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.

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.

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.

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 of A. astacus harbour 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 for A. astacus by 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 endangered A. 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.

During the last eight years several surveys were undertaken to improve the knowledge of the butterfly distribution in Croatia. In this paper we provide additional data for Mt Velebit for some taxa with poorly known distributions like Pieris (napi) balcana Lorković, 1970 as well as the first records for Carterocephalus palaemon, Gegenes pumilio, Lycaena tityrus, Polyommatus escheri, Neptis rivularis, and Charaxes jasius. with the review of recently published data and our contribution, the number of species found on Mt Velebit has increased to 153 in all, which makes Velebit a mountain with a relatively great diversity of butterfly species. additionally, we include records of some species of conservation concern for the neighbouring area of Lika, for Polyommatus damon, P. ripartii, P. admetus, Lycaena dispar, Phengaris alcon (humid meadow ecotype) and Euphydryas maturna. u hrvatskoj su zadnjih desetak godina poduzeta dodatna istraživanja u svrhu poboljšanja znanja o rasprostranjenosti danjih leptira. u ovom radu su za planinu Velebit uz nove podatke za vrste slabije poznatog rasprostranjenja kao što je Pieris (napi) balcana Lorković, 1970 dati su i prvi podaci o nalazima vrsta Carterocephalus palaemon, Gegenes pumilio, Lycaena tityrus, Polyommatus escheri, Neptis rivularis, and Charaxes jasius. s novim nalazima i uz pregled podataka publiciranih u zadnjih osam godina, ukupni broj zabilježenih vrsta na planini Velebitu dosegao je 153 vrsta što čini Velebit jednom od planina s najraznolikijom faunom danjih leptira. Dodatno, u susjednoj Lici opisani su nalazi Polyommatus damon, P. ripartii, Lycaena dispar, Phengaris alcon 'ekotip vlažnih travnjaka' i Euphydryas maturna, redom vrsta od interesa za zaštitu.

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 presence of the Grecian anomalous blue, Polyommatus (Agrodiaetus) aroaniensis (Brown, 1976) in Croatia has been recently published based on external morphology of collected specimens. The aim of this study was to evaluate the credibility of these findings by using DNA barcoding approach as the species exhibit phenotypic variability and can easily be misidentified with morphologically similar species Polyommatus (Agrodiaetus) ripartii. Therefore, we performed a molecular analysis of the mitochondrial gene for cytochrome c oxidase subunit I (COI) of the samples previously published as P. aroaniensis from different localities in Croatia. The results showed that all analysed samples belong to P. ripartii. Additionally, all analysed samples from different Croatian localities (Mt. Mala Kapela, Mt. Lička Plješivica, Mt. Poštak, Mt. Troglav and Mt. Kamešnica) together with previously published population of P. ripartii from Mt. Mosor belong to the same Eurasian clade of P. ripartii. (Brown, 1976), leptira iz porodice plavaca, nedavno je zabilježena u Hrvatskoj na temelju vanjske morfologije prikupljenih jedinki. Cilj ovog rada bio je procijeniti vjerodostojnost tog nalaza korištenjem metode DNA barkodiranja budući da se radi o fenotipski varijabilnoj vrsti koja se lako može zamijeniti s morfološki sličnom vrstom Polyommatus (Agrodiaetus) ripartii. Provedena je molekularna analiza mitohondrijskog gena za citokrom c oksidazu podjedinica I (COI) na uzorcima objavljenih kao vrsta P. aroaniensis s različitih lokaliteta u Hrvatskoj. Rezultati su pokazali da svi analizirani uzorci pripadaju vrsti P. ripartii. Također svi uzorci s različitih lokaliteta (Mala Kapela, Lička Plješivica, Poštak, Troglav i Kamešnica) zajedno s ranije objavljenom populacijom P. ripartii s Mosora pripadaju istoj euroazijskoj grupi vrste P. ripartii. Prisutnost vrste Polyommatus (Agrodiaetus) aroaniensis

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.