PROJECT

(phylogenetic tree building)

Name: Bhoomi jain

Course: BSc life science (II year)
Roll no.: 23583054
Subject: Ecology and Evolution
AIM: Create multiple sequence alignment and construct a phylogenetic tree for the given
sequences through Neighbour joining approach using MEGA software.

THEORY:
Phylogenetic Tree
A phylogenetic tree, phylogeny or evolutionary tree is a graphical representation which shows
the evolutionary history between a set of species or taxa during a specific time. In other words, it
is a branching diagram or a tree showing the evolutionary relationships among various biological
species or other entities based upon similarities and differences in their physical or genetic
characteristics. IN evolutionary biology, all life on Earth is theoretically part of a single
phylogenetic tree, indicating common ancestry.
Phylogenetics is the study of phylogenetic trees. The main challenge is to find a
phylogenetic tree representing optimal evolutionary ancestry between a set of species
or taxa.
Computational phylogenetics (also phylogeny inference) focuses on the algorithms involved in
finding optimal phylogenetic tree in the phylogenetic landscape.
Phylogenetic trees may be rooted or unrooted.
In a rooted phylogenetic tree, each node with descendants represents the inferred most recent
common ancestor of those descendants, and the edge lengths in some trees may be interpreted as
time estimates. Each node is called a taxonomic unit. Internal nodes are generally called
hypothetical taxonomic units, as they cannot be directly observed. Trees are useful in fields of
biology such as bioinformatics, systematics, and phylogenetics.
Unrooted trees illustrate only the relatedness of the leaf nodes and do not require the ancestral
root to be known or inferred.

Significance
1. Phylogenetic trees can be used to study the evolutionary relationships between different
species and to understand the evolutionary processes over time.
2. Phylogenetic trees can be used to study the diversity and distribution of species and to develop
conservation strategies to protect endangered species and ecosystems.
3. Phylogenetic trees can be used to identify the origins of pathogens and to track the spread of
diseases.
4. Phylogenetic trees can also be used in forensics to identify the origins of biological samples
found at crime scenes and to link suspects to crimes.
5. Phylogenetic trees are useful for organizing and classifying organisms and species according
to their DNA sequences and morphological similarities and differences.

Phylogenetic Tree Construction Methods

There are two different methods based on which the phylogenetic tree is constructed.
1. Character-based method
This method is based directly on the sequence characters; therefore, it is also called the discrete
method. The character-based method uses the aligned characters for constructing the
phylogenetic tree. These aligned characters include either the DNA or protein sequences during
the tree inference. The two most popular character-based methods are: Maximum parsimony and
Maximum likelihood.

2 . Distance-based method
This method is based on the amount of the distance or the dissimilarity between the two aligned
sequences. In this method of constructing the phylogenetic tree, the sequence data is transformed
into pairwise distances and then the matrix is used for building a tree.
Both rooted and unrooted trees can be either bifurcating or multifurcating. A rooted bifurcating
tree has exactly two descendants arising from each interior node (that is, it forms a binary tree),
and an unrooted bifurcating tree takes the form of an unrooted binary tree, a free tree with
exactly three Neighbours at each internal node.
In contrast, a rooted multifurcating tree may have more than two children at some nodes and an
unrooted multifurcating tree may have more than three neighbours at some nodes.
 FLOWCHART FOR THE PRACTICAL
1. Open the MEGA window
2. Click the Align Tab → Edit/Build Alignment

3. Select → Create a new alignment → OK

4.MX: Alignment Explorer window opens

5.Select Protein as the Data Type for Alignment.

6. Copy the protein sequences given in FASTA format.

7. Delete the last blank Sequence, if present, from the MX: Alignment Explorer window.
Mega can align multiple sequences using ClustalW as well as MUSCLE.
8. Click Alignment → ClustalW → OK
9.Click Data → Export Alignment → MEGA Format in the MX: Alignment Explorer

10.It will pop up a “Save file as” window. Create a folder Globin on the desk top and save the
file as protien in the new folder.
11.A title window pops up. Write protien and press OK.

12. Click PHYLOGENY tab → Select the approach you want to use for building the tree. For
example, UPGMA.
13. A pop-up window will appear for the data file. MEGA software uses Mega format file
MSA to construct phylogenetic tree.
14. Select protein file from the protein folder created on the desktop.

15. A pop-up window will appear for the analysis preferences. Use default parameters and
continue.
16. After processing tree explorer window will pop-up showing the phylogenetic tree and
explanation of evolutionary relationships of taxa.

17.Save the image of the phylogenetic tree by clicking Image → Save as PNG File (or
the format of your choice).
18. A window for Image export option pops-up. Save the image in the phylogenetic tree
construction folder created earlier. Click OK.
Evolutionary relationships of 7 taxa
The evolutionary history was inferred using the UPGMA method [1] The optimal tree with the
sum of branch length =6.436 is shown. The tree is drawn to scale, with branch lengths in the
same units as those of the evolutionary distances used to infer the phylogenetic tree. The
evolutionary distances were computed using the Poisson correction method (2) and are in the
units of the number of amino acid substitutions per site The analytical procedure encompassed 7
amino acid sequences. The pairwise deletion option was applied to all ambiguous positions for
each sequence pair resulting in a final data set comprising 517 positiones. Evolutionary analyses
were conducted in MEGA12 (3)
1. Sneath PHA. and Sokal RR. (1973) Numerical Taxonomy. Freeman, San Francisco
2. Zuckerkandi E and Pauling L. (1965) Evolutionary divergence and convergence in proteins
Edited in Evolving Geries and Proteins by V. Bryson and H.J. Vogel, pp. 97-166. Academic
Press, New York.
3 Kumar S. Stecher G. Suleski M, Sanderford M., Sharma S, and Tamura K (2024) Molecular
Evolutionary Genetics Analysis Version 12 for adaptive and green computing Molecular Biology
and Evolution 41-1-0
Disclaimer. While every effort has been made to ensure the accuracy of the caption, it is
provided "as is" without warranty of any kind Users are adirsed to thoroughly review the caption
before its use for any purpose and repo authors at www.megausftware.nt. The authors and their
employers disclaim any lability for damages, including but not limited to special or
consequential damages. Addtionaly, the authors expresly disclaim afl other warrantes, whether
expressed implied, including the suitability of the caption text for a specific purpose, use, or
applicatien

INTERPRETATION
1. Grouping of Similar Items:

The diagram shows how closely related the different "SET2" items are based on some metric
(e.g., similarity or distance).

Closer branches indicate more similar groups, while longer branches suggest more dissimilarity.

2. Cluster Formation:

SET2 A and SET2 B are grouped together, indicating they are closely related.

SET2 D and SET2 C are also in a similar cluster.

SET2 E, SET2 G, and SET2 F form another major cluster, suggesting they share similarities.

3. Hierarchical Structure:

The clustering occurs step by step, where smaller clusters merge into larger clusters as we move
leftward.

The largest separation (longest branch) indicates the most significant distinction between groups.