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.

Distribution of endangered Italian gudgeon Romanogobio benacensis (Cypriniformes, Cyprinidae, Gobioninae) with remarks on distinguishing morphological characters.

Distribution data on many freshwater fish species in Croatia are scarce and species identifications are difficult, requiring further detailed studies. This paper presents a report of the Italian gudgeon Romanogobio benacensis from the Mirna River in the Istra Peninsula in Croatia, in the south-east from its previously known distribution range. The identification of R. benacensis in Croatia was supported by a morphological comparison with R. benacensis from Italy and Slovenia, the common gudgeon Gobio gobio, and the Danubian gudgeon Gobio obtusirostris from geographically close locations. A combination of character states (number of scales between anus and anal-fin origin, branched pectoral-fin rays, lateral-line scales, total, abdominal, and caudal vertebrae, and the size and number of lateral blotches) distinguishes R. benacensis from both G. gobio and G. obtusirostris. The phylogenetic analyses using mitochondrial sequences of cytochrome b gene confirmed that specimens from the Mirna River belong to R. benacensis. Also, Reka River system (Adriatic Sea basin) in Slovenia is inhabited by a possibly introduced Danubian gudgeon, G. obtusirostris, and not by R. benacensis.

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 data suggest a multispecies complex of Phoxinus (Cyprinidae) in the Western Balkan Peninsula.

This study aimed to evaluate the phylogeographic distribution of Phoxinus phoxinus sensu lato populations in the Balkan Peninsula using molecular methods. For the western Balkan Peninsula, two species (Danubian P. phoxinus and Adriatic P. lumaireul) have been suggested, but not confirmed by subsequent morphological studies or by genetic data. For the present study, more than 300 specimens were collected from the western Balkans. A partial sequence of the mt cyt b gene analysed using Bayesian and ML methods revealed several well-supported clades, with distances ranging from 4% to 11%. The clades were corroborated by RAG1 sequence analysis. Thus, the molecular analysis points to a multispecies complex of Phoxinus sp. with ranges of the clades extending north-west to south-east along the western Balkan Peninsula. In addition, the dispersion of fish through subterranean water connections in karst is indicated.

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.

Fish migrate underground: the example of Delminichthys adspersus (Cyprinidae).

Complex aquatic systems of karst harbour a rich but little-investigated biodiversity. In Croatia and Bosnia-Herzegovina karst, temporal springs are inhabited by a group of minnow-like fishes that retreat to the associated ground water during dry seasons and spend several months underground. The most abundant species in this group is Delminichthys adspersus (Heckel 1843), which also has the most fragmented distribution range. To determine the population composition and dispersal patterns, and to detect potential underground migration, a large genetic data set comprising 544 specimens of D. adspersus covering most of its distribution area was analysed. Analysis of mitochondrial cytochrome b sequences (∼1000 bp) and eight microsatellite loci showed that D. adspersus comprises at least three subpopulations with gene flow occurring among them. Coalescent-based analysis revealed a complex migration pattern, with several unidirectional dispersal paths, including between temporal springs that share no surface connection. The results of this study suggest the existence of recurrent underground migration of fish in a karst environment and demonstrate the complexity of its hydrological network. The findings are relevant to conservation strategies for endemic karst organisms and karst ecosystems as a whole.

Phylogenetic relationships and biogeographical patterns in Circum-Mediterranean subfamily Leuciscinae (Teleostei, Cyprinidae) inferred from both mitochondrial and nuclear data.

BACKGROUND: Leuciscinae is a subfamily belonging to the Cyprinidae fish family that is widely distributed in Circum-Mediterranean region. Many efforts have been carried out to deciphering the evolutionary history of this group. Thus, different biogeographical scenarios have tried to explain the colonization of Europe and Mediterranean area by cyprinids, such as the "north dispersal" or the "Lago Mare dispersal" models. Most recently, Pleistocene glaciations influenced the distribution of leuciscins, especially in North and Central Europe. Weighing up these biogeographical scenarios, this paper constitutes not only the first attempt at deciphering the mitochondrial and nuclear relationships of Mediterranean leuciscins but also a test of biogeographical hypotheses that could have determined the current distribution of Circum-Mediterranean leuciscins. RESULTS: A total of 4439 characters (mitochondrial + nuclear) from 321 individuals of 176 leuciscine species rendered a well-supported phylogeny, showing fourteen main lineages. Analyses of independent mitochondrial and nuclear markers supported the same main lineages, but basal relationships were not concordant. Moreover, some incongruence was found among independent mitochondrial and nuclear phylogenies. The monophyly of some poorly known genera such as Pseudophoxinus and Petroleuciscus was rejected. Representatives of both genera belong to different evolutionary lineages. Timing of cladogenetic events among the main leuciscine lineages was gained using mitochondrial and all genes data set. CONCLUSIONS: Adaptations to a predatory lifestyle or miniaturization have superimposed the morphology of some species. These species have been separated into different genera, which are not supported by a phylogenetic framework. Such is the case of the genera Pseudophoxinus and Petroleuciscus, which real taxonomy is not well known. The diversification of leuciscine lineages has been determined by intense vicariant events following the paleoclimatological and hydrogeological history of Mediterranean region. We propose different colonization models of Mediterranean region during the early Oligocene. Later vicariance events promoted Leuciscinae diversification during Oligocene and Miocene periods. Our data corroborate the presence of leuciscins in North Africa before the Messinian salinity crisis. Indeed, Messinian period appears as a stage of gradually Leuciscinae diversification. The rise of humidity at the beginning of the Pliocene promoted the colonization and posterior isolation of newly established freshwater populations. Finally, Pleistocene glaciations determined the current European distribution of some leuciscine species.

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.