Rethinking phylogenetics using Caryophyllales (angiosperms), matK gene and trnK intron as experimental platform Sunny Sheliese Crawley Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Biological Sciences Khidir W. Hilu Eric P. Beers Carla V. Finkielstein Jill C. Sible December 2, 2011 Blacksburg, Virginia Keywords: (phylogeny, missing data, caryophyllids, trnK intron, matK, RNA editing, gnetophytes) Copyright 2011, Sunny Sheliese Crawley Rethinking phylogenetics using Caryophyllales (angiosperms), matK gene and trnK intron as experimental platform Sunny Sheliese Crawley ABSTRACT The recent call to reconstruct a detailed picture of the tree of life for all organisms has forever changed the field of molecular phylogenetics. Sequencing technology has improved to the point that scientists can now routinely sequence complete plastid/mitochondrial genomes and thus, vast amounts of data can be used to reconstruct phylogenies. These data are accumulating in DNA sequence repositories, such as GenBank, where everyone can benefit from the vast growth of information. The trend of generating genomic-region rich datasets has far outpaced the expasion of datasets by sampling a broader array of taxa. We show here that expanding a dataset both by increasing genomic regions and species sampled using GenBank data, despite the inherent missing DNA that comes with GenBank data, can provide a robust phylogeny for the plant order Caryophyllales (angiosperms). We also investigate the utility of trnK intron in phylogeny reconstruction at relativley deep evolutionary history (the caryophyllid order) by comparing it with rapidly evolving matK. We show that trnK intron is comparable to matK in terms of the proportion of variable sites, parsimony informative sites, the distribution of those sites among rate classes, and phylogenetic informativness across the history of the order. This is especailly useful since trnK intron is often sequenced concurrently with matK which saves on time and resources by increasing the phylogenetic utility of a single genomic region (rapidly evolving matK/trnK). Finally, we show that the inclusion of RNA edited sites in datasets for phylogeny reconstruction did not appear to impact resolution or support in the Gnetales indicating that edited sites in such low proportions do not need to be a consideration when building datasets. We also propose an alternate start codon for matK in Ephedra based on the presense of a 38 base pair indel in several species that otherwise result in pre-mature stop codons, and present 20 RNA edited sites in two Zamiaceae and three Pinaceae species. iii Acknowledgements I would like to give my most sincere thanks to Dr. Hilu for his mentorship during the course of my graduate studies. He has always been supportive and encouraging and has provided me with so many wonderful opportunities both in the lab and through numerous meetings throughout the years. Thank you for encouraging me to take a semester off to teach Plant Taxonomy and helping that endeavor to be a success. I have enjoy debating our work together and look forward to continuing collaborations. I also thank each member of my committee, Dr. Jill Sible for always listening and providing encouraging feedback along with sage scientific advice, Dr. Carla Finkielstein for all her help with the expression work and always encouraging me to do my best, and Dr. Eric Beers for teaching me plant biology and asking critical questions to help me keep on track. I would also like to thank everyone that has come and gone through the Hilu lab during my tenure. Thanks to Dr. Michelle Barthet-Parker for laying the groundwork of a wonderful and exciting project and setting a great example of how to succeed in graduate school. Thanks to Dr. Sheena Friend-Elliot for taking me under your wing when I first arrived, for getting through all of our classes together both scientific and pedagogical, for troubleshooting the research, for being my traveling companion to the various meetings, for being a wonderful friend and so much more. To Stephanie Voshell for your undying enthusiasm for research and doing the last minute leg-work in my place. To Atia for always having a smile on your face and a joke to share (chicken cookie?). Thanks to Chelsea Black for being a great friend and our PCR miracles. Thank you to the many undergraduate students that have helped with the research through the years, specifically Shelli Newman my “sister” in science, Adrianna Feriolli and the countless hours of PCR and tree building, Dipan Oza my bioinformatics buddy, Emily Steele and the mega-PCR sessions, Keenan Moukarzel for figuring out new programs with me, and Brittany for taking on the yeast expression project. iv Thanks to all the other Derring Hall 5th floor graduate students over the years, Gana, Chen, Catherine, Justin, Nassiba, Shakur, Sarah, and everyone else for your friendship and support. Thanks to all those that have helped me to be a better teacher, specifically Shelli Fowler, Karen DePauw, Art Buikema, Mary Schaeffer, Catherine Sarmadi, Sharon Sible, Chiquita Thomas, Carla Tyler, and Jason Yolitz for teaching me about pedagogy and providing feedback on my teaching through the years. Thank you to my parents Thomas and Laurel Drysdale and my wonderful in-laws Patti and Joe Crawley for their example, support, and encouragement. Thanks to my husband Adam Crawley for enduring the countless hours spent on school work instead of playing and our son Marcus for tolerating my tired days. v Table of Contents ABSTRACT ........................................................................................................................ ii Acknowledgements ............................................................................................................ iv Table of Contents ............................................................................................................... vi List of Figures .................................................................................................................. viii List of Tables ...................................................................................................................... x Chapter 1: Introduction ................................................................................................... 1 Objectives: .................................................................................................................................... 1 References: ................................................................................................................................... 7 Chapter 2: Impact of missing data, gene choice and taxon sampling on phylogenetic reconstruction: the Caryophyllales (angiosperms) ...................................................... 16 Abstract ...................................................................................................................................... 16 Introduction ................................................................................................................................ 16 Materials and Methods ............................................................................................................... 20 Results ........................................................................................................................................ 25 Discussion .................................................................................................................................. 35 Conclusion .................................................................................................................................. 45 Acknowledgments ...................................................................................................................... 46 References: ................................................................................................................................. 46 Chapter 3: Caryophyllales: Evaluating phylogenetic signal in matK and trnK intron ........................................................................................................................................... 57 Abstract ...................................................................................................................................... 57 Introduction ................................................................................................................................ 58 Materials and Methods ............................................................................................................... 60 Results ........................................................................................................................................ 63 Discussion .................................................................................................................................. 76 Conclusion .................................................................................................................................. 87 Acknowledgements .................................................................................................................... 87 References: ................................................................................................................................. 88 Chapter 4: RNA editing of matK in the Gnetales? ....................................................... 95 Abstract