Phylogenetics

1. Phylogenetics

2. Phylogenetic Trees time

3. NODE BRANCH Hypothetical Taxonomic Unit ROOT Operational Taxonomic Unit (OTU) time

4. Information • Branching order (topology) • Relative closeness of different taxa • Branch length • Amount of divergence

5. A B Rooted and unrooted trees C A D B C D E E UNROOTED ROOTED

6. A B Rooted and unrooted trees E A B C D D E C UNROOTED ROOTED

7. Rooted and unrooted trees A A E B B C D D E C UNROOTED ROOTED

8. ROOTED UNROOTED 3 OTUs A A C B B B C A A C B C A A A 4 OTUs C A B B B C C C D D D B D B A A D C C D B A C … 15 rooted trees of 4 OTUs B D

9. Monophyletic & Paraphyletic Birds Crocodiles REPTILES Snakes and lizards Turtles and tortoises Mammals

10. Monophyletic & Paraphyletic • Monophyletic • Natural clade; all of the taxa are derived from a common ancestor • Paraphyletic • Taxonomic group whose most recent common ancestor is shared by another taxon

11. Reconstruct phylogeny from molecular data ACTGTTACCGA ? ACTGTTACCGA ACTGTTACCGA ACTGTTACCGA ACTGTTACCGA

12. Types of phylogenetic analysis methods • Phenetic: trees are constructed based on observed characteristics, not on evolutionary history • Cladistic: trees are constructed based on fitting observed characteristics to some model of evolutionary history Distance methods Parsimony and Maximum Likelihood methods

13. Methods of Tree reconstruction • Distance • Maximum Parsimony • Maximum Likelihood • Bayesian Phylogeny Estimation: Traditional and Bayesian Approaches Nature Reviews Genetics (2003) 4:275

14. Genetic distance • Distance from one sequence to another • Hamming Distance • Count number of differences • Multiple hits – number of events is greater than number of differences • Estimate number of events • Infer tree from genetic distance using Neighbour-joining (NJ) method

15. UPGMA shown for illustrative purposes. Neighbour-joining is preferred method.

16. The algorithm in the text means: find the closest distance between two sequences, cluster those; then find the next closest distance, cluster those; as sequences are added to existing clusters find the average distance between existing clusters • Work through the notation! • UPGMA assumes a molecular clock mechanism of evolution

17. Neighbor-joining: corrects for UPGMA’s assumption of the same rate of evolution for each branch by modifying the distance matrix to reflect different rates of change. • The net difference between sequence i and all other sequences is • ri = Sdik

18. The rate-corrected distance matrix is then • Mij = dij - (ri + rj)/(n - 2) • Join the two sequences whose Mij is minimal; then calculate the distance from this new node to all other sequences using • dkm = (dim + djm - dij)/2 • Again correct for rates and join nodes.

19. Maximum Parsimony (MP) • Find topology requiring smallest number of evolutionary changes • Consider each position (site) in the sequence alignment independently • Not all sites are informative • Informative • Favours one topology over others

20. Informative sites a. A A G A G T T C A b. A G C C G T T C T c. A G A T A T C C A d. A G A G A T C C T a b c a c a d d d b c b

21. Maximum Likelihood (ML) • Likelihood L of a tree is the probability of observing the data given the treeL = P(data|tree) • Find the tree with the highest L value • Results depends on model of nucleotide substitution • Computationally time-consuming

22. Actually, all the other methods discussed implicitly use a simple model of evolution similar to the typical model made explicit in maximum likelihood: • All sites selectively neutral • All mutate independently, forward and reverse rates equal, given by m

23. Also assume discrete generations and sites change independently • Given this model, can calculate probability that a site with initial nucleotide I will change to nucleotide j within time t: • Ptij = dije-mt + (1 - e-mt)gj, where dij = 1 if i = j and dij = 0 otherwise, and where gj is the equilibrium frequency of nucleotide j

24. The likelihood that some site is in state i at the kth node of a tree is Li(k) • The likelihoods for all states for each site for each node are calculated separately; the product of the likelihoods for each site gives the overall likelihood for the observed data • Different tree topologies are searched to find the highest overall likelihood

25. Maximum likelihood is maybe the “gold standard” for phylogenetic analysis; but because of its computational intensity it can only be used for select data and only after much initial fine tuning of many parameters of sequence alignments • Often used to distinguish between several already generated trees

26. Bayesian (B) Phylogeny Estimation • Searches for best trees consistent with both model and data • Incorporates prior knowledge (prior probability) • B maximises probability of tree given data and model • Searches for best set of trees

27. Comparison of methods How much information are they using? • MP, ML, B use actual DNA whereas NJ summarises information into distance matrix • BUT, not all sites are used by MP (“informative” sites only) How can the nature of the data affect the methods? • NJ better for recent divergences • MP works well for a high number of informative sites

28. Comparison of methods How do they cope with lots of sequences? • MP requires comparison of all possible trees • Not possible for large number of taxa • ML is computationally intensive and very slow for large number of taxa • NJ efficient for large number of taxa Anything else? • ML requires explicit assumptions about rate and pattern of substitution (model) • ML may perform poorly if model is incorrect • ML or B may get stuck on local maxima

29. chicken human human mouse mouse rat rat Outgroup rooting of unrooted trees • Outgroup – related sequence that definitely diverged earlier (paleontological evidence)

30. Rate (r) of evolution • K = number of substitutions per site • T = time since divergence • r = K/2T • Rate is expressed as substitutions per site per year Species A Species B T

31. Estimating species divergence times • fossil evidence shows that T1 = 310 mya • What is T2 ? • Only need to have sequences and information on one divergence time Chicken (C) Human (B) Rat (A) T2 T1

32. True tree and inferred tree • There is only one true tree of species relationships • Inferred tree may not be correct • Some genes may not be representative • Tree inference method may have produced an incorrect tree • e.g. parsimony method: may get several equally parsimonious results

33. How credible is the tree? • The tree is a hypothesis of the true relationship • Need some measure of the support for that hypothesis • Note: Bayesian methods simultaneously estimate tree and measures of uncertainty for each branch

34. Standard Error of branches Human Chimp Gorilla Orangutan

35. The bootstrap: randomly sample all positions (columns in an alignment) with replacement -- meaning some columns can be repeated -- but conserving the number of positions; build a large dataset of these randomized samples

36. Bootstrap

37. Then use your method (distance, parsimony, likelihood) to generate another tree • Do this a thousand or so times • Note that if the assumptions the method is based on hold, you should always get the same tree from the bootstrapped alignments as you did originally • The frequency of some feature of your phylogeny in the bootstrapped set gives some measure of the confidence you can have for this feature

38. Applications of phylogenetics • Detection of orthology and paralogy • Estimation of divergence times • Reconstruction of ancient proteins • Identifying residues important to selection • Detecting recombination points • Identifying mutations likely to be associated with disease • Determining the identity of new pathogens

39. 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

40. The Tree of Life • Traditional classification of life into five kingdoms • Bacteria (inc cyanobacteria) • Protista (inc. cilliates, flagellates, amoebae) • Fungi • Plantae • Animalia

41. Archaebacteria • Carl Woese and colleagues • Study relationships by comparing rRNAs • Methanogens were expected to group with other bacteria • BUT, found to be equally distant from bacteria and eukaryotes • Made new taxon - Archaebacteria • Includes many extremophiles • thermophiles • hyperthermophiles • halophiles (salt dependent)

42. The Tree of Life

43. lineage 1 Gene A1 lineage 2 lineage 3 Gene A lineage 1 Gene A2 lineage 2 lineage 3 Where is the root of the Tree of Life? • No possible outgroup (by definition) • Iwabe et al. (1989) • Examined phylogenetic tree of pairs of genes that exist in all organisms • derived from gene duplication that predates lineage divergences

44. Homologous elongation factor genes EF-Tu and EF-G present in all prokaryotes and eukaryotes • Both genes show the same topology Archaea EF-Tu Eucarya Bacteria Archaea EF-G Eucarya Bacteria

45. Changing view ofThe Tree of Life …(Gaucher et al, 2010) based on morphological characteristics (Chatton, 1925) based on DNA sequence analysis (Woese & Fox, 1977) based on phylogenies of hundreds of genes based on membrane architecture & gene indels based on ancient gene duplication Most modern view …

46. Human Chimp Gorilla Orangutan Gibbon Phylogeny of humans and apes • Darwin – Gorilla and Chimpanzee our closest relatives and human evolutionary origins in Africa • Many people preferred anthropocentric idea that humans were special Traditional view

47. So what is the evidence? • Serological precipitation (Goodman 1962) – H, G, C constitute a natural clade, orangutans & gibbons earlier diverging • However, H,G,C relative relationships remained unclear • Most DNA sequence data support ((H,C),G) • Some genes show different relationship Human Chimp Gorilla Orangutan Gibbon

48. Conservation biology – the dusky seaside sparrow • Last one died June 1987 (DisneyWorld) • Discovered 1872 • Ammodramus maritimus nigrescens • Geographically confined to small salt marsh in Florida • 2000 individuals in 1900 • 6 individuals (all male) in 1980 • Conservation program • artificial breeding

49. Conservation genetics • Mating of remaining males with females from closest subspecies available • Female hybrids of first generation then “back-crossed” to original males • Continue as long as original males live • Which species to choose to take the females from??

50. 8 other A. maritimus subspecies • Geographically dispersed along coast • Artificial breeding with Scott’s seaside sparrow (A. m. peninsulae) • Chosen based on Morphological and behavioural similarities • Was this the best choice?