Pier Giorgio Bianco

Allozymic variability and biogeographic relationships in two Leuciscus species complexes (Cyprinidae) from southern Europe, with the rehabilitation of the genus Telestes Bonaparte

Abstract Genetic variability was assessed in eight populations belonging to the Leuciscus souffla complex and in nine populations of Leuciscus cephalus, sampled in France, Italy and Greece and in one population of Leuciscus lucumonis, endemic to Italy. Twenty‐six enzymatic loci were analysed in order to clarify the genetic relationships, the patterns of colonisation of peri‐Mediterranean area and the taxonomic positions. Results indicate a more ancient penetration of the Leuciscus souffia complex in southern Europe (possibly during the Messinian ‘Lago Mare’ phase of the Mediterranean), whereas L. cephalus and L. lucumonis seem to have quite recently reached these regions (about 1Myr). These evidences are discussed in relation to the two main proposed models of primary freshwater fishes colonisation of peri‐Mediterranean area. In addition, the genetic distance suggests a generic separation between the two complexes. The genus Telestes Bonaparte, is rehabilitated to include members of the Leuciscus souffia ...

Phylogeography of bleaks Alburnus spp. (Cyprinidae) in Italy, based on cytochrome b data

Sequence variation of a fragment of the mitochondrial DNA encoding for the cytochrome b gene was used to reconstruct the phylogeography of the two species of bleaks occurring in Italy: the alborella Alburnus arborella in northern Italy and the vulturino Alburnus albidus in southern Italy. The study includes four populations of the alborella and 14 populations of the vulturino. A total of 57 haplotypes were identified; these could not be sorted into two reciprocally monophyletic clusters. Multiple phylogenetic methods and nested clade phylogeographical analysis consistently retrieved three well-supported clades, two of which contained both Northern and Southern Italian haplotypes. A third clade is limited to southern Italy. This clade is tentatively assigned to the vulturino. The placement in the same clade of northern and southern Italian haplotypes is explained in light of the introductions of fishes operated from northern to central and southern Italy. The origin of the vulturino dates back to the last two million years. This divergence time estimate identifies the Pleistocene confluences between adjacent river basins along the Adriatic slope of the Italian peninsula and their subsequent isolation as the cause that triggered the diversification of the genus in the area. The existence of a clade endemic to southern Italy supports the recognition of the area as a new peri-Mediterranean ichthyogeographic district, the borders of which correspond to the northern and southern edges of the vulturino range.

Genetic differentiation and biogeography in southern European populations of the genus Scardinius (Pisces, Cyprinidae) based on allozyme data

Levels of genetic differentiation and variability were assessed in several populations and species belonging to the primary freshwater fish genus Scardinius by means of electrophoretic analyses of 28 enzymatic loci. We studied one population of S. acarnanicus and one of S. graecus, both endemic to Greece, seven populations of S. erythrophthalmus from Greece, Slovenia, northern and central Italy, and the single extant population of S. scardafa, a species endemic to central Italy. Patterns of genetic relationships placed the Greek species basal in the neighbour‐joining (NJ) and maximum parsimony (MP) trees, S. scardafa and S. erythrophthalmus are sister species. Based on the molecular clock, the split between S. acarnanicus and S. graecus occurred around 4.3 Myr ago and the split between (S. acarnanicus, S. graecus) and (S. scardafa, S. erythrophthalmus) around 3.5 Myr ago, both after the Messinian lacustrine ‘Lago Mare’ phase of the Mediterranean. The separation between S. scardafa and S. erythrophthalmus was more recent (0.5 Myr ago) and was probably the result of confluences between adjacent river basins during the Pleistocene. Within S. erythrophthalmus, the Greek population showed a certain degree of genetic differentiation, whereas northern and central Italian populations were nearly indistinguishable. These findings demonstrate the impact of the artificial introduction of this species into central Italy, leading to the near extinction of the endemic S. scardafa.

Speciation pattern of Telestes souffia complex (Teleostei, Cyprinidae) in Europe using morphological and molecular markers*

Gilles, A., Costedoat, C., Barascud, B., Voisin, A., Banarescu, P., Bianco, P. G., Economidis, P. S., Marić, D. & Chappaz, R. (2010). Speciation pattern of Telestes souffia complex (Teleostei, Cyprinidae) in Europe using morphological and molecular markers.—Zoologica Scripta, 39, 225–242.

Threatened fishes of the world: Leuciscus lucumonis Bianco, 1983 (Cyprinidae)

Common name: Cavedano di ruscello (Italian), Brook chub (E). Conservation status: This Italian endemic species has undergone a progressive decline in its original range (Bianco & Taraborelli 1984, Bianco 1991, 1995, Mearelli et al. 1996, Bianco & Ketmaier 2001) since it was first described (Bianco 1983). Leucisus lucumonis is an important biogeographic indicator of the Tuscany-Latium district (Bianco & Ketamaier 2001). At present there are only about 10–12 isolated populations. According to the 2002 IUCN Red List of Threatened Species. www.redlist.org, downloaded on 14 August 2002, the status of this species (erroneously reported as L. lucumontis, Bianco 1983) was listed as at ‘Lower Risk’, (conservation dependent). Today L. lucumonis can be allocated to the endangered category for threatened species as it satisfies paragraph B and point 1 of the IUCN (1996) criterion ‘Severely fragmented distribution’. Identification and origins: The brook chub is a moderate sized species, which rarely reaches 200 mm TL and a maximum age of 5 years. Pharyngeal tooth formula 5.2-2-5. Dorsal fin origin placed behind the anterior origin of pelvics. Meristic counts: dorsal fin 3 unbranched rays and modally 7 branched rays, anal fin 3 unbranched rays and modally 8 branched rays, lateral line scales 40–42 (rarely over 43), 16 circumpeduncular scales. Ground colour of body yellowish, a pale golden iridescent longitudinal band is discernible on the middle part of the sides. Iris light with a brownish semilunar area in the upper half. All fins are greyish with some orange during the spawning season. Caudal fin uniformly greyish, without black tinged free margin as in the common chub Leuciscus cephalus (L.). L. lucumonis seems derived from L. cephalus as a result of vicarious events about 0.5 MYA. (Ketmaier et al. 1998). Drawing by Mariano Caminoli from a 132 mm SL specimen from Tiber River, Zoological Department of Naples University (Cat. N.: IZA 831). Distribution: Main rivers of the Tuscany–Latium district: Tiber R. with about 3–4 reproducing populations; Ombrone River with 3; Arno River probably 3–4, and Serchio River probably extirpated. Abundance: Scarce everywhere. Some moderately structured populations occur only in clean undisturbed brooks. Habitat and ecology: L. lucumonis is a primary, warm-water adapted, obligate riverine cyprinid fish. It lives mainly in brooks, occasionally found in main river courses. It lives together with young of the congeneric L. cephalus and other riverine Tuscany–Latium endemic species: these brooks represent relicts biotopes of biogenetic diversity of rheophilic endemic species of the Tuscany-Latium district (Bianco & Ketmaier 2001). Reproduction: Apparently from April till June. There are no accurate studies on its biology. Threats: The brook chub is very sensitive to pollution, habitat modification and competition with aliens. Its disappearance is mainly due to the introduction of Chondrostoma genei (Bonaparte 1839), a species endemic to northern Italy, heavily stocked in central Italy. This species can colonize small riverine biotopes. Conservation action: River restoration, eradication of introduced species, protection of small brooks, reintroduction of stocks in restored habitats and habitat protection. Conservation recommendation: Avoid any kind of alien introductions especially stocking with ‘white fishes’ (a mixture of mostly unidentified cyprinid and other species of uncertain origins), which often include riverine species, which can compete with L. lucumonis.

A revision of the Rutilus complex from Mediterranean Europe with description of a new genus, Sarmarutilus , and a new species, Rutilus stoumboudae (Teleostei: Cyprinidae)

By combining morphology, ecology, biology, and biogeography with the available molecular (sequence variation of the entire mitochondrial cytochrome b gene; cyt-b) and karyology data, the taxonomy of several species of the Rutilus complex inhabiting southern Europe is revised. Rutilus stoumboudae, new species, is described from Lake Volvi, Greece. It differs from Rutilus rutilus in possessing more total GR and less branched rays in both dorsal and anal fins and in its placement in the cyt-b based phylogeny of the genus. The resurrected genus Leucos Heckel, 1843 (type species Leucos aula, Bonaparte, 1841), which according to molecular data diverged from Rutilus more than 5 million years ago, during the Messinian salinity crisis, includes five species of small size, without spinous tubercles on scales and head in reproductive males, pharyngeal teeth formula 5-5, and all show a preference for still waters. Leucos aula is the Italian species endemic in the Padany-Venetian district: L. basak is widespread in Croatia, Albania, Montenegro and former Yugoslav Republic of Macedonia (FYROM); L. albus, recently described from Lake Skadar, Montenegro, is also found in rivers Moraca and Zeta (Montenegro). L. albus differs from L. basak, its closest relative, in having more scales on the LL and less anal-fin rays; L. panosi is endemic to the western-Greece district, and L. ylikiensis is endemic to lakes Yliki and Paralimni in eastern Greece (introduced in Lake Volvi). Among the nominal species examined, Rutilus karamani, R. ohridanus, R. prespensis and R. prespensis vukovici are all junior synonyms of Leucos basak. Rutilus vegariticus is definitively regarded as junior synonym for R. rutilus. Sarmarutilus n.gen. is a monotypic genus, with Sarmarutilus rubilio as the type species. According to phylogenetic data, Sarmarutilus rubilio is basal to a cluster of species that includes Leucos basak, L. albus, L. aula, L. panosi and L. ylikiensis. Sarmarutilus possibly evolved in pre-Messinian time, in the Lago Mare, entered the Mediterranean area during the Messinian Lago Mare phase of the Mediterranean Sea and survived only in the Tuscany- Latium district. This genus differs from Leucos in having large pearl organs on the central part of head and body scales in mature males and for the habitat preference, being a riverine-adapted species. It differs from Rutilus in pharyngeal teeth formula (5-5 in Sarmarutilus and 6-5 in Rutilus), size (small in Sarmarutilus and large in Rutilus) and for the preferential habitat (riverine vs. still water). Finally, lectotypes for Leucos basak, Leucos aula, and Sarmarutilus rubilio are designated.

Threatened fishes of the world: Gobio benacensis (Pollini, 1816)

Common name: Gobione (Italian). Italian gudgeon (English). Conservation status: This taxon, rehabilitated by Bianco and Taraborelli (1984), and Pizzul et al. (1993), has been confirmed and raised to specific rank (Bianco and Ketmaier 2005). Its inclusion in the genus Romanogobio (Kottelat and Freyhof 2007), is not supported by genetic evidence (Bianco and Ketmaier 2005). The species was quite common in river drainages of the Padano-Venetian district (Delmastro 1982), but has since disappeared from many localities and been replaced by the introduced Danubian species Gobio gobio. This species is currently listed as endangered by the IUCN (Crivelli 2006). Identification: This species can be identified from other Gobio species by possessing 2–3 scales between the anus and anal fin origins, as compared to 4–8 in other European species. Distribution: Endemic in rivers of northern Italy and Slovenia. In the past, the species was subjected to transfers in central Italy (Bianco 1995; Bianco and Ketmaier 2005). Abundance: Very rare due to its replacement by G. gobio where these two species overlap. Habitat and ecology: Warmwater adapted and preferential riverine species which forms small communities especially in the hill stretches of rivers. Moderately structured populations are at least still present in the River Tagliamento, in north-eastern Italy. A small introduced population lives in the River Ombrone in central Italy (Bianco and Ketmaier 2005). Reproduction: Reproduction takes place from April until June. Threats: The major threat is the extensive introduction of G. gobio for angling purposes, which is invasive and tends to replace and eliminate the native species in sympatric conditions. Conservation action: A survey of all existing reproductive populations and their protection in local sanctuaries. Eradication, when possible, of the Danubian species, followed by the re-introduction of G. benacensis. Conservation recommendation: Avoid stocking other fishes, especially cyprinids of exotic origins, were populations of G. benacensis still survive. Environ Biol Fish (2009) 84:39–40 DOI 10.1007/s10641-008-9383-1

Morphogenesis of otoliths during larval development in brook lamprey, Lampetra planeri

Otolith morphogenesis of the brook lamprey, Lampetra planeri, was analysed from larval to adult stages. The brook lamprey remains juvenile for about 4 years, facilitating analysis of otoliths maturation that permits to identify relevant evolutionary traits in this primitive species and to compare our results with more evoluted species of vertebrate taxa. We combined histochemical, immunohistochemical, scanning electron microscopy, elemental analysis and X‐ray diffraction of lamprey otoliths to establish possible relationships between otolithic mass, individual crystals, the otolithic organic substance that binds individual otoconia together and the inorganic elements that mineralize the lamprey otoliths. Histochemical analysis of the otoliths suggests that mineralization occurs gradually, beginning near the apex of the secretory epithelium. Then, the otoconia increase in size by deposition of layers of a dense crystalline substance. Immunohistochemical reactivity of calcium binding proteins indicates that calmodulin, calbindin, S‐100 and parvalbumin are parts of the uncalcified organic mass that holds otoconia together. Imaging of the immunoreactivity of each protein by Confocal Laser Scanning Microscopy in ammocoete at the first year of the larval stage shows weak reaction products which, however, gradually increase in intensity, with peak value in ammocoete at the fourth year of the larval stage.

Isolation and characterization of eight microsatellite loci in the brook lamprey Lampetra planeri (Petromyzontiformes) using 454 sequence data

Eight polymorphic microsatellite loci were developed for the brook lamprey Lampetra planeri through 454 sequencing and their usefulness was tested in 45 individuals of both L. planeri and the river lamprey Lampetra fluviatilis. The number of alleles per loci ranged between two and five; the Italian and Irish populations had a mean expected heterozygosity of 0·388 and 0·424 and a mean observed heterozygosity of 0·418 and 0·411, respectively.

Threatened fishes of the world: Barbus caninus Bonaparte, 1839 (Cyprinidae)

Common name: Barbo di ruscello (Italian). Brook barbel (English). Conservation status: This species has been recently rehabilitated according to morphological (Bianco 1995; 1998) and genetic analyses (Berrebi 1995; Tsigenopoulos et al. 2002). In the past this species was common in most brooks and streams of Alpine tributaries of the Po River drainage (Delmastro 1989), but is now restricted to about 20–25 reproductive populations (Bianco 2003a; Salviati et al. 2004; Giovanni Delmastro pers. observ.). According to IUCN (1996) categories of threatened species, B. caninus should be considered as endangered as it fits in paragraph B and point 1 of the criteria: ‘Severely fragmented distribution.’ There have been local transfers of this species in central Italy (Bianco 1994), but there are no documented cases of successful recruitment in these populations. Identification: Dorsal fin four unbranched and usually seven branched rays; pharyngeal tooth formula: 5,3,2-2,3,5, lateral line 39–51 (usually 41–48); circumpeduncular scales 22–26 (usually 24); total gill rakers 6–12 (usually 7–10). Body with brown or black patches. Peritoneal membrane black, fins greyish or yellowish, the paired fins sometimes red tinged. B. caninus rarely reaches 200–220 mm TL. This species was often confused with populations of B. tyberinus Bonaparte, 1939 which tends to retain the juvenile pigmentation with large black spots as shown in B. caninus or in the ‘meridionalis’ group of strongly riverine species (Bianco 2003b). The population now living in the River Isonzo, in north eastern Italy, did not belong to B. caninus but represents an established population of B. petenyi Heckel, 1848, introduced long ago into the River Soce, the upper Slovenian stretch of the Isonzo River (Bianco 2003a). Drawing by Mariano Caminoli from a specimen 129 mm SL of River Chisone (River Po drainage), Naples University (Cat. No.: IZA 8485). Distribution: B. caninus is the biogeographic indicator of the Padany–Venetian ichthyogeographic district of Northern Italy and Slovenia which includes all tributaries of upper Adriatic drainages, from the River Isonzo in the north, to the River Marecchia in the south. Abundance: Now very localised but there are good populations at a few localities. Habitat and ecology: B. caninus is primary freshwater fish, moderately cold water adapted and obligate inhabitants of rapids. It undertakes upstream migrations during spring and summer, and downstream during the cold season. Reproduction: April till June. Threats: Conservation action: A survey of all existing populations as well as a population dynamic studies is urgently required for this unique Italian endemic species. Conservation recommendation: Avoid introductions of alien Barbus, especially the obligate rapid dwellers which can compete or eliminate local populations as has already happened in River Isonzo were B. caninus has been replaced by the introduced B. petenyi (Bianco & Ketmaier 2001; Bianco 2003a).