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.

Background The noble crayfish (Astacus astacus) is a native European species in decline, with a contracting range and diminishing populations and abundance. Previous studies revealed this species significant genetic diversity in the south-eastern Europe, with populations from the western and the southern part of the Balkan Peninsula being the most divergent. However, sampling of populations from the western part of the Balkans was limited and insufficient for investigating genetic diversity and population divergence for the purpose of conservation planning and management. Thus, the major aim of this study was to fill in this knowledge gap by studying mitochondrial and microsatellite DNA diversity, using 413 noble crayfish from 18 populations from waterbodies in the western part of the Balkan Peninsula. Methods Phylogenetic analysis of studied populations and their mitochondrial diversity were studied using COI and 16S sequences and population genetic structure was described using 15 microsatellite loci. Results Phylogeographic analysis revealed new divergent mitochondrial haplotypes for the populations in the westernmost part of the Balkan Peninsula in the tributaries of the Sava and Drava rivers. Microsatellite data indicated that these populations harbour an important component of genetic diversity within A. astacus. The results suggest that the western part of the Balkans played an important role as microrefugia during the Pleistocene climate fluctuations, allowing the long term persistence of A. astacus populations in this region. These results will also be important to supporting conservation decision making and planning.

Pontastacus leptodactylus is a native European crayfish species found in both freshwater and brackish environments. It has commercial importance for fisheries and aquaculture industries. Up till now, most studies concerning P. leptodactylus have focused onto gaining knowledge about its phylogeny and population genetics. However, little is known about the chromosomal evolution and genome organization of this species. Therefore, we performed clustering analysis of a low coverage genomic dataset to identify and characterize repetitive DNA in the P. leptodactylus genome. In addition, the karyogram of P. leptodactylus (2n = 180) is presented here for the first time consisting of 75 metacentric, 14 submetacentric, and a submetacentric/metacentric heteromorphic chromosome pair. We determined the genome size to be at ~18.7 gigabase pairs. Repetitive DNA represents about 54.85% of the genome. Satellite DNA repeats are the most abundant type of repetitive DNA, making up to ~28% of the total amount of repetitive elements, followed by the Ty3/Gypsy retroelements (~15%). Our study established a surprisingly high diversity of satellite repeats in P. leptodactylus. The genome of P. leptodactylus is by far the most satellite-rich genome discovered to date with 258 satellite families described. Of the five mapped satellite DNA families on chromosomes, PlSAT3-411 co-localizes with the AT-rich DAPI positive probable (peri)centromeric heterochromatin on all chromosomes, while PlSAT14-79 co-localizes with the AT-rich DAPI positive (peri)centromeric heterochromatin on one chromosome and is also located subterminally and intercalary on some chromosomes. PlSAT1-21 is located intercalary in the vicinity of the (peri)centromeric heterochromatin on some chromosomes, while PlSAT6-70 and PlSAT7-134 are located intercalary on some P. leptodactylus chromosomes. The FISH results reveal amplification of interstitial telomeric repeats (ITRs) in P. leptodactylus. The prevalence of repetitive elements, especially the satellite DNA repeats, may have provided a driving force for the evolution of the P. leptodactylus genome.

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.

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Invasive alien crayfish threaten the diversity of freshwater ecosystems and native crayfish fauna. In Europe, this is largely due to transmission of the crayfish plague to susceptible native crayfish. Many invasive species tolerate crayfish plague, but the infection still has the potential to reduce the fitness of a tolerant host due to energy trade-offs between immune response maintenance and life-history traits, such as growth and reproduction. In combination with other unfavourable conditions, such a response could alter further invasion success of an otherwise successful crayfish invader. We examined whether repeated infection with one of the most virulent haplogroups of crayfish plague agent (Aphanomyces astaci) affects growth or survival of the juvenile marbled crayfish (Procambarus virginalis). Juveniles were exposed to i) two levels of pathogen concentrations, and ii) two different feeding regimes under the higher pathogen concentration. In all performed trials, repeated infection reduced growth rates, while the combination of recurring infection and food limitation significantly increased mortality. The average energy cost of the immune response was estimated at 12.07 J/day for individuals weighing 0.3 grams. Since infections were frequent and pathogen concentrations high, results suggest that marbled crayfish is resistant to A. astaci pathogen and its survival is only affected by adding the stress of food limitation. The survival of almost half of the individuals exposed to high pathogen loads and extreme food limitation indicates that chronic infection by crayfish plague is unlikely to be an important factor impeding invasion success of the marbled crayfish, even under harsh conditions. Our results add to the growing body of evidence that marbled crayfish has potential to become one of the most successful freshwater invaders.