Parallel evolution despite low genetic diversity in three-spined sticklebacks.

When populations repeatedly adapt to similar environments they can evolve similar phenotypes based on shared genetic mechanisms (parallel evolution). The likelihood of parallel evolution is affected by demographic history, as it depends on the standing genetic variation of the source population. The three-spined stickleback (Gasterosteus aculeatus) repeatedly colonized and adapted to brackish and freshwater. Most parallel evolution studies in G. aculeatus were conducted at high latitudes, where freshwater populations maintain connectivity to the source marine populations. Here, we analysed southern and northern European marine and freshwater populations to test two hypotheses. First, that southern European freshwater populations (which currently lack connection to marine populations) lost genetic diversity due to bottlenecks and inbreeding compared to their northern counterparts. Second, that the degree of genetic parallelism is higher among northern than southern European freshwater populations, as the latter have been subjected to strong drift due to isolation. The results show that southern populations exhibit lower genetic diversity but a higher degree of genetic parallelism than northern populations. Hence, they confirm the hypothesis that southern populations have lost genetic diversity, but this loss probably happened after they had already adapted to freshwater conditions, explaining the high degree of genetic parallelism in the south.

Endemic fish calling: Acoustics and reproductive behaviour of the Neretva dwarf goby Orsinigobius croaticus.

The Neretva dwarf goby Orsinigobius croaticus (Gobiiformes, Gobionellidae) is an endemic fish native to the freshwaters of the Adriatic Basin in Croatia and Bosnia and Herzegovina, a Mediterranean Biodiversity Hotspot. Due to its limited distribution range, specific karst habitat and endangered status, laboratory studies on reproductive biology are scarce but crucial. Herein, we investigated the sound production and acoustic behaviour of the endangered O. croaticus during reproductive intersexual laboratory encounters, utilising an interdisciplinary approach. We also performed dissections and micro-computed tomography (µCT) scanning of the pectoral girdle to explore its potential involvement in sound production. Finally, comparative acoustic analysis was conducted on sounds produced by previously recorded soniferous sand gobies to investigate whether acoustic features are species-specific. The endemic O. croaticus is a soniferous species. Males of this species emit pulsatile sounds composed of a variable number of short (~15 ms) consecutive pulses when interacting with females, usually during the pre-spawning phase in the nest, but also during courtship outside the nest. Pulsatile sounds were low-frequency and short pulse trains (~140 Hz, <1000 ms). Male visual behaviour rate was higher when co-occurring with sounds and females entered the male's nest significantly more frequently when sounds were present. Characteristic body movements accompanied male sound production, such as head thrust and fin spreading. Furthermore, µCT scans and dissections suggest that O. croaticus shares certain anatomical similarities of the pectoral girdle (i.e. osseous elements and arrangement of levator pectoralis muscles) to previously studied sand gobies that could be involved in sound production. Multivariate comparisons, using sounds produced by eight soniferous European sand gobies, effectively distinguished soniferous (and sympatric) species based on their acoustic properties. However, the discrimination success decreased when temperature-dependent features (sound duration and pulse repetition rate) were excluded from the analysis. Therefore, we suggest both spectral and temporal features are important for the acoustic differentiation of sand gobies.

Metal(loid)s and persistent organic pollutants in yellow European eel from the Rasa River, Croatia.

The anthropogenic impact on the aquatic environment of the Rasa River (Croatia) was investigated through the analysis of seven polybrominated diphenyl ethers (PBDEs), seven polychlorinated biphenyls (PCBs), three DDT isomers, and 22 major and trace elements using yellow European eel (Anguilla anguilla L.) as a biological indicator of contamination. The obtained data indicated generally low contamination status in the surrounding area. Levels of all organic contaminants in muscle significantly increased with lipid content, length, weight and body condition. In both muscle and liver, most metal(loid)s decreased or remained unchanged with increasing size, while at downstream location only several elements (Cd, Cu, Fe, Na, Se, U, V, Zn) accumulated in the liver with fish growth. Spatial analysis revealed higher pressure of Ag, Cd, Cr, Mo, Tl, U, and V at the downstream location, revealing the potentially limited impact of historical coal mining industry on the lower reaches of the Rasa River.

Cast away in the Adriatic: Low degree of parallel genetic differentiation in three-spined sticklebacks.

The three-spined stickleback (Gasterosteus aculeatus) has repeatedly and independently adapted to freshwater habitats from standing genetic variation (SGV) following colonization from the sea. However, in the Mediterranean Sea G. aculeatus is believed to have gone extinct, and thus the spread of locally adapted alleles between different freshwater populations via the sea since then has been highly unlikely. This is expected to limit parallel evolution, that is the extent to which phylogenetically related alleles can be shared among independently colonized freshwater populations. Using whole genome and 2b-RAD sequencing data, we compared levels of genetic differentiation and genetic parallelism of 15 Adriatic stickleback populations to 19 Pacific, Atlantic and Caspian populations, where gene flow between freshwater populations across extant marine populations is still possible. Our findings support previous studies suggesting that Adriatic populations are highly differentiated (average FST  ≈ 0.45), of low genetic diversity and connectivity, and likely to stem from multiple independent colonizations during the Pleistocene. Linkage disequilibrium network analyses in combination with linear mixed models nevertheless revealed several parallel marine-freshwater differentiated genomic regions, although still not to the extent observed elsewhere in the world. We hypothesize that current levels of genetic parallelism in the Adriatic lineages are a relic of freshwater adaptation from SGV prior to the extinction of marine sticklebacks in the Mediterranean that has persisted despite substantial genetic drift experienced by the Adriatic stickleback isolates.

Potentially Toxic Elements in Water, Sediments and Fish from the Karstic River (Rasa River, Croatia) Located in the Former Coal-Mining Area.

The assessment of the environmental quality of a sensitive karst aquatic system under the centuries-long anthropogenic influence of the coal mining industry is important for both improving the quality of water resources and protecting aquatic wildlife and human health. In this study, we investigated the anthropogenic impact on the aquatic environment of the upper and middle course of the Rasa River through the analysis of a suite of metal(loid)s in three aquatic compartments (water, sediment, fish) using inductively coupled plasma mass spectrometry (ICP-MS). Concentrations of inorganic constituents in water were low, while the chemical composition of stream sediments mainly reflected the geological background of the area, indicating the origin of metal(loid)s from predominantly natural sources. Although comparison with PEC-Q values indicated that existing sediment quality conditions could pose a threat to benthic organisms with regard to Cr and Ni, the constant vertical profiles of these elements suggested their natural origin from the weathering of flysch. Element levels in the muscle of targeted fish species were in accordance with the values typical for low-contaminated freshwater systems, while levels of Cd, Pb and Hg were mostly below the European regulatory limits for toxic elements in foods, indicating that the low concentrations of most contaminants in muscles of fish from the Rasa River do not present a risk to humans or other consumers. The obtained data indicated a generally low contamination status of the western part of the Rasa River basin with regard to the analyzed inorganic elements.

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.

Toxicity prediction and effect characterization of 90 pharmaceuticals and illicit drugs measured in plasma of fish from a major European river (Sava, Croatia).

Chemical analysis of plasma samples of wild fish from the Sava River (Croatia) revealed the presence of 90 different pharmaceuticals/illicit drugs and their metabolites (PhACs/IDrgs). The concentrations of these PhACs/IDrgs in plasma were 10 to 1000 times higher than their concentrations in river water. Antibiotics, allergy/cold medications and analgesics were categories with the highest plasma concentrations. Fifty PhACs/IDrgs were identified as chemicals of concern based on the fish plasma model (FPM) effect ratios (ER) and their potential to activate evolutionary conserved biological targets. Chemicals of concern were also prioritized by calculating exposure-activity ratios (EARs) where plasma concentrations of chemicals were compared to their bioactivities in comprehensive ToxCast suite of in vitro assays. Overall, the applied prioritization methods indicated stimulants (nicotine, cotinine) and allergy/cold medications (prednisolone, dexamethasone) as having the highest potential biological impact on fish. The FPM model pointed to psychoactive substances (hallucinogens/stimulants and opioids) and psychotropic substances in the cannabinoids category (i.e. CBD and THC). EAR confirmed above and singled out additional chemicals of concern - anticholesteremic simvastatin and antiepileptic haloperidol. Present study demonstrates how the use of a combination of chemical analyses, and bio-effects based risk predictions with multiple criteria can help identify priority contaminants in freshwaters. The results reveal a widespread exposure of fish to complex mixtures of PhACs/IDrgs, which may target common molecular targets. While many of the prioritized chemicals occurred at low concentrations, their adverse effect on aquatic communities, due to continuous chronic exposure and additive effects, should not be neglected.

Long-term analysis of fish assemblage structure in the middle section of the Sava River - The impact of pollution, flood protection and dam construction.

At the beginning of the 20th century, the middle section of the Sava River in Croatia was unaffected by major human activities and rich in ichthyofauna. The Sava River was important for commercial and recreational fishing for the local population, which still remains today. However, the 1920s mining industry was established in Slovenia, which emitted carbon dust into the Sava River. At the same time, the construction of embankments to mitigate flooding started in the middle section. Furthermore, in the 1980s, the Krsko nuclear power plant (NPP), and in the 2010s, the Krsko hydropower plant (HPP) were built in Slovenia. These activities could have an impact on the composition of fish communities downstream from the major sources of disturbances. Therefore, the main aim of this paper were to analyze the changes in fish assemblages of the Sava River from 1978 to 2017, prior to and after the construction of Krsko NPP and HPP at the Medsave site on the Sava River, 20 km downstream from the major construction operations. Collected data were divided into four sampling periods (SP): SP1, from 1978 to 1980; SP2, from 1991 to 1994; SP3, from 2001 to 2006, and SP4 from 2011 to 2017. Besides alien fish species, water quality and hydromorphological modifications were identified as significant stressors. In SP1 and SP2 limnophilic and eurytopic fish groups were predominant, and 26 different fish species were identified, but in SP3 and SP4 rheophilic fish groups become dominant, and the diversity has declined to 21 species. Threatened species blageon, Telestes souffia seems to be missing from the main course of the Sava River in last 20 years. It can be concluded that disturbances in the fish assemblage pattern have coincided with the presence of multiple stressors of human origin.

Emerging human pathogen Acinetobacter baumannii in the natural aquatic environment: a public health risk?

Bacterium Acinetobacter baumannii is an emerging human pathogen whose presence in the aquatic environment raises the issue of public health risk. Fish colonization represents the potential route of pathogen transmission to humans. The aim was to examine the colonization of A. baumannii to freshwater fish Poecilia reticulata. An extensively drug-resistant A. baumannii was tested at three concentrations in natural spring water. Additionally, 70 fish from the Sava River (Croatia) were screened for the presence of A. baumannii, which was not found in gill swabs or analysed gut. The colonization potential of A. baumannii in freshwater fish is dependent upon its concentration in surrounding water. The low concentration of A. baumannii in natural waters represents low colonization potential of freshwater fish. The risk for public health exists in closed water bodies where there is constant inflow of water polluted by A. baumannii in concentrations above 3 log CFU mL-1.

The Gyrodactylus (Monogenea, Gyrodactylidae) parasite fauna of freshwater sand gobies (Teleostei, Gobioidei) in their centre of endemism, with description of seven new species.

While Gobioidei comprises showcases of (adaptive) radiation, the scientific interest they yielded did not ensure full understanding of goby biodiversity. Even in a well-studied region like Europe, wide knowledge gaps remain. Sand gobies represent one of the few clades whose monogenean parasites have been thoroughly studied. However, in the Balkans, part of the sand gobies' centre of endemism, these parasites were unstudied. We focus on Greek and Croatian freshwater gobies. From five sand goby species, the first parasites are reported, describing seven new Gyrodactylus species. Economidichthys pygmaeus harbours Gyrodactylus benedeni sp. n. and Gyrodactylus dorlodoti sp. n. Its congener E. trichonis hosts G. meelkopae sp. n. Knipowitschia milleri was found to host G. charon sp. n., K. thessala is infected by G. bios sp. n., and K. croatica by G. douglasadamsi sp. n. and G. hellemansi sp. n. Gyrodactylus bubyri was found on its type host K. caucasica. A diverse parasite fauna is expected for a region known for its biodiversity and endemism. The contribution of parasites to species richness in such hotspots is overlooked. The observed species richness per host is rather low compared to the better-studied eastern Atlantic sand gobies. Host vicariance is considered to mediate parasite specificity in this fauna. Some new flatworm species display unique morphological features, such as the remarkable size of the marginal hook sickle proper compared to its foot in the Economidichthys parasites, or a characteristically kinked marginal hook sickle in G. douglasadamsi sp. n. These features reflect their hosts' endemism in the Balkans.

Biomonitoring of heavy metals in fish from the Danube River.

The Croatian part of the Danube River extends over 188 km and comprises 58 % of the country's overall area used for commercial freshwater fishing. To date, the heavy metal contamination of fish in the Croatian part of the Danube has not been studied. The main purpose of this study was to determine heavy metal levels in muscle tissue of sampled fish species and to analyze the measured values according to feeding habits of particular groups. Lead ranged from 0.015 µg(-1) dry weight in planktivorous to 0.039 µg(-1) dry weight in herbivorous fish, cadmium from 0.013 µg(-1) dry weight in herbivorous to 0.018 µg(-1) dry weight in piscivorous fish, mercury from 0.191 µg(-1) dry weight in omnivorous to 0.441 µg(-1) dry weight in planktivorous fish and arsenic from 0.018 µg(-1) dry weight in planktivorous to 0.039 µg(-1) dry weight in omnivorous fish. Among the analyzed metals in muscle tissue of sampled fish, only mercury exceeded the maximal level (0.5 mg kg(-1)) permitted according to the national and EU regulations determining maximum levels for certain contaminants in foodstuffs, indicating a hazard for consumers of fish from the Danube River.

Loaches in a long term study of the Drava River in Croatia.

The diversity, species composition and structure of the fish community were studied in the vicinity of three power plant reservoirs in the middle part of the Drava River in Croatia. Over a period of twenty years in a collection of 25,913 fishes, 56 different fish species belonging to 16 families were recorded. The family Cobitidae is represented by two species: Cobitis elongatoides and Misgurnus fossilis, while the family Balitoridae is represented by only one species, Barbatula barbatula. The presence of the Cobitidae family was recorded in eleven of seventeen investigated localities, while Balitoridae was found in six localities. In the studied fish community, the percentage of C. elongatoides is 1.41% of the total number of individuals, and B. harbatula is 1.02%. M. fossilis is a rare species (0.03 %). Both C. elongatoides and B. barbatula were not caught at the beginning of investigation. C. elongatoides and B. harhatula were first caught in 1985, while M. fossilis was first caught in 1996. The proportion of C. elongatoides in the entire community increased, while the proportion of B. barhatula decreased in later years.

Growth of Cobitis narentana Karaman, 1928 in the Neretva River, Croatia.

Age and growth of Cobitis narentana were examined in the delta of Neretva River in Croatia. Maximum observed length was 100.4 mm for females and 63.5 for males. Five age classes were determined, from 0+ to 4+. For both females (61.0%) and males (53.8%), the greatest proportion of specimens were in the 1+ age category. Growth was faster in males in the first year of life, while females grew at a faster rate than males after the first year. The Fulton condition factor was determined (CF=0.588 for males and CF=0.618 for females). The length-weight relationship was determined for females (W=8xl0(-5)L2.9325) and males (W=2x l0(-5)L2.6431). The parameter b was calculated as less than b=3.0, thus establishing that growth in both males and females was allometric. Growth rate was determined using the von Bertalanffy growth rate curves for females (Lt=101.1[ 1-e(-0.5(t-0.94)]; r2=0.99) and for males (Lt=65.3[1-e(-0.54(t-2.27)]; r2=0.97). The resulting growth rate coefficient (K) was found to be slightly higher in males (0.54) than in females (0.5).