Background: Austropotamobius torrentiumis 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 withinA. 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 ofA. torrentiumand are highly relevant for conservation due to their evolutionary distinctiveness and restricted distribution.

Analysis of Croatian freshwater crayfish populations, aiming to gather new distributional data and complement previous surveys (2005–2011), was performed during 2014–2016, within the frame of Natura 2000 Integration Project. The research included different waterbodies across the whole of Croatia, harbouring both indigenous and non-indigenous crayfish species (ICS and NICS, respectively). Field work was conducted in 117 grid squares with dimensions 10 × 10 km, and up to four waterbodies were surveyed per grid square, making a total of 450 studied sites. Out of those, crayfish were not recorded in 368 sites. In the sites with crayfish presence (i.e., 82), the most frequently observed ICS was Astacus astacus (registered in 33 sites) followed by Austropotamobius pallipes (16 sites), Austropotamobius torrentium (12 sites), and Astacus leptodactylus (5 sites). Concerning NICS, the majority of records were for Orconectes limosus (13 sites), followed by Pacifastacus leniusculus (2 sites), whereas Procambarus fallax f. virginalis was registered in only one locality. Comparisons of obtained data with those from previous surveys showed that NICS are progressively spreading and displacing ICS, as recorded for A. leptodactylus that was almost completely displaced by O. limosus in waterbodies of the east Croatia. Existing ICS populations are under growing anthropogenic preassure, frequently fragmented and isolated. Moreover, this survey showed that in the last decade 55% of A. astacus and 67% of A. pallipes populations disappeared mainly as a consequence of anthropogenic influence onto their habitats. Further monitoring and conservation actions for ICS should be urgently applied to mitigate negative impacts of both NICS and anthropogenic influence.

The noble crayfish,Astacus astacus,is an indigenous European freshwater species. Its populations show significant declines caused by anthropogenic pressure on its habitats, climate change and the spread of invasive species. Diminishing populations’ trends and loss of genetic diversity highlight the need for effective conservation that will ensure their long-term survival. We combined population genetics and species distribution modelling (SDM) to reveal the impact of climate change and invasive species on the noble crayfish, and to guide future conservation programs of current populations. Our study showed that Croatian populations ofA. astacusharbour an important part of species genetic diversity and represent significant genetic reservoir at the European level. The SDM results predicted substantial reductions of suitable habitats forA. astacusby the 2070; only 13% of its current potential distribution is projected to remain stable under pessimistic Representative Concentration Pathway (RCP 8.5) emission scenario. Moreover, most of the populations with high genetic diversity are located in the areas predicted to become unsuitable, and consequently have a high probability of being lost in the future. Further, SDM results also indicated considerable decrease of future habitat suitability for invasive crayfish species in Croatia, suggesting that climate change poses a major threat to already endangeredA. astacus. The obtained results help in the identification of populations and areas with the highest conservation value which should be given the highest priority for protection. In order to preserve present diversity in areas that are predicted as suitable, we propose assisted migration and repopulation approaches, for enhancing populations’ size and saving maximum genetic variability. The result of our research emphasizes once again the benefits of multidisciplinary approach in the modern biodiversity conservation.

The stone crayfish Austropotamobius torrentium (Schrank, 1803) is a native European species distributed in central and southeastern Europe. Recent molecular phylogenetic research has shown that at least seven distinct monophyletic phylogroups exist within A. torrentium, with the highest genetic diversity found within the northern-central Dinaric (NCD) region in Croatia. Genetic divergence for some of these phylogroups was sufficiently large to suggest that they might actually represent cryptic species. Our research studied the morphometric and meristic characteristics of stone crayfish populations in the Žumberak-Samoborsko gorje Nature Park (Croatia) situated in the genetically diverse NCD region. The aim was to test whether there are some morphological features that clearly separate stone crayfish belonging to the three previously identified phylogroups into distinct groups based on morphology. We analysed morphological data of stone crayfish belonging to three distinct populations inhabiting small streams within the Žumberak-Samoborsko gorje Nature Park. Analyses showed that there are significant differences in some of the recorded morphometric and meristic characteristics between the studied populations for both males and females. Multivariate discriminant analyses of the measured morphological features revealed the characteristics that clearly separate populations in a similar way as molecular methods. For males, these were the characteristics describing claws, carapace, and rostrum, and for females those describing carapace, rostrum, and total length. Percentage of correctly classified crayfish per population was high (91–100%) for both sexes. Although isolated stone crayfish populations showed morphological and genetic differences, no significant correlations were obtained between genetic and morphometric distances. Analyses of additional samples and application of different genetic markers are needed to elucidate the relationships between the different populations of the stone crayfish.