DIPLOMARBEIT Titel der Diplomarbeit Species delineation and genetic variation of Hiatella “arctica” (Bivalvia, Heterodonta) in the Mediterranean Sea angestrebter akademischer Grad Magistra der Naturwissenschaften (Mag. rer.nat.) Verfasserin Miriam Oberlechner Matrikel-Nummer: 9703291 Studienrichtung (lt. Biologie Studienblatt): Betreuer: a.o. Univ. Prof. Dr. Gerhard Steiner Wien, am 13. August 2008 Ich komme mir wie ein Mädchen vor, das am Strand spielt und sich damit vergnügt, ein noch glatteres Kieselsteinchen oder eine noch schönere Muschel als gewöhnlich zu finden, während das große Meer der Wahrheit völlig unerforscht vor mir liegt. frei nach Sir Isaac Newton Contents I Abstract ....................................................................................................................... I II Deutsche Zusammenfassung ...................................................................................II 1. Introduction............................................................................................................1 1.1. Morphological and systematic assignment.............................................................1 1.1.1. Bivalvia ..................................................................................................................1 1.1.2. Classification of the Bivalvia................................................................................1 Heterodonta......................................................................................................2 Hiatelloidea.......................................................................................................2 Hiatellidae.........................................................................................................2 1.2. The genus Hiatella.....................................................................................................3 1.2.1. Morphological characteristics .............................................................................3 1.2.2. Geographic range..................................................................................................3 1.2.3. Habitat ...................................................................................................................4 1.2.4. Fossil record ..........................................................................................................5 1.2.5. Nutrition ................................................................................................................5 1.2.6. Development..........................................................................................................5 1.2.7. Taxonomy ..............................................................................................................6 1.3. Differentiation of Hiatella arctica and Hiatella striata...........................................7 1.3.1. Species diagnosis: Shell morphology...................................................................7 1.3.2. Species diagnosis: Habitat choice ........................................................................7 1.3.3. Species diagnosis: Reproductive cycle and larval development .......................8 1.3.4. Species diagnosis: Fossil record...........................................................................8 1.4. Aims of this study......................................................................................................9 1.4.1. Species delineation ................................................................................................9 1.4.2. Genetic variation...................................................................................................9 1.5. Molecular markers .................................................................................................10 2. Material and Methods .........................................................................................11 2.1. Data collection.........................................................................................................11 2.1.1. Sample collection and conservation ..................................................................11 2.1.2. DNA isolation ......................................................................................................12 2.1.3. PCR ......................................................................................................................12 2.1.4. Purification of PCR products ........................................................................... 16 2.1.5. Sequencing reaction ........................................................................................... 16 2.1.6. Alignment and sequence analysis ..................................................................... 16 2.2. Genealogical analyses ............................................................................................ 18 2.2.1. Bayesian Inference ............................................................................................. 18 2.2.2. Network analysis using the statistical parsimony approach .......................... 18 2.3. Genetic variation.................................................................................................... 19 2.3.1. Distance method ................................................................................................. 20 2.3.2. Analysis of molecular variance (AMOVA)...................................................... 20 3. Results ...................................................................................................................23 3.1. Sequence analyses .................................................................................................. 23 3.1.1. Proteins (COI, ATP6, COIII)............................................................................ 23 3.1.2. 16S rRNA............................................................................................................ 24 3.1.3. NC_tRNA (NC_1, NC_2, tRNA Gln) ............................................................... 24 3.1.4. All data................................................................................................................ 25 3.2. Genealogical relationships..................................................................................... 26 3.2.1. Proteins ............................................................................................................... 26 Bayesian Inference ............................................................................................. 26 Network analysis ................................................................................................ 28 3.2.2. 16S rRNA............................................................................................................ 29 Bayesian Inference ............................................................................................. 29 Network analysis ................................................................................................ 31 3.2.3. NC_tRNA............................................................................................................ 32 Bayesian Inference ............................................................................................. 32 Network analysis ................................................................................................ 34 3.2.4. All data................................................................................................................ 35 Bayesian Inference ............................................................................................. 35 Network analysis ................................................................................................ 37 3.2.5. Haplogroups ....................................................................................................... 38 3.3. Genetic variation.................................................................................................... 39 3.3.1. Populations ......................................................................................................... 39 3.3.2. Habitats............................................................................................................... 45 4. Discussion..............................................................................................................49 4.1. Comparison of the different datasets ................................................................... 49 4.2. Species delineation ..................................................................................................50 4.3. Genetic differentiation of populations ..................................................................51 4.4. Correlation between genetic differentiation and geographic distance ..............52 4.5. Genetic differentiation of habitats.........................................................................52 4.6. Gene flow .................................................................................................................53 4.6.1. Dispersal abilities ................................................................................................53 4.6.2. Mode of habit ......................................................................................................53 4.6.3. Habitat distribution in the Mediterranean Sea................................................54 4.6.4. Sea currents.........................................................................................................55 4.6.5. Anthropogenic influence