RECONSTRUCTING THE MOLECULAR PHYLOGENY OF GIANT SENGIS (GENUS RHYNCHOCYON) A Thesis submitted to the faculty of A6 San Francisco State University 3(? In partial fulfillment of zo\5 the requirements for the Degree Ib'oL Master of Science In Biology: Ecology, Evolution, and Conservation Biology by Elizabeth Jane Carlen San Francisco, California August 2015 Copyright by Elizabeth Jane Carlen 2015 CERTIFICATION OF APPROVAL I certify that I have read Reconstructing the Molecular Phylogeny o f Giant Sengis (genus Rhynchocyon) by Elizabeth Jane Carlen, and that in my opinion this work meets the criteria for approving a thesis submitted in partial fulfillment of the requirement for the degree Master of Science in Biology: Ecology, Evolution, and Conservation Biology at San Francisco State University. Research Fellow California Academy of Sciences RECONSTRUCTING THE MOLECULAR PHYLOGENY OF GIANT SENGIS (GENUS RHYNCHOCYON) Elizabeth Jane Carlen San Francisco, California 2015 Giant sengis (genus Rhynchocyon), also known as giant elephant-shrews, are approximately 500 g forest floor mammals that range from Central to East Africa. Previous work on giant sengi taxonomy has focused primarily on pelage color, pelage pattern, and the geographic distributions of the groups. Because there is complex phenotypic variation and large geographic ranges within some species, I chose to use genetic work to evaluate the phylogeny and classification of the genus. Genetic data were used to investigate the four currently recognized species (R. chrysopygus, R. cirnei, R. petersi, and R. udzungwensis) and seven of the eight currently recognized subspecies (R. cirnei cirnei, R. cirnei macrurus, R. cirnei reichardi, R. cirnei shirensis, R. cirnei stuhlmanni, R. p. petersi, and R. p. adersi). I used DNA extracted from fresh and historical museum samples to analyze approximately 4,700 nucleotides (2,685 bases of mitochondrial DNA and 2,019 bases of nuclear DNA) and reconstruct a molecular phylogeny. I also investigated and genetically confirmed the identity of Rhynchocyon sp. sequences published on GenBank, and suggest that the captive Rhynchocyon populations of North American zoos are R. p. adersi. My analyses confirm the current morphological classification, with each currently recognized species forming a monophyletic clade. My phylogeny suggests that hybridization among taxa is not widespread in Rhynchocyon, that the recently reported sengi from the Boni forest of Northern Kenya is genetically similar to R. chrysopygus, and that the subspecies R. c. stuhlmanni should be elevated to full species. I certify that the Abstract is a correct representation of the content of this thesis. ACKNOWLEDGEMENTS I would like to thank my thesis committee J. Dumbacher, G. Rathbun, and D. Blackburn for their support and feedback. This work was financially supported by the California Academy of Sciences, the Biology Department at San Francisco State University, the Graduate Student Council in Biology at San Francisco State University, the Society for the Study of Evolution, and the Society for Integrative and Comparative Biology. G. Rathbun particularly helped with facilitating the inclusion of the Boni Rhynchocyon. B.R. Agwanda of the National Museums of Kenya captured and prepared the Boni Rhynchocyon. S. Adanje of the Kenya Wildlife Service personally imported the Boni Rhynchocyon tissue into the United States of America. S. Musila of the National Museums of Kenya encouraged the inclusion of the Boni Rhynchocyon in this analysis. C. Sabuni collected several tissues of R. p. petersi for this project while working on his dissertation. K. Consolate collected tissues of R. c. stuhlmanni. W. Stanley at the Field Museum of Natural History, N. Duncan at the American Museum of Natural History, and J. Chupasko at the Museum of Comparative Zoology provided museum samples for this project. F. Catzeflis, L. Herwig, G. Rathbun, and J. Dumbacher helped determine the vouchers for the Douady et al. (2003) specimen. W. Wendelen provided a photograph of specimens from within the R. c. macrurus cline, including specimens collected in Chingulungulu, Tanzania (Douady et al. 2003). M. Omura provided photographs of MCZ43732. K. Lengel and S. Eller, at the Philadelphia Zoo, and P. Riger, at the Houston Zoo, helped track down information about the Rhynchocyon zoo specimens. K. Hildebrandt at the Museum of the North, University of Alaska, Fairbanks, A. Sellas at the California Academy of Sciences, and O. Carmi at the California Academy of Sciences helped teach me laboratory techniques. L. E. Olson provided financial support to travel to the University of Alaska Fairbanks to repeat DNA extraction and amplification in an alternate ancient DNA lab (NSF DEB-1 120904 to LEO). R. Bell and E. Stanley helped with phylogenetic analysis. M. Bernal helped with PopART analysis. W. B. Simison provided guidance throughout this project and endless moral support. Finally, I would like to thank my parents who have always supported my decisions. To everyone that supported this project and me, “thank you.” TABLE OF CONTENTS List of Tables.............................................................................................:................................... ix List of Figures..................................................................................................................................x List of Appendices.........................................................................................................................xi Introduction.......................................................................................................................................1 Methods.............................................................................................................................................8 Specimens............................................................ 8 Laboratory Methods...........................................................................................................8 Alignment and Analysis.................................................................................................. 12 Results............................................................................................................................................. 14 Discussion.......................................................................................................................................17 Clarifying the Taxonomic Status of Current Sequences............................................18 Origins of Captive Populations......................................................................................23 Species Diagnosis............................................................................................................25 Current Taxonomic Status of Rhyne hocyon............................................................... 27 Conclusions....................................................................................................................................30 References....................................................................................................................... 49 Appendices..................................................................................................................................... 60 LIST OF TABLES Table Page 1. Data for specimens used for DNA sequencing...................................................................33 2. Primers used for DNA amplification and sequencing................................................35 3. Best fit models for loci sequenced................................................................................ 38 4. Distance matrix for comparing Smit et al. (2011) sequences....................................39 5. Distance matrix for 12s 16s mitochondrial sequences................................................40 ix LIST OF FIGURES Figures Page 1. Rhynchocyon cirnei subspecies ranges..........................................................................41 2. Geographic range of the genus Rhynchocyon..............................................................42 3. MrBayes phylogram of Rhynchocyon 12s 16s mitochondrial region....................... 43 4. TCS allelic networks for Rhynchocyon nuclear loci IRBP and vWF.......................44 5. Nucleotide alignment of Smit et al. (2011) sequences and primers.........................45 6. Nucleotide alignment of Smit et al. (2011) sequences and Homo sapiens.............46 7. MrBayes cladogram for Rhynchocyon 12s 16s mitochondrial region......................47 8. Type locality for Rhynchocyon cirnei hendersoni......................................!............... 48 x LIST OF APPENDICES Appendix Page 1. Rhynchocyon color plates...............................................................................................60 a. Rhynchocyon chrysopygus................................................................................60 b. Rhynchocyon cirnei cirnei............................................................................... 61 c. Rhynchocyon cirnei macrurus.........................................................................62 d. Rhynchocyon cirnei reichardi..........................................................................63 e. Rhynchocyon cirnei shirensis..........................................................................64 f. Rhynchocyon cirnei stuhlmanni...................................................................... 65 g. Rhynchocyon petersi petersi.............................................................................66