Bioinformatica t6-phylogenetics

FBW6-11-2012

Wim Van Criekinge

Inhoud Lessen: Bioinformatica

GEEN LES

Phylogenetics

IntroductionDefinitionsSpecies conceptExamplesThe Tree-of-life

Phylogenetics MethodologiesAlgorithms

Distance MethodsMaximum LikelihoodMaximum Parsimony

RootingStatistical Validation

ConclusionsOrthologous genesHorizontal Gene TransferPhylogenomics

Practical Approach: PHYLIPWeblems

Phylogeny (phylo =tribe + genesis)

Phylogenetic trees are about visualising evolutionary relationships. They reconstruct the pattern of events that have led to the distribution and diversity of life.

The purpose of a phylogenetic tree is to illustrate how a group of objects (usually genes or organisms) are related to one another

Nothing in Biology Makes Sense Except in the Light of Evolution. Theodosius Dobzhansky (1900-1975)

What is phylogenetics ?

Trees

• Diagram consisting of branches and nodes • Species tree (how are my species related?)

– contains only one representative from each species.

– all nodes indicate speciation events

• Gene tree (how are my genes related?)– normally contains a number of genes from a

single species– nodes relate either to speciation or gene

duplication events

Clade: A set of species which includes all of the species derived from a single common ancestor

Species Concepts from Various AuthorsD.A. Baum and K.L. Shaw - Exclusive groups of organisms, where an exclusive group is one whose members are all more closely related to

each other than to any organisms outside the group.

J. Cracraft - An irreducible cluster of organisms, diagnosably distinct from other such clusters, and within which there is a parental pattern of ancestry and descent.

Charles Darwin - "From these remarks it will be seen that I look at the term species, as one arbitrarily given for the sake of convenience to a set of individuals closely resembling each other, and that it does not essentially differ from the term variety, which is given to less distinct and more fluctuating forms. The term variety, again, in comparison with mere individual differences, is also applied arbitrarily, and for mere convenience sake" (Origin of Species, 1st ed., p. 108).

T. Dobzhansky - The largest and most inclusive reproductive community of sexual and cross-fertilizing individuals which share a common gene pool. And later...Systems of populations, the gene exchange between which is limited or prevented by reproductive isolating mechanisms.

M. Ghiselin - The most extensive units in the natural economy, such that reproductive competition occurs among their parts.

D.M. Lambert - Groups of individuals that define themselves by a specific mate recognition system.

J. Mallet - Identifiable genotypic clusters recognized by a deficit of intermediates, both at single loci and at multiple loci.

E. Mayr - Groups of actually or potentially interbreeding natural populations which are reproductively isolated from other such groups.

C.D. Michener - A group of organisms not itself divisible by phenetic gaps resulting from concordant differences in character states (except for morphs - such as sex, age, or caste), but separated by such phenetic gaps from other such units.

H.E.H. Patterson - That most inclusive population of individual biparental organisms which share a common fertilization system.

G.G. Simpson - A lineage of populations evolving with time, separately from others, with its own unique evolutionary role and tendencies.

P.H.A. Sneath and R.R. Sokal - The smallest (most homogeneous) cluster that can be recognized upon some given criterion as being distinct from other clusters.

A.R. Templeton - The most inclusive population of individuals having the potential for phenotypic cohesion through intrinsic cohesion mechanisms (genetic and/or demographic - i.e. ecological -exchangeability).

E.O. Wiley - A single lineage of ancestor-descendant populations which maintains its identity from other such lineages and which has its own evolutionary tendencies and historical fate.

S. Wright - A species in time and space is composed of numerous local populations, each one intercommunicating and intergrading with others.

Species

I. Definitions:

Species = the basic unit of classification

> Three different ways to recognize species:

Definitions:


1) Morphological species = the smallest group that is consistently and persistently distinct (Clusters in morphospace)

species are recognized initially on the basis of appearance; the individuals of one species look

different from the individuals of another

Plant Species

Definitions:


2) Biological species = a set of interbreeding or potentially interbreeding individuals that are separated from other species by reproductive barriers

species are unable to interbreed

Species

Definitions:


3) Phylogenetic species = the boundary between reticulate (among interbreeding individuals) and divergent relationships (between lineages with no gene exchange)

Species

reticulate

divergentPhylogenetic species

recognized by the pattern of ancestor - descendent relationships

boundary

Definitions:


4) Phylogenomics species = ability to transmit (and maintain) a (stable) gene pool

Adresses the Anopheles genome topology variations

Species

• In the tree to the left, A and B share the most recent common ancestry. Thus, of the species in the tree, A and B are the most closely related.

• The next most recent common ancestry is C with the group composed of A and B. Notice that the relationship of C is with the group containing A and B. In particular, C is not more closely related to B than to A. This can be emphasized by the following two trees, which are equivalent to each other:

Branching Order in a Phylogenetic Tree

• A common simplifying assumption is that the three is bifurcating, meaning that each brach node has exactly two descendents.

• The edges, taken together, are sometimes said to define the topology of the tree

More definitions …

Branch node, internal node

Edge, Branch

LeafsTipsexternal node

Outgroups, rooted versus unrooted

An unrooted reptilian phylogeny with an avian outgroup and the corresponding rooted phylogeny. The Ri represent modern reptiles; the Ai, inferred ancestors and the B a bird.

Some definitions …

Phylogenetic methods may be used to solve crimes, test purity of products, and determine whether endangered species have been smuggled or mislabeled: – Vogel, G. 1998.

HIV strain analysis debuts in murder trial. Science 282(5390): 851-853.

– Lau, D. T.-W., et al. 2001. Authentication of medicinal Dendrobium species by the internal transcribed spacer of ribosomal DNA. Planta Med 67:456-460.

Examples

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9841423&dopt=Abstract





– Epidemiologists use phylogenetic methods to understand the development of pandemics, patterns of disease transmission, and development of antimicrobial resistance or pathogenicity: • Basler, C.F., et al. 2001.

Sequence of the 1918 pandemic influenza virus nonstructural gene (NS) segment and characterization of recombinant viruses bearing the 1918 NS genes. PNAS, 98(5):2746-2751.

• Ou, C.-Y., et al. 1992. Molecular epidemiology of HIV transmission in a dental practice. Science 256(5060):1165-1171.

• Bacillus Antracis:

Examples






http://www.sciencemag.org/content/vol296/issue5575/images/large/se2220570002.jpeg

• Conservation biologists may use these techniques to determine which populations are in greatest need of protection, and other questions of population structure: – Trepanier, T.L., and R.W. Murphy. 2001.

The Coachella Valley fringe-toed lizard (Uma inornata): genetic diversity and phylogenetic relationships of an endangered species. Mol Phylogenet Evol 18(3):327-334.

– Alves, M.J., et al. 2001. Mitochondrial DNA variation in the highly endangered cyprinid fish Anaecypris hispanica: importance for conservation. Heredity 87(Pt 4):463-473.

• Pharmaceutical researchers may use phylogenetic methods to determine which species are most closely related to other medicinal species, thus perhaps sharing their medicinal qualities: – Komatsu, K., et al. 2001.

Phylogenetic analysis based on 18S rRNA gene and matK gene sequences of Panax vietnamensis and five related species. Planta Med 67:461-465.

Examples













Tree-of-life

Origin of the Universe 15 billion yrs

Formation of the Solar System 4.6 "

First Self-replicating System 3.5 "

Prokaryotic-Eukaryotic Divergence 2.0 "

Plant-Animal Divergence 1.0 "

Invertebrate-Vertebrate Divergence 0.5 "

Mammalian Radiation Beginning 0.1 "

Some Important Dates in History

Tree Of Life

Tree Of Life

http://tolweb.org/tree?group=life

Tree Of Life

Tree Of Life

What Sequence to Use ?

• To infer relationships that span the diversity of known life, it is necessary to look at genes conserved through the billions of years of evolutionary divergence.

• The gene must display an appropriate level of sequence conservation for the divergences of interest.

.

• If there is too much change, then the sequences become randomized, and there is a limit to the depth of the divergences that can be accurately inferred.

• If there is too little change (if the gene is too conserved), then there may be little or no change between the evolutionary branchings of interest, and it will not be possible to infer close (genus or species

level) relationships.


Carl Woese

recognized the full potential of rRNA sequences as a measure of phylogenetic relatedness. He initially used an RNA sequencing method that determined about 1/4 of the nucleotides in the 16S rRNA (the best technology available at the time). This amount of data greatly exceeded anything else then available. Using newer methods, it is now routine to determine the sequence of the entire 16S rRNA molecule. Today, the accumulated 16S rRNA sequences (about 10,000) constitute the largest body of data available for inferring relationships among organisms.

Ribosomal RNA Genes and Their Sequences

An example of genes in this category are those that define the ribosomal RNAs (rRNAs). Most prokaryotes have three rRNAs, called the 5S, 16S and 23S rRNA.


Namea Size (nucleotides) Location

5S 120 Large subunit of ribosome

16S 1500 Small subunit of ribosome

23S 2900 Large subunit of ribosomea The name is based on the rate that the

molecule sediments (sinks) in water.

Bigger molecules sediment faster than small ones.

The extraordinary conservation of rRNA genes can be seen in these fragments of the small subunit rRNA gene sequences from organisms spanning the known diversity of life:

human ...GTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGCTGCAGTTAAAAAG...

yeast ...GTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGCAGTTAAAAAG...

Corn ...GTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTTAAGTTGTTGCAGTTAAAAAG...

Escherichia coli ...GTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCG...

Anacystis nidulans ...GTGCCAGCAGCCGCGGTAATACGGGAGAGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCG...

Thermotoga maratima ...GTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTACCCGGATTTACTGGGCGTAAAGGG...

Methanococcus vannielii ...GTGCCAGCAGCCGCGGTAATACCGACGGCCCGAGTGGTAGCCACTCTTATTGGGCCTAAAGCG...

Thermococcus celer ...GTGGCAGCCGCCGCGGTAATACCGGCGGCCCGAGTGGTGGCCGCTATTATTGGGCCTAAAGCG...

Sulfolobus sulfotaricus ...GTGTCAGCCGCCGCGGTAATACCAGCTCCGCGAGTGGTCGGGGTGATTACTGGGCCTAAAGCG...

Ribosomal RNA Genes and Their Sequences

Other genes …

• Rate of evolution = rate of mutation• rate of evolution for any macromolecule is

approximately constant over time (Neutral Theory of evolution)

• For a given protein the rate of sequence evolution is approximately constant across lineages. Zuckerkandl and Pauling (1965)

• This would allow speciation and duplication events to be dated accurately based on molecular data

Molecular Clock (MC)

Noval trees using Hox genes

• (a) A traditional phylogenetic tree and

• (a) A traditional phylogenetic tree and

• (b) the new phylogenetic tree, each showing the positions of selected phyla. B, bilateria; AC, acoelomates; PC, pseudocoelomates; C, coelomates; P, protostomes; L, lophotrochozoa; E, ecdysozoa; D, deuterostomes.

• Local and approximate molecular clocks more reasonable– one amino acid subst. 14.5 My– 1.3 10-9 substitutions/nucleotide site/year– Relative rate test (see further)

• ((A,B),C) then measure distance between (A,C) & (B,C)

Molecular Clock (MC)

Rate of Change Theoretical Lookback Time (PAMs / 100 myrs) (myrs)

Pseudogenes 400 45

Fibrinopeptides 90 200

Lactalbumins 27 670

Lysozymes 24 850

Ribonucleases 21 850

Haemoglobins 12 1500

Acid proteases 8 2300

Cytochrome c 4 5000

Glyceraldehyde-P dehydrogenase2 9000

Glutamate dehydrogenase 1 18000

PAM = number of Accepted Point Mutations per 100 amino acids.

Proteins evolve at highly different rates

Phylogenetics







Multiple Alignment Method

• align• select method (evolutionary

model)–Distance–ML–MP

• generate tree• validate tree

4-steps

Some definitions …

• Convert sequence data into a set of discrete pairwise distance values (n*(n-1)/2), arranged into a matrix. Distance methods fit a tree to this matrix.

• The phylogenetic topology tree is constructed by using a cluster analysis method (like upgma or nj methods).

Distance matrix methods (upgma, nj, Fitch,...)



CGT


Since we start with A,p(A)=1


D=evolutionary distance ~ tijdF = dissimilarity ~ (1 – PX(t))

F ~ 1 – d


http://nekrut.uchicago.edu/pics/fig3_3.gif

Unweighted Pair Group Method with Arithmatic Mean (UPGMA)




Distance matrix methods: Summary

http://www.bioportal.bic.nus.edu.sg/phylip/neighbor.html

• The phylogeny makes an estimation of the distance for each pair as the sum of branch lengths in the path from one sequence to another through the tree.

· easy to perform ;

· quick calculation ;

· fit for sequences having high similarity scores ;

• drawbacks : · the sequences are not considered as such

(loss of information) ;

· all sites are generally equally treated (do not take into account differences of substitution rates ) ;

· not applicable to distantly divergent sequences.


• In this method, the bases (nucleotides or amino acids) of all sequences at each site are considered separately (as independent), and the log-likelihood of having these bases are computed for a given topology by using a particular probability model.

• This log-likelihood is added for all sites, and the sum of the log-likelihood is maximized to estimate the branch length of the tree.

Maximum likelihood

Maximum likelihood

• This procedure is repeated for all possible topologies, and the topology that shows the highest likelihood is chosen as the final tree.

• Notes : · ML estimates the branch lengths of the

final tree ; · ML methods are usually consistent ; · ML is extented to allow differences

between the rate of transition and transversion.

• Drawbacks · need long computation time to construct a

tree.

Maximum likelihood

Maximum likelihood

Parsimony criterion • It consists of determining the minimum

number of changes (substitutions) required to transform a sequence to its nearest neighbor.

Maximum Parsimony • The maximum parsimony algorithm searches

for the minimum number of genetic events (nucleotide substitutions or amino-acid changes) to infer the most parsimonious tree from a set of sequences.

Maximum Parsimony

Maximum Parsimony

Occam’s Razor

Entia non sunt multiplicanda praeter necessitatem.

William of Occam (1300-1349)

The best tree is the one which requires the least number of substitutions

• The best tree is the one which needs the fewest changes. – If the evolutionary clock is not constant, the

procedure generates results which can be misleading ;

– within practical computational limits, this often leads in the generation of tens or more "equally most parsimonious trees" which make it difficult to justify the choice of a particular tree ;

– long computation time to construct a tree.

Maximum Parsimony

Maximum Parsimony: Branch Node A or B ?

Maximum Parsimony: A requires 5 mutaties

Maximum Parsimony: B (and propagating A->B) requires only 4 mutations

• The best tree is the one which needs the fewest changes.

• Problems : – If the evolutionary clock is not

constant, the procedure generates results which can be misleading ;

– within practical computational limits, this often leads in the generation of tens or more "equally most parsimonious trees" which make it difficult to justify the choice of a particular tree ;

– long computation time to construct a tree.

Maximum Parsimony

Phylogenetics







· There is at present no statistical methods which allow comparisons of trees obtained from different phylogenetic methods, nevertheless many studies have been made to compare the relative consistency of the existing methods.

Comparative evaluation of different methods

· The consistency depends on many factors, among these the topology and branch lengths of the real tree, the transition/transversion rate and the variability of the substitution rates.

· One expects that if sequences have strong phylogenetic relationship, different methods will show the same phylogenetic tree

Comparative evaluation of different methods

Comparison of methods

• Inconsistency• Neighbour Joining (NJ) is very fast but depends on

accurate estimates of distance. This is more difficult with very divergent data

• Parsimony suffers from Long Branch Attraction. This may be a particular problem for very divergent data

• NJ can suffer from Long Branch Attraction• Parsimony is also computationally intensive• Codon usage bias can be a problem for MP and NJ• Maximum Likelihood is the most reliable but

depends on the choice of model and is very slow• Methods may be combined

Rooting the Tree

• In an unrooted tree the direction of evolution is unknown

• The root is the hypothesized ancestor of the sequences in the tree

• The root can either be placed on a branch or at a node

• You should start by viewing an unrooted tree

Automatic rooting

• Many software packages will root trees automaticall (e.g. mid-point rooting in NJPlot)

• Sometimes two trees may look very different but, in fact, differ only in the position of the root

• This normally involves assumptions… BEWARE!

Rooting Using an Outgroup

1. The outgroup should be a sequence (or set of sequences) known to be less closely related to the rest of the sequences than they are to each other

2. It should ideally be as closely related as possible to the rest of the sequences while still satisfying condition 1

The root must be somewhere between the outgroup and the rest (either on the node or in a branch)

How confident am I that my tree is correct?

Bootstrap values

Bootstrapping is a statistical technique that can use random resampling of data to determine sampling error for tree topologies

Bootstrapping phylogenies

• Characters are resampled with replacement to create many bootstrap replicate data sets

• Each bootstrap replicate data set is analysed (e.g. with parsimony, distance, ML etc.)

• Agreement among the resulting trees is summarized with a majority-rule consensus tree

• Frequencies of occurrence of groups, bootstrap proportions (BPs), are a measure of support for those groups

Bootstrapping - an example

Ciliate SSUrDNA - parsimony bootstrap

Majority-rule consensus

Ochromonas (1)

Symbiodinium (2)

Prorocentrum (3)

Euplotes (8)

Tetrahymena (9)

Loxodes (4)

Tracheloraphis (5)

Spirostomum (6)

Gruberia (7)

100

96

84

100

100

100

• Bootstrapping is a very valuable and widely used technique (it is demanded by some journals)

• BPs give an idea of how likely a given branch would be to be unaffected if additional data, with the same distribution, became available

• BPs are not the same as confidence intervals. There is no simple mapping between bootstrap values and confidence intervals. There is no agreement about what constitutes a ‘good’ bootstrap value (> 70%, > 80%, > 85% ????)

• Some theoretical work indicates that BPs can be a conservative estimate of confidence intervals

• If the estimated tree is inconsistent all the bootstraps in the world won’t help you…..

Bootstrap - interpretation

Jack-knifing

• Jack-knifing is very similar to bootstrapping and differs only in the character resampling strategy

• Jack-knifing is not as widely available or widely used as bootstrapping

• Tends to produce broadly similar results

At present only sampling techniques allow testing the topology of a phylogenetic tree

· Bootstrapping

» It consists of drawing columns from a sample of aligned sequences, with replacement, until one gets a data set of the same size as the original one. (usually some columns are sampled several times others left out)

· Half-Jacknife

» This technique resamples half of the sequence sites considered and eliminates the rest. The final sample has half the number of initial number of sites without duplication.

Statistical evaluation of the obtained phylogenetic trees

Weblems

W6.1: The growth hormones in most mammals have very similar ammo acid sequences. (The growth hormones of the Alpaca, Dog Cat Horse, Rabbit, and Elephant each differ from that of the Pig at no more than 3 positions out of 191.) Human growth hormone is very different, differing at 62 positions. The evolution of growth hormone accelerated sharply in the line leading to humans. By retrieving and aligning growth hormone sequences from species closely related to humans and our ancestors, determine where in the evolutionary tree leading to humans the accelerated evolution of growth hormone took place.

W6.2: Humans are primates, an order that we, apes and monkeys share with lemurs and tarsiers. On the basis of the Beta-globin gene cluster of human, a chimpanzee, an old-world monkey, a new-world monkey, a lemur, and a tarsier, derive a phylogenetic tree of these groups.

W6.3: Primates are mammals, a class we share with marsupials and monotremes; Extant marsupials live primarily in Australia, except for the opossum, found also in North and South America. Extant monotremes are limited to two animals from Australia: the platypus and echidna. Using the complete mitochondnal genome from human, horse (Equus caballus), wallaroo (Macropus robustus), American opossum (Didelphis mrgimana), and platypus (Ormthorhynchus anatmus), draw an evolutionary tree, indicating branch lengths. Are monotremes more closely related to placental mammals or to marsupials?

W6.4: Mammals are vertebrates, a subphylum that we share with fishes, sharks, birds and reptiles, amphibia, and primitive jawless fishes (example: lampreys). For the coelacanth (Latimeria chalumnae), the great white shark (Carcharodon carcharias), skipjack tuna (Katsuwonus pelamis), sea lamprey (Petromyzon marinus), frog (Rana Ripens), and Nile crocodile (Crocodylus niloticus), using sequences of cytochromes c and pancreatic ribonucleases, derive evolutionary trees of these species.

Education

Bioinformatica t6-phylogenetics