Institute of Zoology University of Veterinary Medicine Hannover Phylogeography and population structure of the European tree frog (Hyla arborea) for supporting effective species conservation THESIS Submitted in partial fulfilment of the requirements for the degree DOCTOR OF PHILOSOPHY (PhD) awarded by the University of Veterinary Medicine Hannover by Astrid Krug Bruchsal, Germany Hannover 2012 Supervisor: Prof. Dr. Heike Pröhl Supervision Group: Prof. Dr. Heike Pröhl PD Dr. Heike Hadrys, Dr. Stefan Könemann (until 09.03.2011) Prof. Dr. Miguel Vences 1st Evaluation: Prof. Dr. Heike Pröhl University of Veterinary Medicine Hannover Institute of Zoology PD Dr. Heike Hadrys University of Veterinary Medicine Hannover Division of Ecology and Evolution Prof. Dr. Miguel Vences Technical University of Braunschweig Division of Evolutionary Biology Zoological Institute 2nd Evaluation: Dr. Robert Jehle University of Salford School of Environment & Life Sciences Ecosystems and Environment Research Centre Date of oral exam: 8th of November 2012 Astrid Krug was sponsored by the Scholarship Programme of the German Federal Environmental Foundation (DBU) # 20007/899. Research funds were provided by the German Federal Environmental Foundation (DBU), Heidehof-Stiftung # 57129.01.2/3.10, and “Hans-Schiemenz-Fonds“ - Deutsche Gesellschaft für Herpetologie und Terrarienkunde (DGHT). Table of Contents Table of Contents Summary..……………………………………………………………………………1 Zusammenfassung……………………………………………………………………3 1 General introduction………………………………………...…………………...…5 1.1 Global amphibian decline……………………………………………………………6 1.2 Conservation genetics………………………………………………………………..6 1.3 The European tree frog………………………………………………………………7 1.3.1 Characteristics……………………………………………………………………….7 1.3.2 Distribution………………………………………………………………………….9 1.3.3 Conservation status and major threats……………………………………………...10 1.3.4 Conservation genetics in the European tree frog…………………………………...11 1.4 Aims of the study…………………………………………………………………...12 1.4.1 Phylogeography in Germany and adjacent areas…………………………………...12 1.4.2 Management Units in Lower Saxony and adjacent areas…………………………..12 2 Defining units for conservation management for the European tree frog (Hyla arborea) in Lower Saxony and adjacent areas……………………………14 2.1 Abstract……………………………………………………………………………..15 2.2 Introduction…………………………………………………………………………16 2.3 Materials and methods……………………………………………………………...17 2.3.1 Sample collection and preparation………………………………………………….17 2.3.2 Statistical analysis…………………………………………………………………..20 2.3.2.1 Historic structure: Analysis of mtDNA……………………………………………..20 2.3.2.2 Recent structure: Analysis of microsatellites……………………………………….21 2.3.2.3 Biogeographic zones………………………………………………………………..22 2.4 Results………………………………………………………………………………23 2.4.1 Mitochondrial sequence analysis…………………………………………………...23 2.4.2 Microsatellite analysis………………………………………………………………24 2.4.3 Biogeographic zones………………………………………………………………..29 2.5 Discussion…………………………………………………………………………..30 2.5.1 Genetic structure and conservation units…………………………………………...31 2.5.2 Genetic diversity……………………………………………………………………33 Table of Contents 2.5.3 Future goals…………………………………………………………………………33 2.5.4 Conclusion and implications for conservation management……………………….34 2.6 Acknowledgement………………………………………………………………….34 3 Phylogeographic structure of the European tree frog (Hyla arborea) in its German distribution area…………………………………………………………36 3.1 Abstract……………………………………………………………………………..37 3.2 Introduction…………………………………………………………………………38 3.3 Material and methods……………………………………………………………….39 3.3.1 Sample collection and preparation………………………………………………….39 3.3.2 Statistical analysis…………………………………………………………………..41 3.3.2.1 Analysis of mtDNA in Germany……………………………………………………41 3.3.2.2 Analysis of microsatellites in Germany…………………………………………….41 3.3.2.3 Analysis of mtDNA in the European context……………………………………….42 3.4 Results………………………………………………………………………………43 3.4.1 Analysis of mtDNA in Germany……………………………………………………43 3.4.2 Analysis of microsatellites in Germany…………………………………………….46 3.4.3 Analysis of mtDNA in the European context……………………………………….49 3.5 Discussion…………………………………………………………………………..51 3.5.1 Distinct genetic lineages in the European tree frog? ……………………………….52 3.5.2 Phylogrographic structures of the tree frog in Germany……………………………52 3.5.3 Genetic diversity……………………………………………………………………53 3.5.4 Conclusion………………………………………………………………………….54 3.6 Acknowledgement………………………………………………………………….54 4 General discussion………………………………………………….……………...56 4.1 Future goals…………………………………………………………………………58 5 References………………………………………………………………………….60 6 Appendix…………………………………………………………………………...70 Affidavit……………………………………………………………………………87 7 Acknowledgment…………………………………………………………………..88 List of Abbreviations List of Abbreviations °C degree Celsius µl microliter µM micromolar bp base pairs cyt b cytochrome b DNA deoxyribonucleic acid dNTP’s deoxynucleotide triphosphates ESU evolutionary significant unit IUCN International Union for Conservation of Nature km kilometre min minute mM millimolar mtDNA mitochondrial DNA MU management unit ng nanogram nuDNA nuclear DNA P probability PCR polymerase chain reaction s second SD standard deviation Taq Thermus aquaticus U enzyme unit List of Figures and Tables List of Figures and Tables Figure 1.1: Calling tree frog male Figure 1.2: Distribution map of the European tree frog (Hyla arborea) Figure 2.1: Current distribution of the European tree frog in Lower Saxony and adjacent areas Figure 2.2: Haplotype network cyt b Lower Saxony Figure 2.3: Distribution of cyt b haplotypes in Lower Saxony Figure 2.4: Isolation by distance plots Figure 2.5: LnPD and delta K Figure 2.6: STRUCTURE bar plot for K = 7 Figure 2.7: GENELAND map of estimated cluster membership for K = 7 Figure 2.8: Most important barriers to gene flow Figure 3.1: Haplotype network of cytochrome b Germany Figure 3.2: Haplotype distribution and physical map of Germany Figure 3.3: LnPD and delta K Figure 3.4: Distribution of distinct genetic clusters K = 4 Figure 3.5: Haplotype network of cytochrome b Europe Figure 3.6: Distribution of cyt b haplotypes in Europe Table 2.1: Overview of sample sites Lower Saxony Table 2.2: Pairwise Dest values and pairwise FST values Table 3.1: Overview of sample sites Germany Summary Astrid Krug Phylogeography and population structure of the European tree frog (Hyla arborea) for supporting effective species conservation Many amphibian species around the world are threatened by consequences of habitat degradation and fragmentation. The European tree frog (Hyla arborea) has suffered from dramatic population declines in the last decades and has therefore been categorised as threatened in many Red Data lists. Conservation measures are conducted at many places. To support such measures I conducted molecular studies on two geographic levels to reveal phylogeographic structures and genetic diversity, which are important for effective species conservation management. In Lower Saxony in Germany the current distribution of the tree frog is very patchy with some main occurrences in the lowlands. In order to define management units I sampled 237 individuals at 14 sites (~ 3 - 250 km apart from each other) across the tree frog distribution area in Lower Saxony and adjacent areas. All samples were genotyped with eight microsatellite loci and twelve sites were sequenced for an mtDNA cytochrome b fragment. While all but one of the microsatellite pairwise Dest and FST values showed significant genetic differentiation (Dest: 0 - 0.46, FST: 0 - 0.18), Bayesian analyses suggested seven distinct genetic clusters. The cytochrome b haplotype distribution highlights the former connection of the currently fragmented populations along the river Elbe. However, to reveal genetic structuring at higher geographic levels, as could have been generated e.g. by different postglacial colonisation routes, I conducted the second study with a sampling network of 31 sites across the tree frogs’ distribution area in Germany. 372 individuals were again analysed by mtDNA cytochrome b sequences and eight microsatellite loci. Sequence divergence between sample sites was low, varying between 0 and 0.4 % (overall 0.2 %), and no distinct genetic lineages were found. Nonetheless, a clear North-South partitioning was revealed by both molecular markers with the Central German Uplands as likely barrier. Furthermore, the influence of the major rivers such as Elbe, Rhine, and Danube on the phylogeographic structure was revealed. In general the genetic diversity was relatively high in both studies. Therefore, each of the sampled tree frog occurrences should have the potential to maintain or recover to a stable population size when applying appropriate local conservation measures. For new resettlement 1 Summary projects, the identified genetic structures should be considered when choosing source populations. Where possible, reconnection of originally linked occurrences that are now separated in different conservation units due to habitat fragmentation and genetic drift should be facilitated. 2 Zusammenfassung Astrid Krug Phylogeographie und Populationsstruktur des Europäischen Laubfroschs (Hyla arborea) zur Unterstützung eines effektiven Artenschutzes Weltweit sind viele Amphibienarten, hauptsächlich durch die Folgen von Habitat- Degradierung und Fragmentierung, gefährdet. Der Europäische Laubfrosch (Hyla arborea) hat in den letzten Jahrzehnten immense Bestandsrückgänge erfahren und wurde daher in vielen Roten Listen als gefährdet eingestuft. Naturschutzmaßnahmen werden bereits