1. Lecture 21: Introduction to Phylogenetics November 9, 2015

2. Last Time uSequence data and quantification of variation  Infinite sites model  Nucleotide diversity (π) uSequence-based tests of neutrality  Ewens-Watterson Test  Tajima’s D

3. Today uSignatures of selection  Hudson-Kreitman-Aguade Test  Synonymous versus Nonsynonymous substitutions  McDonald-Kreitman uMolecular clock uIntroduction to phylogenetics

4. Hudson-Kreitman-Aguade (HKA) Test Divergence between species should be proportional to variation within species (polymorphism) Provides a correction factor for mutation rates at different sites Perform test for loci under selection and supposedly neutral loci Loci with less polymorphism than expected are candidates for selective sweeps within a species

5. Hudson-Kreitman- Aguade(HKA) test (Hamilton 266) Typical Gene Selective Sweep Purifying selection in both lineages

6. Polymorphism Divergence Neutral LocusTest Locus A 8 3 20 8 8/20 ≈ 3/8 Slide adapted from Yoav Gilad Hudson-Kreitman-Aguade (HKA) test Polymorphism: Variation within species Divergence: Variation between species

7. Polymorphism Divergence Neutral LocusTest Locus B 8 3 20 19 8/20 >> 3/19 Slide adapted from Yoav Gilad Hudson-Kreitman-Aguade (HKA) test Conclusion: polymorphism lower than expected in Test Locus B: Selective sweep?

8. Sequence Evolution DNA or protein sequences in different taxa trace back to a common ancestral sequence Divergence of neutral loci is a function of the combination of mutation and fixation by genetic drift Sequence differences are an index of time since divergence

9. Molecular Clock If neutrality prevails, nucleotide divergence between two sequences should be a function entirely of mutation rate Expected Time Until Fixation of a New Mutation: Since μ is number of substitutions per unit time uTime since divergence should therefore be the reciprocal of the estimated mutation rate Probability of creation of new alleles Probability of fixation of new alleles

10. Variation in Molecular Clock If neutrality prevails, nucleotide divergence between two sequences should be a function entirely of mutation rate uSo why are rates of substitution so different for different classes of genes?

11. Using Synonymous Substitutions to Control for Factors Other Than Selection d N /d S or Ka/Ks Ratios

12. Types of Mutations (Polymorphisms)

13. uFirst and second position SNP often changes amino acid  UCA, UCU, UCG, and UCC all code for Serine uThird position SNP often synonymous uMajority of positions are nonsynonymous uNot all amino acid changes affect fitness: allozymes Synonymous versus Nonsynonymous SNP

14. Synonymous & Nonsynonymous Substitutions Synonymous substitution rate can be used to set neutral expectation for nonsynonymous rate d S is the relative rate of synonymous mutations per synonymous site d N is the relative rate of nonsynonymous mutations per non-synonymous site  = d N /d S – If  = 1, neutral selection – If  < 1, purifying selection – If  > 1, positive Darwinian selection For human genes,  ≈ 0.1

15. Complications in Estimating d N /d S  Multiple mutations in a codon give multiple possible paths  Two types of nucleotide base substitutions resulting in SNPs: transitions and transversions not equally likely  Back-mutations are invisible  Complex evolutionary models using likelihood and Bayesian approaches must be used to estimate d N /d S (also called K A /K S or K N /K S depending on method) (PAML package) http://www.mun.ca/biology/scarr/Transitions_vs_Transversions.html CGT(Arg)->AGA(Arg) CGT(Arg)->AGT(Ser)->AGA(Arg) CGT(Arg)->CGA(Arg)->AGA(Arg)

16. dn/ds ratios for 363 mouse- rat comparisons interleukin-3: mast cells and bone marrow cells in immune system Hartl and Clark 2007  Most genes show purifying selection (dN/dS < 1)  Some evidence of positive selection, especially in genes related to immune system

17. McDonald-Kreitman Test Conceptually similar to HKA test Uses only one gene Contrasts ratios of synonymous divergence and polymorphism to rates of nonsynonymous divergence and polymorphism Gene provides internal control for evolution rates and demography

18. uAligned 11,624 gene sequences between human and chimp uCalculated synonymous and nonsynonymous substitutions between species (Divergence) and within humans (SNPs) uIdentified 304 genes showing evidence of positive selection (blue) and 814 genes showing purifying selection (red) in humans Bustamente et al. 2005. Nature 437, 1153-1157 uPositive selection: defense/immunity, apoptosis, sensory perception, and transcription factors uPurifying selection: structural and housekeeping genes Application of McDonald-Kreitman Test:

19. Phylogenetics  Study of the evolutionary relationships among individuals, groups, or species  Relationships often represented as dichotomous branching tree  Extremely common approach for detecting and displaying relationships among genotypes  Important in evolution, systematics, and ecology (phylogeography)

20. A B M K I J N L H G F E D C Z Y X W V U T P Q S R O Ç Evolution Slide adapted from Marta Riutart

21. What is a phylogeny? Z Y X W V U T P Q S R O Ç  Homology: similarity that is the result of inheritance from a common ancestor Slide adapted from Marta Riutart

22. Phylogenetic Tree Terms ABCDEFGHIJ ROOT interior branches node terminal branches Leaves, Operational Taxonomic Units (OTUs) Slide adapted from Marta Riutart Group, cluster, clade

23. Bacteria 1 Bacteria 3 Bacteria 2 Eukaryote 1 Eukaryote 4 Eukaryote 3 Eukaryote 2 Tree Topology (Bacteria1,(Bacteria2,Bacteria3),(Eukaryote1,((Eukaryote2,Eukaryote3),Eukaryote4))) Bacteria 1 Bacteria 3 Bacteria 2 Eukaryote 1 Eukaryote 4 Eukaryote 3 Eukaryote 2 Slide adapted from Marta Riutart

24. http://helix.biology.mcmaster.ca How about these? Are these trees different?

25. Rooted versus Unrooted Trees archaea eukaryote Unrooted tree Rooted by outgroup bacteria outgroup root eukaryote archaea Monophyletic group Monophyletic group Slide adapted from Marta Riutart

26. D C B A G E F C B A F E G D Rooting with D as outgroup Slide adapted from Marta Riutart

27. D C B A G E F C B A F E G D C B A F E G D Now with C as outgroup

28. Which of these four trees is different? Baum et al.

29. UPGMA Method uUse all pairwise comparisons to make dendrogram uUPGMA:Unweighted Pairwise Groups Method using Arithmetic Means uHierarchically link most closely related individuals Read the Lab 12 Introduction!

30. Phenetics (distance) vs Cladistics (discrete character states) Lowe, Harris, and Ashton 2004

31. Parsimony Methods uBased on underlying genealogical relationships among alleles uOccam’s Razor: simplest scenario is the most likely uUseful for depicting evolutionary relationships among taxa or populations uChoose tree that requires smallest number of steps (mutations) to produce observed relationships

32. Choosing Phylogenetic Trees uMANY possible trees can be built for a given set of taxa uVery computationally intensive to choose among these Lowe, Harris, and Ashton 2004 n=number of taxa

33. 7 9 8 9 9 9 9 5 8 10 11 Felsenstein 2004 Choosing Phylogenetic Trees uMany algorithms exist for searching tree space uLocal optima are problem: need to traverse valleys to get to other peaks uHeuristic search: cut trees up systematically and reassemble uBranch and bound: search for optimal path through tree space

34. Choosing Phylogenetic Trees u If multiple trees equally likely, select majority rule or consensus u Strict consensus is most conservative approach u Bootstrap data matrix (sample with replacement) to determine robustness of nodes Lowe, Harris, and Ashton 2004 Felsenstein 2004 E A CB D F 60