Phylogenetic Analysis

based on two talks, by

Caro-Beth Stewart, Ph.D.

Department of Biological Sciences

University at Albany, SUNY
c.stewart@albany.edu

and Tal Pupko, Ph.D.

Faculty of Life Science
Tel-Aviv University
talp@post.tau.ac.il

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 What is phylogenetic analysis and why
should we perform it?

Phylogenetic analysis has two major components:

1. Phylogeny inference or tree building

the inference of the branching orders, and
ultimately the evolutionary relationships,
between taxa (entities such as genes,
populations, species, etc.)
2. Character and rate analysis
using phylogenies as analytical frameworks
for rigorous understanding of the evolution of
various traits or conditions of interest

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 Common Phylogenetic Tree Terminology

Terminal Nodes
Branches or
Lineages A Represent the
TAXA (genes,
populations,
B species, etc.)
used to infer
C the phylogeny

D
Ancestral Node
or ROOT of Internal Nodes or E
the Tree Divergence Points
(represent hypothetical
ancestors of the taxa)

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 Phylogenetic trees diagram the evolutionary
relationships between the taxa
Taxon B

Taxon C
No meaning to the
spacing between the
Taxon A taxa, or to the order in
which they appear from
top to bottom.
Taxon D

Taxon E

This dimension either can have no scale (for cladograms),

can be proportional to genetic distance or amount of change
(for phylograms or additive trees), or can be proportional
to time (for ultrametric trees or true evolutionary trees).

((A,(B,C)),(D,E)) = The above phylogeny as nested parentheses

These say that B and C are more closely related to each other than either is to A,
and that A, B, and C form a clade that is a sister group to the clade composed of
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D and E. If the tree has a time scale, then D and E are the most closely related.
 A few examples of what can be inferred
from phylogenetic trees built from DNA
or protein sequence data:

Which species are the closest living relatives of

modern humans?
Did the infamous Florida Dentist infect his
patients with HIV?
What were the origins of specific transposable
elements?
Plus countless others..

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 Which species are the closest living
relatives of modern humans?

Humans Gorillas
Chimpanzees Chimpanzees

Bonobos Bonobos

Gorillas Orangutans
Orangutans Humans

14 0 15-30 0
MYA MYA

Mitochondrial DNA, most nuclear DNA- The pre-molecular view was that the great
encoded genes, and DNA/DNA apes (chimpanzees, gorillas and
hybridization all show that bonobos and orangutans) formed a clade separate
chimpanzees are related more closely to from humans, and that humans diverged
humans than either are to gorillas. from the apes at least 15-30 MYA.
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 Did the Florida Dentist infect his patients with HIV?

Phylogenetic tree DENTIST

of HIV sequences Patient C
from the DENTIST, Patient A
his Patients, & Local Patient G
HIV-infected People:
Patient B
Yes:
The HIV sequences from
Patient E these patients fall within
Patient A the clade of HIV sequences
found in the dentist.
DENTIST
Local control 2
Local control 3
Patient F No
Local control 9

Local control 35
Local control 3
Patient D No
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From Ou et al. (1992) and Page & Holmes (1998)
 A few examples of what can be learned
from character analysis using phylogenies
as analytical frameworks:

When did specific episodes of positive Darwinian

selection occur during evolutionary history?
Which genetic changes are unique to the human
lineage?
What was the most likely geographical location of
the common ancestor of the African apes and
humans?
Plus countless others..
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 The number of unrooted trees increases in a greater
than exponential manner with number of taxa
A B

C A C

B D

C
A D

B E

A C
D

B F E (2N - 5)!! = # unrooted trees for N taxa

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 Inferring evolutionary relationships between
the taxa requires rooting the tree:
B
C
To root a tree mentally,
imagine that the tree is
made of string. Grab the
string at the root and Root D
tug on it until the ends of
the string (the taxa) fall Unrooted tree
opposite the root: A

A B C D

Rooted tree
Note that in this rooted tree, taxon A is
no more closely related to taxon B than Root
it is to C or D.

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Now, try it again with the root at another position:

B
C

Root
Unrooted tree
D

B
C D

Rooted tree

Note that in this rooted tree, taxon A is most

closely related to taxon B, and together they
Root
are equally distantly related to taxa C and D.

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An unrooted, four-taxon tree theoretically can be rooted in five
different places to produce five different rooted trees

2 4
A C
The unrooted tree 1: 1 5

B 3 D

Rooted tree 1a Rooted tree 1b Rooted tree 1c Rooted tree 1d Rooted tree 1e
B A A C D

A B B D C

C C C A A

D D D B B
These trees show five different evolutionary relationships among the taxa!

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 There are two major ways to root trees:
By outgroup:
Uses taxa (the outgroup) that are
known to fall outside of the group of
interest (the ingroup). Requires
some prior knowledge about the
relationships among the taxa. The
outgroup can either be species (e.g.,
birds to root a mammalian tree) or
previous gene duplicates (e.g., outgroup
-globins to root -globins).

By midpoint or distance:
Roots the tree at the midway point A
d (A,D) = 10 + 3 + 5 = 18
between the two most distant taxa in
Midpoint = 18 / 2 = 9
the tree, as determined by branch
10
lengths. Assumes that the taxa are C
evolving in a clock-like manner. This 3 2
assumption is built into some of the B 2
5 D
distance-based tree building methods.
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 Each unrooted tree theoretically can be rooted
anywhere along any of its branches

A C

x =
B D
C
A D

B E
C
A D

B F E (2N - 3)!! = # unrooted trees for N taxa

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 Molecular phylogenetic tree building methods:
Are 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, each having
strengths and weaknesses. 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

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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 A ATGGCTATTCTTATAGTACG
Species B ATCGCTAGTCTTATATTACA
Species C TTCACTAGACCTGTGGTCCA
Species D TTGACCAGACCTGTGGTCCG
Species E TTGACCAGTTCTCTAGTTCG

Distance-based methods: Transform the sequence data into pairwise

distances (dissimilarities), and 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
Species C 0.87 0.59 ---- 0.15 0.40 p distance
(=observed percent
Species D 0.73 1.12 0.17 ---- 0.25 sequence difference)
Species E 0.59 0.89 0.61 0.31 ----

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Example 2: Kimura 2-parameter distance
(estimate of the true number of substitutions between taxa)
Computational methods for finding optimal trees:

Exact algorithms: "Guarantee" to find the optimal or

"best" tree for the method of choice. Two types used in tree
building:
Exhaustive search: Evaluates all possible unrooted
trees, choosing the one with the best score for the method.
Branch-and-bound search: Eliminates the parts of the
search tree that only contain suboptimal solutions.

Heuristic algorithms: Approximate or quick-and-dirty

methods that attempt to find the optimal tree for the method of
choice, but cannot guarantee to do so. Heuristic searches
often operate by hill-climbing methods.

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 Exact searches become increasingly difficult, and
eventually impossible, as the number of taxa increases:

A B

A C
C

B D

C
A D

B E
C
A D

B F E
(2N - 5)!! = # unrooted trees for N taxa

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 Heuristic search algorithms are Rerunning heuristic searches using
input order dependent and can get different input orders of taxa can help
stuck in local minima or maxima find global minima or maxima
Search
for global
Search maximum
for global
minimum GLOBAL GLOBAL
MAXIMUM MAXIMUM
local
maximum

local
minimum GLOBAL GLOBAL
MINIMUM MINIMUM

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Classification of phylogenetic inference methods

COMPUTATIONAL METHOD
Optimality criterion Clustering algorithm
Characters

PARSIMONY

MAXIMUM LIKELIHOOD
DATA TYPE

Distances

MINIMUM EVOLUTION UPGMA

LEAST SQUARES NEIGHBOR-JOINING

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 Parsimony methods:
Optimality criterion: The most-parsimonious tree is the one that
requires the fewest number of evolutionary events (e.g., nucleotide
substitutions, amino acid replacements) to explain the sequences.

Advantages:
Are simple, intuitive, and logical (many possible by pencil-and-paper).
Can be used on molecular and non-molecular (e.g., morphological) data.
Can tease apart types of similarity (shared-derived, shared-ancestral, homoplasy)
Can be used for character (can infer the exact substitutions) and rate analysis.
Can be used to infer the sequences of the extinct (hypothetical) ancestors.

Disadvantages:
Are simple, intuitive, and logical (derived from Medieval logic, not statistics!)
Can be fooled by high levels of homoplasy (same events).
Can become positively misleading in the Felsenstein Zone:

[See Stewart (1993) for a simple explanation of parsimony analysis, and Swofford
et al. (1996) for a detailed explanation of various parsimony methods.]
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Branch and Bound

Tal Pupko, Tel-Aviv University

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 There are many trees..,

We cannot go over all the trees. We will try to find

a way to find the best tree.
There are approximate solutions But what if we
want to make sure we find the global maximum.

There is a way more efficient than just go over all

possible tree. It is called BRANCH AND BOUND
and is a general technique in computer science,
that can be applied to phylogeny.

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 BRANCH AND BOUND

To exemplify the BRANCH AND BOUND (BNB)

method, we will use an example not connected to
evolution. Later, when the general BNB method is
understood, we will see how to apply this method
to finding the MP tree. We will present the
traveling salesperson path problem (TSP).

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 THE TSP PROBLEM
(especially adapted to israel).
A guard has to visit n check-points whose location
on a map is known. The problem is to find the
shortest path that goes through all points exactly
once (no need to come back to starting point).

Nave approach: (say for 5 points). You have 5

starting points. For each such starting point you
have 4 next steps. For each such combination of
starting point and first step, you have 3 possible
second steps, etc. All together we have 5*4*3*2*1
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= 5! .
 THE TSP TREE

1 2 3 4 5

2 3 4 5 1 3 4 5 1 2 4 5 1 2 3 5 1 2 3 4

245 145 125 124

45 25 24

54 52 42

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 THE SHP NAVE APPROACH

Each solution can be represented as a

permutation:

(1,2,3,4,5)
(1,2,3,5,4)
(1,2,4,3,5)
(1,2,4,5,3)
(1,2,5,3,4)

We can go over the list and find the one giving the
highest score.
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 THE SHP NAVE APPROACH

However, for 15 points, for example, there are

1,307,674,368,000

The rate of increase of the number of solutions is

too fast for this to be practical.

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 A TSP GREEDY HEURISTIC

Start from a random point. Go to the closest point.

Go to its closest point, etc.etc.
This approach doesnt work so well

(but a reasonably close heuristic, based on simulated

annealing, will be presented in a couple of lectures.)

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 BNB SOLUTION TO SHP

1 2 3 4 5

2 3 4 5 1 3 4 5 1 2 4 5 1 2 3 5 1 2 3 4

Score here
Shortest path 245 145 125 124
already 16:
found so far = no point in
15 45 25 24 expanding
the rest of
54 52 42 the subtree
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 Back to finding the MP
tree

Finding the MP tree is NP-Hard (will see shortly)

BNB helps, though it is still exponential

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 The MP search tree
1

4 is added to branch 1. 3

1 1 1
4 3 4 3 3
4
2 2 2

5 is added to branch 2.
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 The MP search tree
30
4 is added to branch 1.

43 55 39

52 54 52 53 58 61 56 59 61 69 53 51 42 47 47

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 MP-BNB
30
4 is added to branch 1.

43 55 39

52 54 52 53 58 61 56 59 61 69 53 51 42 47 47

Best (minimum) value = 52

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 MP-BNB
30
4 is added to branch 1.

43 55 39

52 54 52 53 58 61 56 59 61 69 53 51 42 47 47

Best record = 52
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 MP-BNB
30
4 is added to branch 1.

43 55 39

52 54 52 53 58 61 56 59 61 69 53 51 42 47 47

Best record = 52
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 MP-BNB
30

43 55 39

52 54 52 53 58 53 51 42 47 47

Best record = 52
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 MP-BNB
30

43 55 39

52 54 52 53 58 53 51 42 47 47

Best record = 52
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 MP-BNB
30

43 55 39

52 54 52 53 58 53 51 42 47 47

Best record = 52 51
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 MP-BNB
30

43 55 39

52 54 52 53 58 53 51 42 47 47

Best record = 52 51 42
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 MP-BNB
30

43 55 39

52 54 52 53 58 53 51 42 47 47

Best record = 52 51 42
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 MP-BNB
30

43 55 39

52 54 52 53 58 53 51 42 47 47

Best record = 52 51 42
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 MP-BNB
30

43 55 39

52 54 52 53 58 53 51 42 47 47

Total # trees visited: 14 Best TREE.

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 Order of Evaluation Matters
30 The bound
Evaluate all 3 first after searching
this subtree
will be 42.
43 55 39

53 51 42 47 47

Total tree visited: 9

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 And Now

Maximum Parsimony is
Computationally Intractable

Felsensteins Dynamic Programming

Algorithm for tiny maximum likelihood

and more, time permitting

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