DNA-based methods for freshwater biodiversity conservation - Phylogeographic analysis of noble crayfish (Astacus astacus) and new insights into the distribution of crayfish plague DISSERTATION zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften Fachbereich 7: Natur- und Umweltwissenschaften der Universität Koblenz-Landau Campus Landau vorgelegt am 16. Januar 2013 von Anne Schrimpf geboren am 21. September 1984 in Frankfurt am Main Referent: Prof. Dr. Ralf Schulz Koreferent: Prof. Dr. Klaus Schwenk - This thesis is dedicated to my grandparents - Content CONTENT CONTENT ............................................................................................................... 5 ABSTRACT ............................................................................................................ 8 ZUSAMMENFASSUNG ........................................................................................ 10 ABBEREVIATIONS .............................................................................................. 13 GENERAL INTRODUCTION ................................................................................ 15 Conservation of biological diversity ........................................................................ 15 The freshwater crayfish ............................................................................................ 17 General ............................................................................................................... 17 The noble crayfish (Astacus astacus) ................................................................. 19 Threats to native crayfish .................................................................................... 20 Genetic Methods ........................................................................................................ 24 OBJECTIVES ....................................................................................................... 27 THESIS OUTLINE ................................................................................................ 29 SUMMARY OF RESULTS .................................................................................... 33 European crayfish species (Astacus astacus) ....................................................... 33 Crayfish plague (Aphanomyces astaci) ............................................................... 35 GENERAL DISCUSSION ..................................................................................... 37 Conservation considerations - native crayfish ....................................................... 37 Conservation considerations - crayfish plague ...................................................... 40 Synthesis of discussion ............................................................................................ 42 CONCLUSION ...................................................................................................... 43 REFERENCES ..................................................................................................... 44 APPENDIX ............................................................................................................ 51 Appendix 1: The first large-scale genetic analysis of the vulnerable noble crayfish Astacus astacus reveals low haplotype diversity in central European populations ..... 53 5 Content Appendix 2: Noble crayfish (Astacus astacus) with migratory background – natural versus human-mediated processes ............................................................................. 73 Appendix 3: Crayfish plague agent detected in populations of the invasive North American crayfish Orconectes immunis (Hagen, 1870) in the Rhine River, Germany ................................................................................................................................... 107 Appendix 4: Invasive crayfish and crayfish plague on the move: first detection of the plague agent Aphanomyces astaci in the Romanian Danube ................................... 119 Appendix 5: Crayfish plague pathogen detected in the Danube Delta – a potential threat to freshwater biodiversity in southeastern Europe ........................................... 139 Appendix 6: Absence of the crayfish plague pathogen (Aphanomyces astaci) facilitates coexistence of European and American crayfish in central Europe155 ERKLÄRUNG ..................................................................................................... 175 ACKNOWLEDGMENTS ..................................................................................... 176 CURRICULUM VITAE ........................................................................................ 178 FIGURES Figure 1 Noble crayfish (Astacus astacus). .................................................................... 20 Figure 2 Narrow-clawed crayfish (Astacus leptodactylus). ............................................. 22 Figure 3 Spiny-cheek crayfish (Orconectes limosus) and Calico crayfish (O. immunis). 23 Figure 4 Overview of the different Appendices of this thesis. ........................................ 29 Figure 5 Phylogenetic tree. ............................................................................................ 34 Figure 6 Hypothetical migration routes of noble crayfish. .............................................. 35 TABLES Table 1 List of crayfish and crayfish pathogen species included in this thesis. ............ 18 Table 2 Results of the sequence analysis. .................................................................... 33 Table 3 The common assumption and the outcome of this thesis. ............................... 40 6 7 Abstract ABSTRACT Worldwide one third to one half of the freshwater crayfish species are threatened with population decline or extinction. Besides habitat deterioration, pollution, and other man-made environmental changes, invasive species and pathogens are major threats to the survival of European crayfish species. Freshwater crayfish are the largest freshwater invertebrates and strongly influence the structure of food webs. The disappearance of crayfish from a water body may change the food web and could have dramatic consequences for an ecosystem. One goal in modern species conservation strategies is the conservation of genetic diversity, since genetic diversity is an advantage for the long-term survival of a species. The main aim of my thesis was to reveal the genetic structure and to identify genetic hotspots of the endangered noble crayfish (Astacus astacus) throughout Europe (part 1 of my thesis). Since the most significant threat to biodiversity of European crayfish species is the crayfish plague pathogen Aphanomyces astaci I studied new aspects in the distribution of A. astaci (part two of my thesis). The results serve as a basis for future conservation programs for freshwater crayfish. In the first part of my thesis I conducted a phylogeographic analysis of noble crayfish using mitochondrial DNA and nuclear microsatellite data. With these methods I aimed to identify its genetic hotspots and to reconstruct the recolonization history of central Europe by this species. I detected high genetic diversities in southestern Europe indicating that noble crayfish outlasted the cold climate phases during the Pleistocene in this region (Appendix 1). Because of the high genetic diversity found there, southeastern Europe is of particular importance for the conservation of noble crayfish. The mitochondrial DNA analysis points to a bifurcated colonization process from the eastern Black Sea basin to a) the North Sea and to b) the Baltic Sea basin (Appendix 2). A second independent refugium that was localized on the Western Balkans did not contribute to the colonization of central Europe. Furthermore, I found that the 8 Abstract natural genetic structure is dissolved, probably due to the high human impact on the distribution of noble crayfish (e.g. artificial translocation). In the second part of this thesis using real-time PCR I identified calico crayfish (Orconectes immunis) as the fourth North American crayfish species to be carrier of the agent of the crayfish plague (Appendix 3). Furthermore I detected the crayfish plague pathogen in American spiny-cheek crayfish (Orconectes limosus) and native narrow-clawed crayfish (Astacus leptodactylus) in the lower Danube in Romania (Appendix 4). The distribution of infected spiny- cheek crayfish poses a threat to the native biodiversity in southeastern Europe and shows the high invasion potential of this crayfish species. Moreover, I found that even the native narrow-clawed crayfish in the Danube Delta, about 970 km downstream of the current invasion front of American crayfish, is a carrier of A. astaci (Appendix 5). This finding is of high importance, as the native species do not seem to suffer from the infection. In Appendix 6 I elucidate demonstrate that the absence of the crayfish plague agent is the most likely explanation for the coexistence of populations of European and American crayfish in central Europe. In my thesis I show that the common assumption that all North American crayfish are carrier of A. astaci and that all native crayfish species die when infected with A. astaci does not hold true. The studies presented in my thesis reveal new aspects that are crucial for native crayfish conservation: 1) The genetic diversity of noble crayfish is highest in southeastern Europe where noble crayfish outlasted the last glacial maximum in at least two different refugia. 2) Not all American crayfish