCATCACT +ATAACAAGCTTCCACTGACTAATAGCCTGAGTTGGACTAGAACTTAATACATTAGCAATT +ATCCCAATTATTTCAACATTACACCACCCCCGGGCCACAGAAGCCTCAACAAAGTATTTC +CTCACTCAAGCAGCAGCCTCTGCCATAATTTTATTTTCTAGCATAATTAATGCCTGACAC +ACGGGAACATGGGACATTACTCAAATATCTTACACCCCCTCCAACATCATTTTAACAATA +GCACTAGCCATAAAGCTAGGACTTGCCCCAATGCACTTTTGACTTCCAGAAGTCCTACAA +AACTCAACCCTTATTACTGCTTTTATTATTTCTACATGACAAAAACTTGCCCCAATATCA +CTAGTCTACTTGACTATTAATAACCTTCCACCAATAATTCTGCTTACCCTAGGAGTTATT +TCCTCTACTATGGGCGGATGAGCAGGACTAAACCAAACACAAACTCGAAAAATTATAGCA +TACTCATCAATTGCACACCTGGGCTGAATGGCCTCTATTGCCTCAATCATAACAAACATT +ATAATCATGAATCTAATGATTTATTTAATTATAACAGCGGCCCTATTCTTCTCATTAATT +TATTCTAAGTCAAAAACAATTAAAGACACAACATCTGCCTGAACAATATCCCCCACCCTA +ACTATTACAATAATGTTAACCCTTTTATCACTTGGAGGATTACCTCCCCTAACAGGATTT +TTACCAAAATGACTAATCCTAGAAGAANNNNNNNNNNNNNNNCTTGCCCCATTAGCCACC +ACCNTAGCAATATCCGCTCTTCTAAGCCTATTCTTTTACCTACGCCTGGCCTATACAACA +ACACTAACACTTTCACCAAATACACTTCAAATAAAATTTAAATGACGATTTAAACCAACC +ACTACACCACCACTAATAATCT--TTT---CATCTATAGCCGTTTTTTTACTACCACTTA +CACCTTTAATCTTATTGTAAAA---ACTTAGGATAAGATAATAAACCAAGAACCTTCAAA +GTTCAAAATAAGGGTGAAAGTCCC-TTAGTTTTTGTAAGACTTGTGAAACATTAATTCAC +ATCTCATGAATGCAACTCAAGCACTTTTATTAAGCTAAAGCCTC-CCTGAA-TAGGCGGG +CTTTGATCCCACGAAAAATTAATTAACAGCTAACTACCCATTCCAGC-GGGCTTCTATTC +GCTTCTCCCGTACGGGAG---------AAGCCCCGGAGCCCCTTA--GGGCTCTTTTTCA +AACTTGCATTTTGACGTGAAAGCCACTTCA-AGGCT---GTGACAAAGAAAGGATTCAAA +CCAATATAAGTAAGTTTACAGCCTACCGCCTAATATT-CGGCCACTTTACCTGTGTCTAT +TAATC----------------- +>A.limifrons +ATGAGCCCATCAATTTTTATAATTATTTTATCAAGTCTAGCCACAGGGACAATTATTATT +ATAACAAGCCACCATTGACTTTTTGCCTGACTTGGCTTAGAAATAAACACTTTAGCAATT +ATCCCAACTATTTCTACTACCCATAACCCACGATCAACAGAAGCAGCTACAAAGTATTTT +TTAACACAAGCAGCAGCCTCCGCCTTAATTTTATTTTCAAGTACAATTAATGCTTGACAA +ACAGGAACCTGAGATATTACTAACACACTATCTACACCCGCACACATTTTATTGACCCTA +GCACTAGCAATAAAACTAGGACTTGCCCCAATACACTTCTGACTGCCAGAGGTATTACAA +GGCGTTACATTAGTTACTGCACTTATTATTTCTACCTGACAAAAACTCGCACCACTAACC +CTAATTTACTTAACAGTTAATAATCTATCAACAACAATTCTATTAGTTATGGGACTATTA +TCTGCCATTGTAGGCGGATGGGGCGGACTTAATCAAACACAAACTCGTAAAATCATAGCA +TATTCATCAATTGCTCACCTAGGCTGAATACTCTCTATTTCATCCATCATAGTTAATATT +ATACTTTTAAACTTANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +NNNNNNNNNNNNAAAACAATCCAAGACACAATAATGACATGAACACTCTCACCCATACTA +ACCTCTATAATAATAACTTTACTATTGTCATTAGGTGGTTTACCACCTTTAACCGGATTT +ATACCAAAATGGCTAATCTTACAAGGACTCGTTGCCCAAAACCTTACCTCAGTAGCCATT +ATATTNNCAATCGCCACCTTACTTAGCTTATTTTTTTACTTACGATTAGCTTATACAACA +GCCACTACACTTTCCCCAAAGCNATTACAAACAAAACTCAAATGACGATTAAAACCAATA +TTACCAACATTACTTGTGGCAGCCTCA-T-CTATAATANGCCACCTTTTTTCTTCCTATA +CCCCCCTATTTTTACTATA------AAAATTTAGGT-TAACTAAACCAAGGGCCTTTAAA +ACCCTAAATAAGGACACTCACCCCTTAATTTTTGATAAGACCTATAAATGACTTATTTAT +ATCTCCTGAATGCAANNNAACAACTTTCATTAAGCTAAAGCCTCTCTGAGA--AACGGNN +CCTTGANNNNNN-AAAAATTAGTTAACACCTAATTACCCTAACCCAC-GGGCTTCTACTC +GCTTCTCCCGTACGGAAG---------AA-CCCCNNGACCCCTTA-TGGGCTCTCCTNCA +AACTTGCATTTTGACGTAATTCACTTCGAN-GCT-----TTGATAAAGAAAGGAATTAAA +CCAATGTNTACAGGACTACAGCCTGCCGCCTTACACT-CGGCCACTTTACCTGTGTTTAT +TAATCGTTGA------------ +>A.lineatopus +ATGAGCCCTGCTATCTTTTCAATTGTCATATCCAGCCTAGCAACAGGCACTATTATTACC +ATAACAAGCCACCACTGACTATTAGCCTGAATAGGGTTAGAAATAAATACCCTAGCAATT +ATTCCAATTATTTCTATTACCCACAACCCCCGAGCCACAGAAGCCGCCACAAAGTACTTC +TTAACACAAGCAGCAGCCTCAGCCATAGTTTTATTTGCAAGCATAACTAATGCTTGACAA +ACAGGAACATGAGATATTACTAGCCTATCATATATGCCCTCACATATTTTATTAACTATA +GCATTAGCTATAAAACTAGGGCTTGCCCCAATGCACTTCTGACTACCAGAAGTGCTACAA +GGAACAGCCCTACCAACTTCACTTATTATTACTACTTGACAAAAACTTGCCCCAATGTCC +CTAATTTATATAACTATTAATAGTTTATCAACCATAATTCTAATNTCACTAGGCCTACTA +TCCTCAATTATTGGAGGATGGGGAGGACTAAACCAAACACAAACTCGAAAAATTATAGCA +TTTTCATCAATCGCCCACCTAGGATGAATAATTACTATTTTTTCACTCATAACAAATATT +ATAATCTTAAACCTAACAATTTACTTAATCATAACAACAGCAATATTTTGCTCACTAATC +TTATCAAAATCAAAAACAATACAAGATATTATGATAACTTGAACAATCTCACCCACCCTT +ACTACCCTAATAATAATTTTACTCCTCTCCCTGGGCGGCCTACCACCACTAACCGGATTT +ATACCAAAATGATTAATTCTAGAAGAATTGGTTGCCCAAAACCTTATCCCAACAGCTACT +ATTATAGCAATGTCGGCACTCCTCAGCCTATTTTTTTATTTACGACTTACCTATGTAACT +GNACTTACACTCTCCCCAAACACAGCACAAACAAAATTCAAATGACGACTTAAACCAAAC +TCACCAAAACTGTTCATAATAGCTTC--CTCCAGCAATATCAATATTTTTACTTCCCTAA +CCCCCCTTATATTACTATAAAA---ACTTAGGAT----AATTAAACCAAGGGCCTTCAAA +GCCCTAAACAAGGGCACTA-CCCCCTTAGTTTTTGTAAGACTTATGGACAACTACACCAT +ATCTTCTGAATGCAACTCAAACACTTTAATTAAGCTAAAGCCTCTTCCAAA-TAGGGGGG +CCTTGATCCCACAA-TAAACTAGTTAACAGCTAATCGCCCAAGCCAGCGGGCTTCTATTT +GCTTCTCCCGTACGGGAG---------AA-GCCCCGGAGCCTTTA-AGGGCTCCTTTTCA +AACTTGCATTTTGATGTGAATCACTTCGGG-GCT-----GTGATAAAGAAAGGAATTAAA +CCAATGTAAATAGGACTACAGCCTACCGCCTACCATT-CGGCCACTTTACCTGTGTTTAT +TAATCGTTGA------------ +>A.lineatus +ATGAGTCCAACAATTTTTATAATTATAGTGATAAGCCTAACAACCGGGACTATTATTACC +TTAACAAGCCACCACTGACTTATGGCCTGAATAGGCCTGGAAATAAACACCTTAGCAATT +ATTCCAATCATTTCCTACACCCATAACCCACGATCCACCGAAGCTGCCACAAAATATTTC +CTAACCCAAGCAGCAGCCTCCGCTACAATTTTATTTTCAAGTATAACTAATGCCTGACAA +ACAGGCACATGAGANNNNACCAATATATCTTACGAACCATCATGCATTCTATTAACAACA +GCACTAGCAATAAAACTAGGACTCGCCCCCATACACTTCTGACTACCAGAAGTCGTTCAA +GGAACAACACTAACTAATGCATTAATCATCACCACCTGACAAAAACTTGCACCAATAGCC +CTAATTATGCTTACCATTAACAACCTATCAACAACAATTTTACTACTAATAGGACTACTT +TCCGCAATCATTGGTGGATGGGGAGGACTAAACCAGACACAGACCCGAAAAATCATGGCC +TACTCATCAATCGCACACCTCGGATGAATAATTACCATTTCATCAATCATAACAAACATC +ATACTAATTAACCTACTTATTTACTTAATTATAACCACAGCAATGTTCTGCCTACTAATT +TCATCCAAATCAAAAACAATCCAAGATACAACAACAACATGAACACTATCACCAACACTA +ACTTCAATAGCATCAATTTTACTTTTATCCCTAGGAGGACTGCCACCATTAACCGGGTTT +GTTCCAAAATGATTAATCCTAGAAGAATTAGTAACCCAAAACCTAATCCCAACAGCTATT +ATTATAGTAATATCATCGTTACTTAGCTTATTCTTCTACCTACGACTAACATATACAATA +ACCATAACACTGTCCCCTAACACAACACAAACAACATTTAAATGACGATCTAACCCTAAC +TTTAACTCTTCATTAATTATAATTTC--ATCAACAATATCGATCTTTTTACTTCCAATAA +CCCCATTAATACTACTATAAAA---ACTTAGGTTA---ACACAAACCAAGGGCCTTCAAA +GCCCTAAATAAGGATGACC-TCCCCTTAGTTTTTGTAAGACTTATGA-AAACTACTTCAT +ATCTTCTGATTGCAACTCAAACACTTTTATTAAGCTAAAGCCTTCACCTGAACAGGAGGG +CCTCGATCCCACAA-AAATTAGTTAACAGCTAATTACCCAAACCAGCGGG-CTTCTATTC +GCTTCTCCCGTACGGGAG---------AAGCCCCGGAGCCCCTTA-AGGGCTCATTTTCA +AACTTGCATTTTGACGTGAATCACTTCGGG-ACT-----TTGATAAAGAAAGGAATTAAA +CCAATGTGAATAGGTCTACAGCCTACCGCCTCTCATT-CGGCCACTTTACCTGTGTCTAT +TAATCGTTA------------- +>A.loysiana +ATGAACCCAGTCGTTATTNNNATTCTTCTATCAAGCCTAGCAACTGGTACTATTATTACT +ATAACTAGCTATCACTGATTAATAGCCTGAGTTGGACTAGAAATAAACACATTAGCAATT +ATTCCAATCATTTCAACAACTCACCACCCACGAGCCACAGAAGCAGCCACAAAATACTTC +TTAACCCAAGCTGCAGCCTCCGCCCTAATCTTATTTTCAAGTACAATTAATGCTTGATAC +TCAGGCTCATGAAATATTACCCAAATACTAACCTTCCCATCACACATTCTCTTAGCCATA +GCACTGGCCATAAAATTAGGCCTAGCCCCACTTCACTTCTGACTACCAGAAGTTATTCAA +GGATCAACCATAATTACTGCATTTNTTATTACCACATGACAAAAACTAGCCCCTATGTCT +CTTATTTTCATAACCTCAAACAATATGCCCACAACAATCTTTCTGCTTATAGGACTTCTT +TCCTCATTCACCGGAGGGTGGGGCGGACTAAACCAAACACAGACCCGAAAAATTATAGCC +TACTCATCAATCGCCCATCTAGGATGAATCGCCTCCATTTCCTCAATTATAACAAACATT +ATAATTATTAACCTACTAATCTACCTAATTATAACGACCTCCATATTTTTAACCCTTATT +TTTTCTAACTCAAAAACAATCCAAAACACAGCAATTAATTGAACACTTTCTCCAACACTT +ACAACTATTATTCTACTTTCACTCCTATCACTAGGAGGACTACCACCCCTAACAGGATTT +ATACCAAAGTGACTAGTTATAGAAGAACTAATTTTACAAAACTTCTTTTTATTAGCCAGT +ATAATAGCCCTATCATCTCTACTCAGCCTATTTTTTTACCTCCGACTAACCTACACTACC +ACACTAACACTTTCCCCAAACACAACACAAACAAAATTTAAATGACGATTTTACCCTAAC +ACAAANACAACCCCAATAATAA--TCTCAGCTACAATCTCTATTTTCCTTCTACCACTAA +CCCCACTCATTTTATCATAAAN---ACTTA--GGATAAACA-AAACCAAGAGCCTTCAAA +GCTCAAAATAGGAGTGCAATCCCCCTAGTTTTTGT-AAGACCTGTGAAATACTATTACAC +ATCTTCTGAATGCAACTCAAACACTTTTATTAAGCTAAAGCCTC-CCTGAA-TAGGCGGG +CCTTGATCCCACGACAAATTAATTAACAGCTAATTACCCTAACCAGC-GGGCTTCTATTC +GCTTCTCCCGTACGGGAG---------AAGCCCCGGAGCCTTTT--ATGGCCCTTCTTCA +AACTTGCATTTTGACGTGAAAACAC-TACG-AGACT---CTGATAAAGAAAGGAATCAAA +CCAGTGTAAGTAGGTCTACAGCCTACCGCCTAACATT-CGGCCACTTTACCTGTGTTTAT +TTACCGTTGA------------ +>A.equestris2 +------------------------------------------------------------ +ATAACAAGTTATCATTGACTTATGGCCTGAATTGGACTAGAAATAAACACATTAGCAATT +ATTCCAATTANATTTACTATACACAACCCACGAGCCACAGAAGCTGCCACAAAGTACTTC +CTAACACAAGCATCAGCATCAGCCATAATTTTATTTTCAAGCACAATTAACGCCTGACAA +ACAGGGACCTGAGACATTACTAACCTCTCATCCGAACCAGCACACATTTTATTAACCATT +GCACTAGCAATAAAACTAGGCCTAGCCCCAATACACTTTTGACTACCAGAAGTACTACAA +GGGTCAACCATAACCACAGCACTAGTTATCTCTACTTGACAAAAACTGGCCCCAATAGCC +CTAATTTATCTGAACATTAATAATCTATCAACAGCTGTCCTAATCTTACTAGGACTCTTA +TCATCAATTATTGGGGGCTGAGGGGGACTTAATCAAACACAAACCCGAAAAATTATGGCA +TTTTCATCAATTGCACACCTAGGGTGAATAATTACTGTTTCATCTATTATAACTAATATC +ATGATTATAAACTTAATAATCTACTTAATTATAACTACAGCCCTATTTTACTCATTAATT +TTATCCAAATCAAAAACTATTCAAGATACAACAACAGCCTGAANACTATCACCCACCCTC +ACCACTATAATAATAATTTTACTTCTGTCCTTAGGCGGACTACCACCATTAACAGGATTC +ATGCCTAAATGACTTATTTTAGAAGAACTAATTATTCAAAACCTTATCCCAACAGCCACT +ACTTTAGCAGTATCCACACTACTAAGCCTATTTTTTTACNTACGGCTTGCCTACACAACA +ACACTCACCCTATCCCCAAACACTATTCAGACAAAATTTAAATGACGATTTAAATTAAAA +TTTACAACATTTTTTATAATGCCACT--TTCCACAATTTCAATTCTTCTCCTTCCTCTTA +CCCCGCTAATACTATTATAAAA---ACTTAGGATNN-NNNNNAAACCAAGGGCCTTCAAA +GCCCTAAATAAGGGNCCCA-CCCCCTTAGTTTTTGTAAGACTTATGGGTGTCTACTCCAT +ATCTCCTGAATGCAAATCAAACACTTTTATTAAGCTAAAGCCTTGACCAAA-TAGGTGGG +CCTTGATCCCNNAA-AAATTAGTTAACAGCTAATTACCCAAACCAGA-GGGCTTCCATTT +GCTTCTCCCGTACGGGAG---------AA-CCCCNGGAGCCTTTC-AGGGCTCATTTTCA +AACTTGCATTTTGATGTGAATCACTTCGGG-GCT-----G--ATAAAGAAAGGAATTAAA +CCAATGTAAATAGGACTACAGCCTACCGCCTACCATT-CGGCCACTTT------------ +---------------------- +>A.lucius +ATGAGCCCAACAATTTATATAATCACTTTATCAAGCCTAGCAACAGGGACTATTATCACC +ATAACAAGCTACCACTGACTAATAGCCTGAGTGGGGTTAGAAATCAACACACTATCAATT +ATTCCAATCATTTCAATAATACATCACCCACGATCCACAGAAGCTGCCACAAAATATTTC +TTAACCCAAGCAGCAGCCTCAGCTCTAATTTTATTTTCTAGTACAACACACGCATGATAT +TCAGGCACATGAGATATTACCCAAACATCAAATATTACCTCCAACATTCTTCTAACAATA +GCACTTACAATAAAATTGGGCCTAGCCCCATTACACTTTTGATTACCAGAAGTAGTTCAA +GGATCGACTATAAATACAGCCTTTATCATTATCACATGACAAAAACTAGCCCCAATATCA +CTTATTTACATAACTATAAACAACTTATCCACAACAATTTTATTAATCATAGGATTAATA +TCATCAATAATTGGAGGCTGAGGCGGCCTAAACCAAACACAAGTCCGAAAAATTATAGCC +TACTCATCAATTGCACATCTTGGATGAATAGCAACAATTTCAACAATTATAACTAATATT +TTAGTTTTAAACCTATTAATTTACCTAATTATAACAACATCTATATTTCTTTTATTAATT +TTAACCAATTCTAAAACAATTCAAGACATATCAACTACCTGAACTCTTTCACCAACCCTA +CTAATTATAATAATACTAACACTATTATCACTAGGCGGCCTACCCCCTCTAACAGGATTT +ATACCAAAATGATTAATCTTAGAAGAGCTAACAACCCAAAACTTAACCCCACTAGCTACC +CTTATAGCACTATCAGCCCTATTAAGCTTATTCTTTTACCTACGATTAGCTTACACCACA +GCCCTTACACTATCTCCAAACACACTACAAACAAAATTTAAATGACGATTTACCCAAAAT +ATAGCAACACCAACAACAATTA--TTACCACTACAATAACAATTTTTTTGCTCCCTTTAA +CACCTCTTATACTCTCATAAAA---ACTTA--GGAT-AATTCAAACCAAGGGCCTTCAAA +GCCCAAAATAAGG-GCTAATCCCCTTAGTTTTTGC-AAGACTCATGTAATATTAATACAC +ATCTCCTGAATGCAACTCAAACACTTTAATTAAGCTAGAGCCTC-TCTGAA-TAGGCGGG +CCTTGATCCCGCGACAAATCAATTAACAGCTAATTACCCAAACCAGC-GGGCTTCTATTC +TTCTCCCG--TACGGGAG---------AAGCCCCGGAGC-CTTTTA-GGGCTCTTCTTCA +AACTTGCAATTTGATGTAAGTCACTTCGGA-GCT-----GTGATAAAGAAAGGAGTTGAA +CCAATGTAAATAGGACTACAGCCTACCGCCTATCAGT-CGGCCACTTTACCTGTGTTTAT +TAATCGTTGA------------ +>A.marcanoi +ATGAGCCCAACAATTTTTTCAATTATGCTATCGAGTCTAGCAACAGGCACCATTATTACT +ATAACAAGCTTTCACTGACTAATGGCCTGAGTCGGCTTAGAAATTAATACGCTAGCTGCT +ATTCCAATTATTTCAATACAACACCACCCTCGATCAACAGAAGCAGCCACAAAATACTTT +TTAACACAAGCAACTGCATCCTCCTTAATTTTATTTTCAAGTATGATTAATGCTTGGCAT +ACAGGAACATGAGACATTACTCAAATTTCAACTACCCAATCATGCACCCTTCTTACAATA +GCAATCTCAATAAAGCTAGGCTTAGCCCCACTACACTTCTGACTACCAGAGGTTCTCCAA +GGTTCAACAATATTTACTGCTTTAATTATTATAACATGACAGAAACTAGCCCCCATATCC +CTACTTTTCCTCACTATAAACAACCTATCCACATCAATACTCTTAACAATTGGACTTGTA +TCAACAATAGTTGGCGGATGGGCAGGATTGAATCAGACACAAACACGAAAAATTATAGCA +TACTCATCAATTGCGCATCTTGGCTGAATAGCCCCTATTGCCTCAATTATAACAAACATT +CTTATTATAAATTTACTTATTTACTTACTTCTTACAACGGCCCTATTTTCCTCTCTAACA +CTATCTAAATCAAAGACAATTCAAGATACATCAACAACCTGAACAATATCACCATATTTA +ACTATTATAACCATACTTATACTTCTTTCATTAGGCGGACTACCGCCACTTACAGGATTC +ATCCCAAAATGATTAGTATTAGAAGAACTAGTTACTCAAAACCTACTCCCACTAGCTACT +ATTATAGCAATAGCAGCTCTCCTTAGTCTTTTCTTTTATTTACGCCTAACCTATGCCACA +GCACTAACACTTTCACCGAATACAATACAAACTAAATTTAAATGACGATTTAAACCTAAT +CACCCATCCGCAACAACAGCAC--TAACTTGTACAATCGCCATTTTTTTACTCCCAATAA +CCCCTCTTATTTTGCTATAAAA---ACTTA--GGAT-AA-ACAAACCAAGGGCCTTCAAA +GCCCGAAATAAGGGTGCAAGTCCCCTAGTTTTTGT-AAAACTTGTACTATACTATAGCAC +ATCTCTTGAATGCAACTCAAGCACTTTTATTAAGCTAAAGCCTC-CCTGAA-CAGACGGG +CCTCGATCCCGCGACAAATTAATTAACAGCTAACTGCCCAAACCAGC-GGGCTTCAATTC +GCTTCTCCCGTACGGGAG---------AAGCCCCGGAGCCTTTTA--GGGCTCATCTTTA +AATTTGCATTTTAACGTATAA-CACCTCGGGAC------TTTGATAAAGAAGGGGTTTAG +CCCTCATGGGTGGAGCTACAAGCCACCGCCTATTATT-CGGCCACTTTACCTGTGTTTTT +TACTCGTTGA------------ +>A.maynardi +ATGAGCCCACCATCNTATANAATTATTTTATCAAGCCTAGCAACCGGCACCATTATTACC +ATAACCAGCTATCACTGACTTATAGCCTGAGTCGGCCTAGAAATAAACACCTTAGCAATC +ATCCCAATCATCTCAACAATACATCACCCACGGTCTACAGAAGCAGCTACAAAATACTTC +TTAACCCAAGCTGCAGCCTCAGCTCTAATTTTATTTTCAAGCATAACAAATGCTTGAAGC +TCAGGCTCATGAGATATCACTCAAACATTAACCTCCCCCTCACATATTCTATTAACTATA +GCACTAGCTATAAAGCTCGGACTAGCCCCAGTACACTTCTGACTACCAGAGGTAATCCAA +GGCTCTTCCATAATTACAGCCTTCATTATCACCACATGACAAAAACTTGCCCCAATCTCA +CTTATCTTNATAACCATAAACAACCTATCTACTACAATCNNNTTATTAATAGGACTTCTA +TCTTCACTCATTGGAGGATGGGGAGGACTTAACCAAACACAAACCCGAAAAATCATAGCA +TACTCATCAATTGCACATCTAGGATGAATAGCAACAATTTCTTCAATCATAACAAATATT +CTCATTATGAACTTATTAGTCTATCTTATTATAACAACCTCAATATTTCTCTCTCTTATC +ATCCCTAAATCAAAAACAATCCAAAACACAACATCCACCTGAACACTATCCCCAACACTT +ACAATTATTATGATACTCTCACTCCTCTCCTTAGGAGGACTACCCCCACTAACAGGGTTT +GTACCAAAATGACTTATCATAGAAGAACTAATTTTGCAGGACTTCAACTTACTAGCCATC +ATAATAGCAATATCCTCCTTACTAAGCCTATTTTTTTACTTACGACTAACTTATGCAACC +ACACTTACACTATCTCCAAATACAACCCAAACAAAATTTAAATGACGATTTTACCCAAGC +ACACCAACCATCTTTCTACCTA--TTCCTGCTGCAATCTCCATCTTCCTACTCCCCCTAA +CCCCACTAATTTTGTTATAAAA---ACTTA--GGATCAACT-AAACCAAGAGCCTTCAAA +GCTCAAAATAGGGATGAAAACCCCCTAGTTTTTGT-AAGACTTGTGAAATATTATAACAC +ATCTCCTGAATGCAACTCAAGCACTTTAATTAAGCTAAAGCCTC-TCTGAA-CAGGCGGG +CCTTGATCCCACAAAAAATTAATTAACAGCTAATTACCCAAACCAGC-GGGCTTCTATTC +GCTTCTCCCGTAACGGGA---------GAAGCCCCGGAGCCTTT--AGGGCTCCTTNTCA +NACTTGCATTTTGACGTGAAACACT-TCG---GGGC---TTGATAAAGAAGGGAATTANN +CCCCTGTGAATAGGACTACAGCCTACTGCCTGACACT-CGGCCACTTTACCTGTG----- +---------------------- +>A.microtus +ATGAGCCCGATAACTGACACAATCATTATTTCCAGCCTTACAACCGGCACNNNNNNNACT +ATAACCAGCTACCATTGATTGGTAGCTTGACTTGGACTAGAGATCAACACATTGGCAATC +ATTCCAATCATCTCAATACAACACCACCCCCGATCCACAGAAGCTGCCACAAAATACTTC +CTCACACAAGCAGCAGCCTCTGCCATAGTCTTATTCGCAAGCACAACCAACGCTTGACAT +ACAGGAACATGAGACATTACCCAAATAACCACCCCTCATTCCAACACTCTTCTTACCATG +GCACTCTCAATAAAACTAGGATTAACCCCCGTACACTTCTGATTGCCAGAAGTACTACAA +GGATCAACCATAACTACAGCACTCATTATTACAACATGACAAAAACTAGCTCCCATATCC +CTAATTTATCTGACCATAAACAACCTCTCCCCAACAATCCTTATCACATTGGGGCTTCTA +TCTTCAATAGTAGGGGGGTGAGCGGGCCTAAACCAAACACAAACACGAAAAATTATAGCA +TACTCATCAATCGCTCACCTGGGATGAATAGCAGCAGTATCATCAATTATAACAAACATC +CTAACACTAAATTTATTTATCTACTTAATAATAACAACAGCCTTATTTTATTCACTAATT +TTATCAAAAACTAAAACAATCCAAGACACAACAACCTCTTGAGCAACCTCCCCCATACTA +ACAACTATCATAATGCTTACCCTTTTGTCATTAGGAGGCCTACCTCCACTTACAGGCTTT +ATACCAAAATGACTAATTCTAGAAGAACTAATGATACAAAACTTAGCCCCCCTAGCAATT +GTTATAGCANNNACATCACTACTAAGCCTTTTTTTCTACCTACGACTAACCTACACAACC +ACACTTACTCTATCTCCAAATACAGTACAAACAAAACACAAATGACGATTTAAACCTTCA +ACTACCATACTTCCACTAACCA--TCATAACACCCGCAACCCTACTTCTATTACCAATAA +CACCCCTAGTTATTTGCTAGAN---ACTTA--GGAT-AAACCTAAACCAAGAGCCTTCAA +AGCTCAANATATGAACTAGCCCTCATAGTTTCTGC-AAGACTTGTGAAACATTAACACAC +ATCTCTTGAATGCAACTCAAGCACTTTAATTAAGCTAAAGCCTC-TCTGAA-TAGGCGGG +CCTTGATCCCACGATATATTAATTAACAGCTAACTACCCAAACCAGC-GGGCTTCTATTC +GCTTCTCCCGTACGGGAG---------AAGCCCCGGAGCCTTTTTAGAGGCTCTTCTTCA +AATTTGCATTCTGACGTGGAACACCCCAGG-GCT-----GTGATAAAAAGAGGAATTAAA +CCCCTATTAATAGGACTACAGCCTACTGCCTTACATT-CGGCCACTTTACCTGTGTTTAT +TAATCGCTGA------------ +>A.occulatus +ATGAGCCCCAATAATCTACTTAATAGTTTAATTAGCTTATTTATANNNACAACACTAGTA +ACCACTAGCCACCACTGATTATTAGCGTGAGTTGGCTTGGAAATTAACACACTTGCAGCT +ATTCCACTTATCTCAACAAAACATCACCCCCGAGCTACAGAATCAGCCACAAAATACTTT +TTAATTCAAGCAGCAGCCTCAGCTACAATCTTATTTTCAAGTACCATTAATGCTTGACAC +ACAGGCTCATGAGACATTACCCAAATAACCACAAACCCATCCAATATTTTACTAACTATA +GCCCTTGCCATAAAACTAGGCCTAGCCCCAACACACTTCTGACTCCCAGAAGTACTTCAA +GGGTCAACTATAGACACTGCCCTAATTATTACAACATGACAAAAAATTGCCCCAATAACC +CTAATTTACCTCACCATAAACAACCTATACCCAAGCATTCTAATAACAATAGGCCTATTA +TCTACAATAATCGGGGGTTGAGGAGGCCTAAACCAAACACAAACCCGAAAAATTATAGCA +TACTCATCAATTGCACACTTGGGTTGAATGGCCGTAATTTCCACCCTAATAACAAACATC +TTTATTATAAACCTAATTATGTATCTAATTATAACAACAACCGCATTCTGGGCCCTAATC +TGCTTAGAATCTAAAACTGTAAAAGACACAACAACTGCCTGAACAACCTCACCAACCATC +ACTTTAACACTTCTTATTTCCCTCTTATCCCTAGGAGGCCTTCCCCCTCTCTCTGGATTC +TTACCAAAATGACTAATCCTAGAAGAACTTACAAACCAAAATCTACTACCATTAACCACC +ACTATAGCAGTCTCCTCCCTGCTAAGCCTGTTTTTTTACCTACGACTTACCTATAATACA +ACACTAACAATAGCCCCAAACACAATACAAACAAAATTCAAGTGACGATTTAAACCTAAT +AACCCACCCTTATTATTAACAG--CTACTGCCCCACTAACCACTCTACTACTCCCGCTTA +CACCTCTTATATTAATATAGAA---ACTTA--GATT-TAAACAAACCAAGAGCCTTCAAA +GCTCAAAATATGGGTGTAACCCCCATAGTTTCTGT-AACACTTACAAAACTTTAATTTAC +ATCTTTTGATTGCAACTCAAACACTTTTATTAAGCTAAAGCCTC-CTTGAA-TAGTCGGG +CTTCGATCCCACGACAAATTAATTAACAGCTAACTGCCCAAACCAGC-GGGCTTCTACTC +GCTTCTCCCGTACGGGAG---------AAGCCCCGGAGCCTTTTG--GGGCTCCTTTTCA +AACTTGCATTTTGACGTGAAAACACTTCGA-GACT----TTAATAAGNAAAGGAATCAAA +CCCCNGNAAATAGGACTACAGCCTACCGCCTAACACT-CGGCCACCTTACCTGTGTCTAT +TAATCGTTA------------- +>A.olssoni +ATGAACCCCACCATCTCCATAAATTATCTATCAAGCCTAGCAACAGGAACAATTATTACT +ATGACCAGCTTTCATTGATTAATAGCATGAATTGGATTAGAAGTCAACACACTAGCAATT +ATTCCAATCATCTCAGCCCCTCACCACCCACGATCAACAGAAGCTGCAACAAAATACTTT +CTCACACAAGCAGCTGCCTCCGCTATAATTCTATTTGCCAGTATAATTAACGCCTGACAA +ACAGGCACATGAGACATTACTCAACTGTCAACCACCCCTGCCCACACCCTCCTAACTATA +GCACTAGCCATAAAACTAGGACTTGCCCCACTTCACTTCTGATTACCAGAAGTTATTCAA +GGTGCCACCCTTCCCACTGCTTTCATCATTGTAACATGGCAAAAACTTGCTCCTATATCA +CTTATTTTTTTAACCATAAACAACCTAAACCCAACAATTTTACTTCTCCTTGGACTTCTA +TCTTCTACAGTAGGAGGATGAGCCGGATTAAACCAAACACAAACCCGAAAAATTATAGCC +TATTCATCAATTTCACACTTAGGCTGAATGGCCGCAATCTCCTCTATTATAACTAATATT +CTTATTATAAACCTAACCCTTTATTTAATTATAACAACAACCATGTTTTATACCCTAATT +TTAACTAAATCCAAAACAATCCAAGACTCAACCCTCACCTGATCACTTTCACCAGTACTA +ACAATTATTATAATACTAACACTCCTCTCACTAGGAGGACTACCTCCCCTAACCGGATTT +TTACCAAAATGAATAATCCTAGAAGAACTTACTACCCAAAACCTAACCCCACTGGCCACC +ATAATAGCCATTTCAGCTTTACTAAGCCTATTTTTTTACCTACGCCTAACTTATACAACA +ACCTTAACACTCTCCCCAAACACAATACAAACAAAACTCAAATGGCGATTTAAATTAAAT +AAACCAACAACACTTATACTAA--TAACTTCTACAATTACCACCTTCTTACTCCCCCTAT +CACCACTCATTTTAATATAAAA---ACTTA--GGTT-TAACCAAACCAAGAGCCTTCAAA +GCTCAAAATAAGGGTCAAACACCCTTAGTTTTTGT-AAGACTTGTGAAATACTAATACAC +ATCTTCTGAATGCAACTCAAACACTTTAATTAAGCTAAAGCCTC-TCTGAA-TAGGCGGG +CCTCGATCCCGCGACAAATTAATTAACAGCTAACTACCCAAACCAGC-GGGCTTCTACTC +GCTTCTCCCGTACGGGAG---------AAGCCCCGGAGCCG-TTTG-GGGCTCTTCTTCA +AACTTGCATTTTGATGTGAAACACTTCGGG-ACT-----TTGATAAAGAAAGGAATTAAA +CCAATGTTAATAGGACTACAGCCTACTGCCTATCATT-CGGCCACTTTACCTGTGTTCAT +TAAT------------------ +>A.ophiolepis +ATGAGCCCAACAATCTTTATAATTATTTTATCAAGTCTTGCAACTGGTACAATTATTACT +ATAACTAGTTATCACTGACTATTAGCCTGAATCGGCCTAGAAATTAATACCTTATCAATT +ATCCCACTTATTTCAACAACCCACCATCCACGAGCCACAGAAGCCGCTACCAAGTATTTT +CTTACACAAGCAGCAGCTTCGGCCATAATTTTGTTTTCTAGTATAACTAATGCATGAGAG +ACAGGCACATGAGATATTACCCAGCTATCATCAACACCATCACACATCTTTCTTACAATA +GCATTAGCAATAAAACTAGGTCTAGTCCCAGTACACTTTTGATTACCAGAAGTCCTACAA +GGGTCAACCCTAACAACTGCACTTATTATTACTACATGACAAAAACTTGCACCAATATCT +TTAGTCTACTTAACCATTAATTCCCTTTCAACAACAGTTTTATTATTAATGGGACTCATA +TCCACTGTTATTGCTGGATGAGCAGGACTTAACCAAACACAAACACGAAAAATTATAGCA +TACTCATCAATTGCTCACCTTGGATGAATAGTCGCTGTTTCTTCTATTATAATAAACCTC +ATAGTAATAAATCTAATTATTTATTTAATCATGACCTCTGCCCTATTTCAAGCACTAATT +GTCTCTCAATCAAAAACAATTCAAGATACAACAATGACATGAGCACTATCACCAACCATT +TCCATTTTAACCATAGCACTTCTACTATCACTTGGAGGCTTACCACCACTAACAGGATTT +ATCCCTAAATGATTAATTCTAGAAGAACTAGTAACCCAAAACCTCATTCCAACAGCAACA +ATAATAGCCCTATCTGCCTTACTAAGCCTTTTCTTTTACTTACGCCTAACTTATACCACA +ACACTAACACTATCACCAAACACAACTACTACAAAATTTAAATGACGACTTAAACCAAAT +CAACCTACACAACCCATACTTATTGC--TACCACAACCTCTATCCTTCTACTTCCACTAG +CACCACTCATGCTTATATAAAATAAAAACTTAGGAT-AACCTAAACCAAGGGCCTTCAAA +GCCCTAAATAAGGGATAGA-CCCCCTTAGTTTTTGTAAGACTTGTGAAATATTAATTCAC +ATCTCATGAATGCAACTCAAGCACTTTTATTAAGCTAAAGCCTTTAGGAAT--GGGCGGG +CCTTGATCCCGCAAAAAATTAATTAACAGCTAATTACCCAAACCAG-CGGGCTTCCATTC +GCTTCTCCCGTACGGGAG---------AA-GCCCCGGAGCCTTTT-AGGGCTCCTCTTCA +AACTTGCATTTTGATGTGGGTCACTTCGGG-GCT-----TTGATAAAGAAAGGAATTGAA +CCAATGTTAGTAGGACTACAGCCTACCGCCTATTACT-CAGCCACTTTACCTGTGTCTAT +TAATCGTTGA------------ +>A.paternus +ATGAGCCCATTTATTCTTACAATTATTTTTTCAAGCTTAGCAACAGGCACAATTATTACT +ATAACCAGCTACCACTGACTTATGGCCTGAATTGGATTAGAGATAAACACACTAGCAATT +ATTCCCATTATCTCAACAACACATCACCCACGAGCCACAGAAGCTTCCACAAAATATTTT +CTTACACAAGCTGCAGCCTCTGCCTTAATTTTATTTTCTAGTATAACCAATGCATGACAT +ACGGGATCTTGAGACATTACTCAAATATTATTTTCTCCATCAAACACCATGCTAACCATA +GCCCTAGCCATAAAACTAGGGTTAGCTCCACTTCACTTCTGGCTACCAGAAGTAATTCAA +GGATCAACAATACTAACTGCCTTTATTATTACTACATGACAAAAACTAGCCCCAATATCT +CTAATTTTTTTAACTATAAATAGTATGTCCACAACAATTCTTCTACTAATGGGAGTCTTA +TCCTCACTTGTTGGTGGTTGAGGGGGGTTGAACCAAACACAGACCCGAAAAATCATAGCA +TACTCATCAATTGCCCACCTCGGATGAATAGCTACAATTTCATCTATTATAACAAACATC +TTAATTTTAAACCTAACTATTTACCTAATTATAACAACTTCCATATTTTTTTCCCTAATT +ATTTCTAAGTCAAAAACAATCCAAAACACAGCGGAGACCTGAACATTTTCACCCATACTA +ACGATTATAATAATACTTTCACTTCTTTCACTAGGGGGACTACCACCACTAACTGGATTT +GTACCAAAGTGGCTAGTTATAGAAGAATTAATTTTACAAAATTTTAATCTTCCAGCTATT +TTAATAGCTATATCTTCCCTACTAAGCCTATTTTTTTACCTTCGAATTTCCTACACAGCC +ACACTCTCACTATCACCTAATACAACTCAAATAAAATTCAAATGACGATTTTATCCAAAA +ACTACAACCACCTTAACCTCTT--CCACCACAACAATAACAATCTTCCTCCTACCACTTA +CACCCATAATTTTATTATAAAA---ACTTA--GGATCAAAT-AAACCAAGGGCCTTCAAA +GCCCAAAATATGGGTTAAACACCCATAGTTTTTGT-AAGACTTGTGAAACACTATTACAC +ATCTCCTGAATGCAACTCAAGCACTTTTATTAAGCTAAAGCCTC-ACTGAA-TAGGCGGG +CTTTGATCCCACGACAAATTAATTAACAGCTAATTACCCAAACCAGC-GGGCTTCTATTC +GCTTCTCCCGTCGGTGAAGAAACGGGAGAAGCCCCGGAGCCTTT--AGGGCTCTTCTTCA +AACTTGCATTTTGATGTGGAACAC--TTCG-GGACT---TTGATAAAGAAAGGAATTAAA +CCAATGTAAATAGGACTACAGCCTACTGCCTAACACT-CGGCTACTTTACCTGTGTTTAT +TAACCGTTGATTCTTCTCAACC +>A.luciae +------------------------------------------------------------ +-----CAGCCACCATTGATTAATAGCTTGAATTGGATTAGAAATCAACACACTAGCCATT +ATTCCTATTATCTCAATACAACATCACCCACGAACTACAGAAGCTGCCACAAAATACTTT +TTAACCCAAGCAGCAGCATCTGCCATAATCCTTTTTGCAAGCACAACAAACGCTTGATTT +ACAGGAACATGAAATATTACCCAAATATCAACTACCCCCTCCCATATCTTACTAACTATA +GCACTAGCAATAAAACTAGGGCTAGCCCCAACACACTTCTGACTCCCAGAAGTACTACAA +GGCTCAACCATAACCACCGCCCTAATCATTACCACATGACAAAAACTTGCCCCCATATCC +CTAATTTACATAACCATAAACAACCTATCACCACTAATACTACTTCTACTAGGACTTATA +TCCTCAATCATTGGAGGCTGGGGCGGATTAAATCAAACACAAACACGAAAAATCATAGCA +TATTCATCCATCGCCCACCTAGGCTGAATAGCAGCCATTACATCAATTATAACAAATATT +TTAATCTTAAACTTACTAATTTACCTTGTCATAACAATAACAATATTTTATTCCCTTATT +TTACTAAAATTAAAAACAATCCAAGATACATCCACCTCCTGATCAACCTCCCCCACATTA +ACCACTACTACAATACTAACCCTTCTCTCACTGGGGGGCCTTCCCCCACTCACTGGATTT +ATGCCAAAATGACTTATTTTAGAAGAGCTAACCATACAAAACCTAGTTATACTAGCAACT +ATAATAGCACTAGCATCCTTACTAAGCCTATTCTTCTACTTACGCCTAAGCTACACAACA +ACCCTTACATTATCCCCAAATACCATCCAAACAAAACATAAATGACGATTTAAATCACCA +ACCACCACACACCCACTAATTA--CACTAATCCCTATAACTACCCTTATACTACCACTAA +CACCAACAATACTATCATAGAA---ACTTA--GGAC-TAACCAAACCAAGAGCCTTCAAA +GCCCTAAACATGAGTCTAACCCTCATAGTTTCTGT-AAGACTTGTGAGATACTAACCCAC +ATCTAATGAATGCAACTCAAACACTTTAATTAAGCTAAAGCCTT-CCTGAG-TAAGCGGG +CCTTGATCCCGCGATATATTAATTAACAGCTAACTACCCAAACCAGC-GGGCTTCTACTC +GCTTCTCCCGTACGGGAG---------AAGCCCCGGAGCCTTTTA--GGGCTCTTCTTTA +AACTTGCATTTTAATGTGTAACACCCCGGG-ACCTCATGGTGGTAAAGAGAGGAATTAAA +CCCCTTTAAATAGGACTACAGCCTACCACCTAGCATT-C--------------------- +---------------------- +>A.paternus2 +ATGAGCCCATTTATTTTTACAATTATTTTTTCAAGCTTAGCAACAGGCACAATTATTACT +ATAACCAGCTACCACTGACTTATAGCCTGAGTTGGATTAGAAATAAACACACTAGCAATT +ATTCCCATTATCTCAACAACACATCACCCACGAGCCACAGAAGCTTCCACAAAATATTTT +CTTACACAAGCTGCAGCCTCTGCCTTAATTTTATTTTCTAGTATAACCAATGCATGACAT +ACGGGATCTTGAGACATTACTCAAATATTATTTACTCCATCAAACACCATGCTAACCATA +GCCCTAGCCATAAAACTAGGGTTAGCTCCACTTCACTTCTGGCTACCAGAAGTAATTCAA +GGATCAACAACACTAACCGCCTTTATTATTACTACATGACAAAAACTAGCCCCAATATCT +CTAATTTTTTTAACTATAAATAGTATGTCCACAACAATTCTTCTACTAATGGGAGTCTTA +TCCTCACTTGTTGGTGGTTGAGGGGGGTTAAACCAAACACAGACCCGAAAAATCATAGCA +TACTCATCAATTGCTCACCTCGGATGAATAGCTACAATTTCATCTATTATAACAAACATC +TTAATTTTAAACCTAACTATTTACCTAATTATAACAACTTCAATATTTTTTTCCCTAATT +ATTTCTAAGTCAAAAACAATCCAAAACACAGCAGAGACCTGAACATTTTCACCCATACTA +ACGATTATAATGATACTTTCACTTCTTTCACTAGGGGGACTACCACCACTAACTGGATTT +ATACCAAAATGGCTAGTTATAGAAGAATTAATTTTACAAAATTTTAATCTTCCAGCTATT +TTAATAGCTATATCTTCCCTACTAAGCCTATTTTTCTACCTTCGAATTACCTACACAGCC +ACACTCTCACTATCACCTAATACAACTCAAATAAAATTCAAATGACGATTTTACCCAAAA +ACTACAACCACCTTAACCTCTT--CCACCACAACAATAACAATCTTCCTCCTACCACTTA +CACCCATAATTTTATTATAAAA---ACTTA--GGATTAAAT-AAACCAAGGGCCTTCAAA +GCCCAAAATATGGGTTAAACACCCATAGTTTTTGT-AAGACTTGTGAAANACTATTACAC +ATCTCCTGAATGCAACTCAAGCACTTTTATTAAGCTAAAGCCTC-ACTGAA-TAGGCGGG +CTTTGATCCCACGACAAATTAATTAACAGCTAATTACCCAAACCAGC-GGGCTTCTATTC +GCTTCTCCCGTAC----------GGGAGAAGCCCCGGAGCCTTT--AGGGCTCTTCTTCA +AACTTGCATTTTGATGTGGAACAC--TTCG-GGACT---TTGATAAATAAAGGAATTANA +CCAATGTAAATAGGACTACAGCCTACTGCCTAACACT-CGGCTACTTTACCCGTGTTTAT +TAACCGTTAT------------ +>A.pumilis +ATGAACAGCCCAACCATTTTTTTTCTACTATCAAGCCTTCAACCCGGCACCACTATTACC +ATAACCAGCCATCACTGACTTATGGCCTGAGTAGGGCTAGAAATAAACACACTAGCTATT +ATCCCAATTATTTCAACAATACACCACCCACGAGCCACAGAAGCATCTACAAAATACTTC +TTAACCCAAGCTGCAGCTTCCGCACTAATCTTATTCTCAAGCATAATTAGTGCCTGATAT +ACAGGCTCATGAAATATTACCCAAACATTAACCTATCCATCACACATCTTACTGACCATG +GCACTAGCCATAAAACTTGGCCTCGCCCCACTACACTTCTGACTACCGGAAGTAATTCAA +GGATCAACCATACCCACAGCCTTCATTATTACCACATGACAAAAACTAGCCCCTATGTCA +CTTATCTTTTTAACCACAAACAATATATCCACAACAATTTTTTTAGCCTTAGGACTTTTA +TCCTCATTAATTGGAGGATGAGGAGGCCTAAACCAAACACAAACCCGAAAAATTATAGCC +TATTCATCAATTGCCCATTTAGGATGAATAGCCTGTATCTCTTCAATTATAACAAACATT +TTAATTCTAAACCTATTAATTTACCTAATTATAACAAGTTCAATATTTATAACCCTTATT +TTATCTAAATCTAAAACCATTAAAGATACAACAACCACCTGAACACTCTCCCCCACACTT +ACAGTAATTATAATACTCTCCCTCCTTTCATTAGGGGGCCTCCCACCACTAACAGGATTT +TTACCTAAATGACTAATTTTAGAAGAATTAATTTTACAAGACTTTCTCTTATTAGCCACA +ATAATAGCCTTAACATCACTATTTAGCCTGTTCTTCTACTTACGACTAACTTATACAACC +CTTCTTACACTCTCACCAAACACAACCCAAACAAAATTTAAATGACGATTTTTCCCTAAT +ACAAAAACAACCCCCACCATAA--TTTTCCTAACAATCTCCACCCTCTTACTTCCCTTAA +CCCCCCTCATCTTACTTTAAAN---ACTTA--GGATA-ACT-AAACCATGAACCTTCAAA +GCTCAAAATAAGGGTGTAAACCCCTTAGTTTTTGT-AAGACTTGTGAAATACTAATTCAC +ATCTCCTGAATGCAACTCAAACACTTTTATTAAGCTAAAGCCTC-CCTGAA-TAGGCGAG +CCTTGATCTCACAACAAATTAATTAACAGCTAATTACCCAAACCAGC-GGGCTTCTATTC +TCTTCTCCCGTACGGGAG---------AAGCCCCGGAGCCTT-T--ATGGCTCATCTTCA +AACTTGCAATTTGACGTAAAATCAC-TACA-GAGCT---TTGATAAAGAAAGGAATTAAA +CCAATGTAAGTAGGACTACAGCCTACCACCTAGCACT-CGGCCACTTTACCTGTGTTTAT +TAATCGTTGA------------ +>A.richardi +ATGAGCCCTGCAATATACACAATCATCTTATCCAGCCTTGCAACAGGTACTATTATTACT +ATGTCAAGTTACCACTGATTAATAGCTTGANTTGGCTTAGAAATTAATACACTAGCAATT +ATTCCAATTATTTCTATACAACACCACCCACGGTCTACAGAAGCCGCTACAAAATACTTC +TTAATCCAAGCAGCAGCTTCAGCCATAATTTTNTTTGCAAGCACAACAAATGCCTGACAT +ACAGGCACATGAAATATTATTCANATATCTACTACCCCTTCACACATTATATTAACCCTA +GCACTATCAATAAATTTGGGACTAGCTCCAATACTCTTTTGCGTTCCAGAAGTCTTACAA +GGATCAACCATATCTNCTGCCTTTNNNNNNACAACATGACAAAAACTTGCCCCAATATCC +TTAATTTTAATAACTGCAAACAACCTATCCCCATTAATTTTACTTCTACTTGGACTCACT +TCCTCAATGATAGGAGGATGAGGAGGACTAAATCAAACACAAATACGAAAAATCATAGCA +TTTTCATCAATTGCACACCTAGGATGAATAATAACAGNAGCATCAATCACAACAAATATT +ATAANNNNAAANNNNCTAATTTATTTAATTATAACCTCCACTATATTTTTATCCCTAATT +ATACTAAAAATTAACACAATTCAAGACTCCTCAACCTCATGAACAACCTCTCCCATACTA +ACTATTATAATAATATTAACCCTATTATCACTAGGAGGGCTCCCTCCTCTTACTGGGTTT +ATACCAAAATGACTCATTCTAGAAGAACTGACAACACAAAACCTAACCGTATTAGCAACT +ATAATAGCACTAGCTTCACTACTAAGCCTATTCTTTTACTTACGACTTAGTTATACTACT +ANTCTTACTCTATCCCCAAATACAACTCANANAANACATAAATGACGGTTCCAACCATTA +ACAACTACATCCCCACTAATAA--TTTTAACCCCTATAACCATTCTATTACTACCAATAA +CACCAATAATATTATTATAGAA---ACTTA--AGAT-TAA-TAAACTAAGGACCTTCAAA +GCCCTAAATATGAGTTCAACTCTCATAGTTTCTGT-AAGACTTGTGAAATATTAATTCAC +ATCCTCTGAATGCAACTCAACTACTTTAATTAAGCTAAAGCCTT-TCTAAG-TAGGCGGG +CTTTGATCCCACGACAAATTAATTAACAGCTAACTACCCAAACCAGC-GGGCTTCTACTC +GCTTCTCCCGTACGGGAG---------AAGCCCCGGAGCCTTTTA--GGGCTCATCTTTA +AATTTGCATTTTAATGTGGAACACTTCAGG-ACTA----TNGATAAAAAAAGGAATTGAA +CCTCTATAAATAGGACTACAGCCTACTGCCTAACACT-CGGCCATTTTACCT-------- +---------------------- +>A.sagrei +ATGAGCCCAACAATCTTTATAATTATCATACTAAGTCTTGCAACTGGTACAATTATTACT +ACTACTAGCCACCACTGACTATTAGCCTGAATCGGCCTAGAAATTAATACCCTCTCAATT +ATTCCAATTATTTCAATAACCCACCACCCACGATCCACAGAAGCCGCTACCAAGTACTTT +CTGACACAAGCAGCAGCCTCCGCCCTAATTTTATTTTCCAGTATAACTAATGCATGAGAA +ACAGGTACATGAGACATTACACAACTCTCATCAACACCATCACACATCTTATTAACAGTA +GCACTAGCAATAAAACTAGGCCTCGTACCAATACACTTTTGACTGCCAGAAGTACTACAA +GGGTCAAGCCTAATAACTGCACTTATTATTACTACATGACAAAAAATCGCACCTATAACT +TTAATTTATCTAACAATTAACTCCCTCTCAACAACAATTCTGTTATTTATGGGACTTACA +TCCTCTATCATTGCAGGATGAGCTGGACTTAATCAAACACAAACACGGAAAATCATGGCA +TACTCATCAATTGCTCACCTAGGATGAATAATTGCTATTTCCTCTATTATGACGCATCTT +ATAGTAATAAACCTAATAATTTACTTAATTATAACCTCAGCCCTATTCCAAGCACTAATT +TGCTCTAAATCAAAAACAATCCAAGACACAACAACAACCTGAGCCCTATCACCAGTTATT +GCTACCATAACCATAGCACTATTATTATCATTAGGAGGCCTTCCACCATTAACTGGTTTT +ATACCAAAATGACTAATTCTAGAAGAACTAGTAACCCAAAATCTCATTCCAATAGCAACA +GTAATAGCCCTGTCCGCTCTACTAAGCNNNTTTTTTTATCTACGCCTAACATATACTACA +ACACTAACACTTTCCCCAAACACAACCGCTACAAAATTTAAATGGCGACTTAAACTAAAC +CAACCTACACCACTAATGCTAATTAC--TGCTACAACCTCTATTCTTTTACTTCCACTAA +CACCACTAATACTTCTATA-----AAAACTTAGGAT-AACCTAAACCAAGGGCCTTCAAA +GCCCTAAATAAG--CTAGA-CGCCCTTAGTTTTTGTAAGACTTGTGAAAAACTAATTCAC +ATCTCATGAATGCAACTCAAGCACTTTTATTAAGCTAAAGCCTT-ATGAAT--GAGCGGG +CCTCGATCCCACAAAAAATTAATTAACAGCTAATTACCCAAACCAG-CGGGCTTCCATTC +GCTTCTCCCGTACGGGAG---------AA-NNNNCGGAGCCTTTT-AGGGCTCCTTTTCA +AACTTGCATTTTGACGTGGGTCACTTCGGG-GCT-----TTGATAAAGAAAGGAATTGAA +CCAATGTTAGTAGGACTACAGCCTACCGCCTATCACT-CGGCCACTTTACCTGTGTCTAT +TAATCGTTGA------------ +>A.sheplani +ATGAGCCCAACCATCTACACAATTATTTTATCAAGCCTAGCAACCGGCACTATTATTACA +ATATCTAGTTATCACTGACTAATGGCCTGAATCGGCCTAGAGATTAATACACTAGCAATA +ATCCCGATCATCTCAACAACACATCACCCACGATCTACAGAAGCAGCCACAAAATATTTT +TTAATTCAAGCTGCAGCCTCCGCCCTAATTTTGTTCTCAAGTATAACTAACGCCTGATAT +ACAGGCTCCTGGGACATTACCCAAATAGTAAACAAGCCCTCAAATATTGCACTAACAGTA +GCACTGGCCATAAAACTAGGACTGGCCCCACTTCACTTTTGATTACCAGAGGTAGTTCAA +GGATCAACCACAACCACAGCACTTATTATTATAACCTGACAAAAACTAGCCCCCATATCA +CTGATTTTTCTTACCATAAATAACTTATCTACAACAGCCCTTCTACTGATAGGACTTCTG +TCTTCAATAATTGGGGGCTGAGGGGGCTTAAACCAAACACAAGTACGAAAAATTATAGCA +TACTCATCAATTGCCCACCTAGGGTGGATAGCCTCCGTTTCCTCAATTATAACAAACCTT +TTAATCATAAACCTGGTAATTTATATTATTGTAACAACATCCACCTTCTTCCCCCTAATT +ACTTCCAAATCTAAGACAATTCAAAATATAACTTCAACTTGAACACTCTCCCCCACACTA +ATTGTTGTTATAATATTATCCCTCCTGTCCTTAGGGGGCCTGCCCCCTTTAACTGGGTTT +ATTCCAAAGTGACTTATTATGGAAGAACTAATTCTACAAAACTTTAACCTATTTACCACT +ATAATAGCCCTGTCATCTTTATTAGGCCTCTTCTTTTACCTACGAATAGTCTACTTAACT +ACACTTACACTTTCCCCAAACGTGGCCCAAACAAAATTCAAATGACGATTTTATCCAAAT +ACGACAACCAACTTTTTCTCAG--CATTTTTCTTAATTTCTACCCTTCTTCTTCCCCTCA +CACCACTAATTTTAATATAAAA---ACTTA--GGATAAAT--AAACCAAGAGCCTTCAAA +GCTCAAAACATGGGTAAAACCCCCATAGTTTTTGT-AAGACCTGT-ACATACTATTATAC +ATCTCCTGAATGCAACTCAAGCACTTTAATTAAGCTAAAGCCTC-TCTAAG-TAGGCGGG +CTTTGATCCCGCGACAAATTAATTAACAGCTAACTACCCAAACCAGC-GGGCTTCTACTT +GCTTCTCCCGTACGGGA----------GAAGCCCCGGAGCCTTT--ATGGCTCTTTTTCA +AACTTGCATTTTGACGAGAAACCAC-TACA-GGGCT---CTGATAAAGAAAGGAGTTAAA +CCAATGTTAGCAGGACTACAGCCTGCCGCCTTACACT-CGGCCACTTTACCTGTGTTTAT +TACCCGTTGA------------ +>A.strahmi +ATGAGCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNACT +ATAACAAGCTACCACTGACTCATAGCCTGAATTGGACTAGAAATTAACACCTTAGCCATT +ATTCCAATTATTTCTATACAACACAACCCACGATCAACGGAAGCCGCAACAAAATACTTC +CTAACTCAAGCAGCTGCATCCTCCCTAATTTTATTCGCAAGCCTATTTAACGCCTGACAA +GTAGGCACATGAGATATTACACAACTATCAACTACACAAGCACACATTCTTTTGACTATG +GCCCTATCAATAAAACTAGGCCTAGCCCCACTACACTTTTGATTACCAGAAGTAATTCAA +GGATCTACTATATTTACTGCCTTAATTATTACAACATGACAAAAACTAGCCCCAATATCT +CTTATTTTCCTCACCGCAAACAACTTATCAACGACAATTCTTTTAACAATAGGACTTCTG +TCTGCAATAATCGGCGGATGAGCAGGACTTAACCAAACACAAACCCGAAAAATTATAGCA +TACTCATCAATTGCTCACCTTGGATGAATATCCACAATTGCCTCAATCTATACAAACATT +TTTATTATAAACTTATTTATTTACTTACTACTAACAACAGCCATATTTTACTCCCTAATT +TTTTCAAAATCAAAAACAATTCAAGATACATCAGTCACTTGAACAACTTCCCCATATTTA +ATGATTTTAACAACATTAACATTACTTTCATTAGGTGGACTTCCTCCCCTAACAGGATTT +ATCCCAAAATGACTCGTACTAGAAGAACTAACAACTCAAAATCTAATCCCTTTAGCCACT +GTCATAGTAATAGCCGCTCTCCTCAGCCTATTTTTTTACCTACGCTTAGCCTATACTACA +ANACTTACACTTTCCCCAAACACAANACAAACTAAATTTAAATGACGATTTAAACCTAAC +ATTAAAACCCCCCTAATAACTG--CAACCCTTGTGGTTTCAATATTTTTACTTCCAATAT +CCCCAATACTTCTACTTTAAAG---ACTTA--GGAT-AATTAAAACCAAGAGCCTTCAAA +GCTCAAAATAAGGGTGCAACCCCCTTAGTTTTTGT-AAGACTTGCACCATACTATAATGC +ATCTCCTGAATGCAACTCAAGCGATTTAATTAAACTAAAGCTTT-ACTGAA-CAGGCGGG +CCTCGATCCCACGATAAATTAATTAACAGCTAATTACCCAAACCAGC-GGGCTTCAATTC +GCTTCTCCCGTACGGGAG---------AAGCCCCGGAGCCTTTTA--GGGCTCATCTTTA +AACTTGCATTTTAATGTAGTAACACTTCGGGGC------TTTGGTACAGAAAGGAATTAA +CCAATGTGAGTAGGACTACAGCCTACCGCCTGACATT-CGGCCACTATACCTGTGTACCT +TAATCGTTGA------------ +>A.stratulus +ATGAGCCCTATAATTTACACAATCATTTTGTCAAGCCTAGCAACAGGGACAATTATTACC +ATAACCAGCTACCACTGATTAATAGCTTGAATAGGCCTAGAACTTAATACTCTAGCAATT +ATTCCATTATCTCATACAACACACAACCCACGATCTACAGAAGCCGCAACAAAATACTTC +TTAACACAAGCAGCAGCATCTGCTATAATCTTGTTTTCCAGCATAACCAACGCCTGCTTT +ACGGGCATATGAGACATTACTCAAATATCTTATTTACCATCTAATATTCTTCTAACCATG +GCACTAGCCATAAAACTCGGCCTAGCACCAGTGCACTTCTGATTTCCAGAAGTCTTACAA +GGCTCAACCCTACTTACCGCTTTTATTATTTCAACATGACAAAAACTGGCCCCAATATCA +CTAATTTTTTTGACAATAAGCAATCTTCCACCTATAATTTTATTAACTTTAGGACTTGTT +TCCTCTATTATTGGTGGGTGAGGCGGACTTAACCAAACACAAACCCGAAAAATCATAGCA +TACTCATCAATCGCACACCTAGGATGAATAGCTGTAATTTCCTCAATTATAACAAACATT +ATAATTATAAACCTTTTAGTTTATTTAATAATAACAACAGCCCTATTTTTAGCCCTAATT +TTATCTAAATCAAAAACAATTCAAGATACAACAAACACTTGAACAATATCCCCCACTTTA +ACTATTATTATGATACTTCTACTCCTCTCACTTGGTGGCCTGCCCCCCTTAACAGGGTTT +TTACCGAAATGATTAATTTTAGAAGAACTAATAACCCAAAACCTTATTCCACTAGCAACA +CTTATAGCACTTACAGCCCTTCTTAGCTTATTCTTTTATCTTCGCCTAGCTTACACAACA +ACCTTAACACTTTCTCCAAACACACTACAAATAAAGTTTAAATGACGATTTAAACCAGCC +ACCAAGGCCTACCTAATATTAT--TTT---CAACCCTAGCTATTTTTCTTTTACCACTTA +CACCACTAATTTTACTATAAAA---ACTTAGGATAAT--CTTAAACCAAGAGCCTTCAAA +GTTCAAAATAAGGGTGCGATTCCC-TTAGTTTTTGTAAGACTTGTAAAATACTAATTTAC +ATCTCATGAATGCAACTCAAGCGCTTTTATTAAACTAAAGCCTC-CCTGAA-CAGGCGGG +CCTTGATCCCACGACAAATTAATTAACAGCTAACTGCCCTAACCAGC-GGGCTTCTATTC +GCTTCTCCCGTACGGGAG---------AAGCCCCGGAGCC-TTTA--AGGCTCTTCTTCA +AACTTGCATTTTGACGTGAAAACACTACAG-GGCTT---TTAGTAAAGAAAGGGATTAAA +CCAATGTAAGTAAGTTTACAGCTTACCGCCTAACACT-CAGCCACTTTACCTGTGTTTAT +TAATCGTTGA------------ +>A.vanidicus +ATGAGCCCAACAATTTATACAACTATTCTAACCAGTCTTGCCACCGGAACAATTATTACA +ATAACCAGCCACCACTGATTAATAGCCTGANTCGGATTAGAAATNAATACATTAGCAATA +ATCCCAACTATCTCAACAATGCACCACCCTCGATCAACTGAAGCTGCCACAAAATACTTC +TTAACTCAGGCAGCTGCCTCAGCCTTAATTCTCTTTTCTAGTATAACTAACGCCTGACAA +ACAGGCTCCTGAGATATTACCCATCTATCATCAACCCCAGCACATATTTTATTAACCATG +GCCCTAGCAATAAAATTAGGCCTGGCCCCATTTCACTTTTGATTACCAGAAGTCCTACAA +GGATCTGCTATAAATACTGCACTAATCATTACTACTTGACAAAAGCTAGCTCCAATATCT +CTAATTTTCTTGACCGTAAATAACCTATCTACAACAATTCTTTTAACAATGGGACTTATA +TCTTCACTAATCGGAGGGTGGGCCGGACTAAATCAAACACAAACCCGAAAAATTATAGCT +TTCTCATCAATCGCCCACCTAGGATGAATAGCAGCTATCTCTTCTATTATAACAAACATT +TTAATTCTAAACCTACTTATTTACCTAATTATAACAACCTGCATGTTTTGCCTACTTATC +ATTACAAAATCTAAAACAATTCAAAACTCAACCATTATTTGAACACTATCCCCCACAATT +ACAATTATCATAATACTTTCACTTCTTTCTCTGGGGGGTTTACCACCACTTACTGGGTTC +CTACCAAAATGATTTATTGTAGAAGAACTCATTACTCAAAACTTAATNACAGTTATTACC +ATTATAGCACTATCATCTCTACTTAGCTTATTCTTCTATATCCGACTAGCCTACACTACC +ACACTAACACTTTCTCCTAATACAACCCAAACAAAGTTCAAGTGACGATTTTACCCTAAC +ACAAAATCTACCACAATTATAA--TCCCTGCAATAATTACTATCTTCATACTACCCTTAA +CACCAACAATCTTAATATAAAG---ACTTA--GGATAAACA--AACCAAGAGCCTTCAAA +GCTCCAAACAAGGGTGCAACCCCCTTAGTTTTTGT-AAGACCTGT-AAATACTAATTTAC +ATCTCCTGAATGCAACTCAAGCGCTTTAATTAAGCCAAGACCTA-ACTGAA-TAGACGGG +CTTCGATCCCGCGATAAGTTAATTAACAGCTAACCACCCAAACCAGC-GGGCTTCAATTC +TGCTTCTCCCGTACGGGA--------GAAGCCCCGGAGCCTTTA--GGGGCTCTTCTTCA +AACTTGCAATTTGACGTAAGTCACTTCGGA-GCTGT---GTGATAAAGAAAGGAATTAAA +CCAATATTAGTAGGACTACGGCCTACCGCCTATCATT-CGGCCACTTTACCAGTGTTTAT +TAATCGTTGA------------ +>A.vermiculatus +ATGAGCCCAACAATCTACTCAATTATTTTATCTAGCCTAGCAACAGGCACCATCATTACT +ATAACTAGCTACCATTGACTAATGGCCTGAATTGGGCTAGAAATCAACACACTAGCAATC +ATTCCAATTATTTCAACTAAATACCACCCACGATCCACAGAAGCTGCCACAAAATACTTC +CTCACACAAGCAGCAGCCTCTGCTATAATTTTATTTTCAAGCACAACTAATGCTTGATTA +ACAGGCACATGAGACATCACCCAAATCTCAACTACCCCTTCTCACATTCTACTAACAGCA +GCACTAGCTATAAAACTAGGACTAGCCCCTATACATTTTTGACTCCCAGAAGTTCTACAA +GGATCAACTATAAAAACAGCTTTTATTATTACAACATGACAAAAACTTGCTCCAATATCA +TTAATTTATTTAACCGTAAACAACCTATCTACAACAATTCTTCTTTCACTAGGATTATTA +TCATCCATAGTAGGAGGATGGGGAGGTTTAAATCAAACACAAATACGAAAAATCATAGCA +TACTCATCAATTGCTCACCTAGGATGAATAGCCACAATTTCATCAATCATAACCAACCTT +CTAATAATAAATTTAATAATCTATTTAATTATAACAACAACCATATTTTGAGCCCTAATC +TTGTCAAAATCCAAGACCATCCAAGACACAACAACCACCTGAACATTTTCACCATCTTTA +ACCATCATTACAATACTAACACTCCTTTCACTTGGTGGCCTACCCCCACTAACTGGATTT +ATGCCAAAATGACTTATCCTAGAAGAATTAACAACCCAAAACTTCACTCCACTAGCAACT +ATTATAGCACTATCCGCTTTACTAAGCCTATTTTTTTATTTGCGACTTACCTACACTACA +ACTTTAACTATGTCCCCAAACACCACAAAAACCAAATTTAAATGACGATTTAAACCATCA +ATAATAACACCACTACTAACAA--TAACCACACCAATGACTATTTTTTTTCTTCCTCTAA +CACCCCTTATCTTAATATAGAA---ACTTAGGATTA-ATATTAAACCAAGGGCCTTCAAA +GCCCAAAATATGGGTATAACACCCATAGTTTCTGC-AAGACCTGTGAAATACTAATTCAC +ATCTTATGAATGCAACTCAAACACTTTAATTAAGCTAAAGCCTT-ACTGAA-TAGGCGGG +CCTTGATCCCACAACAAATTAATTAACAGCTAATTACCTAAACCAGC-GGGCTTCTATTC +GCTTCTCCCGTACGGGAG---------AAGCCCCNNGACCC-TTTA-GGGCTCTTCTTCA +AACTTGCATTTTGACGTGAAACACCTCGAG-ACT-----TTGGTAAGAAAGGGAATTAAG +CCCCTGTAAACAGGACTACAGCCTGACACCTATGCTC-G-GCCACCTTACCTGTGTCCAT +TAATCGTTGA------------ +>A.wattsi +ATGAGCCCAGCAATTCACATAATTATCTCATCAAGCCTAGCAACAGGCACCATTATTACC +ATGAACAGCCACCACTGATTAATGGCCTGACTTGGGCTAGAAATTAACACACTAGCAATT +ATTCCAATAATCACATCACCACATCACCCACGGTCCACAGAAGCCGCCATAAAATACTTC +TTAACACAAGCAGCAGCCTCGGCCATAATCTTATTTTCTAGCATAATTAACGCCTGATAT +CTGGGGACATGAGACATCACCCAACTATCTTACACCCCGTCAGTTATTCTTTTAACCATA +GCATTAGCCATAAAACTAGGCCTAGCCCCCACACACTTCTGACTTCCAGAAGTACTTCAA +GGCTCAACCCTACCAACAGCCTTTATCATTGTAACATGACAAAAACTCGCCCCAATATCC +CTAATCTTCTTAACCATTAATAACCTTAACACCCCCACACTATTAGTATTAGGCATTTTA +TCCTCCATTATCGGAGGCTGAGGCGGTCTTAATCAAACATATATTCGAAAGCTCATGGCA +TACTCATCAATTGCACACCTTGGATGAATGGCTTCTATTTCCTCAATCATAACAAACATT +ATAATCATAAATTTACTAGTCTATCTAATTATAACAATGGCCCTATTTTGCTCCCTCATT +GTGTCCAATTCAAAAACAATTCAGGATACAACAACTGCCTGAACAGCTTCACCAGTATTA +ACTGCCTCCATACTACTTTNNCTTCTATCACTAGGGGGCCTCCCACCTCTAACAGGGTTC +GCACCAAAATGATTAATCCTAAATGAACTAGTAGCCCAAAACTTAGTTCCCCTAGCCACT +GCAATGGCTATGTCCGCATTACTAAGTCTATTCTTTTATCTTCGTCTCACCTATACAACA +ACACTCACACTAGCACCAAACACAACACAAATAAAATTTAAATGACGACTTTATCCAGCA +ACATTTACACCAGTTTTAACAA--TTTCAACCACACTAGCCGCGCTTCTTCTACCAATAA +CCCCGCTAATCTTATTATAAGA---ACTTAGGATAA----CTAAACCAAGAGCCTTCAAA +GCCCAAAATAGGGGTGAAACTCCCCCAGTTCTTGCTAAGACCTGTGAAATTTTAATTCAC +ATCTTCTGAATGCAACTCAAACACCTTCATTAGGCTAAGACCTT-ACTGAA-TAGGCGGG +CCTTGATCCCACAACAAATTAATTAACAGCTAATTACCCTAGCCAGC-GGGCTTCTATTC +GCTTCTCCCGTACGGGAG---------AAGCCCCGGAGCCCTTTT--GGGCTCTTTCTCA +AACTTGCATTTTGACGTGGATCACTGC--G-GAACT---GTGATAAAGAAAGGAATCNAA +CCAATGTAAGTAGGACTACAGCCTACTGCCTAGCACT-CGGCCACTTTACCTGTGTCTAT +TAATCGTTGA------------ +>Chamaeleolis.barbouri +ATGAGCCCAATTATACATACAATTATTTTATCAAGCCTAGCAACAGGCACAATTATTACT +ATAACTAGCTTTCACTGACTAATAGCATGAGTCGGCCTAGAAATCAACACCCTAGCAATG +ATTCCCATTATTTCAGCACCCCATCACCCCCGCTCCACAGAAGCAGCTACAAAATATTAC +CTCACACAAGCAGCAGCCTCCTCTATAATTTTACTCTCTAGCACACTCAACGCATGACAA +ACAGGATCATGAGATATTACTCAACCATCCACAACCCCCTCCCACATCATCTTAACAATA +GCCCTTGCTATAAAACTAGGCCTAGCCCCTTTCCATTTCTGACTGCCAGAAGTCCTACAA +GGATCAACCACACCCACCGTTTTTATTATTACTACATGACAAAAATTAGCCCCAATAACC +CTAATTTTTCTAACATTAAATAACCTATCATCAACAATTCTATTAACATTGGGCTTTTTA +TCTACAATAATTGGAGGTTGAGGGGGACTTAACCAAACACAAATTCGAAAACTCATAGCA +TACTCATCCATTGCACACCTCGGATGAATAGCCACAATTTCTCTAATCTTATCAAATATT +CTTATTATAAACCTACTAATTTACTTAATTATAACAACAACCATATTTTGTCTACTAATT +ATAACTAAATCCAAGACAATCCAAGACATAATAAACACCAAACCACTCTCACCAACCTTA +TCTATTATAATAATATTAACGCTACTTTCACTAGGAGGGCTCCCACCATTAACAGGATTT +TTACCAAAATGACTAATTCTAGAAGAACTTACAACTCAAAATCTAACCCCCCTAGCAATT +ATTATAGCAATATCAACACTACTCAACCTATTTTTTTACCTACGACTTGTATATACAACA +ACCCTTACACTAGCACCAAACACCACACAAGCAAAACTCAAATGACGATTTAAACCAAAC +TTTTCAACCCCTACAACTTTAA--TTTTAATTACATTAACAATCTTTTTACACCCCCTAA +CCCCACTAATATTACAATAAAA---ATTTA--GGTT-TAACTAAACCAGGGGCCTTCAAA +GCCCACAAAAAGGGTGTAAACCCCTTAATTTTTGT-AAGACTTGTGAAATACTAATACAC +ATCTCATGAATGCAACTCAAGCACTTTTATTAAGCTAAAGCCTC-TCTGAA-TAAGCGGG +CCTCGATCCCACGACAAATTAATTAACAGCTAATTACCCAAACCAGC-GGGCTTCTATTC +GCTTCTCCCGTACGGGAG---------AANNCCCGGAGCCT-TTTA-GGGCTCTTCTTTA +AACTTGCATTTTAATGTGAAACACTTCGGG-ACC-----CTGATAAAGAAAGGAGTTAAA +CCAATGTTAGTAGGACTACAGCCTACTGCCTNNCACT-CGGCCACTTTACCTGAGTTTAT +TA-------------------- +>C.guamuhaya +ATGAGTCCAACCATTTACACCATTATTTTATCAAGCTTAGCAACAGGTACAATTATTACC +ATATCTAGCTATCACTGATTAATAGCCTGAGTCGGACTAGAGATCAACACTTTATCTATT +ATTCCTATTGTATCTATACTACACCACCCACGATCCACAGAAGCAGCAACAAAATATTTT +CTTACACAAGCAGCAGCCTCAGCTATAATTTTATTCTCAAGCATAATTAACGCCTGACAA +ACGGGCACATGAGATATTACTCAAATNACCACTATTCCATCCAACACCCTCTTAACCATA +GCACTAGCCATAAAATTAGGCCTAGCCCCAATACACTTCTGACTACCAGAAGTACTACAA +GGATCAACTATAAACACCGCCCTAATTATTACTACATGACAAAAGCTTGCTCCAATATCA +TTAATCTTTATGACCATTAACAACTTATCAACACCAACCCTACTTCTACTAGGAGTTATT +TCCTCATTAATTGGAGGATGAGCCGGACTAAATCAAACGCAAACCCGAAAAATTATGGCA +TACTCATCAATTGCTCATCTGGGATGAATAGCTACAATTTCATCAATTATAACAAATATC +CTACTTATAAATATTTTAATTTACCTAATTTTAACTACATCTATATTTTTAATATTTATT +TTTTCTAAATCCAAAACAATTCAAGACACTGCTACCACTTGGCCCTCTTCACCAGCCCTG +ACCGCTATAATAATAATTACACTACTATCACTGGGCGGCTTGCCCCCACTTACCGGCTTT +ATACCAAAATGAATAATCTTAGAAGAATTAATTAACCAAAACCTACCTGTAGTATCATTA +ATTATGGTAGCATCTACTTTACTAAGCCTATTTTTTTATTTACGACTAACCTACACAACC +TCCCTTACTCTGTCCCCCAATACAACCCAAACAAAATTTAAATGACGACTTAAACCAAAC +ATACAAACCTCTTTAATAATAG--CACTATTTTCAATAACAGTATTTATACTTCCACTAA +CCCCACTAATTTTACTATAAAA---ACTTA--GGAT-AAATTAAACCAAGAGCCTTCAAA +GCTCGAAATAGGAGTTTAAACCTCCTAGTTTTTGT-AAGACTTGTAAAATTCTAATTTAC +ATCTATTGAATGCAACTCAAACACTTTAATTAAGCTAAAGCCTT-ACTGAA-TAAGCGGG +CCTTGATCCCGCGATAAATTAATTAACAGCTAATTGCCCAAACCAGC-GGGCTTTTATTC +GCTTCTCCGTACGGG--A---------GAAGCCCCGGAGC-TTTCA-GGGCTCTTCTTCA +AATTTGCACTTTGATGTGAAACACTTCAGG-ACT-----TTGATAAATAAAGGAATTAAA +CCAATGTAAGTAGGACTACAGCCTACTGCCTAACACT-CGGCCAATTTACCTGTGTC--- +---------------------- +>C.chamaeleonides +------------ATTTATACCATTATTTTATCAAGCTTAGCAACAGGCACAATTATTACT +ATATCTAGCTATCACTGATTAATAGCCTGAGTCGGATTAGAAATCAACACTTTATCTATT +ATCCCTATTGTATCTATATNNCATCACCCACGATCCACAGAAGCAGCAACAAAATATTTT +CTTACACAAGCGGCAGCCTCAGCTATAATTTTATTCTCAAGCATAATTAACGCCTGACAA +ACGGGCACATGAGATATTACTCAAATAACCACTATTCCATCCAACACCCTTTTAACCATA +GCATTAGCCATAAAACTAGGTCTAGCCCCAATACACTTCTGATTACCAGAAGTACTACAA +GGGTCAACTATAGACACTGCCCTAATTATTGCCACATGACAAAAACTTGCTCCAATATCA +CTAATCTTTATAACCATTAACAACTTATCAACACCAACCCTGCTTCTACTAGGAGTTATC +TCCTCATTAATTGGGGGGTGAGCCGGACTAAATCAAACACAAACCCGAAAAATTATGGCA +TACTCATACATTGCTCACCTAGGGTGAATAGCTACAATTTCATCAATTATAACAAACATC +CTACTTATAAATATTTTAATTTACCTAGTTTTAACTACATCTATATTTTCTATATTTATT +TTTTCTAAGTCCAAAACAATTCAAGACACTACTACCACTTGACCCTCTTCACCAGCCCTA +ACCACTATAATAATAATTACACTATTATCACTGGGCGGCTTACCCCCATTTACCGGCTTT +ATACCAAAATGAATAATTTTAGAAGAATTAATTAACCAAAACCTACTTGTAGTATCATTA +ATCATAGTAGCATCTACTTTACTAAGCCTATTTTTTTACTTGCGACTAACCTACACAACC +TCCCTTACTCTATCCCCCAATACAACCCAAACAAAATTTAAATGACGACTTAAACCAAAC +ATACACACCTCTTTAATAATAA--TAATATTTTCAATAACAATATTTATACTTCCACTAA +CCCCACTAATTTTATTATAAAN---ACTTA--GGAT-AAATTAAACCAAGAGCCTTCAAA +GCTCAAAATAGGAGTTTAAATCTCCTAGTTTTTGT-AAGACTTGTAAAATTCTAATTTAC +ATCTATTGAATGCAACTCAAACACTTTAATTAAGCTAAAGCCTT-ACTGAA-TAAGNGGG +CCTTGATCCCGCGAGAAATTAATTAACAGCTAACTGCCCAAACCAGC-GGGCTTTTATTC +TCTTCTCCCGTACGGGAG---------AAACCCCGGAGCC-TTTTA-GGGCTCTTCTTCA +AATTTGCACTTTGATGTGAAATACTTCAGG-ACT-----TTGGTAAAGAAAGGAATTAAA +CCAATGTAAATAGGACTACAGCCTACTGCCTAACACT-CGGCCAATTTACCTATGTCTGT +TAATCGTTA------------- +>Diplolaemus.darwinii +ATGAGCCCAACTACAATAATAATTATTACATCTAGCCTAGCCACGGANACAATCATCACC +GCATCAAGCTACCACTGACTACTGGCCTGAGTAGGCCTAGAACTAAATACACTAGCAATT +CTTCCAATAATTTCAAAATATCACCACCCACGAGCAACAGAAGCTGCAACAAAATATTTC +CTAACACAAGCAGCAGCCTCCGCCATAATCATATTTTCAAGCACACTAAACGCCTGACAA +ACAGGCACATGAAACATCACACAACCAAACAACACAACATCAAGCATCTTACTAACAGTC +GCACTAGCTATAAAACTAGGACTTGTGCCAATACACTTCTGACTACCAGAAGTATTACAA +GGCTCCTCCATAAAAACAGCCCTCATTATTAGTACATGACAAAACGTAGCCCCAATAGCC +CTTATTTTCCTGACATCCAACACACTATCAACCACAATTCTATTATTGATAGCCACACTC +TCCACCATCATTGGTGGCTGAGGCGGATTAAACCAAACACAGCTACGAAAAATTATAGCA +TTCTCATCAATCGCCCATATTGGCTGAATGGCGGCAATCTCAACAATTTCAGAAAACATT +TTAACCCTAAATTTAATACTTTATCTATTAATAACTTCCTCAACCTTCTCAGCACTTATT +TTATCTAAATCAAAAACCATCAAAGACACATCCACATCACTAACAACCTCCACAACACTA +ACCATCATAATAATACTTATGCTACTATCACTAGGAGGACTCCCACCCCTAACAGGCTTT +TTACCAAAGTGATTAGTACTAAAAGAGCTAACAATACAAGGACTACTTATTATTTCCACA +ACCCTAGCCATGTCCTCCCTCCTAAGCCTATTTTTTTACCTACGACTTTCATATACAACA +ACACTTACACTTGCCCCAAATACACTACTAATAAAACACAAATGACGATTCAAACCAATA +TCAAAAACACTTTTAATATCTA--CATCAACACCACTAGCCCTAATATTACTACCAATAA +CCCCACTAATATTATAAGAGAC-----TTAGGCTAACTAACCAAACCAAGGGCCTTCAAA +GCCCAANATAGAGATGAAAACCCTCTAGTTTCTGATAAGACCTGTGTAATACTAAAACAC +ATCTCCTGAATGCAACCCAGACACTTTAATTAAGCTAAGGCCTC-CCTAGA-TAGGGGGG +CTTTGATCCCACGAAACTTTAGTTAACAGCTAAACACCCAAACCAGC-GGGCTTCTATCC +GCTTCTCCCGCACGGGAG---------AANNCCCGGAGCCCTTTAT-GG---GCTCTTCA +AATTTGCATTTTGACGTGCTTACACCACAGGACT-----TTGATAAAAAGAGGAGTTAAA +CCCCTGTAAATAGGACTACAGCCTACCTCCTAAACGCTCGGCCATTTTACCTGTGTTTAT +TAA------------------- +>Phenacosaurus.acutirostrus +ATGAGCCCAGTAACAATAACAATCCTACTCTCCAGCATCGCAGCAGGAACAATTCTTACC +ATATCTAGCTACCACTGGCTCTTGGCTTGACTCGGATTAGAAATTAATACCCTGGCAATC +ATCCCGATTATTTCAAAACAGCATCATCCCCGAGCTACCGAAGCTACAACAAAATATTTC +CTAACACAAGCAGCCGCATCCGCCCTAATCCTATTCTCCAGCACAATTAATGCCTGACAA +ACGGGGACCTGAGATATCACACAAATAACTAATCCAACACCAACCACACTATTAACCGCC +GCCCTGGCCATAAAGCTAGGACTTGCACCCACACACTTCTGACTTCCAGAGGTCCTGCAA +GGCACTACAATAAACACAGCCTTAATTATTACAACATGACAAAAACTAGCCCCAATATCA +CTTATTTACCTAACAATAAACAACCTACCCCCAACAATCCTACTACTTACAGGAATACTG +TCCTCACTCCTAGGCGGCTGGGGAGGACTAAATCAAACACAAACCCGAAAACTAATGGCT +TACTCATCAATCGCCCACCTGGGGTGAATGGCCGTAATTTCCACGATAATACCGAACATT +CTAATTCTTACCCTAACAACATACATTCTAATAACTACTGCAATATTCCTAACACTAATC +TCTACTAAATCAAAAACAATCCAAGATGCATCTACATCATGGACAACTTCTCCAACCATT +ACCACTATATCTCTCCTCATCCTCCTATCCCTTGGAGGCTCCCCCCCTTTAAAAGGCTTT +ATTCCAAAATGACTCATTCTAGAAGAACTCTTCACACAAAACCTAGTCCCAACAGCAACT +ATCATAGCAATATCAGCCCTGCTAAGCCTATTCTTTTATCTCCGACTCACCTACACAACA +ANACTCACAATAGCCCCAAACACAACTCCAACAAAATACAAGTGACGATTCAAAATCTCC +GCACCAAAACTTCTAACAATAA-----CTACCCCCATAGCGCTCCTTCTACTACCAATAA +CCCCCCTACTCACTTCATAGAA---CCTTAGGC----TAATCAAACCTTGGACCTTCAAA +GTCCAAAATAGGAGTGCAAACCCCCTAGTTTCTGC-AAGACCTGCGAACCATTAAAACGC +ATCCTCTGAATGCAACTCAGTCACTTTAATTAAGCTAAGGCCTC-CCTAGA-CAGGCGGG +CCTTGATCCC-GCAAAAATTAGTTAACAGCTAATTACCCAAACCAGC-GGGCTTCCATCC +GCTTCTCCCGTACGGGAG---------AAGCCCCGGAGCCCTTTAG-GGGCTCTTCTCCG +AATTTGCACTTCGGCGTGTTCTCACTACAGGGCT-----GTGGTAAGCAGAGGAATTAAA +CCCCTGTCTATGAGTCTACAGCCCACCGCCTAAACACTCGGCCACCTTACCTGTGTTCAT +CACACGTTAC------------ +>P.nicefori +ATGAGCCCAACAATATATTCAATTATTATCTCTAGCCTTGCAACCGGCACTATCATTACT +ATAACCAGTCACCATTGACTGCTAGCCTGTCTCGGACTAGAAATCAACACACTAGCAATT +GTTCCAACCATTTCAATAAACCACAACCCACGAGCCACAGAAGCCACTATAAAGTACTTT +TTAACACAAACAGCAGCTTCGGCCATAATTTTATTTGCAAGCACAACAAATGCTTGACAC +ACAGGAACATGAGATATTACCCAAATATCAACAACCACCTCTTACATTTTACTTACTCTT +GCATTAGCAATAAAACTAGGACTAGCCCCCCTACACTTCTGACTCCCAGAAGTACTACAA +GGAACTACCCTACCCACAGCCCTAATCATTGTTACATGACAAAAACTTGCCCCTATATCA +TTAATTTACATAACCATTAACAACCTTTCTACAACATTACTACTAACACTAGGATTATTT +TCCTCAATGGTTGGCGGATGAGCTGGACTAAACCAAACACAAACACGAAAAATAATAGCC +TTCTCATCAATCGCTCACTTAGGATGAATATCAACAGTAGCATCAATTATAACAAACATT +CTAATCTTAAATCTAATTATTTATTTAACTATAACAATAACCATATTTTACTCACTTATT +TTATCAAAAGCTAAAACAATTAAAGATACCACCACCTCATGAACACTATCCCCAACATTA +ACAATTATTATAATACTCACTCTTCTCTCACTAGGGGGGCTCCCCCCATTAACCGGGTTT +ATACCAAAATGACTTATTCTAGAAGAACTAACAATTCAAAACCTATGCCCTCTAGCCCTT +TTGATAGCATTAACATCACTATTAAGTTTATTTTTTTACTTACGGCTAACCTACACAACA +ACACTTACACTTTCTCCAAACACCATTTTAACAAAACACAAATGACGATTTAAACCAATA +ACTATAACAACCCCCATAACAA--TTTTAATCCCACTAACAATACTTATACTACCAATAA +CACCAATAATAATCTCTTAGAA---ATTTA--GGCT-AACATTAAACCAAGAGCCTTCAA +AGCTCAAAATATGGACTATA-CCCATAATTTCTGT-AAGACTTGTAAAATATTAATTTAC +ATCTCATGAATGCAACTCAAGCACTTTAATTAAGCTAAAATCTACCATAAG-TAGGCGGG +CTTTGATCCCACGACATATTAATTAACAGCTAACTACCCAAACCAGC-GGGCTTCTACTC +GCTTCTCCCGCACGGGAG---------AAGCCCCGGAGCCTTTTA--GGGCTCTTCTTCA +AATTTGCATTTTGATATGGAACACCTCAGG-GCT-----TTGATAAATGAGGAATTTAA- +-CCCCTTGTGTAGGACTACAAACTACTGCCTTACGTT-CGGCCATTTTACCTGTGTTTCT +TAATCGAT-------------- diff --git a/topics/evolution/tutorials/abc_intro_phylo/data/anolis_Report_and_Final_Tree.iqtree b/topics/evolution/tutorials/abc_intro_phylo/data/anolis_Report_and_Final_Tree.iqtree new file mode 100644 index 00000000000000..a7a3059be9dcb1 --- /dev/null +++ b/topics/evolution/tutorials/abc_intro_phylo/data/anolis_Report_and_Final_Tree.iqtree @@ -0,0 +1,291 @@ +IQ-TREE 2.1.2 COVID-edition built Mar 30 2021 + +Input file name: /mnt/pulsar/files/staging/7756321/inputs/dataset_6092bb0e-7934-473c-8e8f-77054b0192c5.dat +Type of analysis: ModelFinder + tree reconstruction +Random seed number: 204403 + +REFERENCES +---------- + +To cite IQ-TREE please use: + +Bui Quang Minh, Heiko A. Schmidt, Olga Chernomor, Dominik Schrempf, +Michael D. Woodhams, Arndt von Haeseler, and Robert Lanfear (2020) +IQ-TREE 2: New models and efficient methods for phylogenetic inference +in the genomic era. Mol. Biol. Evol., in press. +https://doi.org/10.1093/molbev/msaa015 + +To cite ModelFinder please use: + +Subha Kalyaanamoorthy, Bui Quang Minh, Thomas KF Wong, Arndt von Haeseler, +and Lars S Jermiin (2017) ModelFinder: Fast model selection for +accurate phylogenetic estimates. Nature Methods, 14:587–589. +https://doi.org/10.1038/nmeth.4285 + +SEQUENCE ALIGNMENT +------------------ + +Input data: 55 sequences with 1462 nucleotide sites +Number of constant sites: 377 (= 25.7866% of all sites) +Number of invariant (constant or ambiguous constant) sites: 377 (= 25.7866% of all sites) +Number of parsimony informative sites: 913 +Number of distinct site patterns: 1138 + +ModelFinder +----------- + +Best-fit model according to AIC: GTR+F+R6 + +List of models sorted by AIC scores: + +Model LogL AIC w-AIC AICc w-AICc BIC w-BIC +GTR+F+R6 -37031.233 74312.465 + 0.817 74336.043 + 0.832 74973.410 - 0.0237 +GTR+F+R7 -37031.088 74316.176 + 0.128 74340.548 + 0.0875 74987.696 - 1.88e-05 +TIM2+F+R6 -37036.403 74318.806 - 0.0343 74341.604 + 0.0516 74969.176 + 0.197 +GTR+F+R5 -37037.210 74320.421 - 0.0153 74343.219 - 0.023 74970.791 + 0.088 +TIM2+F+R7 -37036.374 74322.749 - 0.00478 74346.327 - 0.00486 74983.694 - 0.000139 +TIM2+F+R5 -37042.437 74326.875 - 0.000607 74348.907 - 0.00134 74966.669 + 0.691 +TIM3+F+R6 -37070.405 74386.811 - 5.87e-17 74409.609 - 8.82e-17 75037.180 - 3.37e-16 +TIM3+F+R7 -37070.201 74390.401 - 9.74e-18 74413.979 - 9.92e-18 75051.346 - 2.83e-19 +GTR+F+R4 -37074.898 74391.795 - 4.85e-18 74413.828 - 1.07e-17 75031.590 - 5.52e-15 +TN+F+R6 -37075.376 74394.752 - 1.11e-18 74417.166 - 2.02e-18 75039.835 - 8.95e-17 +TIM+F+R6 -37074.693 74395.386 - 8.06e-19 74418.184 - 1.21e-18 75045.756 - 4.63e-18 +TIM3+F+R5 -37076.845 74395.690 - 6.92e-19 74417.723 - 1.53e-18 75035.485 - 7.88e-16 +TN+F+R7 -37075.196 74398.393 - 1.79e-19 74421.579 - 2.22e-19 75054.050 - 7.33e-20 +TIM+F+R7 -37074.525 74399.049 - 1.29e-19 74422.627 - 1.31e-19 75059.994 - 3.75e-21 +TN+F+R5 -37081.842 74403.685 - 1.27e-20 74425.340 - 3.38e-20 75038.192 - 2.03e-16 +TIM+F+R5 -37081.073 74404.146 - 1.01e-20 74426.179 - 2.22e-20 75043.941 - 1.15e-17 +GTR+F+I+G4 -37096.959 74427.917 - 6.95e-26 74448.462 - 3.23e-25 75046.562 - 3.1e-18 +TVM+F+R6 -37095.385 74438.769 - 3.06e-28 74461.955 - 3.79e-28 75094.427 - 1.25e-28 +TVM+F+R5 -37097.648 74439.297 - 2.35e-28 74461.711 - 4.28e-28 75084.379 - 1.9e-26 +TVM+F+R7 -37095.426 74442.853 - 3.97e-29 74466.826 - 3.32e-29 75109.085 - 8.21e-32 +TPM2+F+R6 -37100.987 74445.973 - 8.35e-30 74468.387 - 1.52e-29 75091.056 - 6.75e-28 +TPM2u+F+R6 -37100.987 74445.973 - 8.35e-30 74468.387 - 1.52e-29 75091.056 - 6.75e-28 +TPM2+F+R5 -37103.564 74447.128 - 4.68e-30 74468.784 - 1.25e-29 75081.636 - 7.49e-26 +TPM2u+F+R5 -37103.564 74447.128 - 4.68e-30 74468.784 - 1.25e-29 75081.636 - 7.49e-26 +TPM2+F+R7 -37100.997 74449.993 - 1.12e-30 74473.179 - 1.38e-30 75105.651 - 4.57e-31 +TPM2u+F+R7 -37100.997 74449.994 - 1.12e-30 74473.180 - 1.38e-30 75105.651 - 4.57e-31 +TPM3u+F+R6 -37126.615 74497.230 - 6.18e-41 74519.644 - 1.13e-40 75142.312 - 5e-39 +TPM3+F+R6 -37126.615 74497.230 - 6.18e-41 74519.644 - 1.13e-40 75142.312 - 5e-39 +TPM3u+F+R5 -37129.065 74498.131 - 3.94e-41 74519.786 - 1.05e-40 75132.638 - 6.31e-37 +TPM3+F+R5 -37129.065 74498.131 - 3.94e-41 74519.786 - 1.05e-40 75132.638 - 6.31e-37 +TPM3+F+R7 -37126.341 74500.682 - 1.1e-41 74523.868 - 1.36e-41 75156.340 - 4.5e-42 +TPM3u+F+R7 -37126.342 74500.683 - 1.1e-41 74523.869 - 1.36e-41 75156.341 - 4.5e-42 +HKY+F+R6 -37131.953 74505.906 - 8.08e-43 74527.938 - 1.78e-42 75145.700 - 9.19e-40 +K3Pu+F+R6 -37131.054 74506.107 - 7.3e-43 74528.521 - 1.33e-42 75151.190 - 5.91e-41 +HKY+F+R5 -37134.629 74507.258 - 4.11e-43 74528.540 - 1.32e-42 75136.478 - 9.25e-38 +K3Pu+F+R5 -37133.634 74507.269 - 4.09e-43 74528.924 - 1.09e-42 75141.776 - 6.54e-39 +HKY+F+R7 -37131.734 74509.468 - 1.36e-43 74532.266 - 2.05e-43 75159.838 - 7.82e-43 +K3Pu+F+R7 -37130.809 74509.619 - 1.26e-43 74532.805 - 1.56e-43 75165.276 - 5.16e-44 +GTR+F+G4 -37164.452 74560.904 - 9.22e-55 74581.085 - 5.13e-54 75174.261 - 5.77e-46 +GTR+F+R3 -37179.249 74596.499 - 1.72e-62 74617.780 - 5.51e-62 75225.718 - 3.87e-57 +SYM+R5 -37262.490 74764.980 - 4.47e-99 74786.635 - 1.19e-98 75399.487 - 7.15e-95 +SYM+R6 -37261.122 74766.244 - 2.37e-99 74788.658 - 4.33e-99 75411.326 - 1.92e-97 +TIM2e+R5 -37274.321 74784.641 - 2.4e-103 74805.553 - 9.27e-103 75408.573 - 7.61e-97 +TIM2e+R6 -37273.043 74786.086 - 1.17e-103 74807.741 - 3.1e-103 75420.593 - 1.87e-99 +GTR+F+R2 -37597.901 75429.802 - 1.93e-243 75450.347 - 8.95e-243 76048.447 - 8.6e-236 +TIM3e+R6 -37740.342 75720.684 - 1.32e-306 75742.339 - 3.52e-306 76355.191 - 2.12e-302 +TIM3e+R7 -37740.126 75724.252 - 2.22e-307 75746.665 - 4.05e-307 76369.334 - 1.8e-305 +TVMe+R6 -37741.704 75725.408 - 1.25e-307 75747.441 - 2.75e-307 76365.203 - 1.42e-304 +TVMe+R5 -37745.621 75729.242 - 1.83e-308 75750.524 - 5.88e-308 76358.462 - 4.13e-303 +TVMe+R7 -37741.937 75729.875 - 1.34e-308 75752.673 - 2.01e-308 76380.245 - 7.68e-308 +TIM3e+R5 -37747.544 75731.088 - 7.28e-309 75751.999 - 2.81e-308 76355.020 - 2.31e-302 +TIMe+R6 -37750.759 75741.518 - 3.96e-311 75763.173 - 1.05e-310 76376.025 - 6.33e-307 +TNe+R6 -37752.489 75742.978 - 1.91e-311 75764.260 - 6.12e-311 76372.198 - 4.29e-306 +TIMe+R7 -37750.536 75745.072 - 6.69e-312 75767.486 - 1.22e-311 76390.154 - 5.41e-310 +TNe+R7 -37752.255 75746.511 - 3.26e-312 75768.544 - 7.19e-312 76386.306 - 3.71e-309 +TIMe+R5 -37757.450 75750.900 - 3.63e-313 75771.811 - 1.4e-312 76374.832 - 1.15e-306 +TNe+R5 -37759.321 75752.642 - 1.52e-313 75773.187 - 7.05e-313 76371.287 - 6.77e-306 +K3P+R5 -38328.664 76891.327 - 0 76911.872 - 0 77509.972 - 0 +K2P+R5 -38330.281 76892.562 - 0 76912.744 - 0 77505.919 - 0 +K3P+R6 -38327.530 76893.060 - 0 76914.341 - 0 77522.279 - 0 +K2P+R6 -38329.207 76894.414 - 0 76915.326 - 0 77518.347 - 0 +F81+F+R5 -38740.095 77716.191 - 0 77737.102 - 0 78340.123 - 0 +F81+F+R6 -38739.434 77718.868 - 0 77740.523 - 0 78353.375 - 0 +GTR+F+I -39252.699 78737.399 - 0 78757.580 - 0 79350.756 - 0 +JC+R5 -39408.095 79046.191 - 0 79066.012 - 0 79654.260 - 0 +JC+R6 -39407.610 79049.220 - 0 79069.765 - 0 79667.865 - 0 +GTR+F -41407.684 83045.369 - 0 83065.190 - 0 83653.438 - 0 + +AIC, w-AIC : Akaike information criterion scores and weights. +AICc, w-AICc : Corrected AIC scores and weights. +BIC, w-BIC : Bayesian information criterion scores and weights. + +Plus signs denote the 95% confidence sets. +Minus signs denote significant exclusion. + +SUBSTITUTION PROCESS +-------------------- + +Model of substitution: GTR+F+R6 + +Rate parameter R: + + A-C: 1.4045 + A-G: 4.3329 + A-T: 1.6165 + C-G: 0.7046 + C-T: 7.4429 + G-T: 1.0000 + +State frequencies: (empirical counts from alignment) + + pi(A) = 0.3392 + pi(C) = 0.2607 + pi(G) = 0.1184 + pi(T) = 0.2817 + +Rate matrix Q: + + A -0.6328 0.1737 0.2432 0.2159 + C 0.2259 -1.26 0.03954 0.9942 + G 0.697 0.08712 -0.9177 0.1336 + T 0.26 0.9203 0.05613 -1.236 + +Model of rate heterogeneity: FreeRate with 6 categories +Site proportion and rates: (0.2697,0.01936) (0.1987,0.1952) (0.1537,0.6315) (0.1929,1.365) (0.1619,2.662) (0.02305,7.146) + + Category Relative_rate Proportion + 1 0.01936 0.2697 + 2 0.1952 0.1987 + 3 0.6315 0.1537 + 4 1.365 0.1929 + 5 2.662 0.1619 + 6 7.146 0.02305 + +MAXIMUM LIKELIHOOD TREE +----------------------- + +Log-likelihood of the tree: -37024.7700 (s.e. 781.9510) +Unconstrained log-likelihood (without tree): -9373.5747 +Number of free parameters (#branches + #model parameters): 125 +Akaike information criterion (AIC) score: 74299.5399 +Corrected Akaike information criterion (AICc) score: 74323.1178 +Bayesian information criterion (BIC) score: 74960.4850 + +Total tree length (sum of branch lengths): 10.7102 +Sum of internal branch lengths: 2.7107 (25.3096% of tree length) + +NOTE: Tree is UNROOTED although outgroup taxon 'Anolis.acutus' is drawn at root + ++------Anolis.acutus +| +| +-----A.aeneus +| +-----| +| | +----A.richardi +| +--| +| | +---------A.luciae +| +--| +| | | +------A.agassizi +| | | +--| +| | | | +--------A.microtus +| | +---| +| | +--------------P.nicefori +| +--| +| | | +----------------------Diplolaemus.darwinii +| | +----------| +| | +----------------------Phenacosaurus.acutirostrus +| +--| +| | | +---------A.aliniger +| | | +---| +| | | | +----------A.coelestinus +| | | +--| +| | | | +-------------A.bahorucoensis +| | | +--| +| | | | | +---------A.bartschi +| | | | +----| +| | | | +------A.vermiculatus +| | | +--| +| | | | +-----------A.equestris +| | +--| +| | +-----------------------A.occulatus +| +--| +| | | +------A.etheridgei +| | | +--| +| | | | +---------A.insolitus +| | | +--| +| | | | +-------------A.olssoni +| | +--| +| | +--------------Chamaeleolis.barbouri +| +--| +| | | +--------A.barahonae +| | | +--| +| | | | +--------A.christophei +| | | +--| +| | | | +--------A.cuvieri +| | | +--| +| | | | | +--C.guamuhaya +| | | | +------------| +| | | | +--C.chamaeleonides +| | +--| +| | +--------------A.lucius +| +--| +| | | +-------------A.marcanoi +| | +---------| +| | +-----------A.strahmi +| +--| +| | | +---------------A.ahli +| | | +--| +| | | | | +-----A.ophiolepis +| | | | +---------| +| | | | +----A.sagrei +| | +----| +| | | +-----A.garmani +| | | +----| +| | | | +-----A.grahami +| | | +--| +| | | | +--------A.equestris2 +| | | +--| +| | | | +------------A.lineatopus +| | +--| +| | | +-------------A.humilis +| | | +-------| +| | | | +------------A.limifrons +| | +--| +| | +-------------A.lineatus +| +--| +| | | +--------A.alutaceous +| | | +--------| +| | | | +-----------A.vanidicus +| | +--| +| | | +----A.angusticeps +| | | +-------| +| | | | | +--A.paternus +| | | | +--| +| | | | +--A.paternus2 +| | | +--| +| | | | +----------------A.sheplani +| | | +--| +| | | | | +---A.carolinensis +| | | | +----| +| | | | +----A.maynardi +| | +--| +| | | +------A.loysiana +| | +--| +| | +----------A.pumilis +| +--| +| | | +-----------A.bimaculatus +| | +---| +| | +------------A.wattsi +| +--| +| | | +---------A.brevirostrus +| | +-----| +| | +------A.distichus +| +--| +| | +----------A.krugi ++----| +| +------------A.cristatellus +| ++-----A.stratulus + +Tree in newick format: + +(Anolis.acutus:0.1010470578,((((((((((((((A.aeneus:0.0894217579,A.richardi:0.0771663752):0.0905009536,A.luciae:0.1452269292):0.0505637895,((A.agassizi:0.1016958548,A.microtus:0.1274705993):0.0466408653,P.nicefori:0.2174577338):0.0678983736):0.0434625375,(Diplolaemus.darwinii:0.3250757059,Phenacosaurus.acutirostrus:0.3269305219):0.1596746115):0.0466548353,(((((A.aliniger:0.1391142437,A.coelestinus:0.1557602669):0.0598840477,A.bahorucoensis:0.1976626377):0.0075156245,(A.bartschi:0.1398068405,A.vermiculatus:0.1026256093):0.0701730721):0.0088445438,A.equestris:0.1698075932):0.0120788802,A.occulatus:0.3404603086):0.0198461252):0.0251152781,(((A.etheridgei:0.0991665297,A.insolitus:0.1482517520):0.0540499255,A.olssoni:0.1910514865):0.0088098284,Chamaeleolis.barbouri:0.2095740155):0.0188459879):0.0077203037,((((A.barahonae:0.1260843543,A.christophei:0.1262731407):0.0348296954,A.cuvieri:0.1361832093):0.0309217949,(C.guamuhaya:0.0431099935,C.chamaeleonides:0.0257796446):0.1779877789):0.0345206566,A.lucius:0.2146844398):0.0147942476):0.0193880895,(A.marcanoi:0.1954668346,A.strahmi:0.1658659731):0.1384272979):0.0071850401,((A.ahli:0.2194974552,(A.ophiolepis:0.0874380486,A.sagrei:0.0746247231):0.1427321115):0.0307428243,((((A.garmani:0.0905499626,A.grahami:0.0943764186):0.0703118985,A.equestris2:0.1314905403):0.0198447353,A.lineatopus:0.1838950754):0.0232334317,((A.humilis:0.2006656769,A.limifrons:0.1903931082):0.1159932105,A.lineatus:0.2020567025):0.0284469354):0.0386892413):0.0788578675):0.0071420002,((A.alutaceous:0.1299690048,A.vanidicus:0.1769083322):0.1269197421,((((A.angusticeps:0.0766354706,(A.paternus:0.0125693305,A.paternus2:0.0058013518):0.0519908238):0.1128275458,A.sheplani:0.2418794624):0.0228102911,(A.carolinensis:0.0645999975,A.maynardi:0.0760546166):0.0733086724):0.0301309147,(A.loysiana:0.0985169696,A.pumilis:0.1631220132):0.0511013731):0.0207983825):0.0498971029):0.0462944857,(A.bimaculatus:0.1713426754,A.wattsi:0.1884789044):0.0574997330):0.0334141164,(A.brevirostrus:0.1442087439,A.distichus:0.1088311847):0.0833172684):0.0398851829,A.krugi:0.1583953901):0.0256700857,A.cristatellus:0.1827036810):0.0725247864,A.stratulus:0.0863000570); 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files /dev/null and b/topics/evolution/tutorials/abc_intro_phylo/images/tableOfSitesForSplits.png differ diff --git a/topics/evolution/tutorials/abc_intro_phylo/tutorial.bib b/topics/evolution/tutorials/abc_intro_phylo/tutorial.bib new file mode 100644 index 00000000000000..2774200c69ee55 --- /dev/null +++ b/topics/evolution/tutorials/abc_intro_phylo/tutorial.bib @@ -0,0 +1,189 @@ + +# This is the bibliography file for your tutorial. +# +# To add bibliography (bibtex) entries here, follow these steps: +# 1) Find the DOI for the article you want to cite +# 2) Go to https://doi2bib.org and fill in the DOI +# 3) Copy the resulting bibtex entry into this file +# +# To cite the example below, in your tutorial.md file +# use {% cite Batut2018 %} +# +# If you want to cite an online resourse (website etc) +# you can use the 'online' format (see below) +# +# You can remove the examples below + +@article{Menardo2019, + doi = {10.1371/journal.ppat.1008067}, + url = {https://doi.org/10.1371/journal.ppat.1008067}, + year = {2019}, + month = sep, + publisher = {Public Library of Science ({PLoS})}, + volume = {15}, + number = {9}, + pages = {e1008067}, + author = {Fabrizio Menardo and Sebastian Duch{\^{e}}ne and Daniela Brites and Sebastien Gagneux}, + editor = {Roman Biek}, + title = {The molecular clock of Mycobacterium tuberculosis}, + journal = {{PLOS} Pathogens} +} + +@article{Eldholm2016, + doi = {10.1073/pnas.1611283113}, + url = {https://doi.org/10.1073/pnas.1611283113}, + year = {2016}, + month = nov, + publisher = {Proceedings of the National Academy of Sciences}, + volume = {113}, + number = {48}, + pages = {13881--13886}, + author = {Vegard Eldholm and John H.-O. Pettersson and Ola B. Brynildsrud and Andrew Kitchen and Erik Michael Rasmussen and Troels Lillebaek and Janne O. R{\o}nning and Valeriu Crudu and Anne Torunn Mengshoel and Nadia Debech and Kristian Alfsnes and Jon Bohlin and Caitlin S. Pepperell and Francois Balloux}, + title = {Armed conflict and population displacement as drivers of the evolution and dispersal of + $\less$i$\greater$Mycobacterium tuberculosis$\less$/i$\greater$}, + journal = {Proceedings of the National Academy of Sciences} +} + +@article{Zhou2021, + doi = {10.1093/bioinformatics/btab234}, + url = {https://doi.org/10.1093/bioinformatics/btab234}, + year = {2021}, + month = apr, + publisher = {Oxford University Press ({OUP})}, + volume = {37}, + number = {20}, + pages = {3645--3646}, + author = {Zhemin Zhou and Jane Charlesworth and Mark Achtman}, + editor = {Janet Kelso}, + title = {{HierCC}: a multi-level clustering scheme for population assignments based on core genome {MLST}}, + journal = {Bioinformatics} +} + +@article{Goig2018, + doi = {10.1101/403824}, + url = {https://doi.org/10.1101/403824}, + year = {2018}, + month = aug, + publisher = {Cold Spring Harbor Laboratory}, + author = {Galo A Goig and Silvia Blanco and Alberto L. Garcia-Basteiro and I{\~{n}}aki Comas}, + title = {Contaminant {DNA} in bacterial sequencing experiments is a major source of false genetic variability} +} + +@article{Ford2013, + doi = {10.1038/ng.2656}, + url = {https://doi.org/10.1038/ng.2656}, + year = {2013}, + month = jun, + publisher = {Springer Science and Business Media {LLC}}, + volume = {45}, + number = {7}, + pages = {784--790}, + author = {Christopher B Ford and Rupal R Shah and Midori Kato Maeda and Sebastien Gagneux and Megan B Murray and Ted Cohen and James C Johnston and Jennifer Gardy and Marc Lipsitch and Sarah M Fortune}, + title = {Mycobacterium tuberculosis mutation rate estimates from different lineages predict substantial differences in the emergence of drug-resistant tuberculosis}, + journal = {Nature Genetics} +} + +@article{6ef38333-9ecd-3f40-8f3e-5f3d6df3c4b4, + ISSN = {00027685, 19384211}, + URL = {http://www.jstor.org/stable/4444260}, + author = {Theodosius Dobzhansky}, + journal = {The American Biology Teacher}, + number = {3}, + pages = {125--129}, + publisher = {[University of California Press, National Association of Biology Teachers]}, + title = {Nothing in Biology Makes Sense except in the Light of Evolution}, + urldate = {2023-11-12}, + volume = {35}, + year = {1973} +} + +@article{PMID:27572647, + Title = {A new view of the tree of life}, + Author = {Hug, Laura A and Baker, Brett J and Anantharaman, Karthik and Brown, Christopher T and Probst, Alexander J and Castelle, Cindy J and Butterfield, Cristina N and Hernsdorf, Alex W and Amano, Yuki and Ise, Kotaro and Suzuki, Yohey and Dudek, Natasha and Relman, David A and Finstad, Kari M and Amundson, Ronald and Thomas, Brian C and Banfield, Jillian F}, + DOI = {10.1038/nmicrobiol.2016.48}, + Volume = {1}, + Month = {April}, + Year = {2016}, + Journal = {Nature microbiology}, + ISSN = {2058-5276}, + Pages = {16048}, + Abstract = {The tree of life is one of the most important organizing principles in biology(1). Gene surveys suggest the existence of an enormous number of branches(2), but even an approximation of the full scale of the tree has remained elusive. Recent depictions of the tree of life have focused either on the nature of deep evolutionary relationships(3-5) or on the known, well-classified diversity of life with an emphasis on eukaryotes(6). These approaches overlook the dramatic change in our understanding of life's diversity resulting from genomic sampling of previously unexamined environments. New methods to generate genome sequences illuminate the identity of organisms and their metabolic capacities, placing them in community and ecosystem contexts(7,8). Here, we use new genomic data from over 1,000 uncultivated and little known organisms, together with published sequences, to infer a dramatically expanded version of the tree of life, with Bacteria, Archaea and Eukarya included. The depiction is both a global overview and a snapshot of the diversity within each major lineage. The results reveal the dominance of bacterial diversification and underline the importance of organisms lacking isolated representatives, with substantial evolution concentrated in a major radiation of such organisms. This tree highlights major lineages currently underrepresented in biogeochemical models and identifies radiations that are probably important for future evolutionary analyses.}, + URL = {https://www.nature.com/articles/nmicrobiol201648.pdf}, +} + +@article{PMID:25275450, + Title = {Phylogenetic distribution of extant richness suggests metamorphosis is a key innovation driving diversification in insects}, + Author = {Rainford, James L and Hofreiter, Michael and Nicholson, David B and Mayhew, Peter J}, + DOI = {10.1371/journal.pone.0109085}, + Number = {9}, + Volume = {9}, + Year = {2014}, + Journal = {PloS one}, + ISSN = {1932-6203}, + Pages = {e109085}, + Abstract = {Insects and their six-legged relatives (Hexapoda) comprise more than half of all described species and dominate terrestrial and freshwater ecosystems. Understanding the macroevolutionary processes generating this richness requires a historical perspective, but the fossil record of hexapods is patchy and incomplete. Dated molecular phylogenies provide an alternative perspective on divergence times and have been combined with birth-death models to infer patterns of diversification across a range of taxonomic groups. Here we generate a dated phylogeny of hexapod families, based on previously published sequence data and literature derived constraints, in order to identify the broad pattern of macroevolutionary changes responsible for the composition of the extant hexapod fauna. The most prominent increase in diversification identified is associated with the origin of complete metamorphosis, confirming this as a key innovation in promoting insect diversity. Subsequent reductions are recovered for several groups previously identified as having a higher fossil diversity during the Mesozoic. In addition, a number of recently derived taxa are found to have radiated following the development of flowering plant (angiosperm) floras during the mid-Cretaceous. These results reveal that the composition of the modern hexapod fauna is a product of a key developmental innovation, combined with multiple and varied evolutionary responses to environmental changes from the mid Cretaceous floral transition onward.}, + URL = {https://europepmc.org/articles/PMC4183542}, +} + +@article{10.1080/106351599260283, + author = {Jackman, Todd R. and Larson, Allan and de Queiroz, Kevin and Losos, Jonathan B.}, + title = "{Phylogenetic Relationships and Tempo of Early Diversification in Anolis Lizards}", + journal = {Systematic Biology}, + volume = {48}, + number = {2}, + pages = {254-285}, + year = {1999}, + month = {06}, + abstract = "{We examine phylogenetic relationships among anoles using mitochondrial DNA sequences from the NADH dehydrogenase subunit 2 gene (ND2) and five transfer-RNA genes representing 1,455 alignable base positions and 866 phylogenetically informative characters (parsimony criterion). We also present 16 morphological characters for phylogenetic analysis. Our analyses yielded poorly-supported nodes deep in the anole tree but many well-supported nodes for more recent phylogenetic divergences. We test the hypothesis that the major clades of anoles form a hard polytomy and present a general statistical framework for testing hypotheses of simultaneous branching of lineages by using molecular sequence data. Our results suggest that rapid diversification early in the evolutionary history of anoles explains why numerous researchers have had difficulty reconstructing well-supported dichotomous phylogenetic trees for anoles.}", + issn = {1063-5157}, + doi = {10.1080/106351599260283}, + url = {https://doi.org/10.1080/106351599260283}, + eprint = {https://academic.oup.com/sysbio/article-pdf/48/2/254/19502216/48-2-254.pdf}, + } + + @article{10.1093/oxfordjournals.molbev.a040454, + author = {Saitou, N and Nei, M}, + title = "{The neighbor-joining method: a new method for reconstructing phylogenetic trees.}", + journal = {Molecular Biology and Evolution}, + volume = {4}, + number = {4}, + pages = {406-425}, + year = {1987}, + month = {07}, + abstract = "{A new method called the neighbor-joining method is proposed for reconstructing phylogenetic trees from evolutionary distance data. The principle of this method is to find pairs of operational taxonomic units (OTUs [= neighbors]) that minimize the total branch length at each stage of clustering of OTUs starting with a starlike tree. The branch lengths as well as the topology of a parsimonious tree can quickly be obtained by using this method. Using computer simulation, we studied the efficiency of this method in obtaining the correct unrooted tree in comparison with that of five other tree-making methods: the unweighted pair group method of analysis, Farris's method, Sattath and Tversky's method, Li's method, and Tateno et al.'s modified Farris method. The new, neighbor-joining method and Sattath and Tversky's method are shown to be generally better than the other methods.}", + issn = {0737-4038}, + doi = {10.1093/oxfordjournals.molbev.a040454}, + url = {https://doi.org/10.1093/oxfordjournals.molbev.a040454}, + eprint = {https://academic.oup.com/mbe/article-pdf/4/4/406/11167444/7sait.pdf}, + } + @article{10.1093/oxfordjournals.molbev.a040056, + author = {Rzhetsky, A and Nei, M}, + title = "{Theoretical foundation of the minimum-evolution method of phylogenetic inference.}", + journal = {Molecular Biology and Evolution}, + volume = {10}, + number = {5}, + pages = {1073-1095}, + year = {1993}, + month = {09}, + abstract = "{The minimum-evolution (ME) method of phylogenetic inference is based on the assumption that the tree with the smallest sum of branch length estimates is most likely to be the true one. In the past this assumption has been used without mathematical proof. Here we present the theoretical basis of this method by showing that the expectation of the sum of branch length estimates for the true tree is smallest among all possible trees, provided that the evolutionary distances used are statistically unbiased and that the branch lengths are estimated by the ordinary least-squares method. We also present simple mathematical formulas for computing branch length estimates and their standard errors for any unrooted bifurcating tree, with the least-squares approach. As a numerical example, we have analyzed mtDNA sequence data obtained by Vigilant et al. and have found the ME tree for 95 human and 1 chimpanzee (outgroup) sequences. The tree was somewhat different from the neighbor-joining tree constructed by Tamura and Nei, but there was no statistically significant difference between them.}", + issn = {0737-4038}, + doi = {10.1093/oxfordjournals.molbev.a040056}, + url = {https://doi.org/10.1093/oxfordjournals.molbev.a040056}, + eprint = {https://academic.oup.com/mbe/article-pdf/10/5/1073/11176298/11RZHE.PDF}, + } + + @article{10.1093/molbev/msaa015, + author = {Minh, Bui Quang and Schmidt, Heiko A and Chernomor, Olga and Schrempf, Dominik and Woodhams, Michael D and von Haeseler, Arndt and Lanfear, Robert}, + title = "{IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era}", + journal = {Molecular Biology and Evolution}, + volume = {37}, + number = {5}, + pages = {1530-1534}, + year = {2020}, + month = {02}, + abstract = "{IQ-TREE (http://www.iqtree.org, last accessed February 6, 2020) is a user-friendly and widely used software package for phylogenetic inference using maximum likelihood. Since the release of version 1 in 2014, we have continuously expanded IQ-TREE to integrate a plethora of new models of sequence evolution and efficient computational approaches of phylogenetic inference to deal with genomic data. Here, we describe notable features of IQ-TREE version 2 and highlight the key advantages over other software.}", + issn = {0737-4038}, + doi = {10.1093/molbev/msaa015}, + url = {https://doi.org/10.1093/molbev/msaa015}, + eprint = {https://academic.oup.com/mbe/article-pdf/37/5/1530/33386032/msaa015.pdf}, +} \ No newline at end of file diff --git a/topics/evolution/tutorials/abc_intro_phylo/tutorial.md b/topics/evolution/tutorials/abc_intro_phylo/tutorial.md new file mode 100644 index 00000000000000..2d50792b7a7142 --- /dev/null +++ b/topics/evolution/tutorials/abc_intro_phylo/tutorial.md @@ -0,0 +1,1319 @@ +--- +layout: tutorial_hands_on +title: Phylogenetics - Back to basics +zenodo_link: 'https://zenodo.org/records/11089653' +tags: + - phylogenetics + - evolution +level: Intermediate +questions: +- What information can I get from a phylogenetic tree? +- How do I estimate a phylogeny? +- How can I tell whether the phylogeny is reliable? +- How do phylogenetic models work? +objectives: +- Describe the basic concepts behind phylogenetic trees and how they are made +- Read and interrogate a phylogeny encountered in the literature +- Use standard tools to align a set of molecular sequences +- Assess the quality of a molecular sequence alignment and be able to modify it appropriately for phylogenetic analysis +- Use standard tools to estimate a phylogenetic tree based on a set of aligned molecular sequences +- Assess the reliability of estimated phylogenies with bootstrapping +- Explore phylogenetic signal using phylogenetic networks +time_estimation: 4H +subtopic: phylogenetics +priority: 0 +contributions: + authorship: + - mcharleston + editing: + - adamtaranto + - burkemlou + - PatCapon39 + +--- + + +Phylogenetics is essential for comparing biological species and understanding biodiversity for conservation. This tutorial discusses the basic principles and methods of phylogenetic inference and what you can learn from phylogenetic estimation. It is intended to help you make informed decisions about which methods to use in your research. + +{% include _includes/slides-youtube-selector.html id="dmCCQRliEMk" title="Introductory Lecture: Welcome and why phylogenetics?" slides="topics/evolution/tutorials/abc_intro_phylo-intro" %} + +Using real-life data and standard tools that are (mostly) available in Galaxy, the tutorial demonstrates the principles behind a variety of methods used to estimate phylogenetic trees from aligned sequence data or distance data. + +This is not just a "how to" tutorial, but is instead aimed at giving you a better understanding of the principles of phylogenetics and how the methods work. Maybe you've even built phylogenetic trees before but want to know more about the principles behind the tools. + +This tutorial does not cover workflows for taking read data to phylogeny or Bayesian methods. We've included recommended reading and tutorials on these topics in the resources section. + +We’ve designed this tutorial with flexibility in mind and so that you can focus on the sections that are relevant to you. It includes videos that explore key concepts, written descriptions of each concept, and exercises that demonstrate the methods in action using tools (mostly) available in Galaxy. The exercises are beginner level, but you should know how molecular sequence data is produced and what it looks like. Depending on how you like to learn, you can choose to watch the videos, read the text, work through the exercises, or a combination of the three! + +This tutorial is adapted from a 2019 workshop run by the Australian BioCommons and Professor Michael Charleston (University of Tasmania). + +> +> +> In this tutorial we will deal with: +> +> 1. TOC +> {:toc} +> +> **We will not cover:** +> +> * Workflows from read data to sequences +> * How to get an alignment (much: will use automated methods) +> * Bayesian phylogenetics: MCMC / BEAST / MrBayes +{: .agenda} + + + +## What is a Phylogeny? +![Hand drawn phylogenetic tree from Charles Darwin’s notebook with clades A, B, C, D branching from a common ancestor. Prefaced with handwriting that reads ‘I think’ and annotated with other illegible notes.](images/Darwin_tree.png "Charles Darwin's first sketch of an evolutionary tree. Source: Wikimedia commons"){: width="20%"} + +A *phylogenetic tree*, also called a *phylogeny*, is usually a tree-like structure, like [Darwin's famous sketch](#figure-1). The leaves or tips of the tree represent extant (living/existing) taxonomic entities like species, genera, or strains (in general called "taxa"). The lines connecting taxa describe the evolutionary relationships between them. +The intersections of lines correspond to hypothetical ancestral taxa. They represent branching events when species split into two new species, or a strain developed a phylogenetic important offshoot strain, etc. + +The phylogeny of a group of taxa is the best representation of their evolutionary relationships. +It is also the main basis on which we can build statistics comparing species: without the phylogeny, comparing species (or strains, genera, etc.) is not meaningful. + +As Theodosius Dobzhansky famously wrote, **"Nothing in biology makes sense except in the light of evolution"** {% cite 6ef38333-9ecd-3f40-8f3e-5f3d6df3c4b4 %}. + + +## Why we need phylogenetics + +There are many ways in which we can use phylogenetic analyses, from the most fundamental understanding of the evolutionary relationships that exist between a set of species, as in [Charles Darwin's famous sketch](#figure-1), to families: + +![Circular phylogenetic tree of hexapods (insects). Clades are colour coded and labelled with common names e.g. ‘Fleas’. Silhouettes of representative species are shown around the outside of the tree.](./images/Hexapoda_phylogenetic_tree.png "Phylogeny of extant hexapod families. {% cite PMID:25275450 %} reproduced under Creative Commons Attribution 4.0 International License") + + +and much bigger projects across all of life: + +![Large and complex phylogenetic tree that includes 92 named bacterial phyla, 26 archaeal phyla and all five of the Eukaryotic supergroups. Major lineages are assigned arbitrary colours and named, with well-characterized lineage names, in italics.](./images/nmicrobiol201648_Fig1_HTML.webp "A modern view of the tree of life based on sequenced genomes. {% cite PMID:27572647 %} reproduced under Creative Commons Attribution 4.0 International License"){: width="800"} + + + +Aside from gaining a fundamental understanding of biology, other reasons for inferring phylogenetic relationships include: + +- Designing vaccines, for example for SARS-CoV2 and influenza; +- Measuring phylogenetic diversity for guiding conservation efforts; +- Understanding coevolution; for example, around 70% of emergent human diseases have come from other species; +- Dating major evolutionary events to study the effects of environmental change on different species. + +> Gene trees, species trees reconciliation problem +> It's worth noting that getting the phylogeny from a set of genes – what we often call a **gene tree** – might *not* give us the true phylogeny of the species that house those genes, *even if we get everything right!* +> +> This happens because there are other processes that can influence the so-called "gene tree" such as: +> - lateral gene transfer events +> - gene duplication +> - gene loss and incomplete lineage sorting +> - recombination +> +> **The situation where gene trees and species trees differ is often called the "gene tree / species tree reconciliation problem".** +> +> While it is a fascinating topic, it is beyond the scope of this tutorial. Today we will work under the assumption (which is reasonable for the dataset we will use) that the gene tree will reflect the species relationships. +> +{: .comment} + +## Terminology +{% icon video %} From the root to the tips, watch the video to explore common features of phylogenetic trees. See how these features relate to evolutionary patterns and how outgroups can be used to find the root or common ancestor of extant species. + +{% include _includes/slides-youtube-selector.html id="gnWlMbGPYx8" title="Lecture: Phylogenetics Terminology" slides="topics/evolution/tutorials/abc_intro_phylo-terminology" %} + +Before we start building trees, let's define some terms. + +![Schematic of a phylogenetic tree where features such as nodes/taxa, edges/branches are annotated and colour coded. The root of the tree is at the top of the image and the tree branches into two clades as you move towards the bottom of the image. The clades are formed of hypothetical common ancestors and five extant taxa which are labelled as the ‘in group’. Two additional taxa are appended to the right hand side of the tree and are labelled as the outgroup.](images/TreeAnatomyWithOutgroup.png "Anatomy of a phylogenetic tree."){: width="100%"} + + + +It's common to call **phylogenetic tree** a **phylogeny**. + +Mathematically, a **tree** is a kind of **graph**, which has objects called **nodes** or **vertices** (lavender, white, and blue boxes in the figure above), connected in pairs by things called **edges** (green and orange lines in the figure above). + +Trees are a natural way to think about phylogenetic relationships. The nodes correspond to **taxa**, and the edges, also called **branches**, show the relationships between them, where taxa could be species, or lineages, genera, populations, or even individuals if we are considering something like a genealogy. + +Nodes with only one edge attached to them are called **leaves** (or **tips**; in white above) and correspond to taxa with no descendant taxa in the tree. These taxa might be from fossils, or, be currently living, in which case they’re referred to as **extant**. + +Internal nodes (in lavender above) correspond to hypothetical common ancestors of the extant taxa: the set of descendants that each one has determines the tree. + +Many phylogenies have a special node assigned as the common ancestor of all the taxa represented by the leaves in the tree. This node is called the **root** (in blue above). When this is the case, a natural direction is implied from the root to the tips, going forward in time. We call such trees and phylogenies **rooted**; if there is no root, they are called **unrooted**. + +*The majority of phylogenetic inference methods produce unrooted trees, but rooted trees are more useful.* + +In a rooted phylogeny, all the leaves that are descendant from any given node form a **monophyletic clade**, or often just “**clade**” (monophyletic means “one tribe (of) origin” from the Greek). + +One way to determine where the root of a tree belongs is to include an **outgroup** in the data, which is a set of taxa that are definitely not within the clade of interest (which is then called our **ingroup**) but which share a common ancestor with that clade. A good outgroup won’t be too distantly related to our ingroup, because if it’s too distant, choosing where it should connect to the ingroup will be hard, ultimately resulting in a guess. + +You can see in the diagram above that the connection of the ingroup to the outgroup could be from multiple locations. Once the unrooted tree is created, using combined data from ingroup and outgroup taxa, we can confidently say that the root is on the branch connecting our ingroup to our outgroup: + +![A schematic of an unrooted phylogenetic tree. It shows the relationship between an ingroup of 5 taxa on the left and an outgroup of two taxa on the right as a series of boxes connected by edges (lines). The taxa appear at approximately the same level across the image. An arrow points to an edge connecting the ingroup and outgroup where it is likely that a common ancestor exists.](images/TreeAnatomyUnrooted.png "Rooting a tree"){: width="100%"} + +We can then imagine lifting up the unrooted tree at the branch connecting our outgroup and ingroup -- that is our best guess at the hypothetical ancestor of all our taxa and gives us a good indication of the branching order of our ingroup (and the outgroup): + +![A schematic of an unrooted phylogenetic tree. It shows the relationship between an ingroup of 5 taxa on the left and an outgroup of two taxa on the right as a series of boxes connected by edges (lines). An arrow points "Lift here" points to an edge connecting the ingroup and outgroup which is a best guess at the location of the hypothetical ancestor](./images/TreeAnatomyLiftHere.png "'Lifted' tree demonstrating hypothetical ancestor and branching order") + +Phylogeny estimation can be thought of as inferring a collection of compatible hypotheses about **monophyly** -- that is, statements that groups of taxa descendant from a common ancestor are each others' closest relatives in the tree. + +The tree above is called a **binary tree**, because each internal node branches into *two* descendants. It is a very common assumption that trees are binary, and we make that assumption in this tutorial. In fact, it is often very hard to come to a means by which a phylogeny could be truly *non*-binary: in most cases, this is just due to our inability to resolve the tree completely. + +# Building a tree +## Basic Methodology + +So, how do we estimate phylogenetic trees? + +We start with the leaves of the tree which can be living (extant) or older taxa: + +![Five extant taxa (living species) are depicted as five boxes equally spread from left to right. These taxa are contained within a larger blue box which is annotated with the words “Leaves of the tree: these can be extant (living) or older taxa”.](images/WeJustHaveLeaves.png "The 'leaves' of a tree (extant or older taxa) are the starting point for buidling phylogenies"){: width="100%"} + +There are several ways to estimate a tree, such as: + +1. Go with what we think is the case already (this is not recommended!) +2. Attempt to build a tree based on similarity and dissimilarity, with tools such as Neighbor-Joining (NJ) or FastME (we will do this later in the tutorial) +3. Use a score function, such as Parsimony or Maximum Likelihood, to build potential trees and find the best one (we will do this later too) +4. Something else entirely (Perhaps networks or even inferring evolution based on the parasites of your species of interest!). + +**Warning**: Finding an optimal tree is *hard*! + +First and foremost, **phylogenetic inference is a statistic _estimation_ process.** + +Different estimates of the phylogenetic tree relating a given set of species may differ, even if no errors were made. + +It is generally not possible to prove that any tree inferred is *correct* -- since we cannot go back in time and observe speciation events. + +> Common evolutionary assumptions used in phylogenetic estimation +> +> We will (mostly) make these assumptions in this tutorial! +> +> 1. Evolution is “memoryless.” This assumption is that the future evolutionary trajectory of an organism is not affected by its past. This means we can use the powerful mathematics of Markov processes. +> +> 2. Phylogenetic relationships can be correctly represented by a tree! This isn't *always* assumed, but it is very common. Trees are a very attractive representation of evolution, and it is part of our language: "The tree of life" is a common phrase. However evolution is not always explained by a tree-like, "branching" process as other events such as hybridisation and sharing of genetic material can influence envolutionary processes. +> 3. The molecular clock assumption is that sequences in a clade evolve at about the same rate. This is known to be wrong, but is useful. For instance, there is variation in evolutionary rate between lineages, but if this variation is not significant, we can ignore it and use simpler models, to better leverage the phylogenetic information in the data. +> 4. Lineages don’t interact – once they have speciated, they are independent of each other. This isn't always the case and we know that biological lineages *do* interact with each other -- but our methods are generally not able to manage such complexity. The vast majority of methods make this assumption, particularly if the evolution is also assumed to be tree-like. +> +{: .comment} + + +### Challenges + +Phylogenetic Inference is **Hard**. +One of the things that make it hard is the sheer number of possible trees that can describe relationships among species. + +The number of rooted binary trees grows as 1, 3, 15, 105, 945, 10395... in fact the formula for this number for $$n$$ taxa (we use `taxa' in general, since not all phylogenetic inference is at the species level) is $$(2n-3)!! = (2n-3)(2n-5)...(3)(1),$$ which grows as fast as $$2^{n}n!$$. + +The table below gives you an idea of the scale we are dealing with. + +*The Number of Unrooted Binary Trees* + +| *n* | # trees | notes | +|--------------|------------|-------------| +| 3 | $$3$$ | trivial to check | +| 4 | $$15$$ | enumerable by hand | +| 5 | $$105$$ | enumerable by hand on a rainy day | +| 6 | $$945$$ | enumerable by hand during lockdown | +| 7 | $$10395$$ | easily searched by computer | +| 8 | $$135135$$ | a bit more than the number of hairs on your head | +| 9 | $$2027025$$ | population of Sydney living west of Paramatta | +| 10 | $$34459425$$ | comparable with the number of possible tickets in a typical lottery | +| 20 | $$\approx 8.2\times 10^{21}$$ | getting slow for computers even with branch-and-bound | +| 48 | $$\approx 3.21\times 10^{70}$$ | number of particles in the universe-ish | +| 136 | $$\approx 2.11\times 10^{267}$$ | number of trees to choose from in the first "Out of Africa" data set | +|---|---|---| + + + +## Calculating distances + +Building a tree begins with a set of *distances*, which record how different the taxa are from each other. +Distances have very desirable properties, that can be summarised as follows: for any objects $$ x $$, $$y$$, $$z$$, writing $$d(x,y)$$ means the distance from $$x$$ to $$y$$ etc. These properties are: + * *non-negativity* -- distances can never be negative, and in fact we treat two things as identical if they have a distance of 0 between them. + * *symmetry* -- the distance from $$x$$ to $$y$$ is the same as the distance from $$y$$ to $$x$$; that is, $$d(x,y) = d(y,x)$$. + * the *triangle inequality* -- there are no short-cuts! The distance from $$x$$ to $$z$$ is always *at most* the distance from $$x$$ to $$y$$ plus that from $$y$$ to $$z$$; that is, $$ d(x,y) + d(y,z) \leq d(x,z)$$. + + In phylogenetics terms, we like distances to represent something like time and we can assign lengths to branches (see the [Tree Anatomy diagram](#figure-4) above). + +Distances can be calculated based on a variety of data. Here is a flow-chart of the process: + +![Flow chart illustrating how sequence alignment data or dis/similarity measures are used to calculate and calculate phylogenetic distances. Colours and shapes are used to differentiate different sections of the flowchart, guiding the viewer through each step from left to right. The flowchart begins with Sequence Alignment or Dis/Similarity Measures. These are used to form a distance matrix (D) which is used to select two nodes (x and y) forming a new node z. The distance matrix is updated with the new node z until no further nodes can be formed.](images/TreeConstruction.drawio.png "Tree construction flow-chart"){:width="100%"} + +The blue boxes on the left show some of the input data forms. The most commonly used kind of data in modern phylogenetics is *aligned molecular sequences* -- typically, DNA, RNA, or Amino Acids (AA) from equivalent (homologous) genes in the species of interest. We focus on this form of molecular phylogenetics in this tutorial. + +Other input data forms are distances or dissimilarity measures based on molecular-based measures like DNA-DNA hybridisation, gene presence/absence, and morphology (physical dimensions). We will not discuss this kind of data in this tutorial. + +Aligned sequences can be converted into distances (green box above), using models for how the sites (i.e. specific nucleotides or amino acids) have evolved. +These distances can be expressed as a matrix _D_, which becomes the input for distance-based methods. + +Distance-based methods (orange boxes) use algorithms to select a pair of taxa, or clades, to join together to make a new clade. Once that decision is made, the two taxa / clades that have been joined are replaced with the clade that the two of them make together as seen in the [figure below](#figure-7). + +![Schematic illustrating how taxa are combined into clades. The relationship between three taxa and two clades are represented by green lines. The image consists of two main steps: Taxa 1 and 2 are combined into Inferred Clade 1. Clade 1 and Taxon 3 are then combined to form a new clade. A dashed green line indicates that this process continues until all taxa and clades are joined.](images/JoiningCladesForTreeConstruction.png "Joining clades for tree construction"){: width="60%"} + + +## Let's build our first tree + +In this tutorial we will use a set of *Anolis* lizard DNA sequences, from {% cite 10.1080/106351599260283 %} to trial some phylogenetic methods. +The sequences are from the mitochondrial NADH dehydrogenase subunit 2 gene (ND2) and five transfer RNA (tRNA) genes, with an average sequence length of 1419.109 and a range of [1329,1727]. + + + + + + + +We are using a relatively small set of sequences to ensure the methods run quickly for the purposes of the tutorial. + +In the real world, a phylogenetic analyses often span hundreds, or even thousands, of taxa. Phylogenetic estimation on this many sequences is computationally very intensive, and can take weeks of time even on a high-performance computer. + +### Get the data + + + + +> Obtain your data +> +> 1. Make sure you have an empty analysis history. Give it a name. +> +> {% snippet faqs/galaxy/histories_create_new.md %} +> +> 2. Import the file anolis-raw.fst from [Zenodo](https://zenodo.org/records/11089653/files/anolis-raw.fst) or from the shared data library. +> ``` +> https://zenodo.org/records/11089653/files/anolis-raw.fst +> ``` +> You may need to refresh your history to show the data. +> If the paste/fetch link does not work for you, you can download it to your own computer and then upload it using the "Upload" button at the top of the left panel in Galaxy. +> +> {% snippet faqs/galaxy/datasets_import_via_link.md %} +> +{: .hands_on} + +You can click on the 'eye' icon {% icon galaxy-eye %} on the right to see the unaligned data (go ahead!) but the view isn't very informative. This is the raw FASTA file, with the symbols A, C, G, T representing nucleotides. You can see that the sequences are different lengths. + +**FASTA** or **Fasta** format (pronounced to rhyme with pasta) format is is commonly used as input to phylogenetic inference programs and has a very simple structure, as follows: + +1. Each sequence has a name, which appears on its own line after a '>' sign +2. The next line(s) contain the sequence; continuing either until the next sequence name line, or the end of the file. + +The example below has four sequences in it named Taxon_1, Taxon_2, etc, each with a short set of characters representing DNA sequences. + + +``` +>Taxon_1 +ACTGCGTTAGGTCTAGCC +>Taxon_2 +GATCTACTGCTTTAGGTTGAGCC +>Taxon_3 +ACTGCTCTAGCACTGAGCCCA +>Taxon_4 +ACTTGGCGTAGCCGGAGGCC +``` + + + + + +> FASTA format for multiple sequence alignments +> +>The Fasta format can also include symbols such as a question mark '?' for missing data, or hyphen '-' to indicate an insertion or deletion events, collectively *indels*. It is common to represent a multiple sequence alignment in Fasta format with these symbols. It is also a common format for storing high-throughput read data, but *without quality scores* -- if you want to include read quality you would use FASTQ format. +{: .tip} + +> View your data +> Let's view the unaligned sequence in a more understandable form. +> +> 1. Click on the title of your file to see the row of small icons for saving, linking etc: +> ![Screenshot of Galaxy icons. Seven small blue icons are shown on a green background. From left to right they are: floppy disk, link, information, redo, bar chart, flow chart and a question mark.](./images/LittleJobIcons.png) +> +> 2. Click on the **visualise icon** {% icon galaxy-visualise %} and then select the **Multiple Sequence Alignment tool**. +> You should see something like this: +> +> ![Screenshot of sequence visualisation output from Galaxy. Fifteen Anolis DNA sequences are arranged in rows. The nucleotides are colour coded and arranged in columns: A(blue), T (green), C (pink), G (orange). The top half of the image shows approximately 50 bases of each sequence. The lower half of the image shows a zoomed out heatmap-like image of a larger portion of the sequences.](./images/UnalignedAnolis.png){:width="600"} +> +> Play around with the view. You can change colour schemes and add or remove various elements. Good colour schemes for nucleotide data are "Clustal2" and "nucleotide". +> +> If you cannot see the slider at the top (for moving left or right in the view) you will need to check the "Show residues indices" in the "Vis. elements" drop-down menu. +> +> Do not adjust the scale slider! Currently, this breaks the page. +{: .hands_on} + +> Understanding the FASTA file +> 1. How many sequences are there in your data? +> 2. How long is the longest sequence, and what is it? +> 3. What about the shortest sequence? +> +> > +> > There are 55 sequences. The longest is from Anolis paternus with length 1729 nucleotides; the shortest is A. luciae with length 1252. +> {: .solution} +{: .question} + +# Sequence Alignment + +{% icon video %} Watch the video to find out +[0:00] What ‘alignment’ really means and why it is useful for phylogenetics; [6:22] What makes a good alignment; [12:15] How pairwise sequence alignment and dynamic programming work; [33:47] How the multiple sequence alignment algorithms work; and to +[40:00] explore multiple sequence alignment of the Anolis sequences with SeaView. + +{% include _includes/slides-youtube-selector.html id="FUc2A2r1LGY" title="Lecture: Sequence alignment" slides="topics/evolution/tutorials/abc_intro_phylo-msa" %} + + +Molecular sequences must be aligned before they can be used to build phylogenies. + +Aligning sequences amounts to finding the nucleotide positions (sites) that we can be confident have the same evolutionary history: they correspond to each other across species and can be considered to have evolved from the same common ancestor. +A good clue to identify these sites, which are called homologous, is that they are well conserved, with only a few changes. + +Below is an example of an alignment. On the left are the sequences in Fasta format. +In the middle we see an alignment of those sequences, which has gaps in it that help line up the sites so that more of them agree. +A sign of a "good" alignment is one in which the colours line up vertically. The overhanging parts can be removed as seen in the "trimmed version" on the right. + +![Table containing DNA sequence from four taxa arranged in rows. Three columns show the sequences in ‘Unaligned’, ‘Aligned’ and ‘Trimmed’ states. The nucletodies are colour coded A (green), T (pink), C (blue) and G (orange). In the “Unaligned” column, the sequences are continuous. In the ‘Aligned’ column gaps are introduced and represented as dashes so that the sequences align vertically. In the “Trimmed” column overhanging portions of the sequences are greyed out to indicate that they would be trimmed.](./images/ToyAlignmentAndTrim.png "Sequence alignment"){:width="700"} + +While aligning *two* sequences is "easy", in the sense that an optimal alignment between two sequences can be found in a reasonable amount of time, optimally aligning multiple sequences is *computationally intractable*. Multiple sequence alignment is a complex process and many methods have been developed to tackle this challenge. + +## Aligning sequences with MAFFT + +Today you will be aligning sequences using a modern multiple alignment program called **MAFFT**, which is available on Galaxy. + + + + +> Sequence alignment with MAFFT +> +> 1. In Galaxy, search for and select the {% tool [MAFFT](toolshed.g2.bx.psu.edu/repos/rnateam/mafft/rbc_mafft/7.508+galaxy1)%} from the tool finder on the left, and run it with the following parameters: +> - {% icon param-file %} In the MAFFT tool, the **Sequences to align** field should already be filled with your unaligned data. If it isn't, select it using the drop-down menu. +> - In the **Data type** field select "_Auto detection_". +> - Leave the **MAFFT flavour** as "_fftns_". This is a good default. +> - In the **Matrix selection** select "_No matrix_". +> 2. Click "**Run tool**". +{: .hands_on} + +Here is a visualisation of the resulting alignment. Note that the colours are now vertically aligned: + +![Screenshot of aligned sequences. Multiple DNA sequences are represented in rows and aligned vertically. Vertical columns of purple (G), blue (C), red (T) and green (A) indicate alignment of nucleotides across sequences. Dots replace the letters as an abbreviation where all nucleotides are the same in a given column.](./images/MEGA_alignment.png "Aligned sequences"){:width="600px"} + +> Inspecting your alignment +> +> 1. Click on the title of the completed MAFFT job to show the row of small icons for saving, linking etc. +> ![Screenshot of Galaxy icons. Seven small blue icons are shown on a green background. From left to right they are: floppy disk, link, information, redo, bar chart, flow chart and a question mark](./images/LittleJobIcons.png){:align="center"} +> 2. Click on the **visualise icon** {% icon galaxy-visualise %} +> 3. You will be presented with a couple of options. Select "**Multiple Sequence Alignment**". +> *Your alignment is displayed!* +> The colour scheme is horrible to start with because it's not automatically detecting the data as DNA sequences. Click on the "Color scheme" button and select "**Clustal2**" for a nicer picture. +> 4. You can click and drag the display of nucleotides in the upper panel, and when you go far enough to the right you'll see some gaps have been introduced by MAFFT. On either side of each gap you should see that the nucleotides match up pretty well. This is because the MAFFT algorithm has identified that these site are homologous, and that either an insertion event has happened for a group of sequences, or a deletion happened in the others. +> 5. You may be tempted to play around with "Vis. elements" and show the scale slider - don't! It's currently broken and will crash your browser tab :(. +{: .hands_on} + +You should ALWAYS visually check your alignment to see if it makes sense. +A tool you can use on your own computer is [SeaView](https://doua.prabi.fr/software/seaview). + + + +# Distance-based phylogenetic inference +{% icon video %} Watch the video to delve into the mathematics of building phylogenetic trees from distances. [0:00] Why use distances and how they relate to the structure of a phylogenetic tree +[3:05] Types of distances and distance matrices +[11:31] The mathematics behind the Jukes-Cantor/JC69 and HKY85 models [16:04] An example of building a tree from ultrametric distances [21:01] Building a tree from non-clocklike distances with the Neighbour-joining method [25:59] Real life data and limitations for building trees from distances. + +{% include _includes/slides-youtube-selector.html id="t68H2Ilc4mI" title="Lecture: Phylogenetic Trees" slides="topics/evolution/tutorials/abc_intro_phylo-trees" %} + +We are going to build our first tree, using a very common method called _Neighbor-Joining_. This method was created in the 1980s by Saitou & Nei ({% cite 10.1093/oxfordjournals.molbev.a040454 %}). + + + +## Building a Neighbor-Joining Tree + +The Neighbor-Joining (NJ) algorithm is a standard method that takes a set of distances between taxa as input, and sequentially connects them into larger and larger clusters until all taxa have been joined. + +NJ is rarely used as a complete tool for phylogenetic analysis. Although it is quite accurate and fast, there are other fast methods that can be then applied to modify the NJ tree and create a better one. + +The FastTree2 program that we are using does this. First it creates a "rough" NJ tree, and then modifies it to optimise a quantity called *Minimum Evolution* (ME) (more on this later). A detailed description of how FastTree works is available from [Microbes online](http://www.microbesonline.org/fasttree/). + +> Build a Neighbour-Joining Tree with FastTree +> +> 1. Search for the {% tool [FastTree](toolshed.g2.bx.psu.edu/repos/iuc/fasttree/fasttree/2.1.10+galaxy1) %} in the tool finder on the left, and run it with the following parameters: +> +> - *"Aligned sequences file (FASTA or Phylip format)"*: `fasta` +> - {% icon param-file %} *"FASTA file"*: `outputAlignment` (output of **MAFFT** {% icon tool %}) +> - *"Protein or nucleotide alignment"*: `Nucleotide` +> - *"Nucleotide evolution model"*: `Jukes-Cantor + CAT` +> - *"Show advanced options"*: `Yes` +> - *"Use constant rates?"*: `Use constant rates.` +> - *"Turn off maximum-likelihood."*: `Yes` +> +> 2. Click on "Run tool". +> +{: .hands_on} + + + + +It won't take very long for FastTree to build your tree. +But when it's done, how can you see it? + +Clicking on the 'eye' icon {% icon galaxy-eye %} of the output doesn't at first appear to be very illuminating: it's just a parenthesised list of taxon names and numbers. +This is **Newick Format**, and it's worth knowing at least a little of what it means. + +* Each matched pair of parentheses denotes a **cluster** or **subtree**: "(A,B)" means that A and B are each others' closest relatives (also called _sister taxa_). +* A number after a cluster (so, after a closing parenthesis) is a **label** for that cluster. In the output from FastTree, this label is an indicator of the support for that branch. +* If there is a colon ':' followed by a number, then this is the **branch length** for the subtree. + + +![Two rooted phylogenetic trees (a) and (b) are shown. Both trees have three taxa, labelled A, B and C as the leaves and a root y. Taxa A and B form a clade and connect to y via the internal node x. Taxon C connects directly to y. Distances between nodes are shown as numbers. For tree (a): y:C = 6 ; y:x = 2; x:A = 3 ; x:B =2. For tree (b): y:C = 5 ; y:x = 3; x:A = 3 ; x:B =2](./images/NewickExplained.png "Newick Format") + +The rooted, 3-taxon trees above have three taxa, labelled A, B and C. Two of the internal nodes have been labelled (x and y), but it isn't necessary to do so in general (for example, if you wanted to use the label for something like support of each branch, as does FastTree). + +In both trees, A and B are sister taxa, and branch lengths are indicated near each branch: you can see how the branch lengths are above each cluster, including the individual taxa (the "leaves" of the tree), but not above the root. + +The Newick format for the tree on the left is "((A:3, B:2)x:2, C:6)y;" and for the one on the right it is "((A:3, B:2)x:3, C:5)y;". The number after each colon is the length of the branch above it (closer to the root). + +Note that these two trees are very similar: they only differ in the position of the root (y), either being distance 2 from node x, or being 3 from it. The distance between any two nodes in the tree is the sum of the branch lengths on the path connecting them, so for the trees above, the distance matrix is + +| | A | B | C | +| --- | :--: | :--: | :--: | +| A | 0 | 5 | 11 | +| B | 5 | 0 | 10 | +| C | 11 | 10 | 0 | + +Ideally, these will reflect the actual input distances, but such distances are based on messy real data, and do not necessarily obey this ideal. +That is why methods like FastTree are employed to find a tree with the best possible agreement between the distance inferred, and that calculated from sequence data. + +> Visualising a tree +> To visualise your tree in Galaxy: +> 1. Search for the {% tool [Newick Display](toolshed.g2.bx.psu.edu/repos/iuc/newick_utils/newick_display/1.6+galaxy1) %} in the tool finder on the left, and run it with the following parameters: +> +> - *"Newick file"*: {% icon param-file %}: `tree.nhx` (output of **FastTree** {% icon tool %}) +> - *"Branch support"*: `Display branch support` +> - *"Branch length"*: `Display branch length` +> - *"Choose an ouput format"*: `PNG` +> 2. Click on the **display icon** {% icon galaxy-eye %} next to the title of the completed Newick Display job to display your tree. +> +> +> +> +> Notice that there are quite a lot of long branches adjacent to the extant taxa (leaves) and that these branches are much shorter near the centre of the tree. +> Note: **Short branches are much harder to get right.** +> +> Additional visualisation options +> 1. To build a radial tree, rerun Newick Display and choose the *"Draw a radial tree"*: `Yes` option. +> 2. Alternatively you can visualise your tree outside of Galaxy by downloading your FastTree output and using software such as [SplitsTree](https://software-ab.cs.uni-tuebingen.de/download/splitstree4/welcome.html) or [FigTree](http://tree.bio.ed.ac.uk/software/Figtree/). These tools provide additional options for interactively exploring and customising the appearance of your tree. +> +>(If you wish you may of course re-run FastTree and allow it to seek a Maximum Likelihood tree -- maybe once you've learned more about Maximum Likelihood.) +> +> +{: .hands_on} + + +# Searching for the "best" tree + +The other way we can estimate a phylogeny is by choosing some kind of score of "goodness" and then searching the entire set of possible trees for the tree (or trees) that optimises this score. +Note that such scores are "surrogates for truth" in that we *hope* the optimal score will correspond to the true tree, but it is not necessarily the case. In many analyses we therefore use *multiple* methods, in the hope that they will give us the same answer. Minimum Evolution (ME), Maximum Parsimony (MP), and Maximum Likelihood (ML) are common such score functions. + +> Data vs Method +> If your conclusion changes based on your reasonable choice of analytical method, then perhaps your data are not adequate. +{: .comment} + + +## Minimum Evolution (ME) + +Minimum Evolution is the idea that the sum of the branch lengths should be as small as possible to still account for the distances between the leaves of the tree, in that the sum of squared differences between the distances implied by the tree and the observed distances from the data, is minimised. You can read more about this in an article by [Rzhetsky and Nei](https://academic.oup.com/mbe/article/10/5/1073/1037508){%cite 10.1093/oxfordjournals.molbev.a040056 %}. + +There are some variations on this ME criterion and FastTree uses an approximation of one of them to find good trees. + +## Maximum Parsimony (MP) and Parsimony Length + +Most tree estimation methods output trees with branch lengths that correspond to the amount of evolutionary "work" that has to be done to turn one sequence into another. + +This can be given as the *minimum number of character state changes required* -- the so-called *parsimony length* -- to convert the (hypothetical) sequence at one end of a branch to that at the other end. +The Maximum Parsimony method is based on this approach. + + +Finding the parsimony length of a site pattern is easy and fast due to a clever algorithm created by Walter Fitch; hence, finding the score (the "goodness") of a tree is fast. But finding the tree that minimises this score is still computationally intractable, because the space of trees is so huge. + +The Maximum Parsimony method for finding the "best" tree is to search tree space for the tree (or trees) that *minimises* the parsimony length of any tree for that alignment. Note that when parsimony is *maximised*, this means the fewest possible changes required, so the *minimum* length. + +We do not use the Maximum Parsimony method in this tutorial. + +## Maximum Likelihood (ML) +{% icon video %} Watch the video to learn about [0:00] the concept of likelihood and how it lends statistic rigour to phylogenetic analysis as well as [26:45] how it is applied in models like Jukes-Cantor, HKY85 and GTR models to select the “best tree” from your data. [34:16] Wander through tree space to find the best tree using Nearest Neighbour Interchange, Subtree Pruning and Regrafting, and Tree Bisection and Reconnection tree perturbations and [45:40] go “hill climbing” with tree-search algorithms. [48:52] Finally, explore the output of IQTree for the Anolis dataset used in the tutorial. + +{% include _includes/slides-youtube-selector.html id="0WzQLAl9h8Q" title="Lecture: Maximum Likelihood & Trees" slides="topics/evolution/tutorials/abc_intro_phylo-mle-trees" %} + +Likelihood is the most statistically defensible phylogenetic inference method. +It is based on the idea that the tree that has the highest probability of producing the sequences at the tips of the tree is the tree that is the "most likely" to be correct: this is called the Maximum Likelihood (ML) Tree. + +Likelihood is *not* the same as probability, though they are often confused with each other. However, it is *proportional* to the probability that the tree is correct, out of the set of possible trees and models you are considering. + +One major, almost ubiquitous, assumption about molecular sequence data is that each site evolves independently of all other sites. Biologically, this isn't always the case, but in practice, this makes things much more tractable, and we still have a good chance of getting the tree(s) right. + +Another assumption we make is that the substitution rate -- the rate at which changes of nucleotide at a given position in the sequence happen -- is only dependent on the current state, i.e., we do not care about how a sequence came to be what it is, only what the sequence is now, to determine what are the probable evolutions of it. +This seems much more biologically reasonable and makes this into a Markov process, which in turn enables a lot of calculations to be made simply. + +### Searching for trees and their branch lengths + +When dealing with nucleotides A, C, G, T, there are 4x4 rate matrices with different names: Jukes-Cantor (JC69) with one parameter, Hasegawa-Kishino-Yano (HKY85) with five parameters, and many more, that define the rates at which nucleotides change. +There are other 20x20 matrices for amino acids, and even 64x64 matrices for codons. + +To convert from a rate to a probability, hence giving us a likelihood, requires that we have a branch length. Then, we can calculate the probability under a given model, and after a specific time interval, of going from one nucleotide to another. We multiply these site probabilities to calculate the probability of going from an entire sequence to another. +Thus, looking for the optimal tree under likelihood requires we also search for the best-fit **branch lengths**, as well as looking for the best **tree**. + +Maximum Likelihood is therefore the **slowest** tree inference method we discuss in this tutorial. + +### Models of sequence evolution + +*If you are in a hurry to do the phylogenetic analysis you can skip this section and go to the next [Hands-on: running IQ Tree](#estimating-a-maximum-likelihood-tree-with-iqtree).* + + +> Likelihood models +> Likelihood is based on probability, so requires we choose a probabilistic model for the evolution of sequences. +> The simplest model for DNA is that each nucleotide has the same rate of change, and that all nucleotides appear with equal frequency (called the base frequencies) of 25%, 25%, 25%, 25%. This is the Jukes-Cantor (JC69) model published in 1969, and this model has just one parameter. +> +> More biological realism allows for different nucleotide proportions outside the uniform 25% rate. This is the Felsenstein 1981 model, known as F81, and it has three more parameters for the rates (not four: given the first three base frequencies, this defines the other one). +> +> A next level of sophistication is the Hasegawa-Kishino-Yano model (HKY85) published in 1985, which acknowledges that transitions (changes of state within the purines A, G or within the pyrimidines C, T) occur more readily than transversions (changes from purine to pyrimidine or vice versa). +> Hence the HKY85 model has an additional parameter of these different types of subtitution: it can be represented by the substitution rate matrix below:\ +> \ +> $$ +> Q_{HKY} = \left[\begin{array}{c@{ }c@{ }c@{ }c} +> \ * & \kappa\pi_{G} & \pi_{C} & \pi_{T} \\ +> \kappa\pi_{A} & * & \pi_{C} & \pi_{T} \\ +> \pi_{A} & \pi_{G} & * & \kappa\pi_{T} \\ +> \pi_{A} & \pi_{G} & \kappa\pi_{C} & * \\ +> \end{array}\right] $$ +> +> \ +> In the above, the $$\pi$$ symbol is used for the base frequencies, and a $$\kappa$$ symbol is used for the transition/transversion ratio parameter. The asterisk "*" is a short-hand to mean "the sum of everything else in the row." +> +> A more general model still is the *General Time-Reversible* model (GTR), in which each substitution type has its own rate. It still keeps the property that a substitution from $$x$$ to $$y$$ has the same probability as one from $$y$$ to $$x$$ (this comes from the "reversible" property) but otherwise all rates are independent of each other:\ +> \ +> $$ +> Q_{GTR} = \left[\begin{array}{c@{ }c@{ }c@{ }c} +> \ * & \alpha\pi_{G} & \beta\pi_{C} & \gamma\pi_{T} \\ +> \alpha\pi_{A} & * & \delta\pi_{C} & \epsilon\pi_{T} \\ +> \beta\pi_{A} & \delta\pi_{G} & * & \eta\pi_{T} \\ +> \gamma\pi_{A} & \epsilon\pi_{G} & \eta\pi_{C} & * \\ +> \end{array}\right] +> $$ +> +> \ +> A further level of sophistication is the recognition that some sites may be constrained from changing at all. For example, there may be some that have a critical role in fixing the correct amino acid for a protein to function. This addition to the above methods is known as "invariable" sites and is usually represented by a "+I" appended to the model name. +> +> The last level of sophistication is that some sites may evolve faster than others, even if they are under the same kind of model with the same parameters in the matrix $$Q$$. +> The most common way to allow for this is to imagine that the relative rate for a particular site is drawn from a Gamma $$\Gamma$$ probability distribution, which has some nice properties like allowing most sites to change very slowly and permitting some to change rapidly. +> This is usually denoted by a "+$$\Gamma$$" or "+G" appended to the model name. +> +> There are **many** more models, with many more parameters and constraints. Finding the best one to fit a data set is a complex task! +> +> Fortunately there are tools that help determine the most appropriate model for a given data set, such as the Akaike Information Criterion (AIC) and some variations of that. +> +> The program IQTree, which we use later, performs a step to determine which model is most appropriate for your data set, based on AIC and other schemes to avoid over-fitting while still having as good a fit to your data as possible. +> In that step, trees, and their likelihoods based on your data, are estimated for many different models. Each yields a likelihood score but rather than simply take the model that maximises the likelihood, over-complex models are penalised, to avoid over-fitting. One such penalty function is the AIC; there are others. +> +> There are whole books describing this process, and it's clearly well beyond the scope of this tutorial to go into such depth, but now you should have some appreciation of what is going on behind the scenes when an Maximum Likelihood method is looking for the best model for your data. +> +{: .details} + + + +# Assessing the Quality of trees + +A tree-building method will, of course, build you a tree. + +But what if your data are not even *from* a tree? Or, what if the data are from sequences that are so distantly related that they are virtually independent of each other and are essentially random? + +It is important that, once you have estimated a tree, you assess how reliable it is. + +Remember that a phylogeny is a *collection of hypotheses of relatedness*. Each branch separates some of the taxa from the others, and if the branch is above a subtree it corresponds to a hypothesis that the taxa in that subtree are *monophyletic* with respect to the other taxa in the rest of the tree (as shown in [Figure 1](#figure-1).). + +This means that it is meaningful to assess the reliability of *branches* of your tree, in addition to just assessing it overall. + + +## Resolution + +A good phylogenetic tree is one that is *well resolved* -- that is, every time a lineage branches, it forms two new branches. +Equivalently, every internal node has three edges touching it. + +An unresolved node *may* be a true representation of the branching pattern of a group of lineages. For example, this is generally the case for very rapid diversification, such as during an island radiation (species arrives in new place with lots of niches; diversifies incredibly quickly). + +In phylogenetics unresolved nodes are more often due to a lack of resolving power in the data, so the phylogenetic method cannot choose the branch ordering: + +![Four simple phylogenetic trees composed of three taxa (A, B, C) at the bottom that are connected to a common ancestor at the top. The first three trees group the taxa into two clades but differ in the branching patterns. Arrows indicate that they are so similar that there is not enough information to choose between them. The fourth tree is ‘unresolved’ where taxa A, B, C remain in a single clade derived from a common ancestor.](./images/Unresolved.png "Unresolved nodes"){: width="400px"} + +If there are many unresolved branches in the phylogeny, this is an indication that there is not enough information in your data: you'll need to obtain more. + +## Bootstrapping + +A very common (and useful) method for dealing with unresolved branches is called *bootstrapping*, which is a technique that has a solid basis in statistics (not just phylogenetics). The idea is that one resamples with a replacement data set to create a "pseudoreplicate" that is analysed in the same way as the original data. This process is then repeated many times to create a distribution. Bootstrapping is known to be a is a good way to measure the internal consistency of a data set, and its use in phylogenetics is well established. + +The naive method for bootstrapping is called "non-parametric" and works by effectively resampling the patterns at each site in the alignment, creating a pseudo-alignment of the same total number of sites, then re-building the tree. + +IQTree has a very - *ultra* - fast bootstrapping method that is cleverer and works a bit better than the naive method. When we use IQTree in the next part of the tutorial, we will also do bootstrapping on the tree. See {% cite 10.1093/molbev/msaa015 %} for details on the method. + +Bootstrapping can be done on any inference method. We will use the likelihood method that searches for the tree and branch lengths that maximises the likelihood for (1) our actual data, and then (2) for each of the pseudoreplicates, noting for each of these which branches occur in the best trees found. + +By keeping track of which branches occur in the best trees found for each of the pseudoreplicates, we can note how often the branches in the best tree for our *actual* data occur in the resampled data. If they occur a lot -- say, 80% of the time or more -- then we can be fairly sure that that branch is well supported by the data. + +Bootstrap values therefore appear for each branch, and are most often expressed as a percentage or proportion. Branches at the leaves that occur in every possible tree so these would get 100% bootstrap values every time, and don't tell us anything. + +(Note: FastTree does not do bootstrapping natively, but can in conjuction with other tools ([see details](http://meta.microbesonline.org/fasttree/ )). It's fiddly to do this so we will not try it in this tutorial.) + +## Estimating a Maximum Likelihood tree with IQTree + +IQTree is a state-of-the-art cross-platform program that uses maximum likelihood to find optimal phylogenetic trees. +It can perform model selection and bootstrapping. +And it's on Galaxy! + +While this is running you might use your time to read the [Models of sequence evolution](#models-of-sequence-evolution) and [Bootstrapping](#bootstrapping) sections above. + +> Estimating a Maximum Likelihood tree with IQTree +> +> 1. Find the {% tool [IQTree](toolshed.g2.bx.psu.edu/repos/iuc/iqtree/iqtree/2.1.2+galaxy2) %} program in the tool finder, and run it with the following parameters: +> +> - In *"General options"*: +> - {% icon param-file %} *"Specify input alignment file in PHYLIP, FASTA, NEXUS, CLUSTAL or MSF format."*: your aligned sequence data (output of **MAFFT** {% icon tool %}) +> - *"Specify sequence type as either of DNA, AA, BIN, MORPH, CODON or NT2AA for DNA, amino-acid, binary, morphological, codon or DNA-to-AA-translated sequences"*: `DNA` +> - In *"Modelling Parameters"*: +> - In *"Rate heterogeneity"*: +> - *"Write maximum likelihood site ratios to .mlrate file"*: Yes +> - In *"Bootstrap Parameters"*: +> - In *"Ultrafast bootstrap parameters"*: +> - *"Specify number of bootstrap replicates (>=1000)."*: `1000` +> +> +> +> +> 7. Click "Run tool". +> 8. To visualise your tree in Galaxy, search for the {% tool [Newick Display](toolshed.g2.bx.psu.edu/repos/iuc/newick_utils/newick_display/1.6+galaxy1) %} in the tool finder on the left, and run it with the following parameters: +> +> - *"Newick file"*: {% icon param-file %}: `MaxLikelihoodTree` (output of **IQTree** {% icon tool %}) +> - *"Branch support"*: `Display branch support` +> - *"Choose an ouput format"*: `PNG` +> 9. Click on the **display icon** {% icon galaxy-eye %} next to the title of the completed Newick Display job to display your tree. +> +> Alternatively you can visualise your tree outside of Galaxy by downloading your IQTree output and using software such as [SplitsTree](https://software-ab.cs.uni-tuebingen.de/download/splitstree4/welcome.html) or [FigTree](http://tree.bio.ed.ac.uk/software/Figtree/). These tools provide additional options for interactively exploring and customising the appearance of your tree. +> +> +> ![Screenshot of a phylogenetic tree of Anolis species. The root of the tree is on the left and the species are listed vertically on the right. The tree consists of multiple branching events and clades and includes bootstrap values in red.](./images/Newick_Display_IQTree.png "The resulting tree found by IQTree, displayed using Newick Display.") +> +>Observe that the bootstrap values (in red) in the IQTree output for deep branches are not as high. +> +>Note that bootstrap values for UFBoot (provided by IQTree) are actual estimates of the probability that the branch is correct, so are not quite the same as traditional "naive" bootstrap values. +{: .hands_on} + +With the visualisation open answer the following questions: +> How well supported is your tree? +> 1. What are the bootstrap values near the root of the tree? Do you think those branches are well supported? +> 2. Which do you think is the biggest well-supported clade? +> 2. Are there some nodes that would be better left unresolved? +> 3. Is your tree "probably right"? -- or 80% right? +> +> > +> > Near the base of the tree the bootstrap values are high: mostly around 100%. These appear to be well supported. Since they're near the root, there are some very large well-supported clades, e.g., all the way from _A. disticus_ near the top, down to _A. lineatus_ near the bottom. There are a number of poorly supported clades _within_ this clade though, with some low bootstrap values; the smallest of these is just 29% for the clade from _C. barbouri_ to _Diplolaemus darwinii_. +> > Overall the tree looks well supported, but it's probably wrong _somewhere_. It would be worth seeking more data, molecular or morphological, to resolve those poorly supported clades. +> > +> {: .solution} +{: .question} + + + +### Report on the final tree + +Look at the IQTree Report file. + +In that you will see a *long* list of models that have been tested, with the favoured one at the top. + +You will also see the Newick Format of the best tree found. When I ran it, the best model was "GTR+F+R6", which means the General Time-Reversible + +> Understanding the IQ-Tree report +> 1. What is the second-most favoured model? +> 2. How many relative rates of evolution have been estimated, and what are these rates? +> +> > +> > 1. The second-best supported model is GTR + F + R7: it's almost identical to the first model, but with one more rate category. +> > 2. There are 6 relative rates, in the table with columns Category, Relative_rate and Proportion: the rates are 0.01936 (very slow), 0.1952, 0.6315, 1.365, 2.662, and the fastest, 7.146. Very few sites are estimated to be in this category - about 2.3% - whereas about 27% of the sites are deemed to be in the slowest category. +> {: .solution} +{: .question} + +> Guide to the report +> The report below shows a long list of models that have been tested, and these are sorted by their AIC score. +> The columns comprise the name of the model, the log-likelihood (that is, the log of the likelihood), of the best-fit tree using that model, then six more columns that are the penalised according to the number of parameters each model has, and an overall weighting of the posterior probability of each model. In the table below, the GTR+F+R6 model has approximately 81.7% of the probability mass based on AIC, but in terms of BIC, it is about 2.37% (guide at the bottom of the table). +> +> It next shows details of the model selected, with relative rate parameters for the various substitutions (A-C is from A to C, etc.); estimated nucleotide frequencies, and then the rate categories. +> In this analysis the best-fit model is GTR+F+R6: +> "Model of rate heterogeneity: FreeRate with 6 categories" +> ... which allows for six categories of relative rates, and shows the proportion of sites estimated to be in each category. +> +> The last thing in the report is the tree! First in the nostalgic ASCII-format "drawing," and then the same tree in Newick format. +> +> 
+> IQ-TREE 2.1.2 COVID-edition built Mar 30 2021
+>
+> Input file name: /mnt/pulsar/files/staging/7756321/inputs/dataset_6092bb0e-7934-473c-8e8f-77054b0192c5.dat
+> Type of analysis: ModelFinder + tree reconstruction
+> Random seed number: 204403
+>
+> REFERENCES
+> ----------
+>
+> To cite IQ-TREE please use:
+>
+> Bui Quang Minh, Heiko A. Schmidt, Olga Chernomor, Dominik Schrempf,
+> Michael D. Woodhams, Arndt von Haeseler, and Robert Lanfear (2020)
+> IQ-TREE 2: New models and efficient methods for phylogenetic inference
+> in the genomic era. Mol. Biol. Evol., in press.
+> https://doi.org/10.1093/molbev/msaa015
+>
+> To cite ModelFinder please use:
+>
+> Subha Kalyaanamoorthy, Bui Quang Minh, Thomas KF Wong, Arndt von Haeseler,
+> and Lars S Jermiin (2017) ModelFinder: Fast model selection for
+> accurate phylogenetic estimates. Nature Methods, 14:587–589.
+> https://doi.org/10.1038/nmeth.4285
+>
+> SEQUENCE ALIGNMENT
+> ------------------
+>
+> Input data: 55 sequences with 1462 nucleotide sites
+> Number of constant sites: 377 (= 25.7866% of all sites)
+> Number of invariant (constant or ambiguous constant) sites: 377 (= 25.7866% of all sites)
+> Number of parsimony informative sites: 913
+> Number of distinct site patterns: 1138
+>
+> ModelFinder
+> -----------
+>
+> Best-fit model according to AIC: GTR+F+R6
+>
+> List of models sorted by AIC scores:
+>
+> Model                  LogL         AIC      w-AIC        AICc     w-AICc         BIC      w-BIC
+> GTR+F+R6         -37031.233   74312.465 +    0.817   74336.043 +    0.832   74973.410 -   0.0237
+> GTR+F+R7         -37031.088   74316.176 +    0.128   74340.548 +   0.0875   74987.696 - 1.88e-05
+> TIM2+F+R6        -37036.403   74318.806 -   0.0343   74341.604 +   0.0516   74969.176 +    0.197
+> GTR+F+R5         -37037.210   74320.421 -   0.0153   74343.219 -    0.023   74970.791 +    0.088
+> TIM2+F+R7        -37036.374   74322.749 -  0.00478   74346.327 -  0.00486   74983.694 - 0.000139
+> TIM2+F+R5        -37042.437   74326.875 - 0.000607   74348.907 -  0.00134   74966.669 +    0.691
+> TIM3+F+R6        -37070.405   74386.811 - 5.87e-17   74409.609 - 8.82e-17   75037.180 - 3.37e-16
+> TIM3+F+R7        -37070.201   74390.401 - 9.74e-18   74413.979 - 9.92e-18   75051.346 - 2.83e-19
+> GTR+F+R4         -37074.898   74391.795 - 4.85e-18   74413.828 - 1.07e-17   75031.590 - 5.52e-15
+> TN+F+R6          -37075.376   74394.752 - 1.11e-18   74417.166 - 2.02e-18   75039.835 - 8.95e-17
+> TIM+F+R6         -37074.693   74395.386 - 8.06e-19   74418.184 - 1.21e-18   75045.756 - 4.63e-18
+> TIM3+F+R5        -37076.845   74395.690 - 6.92e-19   74417.723 - 1.53e-18   75035.485 - 7.88e-16
+> TN+F+R7          -37075.196   74398.393 - 1.79e-19   74421.579 - 2.22e-19   75054.050 - 7.33e-20
+> TIM+F+R7         -37074.525   74399.049 - 1.29e-19   74422.627 - 1.31e-19   75059.994 - 3.75e-21
+> TN+F+R5          -37081.842   74403.685 - 1.27e-20   74425.340 - 3.38e-20   75038.192 - 2.03e-16
+> TIM+F+R5         -37081.073   74404.146 - 1.01e-20   74426.179 - 2.22e-20   75043.941 - 1.15e-17
+> GTR+F+I+G4       -37096.959   74427.917 - 6.95e-26   74448.462 - 3.23e-25   75046.562 -  3.1e-18
+> TVM+F+R6         -37095.385   74438.769 - 3.06e-28   74461.955 - 3.79e-28   75094.427 - 1.25e-28
+> TVM+F+R5         -37097.648   74439.297 - 2.35e-28   74461.711 - 4.28e-28   75084.379 -  1.9e-26
+> TVM+F+R7         -37095.426   74442.853 - 3.97e-29   74466.826 - 3.32e-29   75109.085 - 8.21e-32
+> TPM2+F+R6        -37100.987   74445.973 - 8.35e-30   74468.387 - 1.52e-29   75091.056 - 6.75e-28
+> TPM2u+F+R6       -37100.987   74445.973 - 8.35e-30   74468.387 - 1.52e-29   75091.056 - 6.75e-28
+> TPM2+F+R5        -37103.564   74447.128 - 4.68e-30   74468.784 - 1.25e-29   75081.636 - 7.49e-26
+> TPM2u+F+R5       -37103.564   74447.128 - 4.68e-30   74468.784 - 1.25e-29   75081.636 - 7.49e-26
+> TPM2+F+R7        -37100.997   74449.993 - 1.12e-30   74473.179 - 1.38e-30   75105.651 - 4.57e-31
+> TPM2u+F+R7       -37100.997   74449.994 - 1.12e-30   74473.180 - 1.38e-30   75105.651 - 4.57e-31
+> TPM3u+F+R6       -37126.615   74497.230 - 6.18e-41   74519.644 - 1.13e-40   75142.312 -    5e-39
+> TPM3+F+R6        -37126.615   74497.230 - 6.18e-41   74519.644 - 1.13e-40   75142.312 -    5e-39
+> TPM3u+F+R5       -37129.065   74498.131 - 3.94e-41   74519.786 - 1.05e-40   75132.638 - 6.31e-37
+> TPM3+F+R5        -37129.065   74498.131 - 3.94e-41   74519.786 - 1.05e-40   75132.638 - 6.31e-37
+> TPM3+F+R7        -37126.341   74500.682 -  1.1e-41   74523.868 - 1.36e-41   75156.340 -  4.5e-42
+> TPM3u+F+R7       -37126.342   74500.683 -  1.1e-41   74523.869 - 1.36e-41   75156.341 -  4.5e-42
+> HKY+F+R6         -37131.953   74505.906 - 8.08e-43   74527.938 - 1.78e-42   75145.700 - 9.19e-40
+> K3Pu+F+R6        -37131.054   74506.107 -  7.3e-43   74528.521 - 1.33e-42   75151.190 - 5.91e-41
+> HKY+F+R5         -37134.629   74507.258 - 4.11e-43   74528.540 - 1.32e-42   75136.478 - 9.25e-38
+> K3Pu+F+R5        -37133.634   74507.269 - 4.09e-43   74528.924 - 1.09e-42   75141.776 - 6.54e-39
+> HKY+F+R7         -37131.734   74509.468 - 1.36e-43   74532.266 - 2.05e-43   75159.838 - 7.82e-43
+> K3Pu+F+R7        -37130.809   74509.619 - 1.26e-43   74532.805 - 1.56e-43   75165.276 - 5.16e-44
+> GTR+F+G4         -37164.452   74560.904 - 9.22e-55   74581.085 - 5.13e-54   75174.261 - 5.77e-46
+> GTR+F+R3         -37179.249   74596.499 - 1.72e-62   74617.780 - 5.51e-62   75225.718 - 3.87e-57
+> SYM+R5           -37262.490   74764.980 - 4.47e-99   74786.635 - 1.19e-98   75399.487 - 7.15e-95
+> SYM+R6           -37261.122   74766.244 - 2.37e-99   74788.658 - 4.33e-99   75411.326 - 1.92e-97
+> TIM2e+R5         -37274.321   74784.641 - 2.4e-103   74805.553 - 9.27e-103   75408.573 - 7.61e-97
+> TIM2e+R6         -37273.043   74786.086 - 1.17e-103   74807.741 - 3.1e-103   75420.593 - 1.87e-99
+> GTR+F+R2         -37597.901   75429.802 - 1.93e-243   75450.347 - 8.95e-243   76048.447 - 8.6e-236
+> TIM3e+R6         -37740.342   75720.684 - 1.32e-306   75742.339 - 3.52e-306   76355.191 - 2.12e-302
+> TIM3e+R7         -37740.126   75724.252 - 2.22e-307   75746.665 - 4.05e-307   76369.334 - 1.8e-305
+> TVMe+R6          -37741.704   75725.408 - 1.25e-307   75747.441 - 2.75e-307   76365.203 - 1.42e-304
+> TVMe+R5          -37745.621   75729.242 - 1.83e-308   75750.524 - 5.88e-308   76358.462 - 4.13e-303
+> TVMe+R7          -37741.937   75729.875 - 1.34e-308   75752.673 - 2.01e-308   76380.245 - 7.68e-308
+> TIM3e+R5         -37747.544   75731.088 - 7.28e-309   75751.999 - 2.81e-308   76355.020 - 2.31e-302
+> TIMe+R6          -37750.759   75741.518 - 3.96e-311   75763.173 - 1.05e-310   76376.025 - 6.33e-307
+> TNe+R6           -37752.489   75742.978 - 1.91e-311   75764.260 - 6.12e-311   76372.198 - 4.29e-306
+> TIMe+R7          -37750.536   75745.072 - 6.69e-312   75767.486 - 1.22e-311   76390.154 - 5.41e-310
+> TNe+R7           -37752.255   75746.511 - 3.26e-312   75768.544 - 7.19e-312   76386.306 - 3.71e-309
+> TIMe+R5          -37757.450   75750.900 - 3.63e-313   75771.811 - 1.4e-312   76374.832 - 1.15e-306
+> TNe+R5           -37759.321   75752.642 - 1.52e-313   75773.187 - 7.05e-313   76371.287 - 6.77e-306
+> K3P+R5           -38328.664   76891.327 -        0   76911.872 -        0   77509.972 -        0
+> K2P+R5           -38330.281   76892.562 -        0   76912.744 -        0   77505.919 -        0
+> K3P+R6           -38327.530   76893.060 -        0   76914.341 -        0   77522.279 -        0
+> K2P+R6           -38329.207   76894.414 -        0   76915.326 -        0   77518.347 -        0
+> F81+F+R5         -38740.095   77716.191 -        0   77737.102 -        0   78340.123 -        0
+> F81+F+R6         -38739.434   77718.868 -        0   77740.523 -        0   78353.375 -        0
+> GTR+F+I          -39252.699   78737.399 -        0   78757.580 -        0   79350.756 -        0
+> JC+R5            -39408.095   79046.191 -        0   79066.012 -        0   79654.260 -        0
+> JC+R6            -39407.610   79049.220 -        0   79069.765 -        0   79667.865 -        0
+> GTR+F            -41407.684   83045.369 -        0   83065.190 -        0   83653.438 -        0
+>
+> AIC, w-AIC   : Akaike information criterion scores and weights.
+> AICc, w-AICc : Corrected AIC scores and weights.
+> BIC, w-BIC   : Bayesian information criterion scores and weights.
+>
+> Plus signs denote the 95% confidence sets.
+> Minus signs denote significant exclusion.
+>
+> SUBSTITUTION PROCESS
+> --------------------
+>
+> Model of substitution: GTR+F+R6
+>
+> Rate parameter R:
+>
+>   A-C: 1.4045
+>   A-G: 4.3329
+>   A-T: 1.6165
+>   C-G: 0.7046
+>   C-T: 7.4429
+>   G-T: 1.0000
+>
+> State frequencies: (empirical counts from alignment)
+>
+>   pi(A) = 0.3392
+>   pi(C) = 0.2607
+>   pi(G) = 0.1184
+>   pi(T) = 0.2817
+>
+> Rate matrix Q:
+>
+>   A   -0.6328    0.1737    0.2432    0.2159
+>   C    0.2259     -1.26   0.03954    0.9942
+>   G     0.697   0.08712   -0.9177    0.1336
+>   T      0.26    0.9203   0.05613    -1.236
+>
+> Model of rate heterogeneity: FreeRate with 6 categories
+> Site proportion and rates:  (0.2697,0.01936) (0.1987,0.1952) (0.1537,0.6315) (0.1929,1.365) (0.1619,2.662) (0.02305,7.146)
+>
+>  Category  Relative_rate  Proportion
+>   1         0.01936        0.2697
+>   2         0.1952         0.1987
+>   3         0.6315         0.1537
+>   4         1.365          0.1929
+>   5         2.662          0.1619
+>   6         7.146          0.02305
+>
+> MAXIMUM LIKELIHOOD TREE
+> -----------------------
+>
+> Log-likelihood of the tree: -37024.7700 (s.e. 781.9510)
+> Unconstrained log-likelihood (without tree): -9373.5747
+> Number of free parameters (#branches + #model parameters): 125
+> Akaike information criterion (AIC) score: 74299.5399
+> Corrected Akaike information criterion (AICc) score: 74323.1178
+> Bayesian information criterion (BIC) score: 74960.4850
+>
+> Total tree length (sum of branch lengths): 10.7102
+> Sum of internal branch lengths: 2.7107 (25.3096% of tree length)
+>
+> NOTE: Tree is UNROOTED although outgroup taxon 'Anolis.acutus' is drawn at root
+>
+> +------Anolis.acutus
+> |
+> |                                              +-----A.aeneus
+> |                                        +-----|
+> |                                        |     +----A.richardi
+> |                                     +--|
+> |                                     |  +---------A.luciae
+> |                                  +--|
+> |                                  |  |      +------A.agassizi
+> |                                  |  |   +--|
+> |                                  |  |   |  +--------A.microtus
+> |                                  |  +---|
+> |                                  |      +--------------P.nicefori
+> |                               +--|
+> |                               |  |          +----------------------Diplolaemus.darwinii
+> |                               |  +----------|
+> |                               |             +----------------------Phenacosaurus.acutirostrus
+> |                            +--|
+> |                            |  |               +---------A.aliniger
+> |                            |  |           +---|
+> |                            |  |           |   +----------A.coelestinus
+> |                            |  |        +--|
+> |                            |  |        |  +-------------A.bahorucoensis
+> |                            |  |     +--|
+> |                            |  |     |  |    +---------A.bartschi
+> |                            |  |     |  +----|
+> |                            |  |     |       +------A.vermiculatus
+> |                            |  |  +--|
+> |                            |  |  |  +-----------A.equestris
+> |                            |  +--|
+> |                            |     +-----------------------A.occulatus
+> |                         +--|
+> |                         |  |        +------A.etheridgei
+> |                         |  |     +--|
+> |                         |  |     |  +---------A.insolitus
+> |                         |  |  +--|
+> |                         |  |  |  +-------------A.olssoni
+> |                         |  +--|
+> |                         |     +--------------Chamaeleolis.barbouri
+> |                      +--|
+> |                      |  |           +--------A.barahonae
+> |                      |  |        +--|
+> |                      |  |        |  +--------A.christophei
+> |                      |  |     +--|
+> |                      |  |     |  +--------A.cuvieri
+> |                      |  |  +--|
+> |                      |  |  |  |            +--C.guamuhaya
+> |                      |  |  |  +------------|
+> |                      |  |  |               +--C.chamaeleonides
+> |                      |  +--|
+> |                      |     +--------------A.lucius
+> |                   +--|
+> |                   |  |         +-------------A.marcanoi
+> |                   |  +---------|
+> |                   |            +-----------A.strahmi
+> |                +--|
+> |                |  |       +---------------A.ahli
+> |                |  |    +--|
+> |                |  |    |  |         +-----A.ophiolepis
+> |                |  |    |  +---------|
+> |                |  |    |            +----A.sagrei
+> |                |  +----|
+> |                |       |             +-----A.garmani
+> |                |       |        +----|
+> |                |       |        |    +-----A.grahami
+> |                |       |     +--|
+> |                |       |     |  +--------A.equestris2
+> |                |       |  +--|
+> |                |       |  |  +------------A.lineatopus
+> |                |       +--|
+> |                |          |          +-------------A.humilis
+> |                |          |  +-------|
+> |                |          |  |       +------------A.limifrons
+> |                |          +--|
+> |                |             +-------------A.lineatus
+> |             +--|
+> |             |  |           +--------A.alutaceous
+> |             |  |  +--------|
+> |             |  |  |        +-----------A.vanidicus
+> |             |  +--|
+> |             |     |                +----A.angusticeps
+> |             |     |        +-------|
+> |             |     |        |       |  +--A.paternus
+> |             |     |        |       +--|
+> |             |     |        |          +--A.paternus2
+> |             |     |     +--|
+> |             |     |     |  +----------------A.sheplani
+> |             |     |  +--|
+> |             |     |  |  |    +---A.carolinensis
+> |             |     |  |  +----|
+> |             |     |  |       +----A.maynardi
+> |             |     +--|
+> |             |        |  +------A.loysiana
+> |             |        +--|
+> |             |           +----------A.pumilis
+> |          +--|
+> |          |  |   +-----------A.bimaculatus
+> |          |  +---|
+> |          |      +------------A.wattsi
+> |       +--|
+> |       |  |     +---------A.brevirostrus
+> |       |  +-----|
+> |       |        +------A.distichus
+> |    +--|
+> |    |  +----------A.krugi
+> +----|
+> |    +------------A.cristatellus
+> |
+> +-----A.stratulus
+>
+> Tree in newick format:
+>
+> (Anolis.acutus:0.1010470578,((((((((((((((A.aeneus:0.0894217579,A.richardi:0.0771663752):0.0905009536,A.luciae:0.1452269292):0.0505637895,((A.agassizi:0.1016958548,A.microtus:0.1274705993):0.0466408653,P.nicefori:0.2174577338):0.0678983736):0.0434625375,(Diplolaemus.darwinii:0.3250757059,Phenacosaurus.acutirostrus:0.3269305219):0.1596746115):0.0466548353,(((((A.aliniger:0.1391142437,A.coelestinus:0.1557602669):0.0598840477,A.bahorucoensis:0.1976626377):0.0075156245,(A.bartschi:0.1398068405,A.vermiculatus:0.1026256093):0.0701730721):0.0088445438,A.equestris:0.1698075932):0.0120788802,A.occulatus:0.3404603086):0.0198461252):0.0251152781,(((A.etheridgei:0.0991665297,A.insolitus:0.1482517520):0.0540499255,A.olssoni:0.1910514865):0.0088098284,Chamaeleolis.barbouri:0.2095740155):0.0188459879):0.0077203037,((((A.barahonae:0.1260843543,A.christophei:0.1262731407):0.0348296954,A.cuvieri:0.1361832093):0.0309217949,(C.guamuhaya:0.0431099935,C.chamaeleonides:0.0257796446):0.1779877789):0.0345206566,A.lucius:0.2146844398):0.0147942476):0.0193880895,(A.marcanoi:0.1954668346,A.strahmi:0.1658659731):0.1384272979):0.0071850401,((A.ahli:0.2194974552,(A.ophiolepis:0.0874380486,A.sagrei:0.0746247231):0.1427321115):0.0307428243,((((A.garmani:0.0905499626,A.grahami:0.0943764186):0.0703118985,A.equestris2:0.1314905403):0.0198447353,A.lineatopus:0.1838950754):0.0232334317,((A.humilis:0.2006656769,A.limifrons:0.1903931082):0.1159932105,A.lineatus:0.2020567025):0.0284469354):0.0386892413):0.0788578675):0.0071420002,((A.alutaceous:0.1299690048,A.vanidicus:0.1769083322):0.1269197421,((((A.angusticeps:0.0766354706,(A.paternus:0.0125693305,A.paternus2:0.0058013518):0.0519908238):0.1128275458,A.sheplani:0.2418794624):0.0228102911,(A.carolinensis:0.0645999975,A.maynardi:0.0760546166):0.0733086724):0.0301309147,(A.loysiana:0.0985169696,A.pumilis:0.1631220132):0.0511013731):0.0207983825):0.0498971029):0.0462944857,(A.bimaculatus:0.1713426754,A.wattsi:0.1884789044):0.0574997330):0.0334141164,(A.brevirostrus:0.1442087439,A.distichus:0.1088311847):0.0833172684):0.0398851829,A.krugi:0.1583953901):0.0256700857,A.cristatellus:0.1827036810):0.0725247864,A.stratulus:0.0863000570);
+>
+> TIME STAMP
+> ----------
+>
+> Date and time: Fri Dec  8 00:36:23 2023
+> Total CPU time used: 243.654 seconds (0h:4m:3s)
+> Total wall-clock time used: 56.1638 seconds (0h:0m:56s)
+>
+> +{: .details} + + +# Phylogenetic Networks + + + +{% icon video %} Watch the video to see how SplitsTree4 can be used to explore the evidence supporting different phylogenetic splits in data from Anolis species using networks built with uncorrected P, Jukes-Cantor and HKY85 models. + +{% include _includes/slides-youtube-selector.html id="MvZhj3cSVhA" title="Introductory Lecture: Welcome and why phylogenetics?" slides="topics/evolution/tutorials/abc_intro_phylo-networks" %} + + +Perhaps surprisingly, phylogenetic estimation doesn't have to start with a tree. Instead you can start with a *phylogenetic network*. + +Phylogenetic networks have two purposes: + +1. to show an estimate of the evolutionary history that is not strictly branching, so, involving horizontal gene transfer or hybridisation events. + + or + +2. to show conflicting phylogenetic signal in the data set, suggesting different possible trees. + +In this tutorial we use networks for the second purpose because we are working under the assumption that there is a true tree, which is the actual evolutionary history of our species of interest. + +To understand what this kind of phylogenetic network is, and how to interpret one, we need to think about what the branches of a tree _do_. Branches *split* the taxa into two groups: those on one side of the branch, and those on the other. We are thinking about unrooted trees in this context, so the two branches coming from the root constitute a single split. + +Thus every branch of a tree can be thought of as a _split_ and a tree is a collection of *compatible* splits: where by "compatible" we just mean that they can be on the same tree. + + +So our tree search is an attempt to find a "best" set of _splits_ that are all compatible, and which together explain the evolutionary relationships among the taxa of interest. + +However, the data may support multiple splits that are _not_ compatible. For example, we might have a set of nucleotide sequence data like this: + +![A table displaying DNA sequences for four different taxa. Each row represents a taxon, and the columns ‘sequence’, ‘constant’, ‘singleton’, ‘12|34’, ‘23|14’, ‘13|24’ display portions of the DNA sequences at different sites. Constant: Contains shorter sequences that are consistent across all taxons at sites 1-4. Singleton: Displays unique sequences specific to each taxon at sites 5-8; 12|34: Displays differences in the sequence at sites 9-12; 23|14: Displays differences the sequence at sites 13 and 14; 13|24: Displays differences in sequence at site 15.](./images/tableOfSitesForSplits.png) + + + +In this table the sequence is listed in full in the second column, then broken up into different categories in subsequent columns. + +The first four sites are _constant_, so don't tell us anything much about the phylogeny. + +The next four _singleton_ sites segregate one taxon from the others (which is again not terribly useful in terms of resolving the branching in the phylogeny). + +The so-called "parsimony informative" sites start at site 9. These sites tell us about the support for the _internal_ branches of the tree. + +Sites 9-12 suggest splitting the taxa into (1,2) vs (3,4). We write this as a split $$12\vert34$$ for brevity, or even just 12. + +The next two sites, numbers 13 and 14, suggest the split (2,3) vs (1,4), which we could write as $$23\vert14$$ or $$14\vert23$$ or just 14. + +The last site suggests that taxa 1 and 3 should go together. + +![Image of a phylogenetic split in a network of four taxa. Taxa 1 and 2 appear on the left of the image and taxa 4 and 3 on the right. The taxa are connected to a rectangle in the centre of the image that represents the split. Two sets of parallel lines are highlighted. The green, horizontal lines separate taxa 1 and 2 from 3 and 4. Blue, vertical lines separate taxa 1 and 3 from 2 and 4.](./images/SimpleSplits.png "Splits in a simple phylogenetic network"){: align="center"} + +In the above figure we have four taxa 1,2,3,4, in a phylogenetic network. +The pairs of parallel lines correspond to two of the _splits_ that could separate taxa from each other, and which could (separately) appear in phylogenetic trees. + +The green, horizontal branches separate taxa 1 and 2 from 3 and 4; the split $$12\vert34$$. These are longer than the blue, vertical ones, which separate taxa 1 and 3 from 2 and 4: the split $$13\vert24$$. + +The beauty of the network is that both thes pieces of information can be shown on the same figure, despite them not being compatible -- they can't both be on the same tree. + + +## Building a Neighbor-Net phylogenetic network + +> Build a Neighbor-Net with Splitstree +> +> 1. Download and install [SplitsTree 4](https://software-ab.cs.uni-tuebingen.de/download/splitstree4/welcome.html) or [SplitsTree 6 CE](https://software-ab.cs.uni-tuebingen.de/download/splitstree6/welcome.html) (Community Edition) on your own computer. +> +> Note: SplitsTree 4 is an older version, but works well. SplitsTree 6 is in development but appears to work equally well (at the time of writing). They do the same thing, and we only use tools common to both versions. Installing either version takes a minute or two. +> 2. Download the aligned data .FASTA file (the output of MAFFT) to your own computer. +> 3. Start up SplitsTree and open the file. Within moments you should see something like this (using SplitsTree 4): +> ![Screenshot of a phylogenetic network of Anolis species. The root of the network is at the centre of the image and clades radiate outwards forming a circular network.](./images/ST4-default.png) +> Click on some of the interior branches, which will highlight sets of parallel lines, that correspond to each split that is shown in the network. +> This network shows a number of deep branches that are quite well resolved, in the sense that they have very long, thin parallelograms, but there is a jumble of very small parallelograms in the centre of the network, indicating that there is not a lot of information in the data to determine the early branching order of the _Anolis_ phylogeny. +> +> 4. Note that the initial distance measure is using "Uncorrected P" distance, which is simply the proportion of sites that differ between each pair of sequences. It is possible to use more sophisticated models for maximum likelihood, such as Jukes-Cantor and HKY85. +> Click on the **Distances menu** and select "HKY85". A dialog box will appear with some options. Click "Apply" and get a network like this: +> ![HKY85 Network. Screenshot of a phylogenetic network of Anolis species. The root of the network is at the centre of the image and clades radiate outwards forming a circular network.](./images/ST4-HKY85.png) +> The above shows you that the basic structure of the network does not change very much using different distance measures. This is a good thing because it means reasonable assumptions you make about your data are not drastically changing the results. +> 5. Finally, perform a bootstrap analysis on this network. Click on the **Analysis menu** and select Bootstrap. Leave the default number of replicates as 1000 and click "run". +>After a few seconds (less than a minute) you should see something like this: +> ![HKY85 Network with Bootstrap. Screenshot of a phylogenetic network of Anolis species. The root of the network is at the centre of the image and clades radiate outwards forming a circular network. The network is annotated with bootstrap values.](./images/ST4-HKY85-BS.png) +> This network shows the percentage of times out of those 1000 replicates that each split was in the network created for each replicate. +>Zoom in (there is a magnifying glass tool at the top) and scroll around the figure to see which are the strongly supported *splits*, which should correspond to the well supported bootstrap values in the trees you inferred above. +{: .hands_on} + + + + +# Troubleshooting + +Here are a few things that can catch us out: + +>Long Branch Attraction (LBA): + >Be wary of long branches that come out together in the estimated phylogeny. This can be the result pairs of sequences that are very different from the rest, so match each other “by chance” more than they match the rest. +> +>**Fix:** break up these long branches by adding in some taxa that are closely related to one or the other; remove one long branch at a time to see where the remaining one fits best; consider other methods that are more robust to LBA. +{: .tip} + +>Very “Gappy” Sequences: +>Sequences that are hard to align might contain many gaps and many equally “good” alignments. +> +>**Fix:** Try different multiple alignment programs; consider using “alignment-free” methods such as k-mer distances; remove very problematic regions using programs such as GBlocks (also available on Galaxy). +{: .tip} + +>Low resolution: +>Low bootstrap support or lots of conflict in a network. +> +>**Fix:** Look at which sites support which splits (internal branches); consider sliding window approaches to identify recombination, or check that your sequences don’t span regions with different selection pressures; consider using PartitionFinder or similar methods to work out which sets of sites have similar evolutionary dynamics. +{: .tip} + +>The gene trees are different! +>**Fix:** They might not need fixing: it might just be that the genes’ evolutionary histories aren’t the same as those of the species that host them. Look at all the gene trees and see what other events might have led to the differences between them. +{: .tip} + +>I can’t find an outgroup! +>**Fix:** Consider mid-point rooting: it is in most cases pretty good. +{: .tip} + +# Summary + +Phylogenetics provides the statistical framework that is essential for comparing biological organisms. A phylogenetic tree provides a best estimate of the evolutionary relationships between species (taxa) of interest, which is the framework we need to compare them. + +A key component of molecular phylogenetics is creating an alignment -- this is a complex process that can have different outcomes using different methods. It is always a good idea to look at the alignment to see if it makes sense: has the alignment program included the insertion/deletion events which are consistent with a phylogenetic relationship between the species? + +While estimating phylogenetic trees is computationally challenging, there are a range of well-validated methods to use, constructing them based on distances, molecular sequences, and other data; these can also be used to create phylogenetic networks that represent the complexities in the data. There is an extensive range of scoring functions by which we can compare trees, including: Minimum total distance (minimum evolution); the maximum amount of evolutionary "work" to account for the data (maximum parsimony); or by finding the model that has the highest probability of giving us the data we saw (maximum likelihood). + +It's important to remember that these are all statistical estimates, so they may differ, and it is good advice to use multiple methods to confirm general phylogenetic relationships rather than choose a single method. + +We can use Bootstrapping to calculate a measure of confidence for clades in trees based on molecular sequences. Bootstrapping involves representative independent re-samples of the input alignment which are analysed again using the same methods, 100s to 1000s of times, to gauge how reliable each branch is, in the sense of how often the branch is part of the best tree found for each re-sample. This isn't a measure of confidence in the statistical sense, but a rule of thumb of 80% or more being "good support" is common in the literature -- remembering that as sequences get longer and longer, bootstrap support values go up, no matter how poor the data are! + +It's worth noting that this tutorial is only focusing on the estimation of individual best trees rather than taking a Bayesian approach in which a posterior probability of trees is created, using programs like [MyBayes](https://nbisweden.github.io/MrBayes/index.html) or [BEAST](http://www.beast2.org). These programs use even more computational resources and can run for many days or weeks as they wander around "tree space", building up a representative distribution of the probability that each tree is "right". This computational burden is one of the main reasons we have not gone into Bayesian methods in this tutorial. Still, many good examples are available online on the web pages for the above programs. + +We hope that this tutorial and accompanying videos have given you a better understanding of the principles of phylogenetics and how these methods work, which will help you make informed decisions about the phylogenetic analyses you perform. + +See the resources below to learn more about phylogenetics. + +# Resources +To develop a deeper understanding of phylogenetic trees, there is no better way than estimating phylogenies yourself --- and work through a book on the topic at your own pace. + +## Books +- *Phylogenetics in the genomics era*, 2020. An [open access book](https://hal.inria.fr/PGE) covering a variety of contemporary topics. +- *Tree Thinking*, 2013, by David A. Baum & Stacey D. Smith +- *Molecular Evolution*, 2014, by Ziheng Yang + +## Useful links +- [MEGA Software](https://megasoftware.net/) +- [Tutorial on how to read a tree, with a virus example](https://artic.network/how-to-read-a-tree.html) +- [Tree Of Life web project](http://tolweb.org) +- [Phylogenetic Inference in the Stanford Encyclopedia](https://plato.stanford.edu/entries/phylogenetic-inference/) +- [MrBayes](https://nbisweden.github.io/MrBayes/index.html) +- [BEAST](http://www.beast2.org/) +- [SplitsTree](https://software-ab.cs.uni-tuebingen.de/download/splitstree4/welcome.html) or [FigTree](http://tree.bio.ed.ac.uk/software/Figtree/) can be used to visualise phylogenetic trees outside of Galaxy. These tools provide additional options for exploring and customising your trees. + +## How to cite the tools used in this tutorial + +If you use, extend or reference Galaxy in your published work, please [cite the Galaxy publication and acknowledge the public Galaxy platforms you use (e.g. Galaxy Australia)](https://galaxyproject.org/citing-galaxy/). + +Citations for tools used in a Galaxy workflow can be downloaded from the {%icon galaxy-history-options%} dropdown in your Galaxy history. + + + + + diff --git a/topics/evolution/tutorials/abc_intro_phylo/workflows/index.md b/topics/evolution/tutorials/abc_intro_phylo/workflows/index.md new file mode 100644 index 00000000000000..e092e0ae66ddd4 --- /dev/null +++ b/topics/evolution/tutorials/abc_intro_phylo/workflows/index.md @@ -0,0 +1,3 @@ +--- +layout: workflow-list +--- diff --git a/topics/evolution/tutorials/abc_intro_phylo/workflows/main_workflow-tests.yml b/topics/evolution/tutorials/abc_intro_phylo/workflows/main_workflow-tests.yml new file mode 100644 index 00000000000000..9f1b27001dc7b1 --- /dev/null +++ b/topics/evolution/tutorials/abc_intro_phylo/workflows/main_workflow-tests.yml @@ -0,0 +1,11 @@ +# GTN_RUN_SKIP_REASON: No easy way to test workflows with interactive tools. +- doc: Test outline for main_workflow.ga + job: + Input alignment: + class: File + location: https://zenodo.org/records/10415242/files/anolis-raw.fst + outputs: + Best-scoring ML Tree: + class: '' + Info: + class: '' diff --git a/topics/evolution/tutorials/abc_intro_phylo/workflows/tree-building-tests.yml b/topics/evolution/tutorials/abc_intro_phylo/workflows/tree-building-tests.yml new file mode 100644 index 00000000000000..aef635d6f8dc34 --- /dev/null +++ b/topics/evolution/tutorials/abc_intro_phylo/workflows/tree-building-tests.yml @@ -0,0 +1,19 @@ +- doc: Test outline for tree-building.ga + job: + Input FASTA Sequence File: + class: File + location: https://zenodo.org/records/10415242/files/anolis-raw.fst + filetype: fasta + outputs: + outputAlignment: + asserts: + has_text: + text: '((' + output: + asserts: + has_text: + text: '((' + treefile: + asserts: + has_text: + text: '((' diff --git a/topics/evolution/tutorials/abc_intro_phylo/workflows/tree-building.ga b/topics/evolution/tutorials/abc_intro_phylo/workflows/tree-building.ga new file mode 100644 index 00000000000000..7fa5657b1e7ed6 --- /dev/null +++ b/topics/evolution/tutorials/abc_intro_phylo/workflows/tree-building.ga @@ -0,0 +1,223 @@ +{ + "a_galaxy_workflow": "true", + "annotation": "Tree building for \"Phylogenetics - Back to basics\"", + "comments": [], + "creator": [ + { + "class": "Person", + "identifier": "0000-0001-9760-8992", + "name": "Helena Rasche" + }, + { + "class": "Person", + "identifier": "0000-0001-8385-341X", + "name": "Michael Charleston" + } + ], + "format-version": "0.1", + "license": "MIT", + "name": "Tree Building (imported from uploaded file)", + "steps": { + "0": { + "annotation": "This should be a fasta formatted file with multiple 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