University of Alberta Genetic Studies of North American Wapiti Subspecies Renee Otti Polziehn 0 A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy Depatment of Biological Sciences Edmonton, Alberta Fall 2000 NationaI Library Bibliothèque nationale I*I of Canada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395. rue Wellington Ottawa ON K1A ON4 Ottawa ON K1A ON4 Canada Canada Your lF& Votre reference Our fiie Narre reference The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sel1 reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la fome de rnicrofiche/filrn, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or othenvise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. Abstract Many species have seen changes to their classification since molecular information was made available. In this study, both mitochondnal (control region) and nuclear (microsatellite) loci were employed to support the divisions among subspecies of North Amencan wapiti (Cerwrts elaplzris ssp). The genetic markers were chosen on the basis that their higher rate of mutation would be needed to detect diversity among these highly related organisms. To put the observed genetic diversity arnong North American wapiti into perspective, the genetic differentiation was cornpared at a population, subspecies, and species leveI. Overall, North Amencan wapiti populations express IittIe genetic variation, however isolated populations such as the Tule and Roosevelt wapiti were differentiated at both nuclear and mitochondrial markers. In contrast, the Rocky Mountain and Manitoban wapiti did not receive support for their subspecies designation from either set of markers. The translocation and migration of anirnals likely enhanced the similarity of genotypes among the Rocky Mountain and Manitoban wapiti. Their similar distribution of microsatellite genotypes and mitochondrial haplotypes suggests the separation among North American subspecies was recent. When the cornparison of mitochondrial DNA was extended to include Asian wapiti and European red deer, a division of haplotypes was detected along the Gobi Desert and Hirnalayan Mountains. The division of mitochondrial haplotypes corroborates the classification that recognized red deer as Cervrcs elaphus and wapiti as C. canadensis. Acknowledgements First and foremost, 1need to thank my supervisor, Curtis Strobeck for providing me with the opportunity to fulfill my goals to cornplete a doctoral thesis. I am gateful for his openness to explore new techniques and to ask new questions. None of this work would have been possible without the help of many people who collected sarnples. Therefore 1 need to thank the numerous anonymous park wardens frorn Elk Island, Banff, Jasper, Kootenay, Riding Mountain and Yoho National Parks for sending samples to the repository at the University of Alberta. Additional samples from North Amenca animals were provided by Didi Hawk (Wyoming Fish and Wildlife), Frank Mallory (Laurentian University), Joe Ham (Carnbrian College), Bob McClymont (Alberta Fish & Wildlife), Tom Packer (Alberta Fish & Wildlife) and Steven Fain (Forensic Laboratory of the United States Fish and Wildlife). Cornparisons with Asian subspecies would not have been possible without the help of Yan Lu (Beijing Zoo), Jianjun Peng (Institute of Zoology, Chinese Academy of Sciences, Beijing, China), Yegus Cheng, Jon Winters (Purdue University), Ray Ference (Elk Valley Processors), Carmi Penny and staff of the San Diego Zoo. Funding for this research was supplied by a service contract with Parks Canada and NSERC grant. 1 wouId Iike to thank Nusha Keyghobadi, Michael Proctor, Greg Wilson, Corey Davis, Ken Zitlau, and Chris KyIe, who have helped me with the various revisions of this thesis, and hopefully it is service that 1may reciprocate. 1 would also like to thank my examiners and cornmittee members, Andrew Beckenbach, Bob Hudson, Sean Graham, and Allan Good for their final comments. Without the contributions of John Coffin, David Paetkau, and Isabelle Delisle, 1am sure my work in lab would also have gone much slower. Finally, 1 am especially grateful to Roger Marchand and my family for their support and to rny colleges that have made me laugh. Table of Contents 1 . General Introduction ....................................................................... 1 Defining a species and subspecies ............................................... 1 Genetics in general ..................................................................3 Information gained from mitochondnal DNA .............................. -4 Wapiti as the species of interest .............................. .. .................. 5 Taxonomy of Cemus elaplzus ................................................... 6 Genetic cornparisons among Centus elaphus ................................. -7 Objectives of project .............................................................. 8 Gerzetic variation within szlbspecies ..................................... 8 Genetic variation anzong subspecies ..................................... 9 References .......................................................................... 9 2. Phylogenetic Status of North American Wapiti Subspecies (Cervus elaphts spp) .................................................................. 13 Introduction ........................................................................ 13 Materials and Methods ............................................................ 18 Collection ................................................................... 18 Isolntiorz arzd ar?zpZij7catiorr (polyiirerase clzain reaction) ............ 19 Phylogerzetic analysis ...................................................... 20 Divergence ........ .. ........................................................ 20 Results .............................................................................. 20 Discussion ......................................................................... 28 References ........................................................................ -31 3 . Microsatellite analysis of North American wapiti (Cervrts elaplrrcs) populations ........................................................ 34 Introduction ........................................................................ 34 Materials and Methods ............................................................ 38 Sanzple collecrio~z.......................................................... 38 A ~~iplzficarioizof nzicrosarellite loci ...................................... 3 8 Statisrical rnethods ......................................................... 39 ResuIts .............................................................................. 41 Measru-es of genetic divers@ ............................................ -41 Relatio~zshipsanlong popitlations ........................................ 42 Discussion ........................................................................ -47 Summary ................................................................................ lû4 Mitochondrïal and microsatellite DNA ........................................ 104 Classification ..................................................................... 105 Pop~datioizlevel ............................................................ 105 Sttbspecies level ........................................................... 106 Species levez ................................................................ 107 Future ............................................................................. -108 References ........................................................................ -109 6. Addendum .Comparative sequences from the SRY gene from North American ungulates .......................................................... 111 Introduction ....................................................................... 111 Materials and Methods ........................................................... 112 Results ............................................................................ -113 Discussion ........................................................................ 116 References ......................................................................... 117 List of Tables Table 2.1 . Primers employed in the sequencing of the control region .................... 19 Table 2.2 . Control region sequences .......................................................... 21 Table 2.3 . Nucleotide substitutions in the control region .................................. 23 Table 2.4 . Divergence of mtDNA d-loop sequences ........................................ 25 Table 3- 1. Measures of genetic diversity among wapiti populations .................... 41 Table 3.2 . Genetic distances among wapiti populations .................................. 43 Table 3.3 . The numbers