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Introduction to Phylogenies. for immunologists 2013. Dr Laura Emery Laura.Emery@ebi.ac.uk www.ebi.ac.uk /training. Objectives. After this tutorial you should be able to… Use essential phylogenetic terminology effectively
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Introduction to Phylogenies for immunologists 2013 Dr Laura Emery Laura.Emery@ebi.ac.uk www.ebi.ac.uk/training
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
Outline • Introduction • Aspects of a tree • Topology • Branch lengths • Nodes • Confidence • Simple phylogenetic interpretation • Including homology, gene duplication, co-evolution
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
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
Phylogenies explain genealogical relationships • Family tree
Aspects of a tree • Topology (branching order) • Branch lengths (indication of genetic change) • Nodes • Tips (sampled sequences known as taxa) • Internal nodes (hypothetical ancestors) • Root(oldest point on the tree) • Confidence (bootstraps/probabilities)
1. Topology The topology describes the branching structure of the tree, which indicate patterns of relatedness. A A C B C A B B A B A B C B C C C A These trees display the sametopology These trees display differenttopologies
Topology Question Are these topologies the same? Answer = yes
Topology Question II Which of these trees has a different topology from the others? C F F F F A B E D D A A C E B D A A E B B C D C F E E C D B
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) 0.8 1.2 0.5 0.6 0.5 0.5
A scale bar can represent branch lengths These are alternative representations of the same phylogeny 0.8 1.2 0.5 0.6 0.5 0.5 0.5
Alternative representations of phylogenies All of these representations depict the same topology Branch lengths are indicated in blue Red lengths are meaningless Newick format
Not all trees include branch length data CladogramPhylogram
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) A B C E D
3. Nodes • Nodesoccuratthe ends of branches • There are three types of nodes: • Tips (sampled sequences known as taxa) • Internal nodes (hypothetical ancestors) • Root (oldest point on the tree) A B C D E Figures Andrew Rambaut
The root is the oldest point on the tree present • 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 A B C D E past Figures Andrew Rambaut
Trees can be drawn in an unrooted form Rooted Unrooted These are alternative representations of the same topology A B C D E A D B E C
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
How to root a tree Midpoint rooted • Midpointrooting • Assume constant evolutionary rate • Often not the case! • Outgrouprooting • 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 Unrooted Outgroup rooted Recommended
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 notsister taxa (each other's closest relatives).
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 100 0.99 63 0.81 85 0.93 probabilistic
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
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? 91 63 A B C D E F 100 84 • Note: high bootstrap values do not always mean that we have confidence in a branch. False confidence can be generated under some phylogenetic methods
Part two: Phylogenetic interpretation for immunologists 2013 Dr Laura Emery Laura.Emery@ebi.ac.uk www.ebi.ac.uk/training
Phylogenetic interpretation skill set • Tree-thinking skills • relatedness, confidence, homology • Knowledge of phylogenetic methods and their limitations • Knowledge of biological processesaffecting sequence evolution • gene duplication, recombination, horizontal gene transfer, population genetic processes, and many more! • Knowledge of the data you wish to interpret Covered in introduction to phylogenies
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
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
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
Teleost MHC class II phylogeny • Can you spot any MHC class II gene duplication events? Harstad et al BMC Genomics 2008
Immunology related genes have atypical patterns of molecular evolution • Immunology genes have a high dN/dS ratio indicative of positive selection • Rapid evolutionary rate • Difficult to align • Violate assumptions of many phylogenetic models Park et al2012. Scientific Reports
Example: influenza haemagglutinationphylogeny and immunological mapping Smith et al 2004. Science
Phylogenetics can inform us of host-pathogen interactions and co-evolution • "Mirror" phylogenies are indicative of host-parasite vertical inheritance Jigginsweb page: http://www.gen.cam.ac.uk/research/jiggins/research.html
What does this phylogeny tell us about Human Cytomegalovirus (HCMV)? Baboon Simian Rhesus Chimp Human Rat Murine Nicholson et al 2009. Virol J
T-cell receptors and immunoglobulin chains are homologous Richards et al 2000
An extremely brief introduction to methods, analyses, & pitfalls
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
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
The problem of multiple substitutions A * • More likely to have occurred between distantly related species • > We need an explicit model of evolution to account for these hidden mutations * G A * * A T
Methodological approaches • Distance matrix methods (pre-computed distances) • UPGMA assumes perfect molecular clock Sokal & Michener (1958) • Minimum evolution (e.g. Neighbor-joining, NJ) Saitou & Nei (1987) • Maximum parsimony Fitch (1971) • Minimises number of mutational steps • Maximum likelihood, ML • Evaluates statistical likelihood of alternative trees, based on an explicit model of substitution • Bayesian methods • Like ML but can incorporate prior knowledge
Phylogenetic analyses are not straightforward Phylogenetic Result(s) Formulate hypotheses • Data assessment • - known biology • - additional data (e.g. geography) Decide upon and implement method Investigate unexpected and unresolved aspects further - consider including more data Answered your question? No No Yes Can you validate this? Final phylogeny and analysis Yes
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-MiekeVandammeEds, Cambridge University Press, Cambridge. • Inferring Phylogenies (2003) Joseph Felsenstein, Sinauer. • Molecular Evolution (1997) Wen-Hsiung Li , Sinauer
Phylogenetics at the EBI • Clustal phylogeny currently available • RAxML coming soon… • www.EBI.ac.uk/tools/phylogeny
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) FundingEMBL member states and…
Thank you! www.ebi.ac.uk Twitter: @emblebi Facebook: EMBLEBI