An integrative approach highlights the discrepancy in the genetic, phenotypic, and presumptive taxonomic structure of Phoxinus (Actinopterygii, Leuciscidae, Phoxininae) in Bulgaria.

The present drainage network of Bulgaria is the result of a complex Neogene and Quaternary evolution. Karst, which has developed on 23% of the territory, further complicates the hydrological pattern. Fresh waters of Bulgaria drain into the Black Sea and the Aegean Sea basins and can be roughly divided into the Danube (Middle and Lower Danube), non-Danube Black Sea, East Aegean, and West Aegean hydrological regions. Phoxinus, a small leuciscid fish, has a mosaic distribution in all four of these regions, inhabiting small mountainous and semi-mountainous streams. Based on morphology, it was identified as three species, Phoxinus phoxinus in the Danube, Phoxinus strandjae in the non-Danube, and Phoxinus strymonicus in West Aegean region. Later, molecular data revealed Phoxinus csikii and Phoxinus lumaireul in the Middle Danube and P. csikii in the Lower Danube. Phoxinus has been the focus of many studies, showing a high molecular and morphological diversity, which is not entirely consistent with previous morphology-only-based taxonomic concepts. In this study, molecular (a mitochondrial marker and a nuclear marker) and morphological data from both historical and recently sampled collections were analysed to assess the applicability of the integrative approach in Phoxinus. The results showed a significant influence of the complex paleo- and recent hydrology on the currently observed genetic structure of the considered populations and species. Furthermore, the study also demonstrated a strong influence of phenotypic plasticity on the morphological analysis of Phoxinus and the lack of a clear differentiation between P. csikii and P. strandjae. A barcoded specimen was designated as neotype to fix the species named P. strandjae in the current taxonomic concept. Finally, a significant discordance between genetically delimited clades and phenotypic groups did not allow a proper delineation of the species distributed in Bulgaria, demonstrating that more molecular markers are needed for further taxonomic study of the Phoxinus complex.

Alien parasites on an alien fish species: monogeneans from the black bullhead Ameiurus melas (Siluriformes) in the Lake Srebarna Biosphere Reserve, Bulgaria, with the first record of Gyrodactylus nebulosus in the Palaearctic.

Black bullhead Ameiurus melas (Actinopterygii: Siluriformes) is an alien fish species of North American origin, which has expanded its invasive geographical range in Europe. In 2017-2019, 32 black bullhead specimens from the Lake Srebarna Biosphere Reserve, Bulgaria, were examined for monogenean parasites. Two species of monogeneans were recorded and identified on the basis of morphological and molecular data: Ligictaluridus pricei (Ancyrocephalidae), with prevalence 100% and intensity 2-32 (mean 13.3 ± 6.8), and Gyrodactylus nebulosus (Gyrodactylidae), with prevalence 72.0% and intensity 1-15 (mean 7.4 ± 4.3). Partial 18S rDNA and the ITS1 region of L. pricei were sequenced. For G. nebulosus, sequenced genes included the partial 18S rDNA and the entire ITS1-5.8S rDNA-ITS2 region as well as the mitochondrial COI gene. Both recorded monogenean species are specific parasites of North American ictalurid fishes and alien to Europe. The present study is the first record of L. pricei from Bulgaria and the first record of G. nebulosus from Europe and the Palaearctic Region.

Description of a new species of Phoxinus from the upper Krka River (Adriatic Basin) in Croatia (Actinopterygii: Leuciscidae), first discovered as a molecular clade.

Using morphometric, meristic and qualitative characters, we confirmed a specific status of a clade discovered based on molecular data only. The newly described species Phoxinus krkae has a very local distribution in the upper Krka River but is easily morphologically distinguished from geographically neighbouring species with much wider ranges: Phoxinus lumaireul, Phoxinus marsilii and Phoxinus csikii. Phoxinus krkae is well differentiated in statistical analyses and differs from other species of Phoxinus in northern Adriatic Sea drainages and right-bank tributaries of the middle and lower River Danube by a combination of characters, none of them unique: caudal peduncle length 1.9-2.2 times minimum depth of caudal peduncle; upper lip not projecting beyond lower lip; rostral fold absent; total number of scales in lateral series 74-84; lateral line incomplete and interrupted, commonly 17-49 (< 60% of total number of scales in lateral series) with no or few canal segments on posterior body and caudal peduncle; scales on belly extending forward to over middle of distance between pelvic-fin base and pectoral-fin origin; patches of breast scales commonly connected by scattered scales or forming 1-3 regular rows of scales; total vertebrae commonly 38-39 (22 + 17, 22 + 16 or 21 + 17). The study reveals unrecognised morphological diversity within the group and provides the groundwork for future taxonomic study in Phoxinus in the Adriatic region and the Danube Basin.

A recent record of Romanogobioantipai (Actinopterygii, Cyprinidae, Gobioninae) from the Danube River in Bulgaria.

The Danube delta gudgeon, Romanogobioantipai, has been considered to be extinct because there were no reliable recent observations. The latest record confirmed by a voucher specimen dating from 1992. We report here on a specimen of R.antipai collected in 2016 in the Bulgarian sector of the Danube main stream using a bottom drift net at a depth of 8 m. The species determination is supported by morphological examination including discriminant and cluster analyses in comparison with three syntypes and five non-type specimens of R.antipai, samples of the R.kesslerii species complex and R.vladykovi. Romanogobioantipai most clearly differs from both R.kesslerii and R.vladykovi by proportional measurements (caudal peduncle depth, head width, eye horizontal diameter, and interorbital width), from R.kesslerii also by the number of scales above and below the lateral line (6 and 4, respectively, (vs. commonly 5 and 3), and from R.vladykovi, also by 8½ branched dorsal-fin rays (vs. 7½) and the vertebral caudal region longer than the abdominal vertebral region (abdominal+caudal vertebrae 19+21 or 20+21, vs. commonly 20+20 or variants with a caudal region shorter than the abdominal one). The possibility that R.antipai represents a deep-water cophenotype of either R.kesslerii or R.vladykovi, cannot be excluded. The new record demonstrates that R.antipai is still extant in the lower Danube but may be restricted to greater depths in the main channel and the deltaic branches.

High cryptic diversity of bitterling fish in the southern West Palearctic.

South-east Europe, along with the adjacent region of south-west Asia, is an important biodiversity hotspot with high local endemism largely contributed by contemporary continental lineages that retreated to southern refugia during colder Quaternary periods. We investigated the genetic diversity of the European bitterling fish (Rhodeus amarus) species complex (Cyprinidae) across its range in the western Palearctic, but with a particular emphasis in the region of Balkan, Pontic and Caspian refugia. We genotyped 12 polymorphic microsatellite loci and a partial sequence of mitochondrial gene cytochrome b (CYTB) for a set of 1,038 individuals from 60 populations. We used mtDNA sequences to infer phylogenetic relationships and historical demography, and microsatellite markers to describe fine-scale genetic variability and structure. Our mtDNA analysis revealed six well-supported lineages, with limited local co-occurrence. Two lineages are distributed throughout central and western Europe (lineages "A" and "B"), with two zones of secondary contact. Another two lineages were restricted to the Ponto-Aegean region of Greece (lineages "C" and "D") and the final two lineages were restricted south of the Caucasus mountains (lineage "E" from the Black Sea watershed and lineage "F" from the Caspian watershed). A signal of recent expansion was revealed in the two widespread lineages and the Ponto-Aegean lineage "C". The geographic distribution of clusters detected by nuclear microsatellites corresponded well with mitochondrial lineages and demonstrated finely sub-structured populations. A profound population structure suggested a significant role of genetic drift in differentiation among lineages. Lineage divergence in the Ponto-Aegean and Caspian regions are substantial, supporting the validity of two described endemic species (Rhodeus meridionalis as lineage "D" and Rhodeus colchicus as lineage "E") and invite taxonomic evaluation of the other two southern lineages (Thracean "C" and Caspian "F").

A multilocus assessment of nuclear and mitochondrial sequence data elucidates phylogenetic relationships among European spirlins (Alburnoides, Cyprinidae).

The phylogenetic relationships and taxonomy of the spirlins in the genus Alburnoides are examined by comparative sequencing analysis of mitochondrial and nuclear markers. Molecular analyses revealed 17 Eurasian lineages divided into two main clades, termed the Ponto-Caspian and European in accordance with the lineage distribution. The indel diagnostics of ß-actin and S7 markers and translation of cyt b to the amino acid chain were evaluated as a reliable identifying tool for most of the recognised lineages. Lineage richness is closely connected with the existence of known glacial refugia in most cases. The underestimation of species richness in the genus Alburnoides is confirmed: the genetic analyses support the validity of 11 morphologically accepted species; apart from them, four phylogenetic lineages requiring descriptions as separate species were revealed. The distribution area of the nominotypical species A. bipunctatus s. stricto is newly defined. Two diverging phylogenetic lineages, A. ohridanus, and A. prespensis complex, were observed in the Southeast Adriatic Freshwater Ecoregion, confirmed as a hotspot of endemic biodiversity. A. ohridanus demonstrates high divergence from the A. prespensis complex, represented by three similar mitochondrial lineages with the same nuclear haplotypes and sympatric occurrence. The range restricted endemism was confirmed for at least seven species. The Albanian river systems, as well as the wider Ponto-Caspian basin exhibit complications among definite species delineations and gaps in understanding of microevolutionary processes; these areas require further investigations.