Ecological and Evolutionary Genetics of Puffinus spp. Jeremy James Austin, BSc. (Hons.) Submitted in fulfllment of the requirements for the degree of Doctor of Philosophy, University of Tasmania (October, 1994) Statements I declare that this thesis contains no material which has been accepted for the award of any other degree or diploma in any tertiary institution and, to the best of my knowledge and belief, this thesis contains no material previously published or writtenby another person, except where due reference is made in the text This thesis is not to be made available for loan or copying for two years following the date this statement is signed. Following that time the thesis may be made available for loan and limited copying in accordance with the Copyright Act 1968 Signed f!td4- . This thesis may be made available for loan. Copying of any part of this thesis is prohibited for two years from the date this statement was signed� after that time limited copying is permitted in accordance with the Copyright Acy 1968. Signed Date Summary distinct molecular genetic techniques were applied at different levels in the evolutionary hierarchy to investigate the reproductive ecology, population biology and systematics of Three species in the shearwater genus Puffinus, with particular emphasis on the short-tailed shearwater, P. tenuirostris, or Tasmanian muttonbird. Genetic relationships between mated pairs of adult short-tailed shearwaters and the single offspring in the nest were analysed by multilocus DNA fingerprinting. The human polycore minisatellite probe, 33.6, revealed sufficient variation in shearwater DNA to allow individual­ specific identification. In addition this probe hybridised to a large minisatellite restriction­ fragment derived from the female W chromosome, which allowed the identification of sex of adults and nestlings in this sexually monomorphic species. Analysis of DNA fingerprint profiles from 107 nestlings and one or both of the attendant adults in each case, in two independent studies, revealed 13 cases where a nestling was not related to one of the attendant adults. Although four of these unrelated adults could be accounted for by sampling errors, the remaining nine cases all involved the male in each nest and were more likely to have resulted from extra-pair copulations involving the attendant female and an unknown, extra-pair male. These results suggest that although short-tailed shearwaters exhibit strong pair fidelity and social monogamy, some birds are engaging in an alternative mating strategy that may substantially enhance both male and female reproductive success. Future estimates of life-time reproductive success in this species will have to allow for the small percentage of paired males that are unrelated to the nestlings that they are providing care for. Restriction enzyme analysis of mitochondrial DNA (mtDNA) was used to examine mtDK-\ variation among 335 short-tailed shearwaters from 11 breeding colonies across southeastern Australia, and assess population genetic structure as a genetic test of the observed stri..:t breeding and natal philopatry exhibited by this species. Eleven 6/5.33-base and four 4-basc restriction enzymes revealed 25 and 48 mtDNA haplotypes in two overlapping surveys 215 individuals from seven colonies and 231 individuals from eight colonies, respectively·. of low mean sequence diversity among individuals (0.247%) and lack of spatial structuring :\. mtDNA haplotypes suggests a lack of population genetic structure and a reduced ance:sr.:-.:.1 :f 11 Summary population size during the Pleistocene glaciation, followed by a population and range expansion to current levels. Intracolony mtDNA diversities in three recently established colonies, and in one colony that has experienced a recent bottleneck were comparable to mtDNA diversities within larger and older colonies. This suggests that, despite strict philopatry in those colonies, colony founding and recovery from population reduction occurs via immigration of a large number of individuals. Phylogenetic relationships among 19 extant species and subspecies within the genus Puffinuswere examined using partial sequences from the mitochondrial cytochrome b gene. Nucleotide variation in a 307 bp fragment of this gene was sufficient to distinguish all taxa, except in one case, and contained phylogenetic information resolve both shallow and deep phylogenetic relationships. Phylogenetic analysis of these sequences revealed a deep to phylogenetic split amongst Puffinus species with one clade containing the larger, less aquatic and highly migratory Southern Hemisphere species and the second the smaller, more aquatic, less migratory and more northerly distributed forms. Within each clade, several currently recognised taxonomic subgroups were resolved, which have evolved via a polytomous or rapid series of speciations from a Southern Hemisphere or North Atlantic ancestor. Secondary dispersal has seen representatives of the second clade distributed widely throughout the major ocean basins. The phylogenetic hypothesis based on molecular data is generally concordant with trees based on morphological characters. Lack of congruence between the morphological and molecular trees and unexpected phylogenetic relationships among taxa were explained by introgressive hybridisation between two taxa or lineage sorting from a recent common ancestor, an error in one morphological tree, and a more parsimonious interpretation of a prevous evolutionary scenario for the genus. The phylogenetic results suggest a taxonomic revision of the subgroup Neonectris, which currently is a paraphyletic group, and supports previous suggestions that the two Mediterranean subspecies of the shearwater, P. puffinus. yelkouan and P. p. mauretanicus should be elevated to the level of �1a.:1x species and separate from the Atlantic form, P. p. puffinus. ill Acknowledgements I extend my sincere thanks to my supervisor, Robert White, who provided the considerable logistical and academic support and encouragement needed to see this project to a conclusion. Thankyou to Jenny Ovenden for her role in initiating the project and constructive criticism of the population genetic component of the manuscript. I am extremely grateful to Dr David Parkin and the Genetics Department at the University of Nottingham, UK, who generously provided space, facilities and support during my two visits there. Special thanks to Roy Carter, Jamie Darwin, Rob Dawson, Celia May and Jon Wetton for practical assistance with things DNA. all I thank the following people who either assisted with collection of samples, collected samples on my behalf or provided samples from their own collections: Greg Austin; Lisa Ballance (SW Fisheries Science Center, USA); J. A. Bartle and Noel Hyde (National Gallery and Museum, New Zealand); Walter Boles (Australian Museum); Mike Bingham (Falklands Art Conservation, Falklands Islands); Mike Crowley; Charles Daugherty (Victoria University, New Zealand); Rob Dawson (University of Nottingham, UK); Matt Edmunds, Tim Mel Lorkin, Justin Walls (Zoology Department, University of Tasmania); Peter Fullagar Lamb, (CSIRO Wildlife, Australia); John Gerwin and Dave Lee (North Carolina State Museum of Natural Sciences, USA); Juan Salvador Aguilar Gonzalez and Sebastian Pons (Spain); Richard Griffiths (University of Oxford, UK); Brian Bell, M. J. Imber, Ian Millar, Janice Molloy, Richard Parish and G.A. Taylor (Department of Conservation, New Zealand); Cameron Leary and Dean Hiscox (Lord Howe Island Board); Juan Luis Rodriguez Luengo (Viceconsejerfa de Medio Ambiente, Vivero Forestal, Tenerife, Spain); Donna O'Danicl (United States Fish and Wildlife Service, USA); Myriam Preker (Heron Island Research Station, The University of Queensland, Australia); Tineke Prins (Universiteit v:m Amsterdam, Instituut voor Taxonomische ZoOlogie [ZoOlogisch Museum], The Netherl::u'1ds>; Diana Reynolds (Louisiana State University, Museum of Natural Science, USA); Betty Schreiber (Natural History Museum of Los Angeles County, USA); Irynej Skira and �ig::l A..rL-::e Brothers (Parks, Wildlife and Heritage, Tasmania); Thomas Telfer (Hawaii Departme.:Jt Land and Natural Resources, Division of Forestry and Wildlife, Hawaii); Gary .:f lV Voc-lk:r Acknowledgements (University of Washington, Burke Museum, USA); Carol Williams (Binalong Bay Land Care-Ocean Care) andRichard Zotier (France). My thanks to Darren Brasher, Adam Smolenski, Tom Krasnicki and Jon Waters for assistance in the lab. Wayne Kelly, Alan Dumphy, Kit Williams, Ron Mawbey, Richard Holmes, Kate Hamilton, Brenda Bick and Sherrin Bowden all supplied help along the way, for which I am grateful. Special thanks to Barry Rumbold who almost always got things here on time. I am also grateful to the Department of Agricultural Science and the Molecular Biology Unit at the University of Tasmania for allowing me access to their equipment and laboratory space. Special thanks must go to Irynej Skira for his assistance and advice on all things short-tailed, and to Matt Edmunds whose assistance in the field and friendship over the last three years has been greatly appreciated. Thanks Matt. Collections from live birds for these studies were made under permits from the Tasmanian Department of Parks, Wildlife and Heritage; the Queensland National Parks and Wildlife Service; the Victorian Department of Conservation and Environment; the New South Wales National Parks andWildlife Service; and the United States Fisheries and Wildlife Service. work on animals had University