Molecular Ecology of the Kentish Plover Charadrius alexandrinus CLEMENS KÜPPER A thesis submitted for the degree of Doctor of Philosophy University of Bath Department of Biology & Biochemistry August 2008 COPYRIGHT: Attention is drawn to the fact that copyright of this thesis rests with its author. A copy of this thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and they must not copy it or use material from it except as permitted by law or with the consent of the author. This thesis may be made available for consultation within the University Library and may be photocopied or lent to other libraries for the purposes of consultation. Contents 3 Acknowledgements 4 Summary 5 Chapter I: Introduction 17 Chapter II: Characterisation of 36 polymorphic microsatellite loci in the Kentish plover (Charadrius alexandrinus) including two sex-linked loci and their amplification in four other Charadrius species 18 Chapter III: Enhanced cross-species utility of conserved microsatellite markers in shorebirds 65 Chapter III: Supplementary material 72 Chapter IV: Kentish versus snowy plover: Phenotypic and genetic analyses of Charadrius alexandrinus reveal divergence of Eurasian and American subspecies 108 Chapter V: Overdominance and underdominance of conserved microsatellite markers are associated with offspring survival in Kentish plover Charadrius alexandrinus 136 Chapter V: Supplementary material 164 Chapter VI: Conclusions 174 Appendix I: Breeding ecology of Kentish plover Charadrius alexandrinus in an extremely hot environment 192 Appendix II: Parental cooperation in an extreme hot environment: incubation behaviour in Kentish plover Charadrius alexandrinus 212 Appendix III: Sexual conflict over parental care: a case study of shorebirds 213 Appendix IV: Practical guide for investigating breeding ecology of Kentish plover Charadrius alexandrinus Acknowledgements The research for my PhD has been the team effort for the last years. I benefited from being supported by genuinely great people who helped me achieving my tasks. Special thanks to Tamás Székely for his truly inspirational supervision and guidance. Our work relationship has been an evolutionary process itself, definitively never boring. You pushed me further than I sometimes wanted to go and yet, gave me time and space to explore the questions I wanted to explore. I’d also thank the following people who have contributed to this work or (sort of) sane and kept my spirit up during this time in one way or another. Monif Al-Rashidi, Araceli Argüelles-Tico, Jakob Augustin, Donald Blomqvist, Marcos Bucio-Pacheco, Terry Burke, Fiona Burns, Debs Dawson, Martin Deschauer, Jordi Figuerola, Richard ffrench-Constant, Gabriel Garcia-Peña, Wolfgang and Sabine Gfreiner (and family), Martin Gottsacker, Father Bill McCoughlin, Freya Harrison, Gavin Horsburgh, Richard James, Salim Javed, Tim Karr, János Kis, Andy Krupa, Andras Kosztolányi, Bernadett Küpper, Gabriele and Siegfried Küpper, Miriam, Johanna, Josef, Magdalena, Samuel, Daniel and Claudia Küpper, Peter Long, Medardo Lopez-Cruz, Lydia Lozano-Angulo, Maria Elena Maranelli, Erick Gonzalez-Medina (and family), Marc O’Connell, Rotary Club La Cruz de Elota, Klaus Reinhardt, Alejandro Serrano-Meneses, James St Clair, Ian Stewart, John Strassmeyer, René van Dijk, Thijs van Overveldt, Matthew Wills, Xico Vega-Pico, Tuluhan Yilmaz, Richard Young, Sama Zefania Each of you would deserve his own chapter in this thesis; I don’t know where I would be without you. Going to tropical beaches and study beautiful birds – what can I ask more for?! I thank God every day for my privileged life. 3 Summary Molecular ecology has already provided profound insights into behaviour, ecology and systematics of organisms improving our understanding of the relationship between genetic variation and biodiversity. The objectives of my PhD were to develop new genetic markers and use these markers to address fundamental issues in evolutionary biology using shorebirds as model organisms. Shorebirds are part of the ancient avian Charadriiformes order and are characterised by extraordinary ecological and behavioural diversity. However, due to the lack of appropriate genetic markers the molecular ecology of many shorebirds has not been investigated previously. Therefore, first, I developed polymorphic microsatellite markers from genomic libraries for a behaviourally diverse shorebird, the Kentish plover Charadrius alexandrinus (Chapter II). Second, using the genomic data-bases I expended this work to develop further markers that cannot only be used in the Kentish plover, but also a large number of other shorebird species (Chapter III). Third, I investigated population differentiation and genetic diversity of Eurasian and American Kentish plover populations using the newly developed microsatellite markers and further mitochondrial markers (Chapter IV). The genetic differences between Eurasian and American populations that are mirrored by phenotypic differences call for a reconsideration of the current taxonomic status of the species; Eurasian and American populations should be recognised as belonging to two separate species. Finally, I asked how genetic diversity influences the fitness of precocial Kentish plover young (Chapter V). I found that survival of chicks until fledging was associated with genetic diversity (measured as heterozygosity) at three of eleven marker loci. Genetic diversity at one marker locus had a positive effect on survival whilst it had negative effects at two loci. The results of my PhD have brought up many new questions and I propose promising lines that need to be explored in the future (Chapter VI). 4 Chapter I Introduction 5 Background Molecular ecology, the application of molecular genetic methods to problems in ecology, is a broad but still young scientific discipline (Beebee & Rowe 2004). Most research in molecular ecology is centred around the molecule of life, deoxyribonucleic acid (DNA), attempting to link its variation with variation in ecology, behaviour and evolutionary history of individuals, populations and species. Once DNA sequences are known, ecologists and evolutionary biologists can answer many questions for which morphological, behavioural or ecological data cannot provide complete answers. Molecular ecology is bringing new insights into systematics, improving our understanding of the tree of life and how the present species phylogenies have evolved. It provides the tools for examining the coherence of populations, gene-flow between fragmented demes, and enables conservation biologists to identify genetic threats and model the survival prospects of populations. Molecular genetic tools have helped researchers to quantify reproductive success of individuals, reconstruct pedigrees and identify beneficial alleles and/or allele combinations to understand the interplay of natural and sexual selection. Its findings enhance our understanding of biodiversity and can help to conserve and protect it (Karp et al. 1997, Frankham et al. 2002, DeSalle & Amato 2004). In molecular ecology various genetic markers are employed (Beebee & Rowe 2004). Popular markers currently used include allozymes, protein variants of a gene that can be separated by gel electrophoresis, mitochondrial DNA markers, haploid markers with intermediate variability that are inherited exclusively through maternal lineages, amplified fragment length polymorphism (AFLP) markers, restriction-digested DNA annealed to specific linkers which results in specific amplification band patterns, single- nucleotide polymorphism markers, homologous DNA fragments that differ in their sequence in a single base, minisatellites, 10-60 bp repeated long sequence motifs and microsatellites, 1-6 bp frequently repeated highly variable short sequences. Each marker has its strengths and limitations. To answer different questions the variability of different markers is exploited. Many markers were initially considered to be selectively neutral, although this assumption turned out to be often incorrect. For example, many microsatellites which are usually considered to be selectively neutral have been found to be located in expressed genes or in DNA regions with regulatory functions where their variability can modify functionality (Li et al. 2002, 2004). 6 Microsatellites In my PhD I focussed on the application of microsatellite markers in shorebirds (plovers, sandpipers and allies, see below). Microsatellites (or simple sequence repeats) are the most versatile markers currently used in molecular ecology and can be applied to identify individuals, populations and species. Microsatellites are often found in non- coding regions of eukaryotic genomes (Ellegren 2000, Li et al. 2002). Variability is introduced by mutations and microsatellites exhibit high mutation rates of 10-3 – 10-7 allele changes per generation. However, these mutation rates differ between species and loci (Ellegren 2000, Buschiazzo & Gemmell 2006). The unique mutation process of a microsatellite involves changes of the repeat number by replication slippage or interchromosomal exchange (Buschiazzo & Gemmell 2006). Replication slippage, considered by most authors as the main mutation process, occurs when DNA strands are not properly aligned during the replication process. A new repeat is either added (when a loop forms in the newly synthesised strand), or deleted (when loops form in the existing template strands, Ellegren 2000, Buschiazzo & Gemmell 2006).