Genomic insights into the conservation status of the Idle Crayfish Austropotamobius bihariensis Pârvulescu, 2019: low genetic diversity in the endemic crayfish species of the Apuseni Mountains.

BACKGROUND: Biodiversity in freshwater ecosystems is declining due to an increased anthropogenic footprint. Freshwater crayfish are keystone species in freshwater ecosystems and play a crucial role in shaping the structure and function of their habitats. The Idle Crayfish Austropotamobius bihariensis is a native European species with a narrow distribution range, endemic to the Apuseni Mountains (Romania). Although its area is small, the populations are anthropogenically fragmented. In this context, the assessment of its conservation status is timely. RESULTS: Using a reduced representation sequencing approach, we identified 4875 genomic SNPs from individuals belonging to 13 populations across the species distribution range. Subsequent population genomic analyses highlighted low heterozygosity levels, low number of private alleles and small effective population size. Our structuring analyses revealed that the genomic similarity of the populations is conserved within the river basins. CONCLUSION: Genomic SNPs represented excellent tools to gain insights into intraspecific genomic diversity and population structure of the Idle Crayfish. Our study highlighted that the analysed populations are at risk due to their limited genetic diversity, which makes them extremely vulnerable to environmental alterations. Thus, our results emphasize the need for conservation measures and can be used as a baseline to establish species management programs.

Living on the edge: Crayfish as drivers to anoxification of their own shelter microenvironment.

Burrowing is a common trait among crayfish thought to help species deal with adverse environmental challenges. However, little is known about the microhabitat ecology of crayfish taxa in relation to their burrows. To fill this knowledge gap, we assessed the availability of oxygen inside the crayfish shelter by series of in-vivo and in-silico modelling experiments. Under modeled condition, we found that, except for the entrance region of the 200 mm, a flooded burrow microenvironment became anoxic within 8 h, on average. Multiple 12-hour day-night cycles, with burrows occupied by crayfish for 12 h and empty for 12 h, were not sufficient for refreshing the burrow microenvironment. We then examined the degree to which crayfish species with different propensities for burrowing are tolerant of self-created anoxia. From these experiments, primary and secondary burrowers showed best and most consistent tolerance-exhibiting ≥ 64% survival to anoxia and 25-91% survival of ≥ 9 h at anoxia, respectively. Tertiary burrowers exhibited little to no tolerance of anoxia with 0-50% survival to anoxia and only one species exhibiting survival (2%) of ≥ 9 h at anoxia. Results suggest that moderate to strongly burrowing crayfish can quickly draw down the dissolved oxygen in burrow water but appear to have conserved a legacy of strong tolerance of anoxia from their monophyletic ancestors-the lobsters-whereas tertiary burrowers have lost (or never evolved) this ability.

Genome analysis of the monoclonal marbled crayfish reveals genetic separation over a short evolutionary timescale.

The marbled crayfish (Procambarus virginalis) represents a very recently evolved parthenogenetic freshwater crayfish species that has invaded diverse habitats in Europe and in Madagascar. However, population genetic analyses have been hindered by the homogeneous genetic structure of the population and the lack of suitable tools for data analysis. We have used whole-genome sequencing to characterize reference specimens from various known wild populations. In parallel, we established a whole-genome sequencing data analysis pipeline for the population genetic analysis of nearly monoclonal genomes. Our results provide evidence for systematic genetic differences between geographically separated populations and illustrate the emerging differentiation of the marbled crayfish genome. We also used mark-recapture population size estimation in combination with genetic data to model the growth pattern of marbled crayfish populations. Our findings uncover evolutionary dynamics in the marbled crayfish genome over a very short evolutionary timescale and identify the rapid growth of marbled crayfish populations as an important factor for ecological monitoring.

The future of endangered crayfish in light of protected areas and habitat fragmentation.

The long-term survival of a species requires, among other things, gene flow between populations. Approaches for the evaluation of fragmentation in the frame of freshwater habitats consider only a small amount of the information that combined demography and geography are currently able to provide. This study addresses two species of Austropotamobius crayfish in the light of population genetics, spatial ecology and protected areas of the Carpathians. Advancing the classical approaches, we defined ecological distances upon the rasterised river network as a surrogate of habitat resistance to migration, quantifying the deviations from the species´ suitability range for a set of relevant geospatial variables in each cell of the network. Molecular analyses revealed the populations of the two Austropotamobius crayfish species are clearly distinct, lacking hybridisation. Comparing pairs of populations, we found, in some cases, a strong disagreement regarding genetic and ecological distances, potentially due to human-mediated translocations or the geophysical phenomena of regressive erosion, which may have led to unexpected colonisation routes. Protected areas were found to offer appropriate local habitat conditions but failed to ensure connectivity. The methodology applied in this study allowed us to quantify the contribution of each geospatial (environmental) variable to the overall effect of fragmentation, and we found that water quality was the most important variable. A multilevel approach proved to reveal a better understanding of drivers behind the distribution patterns, which can lead to more adequate conservation measures.

Ecophysiological and life-history adaptations of Gammarus balcanicus (Schäferna, 1922) in a sinking-cave stream from Western Carpathians (Romania).

Freshwater gammarids are known to colonise occasionally sinking-cave streams, providing contrasting morphological, life-history and ecophysiological adaptations compared to their surface conspecifics. In this study, a subterranean and a surface population of the species Gammarus balcanicus was surveyed for one year in a sinking-cave stream from the Western Carpathians (Romania). The results showed that the cave-dwelling population comprised individuals that were significantly larger compared to their surface conspecifics, had larger body-size at sexual maturity and that the females produced fewer, but larger eggs, compared to the population situated outside the cave. The trophic position and the omnivory were significantly higher for the cave-dwelling compared to surface population and the elemental imbalance for C:P molar ratios lower, but similar for C:N. However, the subterranean population did not present troglomorphic characters or longer lifespan as known for other cave-surface paired crustaceans. This, together with the rather extensive hydrological connection of the habitats, suggests active gene-flow between populations and similar response to seasonality for body-size distributions, indicating that the observed ecophysiological and life-history differences are rather the consequence of phenotypic plasticity than the result of genetic adaptation.

A journey on plate tectonics sheds light on European crayfish phylogeography.

Crayfish can be used as model organisms in phylogeographic and divergence time studies if reliable calibrations are available. This study presents a comprehensive investigation into the phylogeography of the European stone crayfish (Austropotamobius torrentium) and includes samples from previously unstudied sites. Two mitochondrial markers were used to reveal evolutionary relationships among haplogroups throughout the species' distributional range and to estimate the divergence time by employing both substitution rates and geological calibration methods. Our haplotype network reconstruction and phylogenetic analyses revealed the existence of a previously unknown haplogroup distributed in Romania's Apuseni Mountains. This haplogroup is closely related to others that are endemic in the Dinarides, despite their vast geographical separation (~600 km). The separation is best explained by the well-dated tectonic displacement of the Tisza-Dacia microplate, which started in the Miocene (~16 Ma) and possibly carried part of the A. torrentium population to the current location of the Apuseni Mountains. This population may thus have been isolated from the Dinarides for a period of ca. 11 m.y. by marine and lacustrine phases of the Pannonian Basin. The inclusion of this geological event as a calibration point in divergence time analyses challenges currently accepted crayfish evolutionary time frames for the region, constraining the evolution of this area's crayfish to a much earlier date. We discuss why molecular clock calibrations previously employed to date European crayfish species divergences should therefore be reconsidered.

Hidden sites in the distribution of the crayfish plague pathogen Aphanomyces astaci in Eastern Europe: Relicts of genetic groups from older outbreaks?

The crayfish plague agent Aphanomyces astaci is one of the world's most threatening invasive species. Originally from North America, the pathogen is being imported alongside American crayfish species, which are used for various purposes. In this study, we investigated the marginal, currently known distribution area of the pathogen in Eastern Europe by sampling narrow-clawed crayfish (Astacus leptodactylus) and spiny-cheek crayfish (Orconectes limosus) populations. In addition, using specific real-time PCR, we tested several marine decapod species, which also occur in brackish waters of the Danube at the West coast of the Black Sea and the Dniester River basin. By sequencing the nuclear chitinase gene, mitochondrial rnnS/rnnL DNA and by genotyping using microsatellite markers, we identified the A. astaci haplogroups of highly infected specimens. The A. astaci DNA was detected in 9% of the investigated A. leptodactylus samples, both in invaded and non-invaded sectors, and in 8% of the studied O. limosus samples. None of the marine decapods tested positive for A. astaci. The results revealed that narrow-clawed crayfish from the Dniester River carried the A. astaci B-haplogroup, while A. astaci from the Danube Delta belonged to the A- and B-haplogroups. In the invaded sector of the Danube, we also identified the A-haplogroup. Microsatellite analysis revealed a genotype identical to the genotype Up. It might be that some of the detected A. astaci haplogroups are relics from older outbreaks in the late 19th century, which may have persisted as a chronic infection for several decades in crayfish populations.

Plasticity in fecundity highlights the females' importance in the spiny-cheek crayfish invasion mechanism.

Invasion is one of the most consequential phenomena affecting the distribution of native species. Few in number of species, European crayfish are losing the competition with introduced North American crayfish. The spiny-cheek crayfish, Orconectes limosus, is an outstanding example, successfully competing against the native narrow-clawed crayfish, Astacus leptodactylus. For four years, we collected data regarding crayfish occurrences, their relative abundance, and the structure of populations in the ongoing colonisation process of O. limosus in the lower Danube. The mature females of both invasive and indigenous crayfish species were analysed with respect to biometry and production of oocytes in relation to the dynamics of invasion. The interspecific comparisons showed no significant differences regarding body size, with an average of approximately 102 mm total length and 31 g wet weight for both species. However, the fecundity of the indigenous species was found to be constant throughout the investigated area, whereas the number of eggs produced by the invasive females was significantly increased at the active front of the invasion. The maximum number of ovarian eggs found was 887 and 1156 in the indigenous species and the invasive species, respectively. We propose the scenario that the invasive species, which carries the deadly crayfish plague, creates an ecological advantage by reducing the populations of indigenous crayfish. Subsequently, the invasive females opportunistically use the available resources to enhance their fecundity, resulting in the acute growth of populations. However, the long-term competitiveness and colonisation success of O. limosus still remain in question.

Zoogeography of epigean freshwater Amphipoda (Crustacea) in Romania: fragmented distributions and wide altitudinal variability.

Inland epigean freshwater amphipods of Romania are diverse and abundant for this region has a favourable geographical position between the Balkans and the Black Sea. Excluding Ponto-Caspian species originating in brackish waters and freshwater subterranean taxa, there are 11 formally recognized epigean freshwater species recorded from this country. They belong to 3 genera, each representing a different family: Gammarus (Gammaridae, 8 species or species complexes), Niphargus (Niphargidae, 2 epigean species) and Synurella (Crangonyctidae, one species). Their large-scale distribution patterns nevertheless remain obscure due to insufficient data, consequently limiting biogeographical interpretations. We provide extensive new data with high resolution distribution maps, thus improving the knowledge of the ranges of these taxa. Gammarus species display substantial altitudinal variability and patchy, fragmented distribution patterns. They occur abundantly, particularly in springs and streams, from lowlands to sub-mountainous and mountainous regions. In the light of recent molecular research, we hypothesize that the complex geomorphological dynamics of the Carpathian region during the Late Tertiary probably contributed to their allopatric distribution pattern. Contrasting with Gammarus, the genera Niphargus and Synurella exhibit low altitudinal variability, broad ecological valences and overlapping distributions, being widespread throughout the lowlands. The current distribution of N. hrabei and N. valachicus seems to be linked to the extent of the Paratethys during the Early Pliocene or Pleistocene. We further discuss the taxonomic validity of two synonymized and one apparently undescribed taxon, and provide an updated pictorial identification key that includes all taxa and forms discussed in our study. The mosaic distribution of epigean freshwater amphipod species in Romania shows that this region is particularly suitable for phylo- and biogeographical analyses of this group.

Invasive crayfish and crayfish plague on the move: first detection of the plague agent Aphanomyces astaci in the Romanian Danube.

Native European crayfish, such as Astacus leptodactylus, are threatened, among other factors, by the crayfish plague agent Aphanomyces astaci, dispersed by invasive North American crayfish. Two of these invaders, Pacifastacus leniusculus and Orconectes limosus, have extended their distribution in the River Danube catchment; the latter was detected for the first time in Romania in 2008. We monitored, at monthly intervals for over 2 yr, occurrence of native A. leptodactylus and invasive O. limosus at 6 sites on the Romanian Danube and checked for the invasive species in 4 of its tributaries. Between January 2009 and March 2011, the relative abundances of O. limosus steadily increased with time, while the native A. leptodactylus dramatically decreased in abundance. O. limosus expanded downstream at a rate of ca. 15 km yr-1; in August 2011, it was already present in the upper 105 km of the Romanian Danube. An agent-specific real-time PCR analyses demonstrated the presence of A. astaci DNA in at least 32% of the analysed invasive (n = 71) and 41% of the native (n = 49) crayfish coexisting in the Danube. Furthermore, A. astaci was also detected in A. leptodactylus captured about 70 km downstream of the O. limosus invasion front (at the time of sampling). Assuming a steady rate of expansion, O. limosus may invade the sensitive Danube delta area in the mid-2060s, even without long-distance dispersal. The crayfish plague agent, however, may reach the delta substantially earlier, through dispersal downstream among populations of native crayfish.