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.

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.

An annotated catalogue of selected historical type specimens, including genetic data, housed in the Natural History Museum Vienna.

Museum collections are an important source for resolving taxonomic issues and species delimitation. Type specimens as name-bearing specimens, traditionally used in morphology-based taxonomy, are, due to the progress in historical DNA methodology, increasingly used in molecular taxonomic studies. Museum collections are subject to constant deterioration and major disasters. The digitisation of collections offers a partial solution to these problems and makes museum collections more accessible to the wider scientific community. The Extended Specimen Approach (ESA) is a method of digitisation that goes beyond the physical specimen to include the historical information stored in the collection. The collections of the Natural History Museum Vienna represent one of the largest non-university research centres in Europe and, due to their size and numerous type specimens, are frequently used for taxonomic studies by visiting and resident scientists. Recently, a version of ESA was presented in the common catalogue of the Fish and Evertebrata Varia collections and extended to include genetic information on type specimens in a case study of a torpedo ray. Here the case study was extended to a heterogeneous selection of historical type series from different collections with the type locality of Vienna. The goal was to apply the ESA, including genetic data on a selected set of type material: three parasitic worms, three myriapods, two insects, twelve fishes, and one bird species. Five hundred digital items (photographs, X-rays, scans) were produced, and genetic analysis was successful in eleven of the 21 type series. In one case a complete mitochondrial genome was assembled, and in another case ten short fragments (100-230 bp) of the cytochrome oxidase I gene were amplified and sequenced. For five type series, genetic analysis confirmed their taxonomic status as previously recognised synonyms, and for one the analysis supported its status as a distinct species. For two species, genetic information was provided for the first time. This catalogue thus demonstrates the usefulness of ESA in providing digitised data of types that can be easily made available to scientists worldwide for further study.

A new species of Enteromius (Actinopterygii, Cyprinidae, Smiliogastrinae) from the Awash River, Ethiopia, and the re-establishment of E. akakianus.

In the present study, populations of small-sized smiliogastrin barbs with a thickened and serrated last simple dorsal-fin ray distributed in the Main Ethiopian Rift were analysed. An integrated approach combining genetic markers and a variety of morphological methods based on a wide set of characters, including osteology and sensory canals, proved to be very productive for taxonomy in this group of fishes. The results showed that Ethiopian Enteromius species with a serrated dorsal-fin ray are distant from the true E. paludinosus (with E. longicauda as a synonym) and the so-called E. paludinosus complex involves several supposedly valid species with two distinct species occurring in the Main Ethiopian Rift area. A new species, Enteromius yardiensis sp. nov., is described from the Afar Depression in the north-eastern part of the Northern Main Ethiopian Rift. Enteromius akakianus is resurrected as a valid species including populations from the Central Main Ethiopian Rift (basins of lakes Langano, Ziway, and Awasa). No genetic data were available for E. akakianus from its type locality. Enteromius yardiensis sp. nov. is clearly distant from E. akakianus from the Central Main Ethiopian Rift by CO1 and cytb barcodes: pairwise distances between the new species and the Ethiopian congeners were 5.4 % to 11.0 %. Morphologically, the new species most clearly differs from all examined Ethiopian congeners by three specialisations which are unique in the group: the absence of the anterior barbel, the absence of the medial branch of the supraorbital sensory canal, and few, 1-3, commonly two, scale rows between the lateral line and the anus.

New data on the western Balkan leuciscids Alburnoides and Alburnus (Teleostei, Leuciscidae) from the Vjosa River, Albania.

The first voucher-confirmed record of Alburnus scoranza and the first morphological description of Alburnoides for the Vjosa River system in Albania are reported with a brief discussion of the diagnostic morphological traits and taxonomic assignment of both species.

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.

Phenotypic diversity in an endangered freshwater fish Squalius microlepis (Actinopterygii, Leuciscidae).

Squalius microlepis was examined from recent and historical collections within the known range of the species with special emphasis on intraspecific variability and variations, and compared to its closest relative species S. tenellus (in total, 193 specimens; 33 absolute and 52 proportional measurements and ratios, and 12 counts including vertebrae). Squalius tenellus was perfectly differentiated in all statistical analyses and can be diagnosed by 76-95 (vs. 64-80) scales in lateral series, 68-83 (vs. 58-77) lateral-line scales, (17)18-20 (vs. 13-16(17)) scales above lateral line, and (7)8-10 (vs. 4-7) scales below lateral line. Squalius microlepis was morphologically heterogeneous, with two phenotypes readily distinguishable (phenotype 1 corresponding to S. microlepis s. str. as defined by its lectotype) by a combination of many characters; those contributing most to the discrimination were number of gill rakers, length of lower jaw (% interorbital width), and head length (% SL). Only phenotype 1 was found in the Ricina-Prolosko Blato-Vrljika karst system; most of the specimens from the lower Matica and the Tihaljina-Trebizat karst system were identified as phenotype 2; the sample from karstic poljes near Vrgorac contained both phenotype 1 and 2, and individuals of intermediate morphology. As very limited molecular data exist on the two phenotypes of S. microlepis, we refrain from any taxonomic conclusions until new molecular approaches (and new markers) are used. We also report on a dramatic reduction of the area of distribution and abundance of S. microlepis in recent years.

Divergence with gene flow between Ponto-Caspian refugia in an anadromous cyprinid Rutilus frisii revealed by multiple gene phylogeography.

The Black and Caspian Seas have experienced alternating periods of isolation and interconnection over many Milankovitch climate oscillations and most recently became separated when the meltwater overflow from the Caspian Sea ceased at the end of the last glaciation. Climate-induced habitat changes have indisputably had profound impacts on distribution and demography of aquatic species, yet uncertainties remain about the relative roles of isolation and dispersal in the response of species shared between the Black and Caspian Sea basins. We examined these issues using phylogeographical analysis of an anadromous cyprinid fish Rutilus frisii. Bayesian coalescence analyses of sequence variation at two nuclear and one mitochondrial genes suggest that the Black and Caspian Seas supported separate populations of R. frisii during the last glaciation. Parameter estimates from the fitted isolation-with-migration model showed that their separation was not complete, however, and that the two populations continued to exchange genes in both directions. These analyses also suggested that majority of migrations occurred during the Pleistocene, showing that the variation shared between the Black and Caspian Seas is the result of ancient dispersal along the temporary natural connections between the basins, rather than of incomplete lineage sorting or recent human-mediated dispersal. Gene flow between the refugial populations was therefore an important source of genetic variation, and we suggest that it facilitated the evolutionary response of the populations to changing climate.

Description of a new species of Alburnus Rafinesque, 1820 (Actinopterygii, Cyprinidae, Leuciscinae) from the Kolpa River in the Sava River system (upper Danube drainage), with remarks on the geographical distribution of shemayas in the Danube.

Alburnus sava, new species, is described from the Kolpa River. The Kolpa is a tributary of the Sava, a major tributary of the Danube River, in the Black Sea basin. Alburnus sava is distinguished from its congeners in the Danube drainage, A. mento and A. sarmaticus, by having 23-27, usually 24-26, gill rakers; the ventral keel usually completely covered by scales (scaleless part maximum 15% of the keel length); 15-16, mode 15, branched pectoral-fin rays; the length of the gill raker at the junction of the arch limbs 65-70% of the length of the opposite outer gill filament; and a relatively long lower jaw (37-40% HL or 112-130% interorbital width). Alburnus sava is a large-sized potamadromous shemaya known to occur in the entire Sava drainage. The taxonomic status of A. mento and A. sarmaticus is confirmed. Alburnus danubicus is discussed and as there are no new arguments, it is kept as a valid species. New details on the distribution of shemayas in the Danube drainage are presented.

Molecular systematics and DNA barcoding of Altai osmans, oreoleuciscus (pisces, cyprinidae, and leuciscinae), and their nearest relatives, inferred from sequences of cytochrome b (Cyt-b), cytochrome oxidase c (Co-1), and complete mitochondrial genome.

Mitochondrial DNA (mtDNA) at the protein-coding Cyt-b gene along with data retrieved from GenBank for Co-1 gene fragments and complete mitochondrial genome (mitogenome) of Altai osmans and the nearest relatives of Leuciscinae fish species were compared for the estimation of variability and phylogenetic tree building. Phylogenetic trees were built by four techniques: Bayesian (BA), maximum likelihood (ML), maximum parsimony (MP), and neighbor-joining (NJ). Resolution of Cyt-b trees for species of two genera (Oreoleuciscus and Phoxinus) was quite distinct at all the approaches. For Tribolodon, the single gene trees were not well resolved; however, the mitogenome tree was resolved. Species identification on per individual basis (DNA barcoding) was high for both Cyt-b and Co-1 genes. The trees built using the data for 13 protein mitochondrial genes revealed a complicated phylogenetic pattern within the subfamily Leuciscinae. Scores of the average p-distances at three taxonomic levels were considerably different: (1) 1.16 ± 0.96, (2) 8.21 ± 1.01, and (3) 16.41 ± 0.85 for Cyt-b and (1) 1.04 ± 0.78, (2) 8.30 ± 0.92, and (3) 10.74 ± 0.79 for 13 protein genes of mitogenome, where (1) is intraspecies, (2) is intragenus, and (3) is intrasubfamily levels. Data on mitogenome distances were summarized for the taxonomic hierarchy for the first time. A concordant increase in distance score with growth of the rank of taxa (having the minimum score at the intraspecies level), both for a single gene and the whole mitogenome, substantiates the concept that speciation in the subfamily Leuciscinae in most cases follows the geographic mode. The distinct clustering of Altai osmans, Oreoleuciscus potanini and O. humilis, in the Cyt-b and Co-1 gene trees with small overall genetic distances, obtained for both genes, allows us to consider these taxa as separate but genetically sister species.

Two new freshwater fish species of the genus Telestes (Actinopterygii, Cyprinidae) from karst poljes in Eastern Herzegovina and Dubrovnik littoral (Bosnia and Herzegovina and Croatia).

Two new species, Telestes dabar and Telestes miloradi, are described on the basis of morphological comparisons of isolated geographical populations of fishes identified earlier as Telestes metohiensis. A lectotype is designated for Telestes metohiensis, whose range is shown to include waters of Gatacko, Cernicko, and Nevesinjsko poljes in Eastern Herzegovina. Telestes dabar from Dabarsko Polje (Eastern Herzegovina) and Telestes miloradi from Konavosko Polje (south Croatia) share with Telestes metohiensis the following combination of characters that distinguish them from the rest of the genus Telestes: pharyngeal teeth in one row, usually 5-4; preoperculo-mandibular canal not communicating with the infraorbital canal; mouth subterminal, the tip of the mouth cleft on or below the level of the ventral margin of the eye; postcleithrum minute or absent; ventral portion of the trunk with a dark stripe on a pale background; and dorsal portion of trunk uniformly dark and bordered ventrally by a dark midlateral stripe. Telestes dabar and Telestes miloradi are distinguishable from Telestes metohiensis in usually having 8½ branched dorsal-fin rays (vs. usually 7½), 9 or 10 gill rakers (vs. 7-10, usually 8), and the dark stripe on the ventral portion of the trunk below the main pigmented area of the back narrow and usually not reaching posteriorly to the caudal peduncle (vs. dark stripe wide and extending posteriorly to the caudal peduncle). Telestes dabar is distinguished from Telestes miloradi by having scales on most of the body situated close to one another and overlapping in a region behind the pectoral girdle and usually on the caudal peduncle (vs. overlapping scales on most of the body); the lateral line usually incomplete and interrupted, with 24-69, usually 54-65, total scales (vs. lateral line usually complete, with 55-67 total scales); scales above and below the lateral line slightly smaller than lateral-line scales (vs. of about equal size); head width 43-52% HL (vs. 48-58% HL); and lower jaw length 10-12% SL or 36-41% HL (vs. 8-10% SL or 33-38% HL). Telestes miloradi, a very local endemic species,is known only by historical samples. Telestes dabar is an abundant fish in Dabarsko Polje, but its range is critically restricted during the dry season by a few permanent sources. Nothing is known about its occurrence in underground karst waters.

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.

Phylogenetic position of the Dalmatian genus Phoxinellus and description of the newly proposed genus Delminichthys (Teleostei: Cyprinidae).

The Dalmatian cyprinid genus Phoxinellus is characterized by reductive characters most likely associated with the environmental conditions of small karstic streams, where all species of this genus occur. Based on 33 morphological traits, nuclear and mtDNA sequences Phoxinellus was found to be paraphyletic and included three not closely related monophyletic units. The scientific name Phoxinellus should therefore be restricted to species having plain coloration, small or absent postcleithrum, no genital papilla and an almost entirely naked body such as P. alepidotus, P. dalmaticus, and P. pseudalepidotus. Species that also have a small or absent postcleithrum and no genital papilla but display a dark stripe from the head to the caudal peduncle, and are entirely covered by distinct, not overlapping scales should be positioned closely to Telestes. Thus, we suggest inclusion of Phoxinellus croaticus, P. fontinalis and Paraphoxinus metohiensis in the genus Telestes. The Phoxinellus species that have a irregularly spotted color pattern, a large postcleithrum, an increased number of precaudal anal-fin pterygiophores, and a large genital papilla in females represent its own evolutionary line closely related to the Balkan species of Pseudophoxinus. For this monophyletic group, we propose to introduce a new genus: Delminichthys. This genus includes the species D. adspersus, D. ghetaldii, D. krbavensis and D. jadovensis.