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1 PierGiorgioBianco GabrieledeFilippo Editors ResearchesonW ildlifeconservation Volume4 A taxonomicstudyonthegenusphoxinus(acthinopterigy,cyprinidae) from ItalyandwesternBalkanswithdescriptionoffournew species: P.ketmaieri,P.karsticus,P.apolonicusandP.likai PierGiorgioBiancoandSalvatoreDeBonis P.likai IGFPublishing

2 Pier Giorgio Bianco Gabriele de Filippo Editors Researches on Wildlife Conservation volume 4 IGF Publishing

3 Reference Bianco P.G. e de Filippo G. (eds.) Res.Wildl.Conserv. 4. IGF Publ., USA. Published by Lulu.com 2015 Istituto di Gestione della Fauna onlus info@gestionefauna.com

4 Content A taxonomic study on the genus Phoxinus (Acthinopterigy, Cyprinidae) from Italy and western Balkans with description of four new species: P. ketmaieri, P. karsticus, P. apollonicus and P. likai Page 1 P. G. Bianco and S. De Bonis

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6 Researches on Wildlife Conservation, vol. 4, 2015, IGF publ. A taxonomic study on the genus Phoxinus (Acthinopterigy, Cyprinidae) from Italy and western Balkans with description of four new species: P. ketmaieri, P. karsticus, P. apollonicus and P. likai Pier Giorgio Bianco * e Salvatore De Bonis Ichthyological laboratory, via Paparelle al Pendino, 5, Naples (Italy) * Correspondence author: gibianco@unina.it Abstract The genus Phoxinus from Italy and western Balkans was analyzed. Five species are recognised: P. Phoxinus, distributed in the rivers of the Alpine slope of the Padano-Venetian, district; P. ketmaieri n.sp, described from Krk island and River Zrmanje, but whose distribution probably includes also the River Cetina and others rivers of the Dalmatian district, the rivers Krka and Neretva. P. karsticus described from the Endorheic-system of the River Trebinje, Bosnia-Herzegovina is another representative of the Dalmatian-Croatian district; P.apollonicus described from the Moraca- Zeta River system, Lake Skadar drainage, is the representative species of the Albanian district. Finally, P. likai shuold be regarded as an endemic species of the extensive endorheic river system of the Lika region and underwater connected rivers of the Bosnia Erzegovina-Croatian district. Studio tassonomico del genere Phoxinus in Italia e nei Balcani occidentali e descrizione di quattro nuove specie P. ketmaieri, P. karsticus, P. apollonicus and P.likai. Il genere Phoxinus da Italia e Balcani occidentali è stato analizzato. Cinque specie sono state identificate: P. phoxinus, distribuito nei fiumi del versante alpino del distretto Padano-Veneto; P. ketmaieri n.sp, descritto per l isola di Krk in Croazia, e il fiume Zrmanje, ma la sua distribuzione potrebbe includere il fiumi Cetina Krka, e Neretva, del distretto della Dalmazia-Croazia; P. karsticus descritto per il sistema carsico del fiume Trebinje in Bosnia-Herzegovina è un altro rappresentante del distretto della Dalmazia-Croazia; P.apollonicus n.sp. descritto per il fiume Moraca, pricipale affluente del lago di Scutari, è la specie rappresentative del distretto Albanese. Infine, P.likai n.sp viene considerato un endemismo del vasto sistema fluviale endoreico della regione della Lika e connessioni carsiche sotterranee dei fiumi della Bosnia Erzegovina e della Croazia adriatica. Étude taxonomique du genre Phoxinus en Italie et dans les Balkans occidentaux et la description de quatre nouvelles espèces P. ketmaieri, P. karsticus, P. apollonicus et P. likai. Le genre Phoxinus de l'italie et les Balkans occidentaux a été analysé. Cinq espèces ont été identifiées: P. Phoxinus, distribué dans les rivières de la pente de la région montagneuse Padano- Veneto; P. ketmaieri décrit pour la 'île de Krk en Croatie, et la rivière Zrmanje, mais la distribution peut comprendre les rivières Cetina Krka et Neretva, dans le district de la Dalmatie et la Croatie; P. karsticus décrit de la endoréiques-système de la rivière Trebinje, Bosnie-Herzégovine est un autre représentant de le district de Dalmatie et la Croatie ; P. apollonicus n.sp. décrit pour la rivière Moraca, principal affluent du lac de Skadar, est les espèces représentatives du district albanais. Enfin, P. likai n.sp est considéré comme une espèce endémique de le réseau fluvial de la région de steppe de Lika et connexions rivières karstiques souterrains de la Bosnie-Herzégovine et de l'adriatique de la Croatie. Key words: Taxonomy, Cyprinidae, Phoxinini, Phoxinus, Italy and western Balkans, new species

7 Introduction Phoxinus is the Palaearctic representative genus of the Holoarctic clade Phoxinini of the subfamily Leuciscinae. The North-American representatives of the clade received considerable attention since they include nearly the whole cyprinid fish fauna of the Nearctic region, with about 300 species (Simons, et al., 2003; Strange & Mayden, 2009; Mayden & Allen, 2015). Among the European Phoxinini, the genus Phoxinus is spread throughout the Palaearctic region, with exception of extreme southern area of the peri-mediterranea peninsular regions and nearly all islands. At present it includes about 15 valid species, ranging from Western Europe to China (Eschmeyer, 2015). A peculiarity of this genus is the presence, in reproductive males, of patches of breasts scales between the pectoral fins, a character shared with genera Margariscus, Couesius, and Clinostomus. But the question of the affinities of Phoxinus with these genera is still debated (Hing-Yu Chen, 1996). The taxonomy of this genus in Europe has been relatively little studied in the recent past when compared to others Palearctic genera such as Rutilus, Barbus, Chondrostoma, and Telestes. Recently, Kottelat (2007) contributed to the our knowledge of the taxonomy of this genus from western and southern peninsular Europe, reporting seven species, three of which new to science, reporting for Italy and western Balkans, a single species Phoxinus lumaieurl Schinz, 1849, but suggesting the potential presence of at least a new species from the Lake Skadar system. The recent molecular analyses of Palandačić et al., (2015) surprisingly found that the genus Phoxinus in the Western Balkans and Italy entails a complex of species with at about seven distinct, molecularly recognizable clades. These results do make sense, because the Western Balkans are characterized by distinct ichthyogeographic districts, each of them characterized by a complex of localized endemic species (Bianco, 1990; Ketmaier et al., 2008; Bianco and Ketmaier, 2015). For this paper, we analyzed the material sampled in these districts available in the collection of the Department of Biology and Zoological Museum of the Naples University, Italy (IZA). The collection is not exhaustive but on a positive note it includes primarily samples collected about years ago, when anthropogenic introductions for re-stocking purposes hadn t yet started in the area. More recently, this phenomenon has had a heavy impact on the original geographic distribution of Phoxinus species (Kottelat, 2007). Material and Methods Most fishes were collected about 30 years ago, previously fixed in formaldehyde 10% and then preserved in ethylic alcohol 60%. All the samples included in the current analyses are preserved in the collection of the Department of Biology and Zoological Museum of Naples University, Italy (acronym: IZA). Figure 1 Map showing the approximate locations of examined materials in Italy and Western Balkans 2

8 Researches on Wildlife Conservation, vol. 4, 2015, IGF publ. Counts, and measurements and the description of the position of patch of breasts scales follow Kottelat (2007), except for the standard length (SL), which was measured from the tip of snout to the upper insertion of the caudal fin. Due to the reduced size of fish, most counts and measurements were taken with the aid of a millimeter digital caliper and a stereoscopic microscope; all measurements were taken from the left side of the body. Pharyngeal bones were dissected mainly from the left side, but in several cases also from the right side. Laboratory illustration has been made with a scanner, Epson perfection 3170 photo, using sensibility from 350 up to 1200 DPI. The sampling localities of the examined specimens are reported in Fig. 1 Systematic accounts. Phoxinus phoxinus (Linnaeus, 1758) (Fig. 2) Figure 2 Female, 49 mm SL, above, and Male, 53 mm sl below of P. phoxinus from the Po river basin. Synonym. Phoxinus lumaireul Schinz, 1849: 331 (type locality, Lake Major). Examined material. IZA 0427, 9 specimens, mm SL, Vicenza, River Bacchiglone, 4 April 1996, P. G. Bianco coll. IZA 00299, 6 specimens, mm SL, stream Chiamogna, western tributary of the Po basin, 2 May 1980, P. G. Bianco coll. - IZA 83134, 9 specimens, mm SL, Fosso Pinzano, River Tagliamento, Friuli, Italy, 15 April 1982, P. G. Bianco coll) IZA uncat., 5 es, mm SL, United Kingdom, River Frame, East Stoke, Dorset, 22 April 1982, A. Wheeler coll. According to Kottelat (2007), the species living in Italy and Western Balkans is Phoxinus lumaireul. A detailed comparison of my samples from upper Adriatic rivers with the extensive neotype description given by Kottelat (2007) for Phoxinus phoxinus and with a sample of P. phoxinus from England shows P. lumaireul be a synonym of P. phoxinus; this conclusion already reported in Bianco (2014) is also supported by the molecular data of Palandačić (2015). The Phoxinus inhabiting water bodies from Croatia to Albany and Montenegro represents a distinct complex of yet undescribed species. 3

9 In the following re-description of P. phoxinus from Italy, we are giving for comparative purposes in parentheses the values reported by Kottelat (2007) for the neotype and additional material. Several diagnostic characters as the number of scales in longitudinal series, the circumpeduncular ones, as well as the pharyngeal teeth formulae, were not reported by Kottelat (2007) but we found that the combination of them are diagnostic especially for the new species described below. Re-description. In Phoxinus phoxinus from Italy the patches of breast scale are separated (Fig 3, 4) (the same), Lateral line incomplete with about pored scales, sometimes reaching or overpass beyond anal base (Fig 2) (lateral line usually reaching beyond anal base); Depth of caudal peduncle % SL (8-11 %SL); in its length ( times), times in body depth ( times body depth), patched of breast scales separated by unscaled area (the same). Lateral line from incomplete to nearly complete (neotype has a nearly complete pored lateral line scales). In addition, P. phoxinus from Italy has scales in longitudinal series and circumpeduncular scales. The pharyngeal teeth formula is Italy is usually 5.2 on both sides, as in P. phoxinus from England (Fig 5). Figure 3 4. Ventral region of a male 54 mm SL from River Bacchiglione showing well separated patches of breast scale (left side) - Ventral view of anterior part of body of a male 53 mm SL from River Tagliamento, showing separated patches of breast scales (right side). Figure 5 A: Left pharyngeal bone, of about 3 mm of length, of a fish 54 mm SL from river Po. B: Left pharyngeal bone of about 3 mm from a fish 53 mm SL from England. 4

10 Researches on Wildlife Conservation, vol. 4, 2015, IGF publ. Total gill rakers, 6-8; branched rays of dorsal, anal and pelvic fin, 7; free margin of dorsal and anal fins straight to slightly convex (Fig 6); dorsal profile of the body, slightly convex; longitudinal stripe extending from the tip of snout to the end of caudal peduncle were, at base of caudal fin, is enlarged in a roundish spot; about vertical blotches accompanying 1-2 myomere of the trunk and of caudal peduncle, sometimes covering the distinctness of the longitudinal stripe. According to this re-description, the Italian populations cannot be distinguished from Phoxinus phoxinus as described by Kottelat (2007). Radmler et al., (2015), stated that according to their geometric morphometrics analyses, population of Phoxinus from Italy and western Balkan show a deeper body and a deeper caudal peduncle when compared with population of central European origins and stated that their results may confirm the validity of P. lumaieurl. This seems incongruent with present data as depth of body and of caudal peduncle are highly variable in the species here described (Table I), and body depth, according to ours results, seems deeper in P. phoxinus from transalpine countries, in contrast with the results of Radmler (2015). In addition, Collin & Fumagalli (2011), according to molecular and geometric morphometrics analyses, founds that shape of body (but also several others phenotypic and genotypic characters) of P. phoxinus is habitat dependant and lacustrine populations show a deeper body and caudal peduncle when compared with the riverine forms. In conclusion, the depth of body and caudal peduncle are adaptive characters in P. phoxinus, and have little value in taxonomy. Figure 6 Female, 53 mm SL, from River Tagliamento Friuli Region showing convex free margin of dorsal and anal fins. Sexual dimorphisms: males in April have nuptial tubercles on the head and fan shaped on the free margin of scales of the body and especially the ventral region of the caudal peduncle (Fig. 7). Pectoral fins are longer than in female and thickened also, on dorsal faces, by minute nuptial tubercles, or unculi aligned along the first six seven branched rays. Anal fin has tubercles, but with the first three rays thickened. All tubercles disappear or just leave some vestigial remains out of spawning season. Small tubercles, fan shaped are on the free margin of the patch of scales between pectoral fins. The scales are embricated and well separated in males in spawning condition, but in females and in non-reproductive fishes, the patches are apparently lacking or deeply hidden in the skin: this features of sexual dimorphisms in males Phoxinus were described in details also by Howes (1985). Colour pattern, In preserved specimens, the colour patterns consist in a blackish, longitudinal stripe extending from the tip of the snout till caudal peduncle, ending in a roundish black spot on the base of caudal fin. Vertical blotches can only be accentuated. All fins are colorless, the dorsal and the caudal fins, with several scattered melanophores. Peritoneal membrane silvery with few scattered melanophores. 5

11 Figure 7 Lateral view of the caudal peduncle of a male 49 mm SL, Phoxinus phoxinus showing minute fan shaped tubercles on the free margin of each scale, and the thickened first 3 rays of the anal fin In alive specimens, according to a colour slide taken on a freshly collected fish in River Astico on October 1995, but not preserved, the ventral part appears silver from the throat and the operculum to the far end of the caudal peduncle, up to the insertion the caudal fin. The longitudinal band is obscured by thick vertical stripes of a dark brown. The paired fins are reddish at their base, the unpaired and caudal, grayish. Phoxinus ketmaieri, new species (Fig 8) Figure 8 Holotype of Phoxinus ketmaieri, male, 43 mm SL, IZA , Isla of Krk Holotype: IZA , Male 43 mm SL, brook on south-eastern slope of Krk island, Baška brook at Baška draga, Croatia, April 1982, R. Hacker coll 6

12 Researches on Wildlife Conservation, vol. 4, 2015, IGF publ. Paratypes: IZA 8823, 2 specimens, mm SL, brook on south-eastern slope of Krk island, Baška brook at Baška draga, Croatia, April 1982, R. Hacker col.-l IZA 8792, 9 specimens, mm SL, River Zrmanje, Obrovac, Croatia, August 1987, P. G. Bianco coll. Diagnosis: Phoxinus ketmaieri is distinguished from other Phoxinus species from Italy and Western Balkans by the low number of scales in longitudinal series 58-61, and number of circumpeduncular scales, Holotype, a male, has a depigmented body but with melanophores traces of the former pigmentation. In a paratype from Zrmanje (Fig 9) the longitudinal stripe extends from the tip of snout to the end of caudal peduncle where it ends in a roundish spot. The holotype has breast blotches of scales separated (Fig 10). The pharyngeal teeth formula is usually 4.1 or 4.2. and meristics are reported in Table I and II. Largest specimens analyzed was a female 46 mm SL from River Zrmanie. Body moderately elongate. Dorsal profile convex sometimes with a slightly pronounced hump, dorsal profile of the head slightly more convex than ventral, snout blunt, mouth slightly subterminal, chin rounded. Figure 9 - Paratype of Phoxinus ketmaieri, 44 mm SL, female, River Zrmanje Description. The outline of the species is shown in figs 6 and 7. Detailed morphometrics and meristics are reported in Table I and II. Largest specimens analyzed was a female 46 mm SL from River Zrmanie. Body moderately elongate. Dorsal profile convex sometimes with a slightly pronounced hump, dorsal profile of the head slightly more convex than ventral, snout blunt, mouth slightly subterminal, chin rounded. Dorsal fin with 3 simple and 7 branched rays; free margin of dorsal and anal fin straight to slightly convex; anal fin with one simple followed by 6-7 branched rays; pectoral fin rounded with one simple and branched rays; in males may nearly reach and overpass the insertion of pelvic fin, in females reaches 2/3 of the distance between the insertion of pelvic and ventral fin; pelvic fin with 3 simple and 7-8 branched rays free margin of anal fin from straight to moderately convex; origin of anal fin from slightly below or at the same level of vertical through posterior insertion of dorsal fin. About scales in longitudinal series, lateral line incomplete with pored scales; in small specimens, less of 30 mm SL, the scales are incipient and only 2-3 are pored, scales embedded but distinct, about circumpeduncular scales. Tubercles in males, fan shaped on the free margin of most scales of the body especially on the lower part of caudal peduncle. Depth of caudal peduncle times in it length; head depth times in its length; eye diameter less than the preorbital distance; body depth about times in SL. Colour pattern. The general color pattern in preserved specimens is yellowish with more or less evident stripes or vertical bands. In most specimens, especially from River Zrmanje, the longitudinal stripe is well marked or confused by vertical reduced stripe which follow 1-2 myomere of the trunk and of the caudal peduncle. Peritoneal membrane, silvery with few scattered melanophores. All fins are almost colourless, with few scattered melanophores. 7

13 Figure 10 Ventral view of anterior part of the body of the holotype of Phoxinus ketmaieri showing separated breast patch of scales. Distribution and habitat. Species of genus Phoxinus are usually considered as coldwater adapted species living in the trout region (Bianco & Ketmaier, 2015). Nevertheless the Phoxinus living in the Zrmanje should be considered as a warm water adapted populations as, at time of collection, the temperature was 22 C, and the fish communities were represented by typical warm water adapted species, namely chubs, bleaks, gobies, river blennies, cobitids, and eels. On the Krk island the species was observed in a small brook not far from the sea: Baška brook at Baška draga. This island is, apparently, the only island around the Mediterranean to host a Phoxinus species. Its presence was firstly recorded by Heckel on Later on the species was recorded several times and collected always from the same brook and preserved at Naturhistorisches Museum Wien. The current conservation status of this unique population is unknown. The distribution would probably include also the River Cetina, R. Zrmanje and probably others rivers of the Dalmatian Ichthyogeographic district, namely, the River Krka, and Neretva. Etymology. From Valerio Ketmaier, a molecular biologist and friend with a long collaborative history with the senior author. Phoxinus karsticus, new species (Fig, 11) Figure 11 Holotype of Phoxinus karsticus, female, 58 mm SL, IZA , Endorheic River Trebinje, Bosnia- Herzegovina. 8

14 Researches on Wildlife Conservation, vol. 4, 2015, IGF publ. Holotype. IZA , 73 mm SL, Endorheic River Trebinje, near Jasenica Village, Bosnia Herzegovina, 24 October 1989, B. Elvira coll. Paratypes. IZA 00314, 4 specimens, mm SL, endorheic river Trebinje, near Jasenica Village, Bosnia Herzegovina, 24 October 1989, B. Elvira coll. Diagnosis. Phoxinus karsticus distinguished from others Phoxinus from Italy and Western Balkans by the high number of scales in longitudinal series 81-86, and number of circumpeduncular scales, 40-42, but also for the size reached of about 91 mm of TL. Holotype has a longitudinal band discolored or reduced. Holotype, and paratypes has patches of breast scales well developed and united in their third front ( Fig 12). Dorsal has three simple rays followed by 7-8 branched rays; 8 branched rays only seldom were found in all others sample here analyzed. The pharyngeal teeth formula from left bone is 5.2 or 4.2. Figure 12 - Ventral view of anterior part of the body of the paratype 49 mm SL, of Phoxinus karsticus showing united patch of breast scales One other character useful to distinguish the species, but not unique are, the position, the anterior origin of anal fin placed in advance of vertical through posterior insertion of dorsal fin, reaching the level of the fifth or sixth branched rays of the dorsal fin. Description. The outline of the holotype, a female, is shown in Fig. 11. Detailed morphometric and meristic data are reported in Table I and II. Largest specimens analyzed was the holotype, a female 73 mm SL with immature eggs mm of diameter. Body quite elongate when compared with others species here analyzed; dorsal profile, straight or moderately convex; dorsal profile of the head and snout slightly convex; snout pointed its dorsal profile is in continuum with that of the head and back; mouth slightly subterminal; eye diameter less than prerbital distance. Dorsal fin with 3 simple and 7-8 branched rays; free margin of dorsal fin straight to slightly convex. Anal fin with three simple and 7 branched rays; free margin of anal fin straight or slightly convex. Apparently the length of anal fin equal in both sex. Pectoral fin rounded with one simple and branched rays; pelvic fin with one simple followed by 7 branched rays. Caudal peduncle times longer than deep; head depth times in its length; eye diameter less to the preorbital distance; body depth about times in SL. 9

15 About scales in longitudinal series, lateral line incomplete with pored scales; about circumpeduncular scales. Tubercles in males incipient (fish were collected in October, outside the reproductive season) on dorsal surface of pectoral fin and scales of body and caudal peduncle; females carry egg of mm of diameter. Colour pattern. The general colour pattern in preserved specimen, is yellowish with more or less evident stripe or vertical bands. In most specimens, especially from Krk Island, the longitudinal stripe is faintly marked or confused by vertical darker bars. All fins are almost colourless, with few scattered melanophores. Distribution and habitat. P. karsticus is probably endemic to the Karstic Popovo Polje-Trebinje endorheic river system. Derivatio nominis: from the karstic area of Popovo Polje placed in westernmost area of Bosnia- Erzegovina region. Phoximus apollonicus, new species (Fig. 13) Figure 13 - Holotype of Phoxinus apollonicus, male 58 mm SL IZA , River Moraca, Montenegro. Holotype. IZA , 58 mm SL, upper River Moraca tributary to Lake Skadar, Montenegro, 26 august 1987, P.G.Bianco coll. Paratypes. IZA 87103, 42 specimens, mm SL, upper River Moraca tributary to Lake Skadar, Montenegro, 26 august 1987, P.G.Bianco coll. Diagnosis. Phoxinus apollonicus is unequivocally distinguished from all others Phoxinus species by the reduced number of teeth on pharyngeal bones. The species usually has four teeth on external row (Fig 14), but also 3 or 2, and 1 or 0 in the inner row. Others characters which, combined, can help to distinguish this species from the others: the holotype has patches of breast scales connected anteriorly (Fig 15); the lateral line is nearly completed, posteriorly reaching the vertical line crossing the upper and the lower insertion of the caudal fin; flank of body with a marked mid-lateral longitudinal stripe extending from tip of snout to the base of caudal fin; a distinct spot at base of the caudal fin; a marked black spot on the opercular bone; depth of caudal peduncle, times in its Figure 14 Left pharyngeal bone of the holotype Phoxinus apollonicus (length 2.5 mm), showing 4 teeth on external and 0 on inner rows). 10 length and in body depth; eye diameter less than the preorbital distance.

16 Researches on Wildlife Conservation, vol. 4, 2015, IGF publ. Description: patches of breast scales absent in specimens less of 30 mm SL, where the scales are still incipient. In largest specimens, breast scales are very difficult to check because deeply hidden into the skin. Apparently, most of the paratypes do not show breast scales, only in few specimens, are connected or separated by unscaled area. The longitudinal strip is well developed in specimens of about mm SL, which are nearly scale-less. In specimens of mm SL, is still unformed. In preserved specimens, the broad longitudinal band extend from tip of snout till the caudal peduncle, where, at the base of the caudal fin, is enlarged in a roundish spot vertical stripe of melanophores, more or less expanded, follow most of myomere of the trunk and the caudal peduncle. Pharyngeal teeth usually 4.1, but also 4.0 and 3.0; in P. apollonicus the principal formula is , with several case of teeth placed on a single outer row (4-4; 4.1-4) (Fig 15); the lateral line is nearly complete, the pored section include about scales, while there are overall about 71 to 79 scales in longitudinal series circumpeduncular scales; above and below the lateral line. Dorsal fin with three simple rays followed by 7 branched rays (rarely 8). Anal fin with constantly three simple rays followed by 7 branched rays. Pelvic fins with one simple followed constantly by 7 branched rays; free margin of dorsal fin from straight to slightly convex; free margin of anal fin from straight to slightly concave. Eye roundish, iris whitish. Gill rakers on the first gill arch, left side, are incipient o very reduced with 5-7 total gill rakers. Figure 15 - Ventral view of anterior part of the body of the holotype, of P. apollonicus showing united patch of breast scales Colour pattern. In preserved specimens all fins are whitish with few scattered melanophores; peritoneum silvery with few scattered melanophores; in living specimens (from a slide taken from a specimens of River Zeta, collected on November 1999, but not preserved) the longitudinal band is masked by very marked and extensive vertical blotches, which, clearly, accompany one or two myomere of the trunk and of the caudal peduncle. All fins are golden yellow; the belly and the lower part of head and the body is brilliant silvery. 11

17 Distribution. River Moraca and its tributary Zeta, both belonging to the Lake Skadar drainage. Possibly P. apollonicus represents the endemic species of the Albanian ichthyogeographic district. Derivatio nominis. From Apollonia, an ancient name to delimitate Albany. Phoxinus likai, new species (Fig 16) Figure 16 Holotype of Phoxinus likai female 47 mm SL, IZA , River Oruca system, Bosnia-Erzegovina. A small sample of five specimens collected in the Karstic area of Lika Valley of Croatia, in a brook belonging to the endorheic river Oruca near Gračac, show characters that cannot be applied to any of the four taxa described above. Indeed, this species has the patches of breast scales united at the centre. This is a very valuable character for species identification for Phoxinus (Kottelat, 2007). In the four species here described, only one other species has patches of breast scales united, Phoxinus karsticus. But this species have a nearly incomplete pored lateral line and a higher number of scales either on longitudinal series or around the caudal peduncle. Based on this evidence, we described the small sample as a new species in the hope to stimulate conservationists and taxonomists to undertake more researches in Croatia on this still poorly investigated genus in Europe. Holotype. IZA , 47 mm SL, Croatia, River Oruca system, near the village of Gračac, 8 December 1999, P.G. Bianco coll. Paratypes. IZA 02263, 4 specimens, mm SL, Croatia, River Oruca system, near the village of Gračac, 8 December 1999, P.G. Bianco coll. Diagnosis. Phoxinus likai is distinguished from Phoxinus phoxinus and the species here described by having patches of breast scales united and continuous across breast ( Fig 17) except P. karsticus, where they are united only on anterior part. It may be distinguished from P. karsticus also for the number of scales in longitudinal series which are in P. karsticus and in P. likai and for circumpeduncular scales, in P. karsticus and in P. likai. Others characters useful for determination, but not unique to this species are the pharyngeal teeth formula, 5.2 or 4.2, shared with P. poxinus but not P. apollonicus; pored lateral line nearly complete, reaching almost the end of the caudal peduncle, in P. ketmaieri and P. phoxinus do not reach more than half of the length of caudal peduncle; eye large, its diameter more than the preorbital distance, but less in the other species; snout short and blunt; longitudinal band from tip of snout to caudal peduncle where it forms a roundish spot; the band is more evident in the second half of the body; the flanks have mid-lateral row of vertical elliptic blocks, more evident on the second half of body; anterior origin of anal fin placed at same level of vertical through posterior insertion of dorsal fin. 12

18 Researches on Wildlife Conservation, vol. 4, 2015, IGF publ. Figure 17- Ventral region of a male of P. likai 49 mm SL showing the united patches of breast scales. Description: General outline of the holotype is shown in Fig 16 and morphometric and meristic data are given in Table I and II. The largest specimen examined was 61 mm of total and 49 mm of SL. Body moderately elongate, dorsal profile more convex than the ventral, dorsal profile of the head straight, snout short and blunt; mouth moderately downturned; chin rounded. Dorsal fin with 3 simple and 7 branched rays, free margin convex; anal fin with three simple and 7 branched rays, its free margin from straight to slightly convex; when depressed it reaches about one half of caudal peduncle length; pectoral fins oval, with one simple and branched rays, when depressed on the flanks it may reach or overpass the insertion of the pelvic fins, in males, in females may reach 2/3 of the pectoral-pelvic fins distance; depth of caudal peduncle about times its length; head depth times its length; eye diameter times the preorbital distance: body depth about times SL. Lateral line incomplete with pored scales; scales on longitudinal series; circumpeduncular scales; pharyngeal teeth hooked at the tip and serrated; about 5.2 or 4.2 teeth on pharyngeal bones. Tubercles are present in males on the head and on the dorsal side of the pectoral fins, which are thickened also by the presence of minute nuptial tubercles aligned along the first six seven branched rays. Colour pattern. In preserved specimens the lateral band extending from the tip of snout to the caudal peduncle is more evident on the second half of the body; vertical blotches are more enlarged on sides of caudal peduncle; a large blackish spot on the opercular bone; ventral region of body and caudal peduncle, which extend about 1/3 of body depth, yellowish; all fins yellowish with few scattered melanophores; peritoneal membrane silvery with few scattered melanophores. Distribution. Probably endemic of the extensive endorheic river system of the Lika region and underwater connected rivers of the Croatian Adriatic slope. Derivatio nominis: from the Lika-Dinaric karstic region of Croatia. Discussion This contribution seems to be complementary to that of Kottelat (2007), who re-analyzed most of the Phoxinus species from transalpine western Europe, but left somehow unsolved the taxonomy of the genus in the western Balkan area, reporting just a single species, Phoxinus lumaieurl, now placed in synonym with Phoxinus phoxinus, distributed in the Padano-Venetian district. In other district of the Western Balkans we found a Phoxinus species complex formed by at least 4 new species; our conclusions are in partial agreement with those based on molecular data of Palandačić et al. (2015). We found, also, that most important diagnostic characters are the meristic counts, principally squamation, the breast patches of scales and the pharyngeal teeth formulae. Several proportional measurements seems to be habitat-dependant and, for 13

19 instance, lacustrine populations show a deeper body and a deeper caudal peduncle when compared with riverine ones (Collin & Fumagalli, 2011). Regarding the teeth on pharyngeal bones, while in Phoxinus phoxinus from the Po basin, and others rivers of the upper Adriatic drainage and England the pharyngeal formula is usually , in Western Balkan we see a progressive lack of teeth along a north to south direction with the tendency to lose one or two teeth on outer and one or both in the inner rows. In the karstic area of Bosnia-Herzegovina, a distinct species was described from River Trebinje, an endorheic system found in the Popovo Polje area. In the Moraca river, Lake Skadar basin, Albanian district, we described the new species Phoxinus apollonicus, mainly according to the dentition of pharyngeal bone and a combination of other characters not exclusive of this species. In conclusion we confirm the finding of Palandačić et al. (2015) that in Italy and Western Balkan a complex of Phoxinus exists. We found five species, but probably additionally species are likely to be described for the area given the molecular evidence of Palandačić et al. (2015) reporting seven different clades. Regarding the biogeographic history of the group, we can postulate that Phoxinus phoxinus probably reached the Padano-Venetian district from the Danubian district, by exploring river captures occurred in quite recent time between opposite drainages of the Alps, or by underground river connections through the extensive karstic Alpine-Dinaric system of Eastern Italy and Western Slovenia and Croatia. An underground dispersal has been already reported in cyprinids (Palandačić et al., 2012). The reason why P. phoxinus did not colonize the Balkan sector of the Padano-Venetian district (namely, Zrmanje and Krka rivers) during the last glacial extended Po basin phase should be sought in the ecological condition present at the time or, more likely, in exclusion by either competition or predation (or a combination of both). As a matter of fact, Phoxinus is absent in all the Adriatic Italian rivers south of the Po river. This distributional pattern implies that Phoxinus did not exploit the extended Po basin as a route for dispersal as some other cold or moderate cold water adapted species such as Salmo farioides, Cottus gobio, Telestes muticellus and the eurythermal Squalius squalus did. These species used this phase of the Po river to reach several Adriatic drainages and the River Zrmanje in Croatia (Bianco, 2014, Bravničar et al., 2015) and, following the orogenesis of the Apennines, to cross the Italian peninsula to reach its Tyrrhenian side ( Tuscany-Latium district). Nowadays we indeed find these species in the head waters of the main rivers of the Tuscany-Latium district: Arno, Serchio, Ombrone. The species Cottus gobio, including a variety of moderately cold water adapted species, did used these routes to reach its current distribution, which encompasses all the afore-mentioned districts (Bianco, 1995). The Phoxinus species flock found in the Western Balkans probably is the result of a combination of penetration through underwater connections in the Karstic area (P. karsticus) and river capture from opposite drainage systems during the orogenesis of the Dinaric mountains (P. apollonicus). Acknowledgement We must thank a secret referee for improving the final version of the manuscript. This article was printed on 15 November 2015 in 30 copies. A copy was sent to each of the following official institutions: Natural History Museum, Cromwell Road, London; Muséum national d'histoire naturelle, 47 Rue Cuvier, Paris; Regional Museum of Natural History, Corso Venezia 55, Milan; Regional Museum of Natural Sciences, Via Giolitti, 36, Turin; Civic Museum of Natural History, Palace Lomellini Carmagnola (To); Civic Museum of Natural History, Via Brigata Liguria 9, Genoa. Free printed copies are available from the senior author or privately on line at Pier Giorgio Bianco Research Gate.com. Copies can be purchased from the bookseller at site Lulu.com 14

20 Researches on Wildlife Conservation, vol. 4, 2015, IGF publ. Bibliography BIANCO P.G., Potential role of the palaeohistory of the Mediterranean and Paratethys basin on the early dispersal of Europe-Mediterranean freshwater fishes. Ichthyological Exploration of Freshwaters, 1: BIANCO P.G., 1995., Factors affecting the distribution of freshwater fishes especially in Italy. Cybium, 19: BIANCO P.G., An update on the status of native and exotic freshwater fishes of Italy. Journal of Applied Ichthyology, 30: BIANCO P.G., KETMAIER V., Nature and status of freshwater and estuarine fisheries in Italy and Western Balkans. In: Freshwater Fisheries Ecology (Ed. John F. Craig), John Wiley & Sons, Ltd., pp BIANCO P.G., AHNELT H., ECONOMIDIS P.S.,1996. The freshwater fish from eastern and large Mediterranean islands with comments on their safety status. Acta Universitatis Carolinae, 40: BRAVNIČAR J., PALANDAČIĆ A., MARIC S., ŠANDA R., SNOJ A., Phylogeny of European Bullhead (Cottus sp.), in western Balkan. XV EIU Congress, Porto, Portugal, Book of abstract, p. 35. COLLIN E., FUMAGALLI L., Evidence for morphological and adaptive genetic divergence between lake and stream habitats in European minnows (Phoxinus Phoxinus, Cyprinidae), Molecular Ecology, 20: ESCHMEYER W.N., Catalog of Fishes. Online version of 2 September 2015, downloaded on 15 september 2015, CHEN, X-Y, 1996, Morphology, Phylogeny, Biogeography and Systematics of Phoxinus (Pisces, Cyprinidae), Bonner Zoologische Monographien, nr 39, 227 p. HOWES G. J.,1985. A revised synonymy of the minnow genus Phoxinus Rafinesque, 1820 (Teleostei: Cyprinidae) with comments on its relationships and distribution. Bulletin of the British Museum (Natural History) Zoology, 48: KETMAIER V., BIANCO, P.G., DURAND J.D., Molecular systematics, phylogeny and biogeography of roaches (Rutilus, Teleostei, Cyprinidae). Molecular Phylogenetics and Evolution, 49: KOTTELAT M., Three new species of Phoxinus from Greece and southern France (Teleostei: Cyprinidae). Ichthyological Exploration of Freshwaters, 18 : MAYDEN R.L., ALLEN J.S., 2015 Molecular Systematics of the Phoxinin Genus Pteronotropis (Otophysi:Cypriniformes) Hindawi Publishing Corporation BioMed Research International, Volume 2015, 6pp. PALANDAČIĆ A., MATSCHINER M., ZUPANČIĆ P., SNOJ A., Fish migrate underground: the example of Delminichthys adspersus (Cyprinidae). Molecular Ecology, 21: PALANDAČIĆ A., BRAVNIČAR J., ZUPANČIĆ P., ŠANDA R., SNOJ A., Molecular data suggest a multispecies complex of Phoxinus (Cyprinidae) in the Western Balkan Peninsula. Molecular Phylogenetics and Evolution, 92 SIMONS A.M., BERENDZEN P.B., RICHARD L., MAYDEN R.L., Molecular systematics of North American phoxinin genera (Actinopterygii: Cyprinidae) inferred from mitochondrial 12S and 16S ribosomal RNA sequences Zoological Journal of the Linnean Society, 39: STRANGE R. M., MAYDEN R.L., Phylogenetic relationships and a revised taxonomy for North American cyprinids currently assigned to Phoxinus (Actinopterygii: Cyprinidae). Copeia 2009:

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