1. for immunologists 2013 Introduction to Phylogenies Dr Laura Emery Laura.Emery@ebi.ac.uk www.ebi.ac.uk/training

2. Objectives After this tutorial you should be able to… Use essential phylogenetic terminology effectively Discuss aspects of phylogenies and their implications for phylogenetic interpretation Apply phylogenetic principles to interpret simple trees This course will not: Provide you with an overview of phylogenetic methods Enable you to use tools to construct your own phylogenies Enable you to evaluate whether a sensible phylogenetic model or method was selected to construct a phylogeny

3. Outline Introduction Aspects of a tree 1.Topology 2.Branch lengths 3.Nodes 4.Confidence Simple phylogenetic interpretation Including homology, gene duplication, co-evolution

4. What can I do with phylogenetics? Deduce relationships among species or genes or cells Deduce the origin of pathogens Identify biological processes that affect how your sequence has evolved e.g. identify genes or residues undergoing positive selection Explore the evolution of traits through history Estimate the timing of major historical events Explore the impact of geography on species diversification

5. What is a phylogenetic tree? A tree is an explanation of how sequences evolved, their genealogical relationships and thus how they came to be the way they are today (or at the time of sampling). Darwin 1837

6. Phylogenies explain genealogical relationships Family tree

7. Aspects of a tree 1. Topology (branching order) 2. Branch lengths (indication of genetic change) 3. Nodes i.Tips (sampled sequences known as taxa) ii.Internal nodes (hypothetical ancestors) iii.Root (oldest point on the tree) 4. Confidence (bootstraps/probabilities)

8. 1. Topology The topology describes the branching structure of the tree, which indicate patterns of relatedness. ABCABCBAC These trees display the same topology ABCCBACAB These trees display different topologies

9. Topology Question Are these topologies the same? Answer = yes

10. Topology Question II Which of these trees has a different topology from the others? ABCFD E AEDFB C BACFD E CABFE D EDFCA B

11. 2. Branch lengths indicate genetic change Longer branches indicate greater change Change is typically represented in units of number of substitutions per site (but check the legend) 1.2 0.6 0.8 0.5

12. A scale bar can represent branch lengths 0.5 These are alternative representations of the same phylogeny 1.2 0.6 0.8 0.5

13. Alternative representations of phylogenies All of these representations depict the same topology Branch lengths are indicated in blue Red lengths are meaningless Newick format

14. Not all trees include branch length data CladogramPhylogram

15. Distance and substitution rate are confounded Branch lengths indicate the genetic change that has occurred We often don’t know if long branch lengths reflect: A rapid evolutionary rate An ancient divergence time A combination of both Genetic change = Evolutionary rate x Divergence time (substitutions/site) (substitutions/site/year) (years) C D EAB

16. 3. Nodes Nodes occur at the ends of branches There are three types of nodes: i.Tips (sampled sequences known as taxa) ii.Internal nodes (hypothetical ancestors) iii.Root (oldest point on the tree) CDEAB Figures Andrew Rambaut

17. The root is the oldest point on the tree The root indicates the direction of evolution It is also the (hypothesised) most recent common ancestor (MRCA) of all of the samples in the tree CDEAB past present Figures Andrew Rambaut

18. Trees can be drawn in an unrooted form Rooted Unrooted These are alternative representations of the same topology CDEAB A B C D E

19. There are multiple rooted tree topologies for any given unrooted tree Most tree-building methods produce unrooted trees Identifying the correct root is often critical for interpretation! * Figure Aiden Budd

20. How to root a tree Midpoint rooting Assume constant evolutionary rate Often not the case! Outgroup rooting The outgroup is one or more taxa that are known to have diverged prior to the group being studied The node where the outgroup lineage joins the other taxa is the root Midpoint rooted Outgroup rooted Unrooted Recommended

21. Root Question This tree shows a cladogram i.e. the branch lengths do not indicate genetic change. Indicate any root positions where bird and crocodile are not sister taxa (each other's closest relatives).

22. Alternative Representations Question

23. 4. Confidence How good is a tree? A tree is a collection of hypotheses so we assess our confidence in each of its parts or branches independently There are three main approaches: Bootstraps Bayesian methods Approximate likelihood ratio test (aLRT) methods 85 63 100 probabilistic 0.93 0.81 0.99

24. What is a monophyletic group? A monophyletic group (also described as a clade) is a group of taxa that share a more recent common ancestor with each other than to any other taxa. monophyletic group

25. Confidence Question Which of the bootstrap values indicates our confidence in the grouping of A, B, C, and D together as a monophyletic group? Do you think we can be confident in this grouping? ABCDEFABCDEF 84 63 91 100 Note: high bootstrap values do not always mean that we have confidence in a branch. False confidence can be generated under some phylogenetic methods

26. for immunologists 2013 Part two: Phylogenetic interpretation Dr Laura Emery Laura.Emery@ebi.ac.uk www.ebi.ac.uk/training

27. Phylogenetic interpretation skill set 1. Tree-thinking skills relatedness, confidence, homology 2. Knowledge of phylogenetic methods and their limitations 3. Knowledge of biological processes affecting sequence evolution gene duplication, recombination, horizontal gene transfer, population genetic processes, and many more! 4. Knowledge of the data you wish to interpret Covered in introduction to phylogenies

28. Simple phylogenetic interpretation question Which is true? A) Mouse is more closely related to fish than frog is to fish B) Lizard is more closely related to fish than mouse is to fish C) Human and frog are equally related to fish

29. Homology is similarity due to shared ancestry Example: limbs and wings Limbs are homologous they share a common ancestor Wings are not homologous they are an analogous as they have evolved similarity independently

30. Gene duplication Gene duplication and subsequent divergence can result in novel gene functions (it can also result in pseudogenes) Genes that are homologous due to gene duplication are paralogous Genes that are homologous due to speciation are orthologous

31. Can you spot any MHC class II gene duplication events? Harstad et al BMC Genomics 2008 Teleost MHC class II phylogeny

32. Park et al 2012. Scientific Reports Immunology genes have a high d N /d S ratio indicative of positive selection Rapid evolutionary rate Difficult to align Violate assumptions of many phylogenetic models Immunology related genes have atypical patterns of molecular evolution

33. Positive selection can lead to ladder-like phylogenies

34. Example: influenza haemagglutination phylogeny and immunological mapping Smith et al 2004. Science

35. Phylogenetics can inform us of host- pathogen interactions and co-evolution "Mirror" phylogenies are indicative of host-parasite vertical inheritance Jiggins web page: http://www.gen.cam.ac.uk/research/jiggins/research.html

36. What does this phylogeny tell us about Human Cytomegalovirus (HCMV)? Nicholson et al 2009. Virol J Human Chimp Rhesus Simian Baboon Rat Murine

37. T-cell receptors and immunoglobulin chains are homologous Richards et al 2000

38. An extremely brief introduction to methods, analyses, & pitfalls

39. There is only one true tree The true tree refers to what actually happened in the evolutionary past All methods attempt to reconstruct the true phylogeny Even the best method may not give you the true tree

40. Phylogenetic Methods: The general approach We want to find the tree that best explains our aligned sequences We need to be able to define “best explains” we need a model of sequence evolution we need a criterion (or set of criteria) to use to choose between alternative trees then evaluate all possible trees (NB: if N=20, then 2 x 1020 possible unrooted trees!) or take a short cut Paul Sharp

41. The problem of multiple substitutions More likely to have occurred between distantly related species > We need an explicit model of evolution to account for these A A AT G * * * * hidden mutations

42. Methodological approaches 1. Distance matrix methods (pre-computed distances) UPGMAassumes perfect molecular clock Sokal & Michener (1958) Minimum evolution (e.g. Neighbor-joining, NJ) Saitou & Nei (1987) 2. Maximum parsimony Fitch (1971) Minimises number of mutational steps 3. Maximum likelihood, ML Evaluates statistical likelihood of alternative trees, based on an explicit model of substitution 4. Bayesian methods Like ML but can incorporate prior knowledge

43. Phylogenetic analyses are not straightforward Data assessment - known biology - additional data (e.g. geography) Decide upon and implement method Phylogeneti c Result(s) Formulate hypotheses Answere d your question? Investigate unexpected and unresolved aspects further - consider including more data Final phylogeny and analysis Can you validate this? Yes No Yes

44. Further Reading Molecular Evolution: A Phylogenetic Approach (1998) Roderic D M Page & Edward C Holmes, Blackwell Science, Oxford. The Phylogenetic Handbook (2003), Marco Salemi and Anne-Mieke Vandamme Eds, Cambridge University Press, Cambridge. Inferring Phylogenies (2003) Joseph Felsenstein, Sinauer. Molecular Evolution (1997) Wen-Hsiung Li, Sinauer

45. Phylogenetics at the EBI Clustal phylogeny currently available RAxML coming soon… www.EBI.ac.uk/tools/phylogeny

46. Acknowledgements People Andrew Rambaut (University of Edinburgh) …and the EBI training team Paul Sharp (University of Edinburgh) Nick Goldman (EMBL-EBI) Benjamin Redelings (Duke University) Brian Moore (University of California, Davis) Olivier Gascuel (University of Montpelier) Aiden Budd (EMBL-Heidelberg) Funding EMBL member states and…

47. Thank you! www.ebi.ac.uk Twitter: @emblebi Facebook: EMBLEBI