Projection of current and future distribution of adaptive genetic units in an alpine ungulate.

Climate projections predict major changes in alpine environments by the end of the 21st century. To avoid climate-induced maladaptation and extinction, many animal populations will either need to move to more suitable habitats or adapt in situ to novel conditions. Since populations of a species exhibit genetic variation related to local adaptation, it is important to incorporate this variation into predictive models to help assess the ability of the species to survive climate change. Here, we evaluate how the adaptive genetic variation of a mountain ungulate-the Northern chamois (Rupicapra rupicapra)-could be impacted by future global warming. Based on genotype-environment association analyses of 429 chamois using a ddRAD sequencing approach, we identified genetic variation associated with climatic gradients across the European Alps. We then delineated adaptive genetic units and projected the optimal distribution of these adaptive groups in the future. Our results suggest the presence of local adaptation to climate in Northern chamois with similar genetic adaptive responses in geographically distant but climatically similar populations. Furthermore, our results predict that future climatic changes will modify the Northern chamois adaptive landscape considerably, with various degrees of maladaptation risk.

Spatial genetic structure of European wild boar, with inferences on late-Pleistocene and Holocene demographic history.

European wildlife has been subjected to intensifying levels of anthropogenic impact throughout the Holocene, yet the main genetic partitioning of many species is thought to still reflect the late-Pleistocene glacial refugia. We analyzed 26,342 nuclear SNPs of 464 wild boar (Sus scrofa) across the European continent to infer demographic history and reassess the genetic consequences of natural and anthropogenic forces. We found that population fragmentation, inbreeding and recent hybridization with domestic pigs have caused the spatial genetic structure to be heterogeneous at the local scale. Underlying local anthropogenic signatures, we found a deep genetic structure in the form of an arch-shaped cline extending from the Dinaric Alps, via Southeastern Europe and the Baltic states, to Western Europe and, finally, to the genetically diverged Iberian peninsula. These findings indicate that, despite considerable anthropogenic influence, the deeper, natural continental structure is still intact. Regarding the glacial refugia, our findings show a weaker signal than generally assumed, but are nevertheless suggestive of two main recolonization routes, with important roles for Southern France and the Balkans. Our results highlight the importance of applying genomic resources and framing genetic results within a species' demographic history and geographic distribution for a better understanding of the complex mixture of underlying processes.

Diversity of Hepatozoon species in wild mammals and ticks in Europe.

BACKGROUND: Hepatozoon spp. are tick-borne parasites causing subclinical to clinical disease in wild and domestic animals. Aim of this study was to determine Hepatozoon prevalence and species distribution among wild mammals and ticks in Europe. METHODS: Samples of wild mammals and ticks, originating from Austria, Bosnia and Herzegovina, Croatia, Belgium and the Netherlands, were tested with PCR to amplify a ~ 670-bp fragment of the small subunit ribosomal RNA gene. RESULTS: Of the 2801 mammal samples that were used for this study, 370 (13.2%) tested positive. Hepatozoon canis was detected in samples of 178 animals (3 Artiodactyla, 173 Carnivora, 1 Eulipotyphia, 1 Lagomorpha), H. martis in 125 (3 Artiodactyla, 122 Carnivora), H. sciuri in 13 (all Rodentia), Hepatozoon sp. in 47 (among which Hepatozoon sp. Vole isolate, all Rodentia) and H. ayorgbor in 4 (all Rodentia). Regarding origin, 2.9% (6/208) tested positive from Austria, 2.8% (1/36) from Bosnia and Herzegovina, 14.6% (173/1186) from Croatia and 13.9% (190/1371) from Belgium/the Netherlands. Of the 754 ticks collected, 0.0% (0/35) Hyalomma sp., 16.0% (4/25) Dermacentor spp., 0.0% (0/23) Haemaphysalis spp., 5.3% (24/50) Ixodes and 1.4% (3/221) Rhipicephalus spp. tested positive for Hepatozoon (4.2%; 32/754), most often H. canis (n = 22). CONCLUSIONS: Hepatozoon canis is most present in mammals (especially in Carnivora such as gray wolves and golden jackals) and ticks, followed by H. martis, which was found merely in stone martens and pine martens. None of the rodent-associated Hepatozoon spp. were detected in the ticks, suggesting the possible implication of other arthropod species or non-vectorial routes in the transmission cycle of the hemoprotozoans in rodents. Our findings of H. canis in ticks other than R. sanguineus add to the observation that other ticks are also involved in the life cycle of Hepatozoon. Now that presence of Hepatozoon has been demonstrated in red foxes, gray wolves, mustelids and rodents from the Netherlands and/or Belgium, veterinary clinicians should be aware of the possibility of spill-over to domestic animals, such as dogs.

Pan-European phylogeography of the European roe deer (Capreolus capreolus).

To provide the most comprehensive picture of species phylogeny and phylogeography of European roe deer (Capreolus capreolus), we analyzed mtDNA control region (610 bp) of 1469 samples of roe deer from Central and Eastern Europe and included into the analyses additional 1541 mtDNA sequences from GenBank from other regions of the continent. We detected two mtDNA lineages of the species: European and Siberian (an introgression of C. pygargus mtDNA into C. capreolus). The Siberian lineage was most frequent in the eastern part of the continent and declined toward Central Europe. The European lineage contained three clades (Central, Eastern, and Western) composed of several haplogroups, many of which were separated in space. The Western clade appeared to have a discontinuous range from Portugal to Russia. Most of the haplogroups in the Central and the Eastern clades were under expansion during the Weichselian glacial period before the Last Glacial Maximum (LGM), while the expansion time of the Western clade overlapped with the Eemian interglacial. The high genetic diversity of extant roe deer is the result of their survival during the LGM probably in a large, contiguous range spanning from the Iberian Peninsula to the Caucasus Mts and in two northern refugia.

The de novo assembly of a European wild boar genome revealed unique patterns of chromosomal structural variations and segmental duplications.

The rapid progress of sequencing technology has greatly facilitated the de novo genome assembly of pig breeds. However, the assembly of the wild boar genome is still lacking, hampering our understanding of chromosomal and genomic evolution during domestication from wild boars into domestic pigs. Here, we sequenced and de novo assembled a European wild boar genome (ASM2165605v1) using the long-range information provided by 10× Linked-Reads sequencing. We achieved a high-quality assembly with contig N50 of 26.09 Mb. Additionally, 1.64% of the contigs (222) with lengths from 107.65 kb to 75.36 Mb covered 90.3% of the total genome size of ASM2165605v1 (~2.5 Gb). Mapping analysis revealed that the contigs can fill 24.73% (93/376) of the gaps present in the orthologous regions of the updated pig reference genome (Sscrofa11.1). We further improved the contigs into chromosome level with a reference-assistant scaffolding method. Using the 'assembly-to-assembly' approach, we identified intra-chromosomal large structural variations (SVs, length >1 kb) between ASM2165605v1 and Sscrofa11.1 assemblies. Interestingly, we found that the number of SV events on the X chromosome deviated significantly from the linear models fitting autosomes (R2  > 0.64, p < 0.001). Specifically, deletions and insertions were deficient on the X chromosome by 66.14 and 58.41% respectively, whereas duplications and inversions were excessive on the X chromosome by 71.96 and 107.61% respectively. We further used the large segmental duplications (SDs, >1 kb) events as a proxy to understand the large-scale inter-chromosomal evolution, by resolving parental-derived relationships for SD pairs. We revealed a significant excess of SD movements from the X chromosome to autosomes (p < 0.001), consistent with the expectation of meiotic sex chromosome inactivation. Enrichment analyses indicated that the genes within derived SD copies on autosomes were significantly related to biological processes involving nervous system, lipid biosynthesis and sperm motility (p < 0.01). Together, our analyses of the de novo assembly of ASM2165605v1 provides insight into the SVs between European wild boar and domestic pig, in addition to the ongoing process of meiotic sex chromosome inactivation in driving inter-chromosomal interaction between the sex chromosome and autosomes.

Traces of past reintroduction in genetic diversity: The case of the Balkan chamois (Mammalia, Artiodactyla).

The translocation of wild animal species became a common practice worldwide to re-establish local populations threatened with extinction. Archaeological data confirm that chamois once lived in the Biokovo Mountain but, prior to their reintroduction in the 1960s, there was no written evidence of their recent existence in the area. The population was reintroduced in the period 1964-1969, when 48 individuals of Balkan chamois from the neighbouring mountains in Bosnia and Herzegovina were released. The main objective of this study was to determine the accuracy of the existing historical data on the origin of the Balkan chamois population from the Biokovo Mountain and to assess the genetic diversity and population structure of the source and translocated populations 56 years after reintroduction. Sixteen microsatellite loci were used to analyse the genetic structure of three source chamois populations from Prenj, Cvrsnica and Cabulja Mountains and from Biokovo Mountain. Both STRUCTURE and GENELAND analyses showed a clear separation of the reintroduced population on Biokovo from Prenj's chamois and considerable genetic similarity between the Biokovo population and the Cvrsnica-Cabulja population. This suggests that the current genetic composition of the Biokovo population does not derive exclusively from Prenj, as suggested by the available literature and personal interviews, but also from Cvrsnica and Cabulja. GENELAND analysis recognised the Balkan chamois from Prenj as a separate cluster, distinct from the populations of Cvrsnica and Cabulja. Our results thus highlight the need to implement genetic monitoring of both reintroduced and source populations of endangered Balkan chamois to inform sustainable management and conservation strategies in order to maximise the chances of population persistence.

Population genetic structure of European wildcats inhabiting the area between the Dinaric Alps and the Scardo-Pindic mountains.

Habitat fragmentation and loss have contributed significantly to the demographic decline of European wildcat populations and hybridization with domestic cats poses a threat to the loss of genetic purity of the species. In this study we used microsatellite markers to analyse genetic variation and structure of the wildcat populations from the area between the Dinaric Alps and the Scardo-Pindic mountains in Slovenia, Croatia, Serbia and North Macedonia. We also investigated hybridisation between populations of wildcats and domestic cats in the area. One hundred and thirteen samples from free-leaving European wildcats and thirty-two samples from domestic cats were analysed. Allelic richness across populations ranged from 3.61 to 3.98. The observed Ho values ranged between 0.57 and 0.71. The global FST value for the four populations was 0.080 (95% CI 0.056-0.109) and differed significantly from zero (P < 0.001). The highest FST value was observed between the populations North Macedonia and Slovenia and the lowest between Slovenia and Croatia. We also found a signal for the existence of isolation by distance between populations. Our results showed that wildcats are divided in two genetic clusters largely consistent with a geographic division into a genetically diverse northern group (Slovenia, Croatia) and genetically eroded south-eastern group (Serbia, N. Macedonia). Hybridisation rate between wildcats and domestic cats varied between 13% and 52% across the regions.

Population structure and genetic diversity of non-native aoudad populations.

The aoudad (Ammotragus lervia Pallas 1777) is an ungulate species, native to the mountain ranges of North Africa. In the second half of the twentieth century, it was successfully introduced in some European countries, mainly for hunting purposes, i.e. in Croatia, the Czech Republic, Italy, and Spain. We used neutral genetic markers, the mitochondrial DNA control region sequence and microsatellite loci, to characterize and compare genetic diversity and spatial pattern of genetic structure on different timeframes among all European aoudad populations. Four distinct control region haplotypes found in European aoudad populations indicate that the aoudad has been introduced in Europe from multiple genetic sources, with the population in the Sierra Espuña as the only population in which more than one haplotype was detected. The number of detected microsatellite alleles within all populations (< 3.61) and mean proportion of shared alleles within all analysed populations (< 0.55) indicates relatively low genetic variability, as expected for new populations funded by a small number of individuals. In STRUCTURE results with K = 2-4, Croatian and Czech populations cluster in the same genetic cluster, indicating joined origin. Among three populations from Spain, Almeria population shows as genetically distinct from others in results, while other Spanish populations diverge at K = 4. Maintenance of genetic diversity should be included in the management of populations to sustain their viability, specially for small Czech population with high proportion of shared alleles (0.85) and Croatian population that had the smallest estimated effective population size (Ne = 5.4).

A Mother's Story, Mitogenome Relationships in the Genus Rupicapra.

Although the two species of chamois (Rupicapra rupicapra and R. pyrenaica) are currently classified as least-concern by the IUCN (International Union for Conservation of Nature), inconsistencies on the subspecies classification reported in literature make it challenging to assess the conservation status of the single subspecies. Previous studies relying on mitochondrial genes, sometimes in combination with nuclear or Y-chromosome markers, reported the presence of clusters corresponding to the geographic distribution but highlighting ambiguities in the genus phylogeny. Here we report novel de novo assembled sequences of the mitochondrial genome from nine individuals, including previously unpublished R. r. balcanica and R. r. tatrica subspecies, and use them to untangle the genus phylogeny. Our results based on the full mitogenome inferred phylogeny confirm the previously reported genus subdivision in three clades and its monophyletic positioning within the Caprinae. Phylogeny and taxonomy of Rupicapra species thus remain controversial prompting for the inclusion of archeological remains to solve the controversy.

Proximate Chemical Composition, Fatty Acid Profile, and Lipid Qualitative Indices of Brown Bear Meat.

Although game meat quality has been under the spotlight in numerous studies, the quality of brown bear (Ursus arctos) meat is still unknown. The aim of this study was to determine the effects of sex and age on the proximate chemical composition, fatty acid profile, and lipid indices of brown bear meat. Nine (n = 9) females and nine (n = 9) males were hunted during the Croatian spring hunting period in 2018. Based on age, bears were divided into two groups: <3 years (n = 9; five females and four males) and 4-6 years (n = 9; four females and five males). For analysis purposes, samples of M.semimembranosus were collected. Age was shown to have an effect on the traits analyzed, while sex-related differences were not found. Brown bear meat has a high fat content (average 6.12%), especially in older bears (~9%). The contents of protein, dry matter, and ash were similar to those of other game species. Monounsaturated fatty acids made up approximately 50% of all fatty acids, with the most abundant being C18:1n-9. More favorable profiles of essential polyunsaturated fatty acids were found in younger bears. The ratio of polyunsaturated and saturated fatty acids was closer to the recommended ratio than the ratio of n-6 and n-3 polyunsaturated fatty acids, and lipid indices were favorable. Further research is needed to determine seasonal changes in brown bear meat quality.

Genetic characterisation of wild ungulates: successful isolation and analysis of DNA from widely available bones can be cheap, fast and easy.

Genetic characterisation of wild ungulates can be a useful tool in wildlife management and in obtaining a greater understanding of their biological and ecological roles in a wider spatiotemporal context. Different ways of optimising methodologies and reducing the costs of genetic analyses using widely available bone tissues collected within regular hunting allocations were examined. Successful isolation and analysis of DNA from widely available bones can be cheap, fast and easy. In particular, this study explored the possibility of using bones for extracting high quality nuclear DNA for microsatellite analysis. The utility of applying a modified demineralisation process using two commercially available DNA isolation kits, which differ significantly in price, was evaluated. The sample sets included bones and, for comparison, muscle tissues from four wild ungulate species: chamois (Rupicapra rupicapra), roe deer (Capreolus capreolus), wild boar (Sus scrofa), and Alpine ibex (Capra ibex). For the recent bones, these results confirmed that the DNA concentrations and microsatellite amplification were sufficiently high, even when using low-cost kits, after prior demineralisation. For old bones, prior demineralisation and use of a specially designed isolation kit led to a more successful extraction of DNA. Besides reducing kit-related costs, low-cost kits are much faster and therefore make genetic analysis more efficient.

MHC Genotyping by SSCP and Amplicon-Based NGS Approach in Chamois.

Genes of the major histocompatibility complex (MHC) code for cell surface proteins essential for adaptive immunity. They show the most outstanding genetic diversity in vertebrates, which has been connected with various fitness traits and thus with the long-term persistence of populations. In this study, polymorphism of the MHC class II DRB locus was investigated in chamois with Single-Strand Conformation Polymorphism (SSCP)/Sanger genotyping and Ion Torrent S5 next-generation sequencing (NGS). From eight identified DRB variants in 28 individuals, five had already been described, and three were new, undescribed alleles. With conventional SSCP/Sanger sequencing, we were able to detect seven alleles, all of which were also detected with NGS. We found inconsistencies in the individual genotypes between the two methods, which were mainly caused by allelic dropout in the SSCP/Sanger method. Six out of 28 individuals were falsely classified as homozygous with SSCP/Sanger analysis. Overall, 25% of the individuals were identified as genotyping discrepancies between the two methods. Our results show that NGS technologies are better performing in sequencing highly variable regions such as the MHC, and they also have a higher detection capacity, thus allowing a more accurate description of the genetic composition, which is crucial for evolutionary and population genetic studies.

Contrasting patterns of sexually selected traits in Mediterranean and continental populations of European mouflon.

The expression of sexually selected traits in highly dimorphic ungulates may be influenced by environmental quality. Variations in habitat conditions can impose different constraints on the allocation of energy resources to male life-history traits, and possibly alter the female preferences for specific features. Here, we compared the horn growth patterns in male European mouflon Ovis aries musimon living in different habitats (Mediterranean vs. continental) but sharing a common genetic origin. We hypothesized that the expression of sexually selected traits such as horn development should be promoted in more favorable habitat conditions (i.e., Mediterranean). Using linear mixed models on data retrieved from individuals harvested under the same hunting regime, we found longer horns and greater individual variance in horn segment length in the Mediterranean population than in the continental one. Furthermore, Mediterranean rams showed no evidence of compensatory horn growth, as opposed to the continental rams. Unexpectedly, horn base circumference was greater in the continental habitat than in the Mediterranean one. The overall results suggest different patterns of investment in horns in the two populations, with seemingly stronger pressure and consequences of sexual selection on mouflon rams living in more favorable environments. Although the role of hunters' selectivity cannot be excluded a priori, our data suggest that the differences in the expression of sexually selected traits in our study populations may be influenced by environmental conditions. Because sexual selection can impose substantial fitness costs on individuals, further investigations on the trade-offs between reproduction and survival would improve our understanding of the dynamics of mouflon populations living in different environmental conditions.

Ungulate management in European national parks: Why a more integrated European policy is needed.

1. Primary objectives of national parks usually include both, the protection of natural processes and species conservation. When these objectives conflict, as occurs because of the cascading effects of large mammals (i.e., ungulates and large carnivores) on lower trophic levels, park managers have to decide upon the appropriate management while considering various local circumstances. 2. To analyse if ungulate management strategies are in accordance with the objectives defined for protected areas, we assessed the current status of ungulate management across European national parks using the naturalness concept and identified the variables that influence the management. 3. We collected data on ungulate management from 209 European national parks in 29 countries by means of a large-scale questionnaire survey. Ungulate management in the parks was compared by creating two naturalness scores. The first score reflects ungulate and large carnivore species compositions, and the second evaluates human intervention on ungulate populations. We then tested whether the two naturalness score categories are influenced by the management objectives, park size, years since establishment, percentage of government-owned land, and human impact on the environment (human influence index) using two generalized additive mixed models. 4. In 67.9% of the national parks, wildlife is regulated by culling (40.2%) or hunting (10.5%) or both (17.2%). Artificial feeding occurred in 81.3% of the national parks and only 28.5% of the national parks had a non-intervention zone covering at least 75% of the area. Furthermore, ungulate management differed greatly among the different countries, likely because of differences in hunting traditions and cultural and political backgrounds. Ungulate management was also influenced by park size, human impact on the landscape, and national park objectives, but after removing these variables from the full model the reduced models only showed a small change in the deviance explained. In areas with higher anthropogenic pressure, wildlife diversity tended to be lower and a higher number of domesticated species tended to be present. Human intervention (culling and artificial feeding) was lower in smaller national parks and when park objectives followed those set by the International Union for the Conservation of Nature (IUCN). 5. Our study shows that many European national parks do not fulfil the aims of protected area management as set by IUCN guidelines. In contrast to the USA and Canada, Europe currently has no common ungulate management policy within national parks. This lack of a common policy together with differences in species composition, hunting traditions, and cultural or political context has led to differences in ungulate management among European countries. To fulfil the aims and objectives of national parks and to develop ungulate management strategies further, we highlight the importance of creating a more integrated European ungulate management policy to meet the aims of national parks.

Microsatellite based assignment reveals history of extirpated mountain ungulate.

During the early 1900s, Northern chamois (Rupicapra rupicapra) populations in the northern Dinaric Mountains were extirpated. During the 1960s and 1970s there were several reintroductions of individuals from two Northern chamois subspecies (Alpine chamois, R. r. rupicapra and Balkan chamois, R. r. balcanica) from neighbouring areas in the attempt to re-establish the population. Accurate taxonomic classification, at subspecies level, of the autochthonous extirpated population was not known. To clarify which subspecies was present before reintroduction, we genotyped four male chamois skulls originating from Velebit Mountain, collected around 25 years before the population local extinction. DNA was successfully extracted from middle layer and outer sheath of horns. Assignment based on microsatellite loci, using both Bayesian clustering in STRUCTURE (with q values between 0.55 and 0.73) and DAPC (with individual membership probabilities of 0.99 and 1.00) indicated higher assessed likelihood for the Alpine subspecies.

Author Correction: Hotspots of recent hybridization between pigs and wild boars in Europe.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Maternal genomic variability of the wild boar (Sus scrofa) reveals the uniqueness of East-Caucasian and Central Italian populations.

The phylogeography of the European wild boar was mainly determined by postglacial recolonization patterns from Mediterranean refugia after the last ice age. Here we present the first analysis of SNP polymorphism within the complete mtDNA genome of West Russian (n = 8), European (n = 64), and North African (n = 5) wild boar. Our analyses provided evidence of unique lineages in the East-Caucasian (Dagestan) region and in Central Italy. A phylogenetic analysis revealed that these lineages are basal to the other European mtDNA sequences. We also show close connection between the Western Siberian and Eastern European populations. Also, the North African samples were clustered with the Iberian population. Phylogenetic trees and migration modeling revealed a high proximity of Dagestan sequences to those of Central Italy and suggested possible gene flow between Western Asia and Southern Europe which was not directly related to Northern and Central European lineages. Our results support the presence of old maternal lineages in two Southern glacial refugia (i.e., Caucasus and the Italian peninsula), as a legacy of an ancient wave of colonization of Southern Europe from an Eastern origin.

Barbary sheep tissues as bioindicators of radionuclide and stabile element contamination in Croatia: exposure assessment for consumers.

Muscle, liver and kidney of 21 Barbary sheep (Ammotragus lervia) from Mosor Mountain, Croatia, were sampled to quantify the activity of caesium and potassium radionuclides and five toxic and ten essential stabile elements in order to establish reference values for this species and to evaluate the potential of Barbary sheep tissues to reflect environmental pollution. We also assessed seasonal diet (botanical composition and dry matter content) of Barbary sheep based on analyses of a rumen content of culled animals. None of the 19 plant species (mostly grasses) identified as part of the Barbary sheep diet is known as a stabile element or radionuclide hyperaccumulator. Measured levels reflected low environmental pollution with arsenic, cadmium, mercury and lead, with levels generally less than those reported for wild herbivorous ungulates. Methodological differences (detection limit of elements in muscle) were shown to hamper interpretation and comparison of the Toxic Contamination Index (TCI) values with those published for other species. There was no homeostasis disturbance of trace elements in Barbary sheep, either due to inadequate intake via food or as an adverse effect due to a high toxic metal(loid) burden. Consumption of the muscle and liver of wild Barbary sheep can be considered safe for the health of adult consumers regarding toxic metal(loid)s and radioactive caesium, though the liver should be avoided as a food item in vulnerable population groups due to the possible adverse effects of cadmium and lead. Otherwise, muscle and liver are a rich source of copper, iron, selenium and zinc for consumers and, as such, can benefit the overall dietary intake of essential elements.

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.