First molecular data on the human roundworm Ascaris lumbricoides species complex from the Bronze and Iron Age in Hallstatt, Austria.

Palaeoparasitological studies can provide valuable information on the emergence, distribution, and elimination of parasites during a particular time in the past. In the prehistoric salt mines of Hallstatt, located in the Austrian Alps, human faeces have been conserved in salt. The aim of this study was to recover ancient DNA of intestinal parasites from these coprolites. Altogether, 35 coprolites from the Hallstatt salt mines, dating back to the Bronze Age mining phase (1158-1063 BCE) and the Iron Age mining phase (750-662 BCE), respectively, were analysed by microscopy and molecular methods. In 91% of the coprolite samples, eggs of soil-transmitted helminths (STH), namely of Trichuris and/or Ascaris were detected by light microscopy. The Ascaris eggs were exceptionally well preserved. For further analysis, DNA was extracted from the palaeofaecal samples and species-specific primers targeting different genes were designed. While amplification of Trichuris DNA remained unsuccessful, sequence data of A. lumbricoides species complex were successfully obtained from 16 coprolites from three different genes, the mitochondrial cytochrome c oxidase subunit 1 gene (cox1), the mitochondrial cytochrome B gene (cytB) and the mitochondrial NADH dehydrogenase subunit 1 gene (nadh1). Importantly, these included two Ascaris sequences from a coprolite from the Bronze Age, which to the best of our knowledge are the first molecular data of this genus from this period.

First Record of Trichobilharzia physellae (Talbot, 1936) in Europe, a Possible Causative Agent of Cercarial Dermatitis.

Several species of avian schistosomes are known to cause dermatitis in humans worldwide. In Europe, this applies above all to species of the genus Trichobilharzia. For Austria, a lot of data are available on cercarial dermatitis and on the occurrence of Trichobilharzia, yet species identification of trematodes in most cases is doubtful due to the challenging morphological determination of cercariae. During a survey of trematodes in freshwater snails, we were able to detect a species in the snail Physella acuta (Draparnaud, 1805) hitherto unknown for Austria, Trichobilharzia physellae; this is also the first time this species has been reported in Europe. Species identification was performed by integrative taxonomy combining morphological investigations with molecular genetic analyses. The results show a very close relationship between the parasite found in Austria and North American specimens (similarity found in CO1 ≥99.57%). Therefore, a recent introduction of T. physellae into Europe can be assumed.

DNA barcoding for species delimitation of the freshwater leech genus Glossiphonia from the Western Balkan (Hirudinea, Glossiphoniidae).

Glossiphoniid leeches are a diverse group and sometimes abundant elements of the aquatic fauna inhabiting various types of freshwater habitats. In this study, we sampled leeches of the genus Glossiphonia from the Western Balkan in order to test the suitability of the mitochondrial cytochrome c oxidase subunit 1 (COI) marker sequence for species delimitation. Morphological analysis revealed the presence of four taxa, G.complanata with two subspecies, G.c.complanata and G.c.maculosa, the latter an endemic of Ohrid Lake, G.nebulosa and endemic G.balcanica. In total, 29 new barcodes of Glossiphonia were sequenced in the course of this study and compared with the available molecular dataset of the latter genus from GenBank/BOLD databases. The applied ASAP distance-based species delimitation method for the analysed dataset revealed an interspecific threshold between 4-8% K2P distance as suitable for species identification purposes of the Western Balkan Glossiphonia species. Our study revealed that morphologically identified taxa as G.nebulosa and G.concolor each consists of more than one clearly different phylogenetic clade. This study contributes to a better knowledge of the taxonomy of glossiphoniid leeches and emphasises future work on the revision of this genus using a standard molecular COI marker in species identification.

Detection of Crenosoma spp., Angiostrongylus vasorum and Aelurostrongylus abstrusus in Gastropods in Eastern Austria.

Canine and feline cardiorespiratory parasites are of utmost relevance in veterinary medicine. Key epizootiological information on major pet metastrongyloids, i.e., Angiostrongylus vasorum and Crenosoma vulpis infecting dogs, and Aelurostrongylus abstrusus and Troglostrongylus brevior infecting cats, is missing from Austria. This study investigated their occurrence in 1320 gastropods collected in the Austrian provinces of Styria, Burgenland, Lower Austria, and in metropolitan Vienna. Metastrongyloid larvae were microscopically detected in 25 samples, and sequence analysis confirmed the presence of metastrongyloids in nine samples, i.e., A. vasorum in one slug (Arion vulgaris) (0.07%), C. vulpis in five slugs (one Limax maximus and four A. vulgaris) (0.4%), A. abstrusus in two A. vulgaris (0.17%), and the hedgehog lungworm Crenosoma striatum was detected in one A. vulgaris. The present study confirms the enzooticity of major cardiorespiratory nematodes in Austria and that canine and feline populations are at risk of infection.

First confirmed record of Trichobilharzia franki Müller & Kimmig, 1994, from Radix auricularia (Linnaeus, 1758) for Austria.

Avian schistosomes are of medical and veterinary importance as they are responsible for the annually occurring cercarial dermatitis outbreaks. For Austria, so far, only Trichobilharzia szidati Neuhaus 1952 was confirmed on species level as causative agent of cercarial dermatitis. Here we present the first record of Trichobilharzia franki Müller & Kimmig 1994 in Austria. The species was detected during a survey of digenean trematodes in Upper Austrian water bodies. Furthermore, we provide DNA barcodes of T. franki as well as measurements of several parasite individuals to indicate the intraspecific diversity. We also recommend the usage of an alternative primer pair, since the "standard COI primer pair" previously used for Schistosomatidae amplified an aberrant fragment in the sequence of T. franki. Overall, our study shows how limited our knowledge about occurrence and distribution of avian schistosomes in Austria is and how important it is to acquire such a knowledge to estimate ecological and epidemiological risks in the future.

Unravelling the hidden biodiversity - the establishment of DNA barcodes of fish-parasitizing Acanthocephala Koehlreuther, 1771 in view of taxonomic misidentifications, intraspecific variability and possible cryptic species.

Acanthocephalans are obligate parasites of vertebrates, mostly of fish. There is limited knowledge about the diversity of fish-parasitizing Acanthocephala in Austria. Seven determined species and an undetermined species are recorded for Austrian waters. Morphological identification of acanthocephalans remains challenging due to their sparse morphological characters and their high intraspecific variations. DNA barcoding is an effective tool for taxonomic assignment at the species level. In this study, we provide new DNA barcoding data for three genera of Acanthocephala (Pomphorhynchus Monticelli, 1905, Echinorhynchus Zoega in Müller, 1776 and Acanthocephalus Koelreuter, 1771) obtained from different fish species in Austria and provide an important contribution to acanthocephalan taxonomy and distribution in Austrian fish. Nevertheless, the taxonomic assignment of one species must remain open. We found indications for cryptic species within Echinorhynchus cinctulus Porta, 1905. Our study underlines the difficulties in processing reliable DNA barcodes and highlights the importance of the establishment of such DNA barcodes to overcome these. To achieve this goal, it is necessary to collect and compare material across Europe allowing a comprehensive revision of the phylum in Europe.

Genetic diversity of Fasciola hepatica in Austria.

The worldwide occurring common liver fluke Fasciola hepatica can infect humans and animals and leads to considerable illness and economic loss annually. The aim of this study was to determine the genetic diversity of F. hepatica in Austria. In total, 31 adult flukes isolated from cattle from various regions in Austria were investigated for their cytochrome oxidase subunit 1 (cox1) and nicotinamide dehydrogenase subunit 1 (nad1) gene sequences. It was shown that Austrian isolates of F. hepatica reveal extensive genetic diversity. To the best of our knowledge, these are the first data on the diversity of F. hepatica in Austria.

Diversity of digenean trematode larvae in snails from Lake Victoria, Kenya: First reports and bioindicative aspects.

This study investigated the occurrence of digenean trematode larvae in snails from the Kenyan part of Lake Victoria. The survey included caenogastropod snails that have received less focus in parasitological studies in Africa: their trematodes are largely unknown. Out of 1145 snail specimens, 149 (13.0%) were infected with Digenea. The highest prevalence (P) was recorded in Melanoides tuberculata (64.5%), followed by Pila ovata (15.4%), Radix natalensis (9.5%), Bulinus ugandae (9.1%), Bellamya unicolor (8.9%), Biomphalaria pfeifferi (7.3%) and Biomphalaria sudanica (4.4%). Morphological and molecular analyses revealed 17 digenean species. Contrary to reports of low diversity of Digenea in caenogastropods, P. ovata harboured 8 species - at least twice as many as in each of the pulmonates. The following taxa are reported for the first time in the Lake Victoria region: Haplorchis pumilio, Thapariella prudhoei, Nudacotyle sp., Renicola sp. and Bolbophorus sp. An unknown cercaria belonging to the genus Haematoloechus is reported from P. ovata: a xiphidiocercaria possessing a long sword-shaped stylet (47-71 µm) which does not match any available literature records. From this study, H. pumilio from M. tuberculata (P = 69.4%), Fasciola gigantica from R. natalensis (P = 1.9%) and Bolbophorus sp. from Bu. ugandae (P = 4.6%) are species of veterinary or medical importance. Snails from the study site with little direct anthropogenic influence had the highest prevalence and diversity of digenean larvae, indicating that environmental conditions influence trematode occurrence.

Genome-wide nuclear data confirm two species in the Alpine endemic land snail Noricella oreinos s.l. (Gastropoda, Hygromiidae).

The Austrian endemic land snail species Noricella oreinos (formerly Trochulus oreinos) occurs in the Northeastern Calcareous Alps at high elevations. Two morphologically highly similar subspecies N. o. oreinos and N. o. scheerpeltzi have been described. First analyses of mitochondrial and nuclear marker sequences indicated a high genetic divergence between them. In the present study, we aimed to assess gene flow between the two subspecies which should help to re-evaluate their taxonomic status. Sequence data and amplified fragment length polymorphism (AFLP) markers of 255 Noricella specimens covering the whole distribution range were analyzed. A clear geographic separation was found within the potential contact zone, the Haller Mauern mountain range. Samples of all western sites were part of the clade representing N. o. scheerpeltzi and almost all samples from the eastern sites clustered with N. o. oreinos. However, within two sampling sites of the eastern Haller Mauern, a few individuals possessed a COI sequence matching the N. o. oreinos clade whereas at the ITS2 locus they were heterozygous possessing the alleles of both taxa. Contrary to the ITS2 results indicating historical and/or ongoing hybridization, AFLP analyses of 202 individuals confirmed a clear separation of the two taxa congruent with the mitochondrial data. Although they occur on the same mountain range without any physical barrier, no indication of ongoing gene flow between the two taxa was found. Thus, we conclude that the two taxa are separate species N. oreinos and N. scheerpeltzi.

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 Cankovic, 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.

Genetic homogeneity of Fascioloides magna in Austria.

The large American liver fluke, Fascioloides magna, is an economically relevant parasite of both domestic and wild ungulates. F. magna was repeatedly introduced into Europe, for the first time already in the 19th century. In Austria, a stable population of F. magna has established in the Danube floodplain forests southeast of Vienna. The aim of this study was to determine the genetic diversity of F. magna in Austria. A total of 26 individuals from various regions within the known area of distribution were investigated for their cytochrome oxidase subunit 1 (cox1) and nicotinamide dehydrogenase subunit 1 (nad1) gene haplotypes. Interestingly, all 26 individuals revealed one and the same haplotype, namely concatenated haplotype Ha5. This indicates a homogenous population of F. magna in Austria and may argue for a single introduction. Alternatively, genetic homogeneity might also be explained by a bottleneck effect and/or genetic drift.

Investigating gene flow between the blind cavefish Garra barreimiae and its conspecific surface populations.

Cave-dwelling taxa often share the same phenotypic modifications like absence of eyes and pigmentation. These "troglomorphic characters" are expressed in the populations of Garra barreimiae from the Al Hoota Cave and nearby Hoti Pit in Northern Oman. Surface morphotypes of this cyprinid species are common throughout the distribution area. Very rarely individuals with intermediate phenotypes can be found. In the present study, potential gene flow between cave and surface populations was tested and population structure within five sampling sites was assessed. Overall, 213 individuals were genotyped at 18 microsatellite loci. We found that the cave populations have lower genetic diversity and are clearly isolated from the surface populations, which seem to be sporadically in contact with each other. The results indicate a recent genetic bottleneck in the cave populations. Thus, it can be assumed that during climatic changes the connection between cave and surface water bodies was disjoined, leaving a subpopulation trapped inside. Nevertheless, occasional gene flow between the morphotypes is detectable, but hybridisation seems only possible in cave habitat with permanent connection to surface water. Individuals from surface sites bearing intermediate phenotypes but cave genotypes imply that phenotypic plasticity might play a role in the development of the phenotype.

Indication for selfing in geographically separated populations and evidence for Pleistocene survival within the Alps: the case of Cylindrus obtusus (Pulmonata: Helicidae).

BACKGROUND: Cylindrus obtusus is one of the most prominent endemic snail species of the Eastern Alps. It is restricted to alpine meadows and calcareous rocky habitats above 1500 m. Peculiar intraspecific differences have been observed in its genital tract in the eastern populations the two mucus glands associated with the love dart sac are highly variable, while almost no variation was observed in the western populations. This raises the question whether the mode and success of reproduction of the respective populations are different. To find out whether these anatomical differences reflect genetic differentiation, which might be an indication for distinct glacial refugia, we investigated a 650 bp fragment of the mitochondrial cytochrome oxidase subunit 1 gene (COI) (280 individuals) and 9 microsatellite loci from samples (487 individuals from 29 populations) covering the whole distribution range of the species. RESULTS: The COI sequences show a geographic differentiation between eastern, central and western populations. The westernmost localities, which were covered under ice sheets during glacial periods, are characterized by extreme low variability. Overall genetic distances among all individuals are small (max. 1.7% p-distance). The microsatellite analysis reveals a high differentiation between populations, implying restriction of gene flow. The highest genetic variability was found in the central populations. Remarkably, nearly all individuals from the eastern populations, which are more variable in their genital morphology, are homozygous in all microsatellite loci, although different alleles were found within populations. CONCLUSIONS: The most peculiar outcome of the study is the strong evidence for selfing in C. obtusus as indicated by the microsatellite data in the easternmost populations. This finding is supported by the deformation of the mucus glands in the same populations. Since mucus glands play an important role in sexual reproduction, it seems plausible that in selfing organisms these structures are reduced. The phylogeographic structure revealed by COI sequences implies that the species has survived the ice ages within the Calcareous Alps. The small genetic distances among all individuals (max. 1.7%) suggest that C. obtusus has experienced severe bottlenecks in the past.

Transcriptomic and Proteomic Analysis of Arion vulgaris--Proteins for Probably Successful Survival Strategies?

The Spanish slug, Arion vulgaris, is considered one of the hundred most invasive species in Central Europe. The immense and very successful adaptation and spreading of A. vulgaris suggest that it developed highly effective mechanisms to deal with infections and natural predators. Current transcriptomic and proteomic studies on gastropods have been restricted mainly to marine and freshwater gastropods. No transcriptomic or proteomic study on A. vulgaris has been carried out so far, and in the current study, the first transcriptomic database from adult specimen of A. vulgaris is reported. To facilitate and enable proteomics in this non-model organism, a mRNA-derived protein database was constructed for protein identification. A gel-based proteomic approach was used to obtain the first generation of a comprehensive slug mantle proteome. A total of 2128 proteins were unambiguously identified; 48 proteins represent novel proteins with no significant homology in NCBI non-redundant database. Combined transcriptomic and proteomic analysis revealed an extensive repertoire of novel proteins with a role in innate immunity including many associated pattern recognition, effector proteins and cytokine-like proteins. The number and diversity in gene families encoding lectins point to a complex defense system, probably as a result of adaptation to a pathogen-rich environment. These results are providing a fundamental and important resource for subsequent studies on molluscs as well as for putative antimicrobial compounds for drug discovery and biomedical applications.

Phylogeography of the land snail genus Orcula (Orculidae, Stylommatophora) with emphasis on the Eastern Alpine taxa: speciation, hybridization and morphological variation.

BACKGROUND: The Central and Southern European mountain ranges represent important biodiversity hotspots and show high levels of endemism. In the land snail genus Orcula Held, 1837 nine species are distributed in the Alps and a few taxa inhabit the Carpathians, the Dinarids and the Western Black Sea region. In order to elucidate the general patterns of temporal and geographic diversification, mitochondrial and nuclear markers were analyzed in all 13 Orcula species. We particularly aimed to clarify whether the Alpine taxa represent a monophyletic group and if the local species diversity is rather the result of isolation in geographically separated Pleistocene glacial refuges or earlier Tertiary and Quaternary palaeogeographic events. In order to test if patterns of molecular genetic and morphological differentiation were congruent and/or if hybridization had occurred, shell morphometric investigations were performed on the Orcula species endemic to the Alps. RESULTS: The phylogenetic trees resulting from the analyses of both the mitochondrial (COI, 12S and 16S) and the nuclear (H4/H3) data sets revealed three main groups, which correspond to the three subgenera Orcula, Illyriobanatica and Hausdorfia. The reconstruction of the historic geographic ranges suggested that the genus originated in the Dinarides during the Middle Miocene and first colonized the Alps during the Late Miocene, giving rise to the most diverse subgenus Orcula. Within the latter subgenus (including all Alpine endemics) almost all species were differentiated by both molecular genetic markers and by shell morphometrics, except O. gularis and O. pseudodolium. CONCLUSIONS: The present study confirms the importance of the Alps as biodiversity hotspot and origin center of land snail diversity. The species diversity in the subgenus Orcula was likely promoted by Miocene to Pliocene palaeogeographic events and the insular distribution of preferred limestone areas. In some cases, speciation events could be linked to the divergence of populations in glacial refuges during the Pleistocene. Sporadic contact between geographically separated and reproductively not yet isolated populations led to intermixture of haplogroups within species and even hybridization and mitochondrial capture between species.

Differentiation in the Trochulus hispidus complex and related taxa (Pulmonata: Hygromiidae): morphology, ecology and their relation to phylogeography.

In this study we investigated the morphology and ecology of representatives of the taxonomically ambiguous genus Trochulus. The main focus was on the T. hispidus complex, which comprises several genetically highly divergent mitochondrial clades, as determined in a parallel molecular genetic study. We analysed shell morphology and anatomical traits and asked whether the clades are differentiated in these characters. In addition, the related species T. oreinos and T. striolatus were investigated and compared with the T. hispidus complex. Finally, we compared the ecological requirements of the taxa. Among the genetic clades of the T. hispidus complex there was no clear morphological differentiation and geographic populations could not be distinguished based on their morphology. The investigated characters of the genital anatomy did not allow discrimination of any of the T. hispidus clades and were not even diagnostic for the group as a whole. The morphotype of T. sericeus is present in all clades and thus cannot be assigned to a genetic group or any specific population. Thus, our morphological data do not provide evidence that any of the mitochondrial T. hispidus clades represent separate species. Concerning interspecific delimitation, the T. hispidus complex was clearly differentiated from T. striolatus and T. oreinos by shell morphological and anatomical characters, e.g. sculpture of shell surface and details of the penis. Finally, the habitat of T. oreinos is different from those of the other two species. In contrast to the lack of correspondence between genetic and morphological differentiation within the T. hispidus complex, related species display intraspecific morphological differentiation corresponding with mitochondrial clades: within T. striolatus there was a slight morphological differentiation between the subspecies T. s. striolatus, T. s. juvavensis and T. s. danubialis. The two subspecies of T. oreinos could be discriminated by a small but consistent difference in the cross-section of the penis. The unequal levels of intraspecific differentiation are caused by different evolutionary histories as a consequence of disparities in ecological demands, dispersal ability and use of glacial refugia: both the T. hispidus complex and T. striolatus are fast-spreading, euryoecious organisms which are able to (re-)colonize habitats and survive under different climate conditions. While the T. hispidus complex probably survived the Pleistocene in several glacial refugia, for T. striolatus one glacial refugium is suggested. Trochulus oreinos differs from the other taxa, as it is a slow disperser with a narrow ecological niche. We suggest that its subspecies spent at least the last glaciation in or close to the presently inhabited areas.

Characterization of 19 new microsatellite loci for the Omani barb Garra barreimiae from 454 sequences.

BACKGROUND: Garra barreimiae is a cyprinid fish from the southeastern Arabian Peninsula, which inhabits regularly desiccating wadis and survives in isolated ponds or underground. In 1984 a cave-dwelling population was found in the Al Hoota cave system and previous genetic analyses revealed some differentiation with limited gene flow between the surface populations and the cave population. Since no suitable markers are available for evaluation of gene flow between the cave population and the adjacent surface populations, we focused on designing and establishing novel microsatellite markers from next generation sequencing data. FINDINGS: 19 microsatellite markers containing di- and tetranucleotide simple sequence repeats were developed from 454 sequences. Forty-four individuals from two surface populations (Wadi Al Falahi and Misfat Al Abriyeen) of G. barreimiae (sampling permission number 13/2012, export permission number 29/2012) were used for analyses and characterization of the loci. On average, the number of alleles per locus is 7.6 (range: 2-20). Two markers displayed indication of linkage disequilibrium in both populations (DL6X, 9XNC). Significant deviation from Hardy-Weinberg equilibrium was observed at four loci in the Misfat Al Abriyeen population (2PUM, 88CM, 1EHE, 3Z7M) and at two loci in the Wadi Al Falahi population (QLIM, 3 N43). Three of the microsatellite loci were significant for null alleles in one of the two populations (Misfat Al Abriyeen: CJHG; Wadi Al Falahi: PH8A, 3ROZ). Expected and observed heterozygosities ranged from 0 to 95.0% respectively from 0 to 95.8% (Wadi Al Falahi) and from 0 to 89.1% respectively from 0 to 95.0% (Misfat Al Abriyeen). Fourteen of these markers were successfully cross-amplified in G. rufa. CONCLUSION: This 19 microsatellite loci provide a useful tool to understand the structure and genetic differences of populations. Moreover, these markers will help to evaluate species delimitation in G. barreimiae and potentially even in related species.

Paraphyly and budding speciation in the hairy snail (Pulmonata, Hygromiidae).

Delimitation of species is often complicated by discordance of morphological and genetic data. This may be caused by the existence of cryptic or polymorphic species. The latter case is particularly true for certain snail species showing an exceptionally high intraspecific genetic diversity. The present investigation deals with the Trochulus hispidus complex, which has a complicated taxonomy. Our analyses of the COI sequence revealed that individuals showing a T. hispidus phenotype are distributed in nine highly differentiated mitochondrial clades (showing p-distances up to 19%). The results of a parallel morphometric investigation did not reveal any differentiation between these clades, although the overall variability is quite high. The phylogenetic analyses based on 12S, 16S and COI sequences show that the T. hispidus complex is paraphyletic with respect to several other morphologically well-defined Trochulus species (T. clandestinus, T. villosus, T. villosulus and T. striolatus) which form well-supported monophyletic groups. The nc marker sequence (5.8S-ITS2-28S) shows only a clear separation of T. o. oreinos and T. o. scheerpeltzi, and a weakly supported separation of T. clandestinus, whereas all other species and the clades of the T. hispidus complex appear within one homogeneous group. The paraphyly of the T. hispidus complex reflects its complicated history, which was probably driven by geographic isolation in different glacial refugia and budding speciation. At our present state of knowledge, it cannot be excluded that several cryptic species are embedded within the T. hispidus complex. However, the lack of morphological differentiation of the T. hispidus mitochondrial clades does not provide any hints in this direction. Thus, we currently do not recommend any taxonomic changes. The results of the current investigation exemplify the limitations of barcoding attempts in highly diverse species such as T. hispidus.

In search of glacial refuges of the land snail Orcula dolium (Pulmonata, Orculidae)--an integrative approach using DNA sequence and fossil data.

Harboring a large number of endemic species, the Alps and the Western Carpathians are considered as major centers of biodiversity. Nonetheless, the general opinion until the turn of the millennium was that both Central European mountain regions did not provide suitable habitat during the Last Glacial Maximum, but were colonized later from southern refuges. However, recent molecular genetic studies provide new evidence for peripheral Alpine refuges. We studied the phylogeography of the calciphilous land snail O. dolium across its distribution in the Alps and the Western Carpathians to assess the amount of intraspecific differentiation and to detect potential glacial refuges. A partial sequence of the mitochondrial COI was analyzed in 373 specimens from 135 sampling sites, and for a subset of individuals, partial sequences of the mitochondrial 16S and the nuclear histone H3 and H4 were sequenced. A molecular clock analysis was combined with a reconstruction of the species' geographic range history to estimate how its lineages spread in the course of time. In order to obtain further information on the species' past distribution, we also screened its extensive Pleistocene fossil record. The reconstruction of geographic range history suggests that O. dolium is of Western Carpathian origin and diversified already around the Miocene-Pliocene boundary. The fossil record supports the species' presence at more than 40 sites during the last glacial and earlier cold periods, most of them in the Western Carpathians and the Pannonian Basin. The populations of O. dolium display a high genetic diversity with maximum intraspecific p-distances of 18.4% (COI) and 14.4% (16S). The existence of various diverged clades suggests the survival in several geographically separated refuges. Moreover, the sequence patterns provide evidence of multiple migrations between the Alps and the Western Carpathians. The results indicate that the Southern Calcareous Alps were probably colonized only during the Holocene.

Wherefrom and whereabouts of an alien: the American liver fluke Fascioloides magna in Austria: an overview.

The giant liver fluke Fascioloides magna, an invasive species originating from North America, was recorded in Austria in the wild for the first time in 2000. Since then, various data concerning the epidemiology in snail intermediate hosts and cervid final hosts have been reported. Galba truncatula acts as snail intermediate host, and red deer, roe deer and fallow deer act as final hosts. G. truncatula is abundant throughout the region, especially along muddy shores of slow-flowing branches of the river system. Prevalence in deer (20-100 %) is much higher than in snails (0.03-0.2 %). Despite medical treatment of parts of the deer population, the parasite has successfully established itself on both sides of the Danube floodplain environments southeast of Vienna. Genetic analysis revealed that the infection of Austrian deer populations apparently originated from foci in the Czech Republic or from populations of Danube tributaries. Areas adjacent southwards, which will soon be joined through wildlife crossings, have not yet evidenced F. magna. Nonetheless, these environments are inhabited by host snails and deer and therefore constitute suitable habitats for F. magna. Invading alien parasites not only threaten native individual hosts but also influence host populations, thus potentially also modifying parasite communities and interactions. The host range of F. magna includes a variety of potential hosts, notably other Lymnaeidae as potential intermediate hosts and various ungulates, including sheep and cattle, as final hosts. Because eradication after medical treatment was unsuccessful, and due to the risk of further spread of the parasite into unaffected regions, enhanced control strategies need to be developed. We recommend assessment of introduction pathways and dispersal, continuous monitoring of host abundance and distribution and the prevalence of flukes in intermediate and final hosts, as well as coordinated and concerted actions with neighbouring countries. This strategy could help to reduce potential negative impacts of this and other invasive parasites on host populations in Europe.