PHYLOGENETIC ANALYSIS

1. Purpose of phylogenetics :
With the aid of sequences, it should be possible to find the genealogical ties between organisms.
Experience learns that closely related organisms have similar sequences, more distantly related
organisms have more dissimilar sequences. One objective is to reconstruct the evolutionary
relationship between species. An other objective is to estimate the time of divergence between
two organisms since they last shared a common ancestor.

2. Disclaimers :
The theory and practical applications of the different models are not universally accepted. With
one dataset, different software packages can give different results. Changes in the dataset can
also give different results. Therefore it is important to have a good alignment to start with.
Trees based on an alignment of a gene represent the relationship between genes and this is not
necessarily the same relationship as between the whole organisms. If trees are calculated based
on different genes from organisms, it is possible that these trees result in different relationships.

3. Terminology :
node : a node represents a taxonomic unit. This can be a taxon (an existing species) or an
ancestor(unknown species : represents the ancestor of 2 or more species).
branch : defines the relationship between the taxa in terms of descent and ancestry.
topology : is the branching pattern.
branch length : often represents the number of changes that have occurred in that branch.
root : is the common ancestor of all taxa.
distance scale : scale which represents the number of differences between sequences (e.g. 0.1
means 10 %differences between two sequences)

How to construct a Phylogenetic tree?

• A phylogenetic tree is a visual representation of the relationship between different
organisms, showing the path through evolutionary time from a common ancestor to different
descendants.
• Similarities and divergence among related biological sequences revealed by sequence
alignment often have to be rationalized and visualized in the context of phylogenetic trees.
Thus, molecular phylogenetics is a fundamental aspect of bioinformatics.
• Molecular phylogenetics is the branch of phylogeny that analyzes genetic, hereditary
molecular differences, predominately in DNA sequences, to gain information on an organism’s
evolutionary relationships.
• The similarity of biological functions and molecular mechanisms in living organisms
strongly suggests that species descended from a common ancestor. Molecular phylogenetics
uses the structure and function of molecules and how they change over time to infer these
evolutionary relationships.
• From these analyses, it is possible to determine the processes by which diversity among
species has been achieved. The result of a molecular phylogenetic analysis is expressed in a
phylogenetic tree.

Phylogenetic Analysis and the Role of Bioinformatics

Molecular data that are in the form of DNA or protein sequences can also provide very useful
evolutionary perspectives of existing organisms because, as organisms evolve, the genetic
materials accumulate mutations over time causing phenotypic changes. Because genes are the
medium for recording the accumulated mutations, they can serve as molecular fossils. Through
comparative analysis of the molecular fossils from a number of related organisms, the
evolutionary history of the genes and even the organisms can be revealed. However, phylogeny
inference are notoriously difficult endeavours because the number of solutions increases
explosively with the number of taxa and the tremendous number of new questions in
evolutionary biology that could be investigated through the use of larger taxon samplings.
But with the development and use of computational and an array of bioinformatics tools, the
ability to analyze large data sets in practical computing times, and yielding an optimal or near-
optimal solutions with high probability are being possible. In response to this trend, much of
the current research in phyloinformatics (i.e., computational phylogenetics) concentrates on the
development of more efficient heuristic approaches.
Steps in Phylogenetic Analysis
The basic steps in any phylogenetic analysis include:
1. Assemble and align a dataset
• The first step is to identify a protein or DNA sequence of interest and assemble a dataset
consisting of other related sequences.
• DNA sequences of interest can be retrieved using NCBI BLAST or similar search tools.
• Once sequences are selected and retrieved, multiple sequence alignment is created.
• This involves arranging a set of sequences in a matrix to identify regions of homology.
• There are many websites and software programs, such as ClustalW, MSA, MAFFT,
and T-Coffee, designed to perform multiple sequence on a given set of molecular data.
2. Build (estimate) phylogenetic trees from sequences using computational methods and
stochastic models
• To build phylogenetic trees, statistical methods are applied to determine the tree
topology and calculate the branch lengths that best describe the phylogenetic relationships of
the aligned sequences in a dataset.
• The most common computational methods applied include distance-matrix methods,
and discrete data methods, such as maximum parsimony and maximum likelihood.
• There are several software packages, such as Paup, PAML, PHYLIP, that apply these
most popular methods.

3. Statistically test and assess the estimated trees.

• Tree estimating algorithms generate one or more optimal trees.
• This set of possible trees is subjected to a series of statistical tests to evaluate whether
one tree is better than another – and if the proposed phylogeny is reasonable.
• Common methods for assessing trees include the Bootstrap and Jackknife Resampling
methods, and analytical methods, such as parsimony, distance, and likelihood.

Bioinformatics Tools for Phylogenetic Analysis

• There are several bioinformatics tools and databases that can be used for phylogenetic
analysis.
• These include PANTHER, P-Pod, PFam, TreeFam, and the PhyloFacts structural
phylogenomic encyclopedia.
• Each of these databases uses different algorithms and draws on different sources for
sequence information, and therefore the trees estimated by PANTHER, for example, may differ
significantly from those generated by P-Pod or PFam.
• As with all bioinformatics tools of this type, it is important to test different methods,
compare the results, then determine which database works best (according to consensus results)
for studies involving different types of datasets.
 Phylogenetic Models
Possible ways of drawing a tree :Trees can be drawn in different ways. There are trees with
unscaled branches and with scaled branches.
Unscaled branches : the length is not proportional to the number of changes. Sometimes, the
number of changes are indicated on the branches with numbers. The nodes represents the
divergence event on a timescale.
Scaled branches : the length of the branch is proportional to the number of changes. The
distance between2 species is the sum of the length of all branches connecting them.
Is is also possible to draw these trees with or without a root. For rooted trees, the root is the
common ancestor. For each species, there is a unique path that leads from the root to that
species. The direction of each path corresponds to evolutionary time. An unrooted tree
specifies the relationships among species and does not define the evolutionary path.
Figure 2: Some possibilities for drawing a tree. (these are just a few examples, there are a lot
of variations possible)
 Tree Building methods

5. Methods of phylogenetic analysis :

There are two major groups of analyses to examine phylogenetic relationships between
sequences :
Phenetic methods : trees are calculated by similarities of sequences and are based on distance
methods. The resulting tree is called a dendrogram and does not necessarily reflect
evolutionary relationships. Distance methods compress all of the individual differences
between pairs of sequences into a single number.
Cladistic methods : trees are calculated by considering the various possible pathways of
evolution and are based on parsimony or likelihood methods. The resulting tree is called a
cladogram. Cladistic methods use each alignment position as evolutionary information to
build a tree.

5.1. Phenetic methods based on distances :

Starting from an alignment, pairwise distances are calculated between DNA sequences as the
sum of all base pair differences between two sequences (the most similar sequences are
assumed to be closely related). This creates a distance matrix. All base changes can be
considered equally or a matrix of the possible replacements can be used.
Insertions and deletions are given a larger weight than replacements. Insertions or deletions of
multiple bases at one position are given less weight than multiple independent insertions or
deletions.
it is possible to correct for multiple substitutions at a single site.

From the obtained distance matrix, a phylogenetic tree is calculated with clustering
algorithms. These cluster methods construct a tree by linking the least distant pair of taxa,
followed by successively more distant taxa. UPGMA clustering (Unweighted Pair Group
Method using Arithmetic averages) : this is the simplest method
Neighbor Joining : this method tries to correct the UPGMA method for its assumption that
the rateof evolution is the same in all taxa.

5.2. Cladistic methods based on Parsimony :For each position in the alignment, all possible
trees are evaluated and are given a score based on the number of evolutionary changes needed
to produce the observed sequence changes. The most parsimonious tree is the one with the
fewest evolutionary changes for all sequences to derive from a common ancestor. This is a
more time-consuming method than the distance methods.
5.3. Cladistic methods based on Maximum Likelihood :This method also uses each position
in an alignment, evaluates all possible trees, and calculates the likelihood for each tree using
an explicit model of evolution (<-> Parsimony just looks for the fewest evolutionary changes).
The likelihood's for each aligned position are then multiplied to provide a likelihood for each
tree. The tree with the maximum likelihood is the most probable tree. This is the slowest
method of all but seems to give the best result and the most information about the tree.

Theoretical problems with evolutionary changes between sequences

Transitions : substitutions from A to G ; G to A ; C to T ; T to C.
Transversions : substitutions from G to C ; C to G ; T to A ; A to T.
Deletions : removal of one or more nucleotides.
Insertion : addition of one or more nucleotides.
Inversion : rotation of 180 °C of a double stranded DNA segment compromised of of 2 or more
base pairs.

The next figure shows that there is a chance that many more mutations occur than visible at a
certain time. Even the best evolutionary models can't solve this problem...

Figure 3: Two homologous DNA sequences which descended from an ancestral sequence and
accumulated mutations since their divergence from each other. Note that although 12 mutations
have accumulated, differences can be detected at only three nucleotide sites. (from
Fundamentals of Molecular Evolution, Wen-Hsiung Li andDan Graur, 1991).
7. Graphical explanation of basic phylogenetic terms

Monophyletic taxon : A group composed of a collection of organisms, including the most

recent common ancestor of all those organisms and all the descendants of that most recent
common ancestor. Monophyletic groups are also called a clade.

Examples : Mammalia, Aves (birds), angiosperms, insects, etc.

Paraphyletic taxon : A group composed of a collection of organisms, including the most

recent common ancestor of all those organisms. Unlike a monophyletic group, a
paraphyletic taxon does not include all the descendants of the most recent common ancestor.

Examples : Traditionally defined Dinosauria, fish, gymnosperms, invertebrates, protists, etc.

Polyphyletic taxon : A group composed of a collection of organisms in which the most

recent common ancestor of all the included organisms is not included, usually because the
common ancestor lacks the characteristics of the group.
Polyphyletic taxa are considered "unnatural", and usually are reclassified once they are
discovered to be polyphyletic.

Examples : marine mammals, bipedal mammals, flying vertebrates, trees, algae, etc.
 Phylogenetic softwares

1. MEGA
MEGA is a useful software in constructing phylogenies and visualizing them, and also for data
conversion. It can easily convert alignment files to other formats such as nexus, paup, phylip,
and fasta, and so on. The MEGA tree explorer is helpful in editing trees very easily, subtrees
can also be selected and edited separately. Some tree image export options are also available.
The input formats are newick, phylip, mega, and nexus. The phylogenetic tree can also be
converted in newick format but it falls short on converting it into other formats such as phylip
which is required in other analyses such as selection analysis.

2. Dendroscope
It is helpful in visualizing large trees and provides several options to export their graphics with
a command line. Several different views are also available, trees can be easily re-rooted and
node labels and branches can be easily formatted. It can export trees in newick and nexus
format. Although users will have to register themselves first to use this feature.

3. FigTree
It is actually designed to visualize trees that are produced by BEAST program. Tip labels and
node labels can be easily edited. It can easily export trees in nexus, newick, and JSON format
with some graphics export options such as emf, pdf, sg, png, etc.

4. Phylotree.js
It is a javascript based library to visualize and annotate trees and offer some other
customizations. It has a wide application in Datamonkey comparative analyses. A user can
upload trees using Phylotree.js where a user can easily select test and reference branches, and
any changes can be mapped to their position on the corresponding structure. It is also good for
comparison of trees with links between leaves known as a tanglegram, where crossings can
represent evolutionary events. It also offers several export options and other built-in features.

5. ggtree
ggtree is an R package for phylogenetic tree visualization and annotation. It also displays
annotation data on the tree apart from visualizing it. Users can annotate trees with their own
data and can easily convert trees into a data frame, and a lot of other features are available
(https://guangchuangyu.github.io/software/ggtree/).