PhylogeneticsReconstructing the Tree of Life

Carol E. LeeUniversity of WisconsinCopyright©2020; do not upload without permission

• In the Speciation lecture, I talked about a “Phylogenetic Species Concept”

–What is a “Phylogeny?”–How do you construct one?–Why on earth should I care?

2

Why you should care:• All biological relationships can be determined by

constructing phylogenies: Even if phylogenies are not always the best way to define species boundaries, they do indeed tell you the genetic and evolutionary relationships among groups and individuals

– Your ancestry

– Diseases—figure out evolutionary origins and evolutionary pathways of disease, like HIV, Ebola, SARS, etc.

– Crops and livestock (food security)—rescue from inbreeding, create new varieties

– Endangered Species— figure out how endangered populations are related and how to perform genetic rescue 3

Tree of Life Web Projecthttp://www.tolweb.org/tree/

Tree of Life 2016Hug et al. 2016 Nature Microbiology

Bacteria

Archaea

Eukarya

Outline

1. What is a phylogeny?

2. How do you construct a phylogeny?The Molecular ClockStatistical Methods

Are Genetic Distancesand fossil recordroughly congruent?

Think about relationships among the major lineages of life and when they appeared in the fossil record

Fossil Record vs Molecular Clock

• Molecular clock and fossil record are not always congruent– Fossil record is incomplete, and soft bodied species are usually

not preserved– Mutation rates can vary among species (depending on

generation time, replication error, mismatch repair)

• But they provide complementary information– Fossil record contains extinct species, while molecular data is

based on extant taxa– Major events in fossil record could be used to calibrate the

molecular clock

Evolutionary History of HIV

Evolutionary AnalysisFreeman& Herron, 2004Time

HIV evolved multiple times from SIV (Simian Immunodeficiency Syndrome)

Charles Darwin (1809 -1882)

On the Origin of Species (1859)

– Living species are related by common ancestry

– Change through time occurs at the population not the organism level

– The main cause of adaptive evolution is natural selection

Darwin envisaged evolution as a tree

The affinities of all the beings of the same class have sometimes been represented by a great tree. I believe this simile largely speaks the truth………The green and budding twigs may represent existing species; and those produced during former years may represent the long succession of extinct species…..….the great Tree of Life….covers the earth with ever-branching and beautiful ramifications

Charles Darwin, On the Origin of Species; pages 131-132

Reconstructing the Tree of Life

The only figure in The Origin of Species

Lamarck proposed a ladder of life

What did people believe before Darwin?

Past Future

Jean-Baptiste Lamarck

• French Naturalist (1744-1829)• “Professor of Worms and

Insects” in Paris

• The first scientific theory of evolution (inheritance of acquired traits)

Lamarck’s View of Evolution

• Continuum between physical and biological world (followed Aristotle)

• Scala Naturae (“Ladder of Life” or “Great Chain of Being”)

Being

Realm of Being

Realm of Becoming

Non-Being

God

Angels

Demons

ManAnimals

Plants

Minerals

What is wrong with a ladder?

• Evolution is not linear but branching

• Living organisms are not ancestors of one another

• The ladder implies progress

What is right with the tree?• Evolution is a branching process• If a mutation occurs, one species

is not turning into another, but there is a split, and both lineages continue to evolve

• So, evolution is not progressive -all living taxa are equally “successful”

• Phylogenies (Trees) reflect the hierarchical structuring of relationships

The only figure in The Origin of Species

The Tree of Life is a Fractal

Genealogical structures • Phylogeny

– A depiction of the ancestry relations between species (it includes speciation events)

– Tree-like (divergent)

• Pedigree– A depiction of the ancestry relations within

populations– Net-like (reticulating)

Four butterflies connected to their parents

offspring

parents

Popu

latio

nIn

divi

dual

s

past

futu

re

Popu

latio

nLi

neag

e/

Spec

ies

Phyl

ogen

y

What happened here?

Lineage-branchingSpeciation

What happened here?

Extinction

A B C

The True History

A B C

A simplified representation

Representation of phylogenies?

Some terms used to describe a phylogenetic tree

Taxon (taxa)Tip

Internal branchInternode

Node (Speciation event)

Root

Outline

1. What is a phylogeny?

2. How do you construct a phylogeny?The Molecular ClockStatistical Methods

• A phylogenetic tree represents a hypothesis about evolutionary relationships

• Each branch point represents the divergence of two taxa (e.g. species)

• Sister taxa are groups that share an immediate common ancestor

What is a Phylogeny?

Sistertaxa

ANCESTRALLINEAGE

Taxon A

Polytomy (unresolved branching point)

Common ancestor oftaxa A–F

Branch point(node)

Taxon B

Taxon C

Taxon D

Taxon E

Taxon F

Molecular Clock• Phylogenies rely on the “Molecular Clock,” namely the fact that Mutations on average, occur at a given rate

• So, on average, more mutational differences between taxa means that they branched from a common ancestor longer ago

• So longer branches on phylogeny often à greater evolutionary distance

Example:Mitochondria: 1 mutation every ~2.2%/million years 31

Phylogeny of 53 humans (Homo sapiens) just based on mtDNA

A different locus might yield a different tree

The horizontal branch lengths reflect genetic distance ≈ # of mutations

Cladogram of mitochondrial cytochrome oxidase II alleles in humans and the African Great Apes (Ruvolo et al. 1994)

A cladogram shows the hierarchical relationships among the taxa, but the branch lengths do not reflect evolutionary time.

This is not a phylogeny, but a cladogram.

Problem: mutation rate can vary among species

• Mutation rate is faster:– Shorter generation time

(greater number of meiosis or mitosis events in a given time)

– Replication Error (e.g. Sloppy DNA or RNA polymerase; poor mismatch repair mechanisms)

Molecular Clock

35

Species

Canislupus

Pantherapardus

Taxideataxus

Lutra lutra

Canislatrans

Order Family Genus

Carnivora

FelidaeM

ustelidaeC

anidae

Canis

LutraTaxidea

Panthera

A A A

BBB

C C C

DDD

E E E

FFF

G G G

Group IIIGroup II

Group I

(a) Monophyletic group (clade) (b) Paraphyletic group (c) Polyphyletic group

A monophyletic clade consists of an ancestral taxa and all its descendants

A

B

CD

E

F

G

Group I

(a) Monophyletic group (clade)

(In the lecture on species concepts we discussed that the “smallest” monophyletic group is a “phylogenetic species”)

Examples of Paraphyletic Groups(not recognized as legitimate groups in the Phylogenetic Species Concept, which only recognizes monophyletic groups)

Synapomorphies

• Synapomorphies are shared derived homologous traits

• They can be DNA nucleotides or other heritable traits

• They are used to group taxa that are more closely related to one another

synapomorphies

Sometimes similar looking traits are not homologous, and are not synapomorphies, but are the result of convergent evolution

How do we construct Phylogenies?

Phylogenetic Methods

• Parsimony: Minimize # steps

• Distance Matrix: minimize pairwise genetic distances

• Maximum Likelihood: Probability of the data given the tree

• Bayesian: Probability of the tree given the data

Parsimony Uses DiscreteCharacters (like mutations, or some heritable trait)

Select the tree with the minimum number of character-state transitions summed across all characters

Fig. 26-15-1

Species I

Three phylogenetic hypotheses:

Species II Species III

I

II

III

I

III

IIIII

III

Parsimony: Example 1

Fig. 26-15-2

Species I

Site

Species II

Species III

I

II

III

I

III

IIIII

III

Ancestralsequence

1/C1/C

1/C

1/C

1/C

4321

C

C C

C

T

T

T

T

T

T A

AA

A G

G

Fig. 26-15-3

Species I

Site

Species II

Species III

I

II

III

I

III

IIIII

III

Ancestralsequence

1/C1/C

1/C

1/C

1/C

4321

C

C C

C

T

T

T

T

T

T A

AA

A G

G

I I

I

II

II

II

III

III

III3/A

3/A

3/A3/A

3/A

2/T2/T

2/T 2/T

2/T4/C

4/C

4/C

4/C

4/C

Fig. 26-15-4

Species I

Site

Species II

Species III

I

II

III

I

III

IIIII

III

Ancestralsequence

1/C1/C

1/C

1/C

1/C

4321

C

C C

C

T

T

T

T

T

T A

AA

A G

G

I I

I

II

II

II

III

III

III3/A

3/A

3/A3/A

3/A

2/T2/T

2/T 2/T

2/T4/C

4/C

4/C

4/C

4/C

I I

I

II

II

II

III

III

III

7 events7 events6 events

Three possible trees

Tree 1

C B

AO

Tree 2

A B

CO

B C

AO

C B AO A B CO

B A CO

Tree 3

Parsimony: Example 2

C B AO

Map the characters (mutations) onto tree 1

12

ABC

O1 2 3 4 5

GGG

T G G A A

CCC

GAA

AAC

AAT

Map the characters (mutations) onto tree 1

ABC

O1 2 3 4 5

GGG

T G G A A

CCC

GAA

AAC

AAT

Total # number of steps = 6

C B AO

12

3

3

45

Actually, there is more than one way to map character 3

ABC

O

3

G

GAA

Either way the character contributes 2 steps to the overall tree length

C B AO

3

3

C B AO

3

3

Map the characters onto tree 2

# steps = 5

ABC

O

1 2 3 4 5

GGG

T G G A A

CCC

GAA

AAC

AAT

A B CO

12

45

3

Tree 3

Length = 6 steps

ABC

O

1 2 3 4 5

GGG

T G G A A

CCC

GAA

AAC

AAT

B A CO

12

453

3

Most parsimonious tree

Which tree had the shortest branch lengths (most parsimonious)?

Tree 1: length = 6

C B

AO

Tree 2: length = 5

B C

AO

C B AO A B CO

B A CO

Tree 3: length = 6

Example from Freeman & Herron, Fig. 4.8

Where do the Whales belong?

Freeman & Herron, Fig. 4.9: Using maximum parsimony, looks like the whales cluster with the hippos (and cows)

Parsimony• Simplest and fastest method of phylogenetic

reconstruction

• Can give misleading results if rates of evolution (rates that mutations occur) differ in different lineages

• Tends to become less accurate as genetic distances get greater

• Could be mislead by reversals, homoplasy: Because with only 4 nucleotides, after a while, same mutations occur repeatedly at a given site (called “saturation”) – “multiple hits (mutations) per site”

Distance MatrixContinuous or

Discrete Characters

Distance Matrix

• Calculate pairwise distances between taxa• Choose the tree that minimizes overall

distances between taxa

proportion sequence distance at 2 genes(hypothetical data)

mouse cat dog dolphin seal

MouseCat 0.05Dog 0.03 0.02Dolphin 0.08 0.15 0.03Seal 0.09 0.23 0.01 0.02

Freeman & Herron, Fig. 4.10: Using genetic distances, looks like the whales again cluster with the hippos (and cows)

Distance Matrix

• Generally more accurate than parsimony

• Like parsimony, it tends to be computationally fast

Z: Probability of the data

Maximum Likelihood (R.A. Fisher)• Probability of the data given the tree• This is a “Frequentist” method: one true answer

(one true tree)

• Draw from the data (probability distribution of DNA sequence data) to find the true tree

• Choose the tree (x, y axis) that maximizes the probability of the observed data (z axis)

Felsenstein, J. 1981. Evolutionary trees from DNA sequences: a maximum likelihood approach. Journal of Molecular Evolution. 17(6):368-76.

x,y: Tree space

Z: Probability of the data

Maximum Likelihood (R.A. Fisher)• Probability of the data given the tree• The aim of maximum likelihood estimation is to find

the parameter value(s) that makes the observed data most likely.

• For example: finding a mean. If you want to have a number that describes the data, like human height, you could find the mean

P(data/tree) = likelihood(tree/data)Tree = hypothesis

Felsenstein, J. 1981. Evolutionary trees from DNA sequences: a maximum likelihood approach. Journal of Molecular Evolution. 17(6):368-76.

x,y: Tree space

• Often yields more accurate tree than parsimony or distance

• Relies on an accurate assumption of which mutations are more probable (A->G more often than A->T or C? i.e. accurate model of molecular

evolution)

• Computationally intensive

Maximum Likelihood(R.A. Fisher)

Bayesian InferenceReverend Thomas Bayes (1702-1760)

• Probability of a tree given the data• Uses prior information on the tree• Does not assume that there is one correct tree• Will modify estimate based on additional information

• Uses Bayes’ Theorem

P(A/B) = P(B/A)P(A)P(B)

Bayesian InferenceReverend Thomas Bayes (1702-1760)

• Uses Bayes’ Theorem

P(A/B) = P(B/A)P(A) = P(tree/data) = P(data/tree)P(tree)P(B) P(data)

P(A) = prior probability, probability of a treeP(A/B) = posterior probability—probably of tree given the dataP(B/A) = the probability B (data) of observing given A (tree), is also known as the likelihood. It indicates the compatibility of the evidence with the given hypothesis.P(B) = probability of the data

Bayesian InferenceReverend Thomas Bayes (1702-1760)

• Probability of a tree given the data:

• Will modify estimate based on additional information: so as you get more data, you update your hypothesis for the tree

• Uses prior information on the tree: this is where you start

• The sequential use of the Bayes' formula (recursive): when more data become available after calculating a posterior distribution, the posterior becomes the next prior

• Does not assume that there is one correct tree

Bayesian Inference

• Like Likelihood, often yields more accurate tree than parsimony or distance

• Computationally more intensive than parsimony or distance matrix, but less intensive than likelihood

• Needs a prior probability for the tree and a model of evolution

• Sufficient Amount of Data: – With enough data most statistical methods usually

yield the same tree (but not always—sometimes there is no single resolved tree)

– Insufficient data would yield a tree that lacks resolution (lacks statistical power)

• Gene trees vs species trees– Evolutionary history of individual genes are not

necessarily the same– Should try to get data from many genes, or the whole

genome

Potential problems of Phylogenetic Reconstruction

Challenges of Phylogenetic Reconstructions

• Different parts of the genome might have different evolutionary histories (different gene genealogies, horizontal gene transfers, allopolyploidy, etc)

• So, there might not be one true tree for a group of taxa, and relationships might be difficult to resolve because they are inherently complex

• Current trend is to use whole genome data to reconstruct phylogenies

• Gain a comprehensive picture of the evolutionary relationships among taxa for the whole genome

• Typically, evolutionary biologists will use a variety of methods to reconstruct a phylogeny. • Maximum likelihood and Bayesian methods are considered

more robust.

• Tree is only as good as the data. Having many homoplastic characters (due to convergent evolution, reversals, etc.) will make the reconstruction less robust• Standard to use Bootstrapping to assess the validity of the

tree

• Understanding statistics is fundamental to understanding evolution• Much of statistics was in fact developed in order to model

evolutionary processes (such as ANOVA, analysis of variance)

Phylogenetic Reconstructions

1. Sometimes the Molecular Clock (based on genetic data) conflicts with the Geological Record. Why would this happen?

(A) Sometimes there are gaps in the geological record, because fossils do not form everywhere, and mutation rate might vary between different species

(B) Radiometric dating relies on chance events in the preservation of isotopes, making the timing events in the geological time scale less accurate than the molecular clock

(C) Mutation rates slow down as you go back in time, making estimation of timing of events less accurate as you go back in time

(D) The molecular clock is calculated from radioisotopes, while the geological record is obtained from fossil data. The two can conflict when fossils end up displaced from their original sedimentary layer

2. You are a medical researcher working on HIV. A novel strain has appeared in Madison, Wisconsin. To determine which drugswould be most effective in treating this new strain (because different strains are resistant to different drugs), you need to determine its recent evolutionary history. You decide to reconstruct the evolutionary history of HIV by using a phylogenetic approach. Thus, you collect samples from patients in various geographic locations and sequence a fragment of RNA. Using parsimony, which is thecorrect phylogeny for HIV-1 based on the data below?

HIV-1, Uganda, Africa ACAUGHIV-1, San Francisco, USA UGAUGHIV-1, Madison, USA UAAGGHIV-1, New York, USA UAAAGHIV-1, Paris ACAUCHIV-2 Africa (ancestral outgroup): ACCUG

3. Which of the following is most TRUE regarding phylogenetic reconstructions?

(A) Phylogenetic reconstruction based on any gene would yield the same tree

(B) Parsimony is the most accurate method for reconstructing phylogenies

(C) Phylogenetic reconstructions based on different genes could yield different phylogenetic trees

(D) Maximum likelihood relies on maximizing distances among taxa

(E) There is always one true tree, and having enough genetic data will inevitably result in one tree

Answers

• 1A• 2C• 3C