In terms of ichthyology, Bosnia and Herzegovina (BiH) is one of the most interesting parts of Southeast Europe, due to its rich biodiversity and high level of endemism. Despite its relevance, the entire territory has been poorly explored. Here, we provide an updated inventory of the current state of knowledge on fishes, including lampreys, from the freshwaters of BiH by hydrographic basin, with recent distributional data and updated taxonomic status reviewed and compared with previous lists. The checklist was compiled based on the existing scientific and grey literature, technical reports, scientific congresses, academic dissertations, and unpublished/personal observations. In total, 123 species including diadromous and euryhaline fishes have been documented in BiH freshwaters to date. Of these, 110 are primarily freshwater. In comparison to the last published monography (Sofradžija 2009), we present a 9% increase in species number (11 species), resulting mainly from taxonomic re-evaluations of existing taxa on the basis of new information and the adoption of a new changes in the taxonomic status of several species. Among the valid primarily freshwater species, 87 are native and 23 are non-native. A total of 38 endemic species have restricted distribution, and are threatened by numerous anthropogenic pressures. Four species are considered endemic only to BiH: Cobitis herzegoviniensis Buj & Šanda, 2014; Phoxinellus pseudalepidotus Bogutskaya & Zupančič, 2003; Telestes dabar Bogutskaya, Zupančič, Bogut & Naseka, 2012; and T. metohiensis (Steindachner, 1901). In total, 75 genera and 34 families are represented: Leuciscidae is represented by 37 species, the Salmonidae by 13, followed by the Cyprinidae, Cobitidae and Percidae, each with eight species. The native species richness follows a pattern similar to that observed in other southern European countries. A national list of endangered species has not yet been proposed to BiH and management strategies for their protection or conservation are also not implemented. Hopefully, this updated checklist will serve as a basis for future research aimed at understanding the origin and status of conservation of the BiH fishes diversity, and supporting effective management and conservation programmes.

The Ohrid trout (Salmo letnica), an endangered salmonid endemic to Lake Ohrid, has repeatedly failed to establish viable populations when introduced outside its native range. A rare exception occurs in Lisina Reservoir, Serbia, where introductions in the late 1970s and 1980s produced a persistent population. Using mitochondrial control region sequences and 12 nuclear microsatellite loci, we tested whether trout resembling S. letnica in Lisina indeed originate from Lake Ohrid, evaluated their genetic similarity to it, and assessed potential hybridization with co-occurring Macedonian trout (Salmo macedonicus) and Atlantic brown trout (Salmo trutta). All analyses confirmed that the Lisina fish belong to S. letnica. A previously unrecorded Ohrid lineage haplotype was detected, likely reflecting stochastic capture during translocation. Microsatellite data revealed modest but significant differentiation between Lisina and Ohrid populations, consistent with a founder effect and subsequent drift. Despite syntopy with co-occurring trout, no introgression into Lisina S. letnica was observed, indicating strong reproductive isolation. The persistence of a genetically distinct and unadmixed S. letnica population outside Lake Ohrid represents an unintended conservation success. While this does not serve as a general endorsement of species translocations, Lisina population could provide a backup resource for future restoration of the declining native Ohrid trout.

Subfamily Leuciscinae, the largest freshwater fish group of the western Palearctic region, has been widely studied, including the phylogenetic relationships between its species and genera. However, until now, one genus completely escaped attention in this regard. Even though several works hypothesised about its evolutionary relationships based on its scarce morphological data, they did not succeed in resolving this puzzle, and their assumptions were often contradictory. Our study aimed to bring insight into the phylogenetic origin and relationships of the enigmatic Caucasian genus Leucalburnus based on the most taxon-dense, multilocus molecular analyses of the subfamily Leuciscinae so far. Moreover, a time-calibrated phylogenetic reconstruction was performed in order to put the emergence of the Leucalburnus lineage in a timeframe. In our analyses, Leucalburnus was consistently recovered as a member of the Alburnus clade, which further comprises Alburnus, Leucaspius and Anaecypris, adding thus one more genus to the clade and providing more evidence of non-monophyly of the genus Alburnus.

One adult specimen of red-mouthed goby Gobius cruentatus Gmelin, 1789 was found in the coastal waters of the Black Sea in Bulgaria. The specimen was caught on 23 September 2023 during an underwater ichthyological survey near the town of Chernomorets. It represents the first record of this species for the western Black Sea coast and for Bulgaria.

The distribution of the neutral component of genetic diversity is the interplay of historical and ongoing processes resulting in the species-specific genetic structure of populations, which can, however, be disrupted by interspecific hybridisation and introgression. In this study, we focused on two species of water frogs, Pelophylax epeiroticus and P. kurtmuelleri, which live in sympatry in the southwestern Balkans, to investigate the rate of hybridisation and population genetic structure using cytogenetic, mitochondrial (ND2) and nuclear DNA (microsatellite) markers. The overall hybridisation rate was 2.6%, with rates reaching up to 10% at specific sites. The course of gametogenesis and the occurrence of later generations of hybrids (beyond the F1 generation) indicate a sexual mode of hybrid reproduction. The bimodal structure of hybrid populations and the rarity of hybrids suggest substantial reproductive isolation between the two species; however, this isolation is unlikely attributable to differences in ecological niche occupation. In P. epeiroticus, sequence variation in the ND2 gene revealed two divergent lineages with a clear geographic pattern that corresponds to the genetic structure in microsatellite markers. In contrast, P. kurtmuelleri populations were not as geographically structured and showed only weak genetic differentiation in both types of markers. Pelophylax epeiroticus was significantly less variable at microsatellite loci compared to P. kurtmuelleri, which, together with the high differentiation of its populations, suggests a stronger influence of genetic drift. We can hypothesise that the differential strength of genetic drift in the two species may lead to unequal interspecific gene flow.

Aim Using a widely distributed and frequently hybridising fish (genus Rutilus) in the Palearctic as a model system, we re-evaluated a series of hypotheses on phylogeny, taxonomy, phylogeography and the role of hybridization in evolutionary history based on the first large-scale analyses of nuclear SNPs together with mtDNA data obtained from all Rutilus species. Location Water systems in Northern Eurasia. Methods Based on nuclear (ddRADseq) data, we applied phylogenomic, population genomic and introgression analyses to assess the diversity of lineages, their distribution and hybridization in the context of the hypotheses tested. Results We identified 22 lineages in three major clades-Rutilus s. str., Leucos and Pararutilus. High diversity of lineages was discovered, with three revived species and three novel lineages of undescribed species in Europe. Evidence for extensive mito-nuclear discordance and ongoing and past inter- and intrageneric introgressive hybridization was found in many lineages. A Caucasian relic lineage and the endangered narrow endemic species 'Rutilus' atropatenus were found to represent an independent lineage (Orthroleucos) with a high level of past introgression with Rutilus. Main Conclusions The genetic structure of Rutilus revealed by nuclear SNPs contrasts markedly with that supported by mtDNA. Numerous mito-nuclear discordances are evidence of the past secondary contacts and highlight the weakness of taxonomy based on mtDNA alone. A super-lineage of mtDNA covering the range of many European, Siberian, Caucasian, and Central Asian Rutilus species (5000 km long) is a result of introgression of mtDNA from a widespread Ponto-Caspian lineage whose distribution likely pulsed with Pleistocene glaciation cycles. Anadromous life histories may have evolved in parallel in different clades and been facilitated by hybridization. The high plasticity, adaptiveness and species diversity of the genus Rutilus may be underlined by their heterogeneous genomes resulting from extensive interspecific hybridization. However, narrow-range endemic species may be threatened by introgressive hybridization.

Interspecific gene flow may profoundly impact genome integrity and adaptive evolution in hybridizing species, leading to novelties such as transgressive traits, supergenes, or, sometimes, the emergence of asexually reproducing lineages. Conventionally, introgression is thought to proceed between reproductively interacting species, mediated by recombining interspecific hybrids, while asexual lineages are considered evolutionary deadlock for genomes trapped in them. Our study on Cobitis loaches in the western Balkan watersheds demonstrates an alternative mechanism where a mix of asexuality and polyploidy facilitates significant introgression from a long-extinct species. Through extensive sampling and cytogenetic and phylogenomic analyses, we identified a sexual species, C. ohridana (OO) coexisting with its asexual hybrid form (OX) originating from hybridization with an extinct species (XX). The diploid OX hybrids pass both parental subgenomes mostly clonally with occasional gene conversions, while triploid OOX hybrids reproduce through meiotic hybridogenesis, producing O-like gametes with localized gene conversions by X alleles. Their mating with the locally dominant sexual species consequently leads to over 4% admixture in both nuclear and mitochondrial genomes. Our findings challenge the view of hybrid asexual lineages as evolutionary dead ends, revealing their significant role as reservoirs of genetic diversity and agents of interspecific gene exchange, even after the extinction of one parental taxon. ### Competing Interest Statement The authors have declared no competing interest.

The Neretva River drainage, a unique biogeographic unit within the Balkans, is a typical karstic river system with complex hydrological connections, hosting many endemic fishes, among which also the Neretva bleak Alburnus neretvae (Cypriniformes: Leuciscidae). It has one of the smallest distribution ranges among its congeners. Moreover, its habitat is especially susceptible to ongoing climate change and human induced alterations, thus the species may face a severe reduction and fragmentation of habitat in the near future. We present the first population genetic study of the Neretva bleak, assessing the current state of the species, based on analyses of mitochondrial gene cytochrome b of 89 individuals from 11 localities across the Neretva drainage, representing five populations. Our results show that the species is undergoing recent population expansion since the end of the Pleistocene and exhibits a high haplotype diversity at most localities. Our results further indicate the existence of genetic structure within this species; pronounced genetic differentiation was revealed between all populations apart from the pairs Trebižat–Ričina and Neretva–Trebišnjica. Although, based on our results, the species is currently in good condition, monitoring and conservation measures should be established with an emphasis on maintaining the genetic diversity within the species.

The Mediterranean and the Middle East represent unique biogeographical regions that significantly shaped the evolutionary history and particular diversity of their associated organisms. However, knowledge on the copepods parasitizing freshwater fishes in these regions is limited. This study aims to investigate the diversity and phylogeny of parasitic copepods in freshwater fishes across the Mediterranean and the Middle Eastern regions. A total of 169 freshwater fish species from the Mediterranean and Middle East were examined for metazoan parasites, yielding over 1000 parasitic copepods. A thorough morphological evaluation combined with molecular analyses of partial fragments of rDNA (18S and 28S) and mitochondrial cytochrome c oxidase subunit I (COI) led to the identification of 7 species of Ergasilidae and 3 species of Lernaeidae. These findings include the descriptions of 2 new species: Ergasilus italicus n. sp. parasitizing South European nase, Protochondrostoma genei (Bonaparte, 1839), in Italy and Pseudolamproglena zahrziensis n. sp. found in yellow barbel, Carasobarbus luteus (Heckel, 1843), in Iraq. New host and geographical records, along with molecular data are provided for 8 previously described species - Ergasilus barbi Rahemo, , Ergasilus briani Markevich, , Ergasilus lizae Krøyer, , Ergasilus rostralis Ho, Jayarajan & Radhakrishnan , Neoergasilus japonicus (Harada, ), Paraergasilus longidigitus Yin, 1954, Lamproglena pulchella von Nordmann, 1832 and Lernaea cyprinacea Linnaeus, 1758.

To date, the phylogeny of Alburnus, one of the most speciose western Palearctic freshwater fish genera, remains unresolved and its monophyly questioned. Here, we present a taxon-dense phylogeny of the genus and related genera, based on mitochondrial and nuclear DNA sequence data. A special focus is put on relationships between the Peri-Adriatic species. Our analyses confirmed the paraphyly of Alburnus with the retrieval of Leucaspius delineatus, Anaecypris punica and Anaecypris hispanica as three separate clades within it. This finding challenges the current taxonomic framework of the genus and highlights the need for a re-evaluation of its classification. The remaining three well-supported clades are currently assigned to Alburnus: Clade I from Levant and the Tigris and Euphrates basins, Clade V with shemayas and Clade VI comprising all remaining species: from Europe, Ponto-Caspian and Aral regions and Anatolia. Most relationships within Clade V and VI were not resolved likely due to rapid diversification in the late Pliocene and Pleistocene. Monophyly of the Peri-Adriatic group, suggested in previous works, was not confirmed. Moreover, Alburnus arborella did not form a monophyletic lineage and displayed extraordinary diversity both in its native and introduced range. Finally, new evidence for introductions of species were provided.

Mitochondrial oxidative phosphorylation (OXPHOS) genes are a system subject to selection under determined environmental constraints despite a neutral evolution model that has long been hypothesized for the mitochondrial genome. In this study, the sequences of ND1, Cytb, and COI OXPHOS genes were analyzed in six populations of the eurythermal and euryhaline killifish A. fasciatus, to detect non-synonymous mutations leading to amino acid changes and to check whether selection acted on them using tests of recombination and selection. The results indicate a high COI and Cytb gene diversity and a high percentage of private haplotypes in all populations. In the Greek population, non-synonymous nucleotide substitutions were observed in the N-terminal region of COI and Cytb. Positively selected sites were also found. The information we obtained from the mitochondrial DNA sequences of A. fasciatus adds to the growing data on selective pressure acting on mitochondrial DNA in non-model species. These results should be explored from the perspective of the local adaptation of eurythermal and euryhaline species and supported using experimental evidence to better understand the interplay between historical climatic events and local adaptation and how each of them contributes to shaping the genetic structure of this species.

Artificial propagation and stocking of brown trout is a standard practice in recreational fishery management. In recent decades, the importance of maintaining intraspecific diversity and protecting locally adapted lineages has been recognized for the species' long-term survival. The first step in selecting donors for stocking involves distinguishing native trout from non-native and introgressed individuals. The established method for discerning Atlantic hatchery strains from the wild populations involves genetic screening of individual diagnostic SNPs and microsatellite assignment tests. This study, using Serbia's Panjica hatchery as an example, illustrates the proper conduct of routine genetic screening for identifying suitable donors for supportive stocking. The broodstock and reference populations were screened using mtDNA control region, LDH nuclear gene, and 12 microsatellite loci to assess the origin, diversity, and inbreeding levels. The analysis revealed only moderate contamination with Atlantic trout and showed the regional origin of the Danubian genes – over 50% of the broodstock was composed of non-introgressed Danubian individuals tracing their origin to the Zapadna Morava River system. Additionally, the study highlighted a considerable discordance between LDH locus and microsatellites in identifying introgressed individuals, raising concerns about the sole reliance on LDH locus for the identification of Atlantic genetic origin in nuclear DNA.

New data on the recently described goby Pomatoschistus nanus Engin & Seyhan, 2017 from the Mediterranean are presented. The species is identified through genetics, morphology and coloration. Data from Malta and Cyprus significantly extend the known distribution area of the species. The Malta data also extends the upper bathymetric limit as well as the maximum known size for the species, currently considered the smallest in the Mediterranean. The morphological variability of P. nanus is also discussed.

During their radiation, certain groups of animals evolved significant phenotypic disparity (morphological diversity), enabling them to thrive in diverse environments. Adaptations to the same type of environment can lead to convergent evolution in function and morphology. However, well-documented examples in repeated adaptations of teleost fishes to different habitats, which are not primarily related to trophic specialization, are still scarce. Gobies are a remarkable fsh group, exhibiting a great species diversity, morphological variability, and extraordinary ability to colonize very different environments. A variety of lifestyles and body forms evolved also in European lineages of gobies. We conducted two-dimensional geometric morphometric and phylomorphospace analyses in European lineages of gobies and evaluated the extent of convergent evolution in shape associated with adaptation to various habitats. Our analyses revealed the change in shape along the nektonic-cryptobenthic axis, from very slender head and body to stout body and wide head. We showed convergent evolution related to mode of locomotion in the given habitat in four ecological groups: nektonic, hyperbenthic, cryptobenthic, and freshwater gobies. Gobies, therefore, emerge as a highly diversified lineage with unique lifestyle variations, offering invaluable insights into filling of ecomorphological space and mechanisms of adaptation to various aquatic environments with distinct locomotion requirements

In the inland waters of the Balkans, many brown trout populations have been severely depleted. Therefore, identifying potential threats to their continued survival and ranking populations based on their biological and evolutionary importance enables setting conservation priorities. To assess the sustainability of the brown trout populations in the territory of Serbia (central Balkans), a modification of the ESHIPPO model was performed. The main modification involves incorporating the investigated populations’ genetic structure into the model. Therefore, the new ESHIPPOsalmo model includes an analysis of biological parameters and the impact of multiple factors, including habitat alterations, invasive species, pollution, human population growth, and over-exploitation. In order to investigate individual levels of influence of the model’s analyzed parameters, a combination of supervised and unsupervised machine learning methods was used. The structure of the model is based on general and easily measurable indicators, which enables its application in any salmonid river in the world. By evaluating the parameters of the ESHIPPOsalmo model, we were able to establish that, of the analyzed populations from 46 localities, 37% have a moderate level of sustainability, 43% low, and 20% critically low.

To elucidate the historical biogeography of a species, the patterns of population divergence must be understood, and the evolutionary history of the species must be accurately known. For brown trout (Salmo trutta complex), estimating divergence times remains a challenge due to the lack of well-defined time calibration points and insufficient phylogeographic coverage in previous studies. The present work aims to improve molecular dating of mitochondrial control region sequences by using a multicalibration framework based on the latest paleogeological evidence for dating the origin of Lake Ohrid and two available Salmo fossils, including the overlooked Salmo immigratus. Our results clearly show that, contrary to common belief, the major divisions within the brown trout occurred in the Late Pliocene, not the Pleistocene. The Pliocene origin suggests that the brown trout lineages did not form because of geo(hydro)morphological changes during glaciation cycles but may be the result of orogeny and drainage evolution. In addition, increased sampling, particularly in Serbia, led to the identification of a new haplogroup (da-int) occupying an intermediate position with respect to da-es and da-bs haplogroups. While the control region can delineate brown trout lineages, its phylogenetic resolution is limited, so even extensive sampling could not further resolve the lineage level polytomies.

Loaches of the genus Sabanejewia are model organisms for many ichthyologic studies focusing on morphological and colour variation. The bright lateral spots—a part of the mimicry of this fish—cause a taxonomic puzzle. Common practice is that morphometric and molecular studies lack habitat assessments of species originating from different environments. In this study, we analysed body shape and colour variation within two species of Sabanejewia in order to reveal whether they are more affected by phylogeny or environmental conditions. The environmental parameters were obtained using satellite mapping tools, body shape using geometric morphometry, and the molecular identification of specimens using an analysis of mitochondrial DNA (sequence of the cytochrome b gene). Generalized linear mixed models were used for the analysis of lateral spot numbers, and principal components analysis to describe the changes in landmark position defined by the permutation tests. Results show a significant impact of the locality elevation and specimen’s sex on the change of the body shape. We identified significant differences in the number of lateral spots, which are considered mimicry variation across an elevation gradient. Morphological differences of Sabanejewia spp. vary with ecological factors along the river continuum.

Despite being one of the most speciose fish families in the Mediterranean Sea, knowledge about the diversity of gobies (Actinopterygii: Gobiidae) in this sea is still unsatisfactory, as documented by recent descriptions of a number of new species. Although very common in shallow water, Gobius incognitus Kovačić & Šanda, 2016, had escaped attention until 2016, when it was discovered. Due to its overall superficial morphological similarity, G. incognitus used to be confused with a much rarer species, Gobius bucchichi Steindachner, 1870, which was considered one of the most common shallow-water gobies in the Mediterranean Sea. In this work, we tested the suitability of the genetic data (mitochondrial gene encoding cytochrome b) for identifying and distinguishing between these two goby species, and assessed the congruency between the distribution records based on genetic data and those based on morphological identification. We analysed material of 304 specimens of G. incognitus and G. bucchichi from 49 localities covering a considerable part of the Mediterranean Sea, Black Sea, and the Atlantic Ocean near Gibraltar, representing 19 geographically well-separated areas. We detected 270 sequences of G. incognitus, and only 34 of G. bucchichi. In both species, a high haplotype variability was observed. The sequence species identity matched morphological identification for all specimens for which vouchers were available. The mean uncorrected p-distance between G. incognitus and G. bucchichi was 13%, while the mean intraspecific distances were much lower (0.63% and 0.68%, respectively). We found 79 fixed mutations between these two species. Data on distribution based on genetic identification are completely congruent with published results based on morphological identification. The results of this study support molecular methods as a reliable tool for distinguishing morphologically similar fish species, which is particularly useful when only tissue is available for determination.

Diplozoidae are common monogenean ectoparasites of cyprinoid fish, with the genus Paradiplozoon being the most diversified. Despite recent studies on Diplozoidae from Europe, Africa and Asia, the diversity, distribution and phylogeny of this parasite group appears to be still underestimated in the Middle East. The objective of this study was to investigate the diversity, endemism and host specificity of diplozoids parasitizing cyprinoid fish from the Middle East, considering this region as an important historical interchange of fish fauna, and to elucidate the phylogenetic position of Middle Eastern Paradiplozoon species within Diplozoidae. Four Paradiplozoon species were collected from 48 out of 94 investigated cyprinoid species. Three known species, Paradiplozoon homoion, Paradiplozoon bliccae and Paradiplozoon bingolensis, were recorded on new cyprinoid host species, and a new species, Paradiplozoon koubkovae n. sp., was recorded on Luciobarbus capito and Capoeta capoeta from the Caspian Sea basin in Iran and Turkey. Paradiplozoon bliccae, exhibiting a wide host range in the Middle East, expressed both morphological and genetic intraspecific variabilities. The four Paradiplozoon species collected in the Middle East were placed in divergent clades, showing the rich evolutionary history of diplozoid parasites in the Middle East. Our study also revealed that two lineages of African diplozoids have a Middle Eastern origin. We stress the importance of applying an integrative approach combining morphological, ecological and molecular methods to reveal the real diversity of diplozoids.