Austropotamobius Torrentium Research Articles

A new perspective on the molecular dating of the stone crayfish with an extended phylogeographic information on the species

Reconstructing the origin and historical biogeography of the Austropotamobius torrentium is hampered by insufficient phylogeographic coverage of the Balkans and deep contradictions in previous molecular dating. The present work extends the phylogeographic coverage to Serbia, a country crucial for understanding the species southward dispersal. Our analysis revealed that the Southern Balkans lineage occurs in most of the country, the Central and southeastern Europe lineage is restricted to the southwest and northeast of the country, while a single population in the north of the country harbors the Lika and Dalmatia lineage, which was previously thought to be restricted to the northern-central Dinarides. Dataset expansion led to revised phylogenetic relationships, which indicated that the Apuseni lineage is not nested within Northern-central Dinarides lineages but arose after the most basal split within Austropotamobius torrentium. This ‘Apuseni first’ phylogeny provides a new perspective for molecular dating, according to which the split between Austropotamobius pallipes and A. torrentium took place in the Late Oligocene, while the formation of the phyletic lineages and the dispersal from the Dinarides to Serbia occurred in the late Miocene and is probably associated with the complex and protracted process of disintegration of the Neogene freshwater lakes in southeastern Europe.

English

Abstract For the stone crayfish, a threatened species listed in the Habitats Directive in Annex II and V, three monitoring methods were evaluated: hand capture by day, hand capture by night and eDNA sampling respectively. The lack of a standardized sampling method in Austria and the simultaneous obligation to monitor and report on the status of protected species requires therefore an evaluation of the different techniques. However, data show that day sampling massively favored the observation of juveniles whereas night sampling obtained higher shares of adults and males. Moreover, organic layer primarily determines the applicability of hand capturing methods. If the share of organic layer exceeded 40%, night sampling was more effective. In brooks with low amount of organic layer, no notable divergences were found. eDNA sampling provided reliable presence-absence data but cannot fully replace hand capturing methods. Data indicate that eDNA detectability depends probably on the discharge level. Below 1.5 L·s-1 eDNA detection failed but provided reliable results in sections with a higher discharge regime. In summary, all three methods are appropriate for crayfish surveillance programs, but must be applied according to the sampling design appropriate for the research questions and with an awareness of their strengths and weaknesses.

Declining populations of Astacus astacus drag European Branchiobdella parasita (Annelida: Clitellata) ectosymbionts to serious biodiversity loss

Branchiobdella parasita is specific ectosymbiont of European freshwater crayfish, including endangered noble crayfish (Astacus astacus). Although numerous information on distribution, biology or ecology were reported, genetic structure of B. parasita in European crayfish populations was not studied truthfully. The available molecular data include only cytochrome oxidase subunit I (COI) barcodes of B. parasita coexisting with noble crayfish in Alpine freshwaters in Austria, as well as stone crayfish (Austropotamobius torrentium) and white-clawed crayfish (Austropotamobius pallipes) in Balkan region. Here we report first genetic diversity and haplotype richness analysis of B. parasita populations on a pan-European scale. Specimens (n = 41) were collected from female noble crayfish from three lakes in northwest Poland (Kołtki Małe, Rosko Duże, Skrzynki Małe) and Elkenrother Weiher Lake in southwest Germany, high-quality COI barcodes were obtained and analysed against existing genetic data. The analysed populations were haplotype rich (h = 23) and had high COI haplotype diversity (Hd = 0.8988, I = 1.4554). Our results indicate that diversification rate of European B. parasita populations highly depends on habitat quality, its connection with surface waters and hosts species and mobility. We revealed that Central European populations inhabiting noble crayfish show significant genetic relationship, presenting common ancestral haplotypes and at the same time high genetic divergence from Balkan populations. Based on our results we suggest that ‘parasita-complex’ of phylogenetically related species with similar body shape and structure of chitin jaws, but with differentiating anatomical structure details are present across the continent.

A new species ofAustropotamobiusSkorikov, 1907 (Decapoda: Astacidae: Astacidae) from the late Miocene (Messinian) of Slovenia, with remarks on the evolution of European crayfishes

AbstractCrayfishes are rather rare in the fossil record, limiting our understanding of their evolutionary history. We describe and discuss a fossil crayfish from the Miocene of Slovenia. This fossil, comprising only the cephalothorax, first pereiopod, and another cheliped, seems closely allied to AustropotamobiusSkorikov, 1907, but also bears an epistome and postorbital carina reminiscent of AstacusFabricius, 1775. For these reasons, it is tentatively assigned to Austropotamobius. It differs from all other Austropotamobius species by the shapes of its epistome cephalic lobe, and postorbital carina, and resemble Austropotamobius torrentium (von Paula Schrank, 1803) most due to its ornamentation. It is therefore described as a new species, Austropotamobius plenicarisp. nov. The new species is associated with brackish water ostracods, most likely reworked from slightly older sediments, suggesting a late Miocene (Messinian) age. The origin of this species and its age show it lived long after the split between Astacus and Austropotamobius, and also probably after the split between the two Austropotamobius species (Au. pallipes and Au. torrentium), and may have been contemporary to, and lived in the area of the diversification of Au. torrentium haplotypes.

Characteristics of the Stone Crayfish Population along a Disturbance Gradient—A Case Study of the Kustošak Stream, Croatia

The stone crayfish, Austropotamobius torrentium, is a native European freshwater species sensitive to habitat alterations such as stream channelization and urban development, putting its populations at risk. This study aims to assess how habitat alteration and subsequent water quality changes affect the stone crayfish population in the Kustošak Stream (Croatia) through three selected sites under different levels of anthropogenic disturbance: (1) undisturbed; (2) recently modified, and (3) long-time modified sites. At each site, crayfish were captured, measured, and marked to estimate population size, structure, and crayfish condition. Additionally, we examined whether water quality (18 measured physicochemical parameters) affects relative crayfish abundance. We also used mitochondrial sequencing and microsatellite genotyping to assess species genetic diversity and population connectivity and to compare results among the sites. The results showed that habitat alteration caused an overall reduction in population abundance and changes in size structure; however, it had not yet resulted in detectable genetic differences. Partial least squares regression showed that crayfish abundance was affected by the physicochemical parameters of water, including, among others, oxygen, calcium ions, nitrates, pH, and water temperature. We discuss our findings in the context of the effects of anthropogenic disturbance on the viability and persistence of this EU priority species.

The Coding Mitogenome of the Freshwater Crayfish Pontastacus leptodactylus (Decapoda:Astacidea:Astacidae) from Lake Vegoritida, Greece and Its Taxonomic Classification.

Pontastacus leptodactylus (Eschscholtz, 1823) (Decapoda:Astacidea:Astacidae) constitutes an ecologically and economically highly important species. In the present study, the mitochondrial genome of the freshwater crayfish P. leptodactylus from Greece is analyzed for the first time, using 15 newly designed primer pairs based on available sequences of closely related species. The analyzed coding part of the mitochondrial genome of P. leptodactylus consists of 15,050 base pairs including 13 protein-coding genes (PCGs), 2 ribosomal RNA gene (rRNAs), and 22 transfer RNA genes (tRNAs). These newly designed primers may be particularly useful in future studies for analyzing different mitochondrial DNA segments. Based on the entire mitochondrial genome sequence, compared to other haplotypes from related species belonging in the same family (Astacidae) available in the GenBank database, a phylogenetic tree was constructed depicting the phylogenetic relationships of P. leptodactylus. Based on the results, the genetic distance between Astacus astacus and P. leptodactylus is smaller than the genetic distance between Austropotamobius pallipes and Austropotamobius torrentium, despite the fact that the latter two are classified within the same genus, questioning the phylogenetic position of A. astacus as a different genus than P. leptodactylus. In addition, the sample from Greece seems genetically distant compared with a conspecific haplotype available in the GenBank database, possibly implying a genetic distinction of P. leptodactylus from Greece.

New data on the Austropotamobius torrentium (Schrank, 1803) from the tributaries of the Bosna River

The crayfish Austropotamobius torrentium is a dominant species of the family Astacidae in the rivers of the Black Sea and Adriatic basins of Bosnia and Herzegovina. Molecular phylogenetic studies in Europe revealed the presence of eight phylogroups. No morphometric and meristic characteristics have been determined that would clearly separate them. The paper presents new data on the morphometric parameters species Austropotamobius torrentium with watercourses Presjenica, Jošanica and Crna Rijeka. This analysis is based on 22 parameters that were measured on 38 crayfish individuals, whose size was larger than 5 cm. Using Anova test, Bonfferoni test and discrimination analysis, it was determined that females differ significantly in a small number of parameters, while males showed greater morphological divergence. The great diversity of habitats of Austropotamobius torrentium in Bosnia and Herzegovina obliges the inclusion of the Dinarides region in molecular phylogenetic studies. This will result in more precise and complete data on Austropotamobius torrentium populations.

Ecological conditions and phenotypic characteristics of Austropotamobius torrentium (Schrank, 1803) in the Subotica and Toplik Rivers

Austropotamobius torrentium (Schrank, 1803) lives in two watercourses, namely Subotica (Sana) and Toplik (Tilava). The influence of the ecological conditions of these rivers on individuals of this species is analyzed based on 22 morphometric parameters. The assessment of abiotic conditions was done through the measurement of physical and chemical water parameters and the analysis of the composition of phytobenthos and macrozoobenthos. Sampling was carried out in June, September, and October 2021. Water quality at the investigated localities (on the basis of abiotic and biotic parameters) is oligo to betamesosaprobic. A total of 37 crayfish were analyzed (20 from Subotica and 17 from Toplik). Female crayfish have shown significant differences in the head width (HEW) and abdominal thickness (ABH). Males differed significantly in a number of parameters, including the weight (W), head shape (HEL and HEW), rostrum length (ROL), carapace width (CPW), and abdominal shape (ABH and ABL). Higher values of the indicated parameters have been found in the males from the Subotica River. The obtained results are influenced by different ecological conditions of their habitats. The Subotica River is a larger watercourse in the lowland belt with a large number of microhabitats, and the Toplik is a small river in the mountain belt, with uniform sediment and a small number of ecological niches of the analyzed species. The general ecological conditions in the water of both watercourses are also affected by aquaculture facilities, where wastewater has a direct influence on the distribution of Austropotamobius torrentium individuals in the watercourses of the Subotica and Toplik Rivers. For more detailed and complete data, it is necessary to analyze a larger number of individuals in both populations, and to include additional morphometric-meristic parameters and molecular-genetic analyses.

Climate change threatens unique genetic diversity within the Balkan biodiversity hotspot – The case of the endangered stone crayfish

The stone crayfish Austropotamobius torrentium is globally endangered due to anthropogenic pressure on its habitats, climate change and the invasive crayfish species, particularly the signal crayfish Pacifastacus leniusculus . Aiming to guide A. torrentium conservation within the hotspot of its genetic diversity in the western Balkan Peninsula, we combined population genetics and species distribution models (SDMs) to reveal the impact of climate change and the invasive P. leniusculus on A. torrentium diversity. Population genetic analyses based on newly developed microsatellites revealed moderate within-population genetic diversity and high differentiation among populations, reflecting isolated populations with limited gene flow. Alongside strong genetic structuring, we discovered high level of inbreeding coefficient indicating homozygote excess within the majority of populations. The SDMs results predicted substantial reductions of suitable habitats for A. torrentium by 2070; 80 % of its currently suitable habitat is predicted to be lost under high‐warming climate change scenario. Obtained results indicated that 44 % of populations with high and/or unique genetic diversity, including three highly divergent and geographically restricted evolutionary lineages, are located in the areas predicted to become unsuitable in the future, highlighting their vulnerability to extinction. Further, SDMs revealed considerable decrease of future habitat suitability for P. leniusculus , suggesting that climate change represents greater threat to A. torrentium . Our study highlights the importance of conserving remnant A. torrentium populations, among other, through assisted migration and population mixing that could help populations overcome the risks of inbreeding and maladaptation, and thus enabling A. torrentium to withstand the ongoing climate change. • Native freshwater crayfish decline is a global conservation concern. • The first study of Austropotamobius torrentium genetic diversity in its hotspot (Balkan Peninsula). • Climate change poses a higher risk to A. torrentium than invasive crayfish species. • Transboundary conservation actions needed for species long-term preservation. • . Conservation strategies should include assisted migration and population reinforcement.

Morphometric characteristics and condition indices of stone crayfish Austropotamobius torrentium (Shrank, 1803) from the Mlinska River

Regarded as a protected species on the IUCN list of endangered species, and classified as data deficient species, the stone crayfish (Austropotamobius torrentium) is one of the five European species from the Astacidae family that lives in freshwater ecosystems across Bosnia and Herzegovina. The aim of this study was to investigate the variability of selected morphometric characteristics of male and female crayfish of the A. torrentium species from the upper reaches of the Mlinska River. The crayfish were sampled in June 2021 when 62 individuals (22 males and 40 females) were caught. They were analyzed fresh in the field and in the laboratory. The treatment included the measurement of 12 morphometric features: total body length (TBL), weight (W), claw length (CLL), claw width (CLW), carapace length (CPL), carapace width (CPW), rostrum length (ROL), rostrum width (ROW), abdominal length and width (ABL, ABW) and telson length and width (TEL, TEW). The mean body length ± SD was 79.05 ± 7.35 in males and 60.54 ± 10.03 mm in females. The mean recorded body weight ± SD was 17.51 ± 8.3 g in males and 7.02 ± 4.41 g in females. Using the linear regression method, a positive correlation was found between body length and weight (♂ R2=0.7644; ♀ R2=0.8222), body weight and claws length (♂ R2=0.876; ♀ R2=0.9661), as well as carapace width and body length (♂ R2=0.7216; ♀ R2=0.8411). Using the T-test, a statistically significant difference of the analyzed morphometric parameters was found between the sexes, which is explained by the pronounced sexual dimorphism of stone crayfish. The calculated values for Condition factor and the Crayfish constant indicate that the males are of better fitness, which is in line with the results of previous research in Europe. Data presented in this paper can serve as a basis for further research of A. torrentium in this area.

Detection of the Endangered Stone Crayfish Austropotamobius torrentium (Schrank, 1803) and Its Congeneric A. pallipes in Its Last Italian Biotope by eDNA Analysis

The stone crayfish, Austropotamobius torrentium, is a European freshwater crayfish. Although this species is relatively widespread throughout the continent, it is undergoing significant declines throughout its range. However, as the decline rates have not been quantified in detail, this species is classified as data deficient by the IUCN Red List of Threatened Species. The present study describes the development and validation of two species-specific assays based on hydrolysis probe chemistry for the detection of A. torrentium and A. pallipes environmental DNA (eDNA) in water samples collected in the Julian Alps of Italy (Friuli Venezia Giulia). The eDNA-based method was applied to 14 sites within the Danubian Slizza basin, known to be inhabited by A. torrentium, but with insufficient information on their distribution. In addition, one station in the Tagliamento River basin was sampled to test the performance of the A. pallipes probe. The presence of A. torrentium is confirmed at 6 out of 15 sites. At four of these sites, A. torrentium is detected for the first time. In contrast, the presence of A. torrentium was not detected at two sites already known to harbour the species. Finally, the presence of A. pallipes was confirmed in the station belonging to the Tagliamento basin. The methodology described, which allows the distinction between the two species, paves the way for the parallel detection of the stone crayfish and the white-clawed crayfish (A. pallipes) through eDNA analysis.

Postembryonic development in freshwater crayfish (Decapoda: Astacidea) in an evolutionary context

Detailed morphology of the first three postembryonic developmental stages (Stages I-III juvenile) in representatives from all four crayfish families, Austropotamobius torrentium (von Paula Schrank, 1803) (Astacidae), Procambarus virginalis Lyko, 2017 (Cambaridae), Cambaroides japonicus (De Haan, 1841 [in De Haan, 1833-1850]) (Cambaroididae) and Cherax destructor Clark, 1936 (Parastacidae) are described and the diagnostic characters for each family are indicated. A phylogenetic tree of freshwater Astacidea, based on these new diagnostic juvenile characters is constructed to suborder, superfamily and family levels, and compared with a molecular phylogenetic tree. The evolutionary history of maternal care in freshwater crayfish is discussed based on particular features of the postembryonic stages of each family. Using comparisons between the phylogenetic tree and global geo-history, the location and timing of the early evolution of maternal care in postembryonic development and the extension of this care are estimated.

Physiological performance of native and invasive crayfish species in a changing environment: insights from Dynamic Energy Budget models.

Crayfish are keystone species important for maintaining healthy freshwater ecosystems. Crayfish species native to Europe, such as Astacus astacus and Austropotamobius torrentium, are facing decline and are increasingly endangered by changing climate and invasions of non-native crayfish, such as Pacifastacus leniusculus and Procambarus virginalis. The success of these invasions largely depends on differences in ontogeny between the native species and the invaders and how changes in the environment will affect the ontogeny. Dynamic Energy Budget (DEB) models can be used to investigate such differences because the models capture dependence of metabolism, and therefore ontogeny, on environmental conditions. We develop DEB models for all four species and investigate key elements of ontogeny and metabolism affecting interspecific competition. We then use the DEB models to predict individual growth and reproduction in current and new conditions that are expected to arise from climate change. Although observations suggest that P. leniusculus poses the major threat to native species, our analysis identifies P. virginalis, in spite of its smaller size, as the superior competitor by a large margin—at least when considering metabolism and ontogeny. Our simulations show that climate change is set to increase the competitive edge of P. virginalis even further. Given the prospects of P. virginalis dominance, especially when considering that it is able to withstand and spread at least some crayfish plague strains that severely affect native species, additional research into P. virginalis is necessary.

English

AbstractWe examined gills (podobranchs, arthrobranchs and pleurobranchs) from five European Astacidae (Austropotamobius pallipes, Austropotamobius torrentium, Astacus astacus, Pontastacus leptodactylus and Pontastacus pachypus) to assess the relationships between their phylogeny and gill morphology and formulae. In addition, we checked for the presence or absence of pleurocoxal lappets. The morphology of the gills and associated structures was similar in all examined European astacid species except for three rudimentary pleurobranchs found in P. pachypus. In European Astacidae and Cambaroididae, the pleurocoxal lappets are absent but are present in the American Astacidae, Cambaridae and Parastacidae. The number of pleurobranchs decreases from the most primitive Parastacidae to the most advanced Cambaridae. Nevertheless, although the most primitive parastacid crayfish and the most advanced Cambaridae all have pleurocoxal lappets, they are absent in European Astacidae and Cambaroididae, which suggests that a reversible gill morphology has occurred in crayfish evolution.

How well does NATURA 2000 protect threatened stone crayfish in Croatia?

The stone crayfish Austropotamobius torrentium (Schrank, 1803) is a threatened native European freshwater crayfish species for which Natura 2000 network represents the most important conservation effort at the European level. In Croatia, there are altogether 25 Natura 2000 sites defined specifically for this species. In the present study, we aimed to assess the effectiveness of Natura 2000 sites in preserving stone crayfish diversity through gap analysis, a GIS-based approach that overlays species distribution data on a map of designated Natura 2000 sites. Our results showed that the existing Natura 2000 network in Croatia encompasses most of the areas with a high diversity of A. torrentium; currently designated sites harbour 73.3% of recorded A. torrentium populations. Future conservation planning efforts, and possible expansion of Natura 2000, should be focused on newly discovered A. torrentium populations that present divergent evolutionary lineages.

EDNA Detection of Native and Invasive Crayfish Species Allows for Year-Round Monitoring and Large-Scale Screening of Lotic Systems

Effective management of both endangered native and invasive alien crayfishes requires knowledge about distribution, monitoring of existing and early detection of newly established populations. Complementary to traditional survey methods, eDNA sampling has recently emerged as a highly sensitive non-invasive detection method to monitor crayfish populations. To advance the use of eDNA as detection tool for crayfish we used a twofold approach: 1) we designed a novel set of specific eDNA-assays for all native (Austropotamobius torrentium, Austropotamobius pallipes, Astacus astacus) and the most relevant invasive crayfish species (Pacifastacus leniusculus, Faxonius limosus, Faxonius immunis) in Central Europe. To ensure specificity each primer pair was tested in silico, in vitro, and in situ; 2) we assessed the influence of spatio-temporal variables (distance to upstream population, season, stream size) on eDNA detection in seven streams using two different detection methods (qualitative endpoint PCR and quantitative droplet digital PCR, ddPCR). The newly developed eDNA assays successfully detected all crayfish species across different lotic and lentic habitats. eDNA detection rate (endpoint PCR) and eDNA-concentration (ddPCR) were significantly influenced by distance and season. eDNA detection was successful up to 7 km downstream of the source population and across all seasons, although detectability was lowest in winter. eDNA detection rate further decreased with increasing stream size. Finally, eDNA-concentration correlated positively with estimated upstream population size. Overall, we provide near operational eDNA assays for six crayfish species, enabling year-round detection, which represents a clear benefit over conventional methods. Due to its high sensitivity, eDNA detection is also suitable for the targeted search of as-yet unrecorded or newly emerging populations. Using quantitative ddPCR might further allow for a rough estimation of population size, provided that the identified spatio-temporal factors are accounted for. We therefore recommend implementing eDNA-detection as a complementary survey tool, particularly for a large-scale screening of data-deficient catchments or a year-round monitoring.

Conservation genetics of the vulnerable stone crayfish using DNA barcoding and microsatellites

The stone crayfish Austropotamobius torrentium is a native European freshwater species with significant population declines caused by anthropogenic pressure onto its habitats, climate change and spreading of non-native invasive crayfish and their pathogens. Large-scale DNA barcoding based on sequencing a short fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene revealed this species represents a highly divergent taxon whose evolutionary heritage is preserved in genetically divergent mtDNA phylogroups, with majority of them having restricted distribution ranges. Guarding in mind vanishing populations trends and potential loss of genetic diversity, effective conservation planning and monitoring are needed for ensuring the persistence and long-term survival of this endangered species. Since genetic data are often critical for defining populations for conservation and management purposes, along with DNA barcoding, we employed microsatellites to examine samples of more than 400 individuals from 17 populations across its entire distribution range in Croatia, known as the stone crayfish diversity hot spot. Microsatellite analyses revealed high level of genetic diversity and differentiation among studied populations that grouped according to their geographical position and mtDNA phylogroup. Almost all sampled populations represent a distinct genetic cluster, showing high level of differentiation and reflecting long periods of isolation. Results of genetic characterisation enabled selection of suitable donor populations for future restocking and reintroduction programs. Combination of DNA barcoding and microsatellites provided good insight into genetic diversity and population structure as well as enabled sound conservation programs for this threatened species in Croatia.

Year-round monitoring and large-scale screening of native and invasive crayfishes in lotic systems

Freshwater crayfish are among the most threatened animal taxa in Central Europe. Effective conservation measures for endangered native and management of invasive alien crayfishes requires knowledge about distribution, monitoring of existing and early detection of newly established populations. eDNA has recently emerged as a promsing, highly sensitive, and non-invasive detection tool in this regard. To evaluate eDNA as detection tool for freshwater crayfish, we developed a novel set of specific eDNA-assays for all native (Austropotamobius torrentium, Austropotamobius pallipes, Astacus astacus) and the most relevant invasive crayfish species (Pacifastacus leniusculus, Faxonius limosus, Faxonius immunis) in Central Europe. To ensure specificity each primer pair was tested in silico, in vitro, and in situ, including a total of 13 lotic and lentic waterbodies (Fig. 1). Moreover, we assessed the influence of spatio-temporal variables (distance to upstream population, season, and stream size) on eDNA detection in seven streams using two different detection methods (qualitative endpoint PCR and quantitative droplet digital PCR, ddPCR). The newly developed eDNA assays successfully detected all crayfish species across different lotic and lentic habitats. Multiple linear mixed-effects analysis revealed a significant effect of distance and season on eDNA detection rate (endpoint PCR) and eDNA-concentration (ddPCR). Year-round detection was successful up to 7 km downstream of the source population, although detectability was lowest in winter. eDNA detection rate further decreased with increasing stream size. Finally, eDNA-concentration correlated positively with estimated upstream population size. Overall, our study provides easily applicable eDNA assays for six crayfish species, enabling year-round detection, which represents a clear benefit over conventional methods. Due to its high sensitivity, eDNA detection is also suitable for the targeted search of as-yet unrecorded or newly emerging populations. Using quantitative ddPCR might further allow for a rough estimation of population size, provided that the identified spatio-temporal factors are accounted for. We therefore recommend implementing eDNA-detection as a complementary survey tool, particularly for a large-scale screening of data-deficient catchments or a year-round monitoring (Chucholl et al. 2021).

Establishing the first DNA barcode reference library for freshwater decapod species (Crustacea: Decapoda) in Republic of North Macedonia

The freshwater ecosystems in the Republic of North Macedonia are considered as biodiversity hotspot on the European level since they provide diverse habitats that correspond with the complex of ecological preferences that many species require. This specially applies to the freshwater crustaceans that, unfortunately, have never been in the focus of a continuous research. In R. North Macedonia, freshwater crustaceans usually inhabit ecosystems exposed to negative anthropogenic impact. Thus, some species may become extinct presenting an irreversible loss for the Macedonian natural heritage. Although DNA barcoding, as a highly effective tool for fast species detection, is already a routine protocol in many taxonomical studies all over the world, there is still no official national DNA barcoding initiative in Republic of North Macedonia. This study employs DNA barcoding based on the ca. 650-bp long standard fragment of the mt COI gene of Astacus astacus, Austropotamobius torrentium, Potamon fluviatile and Potamon ibericum previously identified based on morphological characters, collected from different parts in R. North Macedonia. The ability of the DNA barcoding to rapidly identify all species has been proven. Our research presents the first comprehensive study that employs DNA barcoding as a molecular tool in decapod taxonomy in Republic of North Macedonia, giving the first attempt to establish DNA barcode reference library for freshwater decapod species in the country. We hope that further application of this approach will lead to the construction of DNA barcode reference library for different aquatic biota in the country. Such a library will find purpose in effective and modern bioassessment protocols as well as in phylogenetic research detecting interpopulation genetic variability.

Only one can remain? Environmental and spatial factors influencing habitat partitioning among invasive and native crayfishes in the Pannonian Ecoregion (Hungary)

Biological invasions have increasingly threatened indigenous species, influence metacommunity organisation and consequently, global biodiversity. World-wide expansion of non-indigenous crayfish (NICS) is associated with dramatic changes in species poor indigenous crayfish (ICS) assemblages challenging conservation planning. We analysed long-term changes of crayfish occurrences from the pre-invasion state, through the first appearance of NICS, to their intensive spread in Hungarian waters. Further, we analysed present-day crayfish metacommunity patterns for co-occurrences and influence of spatial and environmental factors. Historic data revealed a marked pre-invasion decline in indigenous noble crayfish Astacus astacus and stone crayfish Austropotamobius torrentium populations, but not in the narrow-clawed crayfish Pontastacus leptodactylus. Historic data provided no direct evidence for the impact of NICS on ICS, rather it supported that NICS often entered areas where ICS had been extinct or were not present at all. Crayfish species extremely rarely co-occurred which could indicate their strong competition and be related to utilization of empty sites by NICS. Crayfish metacommunities were predominantly spatially structured indicating the primary influence of ongoing invasion. Crayfish species also exhibited different environmental preferences mainly along the altitude and temperature gradients. We conclude that the invasion is still in the expanding phase and without an effective conservational program the future of ICS is doubtful in Hungary. Conservation policy should focus on the preservation and reintroduction of the stone and noble crayfishes in highland refugees. Expansion of NICS should be prevented in refugee areas by utilizing possibilities provided by natural and artificial barriers, and education and strict ban should be simultaneously applied to prevent further illegal releases by aquarists.