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.

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.

First record of the stone crayfish in the Romanian lowlands

The stone crayfish is a native European freshwater species with significant population declines caused by anthropogenic pressure onto its habitats, climate change and spreading of non-indigenous invasive crayfish and their pathogens (e.g., Aphanomyces astaci). We present the first record of an Austropotamobius torrentium population in a small stream from the southern lowlands of Romania, in an area without limestone or karst, known as a typical stone crayfish habitat in this country. No additional populations were found in the neighbouring streams. The mtDNA analysis based on sequencing a short fragment of the cytochrome c oxidase subunit I (COI) gene revealed that specimens from this newly discovered population belongs to the most widespread phylogroup of A. torrentium (CSE) and share the same haplotype as the specimens found in Croatia. Unfortunately, this crayfish population has disappeared most likely because of the crayfish plague since pathogen A. astaci was isolated from moribund crayfish. Our study highlights the vulnerability of isolated relic populations in the face of outside stressors.

New insights into the genetic diversity of the stone crayfish: taxonomic and conservation implications

Background Austropotamobius torrentium is a freshwater crayfish species native to central and south-eastern Europe, with an intricate evolutionary history and the highest genetic diversity recorded in the northern-central Dinarides (NCD). Its populations are facing declines, both in number and size across its entire range. By extanding current knowledge on the genetic diversity of this species, we aim to assist conservation programmes. Multigene phylogenetic analyses were performed using different divergence time estimates based on mitochondrial and, for the first time, nuclear DNA markers on the largest data set analysed so far. In order to reassess taxonomic relationships within this species we applied several species delimitation methods and studied the meristic characters with the intention of finding features that would clearly separate stone crayfish belonging to different phylogroups. Results Our results confirmed the existence of high genetic diversity within A. torrentium, maintained in divergent phylogroups which have their own evolutionary dynamics. A new phylogroup in the Kordun region belonging to NCD has also been discovered. Due to the incongruence between implemented species delimitation approaches and the lack of any morphological characters conserved within lineages, we are of the opinion that phylogroups recovered on mitochondrial and nuclear DNA are cryptic subspecies and distinct evolutionary significant units. Conclusions Geographically and genetically isolated phylogroups represent the evolutionary legacy of A. torrentium and are highly relevant for conservation due to their evolutionary distinctiveness and restricted distribution.

Investigations of Variability of Morphometric Characteristics in Stone Crayfish in Order to Preserve Autochthonous Genome

This paper presents the information about morphological variability and sexual dimorphism of the stone crayfish (Austropotamobius torrentium) in the area of Korana River in Mrkonjic Grad. The crayfish were caught by hand made baited traps from October 2018 to May 2019. A total of 46 crayfish were caught, of which 33 males and 13 females. The dimensions of eight morphometric characteristics: body weight (W), body length (TBL), rostrum length (ROL), rostrum width (ROW), claw length (CLL), carapace length (CPL), carapace width (CPW) and abdomen length (ABL), were analyzed, both in males and females. Also, the body condition was determined for all individuales. The measurements results of morphometric characteristics partially matched into the alredy known range of variations. These data presens first ones for the observed area. By using Mann-Whitey U-test, it was noted that there are significant differences between the sexes for W, TBL, CLL, CPL, CPW and ABL. These resuls could be explained by sexual dimorphism of the stone crayfish.

Morphological diversity of the stone crayfish − traditional and geometric morphometric approach

Austropotamobius torrentium is one of four native European crayfish species inhabiting Croatian freshwaters. Existence of eight divergent monophyletic mtDNA phylogroups was described within A. torrentium; six of them are distributed in Croatia, with the highest genetic diversity established in its northern-central Dinaric region. Recent small-scale study of the stone crayfish morphological variability indicated significant differences among different phylogroups. In the present study larger sample size, covering populations from five phylogroups, was analysed with the aim of determining whether there are morphological characteristics that reliably separate stone crayfish from different phylogroups. Aiming this, 245 stone crayfish were analysed through traditional (TM) and, for the first time, geometric morphometric (GM) analyses. Multivariate discriminant analyses included 24 TM characteristics per crayfish, while GM comprised analyses of 22 landmarks on the dorsal side of cephalon. Both methods revealed congruent results, and significant differences among phylogroups in analysed features were obtained, with the cephalon shape contributing the most to crayfish discrimination. Research confirmed that both approaches, combined with statistical methods, are useful in distinguishing and separating crayfish phylogroups. Findings of present study are compatible with the previous molecular findings; stone crayfish present several distinct evolutionary lineages whose species status are currently undefined and require urgent clarification.