Molecular Evolution NUI Maynooth June 2001

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Molecular Evolution NUI Maynooth June 2001
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Aims of the course

• To introduce the theory and practice of
  phylogenetic inference from molecular data
• To provide an introduction to some of the most
  useful methods and computer programmes

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Introduction to the course

• Some basic concepts e.g. phylogeny, monophyly,
  homology analogy
• Exploring patterns in sequence data
• Alignment - ClustalW (done)
• Phylogenetic analysis
• Parsimony
• Distance matrix analysis
• Maximum likelihood
• How robust are phylogenetic hypotheses?

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Phylogenetics (Cladistics)

• Based upon evolutionary relationships i.e. upon
  common ancestry
• Cladogram is a tree diagram which depicts a
  hypothesised evolutionary history
• A Phylogram is a tree which indicates by branch
  length the degree of change believed to have
  occurred along each lineage

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Cladograms and phylograms
Bacteria 1
Bacteria 2
Cladograms show branching order - branch lengths
are meaningless
Bacteria 3
Eukaryote 1
Eukaryote 2
Eukaryote 3
Eukaryote 4
Phylograms show branch order and branch lengths
Bacteria 1
Bacteria 2
Bacteria 3
Eukaryote 1
Eukaryote 2
Eukaryote 3
Eukaryote 4
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Rooting using outgroups
Archaea outgroup
Bacteria 1
Rooted by outgroup
Bacteria 2
Bacteria 3
Eukaryote 1
Eukaryote 2
Eukaryote 3
Root
Eukaryote 4
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How construct a phylogeny?

• What kind of data?
• How to analyse it?

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Richard Owen
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Owens definition of homology

• Homologue the same organ under every variety of
  form and function (true or essential
  correspondence)
• Analogy superficial or misleading similarity
• Richard Owen 1843

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Charles Darwin
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Darwin and homology

• The natural system is based upon descent with
  modification .. the characters that naturalists
  consider as showing true affinity (i.e.
  homologies) are those which have been inherited
  from a common parent, and, in so far as all true
  classification is genealogical that community of
  descent is the common bond that naturalists have
  been seeking
• Charles Darwin, Origin of species
  1859 p. 413

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Homology is...

• Homology similarity that is the result of
  inheritance from a common ancestor -
  identification and analysis of homologies is
  central to phylogenetics

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Phylogenetics

• Sees homology as evidence of common ancestry
• Uses tree diagrams to portray relationships based
  upon recency of common ancestry
• Monophyletic groups (clades) - contain species
  which are more closely related to each other than
  to any outside of the group

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Monophyletic groups
Archaea outgroup
Bacteria
monophyletic groups (clades)
Bacteria
Bacteria
Eukaryote
Eukaryote
Eukaryote
Eukaryote
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How construct a phylogeny?

• What kind of data?
• How to analyse it?

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Fossil primate skulls
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Microbial morphologies - some are complex but
many are simple - for example look at a drop of
lake water
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Linus Pauling
Linus Pauling and his co-workers asked the
questions Where in organisms has the greatest
amount of information about their past history
survived ? How it can be extracted ?
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Molecules can tell us about the past
The sequences of DNA, RNA and protein molecules
are documents of evolutionary history
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What sequences should we use?

• Choice of sequence - appropriate for question
  (fast or slow evolving - close or distant
  relationships).
• Many sequences are a mosaic of different rates
• 16S rRNA different structural regions evolve at
  different rates
• Proteins - synonymous (silent) rate (codon
  position 3) is often faster than nonsynonymous
  (positions 1 2 - changes aa) rate of change
• Transitions occur more readily than transversions

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16S rRNA structure
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Exploring patterns in sequence data

• Do the sequences contain phylogenetic signal for
  the relationships of interest? (too conserved or
  too variable)
• Are sequences saturated for change at the level
  of relationship to be investigated?
• Do sequences manifest biased base compositions
  (e.g thermophilic convergence) or biased codon
  usage patterns which may obscure phylogenetic
  signal

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Saturation in sequence data

• Saturation is due to multiple changes at the same
  site subsequent to lineage splitting
• Models of evolution attempt to infer the missing
  information through correcting for multiple
  hits
• Most data will contain some fast evolving sites
  which are potentially saturated (e.g. in proteins
  often position 3)
• In severe cases the data becomes essentially
  random and all information about relationships
  can be lost

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Multiple changes at a single site
Seq 1 AGCGAG Seq 2 GCGGAC
Number of changes
Seq 1

Seq 2
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Biased base compositions?

• Do sequences manifest biased base compositions
  (e.g thermophilic convergence) or biased codon
  usage patterns which may obscure phylogenetic
  signal

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A case study in phylogenetic analysisDeinococcus
and Thermus

• Deinococcus are radiation resistant bacteria
• Thermus are thermophilic bacteria
• BUT
• Both have the same very unusual cell wall based
  upon ornithine
• Both have the same menaquinones (Mk 9)
• Both have the same unusual polar lipids
• Congruence between these complex characters
  supports a phylogenetic relationship between
  Deinococcus and Thermus

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Guanine Cytosine in 16S rRNA genes
Thermophiles Thermus thermophilus Aquifex
pyrophilus Mesophiles Deinococcus
radiodurans Bacillus subtilis
guanine cytosine at variable sites
72 73 52 50
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A four taxon problem for Deinococcus and
Thermus(Thermus, Deinococcus, Bacillus, Aquifex)

• Aquifex and Bacillus are thermophiles and
  mesophiles, respectively
• No data suggest that Aquifex and Bacillus are
  specifically related to either Deinococcus or
  Thermus
• If all four bacteria are included in an analysis
  the true tree should place Thermus and
  Deinococcus together

Thermus
Aquifex
The true tree
Deinococcus
Bacillus
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Most methods of analysis will be fooled by the
base compositional biases in the data
Aquifex 73 GC
Bacillus 50 GC
Deinococcus 52 GC
Thermus 72 GC
The wrong tree - places taxa which share similar
base compositions together
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Is there a molecular clock?

• The idea of a molecular clock was initially
  suggested by Zuckerkandl and Pauling in 1962
• They noted that rates of amino acid replacements
  in animal haemoglobins were roughly proportional
  to time - as judged against the fossil record

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The molecular clock for alpha-globinEach point
represents the number of substitutions separating
each animal from humans
shark
carp
platypus
number of substitutions
chicken
cow
Time to common ancestor (millions of years)
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There is no universal molecular clock

• The initial proposal saw the clock as a Poisson
  process with a constant rate
• Now known to be more complex - differences in
  rates occur for
• different sites in a molecule
• different genes
• different regions of genomes
• different genomes in the same cell
• different taxonomic groups for the same gene
• there is no universal molecular clock

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Rates of amino acid replacement in different
proteins
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Are there local molecular clocks?

• If there is no universal molecular clock are
  there local clocks?
• Can individual molecular data sets yield useful
  estimates of times of divergence?
• Requires
• demonstration of rate constancy in the data set
  (some kind of relative rate test)
• sufficient external data - fossils - to reliably
  calibrate the clock

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Relative rate test (Wilson Sarich, 1973)
Under a molecular clock the distance (K) from A
to O (the common ancestor of A and B), and B to
O, should be the same We can measure relative
rates for A and B by reference to an outgroup
C KAC - KBC 0 gt0 indicates rate Agt rate B lt0
indicates rate Bgtrate A
O
A
B
C
one can therefore exclude taxa which violate rate
constancy
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Some potential problems with clocks

• Need a good fossil record to calibrate the clock
  - often missing (e.g. for bacteria?)
• Windows on fossil time estimates are often large
• How calculate amount of divergence between 2
  sequences? (use a model - subject of much of the
  present course)

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Using fossils to date splitting events
inferred timing of split
A
B
C
A
B
C
Time
therefore estimates may be very imprecise
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Phylogenetic inferences are premised on

• Phylogenetic inferences are premised on the
  inheritance of ancestral characters, and on the
  existence of an evolutionary history defined by
  changes in these characters
• A tree like model of evolution (paralogy, lateral
  transfer?)

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Gene trees and species trees
ORTHOLOGY
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Paralogy can produce misleading trees
Gene phylogenies
Organism phylogeny
A
a1
b1
B
Misleading tree from incomplete sampling
c1
C
a2
b2
c2
gene duplication
PARALOGY
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The malic enzyme tree contains paralogues
Anas a duck !
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Phylogenetic analysis requires careful thought

• Phylogenetic analysis is frequently treated as a
  black box into which data are fed (often gathered
  at considerable cost) and out of which The Tree
  springs
• (Hillis, Moritz Mable 1996, Molecular
  Systematics)