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The stone crayfish is a native European freshwater species endangered due to anthropogenic pressure on its habitats, invasive alien crayfish species and climate change. Our aims were to assess its genetic diversity in Croatia using molecular phylogenetics and population genetics, and to reveal the impact of climate changes on its diversity using species distribution modelling (SDM). Phylogenetic reconstruction revealed the existence of high genetic diversity, maintained in seven geographically and genetically isolated mtDNA lineages which present distinct Evolutionary Significant Units (ESUs). Population genetic analyses based on microsatellites revealed moderate within-population genetic diversity and isolated populations with limited gene flow. Besides strong genetic structuring concordant with established ESUs, we discovered high level of inbreeding within the majority of populations. The SDM results predicted substantial decrease of suitable habitats for stone crayfish by 2070 ; 80% of its currently suitable habitats are predicted to be lost under high-warming climate change scenario. Moreover, 44% of investigated populations with high and/or unique genetic diversity are located in areas predicted to become unsuitable, thus having a high probability of extinction in the future. Considering ongoing climate change predictions, prompt actions including assisted migration and repopulation should take place in order to preserve the existing diversity of this endangered species.

The stone crayfish (Austropotamobius torrentium) is a native European species endangered by anthropogenically induced alteration of its habitats, climate change and invasive alien crayfish species. Diminishing populations’ trends across its distribution range highlight the importance of developing effective conservation that will ensure its long-term survival. Aiming to guide future conservation programs of the stone crayfish in Croatia, we combined population genetics and species distribution modelling (SDM) to reveal the impact of invasive signal crayfish (Pacifastacus leniusculus) and climate change on the stone crayfish diversity. Population genetic analyses based on microsatellites revealed moderate within-population genetic diversity and high differentiation among populations, reflecting isolated populations with limited gene flow. Alongside strong genetic structuring, we discovered high level of inbreeding coefficient indicating homozygote excess within the majority of populations. The SDM results predicted substantial reductions of suitable habitats for both species by 2080 under two representative concentration pathway (RCP) scenarios: RCP 4.5 and RCP 8.5, respectively describing low‐warming and high‐warming conditions. Obtained results indicated that many populations with high and/or unique genetic diversity are located in the areas predicted to become unsuitable in the future. Further, ecological niche analysis showed that stone crayfish and signal crayfish do not exploit equivalent niches in Croatia. Our study highlights the importance of conserving remnant populations and establishing new populations in areas likely to support stone crayfish in the future (climate change refugia or ark sites), by assisted migration and repopulation approaches that can help populations overcome the risks of inbreeding and maladaptation. The results of our research emphasizes once again the importance of multidisciplinary approach in the modern biodiversity conservation.

Invazivne strane vrste predstavljaju prijetnju raznolikosti slatkovodnih ekosustava i populacijama zavičajnih vrsta slatkovodnih rakova, primarno zbog prijenosa bolesti račje kuge. No, kronična infekcija račjom kugom može i kod tolerantne invazivne strane vrste dovesti do kompromisa u raspodijeli energije između ulaganja u imunosni odgovor i rasta i reprodukcije, što u kombinaciji s drugim nepovoljnim okolišnim uvjetima može utjecati na njezin invazivni uspjeh. U ovom smo istraživanju ispitali da li imunosni odgovor na infekciju patogenom račje kuge negativno utječe na rast i preživljavanje juvenilnih jedinki invazivne strane vrste mramornog raka (Procambarus virginalis). U sklopu eksperimentalnih istraživanja, jedinke su izložene različitim koncentracijama patogena (2) i različitim režimima hranjenja (2). U svim pokusima, ponavljajuća infekcija rezultirala je smanjenim prirastom jedinki. Značajan porast mortaliteta inficiranih jedinki zabilježen je tek pri uvjetima značajne limitacije hrane, u grupi koja je bila vrlo blizu izgladnjivanja. S obzirom da su infekcije bile česte, a koncentracije patogena visoke, rezultati pokazuju kako je mramorni rak otporan na patogen Aphanomyces astaci i kako dodatni stresori (izgladnjivanje) marginalno povećavaju mortalitet inficiranih jedinki. Stoga, kronična infekcija račjom kugom vjerojatno neće imati značajan utjecaj na invazivni uspjeh mramornog raka, čak niti u nepovoljnim uvjetima.

The stone crayfish (Austropotamobius torrentium) and noble crayfish (Astacus astacus) are native European freshwater species with significant population declines caused by anthropogenic pressure on their habitats, invasive crayfish species, and climate change. In order to develop effective conservation for these threatened species, prerequisite is, among others, a sound knowledge of their genetic diversity patterns. The aim of this research was to assess genetic diversity of both native species in the freshwater habitats of Croatia using DNA barcoding based on sequencing a short fragment of the mtDNA cytochrome c oxidase subunit I (COI) gene. In addition, species distribution modelling was used to reveal the impact of ongoing climate change on their genetic diversity. Overall, 392 sequences were obtained for these two species from the study area (118 for the stone crayfish and 274 for the noble crayfish). Phylogenetic reconstruction revealed the existence of high genetic diversity within stone crayfish, maintained in seven distinct mtDNA phylogroups (containing 45 unique COI Croatian haplotypes). Since those geographically and genetically isolated phylogroups are highly relevant for conservation due to their evolutionary distinctiveness and restricted distribution, we declared them as distinct evolutionary significant units. Results for the noble crayfish showed that all Croatian haplotypes belong to two previously discovered genetic lineages, with the indication of a novel lineage (containing 16 unique COI Croatian haplotypes). Overlapping genetic data with potential future distribution of these two keystone species revealed their vulnerability ; most of the populations with high and/or unique genetic diversity are located in the areas predicted to become unsuitable, and consequently have a high probability of being lost in the future. DNA barcoding proved to be a useful tool for studying species genetic diversity and guiding their conservation. Considering ongoing climate extremes and predictions, prompt actions should take place in order to preserve the existing diversity.

Climate change imperils the persistence of numerous keystone species, including European freshwater crayfish. The stone crayfish (Austropotamobius torrentium) is a native European freshwater species endangered due to anthropogenic pressure on its habitats, invasive alien crayfish species and climate change. In order to guide the conservation of the stone crayfish within the hotspot of its genetic diversity in Europe, we combined population genetics and species distribution modelling (SDM) to reveal the impact of climate change and invasive signal crayfish (Pacifastacus leniusculus) on the stone crayfish diversity. Population genetic analyses based on newly developed microsatellites revealed moderate within-population genetic diversity and high differentiation among populations, reflecting isolated populations with limited gene flow. Alongside strong genetic structuring concordant with previously established Evolutionary Significant Units using mtDNA data, we discovered high level of inbreeding, indicating homozygote excess within the majority of populations. The SDM results predicted substantial decreases of suitable habitats for stone crayfish by 2070 ; 80% of its currently suitable habitat is predicted to be lost under high‐warming climate change scenario (RCP8.5). Overlapping genetic data with future habitat suitability revealed that 44% of populations with high and/or unique genetic diversity, including three highly divergent and geographically restricted evolutionary lineages, are located in the areas predicted to become unsuitable in the future. Further, SDMs revealed substantial decrease of future habitat suitability for invasive signal crayfish, suggesting that climate change represents greater threat to stone crayfish. Our study highlights the importance of conserving remnant populations through assisted migration and repopulation approaches that could enable species long-term preservation. The results of our research emphasize the importance of multidisciplinary approach in the modern biodiversity conservation.

Potočni rak, Austropotamobius torrentium (Schrank, 1803), je nativna europska slatkovodna vrsta iz porodice Astacidae koja nastanjuje manje vodotoke na višim nadmorskim visinama. Rakovi porodice Astacidae su ključne vrste slatkovodnih ekosustava, a brojnost njihovih populacija, uključujući i populacija potočnog raka, je pod utjecajem raznih čimbenika koji dovode do značajnog smanjenja broja i veličina populacija. U ovom istraživanju razvijeni su novi mikrosatelitni markeri specifični za potočnog raka te su upotrebljeni u istraživanju genetske raznolikosti populacija ove vrste na području Hrvatske. Od 35 novorazvijenih markera, nakon testiranja, njih četiri su potvrđeni kao prikladni te su, uz četiri prethodno razvijena, korišteni za istraživanje populacijske strukture. Istraživanje je provedeno na 437 jedinki iz 17 populacija korištenjem osam mikrosatelitnih lokusa. Rezultati pokazuju veliku raznolikost populacija koje su grupirane prema geografskoj poziciji i filogrupama određenih na temelju mitohondrijske DNA. Rezultati ovog rada pridonose novim spoznajama o potočnom raku te su primjenjivi u izradi adekvatnih planova zaštite i upravljanja. The stone crayfish, Austropotamobius torrentium (Schrank, 1803), is a native European freshwater species of the family Astacidae that inhabits smaller waterbodies at higher altitudes. Freshwater crayfish are keystone species of freshwater ecosystems. The abundance of their populations, including those of the stone crayfish, is influenced by various factors that lead to a significant decline in the number and size of populations. In this research, new microsatellite markers, specific for A. torrentium, were developed. After testing 35 newly developed markers, four were confirmed as suitable. In addition, four previously developed microsatellite markers were used to study population structure of A. torrentium in Croatia. The study was conducted on 437 individuals from 17 populations using eight microsatellite loci. The results show a large diversity of populations grouped according to their geographical position and to phylogroups determined based on mitochondrial DNA. The results of this research contribute to the knowledge on stone crayfish and are applicable in the development of adequate protection and management plans.

Austropotamobius torrentium is a freshwater crayfish species distributed across central and south-eastern Europe, with the highest genetic diversity recorded in the northern-central Dinarides. Previous studies have shown that evolutionary history of A. torrentium was shaped through different paleohydrologic and paleogeologic events from Miocene onwards. In order to connect current genetic structure of A. torrentium with paleogeographic events divergence time estimates were applied on the large-scale mtDNA dataset. Different calibration approaches were applied, including substitution rates, geological events, and for the first time hydrogeological event of the paleo-Danube and paleo-Tisza rivers connection. We presumed that this event could have had a strong influence onto A. torrentium expansion into nowadays areal. Results obtained by different calibrations yielded mostly congruent results and confirmed complex evolutionary history of A. torrentium. Application

Stone crayfish is a freshwater species that inhabits small waterbodies at higher altitudes of central and south-eastern Europe. Previous research revealed that stone crayfish presents genetically highly diverse species, and recently a new crayfish species, formerly considered stone crayfish, was described. In this comprehensive study we included previous samples and new ones collected from 69 previously unsampled locations in Croatia, Slovenia and Macedonia. In order to assess the genetic diversity of the stone crayfish we analysed mtDNA and nuDNA. Results confirmed Dinaric Karst as a hot spot of the stone crayfish diversity and revealed previously unrecognised genetically divergent populations. Considering the high diversity present within the species, a wide range of species delimitation methods were applied on the mtDNA dataset. Different methods resulted in different numbers of operational taxonomic units within the stone crayfish. The results of this study are a step forward towards the future species description within the stone crayfish species complex.

Biljke su tijekom rasta i razvoja izložene različitim okolišnim uvjetima. Nepovoljni uvjeti mijenjaju fiziološke procese, metabolizam, rast i razvoj biljke te dovode do promjena na staničnoj i molekularnoj razini. Osmotski stres se javlja zbog abiotičkih faktora kao što su povišeni salinitet, suša ili ekstremne temperature. Tijekom takvih uvjeta narušen je vodni potencijal biljke. Kako bi se osmolarnost povećala i smanjio gubitak vode, dolazi do sinteze kompatibilnih osmolita koji su organske molekule niske molekulske mase. Biljke nakupljaju različite spojeve: aminokiseline, tercijarne sulfonijeve spojeve, kvarterne amonijeve spojeve, šećere i šećerne alkohole. Osmoliti se nakupljaju u citoplazmi, gdje održavaju osmotski potencijal i fiziološku aktivnost stanice te je njihova biosinteza pod utjecajem okolišnih signala. Takvi spojevi ne utječu na stanične reakcije, a pomažu u toleranciji stresnih uvjeta stabilizacijom makromolekula i staničnih struktura, detoksifikacijom reaktivnih oblika kisika i održavanjem redoks sustava u stanici. Genskom manipulacijom gena za sintezu osmolita moguće je stvoriti biljke koje akumuliraju osmolite u povišenim koncentracijama te tako poboljšati njihovu sposobnost tolerancije stresnih uvjeta. During growth and development plants are exposed to different environmental factors. Adverse conditions change the physiological processes, the metabolism, growth and development of the plant which affect the organism at a cellular and molecular level. Osmotic stress is a consequence of abiotic factors such as high salinity, drought or extreme temperatures. Those conditions disturb the water potential of the cell. To increase the potential and decrease water loss, compatible osmolytes are synthesized. Osmolytes are low molecular mass organic compounds. Plants accumulate different compounds: amino acids, tertiary sulphonium compounds, quaternary ammonium compounds, sugars and sugar alcohols which accumulate in the cytoplasm. Compatible osmolytes maintain the osmotic potential and physiological activity of the cell and are synthesized under control of environmental cues. Additionally, they stabilize the macromolecule and cell structure, scavenge reactive oxygen species and maintain the cell redox system without affecting the metabolic reactions of the cell. With genetic engineering of osmolytes biosynthetic pathways, it is possible to create plants accumulating osmolytes in high concentration which can improve their stress tolerance.

Potočni rak Austropotamobius torrentium (Schrank, 1803) jedan je od pet nativnih europskih vrsta rakova koje nastanjuju manje vodotokove na većim nadmorskim visinama središnje i jugoistočne Europe. U proteklim desetljećima uočeno je smanjenje populacija potočnih rakova uzrokovano antropogenskim pritiskom na njihova staništa, širenjem stranih vrsta rakova i klimatskim promjenama. Stoga se potočni rak smatra ugroženom vrstom, zaštićenom međunarodnim i nacionalnim zakonima. Poseban naglasak potrebno je staviti na razvoj konzervacijskih planova te upotpuniti saznanja o samoj vrsti, kako bi se razvila učinkovita zaštita. To uključuje i nedavna molekularno filogenetička istraživanja vrste A. torrentium koja su pokazala da unutar vrste postoji namanje 7 različitih monofiletskih haplogrupa s najvećom raznolikosti u sjevernim i središnjim Dinaridima. Dosad neistražene populacije potočnog raka iz Hrvatske, Slovenije i Makedonije uključene su u ovo istraživanje uz uzorke s više od 190 lokaliteta s ciljem da se pomoću mitohondrijskih (16S rRNA i COI) i jezgrinih (ITS2 regija) DNA biljega odrede filogenetski odnosi unutar vrste. Zabilježeno je ukupno 48 16S rRNA i 147 COI haplotipova te veći broj ITS2 alela. Filogenetska rekonstrukcija napravljena na sekvencama sva tri DNA biljega, pokazala je kongruentne topologije te nove haplogrupe nisu pronađene. Raspravljena je primjena dobivenih rezultata u budućim planovima očuvanja vrste A. torrentium. (The stone crayfish Austropotamobius torrentium (Schrank, 1803) is one of five native European crayfish species inhabiting smaller waterbodies at higher altitudes of central and south- eastern Europe. In the last decades severe declines in numbers of stone crayfish populations have been recorded, due to anthropogenic pressure on their habitats, spread of non-native crayfish species and climate change. Hence stone crayfish is considered threatened species, protected by international and national laws. The sound knowledge of species, including information on genetics, is a necessity to develop efficient strategies for its protection. Recent molecularphylogenetic research of A. torrentium revealed that within the species at least 7 distinct monophyletic haplogroups exist, with highest genetic diversity in the north-central Dinaric region. This research aimed to update previous results through the inclusion of previously unstudied populations of stone crayfish from Croatia, Slovenia and Macedonia. Samples from more than 190 localities were analysed either for mitochondrial (16S rRNA and COI) or nuclear (ITS2 region) DNA markers. A total of 48 16S rRNA and 147 COI haplotypes, and several ITS2 alleles were obtained. Phylogenetic relationships were reconstructed using all three datasets with different methods of phylogenetic inference showing congruent topologies and no new haplogroups. Applications of acquired results in the future A. torrentium conservation programs are discussed.)

Additional file 5: Results of species delimitation analyses performed on Austropotamobius torrentium COI dataset applying different methods (ABGD (lumper, splitter), TCS, bPTP, mPTP and GMYC).

Additional file 9: Material and methods extended. The detailed information about gene amplification and sequencing, phylogenetic reconstruction, time of divergence estimates, species delimitation and validation, and analyses of meristic characteristics. Additional file also includes list of used references.

Additional file 9: Material and methods extended. The detailed information about gene amplification and sequencing, phylogenetic reconstruction, time of divergence estimates, species delimitation and validation, and analyses of meristic characteristics. Additional file also includes list of used references.

Additional file 2: TCS phylogenetic network based on Austropotamobius torrentium COI gene.

Additional file 5: Results of species delimitation analyses performed on Austropotamobius torrentium COI dataset applying different methods (ABGD (lumper, splitter), TCS, bPTP, mPTP and GMYC).

Additional file 7: Results of Austropotamobius torrentium meristic characteristics analyses.

Additional file 6: Results of Bayes factor species delimitation (BFD) based Austropotamobius torrentium COI dataset.

Additional file 2: TCS phylogenetic network based on Austropotamobius torrentium COI gene.

Additional file 4: Estimation of divergence times based on Austropotamobius torrentium mitochondrial data set using three molecular clock and four geological calibrations.

Additional file 8: Map of proposed evolutionary significant units (ESUs)/cryptic subspecies for Austropotamobius torrentium. The map depicted in figure was produced in ArcGIS 10.3 program package and finished in the program package FreeHand MXa by authors of this study.

Additional file 4: Estimation of divergence times based on Austropotamobius torrentium mitochondrial data set using three molecular clock and four geological calibrations.

Additional file 6: Results of Bayes factor species delimitation (BFD) based Austropotamobius torrentium COI dataset.

Additional file 8: Map of proposed evolutionary significant units (ESUs)/cryptic subspecies for Austropotamobius torrentium. The map depicted in figure was produced in ArcGIS 10.3 program package and finished in the program package FreeHand MXa by authors of this study.

Additional file 7: Results of Austropotamobius torrentium meristic characteristics analyses.