View source for BIN-PHYLO-Tree analysis

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Analysing Phylogenetic Trees
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(Species trees, gene trees and the importance of naming, Speciation and duplication signatures)
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<b>Abstract:</b><br />
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The analysis of mixed gene trees.
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<b>Objectives:</b><br />
This unit will ...
* ... introduce ;
* ... demonstrate ;
* ... teach how to fetch a species tree from the NCBI taxonomy page;
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<b>Outcomes:</b><br />
After working through this unit you ...
* ... can ;
* ... are familar with ;
* ... have begun to.
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<b>Deliverables:</b><br />
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<li><b>Time management</b>: Before you begin, estimate how long it will take you to complete this unit. Then, record in your course journal: the number of hours you estimated, the number of hours you worked on the unit, and the amount of time that passed between start and completion of this unit.</li>
<li><b>Journal</b>: Document your progress in your [[FND-Journal|Course Journal]]. Some tasks may ask you to include specific items in your journal. Don't overlook these.</li>
<li><b>Insights</b>: If you find something particularly noteworthy about this unit, make a note in your [[ABC-Insights|'''insights!''' page]].</li>
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<b>Prerequisites:</b><br />
This unit builds on material covered in the following prerequisite units:<br />
*[[BIN-PHYLO-Tree_building|BIN-PHYLO-Tree_building (Building Phylogenetic Trees)]]
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=== Evaluation ===
<b>Evaluation: NA</b><br />
<div style="margin-left: 2rem;">This unit is not evaluated for course marks.</div>
== Contents ==


{{Task|1=
*Read the introductory notes on {{ABC-PDF|BIN-PHYLO-Tree_analysis|analysing phylogenetic trees}}.
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==Analysing your tree==
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{{ABC-unit|BIN-PHYLO-Tree_analysis.R}}


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#Copy the tree-string from the R console.
#Visualize the tree online: navigate to the [http://www.trex.uqam.ca/index.php?action=newick&project=trex Trex-online Newick tree viewer]. Visualize the tree as a phylogram. Explore the options.

# A particularly useful viewer is actually Jalview - although this may be more apparent with the larger alignment of '''all''' sequences we'll produce later.
##Open Jalview and load your alignment of all APSES domain proteins.
##Save the Newick-formatted tree.
##In the alignment window, choose '''File &rarr; Load associated Tree''' and load your tree file. You can click into the tree-window to show which clades branch off at what level - it should be obvious that you can identify three major subclades (plus the outgroup). This view is particularly informative, since you can associate the clades of the tree with the actual sequences in the alignment, and get a good sense what sequence features the tree is based on.
##Try the '''Calculate &rarr; Sort &rarr; By Tree Order''' option to sort the sequences by their position in the tree. Also note that you can flip the tree around a node by double-clicking on it. This is especially useful: try to rearrange the tree so that the subdivisions into clades are apparent. Clicking into the window "cuts" the tree and colours your sequences according to the clades in which they are found. This is useful to understand what particular sequences contributed to which part of the phylogenetic inference.


ANALYSIS

* First, the APS and ANK trees should have the same topology, since they are only different parts of the same protein (unless that protein has swapped its domains with another one during evolution). Clearly, that is not the case. The ''basidiomycota'' are reasonably consistent, although their internal ordering is poorly resolved, particularly in the APS tree. The ''ascomycota'' show two major differences, but they are actually consistent between the APS and the ANK tree: SACCE is less similar to all than we would expect from the species tree. And NEUCR is more similar to the ''basidiomycotal'' proteins.

* Consider the scale bars: ANK domains have evolved at about twice the rate of the APS domains. This alone should tell us to be cautious with our interpretations since this shows there are different degrees of selective pressure on different parts of the protein. Moreover the <u>relative rates</u> differ as well. NEUCR's APSES domain has evolved much faster by comparison to other proteins than its ankyrin domain. Has its biological function changed?

* Secondly, both gene trees should follow the species tree. Again, there are differences. But if we exclude SACCE and NEUCR, the remainder actually turns out relatively consistent.

In any case: this is what the data tells us. The big picture is mostly conserved, but there are differences in the details. However: now we know what degree of accuracy we can expect from the analysis.

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==The mixed gene tree==
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You have now practiced how to calculate, manipulate, plot, annotate and compare trees.

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* Now use Rproml to calculate a mixed gene tree based on '''all'' APSES domains. You saved it as <code>APSES.mfa</code>. For the fifty or so domains, each run will take about an hour. Thus run as many <code>random.addition</code> cycles as reasonable during a study break, or overnight. Thus the command will be something like:

<pre>
allApsIn <- read.protein("APSES.mfa")
fullApsTree <- Rproml(allApsIn, path=PROMLPATH, random.addition=3)

#... and don't forget:
save(fullApsTree, file="fullApsTree.rda")
</pre>

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Here are two principles that will help you make sense of the tree.


A: '''A gene that is present in an ancestral species is inherited in all descendant species'''. The gene has to be observed in all OTUs, unless its has been lost (which is a rare event).

B: '''Paralogous genes in an ancestral species should give rise to monophyletic subtrees for each of the paralogues, in all descendants'''; this means: if the MRCA of a branch has e.g. three genes, we would expect three copies of that branch below this node, one for each of the three genes. Each of these subtrees should recapitulate the reference phylogenetic tree of the species, up to the branchpoint of their MRCA. The precise relationships may not be readily apparent, due to the noise and limited resolution we saw above, but the gene ought to be '''somewhere''' in the tree and you can often assume that it is closest to where it ought to be if the topology was correct. In this way you try to reconcile your expectations with your observations - preferably with as small a number of changes as possible.

With these two simple principles (draw them out on a piece of paper if they do not seem obvious to you), you can probably pry your tree apart quite nicely. A few colored pencils and a printout of the tree will help. I would start by identifying all of the Mbp1 RBMs in the tree.

Here is a bit of code that you can use to colour the labels of the Mbp1 RBMs:

<source lang="R">

# You have previously defined the names for Mbp1 RBMs in
# the vector apsMbp1Names. You can use these to check
# which of the tree tipLabels are in that vector and
# then color them red in the plot.

# You'll need to replace <TREE> with whatever you called
# your full tree with all APSES domain proteins.

#First, have a look at the tip labels in your tree:
<TREE>$tip.label

# We'll create a vector of black colours of the same length
# as the tip label vector:
tipColors = rep("#000000", Ntip(<TREE>))

# ... then we replace each one for which the label is
# in apsMbp1Names with "#BB0000" (red)
tipColors[<TREE>$tip.label %in% apsMbp1Names] <- "#BB0000"

#inspect:
tipColors

# ... and then we plot:
plot(<TREE>, tip.color=tipColors,
     cex=0.7, root.edge=TRUE, no.margin=TRUE)


</source>

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===The APSES domains of the MRCA===
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Note: A common confusion about cenancestral genes (MRCA = Most Recent Common Ancestor) arises from the fact that by far not all expected genes are present in the OTUs. Some will have been lost, some will have been incorrectly annotated in their genome (frameshifts!) and not been found with PSI-BLAST, some may have diverged beyond recognizability. In general you have to ask: '''given the species represented in a subclade, what is the last common ancestor of that branch'''? The expectation is that '''all''' descendants of that ancestor should be represented in that branch '''unless''' one of the above reasons why a gene might be absent would apply. Eg. if a branch contains species from ''Basidiomycota'' '''and''' ''Ascomycota'', this means that its MRCA was the ancestor of all fungi.


{{task|1=


* Consider the APSES domain proteins of the fungal cenancestor. What evidence do you see in the tree that identifies them. Note that the hallmark of a clade that originated in the cenancestor is that it contains species from '''all''' subsequent major branches of the species tree. How many of these proteins are there? What arer the names of their SACCE descendants?

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===The APSES domains of MYSPE===
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You have identified the APSES domain genes of the fungal cenancestor above. Accordingly, this defines the number of APSES protein genes the ancestor to MYSPE had. Identify the sequence of duplications and/or gene loss in your organism through which MYSPE has ended up with the APSES domains it possesses today.

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# Print the tree to a single sheet of paper.
# Mark the clades for the genes of the cenancestor.
# Label all subsequent branchpoints that affect the gene tree for MYSPE  with either '''"D"''' (for duplication) or '''"S"''' (for speciation). Remember that specific speciation events can appear more than once in a tree. Identify such events.
# '''Bring this sheet with you to the quiz on Tuesday. Your annotated printout will be worth half of the phylogeny quiz marks.'''

}}

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==Bonus: when did it happen?==

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A very cool resource is [http://www.timetree.org/ '''Timetree'''] - a tool that allows you to estimate divergence times between species. For example, the speciation event that separated the main branches of the fungi - i.e. the time when the fungal cenacestor lived - is given by the divergence time of ''Schizosaccharomyces pombe'' and ''Saccharomyces cerevisiaea'': 761,000,000 years ago. For comparison, these two fungi are therefore approximately as related to each other as '''you''' are ...

A) to the rabbit?<br>
B) to the opossum?<br>
C) to the chicken?<br>
D) to the rainbow trout?<br>
E) to the warty sea squirt?<br>
F) to the bumblebee?<br>
G) to the earthworm?<br>
H) to the fly agaric?<br>

Check it out - the question will be on the quiz.

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==Identifying Orthologs==

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In the last assignment we discovered homologs to ''S. cerevisiae'' Mbp1 in MYSPE. Some of these will be orthologs to Mbp1, some will be paralogs. Some will have similar function, some will not. We discussed previously that genes that evolve under continuously similar evolutionary pressure should be most similar in sequence, and should have the most similar "function".

In this assignment we will define the MYSPE gene that is the most similar ortholog to ''S. cerevisiae'' Mbp1, and perform a multiple sequence alignment with it.

Let us briefly review the basic concepts.

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&nbsp;<br>
;All related genes are homologs.
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Two central definitions about the mutual relationships between related genes go back to Walter Fitch who stated them in the 1970s:
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&nbsp;<br>
;Orthologs have diverged after speciation.

;Paralogs have diverged after duplication.
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[[Image:OrthologParalog.jpg|frame|none|'''Hypothetical evolutionary tree.''' A single gene evolves through two speciation events and one duplication event. A duplication occurs during the evolution from reptilian to synapsid. It is easy to see how this pair of genes (paralogs) in the ancestral synapsid gives rise to two pairs of genes in pig and elephant, respectively. All ''circle'' genes are mutually orthologs, they form a "cluster of orthologs". All genes within one species are mutual paralogs&ndash;they are so called ''in-paralogs''. The ''circle'' gene in pig and the ''triangle'' gene in the elephant are so-called ''out-paralogs''. Somewhat counterintuitively, the ''triangle'' gene in the pig and the ''circle'' gene in the raven are also orthologs - but this has to be, since the last common ancestor diverged by '''speciation'''.

The "phylogram" on the right symbolizes the amount of evolutionary change as proportional to height difference to the "root". It is easy to see how a bidirectional BLAST search will only find pairs of most similar orthologs. If applied to a group of species, bidirectional BLAST searches will find clusters of orthologs only (except if genes were lost, or there are  anomalies in the evolutionary rate.)]]




== Self-evaluation ==
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== Further reading, links and resources ==

{{#pmid: 26323765}}
{{#pmid: 22114356}}
{{#pmid: 19190756}}

Also: [http://www.nature.com/scitable/topicpage/reading-a-phylogenetic-tree-the-meaning-of-41956 Nature-Scitable (2008): '''Reading a Phylogenetic Tree: The Meaning of Monophyletic Groups''']

== Notes ==
<references />

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<div class="about">
<b>About ... </b><br />
&nbsp;<br />
<b>Author:</b><br />
:Boris Steipe <boris.steipe@utoronto.ca>
<b>Created:</b><br />
:2017-08-05
<b>Modified:</b><br />
:2020-09-25
<b>Version:</b><br />
:1.1
<b>Version history:</b><br />
*1.1 2020 updates; remove iTOL (now using taxize::)
*1.0 First live version.
*0.1 First stub
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