The Use of Microsatellites in the Study of Social Structure in Large Mammals: Italian Wolf and Fallow Deer As Case Studies
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The use of microsatellites in the study of social structure in large mammals: Italian wolf and fallow deer as case studies Massimo Scandura PhD Thesis University of Bielefeld, Faculty of Biology Juli 2004 Supervisors: Prof. Dr. Fritz Trillmich - Lehrstuhl für Verhaltensforschung - Universität Bielefeld Prof. Marco Apollonio – Dip. di Zoologia e Antropologia Biologica - Università di Sassari, Italy to Elisa - Table of Contents - Page 1 Summary 2 Acknowledgements 3 Thesis layout 4 Chapter 1 5 - 1.1 Molecular markers: an overview 9 - 1.2 Microsatellites: molecular structure, putative function, and the origin of polymorphism 11 - 1.3 Mutation models and the theory of microsatellite evolution 14 - 1.4 From theory to facts: possible applications 16 - 1.5 Microsatellites to investigate the structure of natural populations 21 Chapter 2 Case 1: Wolf population structure and social organization 22 - 2.1 Aim of the study 23 - 2.2 The biology of the species: an overview 33 - 2.3 Study area 38 - 2.4 Methods 53 - 2.5 Results 69 - 2.6 Discussion 76 Chapter 3 Case 2: Relatedness and heterozygosity in a lekking fallow deer population 77 3.1 Aim of the study 78 3.2 The biology of the species: an overview 90 3.3 Study population 93 3.4 Methods 98 3.5 Results 106 3.6 Discussion 113 Chapter 4 Conclusions 115 References 1 - Summary - The present thesis deals with the application of microsatellite analysis to the study of two species of large mammals, referring to some aspects of their social and mating systems. The Italian wolf (Canis lupus) and the fallow deer (Dama dama) were chosen as case studies, since genetic investigations on their populations result, for different reasons, problematic. The wolf in Italy is a particularly protected species, recovering throughout the peninsula from the effects of a recent bottleneck. Sampling wolves may not rely on capturing or killing them, therefore an alternative, non-invasive, approach was adopted in my study. Scats, shed hairs and blood drops collected on the snow represented the main source of DNA for the analysis. Methodological cares were necessary to obtain reliable wolf genotypes. A set of ten canine microsatellites was employed to achieve unique multilocus genotypes in the population. Fifty two individuals were typed in the period 1998-2003. In some cases, pack composition was determined, confirming that familiar bonds are at the basis of wolf social units. An unpredictable high local differentiation was found among geographic areas. Early dispersal seem to be common in the population, but its effects on the gene flow are not relevant, at least at the study scale. I proposed that most of this dispersal may be unsuccessful or over long distances. The study population, indeed, seem to have reached a high level of local saturation, with clumped pack territories and high reproductive rates, and thus possibly represents a source, from which wolves disperse toward sink areas. The second study involves an enclosed population of fallow deer. Lekking is seldom observed in mammals, and among them, it is more common in ungulate species. Fallow deer is one of the most studied lekking ungulate and the study population has been object of long-term researches on male mating strategies. Mature bucks in the population join into leks during the breeding season: a costly strategy, which apparently does not guarantee high direct benefits (fitness). In this study, for the first time, I used a molecular approach to verify the existence of a genetic basis to lek formation. The recourse to microsatellites enabled to overcome the strong limitation due to the extremely monomorphism of the species, documented in several studies. Though the low variability even at microsatellite loci, the hypotheses of kin selection (territorial bucks in a lek are relatives) and of heterozygosity advantage (territorial bucks in a lek have a high overall heterozygosity) were tested and resulted not supported by data. Hence, future studies should be addressed towards phenotypic variation and consider in detail physiological and ecological factors, in order to clarify the reasons why lekking takes place in fallow deer. 2 - Acknowledgements - This study would not have been possible without the will and the kindness of my supervisors Prof. Fritz Trillmich and Prof. Marco Apollonio, and without the work and the contribution of a number of people. Both studies (on wolf and fallow deer) were based on the combination of genetic and field data. The formers represent my exclusive contribution, whereas behavioural and census data, as well as sample collection, were the result of an extensive work carried out in the last five years by people participating to the two ongoing projects. Specifically, for the collection of samples and field data in the wolf study, I am enormously grateful to Luca Mattioli, Claudia Capitani, Lorenza Mauri, Andrea Gazzola, Elisa Avanzinelli, Paolo Lamberti, Alessia Viviani, Daniela Giustini, Francesca Benvenuti, Andrea Vanni, Sabrina Marsili, Mirko Geri, the provincial guards and all people participating to summer (wolf-howling) and winter (snow-tracking) activities or providing information on the location of wolf packs. Thanks also to Serena Cappelli and Daniela Del Chiaro for their help in laboratory routine. I would like to acknowledge the Provincial Administration of Arezzo, and particularly Gabriele Chianucci, for the financial and logistic support. I am also grateful to the Administration ex-ASFD of Pratovecchio for providing facilities during the study. In the fallow deer study, genetic data were compared to observational data collected at the two leks, during several breeding seasons. With regard to this effort, I express my profound gratitude to Simone Ciuti, Siriano Luccarini, Irene Di Vittorio, Sara Luchetti, Sara Davini, Giuseppe Caleo, Paolo Bongi and many students and volunteers contributing to observation sessions at leks. Concerning sample collection, to the mentioned people I should add my acknowledgements to the warders of the San Rossore estate for their contribution in catching and manipulating animals. I also thank Dave Coltman, Morris Gosling and Kate Byrne for having provided help in testing microsatellite loci, giving access to facilities and unpublished information. For the laboratory activities, I am debtor to Francesca Di Benedetto for her great help during all phases of the study, and to Elena Rossi, a precious help in the management of all administrative jobs. Last but not least, I thank my wife, Tiziana, for the continuous motivation and support provided during last years. The study was financed by the Accademia Nazionale dei Lincei, by the Provincial Administration of Arezzo, and by the Ministry of University and Scientific and Technological Research. 3 - Thesis layout - This thesis is aimed to verify the application of microsatellite analysis to the knowledge of social and mating systems. The manuscript consists of a first chapter describing the nature of microsatellites as molecular markers, their structure and evolution. Advantages and drawbacks in the employment of these DNA regions are also described, with a particular stress to the statistical viewpoint. A further subject concerns possible applications in the study of the biology of wild mammals. The second chapter takes into account the first study case. A set of polymorphic microsatellite loci was used to investigate the structure and the social behaviour of a wolf population. After a review of the known aspects of wolf biology, attention is paid to the local features of the study population, making collecting samples from free ranging individuals difficult. Thus, the adopted non-invasive sampling design is described and results are discussed. Relationships among packs are considered, pointing out the local genetic differentiation over a small geographic scale, due to constraints to gene flow. A second study case is described in chapter 3. The studied species is in this case fallow deer. Dama dama is one of the most variable ungulate species, respect to mating behaviour. The evolution of lekking behaviour in fallow deer is of special interest, because of the possibility the males have to choose, as alternative, every other known mating strategy. The use of highly polymorphic molecular markers, in a genetically impoverished species, enables to collect information to confirm or reject the hypotheses of kin selection and heterozygote preference, verified in other species. Evidences from microsatellites are presented and results discussed. In chapter 4, some conclusions are reported, focusing on the main results of the two studies and highlighting the boost genetic investigations may give to the knowledge of inter-individual relationships within wild populations of large mammals. 4 - Chapter 1 - Introduction 5 1.1 Molecular markers: an overview From the beginning of the ‘DNA era’, the attention of many scientists was addressed to the discovery of molecular tools to employ in several fields going from human health to farming improvement, from genome mapping to the conservation of biological diversity. Since then, different generations of molecular markers were discovered and used for a high number of aims. The intrinsic requirement making each of them useful for several purposes is a sufficient level of variation. In other words, for a given molecular marker is necessary to show among taxa, populations or individuals, at least some detectable difference. The origin of diversity is mostly due to mutation, occurring at different rate in