Phylogeography and taxonomy of the land snail genus Orcula Held, 1837

Die vorliegende Doktorarbeit behandelt die Taxonomie, Phylogenie und Phylogeographie der Landschneckengattung Orcula Held, 1837. Die Gattung Orcula bewohnt kalkreiche Gebirgshabitate Zentral- und Südosteuropas und weist mit neun von 13 Arten die größte Artenvielfalt in den Ostalpen auf. Die hohe Vielfalt in den Alpen wurde von mehreren Autoren auf die geographische Isolation von Populationen in voneinander getrennten eiszeitliche Refugien und auf die Anpassung an sich ändernde Umweltbedingungen zurückgeführt - insbesondere die Ostalpen wurden als Entstehungszentrum und eiszeitliches Refugium vieler Taxa vermutet. Im Rahmen dieser Dissertation führten wir Studien zur Taxonomie der Gattung sowie molekulargenetische Untersuchungen bei allen Orcula-Arten durch. Für die Rekonstruktion vergangener Verbreitungsmuster und die Kalibrierung der phylogenetischen Stammbäume wurden Fossilienbelege mit einbezogen, und bei den Mitgliedern der alpinen Artengruppe wurden morphometrische Analysen durchgeführt. Insbesondere strebten wir an zu klären, ob die hohe Arten- und Unterartenvielfalt in den östlichen Alpen das Resultat von Isolation in eiszeitlichen Refugien während des Pleistozäns ist oder ob sich deren Abstammungslinien bereits davor trennten. Im Typenkatalog stellen wir umfangreiche Daten zu allen bestehenden Arten der Gattung Orcula zur Verfügung. Wir identifizierten 55 nominelle Taxa, für einen Großteil derer wir Referenzen zu den Originalbeschreibungen, Typus-Lokalitäten, Aufbewahrungsorten von Typus-Exemplaren und Sammlungsnummern auflisten. Des Weiteren zeigen wir Abbildungen und Abmessungen von mehr als 30 nominellen Taxa, diskutieren die Gültigkeit mehrerer Unterartnamen und weisen auf Inkonsistenzen in den Auflistungen der Taxa in der Fauna-Europaea und der CLECOM I-Liste hin. Diese Arbeit ermöglichte uns, alle relevanten Taxa zu identifizieren, was entscheidend für die weiteren Untersuchungen war. Die Stammbäume, die aus der Untersuchung sowohl der mitochondrialen (COI, 12S, 16S) als auch der nukleären (H4/H3) Datensätze resultierten, zeigen drei Kladen, die den drei Untergattungen Orcula, Illyriobanatica Páll-Gergely & Deli 2013 und Hausdorfia Páll-Gergely & Irikov 2013 entsprechen. Die durch Fossilien kalibrierten 'Molekulare Uhr'-Analysen und die Rekonstruktion der historischen Verbreitungen deuten darauf hin, daß die Gattung bereits während des Mittleren Miozäns im Dinarischen Gebirge entstanden ist und die Untergattung Orcula die Alpen nicht vor dem späten Miozän besiedelt hat. Die Auftrennung der wichtigsten Linien innerhalb dieser Artengruppe datieren auf das Obere Miozän und das Pliozän - die Isolation in getrennten eiszeitliche Refugien war wahrscheinlich nicht der einzige Faktor, der die Artbildung vorantrieb. Unsere Daten weisen auch darauf hin, daß es zu Hybridisierungen gekommen ist oder diese zwischen einigen 'jüngeren' Arten immer noch auftreten, insbesondere zwischen Orcula pseudodolium Wagner, 1912 und Orcula gularis (Rossmässler, 1837). Die beiden letzteren Arten konnten in den morphometrischen Analysen gut unterschieden werden und wiesen generell unterschiedliche nukleäre H4/H3-Varianten auf, aber fast alle Individuen besaßen ähnliche mitochondriale Sequenzen, was auf die Aufnahme artfremder mitochondrialer DNA ('mitochondrial capture') hindeutet. Komplexe phylogeographische Muster wurden auch bei der am weitesten verbreiteten und auffälligsten Orcula-Art, Orcula dolium (Draparnaud, 1801), gefunden. Die Art besiedelt alle größeren Kalksteingebiete der Alpen und der westlichen Karpaten und umfaßt mehrere morphologisch differenzierte Populationen. Die phylogenetischen Bäume zeigen zumindest vier unterschiedliche alpine Kladen, deren Verbreitungsgebiete nur geringfügig überlappen. Diese Kladen konnten allerdings nicht mit den nominellen Unterarten in Verbindung gebracht werden, außer einer einzigen Klade, die mit dem Verbreitungsareal von Orcula dolium infima Ehrmann, 1933 aus dem Wienerwald (Wien und Niederösterreich) übereinstimmt. Orcula dolium edita Ehrmann, 1933 und Orcula dolium raxae Gittenberger, 1978, die als eiszeitliche Relikte beschrieben wurden, die heute auf höhere Gebirgslagen der östlichen Alpen beschränkt sind, konnten genetisch nicht von den umgebenden Populationen des Tieflandes unterschieden werden. Die phylogeographischen Muster deuten dennoch darauf hin, daß O. dolium das letzte glaziale Maximum in Refugien in den West- und den Ostalpen überdauerte - insbesondere die Ostalpen beherbergen genetisch sehr diverse Populationen. Ungeachtet der wesentlich kleineren Probenanzahl wurde in den Westkarpaten sogar eine noch größere Zahl an genetischen Kladen gefunden, und die Individuen von annähernd der Hälfte der Standorte wiesen stark unterschiedliche mitochondriale Sequenzen auf. Die Rekonstruktion der geographischen Verbreitungsgeschichte deutet darauf hin, daß O. dolium etwa um die Miozän-Pliozän-Grenze in den Westkarpaten entstand und die Alpen erst später besiedelte. Die alpinen Populationen sind allerdings nicht reziprok monophyletisch - die Sequenzmuster weisen auf mehrfache Migrationen zwischen den beiden Gebirgszügen hin. Um die vergangenen Verbreitungsmuster von O. dolium während der letzten glazialen Abschnitte zu rekonstruieren, untersuchten wir auch die pleistozänen Fossilienbelege der Art. Die Daten zeigen klar die Anwesenheit von O. dolium in der Peripherie der Alpen und Westkarpaten sowie im Pannonischen und im Wiener Becken während mehrerer kalter und warmer eiszeitlicher Perioden.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 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. Nonetheless, the phylogeographic patterns indicate that populations of O. dolium outlasted the Last Glacial Maximum in refuges in the Western Alps and in the Eastern Alps - in particular, the latter region harbors genetically highly diverse populations. Despite the considerably smaller sample size, we found an even larger number of genetic clades in the Western Carpathians, and specimens at almost half of the sites showed strongly differing mitochondrial sequences. The reconstruction of the geographic range history suggests that O. dolium originated in the Western Carpathians around the Miocene-Pliocene boundary and settled the Alps later. The Alpine populations, however, are not reciprocally monophyletic, but sequence patterns suggest multiple migrations between the two mountain ranges. In order to reconstruct past distribution patterns of O. dolium during the last glacial periods in lowland areas surrounding the Alps, we also examined the Pleistocene fossil record of the species. The data clearly evidences its presence in the periphery of the Alps and the Western Carpathians, as well as in the Pannonian and Vienna Basins, during several warm and cold glacial periods

The nuclear 18S ribosomal DNAs of avian haemosporidian parasites

Background Plasmodium species feature only four to eight nuclear ribosomal units on different chromosomes, which are assumed to evolve independently according to a birth-and-death model, in which new variants originate by duplication and others are deleted throughout time. Moreover, distinct ribosomal units were shown to be expressed during different developmental stages in the vertebrate and mosquito hosts. Here, the 18S rDNA sequences of 32 species of avian haemosporidian parasites are reported and compared to those of simian and rodent Plasmodium species. Methods Almost the entire 18S rDNAs of avian haemosporidians belonging to the genera Plasmodium (7), Haemoproteus (9), and Leucocytozoon (16) were obtained by PCR, molecular cloning, and sequencing ten clones each. Phylogenetic trees were calculated and sequence patterns were analysed and compared to those of simian and rodent malaria species. A section of the mitochondrial CytB was also sequenced. Results Sequence patterns in most avian Plasmodium species were similar to those in the mammalian parasites with most species featuring two distinct 18S rDNA sequence clusters. Distinct 18S variants were also found in Haemoproteus tartakovskyi and the three Leucocytozoon species, whereas the other species featured sets of similar haplotypes. The 18S rDNA GC-contents of the Leucocytozoon toddi complex and the subgenus Parahaemoproteus were extremely high with 49.3% and 44.9%, respectively. The 18S sequences of several species from all three genera showed chimeric features, thus indicating recombination. Conclusion Gene duplication events leading to two diverged main sequence clusters happened independently in at least six out of seven avian Plasmodium species, thus supporting evolution according to a birth-and-death model like proposed for the ribosomal units of simian and rodent Plasmodium species. Patterns were similar in the 18S rDNAs of the Leucocytozoon toddi complex and Haemoproteus tartakovskyi. However, the 18S rDNAs of the other species seem to evolve in concerted fashion like in most eukaryotes, but the presence of chimeric variants indicates that the ribosomal units rather evolve in a semi-concerted manner. The new data may provide a basis for studies testing whether differential expression of distinct 18S rDNA also occurs in avian Plasmodium species and related haemosporidian parasites

Subgeneric division of the genus Orcula Held 1837 with remarks on Romanian orculid data (Gastropoda, Pulmonata, Orculidae)

The genital anatomy of Orcula jetschini (Romania), O. zilchi (Bulgaria) and O. wagneri (Albania) is described. Based on anatomical features (morphology of the penial caecum), shell characters (sculpture and shape) and unpublished molecular data, the genus Orcula is subdivided into three subgenera. O. zilchi was classified within the monotypic subgenus Orcula (Hausdorfia) subgen. n.; O. jetschini, O. wagneri and O. schmidtii were classified to Orcula (Illyriobanatica) subgen. n. (type species: Pupa Schmidtii), whereas the other Orcula species remain in the nominotypical subgenus. Orcula (Hausdorfia) is known from South-Eastern Bulgaria and North-Western Turkey, Orcula (Illyriobanatica) inhabits Western Romania, North-Western Greece, Albania, Macedonia, Kosovo and Montenegro. The nine species of Orcula (Orcula) are known mainly from the Alps and the Western Carpathians (from Eastern France to Eastern Hungary and Slovakia).The occurrence of only one Orcula species, namely O. jetschini is verified from Romania. Available information suggests that data on the Romanian occurrence of Orcula dolium and O. gularis were based on wrongly identified specimens. Sphyradium dobrogicum (=Orcula dobrogica) is considered as a synonym of S. doliolum

Integrative taxonomy of insular land snails of the genus Sicradiscus Páll‐Gergely, 2013 (Gastropoda, Plectopylidae) with description of a new species

Many land snail taxa have undergone speciation in the Ryukyu Islands and Taiwan in East Asia. We examined the shell, radular, and genital morphology, and mitochondrial phylogeny of two described Sicradiscus species distributed in Miyako Island and Taiwan, and the newly discovered Sicradiscus pallgergelyi sp. nov. from Iriomote Island. Canonical variate analysis based on adult shell measurements indicated that S. pallgergelyi sp. nov. and the Taiwanese Sicradiscus ishizakii had more similar shell measurements, whereas S. pallgergelyi sp. nov. shared common characteristics of shell sculpture with the Japanese Sicradiscus hirasei. The leave-one-out cross-validation results correctly classified 100%, 71.4%, and 88.0% of S. hirasei, S. ishizakii, and S. pallgergelyi sp. nov., respectively. The radular and genital morphology was similar in these three species. Moreover, molecular phylogenetic analyses showed monophyly of the three species, although the Japanese lineages were more closely related to each other than to the Taiwanese species. Accordingly, the characteristics of shell sculpture are common traits of the two Japanese species, and these findings indicate that shell morphology has significantly diverged in Japan. The different apertural callus lengths among the three species may be an adaptation to predators, and shell flatness may reflect interspecific differences in microhabitats

The 18S rRNA genes of Haemoproteus (Haemosporida, Apicomplexa) parasites from European songbirds with remarks on improved parasite diagnostics

Abstract Background The nuclear ribosomal RNA genes of Plasmodium parasites are assumed to evolve according to a birth-and-death model with new variants originating by duplication and others becoming deleted. For some Plasmodium species, it has been shown that distinct variants of the 18S rRNA genes are expressed differentially in vertebrate hosts and mosquito vectors. The central aim was to evaluate whether avian haemosporidian parasites of the genus Haemoproteus also have substantially distinct 18S variants, focusing on lineages belonging to the Haemoproteus majoris and Haemoproteus belopolskyi species groups. Methods The almost complete 18S rRNA genes of 19 Haemoproteus lineages of the subgenus Parahaemoproteus, which are common in passeriform birds from the Palaearctic, were sequenced. The PCR products of 20 blood and tissue samples containing 19 parasite lineages were subjected to molecular cloning, and ten clones in mean were sequenced each. The sequence features were analysed and phylogenetic trees were calculated, including sequence data published previously from eight additional Parahaemoproteus lineages. The geographic and host distribution of all 27 lineages was visualised as CytB haplotype networks and pie charts. Based on the 18S sequence data, species-specific oligonucleotide probes were designed to target the parasites in host tissue by in situ hybridization assays. Results Most Haemoproteus lineages had two or more variants of the 18S gene like many Plasmodium species, but the maximum distances between variants were generally lower. Moreover, unlike in most mammalian and avian Plasmodium species, the 18S sequences of all but one parasite lineage clustered into reciprocally monophyletic clades. Considerably distinct 18S clusters were only found in Haemoproteus tartakovskyi hSISKIN1 and Haemoproteus sp. hROFI1. The presence of chimeric 18S variants in some Haemoproteus lineages indicates that their ribosomal units rather evolve in a semi-concerted fashion than according to a strict model of birth-and-death evolution. Conclusions Parasites of the subgenus Parahaemoproteus contain distinct 18S variants, but the intraspecific variability is lower than in most mammalian and avian Plasmodium species. The new 18S data provides a basis for more thorough investigations on the development of Haemoproteus parasites in host tissue using in situ hybridization techniques targeting specific parasite lineages

Evolution of a dextral lineage by left-right reversal in Cristataria (Gastropoda, Pulmonata, Clausiliidae)

It has long been debated whether mirror image-like similarity in shell morphology between enantiomorphic pairs of dextral and sinistral taxa represents their sister relationship, or each of them is closer related to other congeners with the same coiling direction. The obligate rock-dwelling genus Cristataria Vest, 1867 of the eastern Mediterranean region belongs to the Alopiinae subfamily of door snails (Clausiliidae). Cristataria and a few other genera of this subfamily include enantiomorphic pairs that are conchologically very similar to each other. Dextral C. colbeauiana (Pfeiffer, 1861) and its sinistral counterpart of such an enantiomorphic pair occur nearby one another in southern Turkey. However, the latter has been classified either as the sinistral subspecies C. colbeauiana inversa Szekeres, 1998 or as a form of sinistral C. leprevieri (Pallary, 1922). To examine the phylogenetic relationship of this enantiomorphic pair, we carried out molecular phylogenetic analysis of all the Turkish and two other Cristataria taxa based on both mitochondrial and nuclear DNA markers. Our results show that dextral C. colbeauiana and its sinistral counterpart are closest related to one another. This supports the classification of this enantiomorphic pair as dextral C. colbeauiana colbeauiana and sinistral C. colbeauiana inversa. Our results also reveal that these taxa and C. intersita Nemeth & Szekeres, 1995, sharing a characteristic collar behind the aperture of the shell, represent a monophyletic lineage. By contrast, the Cristataria species of non-collared shells belong to another clade

An integrative taxonomic approach to reveal the status of the genus Pomphorhynchus Monticelli, 1905 (Acanthocephala: Pomphorhynchidae) in Austria

Species of the genus Pomphorhynchus Monticelli, 1905 (Acanthocephala: Pomphorhynchidae) are obligate endoparasites infesting mostly freshwater fish. Morphological identification is challenging due to high intraspecific variations. The use of molecular analyses enabled new insights into the diversity and revealed high cryptic presence and unknown distribution patterns for various European species. In Austria only one species, Pomphorhynchus laevis (Müller, 1776), has been reported so far. We conduct an integrative analysis of Pomphorhynchus in Austria with a combination of morphological and molecular methods. Our results revealed the presence of three species of Pomphorhynchus in Austrian waters: Pomphorhynchus laevis, Pomphorhynchus tereticollis (Rudolphi, 1809) and Pomphorhynchus bosniacus Kiskároly and Čanković, 1967. While P. bosniacus was the predominant species in the Danube, P. laevis was recorded exclusively in Styria. Pomphorhynchus tereticollis occurred mainly in rivers of Styria except for one individual found in the Danube. We document the first occurrence of P. bosniacus and P. tereticollis in Austria. We found a high intraspecific haplotype variation in P. bosniacus suggesting that the species has a longer history in Central and Western Europe. It was previously misidentified as P. laevis, which is also true for P. tereticollis. A large number of hosts examined were infected with only juvenile and cystacanth stages suggesting paratenic infections. Our study highlights the importance of using an integrative taxonomic approach in the identification of species of Pomphorhynchus. Keywords: Acanthocephala, Pomphorhynchus spp., Austria, Fish, Autofluorescence imaging, Integrative taxonomy, 3-D reconstructio

Geographic and host distribution of haemosporidian parasite lineages from birds of the family Turdidae

Background: Haemosporidians (Apicomplexa, Protista) are obligate heteroxenous parasites of vertebrates and blood-sucking dipteran insects. Avian haemosporidians comprise more than 250 species traditionally classified into four genera, Plasmodium, Haemoproteus, Leucocytozoon, and Fallisia. However, analyses of the mitochondrial CytB gene revealed a vast variety of lineages not yet linked to morphospecies. This study aimed to analyse and discuss the data of haemosporidian lineages isolated from birds of the family Turdidae, to visualise host and geographic distribution using DNA haplotype networks and to suggest directions for taxonomy research on parasite species. Methods: Haemosporidian CytB sequence data from 350 thrushes were analysed for the present study and complemented with CytB data of avian haemosporidians gathered from Genbank and MalAvi database. Maximum Likelihood trees were calculated to identify clades featuring lineages isolated from Turdidae species. For each clade, DNA haplotype networks were calculated and provided with information on host and geographic distribution. Results: In species of the Turdidae, this study identified 82 Plasmodium, 37 Haemoproteus, and 119 Leucocytozoon lineages, 68, 28, and 112 of which are mainly found in this host group. Most of these lineages cluster in the clades, which are shown as DNA haplotype networks. The lineages of the Leucocytozoon clades were almost exclusively isolated from thrushes and usually were restricted to one host genus, whereas the Plasmodium and Haemoproteus networks featured multiple lineages also recovered from other passeriform and non-passeriform birds. Conclusion: This study represents the first attempt to summarise information on the haemosporidian parasite lineages of a whole bird family. The analyses allowed the identification of numerous groups of related lineages, which have not been linked to morphologically defined species yet, and they revealed several cases in which CytB lineages were probably assigned to the wrong morphospecies. These taxonomic issues are addressed by comparing distributional patterns of the CytB lineages with data from the original species descriptions and further literature. The authors also discuss the availability of sequence data and emphasise that MalAvi database should be considered an extremely valuable addition to GenBank, but not a replacement

Exo-erythrocytic development of avian haemosporidian parasites in European owls

Avian haemosporidian parasites (Haemosporida, Apicomplexa) are globally distributed and infect birds of many orders. These pathogens have been much investigated in domestic and wild passeriform birds, in which they are relatively easy to access. In birds belonging to other orders, including owls (order Strigiformes), these parasites have been studied fragmentarily. Particularly little is known about the exo-erythrocytic development of avian haemosporidians. The goal of this study was to gain new knowledge about the parasites infecting owls in Europe and investigate their exo-erythrocytic stages. Tissue samples of 121 deceased owls were collected in Austria and Lithuania, and examined using polymerase chain reactions (PCR), histology, and chromogenic in situ hybridization (CISH). PCR-based diagnostics showed a total prevalence of 73.6%, revealing two previously unreported Haemoproteus and five novel Leucocytozoon lineages. By CISH and histology, meronts of several Leucocytozoon lineages (lASOT06, lSTAL5, lSTAL7) were discovered in the brains, heart muscles, and kidneys of infected birds. Further, megalomeronts of Haemoproteus syrnii (lineage hSTAL2) were discovered. This study contributes new knowledge to a better understanding of the biodiversity of avian haemosporidian parasites infecting owls in Europe, provides information on tissue stages of the parasites, and calls for further research of these under-investigated pathogens relevant to bird health