Unravelling the impact of substitution rates and time on branch lengths in phylogenetic trees MSc Biology Thesis Biosystematics (BIS-80430) Wageningen University of Research By Isolde van Riemsdijk St.nr: 910104694070 Supervisor: Lars W. Chatrou 2nd Supervisor: Freek Bakker May 2014 i ii Figure 1: Phylogeny of the Annonaceae constructed with RAxML from the total dataset with clades from left to right: Eupomatia bennettii, Anaxagorea, Ambavioideae, the short branch clade (SBC) alias the Malmeoideae and the long branch clade (LBC) alias the Annonoideae iii iv Contents 1. Preface .............................................................................................................................. vi 2. Summary ........................................................................................................................... vi 3. Introduction ........................................................................................................................ 1 3.1 Genomics and the assumption of a molecular clock .................................................................... 2 3.2 Evolutionary divergence times ................................................................................................ 4 3.3 Using fossil characteristics to construct a calibration prior distribution .......................................... 4 4. Research questions ............................................................................................................. 8 5. Materials and methods ........................................................................................................ 9 5.1 Construction of the DNA sequence supermatrix ......................................................................... 9 5.2 Finding the best possible partition scheme and models ............................................................. 10 5.2.1 Different analyses for LBC and SBC datasets and comparison ............................................... 10 5.2.2 PartitionFinder settings and results ................................................................................... 11 5.3 Phylogenetic tree construction .............................................................................................. 11 5.4 Restriction of nodes with fossils ............................................................................................ 11 5.4.2 Fossils providing deep node ages in the Annonaceae phylogeny ............................................ 12 5.4.3 Fossils providing shallow node ages in the Annonaceae phylogeny ........................................ 14 5.5 Dating analyses with the 4M dataset: BEAST .......................................................................... 18 6. Results .............................................................................................................................. 20 6.1 PartitionFinder: models for the LBC and SBC .......................................................................... 20 6.2 RAxML bootstrap phylogenetic trees ...................................................................................... 20 6.3 BEAST analyses results........................................................................................................ 20 6.3.1 Node age estimates of the fossil exclusion and LBC:SBC ratio analyses .................................. 21 6.3.2 Mean substitution rate estimates for different interesting analyses ........................................ 27 7. Discussion ......................................................................................................................... 30 7.1 First analyses with the data .................................................................................................. 30 7.2 Comparing published ages to results ..................................................................................... 31 7.3 Changing effects of calibration priors in different combinations and data selection ........................ 32 8. Conclusion ........................................................................................................................ 35 9. Future research ................................................................................................................. 36 10. Acknowledgements ......................................................................................................... 38 11. References ...................................................................................................................... 38 Appendix 1: Species table with GenBank numbers and references ................................................. 43 Appendix 2: Starting partition schemes and resulting best schemes for each dataset ........................ 62 Appendix 3: RAxML bootstrap trees for the 4M and total dataset ................................................... 63 Appendix 4: Record of BEAST analysis runs in Tracer v. 1.5 .......................................................... 70 Appendix 5: Maximum clade credibility trees resulting from the BEAST analyses .............................. 74 v 1. Preface I have always had a special interest in evolution. I find it amazing every time to see how simple the processes underlying evolution can be, while the variety of species is so large. I came to work on the problem of the presence of a long branch and short branch clade in the Lentibulariaceae. I learned that the programmes used to investigate phylogenetic trees have become very complicated, but still cannot discern between elapsed time since divergence and substitution rate. To know how, when and in the future maybe even why the divergence of extant plant groups has taken place will shed new light on plant evolution. To solve the problem of the fusion of time since divergence and substitution rate in phylogenetic programs, first it has to be investigated together with the already existing ways of accounting for rate heterogeneity in phylogenetic research. 2. Summary This thesis explores the possibilities to make a distinction between the influences of sequence data in which rate heterogeneity is shown and calibration priors on age and rate estimates. Rate heterogeneity impedes the assumption of a strict molecular clock and causes demand for more complicated models. Annonaceae data have been proved in previous publications to show rate heterogeneity. A dataset of 252 species from the Annonaceae family and four plastid markers (matK, ndhF, psbA-trnH and trnL-trnF) was constructed by combination of already existing datasets. BEAST is used to perform dating analysis taking rate heterogeneity into account. Former dating analyses only used (older) deep node fossils for the Annonaceae clade. This thesis also includes four (younger) fossils assigned to clades situated more shallow in the phylogeny of Annonaceae. Including more fossils increases support of a hypothesis of ages. To identify the influences of the shallow fossil calibrations separately and in combination, all possible younger fossil calibration combinations are used in dating analyses in BEAST. To be able to identify the influence of the sequence data on age and rate estimates also analyses were done including all fossil calibrations but only part of the data. Two extreme situations are created by reducing the short branch clade (SBC) sequences to a minimum of five species in one dataset, remaining all long branch clade (LBC) sequences and vice versa. Mean age estimates and 95 % HPDs are recorded for eight nodes of interest and rate distributions for five analyses are obtained. The results show that the influence of calibration priors is not easy to interpret. Age estimates probably are very much influenced by the amount of sequence data which is constrained by a calibration prior and the properties of those sequences. The age estimates of the total dataset and total prior set are in general older than ages published before. vi 3. Introduction The phylogenetics of Annonaceae have a long history of changes and uncertainties (for a recent summary read Chatrou et al. (2012)). The phylogenetic tree of the Annonaceae is not resolved yet at the species level. Using a large dataset, Chatrou et al. (2012) construced a tree which showed improvement in generic representation and resolution. Still, despite the high amount of sequence data, there are some issues related to resolving the Annonaceae tree. One of them is the observation that there are symptoms of rate heterogeneity within the Annonaceae (Doyle et al., 2004; Pirie & Doyle, 2012). This causes the topology of the tree to have a long branch clade (LBC) and short branch clade (SBC) (Richardson et al., 2004), see for an Figure 2: Annonaceae LBC (green) and example figure 1. Rate heterogeneity makes it SBC (blue) dated in the articles of Richardson et al. (2004) (above, ages ± harder to estimate divergence dates in a reliable 3.6 My) and Couvreur et al. (2011) manner. This effect of rate heterogeneity on (below), age axis in My divergence time estimate is shown by the difference in the result of two studies on dating of the LBC and SBC of the Annonaceae family. Richardson et al. (2004) shows the divergence dates of the two clades to be quite similar. The crown node of the SBC was estimated at 62.5 ± 3.6 Mya and the LBC crown node at 60.2 ± 3.6 Mya with Archaeanthus as calibration point and using NPRS (figure 2). The analysis of Couvreur et al. (2011), done with more data (including the same data as Richardson) but with BEAST resulted in quite different ages.