Novel approaches for large-scale phylogenetics and applications in the context of the amphibian tree of life A thesis submitted to the National University of Ireland for the Degree of Doctor of Philosophy Presented by: Karen Y. Siu Ting Salvatierra M.Sc. Department of Biology NUI Maynooth Maynooth Co. Kildare, Ireland. January 2014 Supervisor: Dr. Davide Pisani B.Sc., Ph.D. (Bristol) Co-supervisor: Dr. Mark Wilkinson B.Sc., Ph.D. (Bristol) Head of Department: Prof. Paul Moynagh Dip. Biology BA (mod.), PhD (Dublin) Table of Contents Index of Figures .............................................................................. IV! Index of Tables ............................................................................... VI! Declaration .................................................................................. VII! Acknowledgments ........................................................................ VIII! Abbreviations .................................................................................. X! Abstract ....................................................................................... XI! Chapter 1. Introduction ...................................................................... 1! 1.1 Large-scale phylogenetics ...................................................................... 1! 1.2 Large-scale phylogenies of Amphibia ....................................................... 2! 1.3 Inferring phylogenies ........................................................................... 5! 1.3.1 Homology ....................................................................................................... 5! 1.3.2 Positional Homology and Multiple Sequence Alignment ................................... 6! 1.3.3 Phylogenetic methods ...................................................................................... 7! 1.3.3.1 Parsimony .............................................................................................................. 8! 1.3.3.2 Character compatibility ........................................................................................ 10! 1.3.3.3 Maximum Likelihood ........................................................................................... 13! 1.3.3.3.1 Assessing best trees ........................................................................................ 14! 1.3.3.4 Bayesian Inference in phylogenetics ...................................................................... 16! 1.3.3.5 Models of DNA evolution .................................................................................... 18! 1.3.3.6 Sequence Saturation ............................................................................................. 24! 1.3.3.7 Consensus and Supertree methods ........................................................................ 28! 1.4 The importance of multiple lines of evidence: microRNAs ........................... 32! 1.4.1 Discovery and function .................................................................................. 32! 1.4.2 Importance as deep-phylogeny markers .......................................................... 33! Chapter 2. Concatabominations – an extension to the a priori method of Safe Taxonomic Reduction. ...................................................................... 35! 2.1 Introduction .................................................................................... 35! 2.2 Methods ......................................................................................... 37! 2.2.1 Safe Taxonomic Reduction ............................................................................ 37! 2.2.2 Recoding strategy ........................................................................................... 39! 2.2.3 Use of Compatibility to identify potential equivalents ...................................... 41! 2.2.4 Implementing concatabominations ................................................................. 42! 2.2.5 Determining which taxa to delete ................................................................... 44! 2.3 Results: Case studies ......................................................................... 46! 2.3.1 Application to a morphological data set .......................................................... 47! 2.3.2 Application to a genomic data set in a supertree context .................................. 53! 2.4 Discussion ...................................................................................... 58! 2.5 Conclusions ..................................................................................... 61! Chapter 3. Placing taxa in the context of a large-scale phylogeny ................. 62! 3.1 Introduction .................................................................................... 62! 3.2 Material and Methods ........................................................................ 66! II 3.2.1 Sampling and DNA extraction ....................................................................... 66! 3.2.2 Data Matrix ................................................................................................... 68! 3.2.3 Saturation Analysis ........................................................................................ 69! 3.2.4 Phylogenetic Analysis: A two-tiered approach ................................................ 70! 3.3 Results ........................................................................................... 73! 3.3.1 Saturation analysis ......................................................................................... 73! 3.3.2 Phylogenetic analyses ..................................................................................... 74! 3.4 Discussion ...................................................................................... 80! 3.4.1 Taxonomy, phylogeny and biogeography ....................................................... 80! 3.4.2 Conservation ................................................................................................. 83! 3.4.3 Incorporating previously unsampled taxa into large-scale phylogenetic analyses .............................................................................................................................. 84! 3.5 Conclusions ..................................................................................... 86! Chapter 4. Investigating the phylogenetic relationships of the extant orders of Amphibia with novel genetic evidence .................................................. 87! 4.1 Introduction .................................................................................... 87! 4.1.1 Conflicting phylogenetic hypotheses of the extant Amphibia ........................... 87! 4.2 Material and Methods ........................................................................ 95! 4.2.1 Materials ....................................................................................................... 96! 4.2.2 Methods ........................................................................................................ 96! 4.2.2.1 Sample preparation ............................................................................................... 96! 4.2.2.2 RNA extraction .................................................................................................... 97! 4.2.2.3 Small RNA libraries generation ............................................................................ 98! 4.2.2.4 Identifying miRNAs ........................................................................................... 101! 4.2.2.5 Shared miRNA analysis ...................................................................................... 103! 4.3 Results .......................................................................................... 104! 4.3.1 Identified shared miRNAs ............................................................................ 104! 4.4 Discussion ..................................................................................... 108! 4.4.1 Batrachia hypothesis and the monophyly of Amphibia. ................................. 108! 4.4.2 Caveats of using miRNA data in phylogenies ................................................ 110! 4.5 Conclusions .................................................................................... 113! Chapter 5. General discussion and conclusions ...................................... 115! 5.1 Large-scale phylogenies and the road to phylogenomics ............................. 116! 5.2 Pragmatic solutions to unequal sampling and the continuous increase of molecular data ................................................................................................... 118! 5.2.1 Targeting problematic areas in a tree ............................................................. 118! 5.2.2 Building up on previous knowledge .............................................................. 119! 5.3 Amphibian phylogenetics ................................................................... 120! Chapter 6. Future work ................................................................... 123! Chapter 7. Bibliography .................................................................. 124! Appendices .................................................................................. 139! Appendix A ......................................................................................... 140! Appendix B. ........................................................................................ 142! Appendix C. .......................................................................................