Department für Ökosystem- und Landschaftsmanagement Fachgebiet Wildbiologie und Wildtiermanagement Genetic characterisation of European grayling populations (Thymallus thymallus L.): Implications for conservation and management Bernhard Gum Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr. Dr. Heinrich H. D. Meyer Prüfer der Dissertation: 1. Priv.-Doz. Dr. Ralph Kühn 2. Univ.-Prof. Dr. Gerhard Müller-Starck 3. apl. Prof. Dr. Oswald Rottmann Die Dissertation wurde am 02.11.2006 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 12.01.2007 angenommen. To my family Contents iii Contents List of figures ……………………………………………………………………...…… vi List of tables ……………………………………………………………………………. vii Preface …………………………………………………………………………….......... viii 1 Introduction ……………………………………………………………………….... 1 1.1 Statement of problem ……………………………………………………............. 1 1.2 Aims of the study ………………………………………………………............... 4 2 Literature review ……….………………………………………………………....... 6 2.1 Biology and ecology ………………….………….…………………………......... 6 2.1.1 Systematics and distribution ……………………………………………… 6 2.1.2 Habitat …………………………………………………………………..... 8 2.1.3 Life history traits …………………………………………………………. 10 Mophology and anatomy …………………………………………………. 10 Egg size and fecundity ……………………………………………………. 11 Spawning habitat and spawning behaviour ………….…………………… 13 Hatching, emergence and fry dispersal ….……………………………….. 14 Age and growth rate ……………………………………………………… 16 Feeding …………………………………………………………………… 18 Habitat requirements, migration and homing ………………………..…... 19 Population size and density ………………………………………………. 20 2.2 Genetics …………………………………………………………………………. 22 2.2.1 General aspects …………………………………………………………… 22 Molecular genetic advances …………………………………...…………. 22 Computational and statistical advances ……………………...………….. 23 2.2.2 Grayling genetics …………………………………………………………. 25 2.3 Endangerment of grayling …………………………………………………......... 27 2.3.1 Anthropogenic and environmental impacts on stream-dwelling fish populations ………………………………………………………........ 28 2.3.2 Bavarian grayling support programme …………………………………… 31 3 Microsatellite variation in Bavarian populations of European grayling ( Thymallus thymallus ): Implications for conservation ...…………………………. 35 3.1 Abstract .…………………………………………………………………………. 35 3.2 Introduction ……………………………………………………………………... 36 3.3 Materials and methods …………………………………………………………... 38 3.3.1 Sample collection …………………………………………………………. 38 3.3.2 DNA isolation and microsatellite analysis ..…………………………........ 38 3.3.3 Data analysis ……………………………………………………………… 41 3.4 Results …………………………………………………………………………... 44 3.4.1 Linkage and Hardy-Weinberg equilibrium tests …………………………. 44 3.4.2 Genetic diversity between and within the drainages ……………………... 44 3.4.3 Genetic differentiation and relationship of populations ………………….. 45 3.5 Discussion ……………………………………………………………………….. 49 3.5.1 Genetic diversity between and within drainages …………………………. 49 3.5.2 Genetic differentiation between drainages ……………………………….. 51 3.5.3 Conservation and management implications ……………………………... 52 3.6 Supplementary material ………………………….……………………………… 53 Contents iv 4 Mitochondrial and nuclear DNA phylogeography of European grayling ( Thymallus thymallus ): evidence for secondary contact zones in Central Europe 54 4.1 Abstract ………………………………………………………………………….. 54 4.2 Introduction ……………………………………………………………………... 55 4.3 Materials and methods …………………………………………………………... 58 4.3.1 Sample collection and DNA isolation ……………………………………. 58 4.3.2 Mitochondrial DNA analysis ……………………………………………... 58 4.3.3 Microsatellite DNA analysis ……………………………………………… 59 4.3.4 Data analysis ……………………………………………………………… 63 4.4 Results …………………………………………………………………………… 65 4.4.1 Mitochondrial DNA diversity and phylogenetic inference ………………. 65 4.4.2 Geographic distribution of mtDNA haplotypes and differentiation among assemblages ………………………………………………………. 70 4.4.3 Microsatellite DNA variation within and between samples ……………… 71 4.4.4 Microsatellite divergence and admixture analysis among assemblages ….. 72 4.4.5 Analysis of molecular variance …………………………………………... 74 4.5 Discussion ……………………………………………………………………….. 76 4.5.1 Diversity and divergence of grayling mtDNA and nuclear DNA ………... 76 4.5.2 Colonization history and postglacial dispersal of Thymallus thymallus ….. 78 4.5.3 Zones of hybridization in central and northern Europe …………………... 79 4.5.4 Comparative phylogeographical inference ……………………………….. 80 4.5.5 Implications for conservation …………………………………………….. 81 4.6 Supplementary material …………………………………………………………. 82 5 Discriminating the impact of recent human mediated stock transfer from historical gene flow on genetic structure of European grayling (Thymallus thymallus L.) …………………………………………………………... 83 5.1 Abstract ………………………………………………………………………… 83 5.2 Introduction …………………………………………………………………….. 83 5.3 Sample collection and stocking history ………………………………………… 85 5.4 Methods ………………………………………………………………………… 91 5.4.1 DNA isolation and genetic analysis ……………………………………… 91 5.4.2 Data analysis ……………………………………………………………… 91 5.5 Results ………………………………………………………………………….. 95 5.5.1 MtDNA diversity and geographical distribution of mtDNA haplotypes … 95 5.5.2 Microsatellite DNA variation within and between samples ……………... 95 5.5.3 Microsatellite divergence and admixture analysis ……………………….. 96 5.5.4 Individual immigrant ancestries ………………………………………….. 98 5.5.5 Discrimination of historical secondary contact from recent human mediated stock transfer …………………………………………………... 98 5.6 Discussion …………………………………………………………………...…. 101 5.6.1 Genetic impact of stocking on genetic structure of European grayling …. 102 5.6.2 Conservation priorities …………………………………………………… 104 6 General Discussion ……………………....................……………………………… 107 6.1 Application of mitochondrial and microsatellite DNA markers in conservation genetics: general considerations ………………….……………… 107 6.1.1 Mitochondrial DNA ……………………………………………………… 107 6.1.2 Microsatellite DNA ………………………………………………………. 109 6.2 Conservation genetics of European grayling …………………………………... 111 Contents v 6.2.1 General aspects ………………………………………………………….. 111 6.2.2 Salmonid population genetic structure based on microsatellite DNA analyses …………………………………………………………… 111 6.2.3 Phylogeograpic inference of European grayling with particular reference to salmonids and other European freshwater fish species ……. 116 6.3 Implications for conservation and management ……………………………….. 122 6.3.1 Contribution of conservation genetics ...………………………………… 122 6.3.2 Contribution of ongoing studies and other fields of research …………… 126 6.4 Perspectives ……………………………………………………………………. 132 7 Summary ……………………...………....................………………………………. 136 8 Zusammenfassung ……………………....................………………………………. 138 9 References ……………………....................……………………………………..…. 141 10 Acknowledgements …………………....................……………………………..…. 165 List of figures vi List of figures Fig. 2.1: Adult male European grayling ( Thymallus thymallus L.) …………………….. 10 Fig. 2.2: Comparison between relative ratio of body height for European grayling from drainages Danube, Main and Elbe ………………………………………. 11 Fig. 2.3: Growth rates of European grayling populations from different drainage systems in Bavaria …………………………………………………………….. 17 Fig. 2.4: Development of annual European grayling catches from 1975 to 1999 averaged over 26 Bavarian rivers of the Alpine Danubian drainage system ….. 21 Fig. 2.5: Co-action of different factors leading to the decline and endangerment of riverine fish populations ……………………………………………………. 29 Fig. 3.1: Sampling locations of grayling ( Thymallus thymallus ) in Bavaria …………… 39 Fig. 3.2: UPGMA dendrogram based on Cavalli-Sforza & Edwards (1967) chord distance for grayling ( Thymallus thymallus ) sampled from the Danube, Rhine/Main and Elbe drainages ……………………………………... 46 Fig. 4.1: Map of sampling locations with pie diagrams showing the distribution of mtDNA lineage frequencies among studied Thymallus thymallus populations .. 57 Fig. 4.2: Dendrogram of grayling mtDNA PCR-RFLP composite haplotypes at the ND-1 and ND-5/6 genes estimated by the Fitch-Margoliash method …………. 67 Fig. 4.3: The estimated 95% plausible set of cladograms and associated nested design for the mtDNA ND-5 sequence data found in Thy. Thymallus ………… 68 Fig. 4.4: N-J tree of the studied European grayling populations based on the number of net nucleotide substitutions per site ( dA) at the mitochondrial ND-1 and ND-5/6 gene regions .…………………………………………………….. 73 Fig. 4.5: Two-dimensional scaling analysis based on 12 microsatellite loci and Nei’s DA distances (Nei et al. , 1983) among pairs of populations ……………. 75 Fig. 5.1: Map of sampling locations with pie diagrams showing the distribution of major mtDNA lineage frequencies among studied European grayling populations …………………………………………………………... 87 Fig. 5.2: Two-dimensional scaling analysis based on 12 microsatellite loci and pairwise estimates of FST among populations …………………………….. 94 Fig. 5.3: Inference of genetic clusters ( K) of the studied grayling populations based on the STRUCTURE algorithm …………………………………………... 97 Fig. 5.4: Histogram of posterior probabilities of individual immigrant ancestries summarized for all grayling populations as determined