PROFESSOR JAYASHANKAR TELANGANA STATE

AGRICULTURAL UNIVERSITY
College of Agriculture, Rajendranagar, Hyderabad- 500030

Presented by,
Ajay
Kumar
Chandra
RAM/14-
97
“The time will come, I believe, though I shall not live to see it, when
we shall have fairly true genealogical trees of each great kingdom
of Nature” - Charles Darwin
 What is Molecular Phylogenetics?

Phylogenetics is the study of evolutionary relationships.

Example - relationship among species crocodiles

birds
Primates lizards
Rodents
snakes
Crocodiles Birds
rodents
primates
Marsupials
Snakes marsupials
Lizards
•Systematics is an analytical approach to understanding the diversity
and relationships of organisms, both present-day and extinct.
•Systematists use morphological, biochemical, and
molecular comparisons to infer evolutionary relationships.
 A Brief History of Molecular
Phylogenetics
1900s
Immunochemical studies: cross-reactions stronger for closely related
organisms
Nuttall (1902) - apes are closest relatives to humans

1960s - 1970s
Protein sequencing methods, electrophoresis, DNA hybridization and
PCR contributed to a boom in molecular phylogeny

late 1970s to present

Discoveries using molecular phylogeny:
- Endosymbiosis - Margulis, 1978
- Divergence of phyla and kingdom - Woese, 1987
- Many Tree of Life projects completed or underway.
 Endosymbiosis: Origin of the Mitochondrion
and Chloroplast
-Purple Bacteria
Other bacteria

Chloropl
asts
Mitochondria

Root Cyanobacteria
Eukaryotes

Archaea

Mitochondria and chloroplasts are derived from the -

purple bacteria and the cyanobacteria respectively, via
separate endosymbiotic events.
 Universal Tree of Life

• Using rRNA sequences: Woese, 1987

• Able to study the relationships of uncultivated organisms, obtained from a
hot
 Phylogeny and classification
• Phylogenetic (cladistic) classification reflects evolutionary history.
• The only objective form of classification
– organisms share a true evolutionary history regardless of
our
arbitrary decisions of how to classify them. Phylogeny
Classification
Genus
Family
Genus
Order
Genus
Family
Genus
Class
Genus
Family
Genus
Order
Genus
Family
Genus
 Phylogenetic concepts
- Relationships are illustrated by a Phylogenetic tree / dendrogram
- The branching pattern is call the tree’s topology
- Trees can be represented in several forms:

Rectangular cladogram Slanted cladogram

 Tree Terminology
Operational taxonomic units (OTU) / Taxa

Internal nodes A

C
Terminal nodes
D
Sisters
Root E

Branches
Polytomy
 Phylogenetic trees
Phylogentic trees: (A) Rooted; (B)
Unrooted

Rooted tree 1 Rooted tree 2 Rooted tree 3 Rooted tree 4 Rooted tree 5

B A A C D

A B D C
B

C C C A A

D
D D B B
These trees show five different evolutionary relationships among the
taxa!
 Rooting and Tree Interpretation
chicken
human
fruit fly
Chicken oak
Human Bones
bacteria
Oak
Cell nuclei
archaea
Fruit fly
Bacteria
Archaebacteria
Oak
Bacteria Archaebacteria Fruit fly
+ Cell nuclei Human

+ Bones Chicken
 How Many Trees?
Unrooted trees Rooted trees
Pair wise Branches Branches
Sequences distances Trees /tree Trees /tree

3 3 1 3 3 4
4 6 3 5 15 6
5 10 15 7 105 8
6 15 105 9 945 10
10 45 2,027,025 17 34,459,425 18

30 435 8.69  1036 57 4.95  1038 58

N N (N - 1) (2N - 5)! 2N - 3 (2N - 3)! 2N - 2
2 2N - 3 (N - 3)! 2N - 2 (N - 2)!
 Scaled vs. Un-scaled trees

Scaled trees: Branch lengths are proportional to the number of

nucleotide/amino acid changes that occurred on that branch.
Unscaled trees: Branch lengths are not proportional to the number of
nucleotide/amino acid changes (usually used to illustrate evolutionary
relationships only).
 Phylogenetic Trees and Timing

•Construction of Phylogenetic

Cenozoic
trees based on shared

65.5
Mesozoic
characteristics.

251
a Phylogenetic,
• Branching timing of divergences.
representing
of

Paleozoic
•Length of a branch in a

542
cladogram reflects the number of
genetic changes. Neoproterozoic

Millions of
years ago
Organism’s evolutionary history is documented
in its genome

AAGACTT -3 mil yrs

-2 mil yrs
AAAAGGGG TTGG
GGAA

CCCCTT CCTTTT
AGGGCA TAGCCC AGCACTT -1 mil yrs
T T

AGGGCAT TAGCCCA TAGACTT AGCACAA AGCGCTT Toda

y
• Comparing nucleic acids to infer relatedness is a valuable tool for
tracing organisms’ evolutionary history.
• Gene duplication increases the number of genes in the
genome, providing more opportunities for evolutionary changes.
Four stages of Phylogenetic
analysis
Molecular Phylogenetic analysis may be described
in four stages:
1. Selection of sequences for analysis
2. Multiple sequence alignment
3. Tree building
4. Tree evaluation
•Sequence alignments can provide clues to evolutionary change by
examining the effect of mutations occurring over time in species with
a common ancestor.
 Examples: sequences for analysis
• Alignment of a portion of the casein gene

•DNA sequence changes in the Cytochrome-c gene

reflect evolutionary distance.
•Calculating the substitution rate (r) for two sequences that
have changed over time.
 Molecular Phylogenetic tree building
methods
• Mathematical and/or statistical methods for inferring the divergence order of
taxa, as well as the lengths of the branches that connect them.
•There are many Phylogenetic methods available today, Most can be classified as
follows:
COMPUTATIONAL METHOD
Optimality criterion Clustering algorithm
Characters

PARSIMONY

MAXIMUM LIKELIHOOD
DATA TYPE
Distances

MINIMUM EVOLUTION UPGMA

LEAST SQUARES NEIGHBOR-JOINING

 Types of data used in phylogenetic inference:
Character-based methods: Use the aligned characters, such as DNA or protein
sequences, directly during tree inference.
Taxa Characters
Species ATGGCTATTCTTATAGTAC
A G
Species ATCGCTAGTCTTATATT
B ACA
Species C TTCACTAGACCTGTGGTCCA
Species TTGACCAGACCTGTGGTCCG
Distance-based
D methods: Transform the sequence data into pairwise distances
TTGACCAGTTCTCTAGTTCG
(dissimilarities),
Species Eand then use the matrix during tree building.
A B C D E
Species A ---- 0.20 0.50 0.45 0.40 Example 1:
Species B 0.23 ---- 0.40 0.55 0.50 Uncorrected
“p”
Species C 0.87 0.59 ---- 0.15 0.40 distance
Species D 0.73 1.12 0.17 ---- 0.25 (=observed percent
sequence difference)
Species E 0.59 0.89 0.61 0.31 ----

Example 2: BKaismeduoran 2el -cptaurraems beyteCr-

B S t e w a rt,and by
Character table
TAX

Salamander
(outgroup)
A

Lamprey

Leopard
Lancelet

Turtle
Tuna
CHARACTERS
Hair

Amniotic (shelled) egg

Four walking

legs

Hinged jaws
Vertebral
column
(backbone)

Cladogram Turtle Leopard

Hair
Salamander
Amniotic egg
Tuna
Four walking legs
La
m
pr
ey
Hinged jaws
L
a
n
c
 Distance Matrix Methods
(matrix calculation)

Reality: Not all sites are free to change, the same sites
change multiple times.
 Distance Matrix Methods
(tree construction)

UPGMA - Unweighted Pair Group Method using

Arithmetic mean.
Step 1: compute the pairwise distances of all the proteins.
Get ready to put the numbers 1-5 at the bottom of your
new tree.

1 2

3
4

5
 Tree-building methods: UPGMA

Step 2: Find the two proteins with the smallest pair wise
distance. Cluster them.

1 2

6
3
1 2
4

5
 Tree-building methods: UPGMA

Step 3: Do it again. Find the next two proteins with the

smallest pairwise distance. Cluster them.

1 2
6 7
1 2 4 5
3
4

5
 Tree-building methods: UPGMA

Step 4: Keep going. Cluster.

1 2
8

7
6
3
4

5
1 2 4 5 3
 Tree-building methods: UPGMA

Step 4: Last cluster! This is your tree.

1 2
8
7
3
4 6
5
1 2 4 5 3
 List of Phylogenetics software
Name Description Methods
It allows manipulation of both DNA
EzEditor and protein sequence alignments Neighbour Joining
for phylogenetic analysis.
Simultaneous Bayesian inference of Bayesian inference, alignment
BAli-Phy
alignment and phylogeny as well as tree search.
Progressive multiple sequence
ClustalW Distance matrix/nearest neighbour
alignment
Analyses trait evolution among groups
BayesTraits Trait analysis
of species
Neighbour-joining, maximum
storage and analysis of all types of parsimony, UPGMA, maximum
BioNumerics
biological data, likelihood, distance matrix
methods
Optimized maximum likelihood
fastDNAml Maximum likelihood
(nucleotides only)
Neighbour-joining, UPGMA,
Geneious provides genome and MrBayes plugin, PHYML plugin,
Geneious
proteome research tools RAxML plugin, FastTree plugin,
 Applications of Phylogenetics
• Forensics:
Did a patient’s HIV infection result from an invasive dental
procedure performed by an HIV+ dentist?
• Conservation:
How much gene flow is there among local populations of
island foxes off the coast of California?
• Medicine:
What are the evolutionary relationships among the
various prion-related diseases?

To be continued…
 Why is phylogeny important?
Understanding and classifying
the diversity of life on Earth.
Testing evolutionary hypotheses:
- Trait evolution
- Coevolution
- Mode and pattern of speci
ation
- Correlated trait evolution
- Biogeography
- Geographic origins
- Age of different taxa
- Nature of molecular evolution
- Disease epidemiology
…And many more applications!