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Survey of softwares for
phylogenetic analysis
ag1805xag1805x
PhylogeneticsPhylogenetics
Phylogenetics - in biology – is the study of the
evolutionary history and relationships among
individuals or groups of organisms (e.g. species, or
populations).
These relationships are discovered through
phylogenetic inference methods that evaluate observed
heritable traits, such as DNA sequences or morphology
under a model of evolution of these traits.
The result of these analyses is a phylogeny (also
known as a phylogenetic tree) – a hypothesis about the
history of evolutionary relationships
Phylogenetic treePhylogenetic tree
Phylogenetic tree also known as
“evolutionary tree” is the
graphical representation of the
evolutionary relationship between
the taxa/genes in question.
A dendrogram is a broad term
for the diagrammatic
representation of a phylogenetic
tree.
The cladogram is a dendogram which explains only
genealogy of the taxa but says nothing about the
branch lengths or time periods of divergence.
The phylogram (additive tree) is a phylogenetic tree that
explicitly represents a number of character changes
(nucleotide/amino acid changes/number of character
variations) through its branch lengths. In case of phylogram
the evolutionary distance between any two taxa is given by
sum of the branch lengths connected them. Though these
trees may be rooted or unrooted, often these trees lack a
root.
A chronogram (ultra metric) is a rooted phylogenetic tree that possesses
all the characteristics of an additive tree, in addition with the
assumption of molecular clock determination of the molecular
divergence time between taxa can be possible. The molecular clock
hypothesis assumes that every site in a protein or coding nucleotide
sequence from all the species evolve at a constant rate. Furthermore,
the chronogram consists of taxa placed equidistant from the ancestor
which cannot be seen in case of phylogram.
Phenetics (taximetrics) infers the relationship between
the taxa that usually involves morphology or other
observable traits as phylogenetic informative markers.
A tree that shows the evolution of the genes is known as
gene tree. While, tree that shows the evolution of species is
known as species' tree
The whole process of construction of the
phylogenetic tree is divided into five different
steps, viz.
Step 1: Choosing an appropriate markers for
the phylogenetic analysis
Step 2: Multiple sequence alignments
Step 3: Selection of an evolutionary model
Step 4: Phylogenetic reconstruction
Step 5: Evaluation of the phylogenetic tree
Process of construction of the phylogenetic treeProcess of construction of the phylogenetic tree
Step 1: Choosing an appropriate markers for theStep 1: Choosing an appropriate markers for the
phylogenetic analysisphylogenetic analysis
Any biological information that can be used to infer the
evolutionary relationship among the taxa is known as a
phylogenetic information marker.
It can be anything like DNA, RNA, protein, RFLP, AFLP,
ISSR, allozymes, and conserved intronic positions, etc.
Identification of conserved genetic loci (coding- or non-
coding) is the first step in analyzing the phylogenetic
relationship.
Both coding (genes) and non-coding genetic region can be
used for the analysis of phylogenetic relationships.
However, selected sequence(s) must satisfy the defined necessary
rules:
(a) the sequence should have a long evolutionary history of
conservation, as this feature facilitates, firstly in the preservation
of long evolution-selection episodes, and secondly, aids in easy
amplification of the target sequences from distant taxa.
(b) conserved, slow evolving genes may be used to resolve the
evolutionary relationship between distantly related species while
fast evolving genes should be choose for the recently evolved
species or intra-species.
(c) amino acid sequences are more informative while inferring the
evolutionary relationship among distantly related taxa, and
conversely, nucleotide information for recently evolved/closely
related species.
(d) the sequences need to be employed in the phylogenetic
analysis should be tested for their usability in a given lineage (for
instance, mitochondrial (cytochrome C oxidase subunit I & II
(CoxI & II)), chloroplast (trnH-psbA, matK, rpoC, rpoB, rbcL),
and nuclear (16S ribosomal RNA) conserved genes are preferred
to use for analyzing animal, plant, and microbial species,
respectively-and are called “barcode genes”).
(e) finally, if, objective is to estimate the divergence periods
between taxa, the selected gene or protein sequences should
essentially follow the molecular clock hypothesis. However,
recently relaxed molecular clock models have also been proposed.
This step follows successful polymerase chain reaction
amplification of the target gene/protein, followed by sequencing
and editing of the sequences for further analysis.
Step 2: Multiple sequence alignmentsStep 2: Multiple sequence alignments
●The main aim of multiple sequence alignment is to compare
the three or more nucleotide or protein sequences and to
provide the basis for calculation of the sequence
diversities/divergences to infer the evolutionary relationship
among the taxa.
●Different models (discussed below) have been proposed based
on various assumptions to calculate the sequence divergences
between the sequences or taxa. Hence, the correct sequence
alignment is mandatory in order to get the true phylogeny that
is representative of the evolutionary relationship among the
taxa.
Step 3: Selection of an evolutionary modelStep 3: Selection of an evolutionary model
Evolutionary models are sets of assumptions about 
the process of nucleotide or amino­acid substitution. 
They describe the different probabilities of change 
from one nucleotide or amino acid to another, with 
the aim of correcting for unseen changes along the
Phylogeny.
Step 4: Phylogenetic reconstructionStep 4: Phylogenetic reconstruction
Two different methodologies are employed by the presently
available programs to generate the dendograms;
(a) clustering methods-where two most closely related taxa
are placed under single inter-node and further add third
taxa considering within internodes taxa as a single group.
In this way, the program progressively adds the other
remaining taxa to yield final phylogenetic tree
(b) second type of methods generate the 'n' number of trees
proportional to the number of taxa involved in the
phylogenetic analysis followed by the selection of best fit
tree topology (increased likelihood or probability) for a
given evolutionary model.
Step 5: Evaluating the phylogenetic treeStep 5: Evaluating the phylogenetic tree
This process can be performed using two
evaluation methods, namely bootstrap
method and interior-branch test.
PracticalsPracticals
Phylogenetic information marker: Cytochrome C
oxidase subunit I (CoxI)
Organisms:
Homo sapiens (Human)
Bos taurus (Bovine)
Danio rerio (Zebrafish)
Sus scrofa (Pig)
Ovis aries (Sheep)
Software  used:  Clustal  Omega  (MSA)  &  Clustal 
W2 phylogeny
Downloading the COX1 sequence from UniProt
>sp|P00395|COX1_HUMAN Cytochrome c oxidase subunit 1 OS=Homo sapiens GN=MT­CO1 PE=1 
SV=1
MFADRWLFSTNHKDIGTLYLLFGAWAGVLGTALSLLIRAELGQPGNLLGNDHIYNVIVTA
HAFVMIFFMVMPIMIGGFGNWLVPLMIGAPDMAFPRMNNMSFWLLPPSLLLLLASAMVEA
GAGTGWTVYPPLAGNYSHPGASVDLTIFSLHLAGVSSILGAINFITTIINMKPPAMTQYQ
TPLFVWSVLITAVLLLLSLPVLAAGITMLLTDRNLNTTFFDPAGGGDPILYQHLFWFFGH
PEVYILILPGFGMISHIVTYYSGKKEPFGYMGMVWAMMSIGFLGFIVWAHHMFTVGMDVD
TRAYFTSATMIIAIPTGVKVFSWLATLHGSNMKWSAAVLWALGFIFLFTVGGLTGIVLAN
SSLDIVLHDTYYVVAHFHYVLSMGAVFAIMGGFIHWFPLFSGYTLDQTYAKIHFTIMFIG
VNLTFFPQHFLGLSGMPRRYSDYPDAYTTWNILSSVGSFISLTAVMLMIFMIWEAFASKR
KVLMVEEPSMNLEWLYGCPPPYHTFEEPVYMKS
>sp|P00396|COX1_BOVIN Cytochrome c oxidase subunit 1 OS=Bos taurus GN=MT­CO1 PE=1 SV=1
MFINRWLFSTNHKDIGTLYLLFGAWAGMVGTALSLLIRAELGQPGTLLGDDQIYNVVVTA
HAFVMIFFMVMPIMIGGFGNWLVPLMIGAPDMAFPRMNNMSFWLLPPSFLLLLASSMVEA
GAGTGWTVYPPLAGNLAHAGASVDLTIFSLHLAGVSSILGAINFITTIINMKPPAMSQYQ
TPLFVWSVMITAVLLLLSLPVLAAGITMLLTDRNLNTTFFDPAGGGDPILYQHLFWFFGH
PEVYILILPGFGMISHIVTYYSGKKEPFGYMGMVWAMMSIGFLGFIVWAHHMFTVGMDVD
TRAYFTSATMIIAIPTGVKVFSWLATLHGGNIKWSPAMMWALGFIFLFTVGGLTGIVLAN
SSLDIVLHDTYYVVAHFHYVLSMGAVFAIMGGFVHWFPLFSGYTLNDTWAKIHFAIMFVG
VNMTFFPQHFLGLSGMPRRYSDYPDAYTMWNTISSMGSFISLTAVMLMVFIIWEAFASKR
EVLTVDLTTTNLEWLNGCPPPYHTFEEPTYVNLK
>sp|Q9MIY8|COX1_DANRE Cytochrome c oxidase subunit 1 OS=Danio rerio GN=mt­co1 PE=2 SV=1
MTITRWFFSTNHKDIGTLYLVFGAWAGMVGTALSLLIRAELSQPGALLGDDQIYNVIVTA
HAFVMIFFMVMPILIGGFGNWLVPLMIGAPDMAFPRMNNMSFWLLPPSFLLLLASSGVEA
GAGTGWTVYPPLAGNLAHAGASVDLTIFSLHLAGVSSILGAINFITTTINMKPPTISQYQ
TPLFVWAVLVTAVLLLLSLPVLAAGITMLLTDRNLNTTFFDPAGGGDPILYQHLFWFFGH
PEVYILILPGFGIISHVVAYYAGKKEPFGYMGMVWAMMAIGLLGFIVWAHHMFTVGMDVD
TRAYFTSATMIIAIPTGVKVFSWLATLHGGAIKWETPMLWALGFIFLFTVGGLTGIVLAN
SSLDIVLHDTYYVVAHFHYVLSMGAVFAIMAGFVHWFPLFTGYTLNSVWTKIHFGVMFIG
VNLTFFPQHFLGLAGMPRRYSDYPDAYALWNTVSSIGSLISLVAVIMFLFILWEAFTAKR
EVLSVELTATNVEWLHGCPPPYHTFEEPAFVQIQSN
>sp|O79876|COX1_PIG Cytochrome c oxidase subunit 1 OS=Sus scrofa GN=MT­CO1 PE=3 SV=2
MFVNRWLYSTNHKDIGTLYLLFGAWAGMVGTALSLLIRAELGQPGTLLGDDQIYNVIVTA
HAFVMIFFMVMPIMIGGFGNWLVPLMIGAPDMAFPRMNNMSFWLLPPSFLLLLASSMVEA
GAGTGWTVYPPLAGNLAHAGASVDLTIFSLHLAGVSSILGAINFITTIINMKPPAMSQYQ
TPLFVWSVLITAVLLLLSLPVLAAGITMLLTDRNLNTTFFDPAGGGDPILYQHLFWFFGH
PEVYILILPGFGMISHIVTYYSGKKEPFGYMGMVWAMMSIGFLGFIVWAHHMFTVGMDVD
TRAYFTSATMIIAIPTGVKVFSWLATLHGGNIKWSPAMLWALGFIFLFTVGGLTGIVLAN
SSLDIVLHDTYYVVAHFHYVLSMGAVFAIMGGFVHWFPLFSGYTLNQAWAKIHFVIMFVG
VNMTFFPQHFLGLSGMPRRYSDYPDAYTAWNTISSMGSFISLTAVMLMIFIIWEAFASKR
EVSAVELTSTNLEWLHGCPPPYHTFEEPTYINLK
>sp|O78749|COX1_SHEEP Cytochrome c oxidase subunit 1 OS=Ovis aries GN=MT­CO1 PE=3 SV=1
MFINRWLFSTNHKDIGTLYLLFGAWAGMVGTALSLLIRAELGQPGTLLGDDQIYNVIVTA
HAFVMIFFMVMPIMIGGFGNWLVPLMIGAPDMAFPRMNNMSFWLLPPSFLLLLASSMVEA
GAGTGWTVYPPLAGNLAHAGASVDLTIFSLHLAGVSSILGAINFITTIINMKPPAMSQYQ
TPLFVWSVLITAVLLLLSLPVLAAGITMLLTDRNLNTTFFDPAGGGDPILYQHLFWFFGH
PEVYILILPGFGMISHIVTYYSGKKEPFGYMGMVWAMMSIGFLGFIVWAHHMFTVGMDVD
TRAYFTSATMIIAIPTGVKVFSWLATLHGGNIKWSPAMMWALGFIFLFTVGGLTGIVLAN
SSLDIVLHDTYYVVAHFHYVLSMGAVFAIMGGFVHWFPLFSGYTLNDTWAKIHFAIMFVG
VNMTFFPQHFLGLSGMPRRYSDYPDAYTMWNTISSMGSFISLTAVMLMIFIIWEAFASKR
EVLTVDLTTTNLEWLNGCPPPYHTFEEPTYVNLK
Multiple Sequence Alignment using Clustal Omega
MSA result page
Phylogenetic tree construction using ClustalW2 Phylogeny
Result page of tree construction
Other softwares for phylogenetic analysisOther softwares for phylogenetic analysis
Description: Phylogenetic 
inference package
Methods: Maximum 
parsimony, distance matrix, 
maximum likelihood
Description: Molecular Evolutionary Genetics 
Analysis
Methods: Distance, Parsimony and Maximum 
Composite Likelihood Methods
T­REX 
Description: Tree inference and visualization, 
Horizontal gene transfer detection, multiple 
sequence alignment
Methods: Distance (neighbor joining), Parsimony 
and Maximum likelihood (PhyML, RAxML) tree 
inference, MUSCLE, MAFFT and ClustalW 
sequence alignments and related applications
Description:  Fast  and  free  multiplatform  tree 
editor
Methods: based Phylip 3.6 package algorithms
BEAST
Description: Bayesian Evolutionary Analysis 
Sampling Trees
Methods: Bayesian inference, relaxed molecular 
clock, demographic history
And many more......
ag1805xag1805x

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