1. 1 General Phylogenetics Points that will be covered in this presentation Tree TerminologyTree Terminology General Points About Phylogenetic TreesGeneral Points About Phylogenetic Trees Phylogenetic AnalysesPhylogenetic Analyses  The importance of Alignments  The different analysis methods  Tree confidence measures

2. 2 Tree Terminology Node: point at which 2 or more branches diverge Internal node: hypothetical last common ancestor Terminal node: molecular or morphological data from which the tree is derived. (These will often be used to represent species or individual specimens and may be referred to as OTUs = Operational Taxonomic Units) Clade: a node (hypothetical ancestor) and all the lineages descending from it internal node terminal node or OTU internal node terminal node or OTU clade

3. 3 Tree Terminology Monophyletic group: a group in which all members are derived from a unique common ancestor Polyphyletic group: a group in which all members are not derived from a unique common ancestor. The common ancestor of the group has many descendants that are not in the group Paraphyletic group: a group that excludes some of the descendants of the common ancestor (a form of polyphyly)

4. 4 General Points About Phylogenetic Trees All branches can rotate freely around a node (i.e. B is not more closely related to C than A, and C is not more closely related to D than E) A B C D E Branch lengths may be be drawn as equal between nodes – “cladograms” (see tree above) (these are used when one is interested only in the branching pattern) Branch lengths may be proportional to the hypothesized distance between nodes – “phylogram” (see tree on left) A B C D E

5. 5 polytomy General Points About Phylogenetic Trees polytomy Fully resolved trees are bifurcating (only two decendant lineages from nodes) A node with more than two decendant lineages is a multifurcating node or a polytomy. Polytomies may be “soft” or “hard” “Soft” = product of data or analysis “Hard” = product of biology

6. 6 polytomy General Points About Phylogenetic Trees Example of a “soft” polytomy: LSU analysis is unable to resolve the relationships of some Ptilophora species. LSU tree Using different data (rbcL) the relationships among Ptilophora species are better resolved. rbcL tree Tronchin et al. 2004

7. 7 Phylogenetic Analyses The Importance of Alignments Phylogenetic trees derived from the analysis of DNA or amino acid sequences are only as good as the data they are based upon. Garbage In = Garbage Out Consequently, sequence alignment is the most important step in phylogenetic analysis. The aligned sites of a sequence must be homologous (or identical by decent = taxa share the same state because their ancestor did). If two taxa share the same state but not by decent it is called homoplasy

8. 8 The Importance of Alignments Phylogenetic Analyses same sites in different sequences need to be homologous inferred insertion/deletion mutations (gaps) area to possibly remove from analyses because of uncertain homology between sites DNA sequences are prone to homoplasy because there are only 4 possible sites (and insertion/deletion mutations[indels] for some loci).

9. 9 Phylogenetic Analyses The Different Analysis Methods See: evolution.genetics.washington.edu/phylip/software.html#methods for a list of software programs Distance methods: based on similarity between OTUs UPGMA – originally used for phenotypic characters in numerical taxonomy. Generally not applied to sequence data because it is highly sensitive to mutation rate changes in lineages, i.e. the data must fit a “molecular clock.” NJ (Neighbor Joining) – algorithm method that will find the “minimum evolution” tree without examining all possible topologies. The accuracy of a distance tree depends on 2 things: 1)How “true” are the distances calculated between taxa (how good is the model of evolution that your distances are based upon). 2) The standard error of the distance measure estimation

10. 10 Phylogenetic Analyses The Different Analysis Methods Optimization methods Parsimony: searching for the tree that requires the least number of mutational steps i.e. the simplest is the best. Maximum Likelihood: searching for the most likely tree (the tree with highest probability) given the OTUs (sequences) and model of evolution i.e. the tree that maximizes the probability of observing the data is the best tree. Bayesian: searching for the best set of trees i.e. the set of trees in which the likelihoods are so similar that changes between them are essentially random.

11. 11 Phylogenetic Analyses Tree Confidence Measures Decay Analysis or Goodman-Bremer Support Values: a test used in parsimony analyses where one determines how many steps less parsimonious than minimal, is a particular branch in your tree no longer resolved in the consensus of all possible trees that length. Most parsimonious tree L = 35 One step less parsimonious L = 36 Two steps less parsimonious L = 37 d1 d2 How meaningful the values are may depend on the tree length.

12. 12 Phylogenetic Analyses Tree Confidence Measures Bootstrapping: A non-parametric test of how well the data support the nodes of a given tree. Determining support is a bit of a statistical problem: Evolution only happened once so there is no underlying distribution to sample in order to develop confidence values. Method: the original analysis is performed multiple times on pseudo-datasets derived by sampling the original dataset with replacement. The number, or fraction, of times that a particular clade is present in the resulting trees is its boostrap value. Bootstrapping is not portable i.e. you can not compare values across studies because changing any parameters will change the values.

13. 13 Tree Confidence Measures Bootstrapping By default most programs will show bootstrap values when they are greater than 50 but, does a bootstrap value of 50 mean anything? For a discussion of this see Hillis & Bull (1993) Systematic Biology 42:182- 192 (they tested bootstrap values based on a known phylogeny). Wilson’s General Rule: 60-80, is there other evidence to support the relationship, be cautious; 80-90, usually pretty solid; 90-100, solid and unlikely to be misleading. Phylogenetic Analyses

14. 14 General Points About Phylogenetic Trees DNA or protein sequence trees are hypotheses of how a particular DNA locus or protein has evolved. We assume that the way the DNA or protein has evolved reflects the way the species has evolved i.e. gene tree = species tree IMPORTANT: This may or may not reflect reality. i.e. You Still Have To Think as molecules do not necessarily trump morphology, development, etc.

15. 15 General Points About Phylogenetic Trees gene tree = species tree gene tree species tree gene tree = species tree AABB C C