The first cavefish in the Dinaric Karst? Cave colonization made possible by phenotypic plasticity in Telestes karsticus.

Cave animals are an excellent model system for studying adaptive evolution. At present, however, little is known about the mechanisms that enable surface colonizers to survive in the challenging environment of caves. One possibility is that these species have the necessary genetic background to respond with plastic changes to the pressures of underground habitats. To gain insight into this process, we conducted a comparative study with the fish species Telestes karsticus, which occurs in a hydrological system consisting of an interconnected stream and a cave. Results showed that T. karsticus resided year-round and spawned in Susik cave, making it the first known cavefish in the Dinaric Karst. Cave and surface populations differed in morphological and physiological characteristics, as well as in patterns of gene expression without any evidence of genetic divergence. To test whether observed trait differences were plastic or genetic, we placed adult fish from both populations under light/dark or constant dark conditions. Common laboratory conditions erased all morphometric differences between the two morphs, suggesting phenotypic plasticity is driving the divergence of shape and size in wild fish. Lighter pigmentation and increased fat deposition exhibited by cave individuals were also observed in surface fish kept in the dark in the laboratory. Our study also revealed that specialized cave traits were not solely attributed to developmental plasticity, but also arose from adult responses, including acclimatization. Thus, we conclude that T. karsticus can adapt to cave conditions, with phenotypic plasticity playing an important role in the process of cave colonization.

Tracing the oomycete pathogen Saprolegnia parasitica in aquaculture and the environment.

Saprolegnia parasitica causes saprolegniosis, a disease responsible for significant economic losses in aquaculture and declines of fish populations in the wild, but the knowledge of its distribution and prevalence in the environment is limited. We developed a fast, sensitive and specific S. parasitica droplet digital PCR (ddPCR) assay and demonstrated its applicability for the detection and quantification of the pathogen in environmental samples: swab DNA collected from the host (trout skin, surface of eggs) and environmental DNA extracted from water. The developed assay was used to assess how abiotic (i.e. physico-chemical parameters of the water) and biotic (health status of the host) factors influence the S. parasitica load in the environment. The pathogen load in water samples was positively correlated with some site-specific abiotic parameters such as electrical conductivity (EC) and calcium, while fluorides were negatively correlated, suggesting that physico-chemical parameters are important for determining S. parasitica load in natural waters. Furthermore, skin swabs of injured trout had significantly higher pathogen load than swabs collected from healthy fish, confirming that S. parasitica is a widespread opportunistic pathogen. Our results provide new insights into various environmental factors that influence the distribution and abundance of S. parasitica.

Comparative trophic ecology of microhabitat-associated guilds of reef fishes in the Adriatic Sea.

Reef ecosystems are characterized by highly heterogenous habitats and functionally diverse fish communities. Few studies have examined how functional diversity differs among habitats within these communities, i.e., species associated with a specific habitat may have similar trophic ecologies meaning that the functional diversity within the community is driven by habitat diversity or, conversely, high functional diversity within each habitat would indicate that resource segregation also occurs at the habitat level. We used stable isotope ratios of carbon and nitrogen to estimate trophic position, resource use and ontogenetic niche shifts of 15 reef fishes associated with four distinct habitat types (cryptobenthic, epibenthic sand, epibenthic rock and hyperbenthic) on the Croatian coast of the Adriatic Sea. Trophic ecology was quite similar across fish assemblages, but there was strong evidence of niche segregation among fish species within each assemblage showing high functional diversity within each microhabitat. The sampled fish community contained benthic and pelagic resource users, along with multiple intermediate generalists. Consumer stable isotope ratios revealed considerable interspecific variation in resource use among fishes within each habitat type. The cryptobenthic fishes were a notable exception to this trend with the narrow range of resource use values, indicating reliance of these species on a single resource. The greatest diversity of trophic positions within a guild was observed in cryptobenthic and rock-associated epibenthic fishes. The majority of observed ontogenetic variation in studied fish species reflected an increase in benthic resource use and trophic position. However, the degree of ontogenetic variation in trophic ecology of studied species, if present, was generally low, showing no dramatic change in the ecology of any species. The size structuring among guilds was considerable, with cryptobenthic fishes the smallest on average and hyperbenthic fishes the largest, despite guilds having similar ranges of trophic positions.

Correlation between acoustic divergence and phylogenetic distance in soniferous European gobiids (Gobiidae; Gobius lineage).

In fish, species identity can be encoded by sounds, which have been thoroughly investigated in European gobiids (Gobiidae, Gobius lineage). Recent evolutionary studies suggest that deterministic and/or stochastic forces could generate acoustic differences among related animal species, though this has not been investigated in any teleost group to date. In the present comparative study, we analysed the sounds from nine soniferous gobiids and quantitatively assessed their acoustic variability. Our interspecific acoustic study, incorporating for the first time the representative acoustic signals from the majority of soniferous gobiids, suggested that their sounds are truly species-specific (92% of sounds correctly classified into exact species) and each taxon possesses a unique set of spectro-temporal variables. In addition, we reconstructed phylogenetic relationships from a concatenated molecular dataset consisting of multiple molecular markers to track the evolution of acoustic signals in soniferous gobiids. The results of this study indicated that the genus Padogobius is polyphyletic, since P. nigricans was nested within the Ponto-Caspian clade, while the congeneric P. bonelli turned out to be a sister taxon to the remaining investigated soniferous species. Lastly, by extracting the acoustic and genetic distance matrices, sound variability and genetic distance were correlated for the first time to assess whether sound evolution follows a similar phylogenetic pattern. The positive correlation between the sound variability and genetic distance obtained here emphasizes that certain acoustic features from representative sounds could carry the phylogenetic signal in soniferous gobiids. Our study was the first attempt to evaluate the mutual relationship between acoustic variation and genetic divergence in any teleost fish.

Ancient connections among the European rivers and watersheds revealed from the evolutionary history of the genus Telestes (Actinopterygii; Cypriniformes).

In order to better understand the complex geologic history of the Mediterranean area, we have analysed evolutionary history, phylogeographic structure and molecular diversity of freshwater fishes belonging to the genus Telestes. As primary freshwater fishes distributed largely in the Mediterranean basin, this genus represents a suitable model system for investigating the historical biogeography of freshwater drainage systems in southern Europe. In this investigation we have included samples representing all Telestes species and based our analyses on one mitochondrial and one nuclear gene. We have investigated phylogenetic structure inside the genus Telestes, estimated divergence times, reconstructed ancestral distribution ranges and described intraspecific molecular diversity. Diversification of Telestes started in the Early Miocene, when the ancestors of T. souffia, lineage comprising T. croaticus and T. fontinalis, and the one comprising T. pleurobipunctatus and T. beoticus got isolated. The remaining species are genetically more closely related and form a common cluster in the recovered phylogenetic trees. Complex geological history of southern Europe, including formation of continental bridges, fragmentation of landmass, closing of the sea corridor, local tectonic activities, led to complicated biogeographical pattern of this genus, caused by multiple colonization events and passovers between ancient rivers and water basins. Especially pronounced diversity of Telestes found in the Adriatic watershed in Croatia and Bosnia and Herzegovina is a consequence of a triple colonization of this area by different lineages, which led to an existence of genetically distinct species in neighboring areas. Significant intraspecific structuring is present in T. souffia, T. muticellus, T. croaticus and T. pleurobipunctatus. Besides in well-structured species, elevated levels of genetic polymorphism were found inside T. turskyi and T. ukliva, as a consequence of their old origin and unconstrained evolutionary history.

Different Histories, Different Destinies‒Impact of Evolutionary History and Population Genetic Structure on Extinction Risk of the Adriatic Spined Loaches (Genus Cobitis; Cypriniformes, Actinopterygii).

The region of Balkans is often considered as an ichthyologic "hot spot", with a great number of species and high portion of endemics living in fresh waters in a relatively small area. The Adriatic watershed in Croatia and Herzegovina is inhabited by six spined loach species (genus Cobitis) whose extinction risk estimations were based solely on their extent of occurrence (and/or area of occupancy) and its fragmentation, and conservation proposals do not consider diversity below species level. In this investigation we employed molecular genetic methods to describe present genetic structure of the Adriatic spined loaches and reveal their demographic history. The divergence of the Adriatic lineages inside the genus Cobitis started in Miocene and lasted until Pleistocene epoch. Geological events responsible for shaping recent diversity of spined loaches in the Adriatic basin are: the Dinarid Mountains upwelling, the evolution of Dinaric Lake system, local tectonic activity, river connections during glaciations and differences in sea level. Even though all the investigated species inhabit karstic rivers located in the same geographic area and that were subject of similar geological events, the results obtained reveal great differences in their genetic diversity and structure and point out the necessity of different conservation measures to ensure their future viability. High level of genetic polymorphism is characteristic for species located more to the south. Two species comprised of more than one population have completely different intraspecific structure; populations of C. illyrica are genetically distinct and represent separate evolutionary significant units, whereas intraspecific structure of C. narentana corresponds to metapopulational pattern. Without population genetic data, evolutionary significant units could be easily misidentified. Furthermore, the obtained results affirm that population genetic measurements are able to detect differences among closely located and related species and estimate extinction risk even more accurately than currently applied IUCN criteria.

Combining morphology and genetics in resolving taxonomy--a systematic revision of spined loaches (Genus Cobitis; Cypriniformes, Actinopterygii) in the Adriatic watershed.

Taxonomic investigation of spined loaches from Dalmatia and Herzegovina was conducted on specimens from 14 localities. The results of the detailed morphological investigations were combined with genetic data (based on one mitochondrial and two nuclear genes) in order to resolve the taxonomic status of each Cobitis population. Among the investigated features of external morphology, the appearance of spots on the caudal fin base turned out to have the greatest diagnostic value. Furthermore, the number of branched fin rays enabled the discrimination of several species. No morphometric character alone could ensure determination of any Cobitis species. Nevertheless, groups of populations that are more similar in their body shapes correspond to mitochondrial phylogenetic lineages. Based on molecular genetic markers, Dalmatian and Herzegovinian spined loaches form independent lineages inside the Adriatic phylogenetic group. Mitochondrial DNA phylogenetic reconstruction revealed six monophyletic lineages, corresponding to six species distributed in the investigated area. The population distributed in Mostarsko blato karstic field in Bosnia and Herzegovina is described as a new species based on a unique combination of morphological characters: a single triangular Canestrini scale; usually 51/2 branched anal fin rays, 61/2 branched dorsal fin rays, 14 branched caudal fin rays; no spots in the surface pigmentation layer on the caudal fin base; scales on the body very small.

Identity of Squalius (Actinopterygii, Cyprinidae) from Istra Peninsula in Croatia (Adriatic Sea basin).

A chub of previously ambiguous identity from the Boljunscica and Pazincica rivers (south-eastern Istra Peninsula) was studied and compared with geographically close Squalius squalus, Squalius zrmanja, and Squalius janae recently described from the Dragonja River drainage in the Adriatic Sea basin in Slovenia. It was shown that the chub from the south-eastern Istra Peninsula differs from all know species of Squalius but one: Squalius janae. Three samples examined from Boljunscica and Pazincica rivers and Squalius janae from its type locality, Dragonja River, show the following characters typical for the latter species: a long head (the head length 27-32% SL); a pointed conical snout with a clearly projecting upper jaw; a long straight mouth cleft, the lower jaw length (39-45% HL) exceeding the caudal peduncle depth; a large eye; commonly 9? branched anal-fin rays; commonly 44 total vertebrae (24+20 or 25+19); bright silvery colouration, scales easily lost; iris, pectoral, pelvic and anal fin pigmentation with yellow shades. The data on the distribution of Squalius chubs in the northern Adriatic basin support the assumption that the range of Squalius janae is determined by the geology of the Trieste Flysch Basin and the Pazin Flysch Basin forming the base of the Istra Peninsula. The distribution pattern of this species does not support a simple model of fish dispersal and a complete connectivity within the whole Palaeo-Po historical drainage. Indeed, it indicates a disrupted surface palaeohydrography that was heavily fragmented by karstification in the whole Dinaric area.