DNA Internal Transcribed Spacer Region Research Articles

Ancient DNA analysis of desiccated wheat grains excavated from a Bronze Age cemetery in Xinjiang

Wheat has been one of the most important crop in Eurasia since the Neolithic period. Understanding the spread of wheat cultivation is crucial to understanding the spread of agriculture as a whole and the interactions between prehistoric populations across the Eurasian continent. However, the routes by which wheat cultivation spread eastwards have been poorly understood to date, due to the scarcity of plant remains recovered from archaeological sites. Desiccated wheat grains excavated from the Xiaohe cemetery in Xinjiang, and dated to the early Bronze Age, show excellent DNA preservation. Here we present an ancient DNA (aDNA) analysis of wheat ( Triticum sp.) grains excavated from Xiaohe and provide the first definitive evidence for bread wheat in China during the Bronze Age. The nuclear ribosomal DNA internal transcribed spacer regions (ITS1 and ITS2) and the intergenic spacer region (IGS) were amplified. The IGS region within the D genome of wheat has a 71 bp insertion that is absent from corresponding regions in the A and B genomes. The results showed that the Xiaohe wheat showed most sequence similarity to hexaploid bread wheat ( Triticum aestivum), including the characteristic insertion into the D genome. The presence of bread wheat at the Xiaohe cemetery is discussed in relation to it having spread into Xinjiang by the Bronze Age, providing new insight into the origins of bread wheat in East Asia.

Molecular phylogenetic analysis of Leibnitzia Cass. (Asteraceae: Mutisieae: Gerbera-complex), an Asian-North American disjunct genus

Abstract Leibnitzia comprises six species of perennial herbs that are adapted to high elevation conditions and is one of only two Asteraceae genera known to have an exclusively disjunct distribution spanning central to eastern Asia and North America. Molecular phylogenetic analysis of Leibnitzia and other Gerbera-complex members indicates that Leibnitzia is monophyletic, which is in contrast with our expectation that the American Leibnitzia species L. lyrata and L. occimadrensis would be more closely related to another American member of the Gerbera-complex, namely Chaptalia. Ancestral area reconstructions show that the historical biogeography of the Gerbera-complex mirrors that of the entire Asteraceae, with early diverging lineages located in South America that were followed by transfers to Africa and Eurasia and, most recently, to North America. Intercontinental transfer of Leibnitzia appears to have been directed from Asia to North America. Independent calibrations of nuclear (ribosomal DNA internal transcribed spacer region) and chloroplast (trnL–rpl32 intron) DNA sequence data using relaxed clock methods and either mean rate or fossil-based priors unanimously support Miocene and younger divergence times for Gerbera-complex taxa. The ages are not consistent with most Gondwanan vicariance episodes and, thus, the global distribution of Gerbera-complex members must be explained in large part by long-distance dispersal. American species of Leibnitzia are estimated to have diverged from their Asian ancestor during the Quaternary (ca. 2 mya) and either migrated overland to North America via Beringia and retreated southwards along high elevation corridors to their present location in southwestern North America or were dispersed long distance.

Multilocus species identification and fungal DNA barcoding: insights from blue stain fungal symbionts of the mountain pine beetle

There is strong community-wide interest in applying molecular techniques to fungal species delimitation and identification, but selection of a standardized region or regions of the genome has not been finalized. A single marker, the ribosomal DNA internal transcribed spacer region, has frequently been suggested as the standard for fungi. We used a group of closely related blue stain fungi associated with the mountain pine beetle (Dendroctonus ponderosae Hopkins) to examine the success of such single-locus species identification, comparing the internal transcribed spacer with four other nuclear markers. We demonstrate that single loci varied in their utility for identifying the six fungal species examined, while use of multiple loci was consistently successful. In a literature survey of 21 similar studies, individual loci were also highly variable in their ability to provide consistent species identifications and were less successful than multilocus diagnostics. Accurate species identification is the essence of any molecular diagnostic system, and this consideration should be central to locus selection. Moreover, our study and the literature survey demonstrate the value of using closely related species as the proving ground for developing a molecular identification system. We advocate use of a multilocus barcode approach that is similar to the practice employed by the plant barcode community, rather than reliance on a single locus.

Identification, distribution and current taxonomy of Botryosphaeriaceae species associated with grapevine decline in New South Wales and South Australia

Background and Aims: Botryosphaeriaceae species are recognised as important pathogens of grapevines both in Australia and overseas. The identity, prevalence and distribution of Botryosphaeriaceae species in vineyards throughout the major winegrowing regions of New South Wales (NSW) and South Australia (SA) was determined to provide a foundation for improved disease prevention and management. Methods and Results: Field surveys from 91 vineyards across NSW and SA resulted in the collection of 2239 diseased wood samples and subsequent isolation of 1258 Botryosphaeriaceae isolates. Morphological identification along with phylogenetic analysis of ribosomal DNA internal transcribed spacer regions (ITS1-5.8S-ITS2) and partial sequences of the translation elongation factor 1-α gene (EF1-α) showed that eight Botryosphaeriaceae species from four phylogenetic lineages occur on grapevines in eastern Australia, including Diplodia seriata, Diplodia mutila, Lasiodiplodia theobromae, Neofusicoccum parvum, Neofusicoccum australe, Botryosphaeria dothidea, Dothiorella viticola and Dothiorella iberica. Conclusions: The prevalence of individual species varied according to geography and climate. Species of Diplodia and Dothiorella, characterised by thick-walled, pigmented conidia were the most prevalent and were distributed widely throughout both NSW and SA. Species with hyaline conidia, such as Neofusicoccum and Fusicoccum, were isolated less frequently and displayed more limited geographic ranges, whilst only a single isolate of Lasiodiplodia was recovered, this being from the northern most region of NSW. Significance of the Study: The identification of eight taxa within the Botryosphaeriaceae, and their distributions throughout south-eastern Australia was established and discussed in context with climate, reported optimum growth temperatures, and more recent taxonomic and nomenclatural revisions. We established a sound base for control strategies based on the prevailing species in Australian viticultural regions.

Two extinct diploid progenitors were involved in allopolyploid formation in the Hordeum murinum (Poaceae: Triticeae) taxon complex

Wall barley ( Hordeum murinum) occurs with three subspecies, naturally distributed from southern Central Asia through the Mediterranean region to northwestern Europe, but now is an invasive weed in many parts of the world. Subspecies glaucum is diploid, while subspp. murinum and leporinum are tetraploids, the latter also occurring with a hexaploid cytotype. Earlier analyses were inconclusive regarding auto- or allopolyploid origins of subspp. murinum and leporinum. We analyzed the phylogeny of the taxon group using amplified fragment length polymorphisms (AFLP), sequences of cloned PCR products of the nuclear ribosomal DNA internal transcribed spacer region (ITS), a part of the nuclear single-copy gene topoisomerase 6 (Topo6) spanning two introns, and sequences of the chloroplast trnL-F region together with length variation at six chloroplast microsatellite loci, including multiple individuals of each subspecies and cytotype, covering the entire natural distribution area of the species. Phylogenetic analyses with all used markers differentiate diploid and polyploids. Sequences of both nuclear regions indicated that diploid subsp. glaucum was involved in tetraploid formation together with a now extinct species belonging to the same Hordeum genome group (Xu). Furthermore, AFLP and ITS analyses suggest that a third, though closely related extinct taxon contributed to hexaploid formation. No method was able to discern tetraploid subspp. murinum and leporinum, which we attribute to the young age of subsp. murinum. None of the used molecular markers revealed a strong geographic pattern of genetic variation that would allow comprehensive phylogeographic analysis, most probably due to the very effective seed dispersal of the taxa.

Host Specialization and Phylogenetic Diversity of Corynespora cassiicola

The fungus Corynespora cassiicola is primarily found in the tropics and subtropics, and is widely diverse in substrate utilization and host association. Isolate characterization within C. cassiicola was undertaken to investigate how genetic diversity correlates with host specificity, growth rate, and geographic distribution. C. cassiicola isolates were collected from 68 different plant species in American Samoa, Brazil, Malaysia, and Micronesia, and Florida, Mississippi, and Tennessee within the United States. Phylogenetic analyses using four loci were performed with 143 Corynespora spp. isolates, including outgroup taxa obtained from culture collections: C. citricola, C. melongenae, C. olivacea, C. proliferata, C. sesamum, and C. smithii. Phylogenetic trees were congruent from the ribosomal DNA internal transcribed spacer region, two random hypervariable loci (caa5 and ga4), and the actin-encoding locus act1, indicating a lack of recombination within the species and asexual propagation. Fifty isolates were tested for pathogenicity on eight known C. cassiicola crop hosts: basil, bean, cowpea, cucumber, papaya, soybean, sweet potato, and tomato. Pathogenicity profiles ranged from one to four hosts, with cucumber appearing in 14 of the 16 profiles. Bootstrap analyses and Bayesian posterior probability values identified six statistically significant phylogenetic lineages. The six phylogenetic lineages correlated with host of origin, pathogenicity, and growth rate but not with geographic location. Common fungal genotypes were widely distributed geographically, indicating long-distance and global dispersal of clonal lineages. This research reveals an abundance of previously unrecognized genetic diversity within the species and provides evidence for host specialization on papaya.

Geographic Distribution and rDNA-ITS Region Sequence Diversity of Waitea circinata var. circinata Isolated from Annual Bluegrass in the United States.

Waitea circinata var. circinata is the causal agent of brown ring patch, an emergent disease of turfgrass in the United States. Forty-two isolates from annual bluegrass were obtained from California, Connecticut, Idaho, Illinois, Massachusetts, New York, Ohio, Oregon, and Rhode Island. Almost all isolates produced white to orange sclerotia (bulbils), 2 to 5 mm in size, that turned dark brown after 21 days on ¼-strength potato dextrose agar. The ribosomal DNA internal transcribed spacer regions and 5.8S region (ITS) were analyzed by restriction fragment length polymorphism (RFLP) analysis using MspI and sequencing to attempt identification of the isolates. Some isolates were heterozygous at the MspI restriction site, results not found in previous reports using the RFLP technique for identification. Four additional nucleotide positions were found to be variable within ITS based on sequence analysis, including two indels and two additional heterozygous positions. A total of 17 ITS haplotypes were found, and there was no obvious relationship between ITS haplotype and the geographic distribution of the isolates. Results of this work indicate that W. circinata var. circinata is present in multiple states and provide an initial understanding of the diversity of the pathogen in the United States.

Phylogenetic Relationships within the <I>Orychophragmus violaceus</I> Complex (Brassicaceae) Endemic to China

A first insight into the phylogenetic analysis of the Orychophragmus violaceus complex was constructed from complete sequences of nuclear ribosomal DNA internal transcribed spacer regions (ITS/5.8S) and chloroplast gene matK. The ITS results strongly supported the division of the O.violaceus complex into two major groups: one is composed of O.hupehensis, O.taibaiensis, the other consists of O.violaceus and O.diffusus. The matK phylogenetic trees showed O.diffusus and O.violaceus were the basal members of the complex. From previous karyotype study and the present results, we considerated that the groups of O.hupehensis and O.taibaiensis should not be allocated in O.violaceus. And two possible ancestral cytotypes were also confirmed by our phylogenetic analysis. Moreover, the biogeographic analysis implied a presumed center of recent differentiation in the region from upper to mid Changkiang River to Qin Mountain.

Optimizing the selection process of yeast starter cultures by preselecting strains dominating spontaneous fermentations

We propose an efficient and time-saving strategy for starter culture selection. Our approach is based on the accomplishment of 3 phases: (i) the selection of yeast strains dominating spontaneous fermentations, (ii) the selection among the dominant strains of those showing the best technological characteristics, and (iii) the final selection among good technological strains of those showing the desired qualitative traits. We applied this approach to wine fermentations, even though the same strategy has the potential to be employed for the selection of any type of starter culture. We isolated and identified yeast strains at the mid- and final stages of 6 spontaneous fermentations carried out in 3 different Spanish wineries. We identified all strains as Saccharomyces cerevisiae by restriction fragment length polymorphism of the ribosomal DNA internal transcribed spacer region, and subsequently distinguished each strain by analyzing the polymorphism of the inter-delta regions. Strains that were detected both at the mid- and final stages of the fermentation were considered dominant. Four dominant strains were finally selected and tested in pilot-scale fermentation, and their performance was compared with that of a commercial wine strain. All dominant strains showed good fitness and resulted suitable to be employed as starter cultures. One of the dominant strains isolated in this study is currently commercialized.

Whole genome amplification of the rust Puccinia striiformis f. sp. tritici from single spores

Rust fungi are obligate parasites and cannot be routinely cultured to obtain sufficient biomass for DNA extractions. Multiple displacement amplification (MDA) was demonstrated in this study for whole genome amplification from single spores of the rust fungus, Puccinia striiformis. The genomic DNA coverage and fidelity of this method was evaluated by PCR amplification and sequencing of two genetic markers: portions of the multi-copy nuclear ribosomal DNA internal transcribed spacer region (ITS) and the single copy β-tubulin gene from two geographical diverse isolates. Our results show that MDA is a valuable tool for whole genome amplification from single spores, and we propose that MDA-amplified DNA can be used for molecular genetic analysis of the wheat yellow rust fungus.

Hybridization and Gene Flow Between Distantly Related Species of Rubus (Rosaceae): Evidence from Nuclear Ribosomal DNA Internal Transcribed Spacer Region Sequences

Abstract Hybridization has long been considered a source of taxonomic complexity in Rubus. This study uses molecular data to examine natural hybridization between R. caesius, a facultatively agamospermous tetraploid of subgenus Rubus (blackberries) and R. idaeus, a sexual diploid of subgenus Idaeobatus (raspberries). Both species are widespread in Europe, and hybridization between them is postulated to have given rise to numerous species, including R. maximiformis and R. picticaulis. We sequenced the nuclear ribosomal DNA internal transcribed spacer (ITS) region of R. caesius, R. idaeus, five putative hybrids from Sweden, and one individual each of R. maximiformis and R. picticaulis from Germany. Rubus caesius differs from R. idaeus at 20 nucleotide sites and one insertion/deletion event. The putative hybrids exhibit nucleotide polymorphism additivity at all or most of these sites indicating that gene flow does occur naturally between R. caesius and R. idaeus. For each of five putative hybrids we analyzed...

Postharvest Black Spot Disease in Broccoli Caused by Alternaria brassicicola

Broccoli in markets frequently suffers from black spot disease and rapidly turns yellow, thus losing commercial value. To clarify the causal pathogen, we isolated micro-organisms from infected broccoli and examined them for their pathogenicity using an inoculation assay. The pathogenic isolates obtained were further examined and identified using morphological and molecular characteristics. For molecular analyses, the nucleotide sequences of ribosomal DNA internal transcribed spacer regions were determined, and specific gene sequences were amplified using PCR. The highly pathogenic isolate obtained was identified as Alternaria brassicicola HLYB-4. Vegetative hyphae of the isolate slowly elongate and produce spores, even during cold storage (5 °C). Optimal hyphal growth was observed at 25 °C. Both ethylene production and chlorophyll degradation of diseased broccoli increased after infection with this isolate, promoting senescence of florets. However, higher temperatures (35 °C) severely restricted growth of the pathogen.

Fungal associates of Pyrola rotundifolia, a mixotrophic Ericaceae, from two Estonian boreal forests

Pyrola rotundifolia (Ericaceae, Pyroleae tribe) is an understorey subshrub that was recently demonstrated to receive considerable amount of carbon from its fungal mycorrhizal associates. So far, little is known of the identity of these fungi and the mycorrhizal anatomy in the Pyroleae. Using 140 mycorrhizal root fragments collected from two Estonian boreal forests already studied in the context of mixotrophic Ericaceae in sequence analysis of the ribosomal DNA internal transcribed spacer region, we recovered 71 sequences that corresponded to 45 putative species in 19 fungal genera. The identified fungi were mainly ectomycorrhizal basidiomycetes, including Tomentella, Cortinarius, Russula, Hebeloma, as well as some ectomycorrhizal and/or endophytic ascomycetes. The P. rotundifolia fungal communities of the two forests did not differ significantly in terms of species richness, diversity and nutritional mode. The relatively high diversity retrieved suggests that P. rotundifolia does not have a strict preference for any fungal taxa. Anatomical analyses showed typical arbutoid mycorrhizae, with variable mantle structures, uniseriate Hartig nets and intracellular hyphal coils in the large epidermal cells. Whenever compared, fungal ultrastructure was congruent with the molecular identification. Similarly to other mixotrophic and autotrophic pyroloids in the same forests, P. rotundifolia shares its mycorrhizal fungal associates with surrounding trees that are likely a carbon source for pyroloids.

Phylogeny and biogeography of Apiaceae tribe Oenantheae inferred from nuclear rDNA ITS and cpDNApsbI–5′trnK(UUU)sequences, with emphasis on the North American Endemics clade

Intergeneric phylogenetic relationships within Apiaceae tribe Oenantheae were investigated using sequence data from the chloroplast DNA psbI–5′trnK(UUU)and nuclear ribosomal DNA internal transcribed spacer regions. One hundred and thirty-one accessions were examined, representing all 17 genera of the tribe and approximately one-half of its species. The cpDNA region includes four intergenic spacers and the rps16 intron and these noncoding loci were analyzed separately to assess their relative utility for resolving relationships. Separate maximum parsimony analyses of the entire psbI–5′trnK(UUU)and ITS regions, each with and without scored indels, yielded concordant trees. Phylogenies derived from maximum parsimony, Bayesian, or maximum likelihood analyses of combined chloroplast and nuclear DNA sequences for 82 accessions were highly resolved, well supported, and consistent. Among the five noncoding loci examined, the trnQ(UUG)–5′rps16 and 3′rps16–5′trnK(UUU)intergenic spacers are the most variable, with the latter contributing the greatest total number of parsimony informative characters relative to its size. The North American genera Atrema , Cynosciadium , Daucosma , Limnosciadium , Neogoezia , Oxypolis , Ptilimnium , and Trepocarpus ally with the western hemispheric and Australasian genus Lilaeopsis in a strongly supported North American Endemics clade that is a sister group to a clade composed primarily of Old World taxa ( Berula sensu lato, Cryptotaenia , Helosciadium , and Sium ). Oxypolis and Ptilimnium are not monophyletic, with the rachis-leaved members of each comprising a clade separate from their compound-leaved congeners. Dispersal-vicariance analysis suggests that the ancestors of the North American Endemics clade probably originated in Canada and the USA or in a broader ancestral area including Mexico and South America.

Phytophthora irrigata, a new species isolated from irrigation reservoirs and rivers in Eastern United States of America

A new species of Phytophthora, previously referred to as the 'Dre I' taxon, is named Phytophthora irrigata. Isolates of P. irrigata morphologically and physiologically resemble Phytophthora drechsleri. They are heterothallic, produce nonpapillate sporangia, and grow well at 35 degrees C. The above two species differ significantly in uniformity of mycelium, presence of chlamydospores, DNA fingerprint, and sequences of multiple nuclear and mtDNA regions. Phytophthora irrigata produces smaller sporangia and a distinct DNA fingerprint. Sequence alignments in the ribosomal DNA internal transcribed spacer regions place Phytophthora fallax and Phytophthora captisoa as its closest relatives. The optimal temperature for culture growth is above 30 degrees C and the maximum temperature is 40 degrees C. This new species is abundant in irrigation reservoirs and natural waterways in Virginia and was also isolated in Pennsylvania.

Changes in frequency of agents of tinea capitis in school children from Western China suggest slow migration rates in dermatophytes

Tinea capitis is a common dermatophyte infection of the scalp of children in Western China, with the gray-patch from being the most prevalent. Twenty years ago, the most widespread etiologic agent was reported to be Trichophyton violaceum, which was later succeeded by Microsporum ferrugineum and Trichophyton schoenleinii. In the framework of our recent study, 97 isolates were collected from patients with clinically suspected tinea capitis. Identification was performed by conventional methods and by sequencing the ribosomal DNA internal transcribed spacer region. In the case of T. violaceum an additional microsatellite primer set (T1) was used. Five species (in order of frequency, Trichophyton violaceum, T. schoenleinii, Microsporum ferrugineum, zoophilic strains of Arthroderma vanbreuseghemii, and Trichophyton tonsurans) were identified. Results of molecular and phenotypic ID of the same strains showed good correspondence. Comparison with earlier data showed that dermatophytes species in former rural societies must have migrated extremely slowly. Preponderance of local transmission from domesticated animals was proven by the occurrence of zoophilic strains of Arthroderma vanbreuseghemii. Etiologic agents in the rural communities of Western China tend to be different from those of the other regions in the country, despite modern communication and traffic.

Development of Nested Polymerase Chain Reaction Detection ofMycosphaerellaspp. and Its Application to the Study of Leaf Disease inEucalyptusPlantations

ABSTRACT Mycosphaerella leaf disease (MLD) is a serious disease of two of the major eucalypt species grown in temperate regions worldwide, Eucalyptus globulus and E. nitens. More than 30 species of Mycosphaerella have been reported on eucalypts worldwide. Accurate, rapid, and early discrimination of Mycosphaerella spp. causing crown damage to E. globulus and E. nitens will assist the development of sustainable management strategies. This study describes the development, and incorporation in a nested polymerase chain reaction (PCR) approach, of specific primers for the detection and identification of Mycosphaerella spp. commonly reported from leaf lesions of E. globulus and E. nitens in Australia. Primer design was assisted by sequence alignment and phylogenetic analysis of 165 nonredundant sequences from the nuclear ribosomal DNA internal transcribed spacer regions of Mycosphaerella and related species. Phylo-genetic analysis revealed very high sequence similarity for two taxon groups, Mycosphaerella grandis and M. parva, and M. vespa, M. ambi phylla, and M. molleriana, and primers were designed to differentiate each of the two groups. Three other species, M. cryptica, M. nubilosa, and M. tasmaniensis, were distinct and distinguished by species-specific primers. In double-blind trials, the detection test accurately and rapidly identified Mycosphaerella spp. in cultures and discriminated against other pathogens that co-occur in or on Eucalyptus leaves, thereby verifying its reliability. The detection test has an internal amplification control in the first-round PCR with fungal-specific primers to raise confidence in test results, particularly to highlight negative results due to PCR inhibition. When applied to DNA extracted from leaf or stem samples either as multiple or single lesions, it detected and identified up to five Mycosphaerella spp. or taxon groups in both positively identified and in young (putative) MLD lesions. The samples were 20 mm(2) or larger in surface area and were collected while undertaking disease rating assessments in an experimental investigation of Eucalyptus plantations and regrowth forest. Using nested PCR detection, Mycosphaerella spp. were positively identified in 2 days, 1 to 5 months earlier than by classical methods, demonstrating the potential application of this detection test to the early discrimination of MLD components in ecological, epidemiological, and genetic investigations.

In Vitro Propagation of Two Perkinsus spp. Parasites from Japanese Manila Clams Venerupis philippinarum and Description of Perkinsus honshuensis n. sp.

Perkinsus species are destructive parasites of commercial Manila clams, Venerupis philippinarum, in Japan, Korea, and Spain. However, in vitro parasite cultures from this important host clam are not available. Tissues of Manila clams collected during April 2002 in Gokasho Bay, Japan harbored Perkinsus sp. parasites at a 97% prevalence (28/29) of moderate- and high-intensity infections. Perkinsus sp. cells in tissue samples were enlarged in alternative Ray's fluid thioglycollate medium, before propagation in DME:Ham's F-12 Perkinsus sp. culture medium. Enlarged parasite hypnospores zoosporulated at high frequencies to release motile zoospores, which gave rise to continuous schizogonic cell lines that also zoosporulated continuously at low frequencies. Four Perkinsus sp. in vitro isolates comprising two distinct morphotypes were cryopreserved, cloned, and archived for public distribution. For three isolates of one morphotype, nucleotide sequences of the ribosomal DNA internal transcribed spacer region, of the large subunit rRNA gene, and of actin genes, were consistent with those reported for P. olseni. Similar sequences from one morphologically unique isolate differed from those of all described Perkinsus species. These results show that at least two Perkinsus spp. infect Japanese Manila clams, and that one represents a new species, Perkinsus honshuensis n. sp.

An overview of fungal community diversity in diseased hop plantations

Samples were taken from several hop fields presenting various symptoms. Fifty-nine pure filamentous fungal strains were isolated and identified through genomic DNA preparations, PCR amplification of the ribosomal DNA internal transcribed spacer region and database interrogations. The most frequent genera were Alternaria (16 isolates) and Epicoccum (14 isolates). The ecosystem was shown to be very diverse, since as many as 27 species belonging to 17 genera were recovered. Furthermore, many of the isolated fungi are known to be involved in phytopathogenesis.

FIRST ANALYSIS OF GENETIC VARIABILITY IN CARINTHIAN POPULATIONS OF THE WHITE-CLAWED CRAYFISH AUSTROPOTAMOBIUS PALLIPES

Several authors have speculated about the nativeness of the white-clawed crayfish (Austropotamobius pallipes) in the Austrian province of Carinthia, since this is the only occurrence of the species within the Danube river system. Phylogeographic analyses based on mitochondrial DNA sequences showed that Carinthian A. pallipes are closely related to specimens from Slovenia, Croatia and north-eastern Italy. These analyses additionally pointed to an absence of variability among populations in Carinthia. In the present study a larger number of individuals sampled from three different brooks in western Carinthia was investigated by analysing sequences of the mtDNA cytochrome c oxidase gene (COI), microsatellite DNA and microsatellite length variation in the nuclear ribosomal DNA internal transcribed spacer region (ITS1). Not only COI sequences revealed low degrees of genetic variability, but also microsatellite loci displayed low allelic diversity and low heterozygosity. Analysis of ITS1 microsatellite length variation in turn showed high levels of intragenomic variability, since single individuals manifested up to seven distinct fragments. Microsatellite data and ITS1 microsatellite length variation analyses also revealed significant differentiation among some population pairs. Since genetic divergence was mainly based on differences in allele frequencies and not on the occurrence of alternative alleles, we assume that Carinthian populations became isolated rather recently and that the occurrence of this species in this region is more likely to be a result of human introduction than of natural postglacial colonisation.