DISSERTATION Titel der Dissertation Phylogeography and taxonomy of the land snail genus Orcula Held, 1837 Verfasser Mag.rer.nat. Josef Harl angestrebter akademischer Grad Doktor der Naturwissenschaften (Dr.rer.nat.) Wien, 2015 Studienkennzahl lt. Studienblatt: A 091 444 Dissertationsgebiet lt. Studienblatt: Ökologie Betreut von: Dr. Elisabeth Haring, Privatdoz. Table of contents PREFACE ......................................................................................................................................... 3 SUMMARY ....................................................................................................................................... 4 GENERAL INTRODUCTION ........................................................................................................... 8 PUBLICATIONS ............................................................................................................................. 10 Harl J, Sattmann H, Schileyko, A (2011) Types of the extant taxa of the landsnail genus Orcula Held 1837 (Gastropoda: Stylommatophora: Orculidae). Archiv für Molluskenkunde: International Journal of Malacology, 140, 175-199. ............................................................................................ 10 Harl J, Páll-Gergely B, Kirchner S, Sattmann H, Duda M, Kruckenhauser L, Haring E (2014) Phylogeography of the land snail genus Orcula (Orculidae, Stylommatophora) with emphasis on the Eastern Alpine taxa: speciation, hybridization and morphological variation. BMC Evolutionary Biology, 14, 223. ............................................................................................................................. 36 Harl J, Duda M, Kruckenhauser L, Sattmann H, Haring E (2014) In search of glacial refuges of the land snail Orcula dolium (Pulmonata, Orculidae) - an integrative approach using DNA sequence and fossil data. PloS one, 9, e96012. ............................................................................................. 63 CONCLUDING DISCUSSION ........................................................................................................ 88 Glacial refuges of the Alpine Orcula species .................................................................................. 88 Systematics of the genus Orcula .................................................................................................... 89 Hybridization within the subgenus Orcula ...................................................................................... 90 Methodological Considerations ...................................................................................................... 91 References of General Introduction and Concluding Discussion ................................................... 93 ACKNOWLEDGEMENTS .............................................................................................................. 99 CURRICULUM VITAE .................................................................................................................. 100 PUBLICATION LIST .................................................................................................................... 102 Preface This doctoral thesis is a cumulative dissertation, compiling three publications, all of which were published in peer-reviewed journals. The publications deal with taxonomy, phylogeny and phylogeography of the land snail genus Orcula Held, 1837. The general introduction of the thesis provides a background on the suitability of land snails for evolutionary questions, in particular regarding the search for glacial refuges. The next section contains my three first-author publications, which are co-authored (in varying combinations) by members of the research group on Alpine land snails, which is housed in the Natural History Museum Vienna, Elisabeth Haring, Helmut Sattmann, Luise Kruckenhauser, Michael Duda, Sandra Kirchner, and two malacologists from other institutions, Barna Páll-Gergely and Anatoly Schileyko. Elisabeth Haring, Helmut Sattmann and Luise Kruckenhauser initiated the project on the diversity of Alpine land snails and participated in the design of my studies. The latter three researchers and my colleague, Michael Duda, were equally involved in fieldwork and sample collection. For all three studies, I performed the main part of the lab work, data analyses, interpretation and writing. Anatoly Schileyko conceived the idea of the type-catalogue and essentially contributed in writing the manuscript. Barna Páll-Gergely's efforts and expertise were crucial for completing the taxon set for the phylogeny of the genus Orcula. Detailed information on contributions of the authors are provided in the 'Authors’ Contributions'. The main part of my thesis is followed by a concluding discussion containing chapters on glacial refuges, systematics and hybridization as well as a section on methodological considerations, mainly about the choice of molecular genetic markers and primer design. The Austrian Science Fund funded the project on Alpine land snails (FWF Proj.-No. P19592-B17; PI Elisabeth Haring) and financed the personnel costs during the first two years of my doctoral thesis (03/2009 to 04/2011). In 2012, I received an award of the 'Theodor Körner Fonds zur Förderung von Kunst und Wissenschaft', and in 2013 a 'PhD Completion Grant' of the University of Vienna. The 'Österreichische Forschungsgemeinschaft' (ÖFG) participated in the financing of the travel expenses to the World Congress of Malacology 2013. The 'Freunde des Naturhistorischen Museums Wien' financially supported several of the collection trips. 3 Summary This doctoral thesis deals with taxonomy, phylogeny and phylogeography of the land snail genus Orcula Held, 1837. The genus Orcula inhabits calcareous mountain habitats of Central and Southeast Europe and shows the largest species diversity in the Eastern Alps with nine out of 13 species. Several malacologists hypothesized that the high diversity in the Alps was the result of geographic isolation of populations in separated glacial refuges and adaption to changing environmental conditions and that particularly the Eastern Alps represented a center of origin and glacial refuge for several taxa. For the present thesis, we studied the taxonomy of the genus and investigated all Orcula species by means of molecular genetics. We integrated fossil data for reconstructing past distribution patterns and for calibrating the phylogenetic trees, and we performed morphometric analyses on members of the Alpine species group. In particular, we aimed at clarifying whether the high species and subspecies diversity in the Eastern Alps is the result of geographical isolation in Pleistocene glacial refuges or if if the lineages already separated in earlier times. In the type catalogue, we provide comprehensive data on all extant taxa of the genus Orcula. We identified 55 nominal taxa, for most of which we provide references to the original descriptions, type localities, places of storage of type specimens and collection numbers. For more than 30 nominal taxa we also present photographs and measurements of type specimens. We discuss the validity of several subspecies names and point out inconsistencies in taxon listings of the Fauna Europaea Checklist and the CLECOM I-list. This work allowed us to identify all relevant taxa and to evaluate their taxonomical status quo, which was crucial for any further investigations. The phylogenetic trees resulting from the analyses of both the mitochondrial (COI, 12S, 16S) and the nuclear (H4/H3) data sets reveal three main clades, corresponding to the three subgenera Orcula, Illyriobanatica Páll-Gergely & Deli 2013 and Hausdorfia Páll-Gergely & Irikov 2013. The fossil calibrated molecular clock analyses and the reconstructions of the historic geographic ranges suggest that the genus originated in the Dinarids during the Middle Miocene and that the subgenus Orcula colonized the Alps not until the Late Miocene. The major splits in the latter species group date back to the Late Miocene and Pliocene, therefore, isolation in separated Pleistocene glacial refuges was most likely not the only factor triggering speciation. Our data also suggest that hybridizations happened or are still ongoing between some of the 'younger' species, particularly between Orcula pseudodolium Wagner 1912 and Orcula gularis (Rossmässler, 1837). The two latter species could be discriminated well in the morphometric analyses and generally provided different nuclear H4/H3 variants, but almost all specimens possessed similar mitochondrial sequences, thus indicating mitochondrial capture. Complex phylogeographic patterns were also found in the most widespread and prominent Orcula species, Orcula dolium (Draparnaud, 1801). The species inhabits all major limestone areas of the Alps and the Western Carpathians and includes several morphologically differentiated populations. The phylogenetic trees reveal at least four distinct Alpine clades, whose 4 distribution areas are overlapping only marginally. These clades, however, could not be related to any of the nominate subspecies, except for a single clade, which matches with the distribution of Orcula dolium infima Ehrmann, 1933 from the Wienerwald (Vienna and Lower Austria). Orcula dolium edita Ehrmann, 1933 and Orcula dolium raxae Gittenberger, 1978, both described as glacial relics, which are currently restricted to high altitudes in the Eastern Alps, were genetically not distinguishable from the surrounding lowland populations.