From Late Miocene to Holocene: processes of differentiation within the Telestes genus (Actinopterygii: Cyprinidae)

Investigating processes and timing of differentiation of organisms is critical in the understanding of the evolutionary mechanisms involved in microevolution, speciation, and macroevolution that generated the extant biodiversity. From this perspective, the Telestes genus is of special interest: the Telestes species have a wide distribution range across Europe (from the Danubian district to Mediterranean districts) and have not been prone to translocation. Molecular data (mtDNA: 1,232 bp including the entire Cyt b gene; nuclear genome: 11 microsatellites) were gathered from 34 populations of the Telestes genus, almost encompassing the entire geographic range. Using several phylogenetic and molecular dating methods interpreted in conjunction with paleoclimatic and geomorphologic evidence, we investigated the processes and timing of differentiation of the Telestes lineages. The observed genetic structure and diversity were largely congruent between mtDNA and microsatellites. The Messinian Salinity Crisis (Late Miocene) seems to have played a major role in the speciation processes of the genus. Focusing on T. souffia, a species occurring in the Danube and Rhone drainages, we were able to point out several specific events from the Pleistocene to the Holocene that have likely driven the differentiation and the historical demography of this taxon. This study provides support for an evolutionary history of dispersal and vicariance with unprecedented resolution for any freshwater fish in this region.     

Isolation and characterization of polymorphic microsatellite loci in the freshwater fishes Telestes souffia and Telestes muticellus (Teleostei: Cyprinidae)

Ten novel polymorphic microsatellites (seven with perfect motifs) were isolated from vairone species (Telestes souffia and Telestes muticellus), which are endangered European cyprinid species. Together with 11 previously published cyprinid-specific loci, five multiplex sets were optimized, allowing the genotyping of 21 polymorphic loci. The level of genetic diversity was assessed in 97 individuals from the two species T. souffia and T. muticellus. We also successfully applied the 21 microsatellites to nine related species. These primers will thus be useful in assessing population structure of the vairone and other cyprinid species, with application for conservation issues and phylogeographical approaches.     

A new endemic cyprinid species from the Danube drainage

Populations of endemic Croatian dace were found to belong to two different species, one of which is first described in this study. Telestes karsticus sp. nov. differed morphologically from Telestes polylepis in the total count of lateral line scales, number of gill rakers and the shape of the posterior margin of the anal fin. Morphological differences were corroborated with mtDNA analyses (with p-distance between T. polylepis and T. karsticus sp. nov. ranging between 3·2 and 4·1%; and the number of substitutions between 37 and 47). The newly described species is geographically very localized. It has been recorded from only four localities around Velika Kapela and Mala Kapela mountains in Croatia.     

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 Gatačko, Cerničko, 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.     

Molecular phylogeny of two lineages of Leuciscinae cyprinids (Telestes and Scardinius) from the peri-Mediterranean area based on cytochrome b data

We examined phylogenetic relationships in two lineages of Leuciscinae cyprinid fishes based on the sequence data of the complete mitochondrial DNA region coding for the cytochrome b gene (1140 bp). Telestes includes obligate riverine, moderately cold water-adapted species whereas Scardinius comprises warm-adapted species living in lowland lakes and still waters of rivers and streams. We also analysed selected representatives of Leuciscus and Phoxinellus because the taxonomic status of some species belonging to these genera is dubious and they could be placed in the genus Telestes. The study includes 18 species, 43 populations, and 111 individuals from 9 of the 14 peri-Mediterranean ichthyogeographic districts. Clades recovered from the phylogenetic analyses do not support previous taxonomic assumptions based on morphology. Telestes, Leuciscus, and Phoxinellus do not form monophyletic assemblages; phylogenetic analyses suggest that L. polylepis, L. turskyi, P. croaticus, and P. metohiensis should be included in Telestes. Similarly, populations of Scardinius erythrophthalmus do not cluster together and the endangered S. scardafa, endemic to central Italy and surviving in a single locality, is nested within them. The radiations of Telestes and Scardinius occurred in different time periods. A major diversification of Telestes is consistent with a sea dispersal during the freshwater Messinian "Lago Mare" phase of the Mediterranean Sea. Cladogenetic events within Scardinius are likely related to the extension and confluence of river drainages in lowlands following multiple lowering of the sea level during the Quaternary glaciations.     

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., Mari?, D. & Chappaz, R. (2010). Speciation pattern of Telestes souffia complex (Teleostei, Cyprinidae) in Europe using morphological and molecular markers.—Zoologica Scripta, 39, 225–242. It is notorious that many species boundaries are erroneously defined. When molecular markers are used, misleading evidence can notably be due to the characteristics inherent to mitochondrial DNA and quantity of markers used but also because of a limited range distribution sampling. European cyprinids biodiversity inventory is still an ongoing task and surprising phylogeographic patterns and phylogenetic relationships are still recovered on account of methodological evolution. This is particularly obvious for the Telestes souffia complex. This species occurs in a fragmented range and species boundaries is greatly debated. In this study, we provide an updated delimitation of the different evolutionary entities constituting this T. souffia complex and propose a taxonomic revision. Morphological and molecular analyses were carried out on 520 specimens coming from 19 localities representing the complete geographical range of this species complex and six related sister species that could potentially interact with it (outgroup ‘sensu lato’). Phylogenetic reconstructions and multivariate analyses demonstrated that the T. souffia complex is constituted of at least three species (T. souffia, Telestes muticellus and Telestes montenigrinus). Data also suggested that T. souffia comprises three subspecies (T. s. souffia, T. s. agassii; T. s. rysela). We also confirm the splitting of the T. souffia sister species Telestes pleurobipunctatus into two distinct species. The Peloponnesian lineage will be referred as Telestes alfiensis and the continental lineage as T. pleurobipunctatus. Morphological and molecular markers displayed some degree of incongruence within T. souffia suggesting that introgressive hybridization has played a role in the evolution of the Telestes genus. However, discordance among data sets could also result from heterogeneous rate of morphological evolution. Finally, we demonstrated that T. muticellus was implicated in two categories of hybridization: an inter‐species hybridization (between T. muticellus and T. souffia) and an inter‐generic hybridization (between T. muticellus and Squalius lucumonis), a phenomenon rarely observed for a same species.     

Early development of the endemic dace Telestes karsticus (Leuciscidae,Cypriniformes) in a Dinaric karst stream in Croatia

Early development of the endemic dace Telestes karsticus is described in a Dinaric karst stream in Croatia. Larvae were acquired from successful rearing in an aquarium. Basic morphometric and meristic measures of larvae were taken during 55 days of observation. The mean total length of larvae at the time of hatching was 6.45 mm. The yolk sac was completely absorbed in all larvae by day 9 post-hatching (PH), when the mouth also opened and independent feeding and intestinal function were observed. Flexion of the urostyle was completed between days 12–16 PH. The caudal fin became completely forked on day 23 PH. Formation of fin rays was complete on day 48 PH when fish fully resembled juvenile specimens. The number of soft ray fins corresponds to the adult. Understanding the early development is particularly valuable in assessing environmental impacts and recovering of this endangered species and it is essential for effective monitoring and its conservation. This is the first report of the early development for the endemic Telestes karsticus, pointing out the importance of the obtained results for the effective conservation of the species.     

Revised classification of former genus Phoxinellus using nuclear DNA sequences

Molecular data inferred from three nuclear DNA regions were used to re-examine the phylogenetic position and taxonomic status of former Phoxinellus taxa. Using either distance method, maximum likelihood or MCMC, phylogenetic tree revealed statistically well supported clade of Delminichthys adspersus, Delminichthys ghetaldii, Delminichthys jadovensis, and Delminichthys krbavensis occupying a sister position to Pelasgus prespensis; furthermore, Phoxinellus pseudalepidotus emerged as a sister taxon to Delminichthys–Pelasgus group. There was a moderate support for sister relationship between Telestes croaticus and Telestes fontinalis, while the position of Telestes metohiensis varied depending on the region and method used. The topology of taxa within Delminichthys was weakly supported and remained pretty much unresolved. Our results confirm a previous notion that the former genus Phoxinellus is not a monophyletic group but rather a grouping of independent lineages.     

A new large barn owl (Aves, Strigiformes, Tytonidae) from the Middle Pleistocene of Sicily, Italy, and its taphonomical significance

A new species of Tytonidae, Tyto mourerchauvireae, is described from the Sicilian cave deposits of Spinagallo, Luparello and Marasà, which have yielded a common vertebrate fossil assemblages referred to the early Middle Pleistocene. T. mourerchauvireae nov. sp. shows a pronounced increase in body size compared to other congeneric taxa. It is larger than the extant Tyto alba and the extinct Tyto balearica and Tyto sanctialbani and comparable in size among the Mediterranean taxa only with the extinct Tyto robusta, which differs in some morphological characteristics of the long bones. The insular adaptations of T. mourerchauvireae nov. sp. and the differences from the other forms of the genus Tyto which spread through the Mediterranean area during Neogene and Pleistocene are discussed. The fossil assemblages of the early Middle Pleistocene of Sicily are dominated by extinct giant Gliridae of the genus Leithia and Maltamys; these taxa are regarded as the primary prey items of T. mourerchauvireae nov. sp. The Gliridae remains were analysed microscopically to detect possible modifications of bones and teeth caused by ingestion and digestion processes. This type of analysis, together with the qualitative study of the fossil assemblages, allows to show the taphonomical importance of T. mourerchauvireae nov. sp. as agent of accumulation in such Sicilian fossil assemblages. Incidentally the taxonomic validity of T. robusta is discussed and confirmed.     

Genetic structure of the vairone Telestes souffia in the eastern part of Lake Constance, central Europe

Examination of the genetic structure of the vairone Telestes souffia based on 10 nuclear markers (microsatellites) revealed little-to-moderate genetic differentiation between geographically adjacent populations in the eastern part of Lake Constance in central Europe. Results emphasize the critically endangered status of this freshwater fish in the upper Rhine River system.     

Genetic variability and phylogeography of the cyprinid Telestes muticellus within the Italian peninsula as revealed by mitochondrial DNA

A phylogeographic analysis of 12 populations of Telestes muticellus was performed on the basis of mitochondrial DNA sequences of the cytochrome b gene. The main aims were to determine phylogenetic relationships from congener species and to highlight the patterns of genetic differentiation within the Italian peninsula. The results obtained showed a significant divergence with the congener species, indicating a possible Miocene origin for the genus. Divergence was found to be low for T. muticellus , yet distinct lineages resulted as being allopatric within the two main Italian biogeographic districts, the Padano-Venetian and the Tuscano-Latium, suggesting the existence of recent vicariance coupled with past demographic regression. The high level of genetic differentiation found between populations highlighted the low dispersal ability of this rheophilic Cyprinid, whose migration was mainly related to connections between the upper parts of river basins. The distribution of sequence characteristics found suggests that particular attention should be paid to the conservation management of this species.     

Phylogeographical structure of vairone Telestes muticellus (Teleostei, Cyprinidae) within three European peri-Mediterranean districts

The phylogeographical pattern of vairone ( Telestes muticellus ) populations was assessed to test the biogeographical distinction of three peri-Mediterranean ichthyogeographical districts: Padano-Venetian (PV), Tuscano-Latium (TL) and southern France (SF), evaluating the role of Alpine and Apennine barriers in shaping distinct evolutionary lineages. A fragment of mitochondrial DNA (mtDNA) cytochrome b (cyt b ) was sequenced in 153 specimens from 14 north-western Italian populations, collected in 10 tributaries of Po River and in four rivers on the Tyrrhenian slope of Ligury, and 32 haplotypes were identified. The phylogenetic analyses confirmed the presence of two distinct clades, an 'Italian clade' ( T. muticellus ) and a 'French clade' ( T. souffia ), showing an average genetic distance of 12.9% (± 1.3 SD) and allopatric distribution. The Nested Clade Analysis (NCA) and the Analysis of Molecular Variance ( amova ) revealed an isolated gene pool in eastern basins on the Tyrrhenian slope of Ligury. The phylogeographical findings suggest: (i) the lack of permeability of Alpine barrier towards the dispersion across Italian and French hydrographical systems; and (ii) partial permeability of Mediterranean Alps and Apennines through river captures crossing lower watersheds.     

Genetic variability and differentiation in Leuciscus (Telestes) souffia. Taxonomic and conservation inferences

Genetic variability of eight populations of Leuciscus (Telestes) souffia from France, Italy and Slovenia, and one of Leuciscus (Squalius) carolitertii from Spain was analysed using allozymic electrophoresis. For L (Telestes) souffia the parameters of variability ranged from P = 1 8.2 to 48.5 % and H_o = 0.023 to 0.092, values included in the global averages found for freshwater fishes. The phylogenetic relationships among these populations lead us to consider the souffia species belonging to the genus Telestes, and to subdivide it into two species: souffia (with at least two subspecies: souffia in France and agassizi in Slovenia), and muticellus. The genetic structure found within the French populations showed a certain differentiation between the Hérault Basin and the Var/Argens populations, recommending the preservation of these entities that could represent different adapted genetic pools. In the same way, the Italian region should be studied in detail, since the populations analysed from Bevera and Staffora Rivers (T. muticellus) showed evidence of a break of genetic flow (including a diagnostic locus between them).     

Morphology,diet, and stable carbon isotopes: On the diet of Theropithecus and some limits of uniformitarianism in paleoecology

Geladas were long supposed to be the only living primates feeding almost entirely on graminoids and accordingly display dramatic dental and manual adaptive traits. A recent study of Theropithecus gelada, the first in a relatively undisturbed habitat, revealed a more diverse diet, also incorporating large quantities of forbs. The peculiar adaptive traits of T. gelada are also observed in extinct Theropithecus as early as 3.7 Ma. Stable carbon isotopic data of extinct Theropithecus from eastern Africa indicate that specimens older than 3 Ma consumed a significant proportion of C₃ plants (on average ca. 40% of total food intake) whereas specimens younger than 2 Ma consumed more C₄ plants (on average ca. 80%). Recent paleobotanical evidence suggests that C₃ herbaceous plants were still present in non‐negligible proportions in Plio‐Pleistocene lowland tropical ecosystems. Together, the shared morphological adaptive traits of extant and extinct Theropithecus and the varied diets of extant T. gelada suggest that the paleodiets of Theropithecus may have been dominated by herbaceous plants, comprising both C₃ forbs and graminoids and C₄ graminoids. The changes in stable carbon isotopes could correspond to a replacement of C₃ plants by C₄ plants within the herbaceous strata rather than a shift from C₃ woody vegetation to C₄ graminoids. This synthesis highlights the need for a more exhaustive knowledge of the ecology of extant species to achieve meaningful paleodietary and paleoenvironmental reconstructions. A strong selectivity for food resources that are rare in the landscapes (as in T. gelada) should also be considered when interpreting stable carbon isotopes of extinct African mammals (and notably hominids).     

Seasonal profile of the diet of the dace Telestes karsticus Marčić & Mrakovčić, 2011 (Cyprinidae,Leuciscinae) endemic to Sušik Creek,Croatia

The first data on the feeding ecology of the endemic Telestes karsticus are presented for specimens sampled monthly (July 2007–July 2008) from the Su?ik Creek in the Lug Polje field, Croatia. Macrozoobenthos density was calculated and compared with the stomach contents of T. karsticus and the seasonal profile of the diet was examined. The ratio of gut length vs. standard length was 0.9 ± 0.12. Gut contents revealed that benthic invertebrates accounted for the majority of food consumed, although terrestrial arthropods, plant material, algae and fish were also found. Since this is the only fish species in the investigated creek, T. karsticus can be concluded to be cannibalistic. The proportion of empty guts found in all seasons was small, indicating year round feeding. The number and diversity of prey species was highest in spring and lowest in summer. Ivlev's electivity index was very high for Cladocera, Coleoptera, Trichoptera, Heteroptera and Hymenoptera in all seasons, suggesting positive selection for these prey groups, as opposed to Oligochaeta, Isopoda and Bivalva, which showed negative values in all seasons. For Actinopterygii, the Ivlev coefficient of selection was highly negative in spring, and highly positive in summer. Plant matter was detected in stomach contents in all seasons, with the highest share found in summer when other prey species were less available. The karstic dace is a generalised feeder, consuming the most available prey, and therefore can be classified as a euryphagous omnivore.     

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.     

The Miocene tortoise Testudo catalaunica Bataller, 1926, and a revised phylogeny of extinct species of genus Testudo (Testudines: Testudinidae)

We provide a taxonomic review of the extinct testudinid Testudo catalaunica, based on published and unpublished material from several Miocene (late Aragonian and early Vallesian) sites of the Vallès‐Penedès Basin (north‐east Iberian Peninsula). We show that Testudo catalaunica irregularis is a junior subjective synonym of T. catalaunica, and further provide an emended diagnosis of the latter based on newly reported material. Contrary to some recent suggestions, this emended diagnosis discounts an alternative attribution of T. catalaunica to Paleotestudo. The latter is merely recognized as a subgenus of Testudo, based on a cladistic analysis that assessed the phylogenetic position of all extant and most extinct species of Testudo currently recognized as valid (including T. catalaunica). Our phylogenetic analysis (which recovers the molecular phylogeny of extant Testudo s.l.) supports a taxonomic scheme in which the three extant subgenera of Testudo are represented in the fossil record. Testudo s.s. is retrieved as the sister taxon of Testudo (Agrionemys) + [Testudo (Paleotestudo) + Testudo (Chersine)]. The extinct Testudo (Paleotestudo) is therefore the sister taxon of the Testudo (Chersine) clade. The latter subgenus reveals as the most diverse clade of Testudo s.l. in the fossil record, with T. catalaunica + Testudo steinheimensis constituting a subclade distinct from that including Testudo hermanni.     

Influence of introduced fish Percottus glenii (Odontobutidae, Pisces)

Monitoring of 28 waterbodies has been carried out since 1994 in the region of reserve "Lake Glubokoe" (Moscow region, Russia). It was revealed that species diversity as well as abundance of larval amphibians correlate negatively with presence of introduced fish, rotan, Perccottus glenii (Odontobutidae). Newts (Triturus cristatus, T. vulgaris) and frogs (Rana temporaria, R. arvalis, R. lessonae) as a rule are not capable to breed in waterbodies colonised by rotan. In contrast, toads (Bufo bufo) breed successfully in such sites. Larvae of toads are comparatively less edible for rotan and pass their metamorphosis. Persistence of amphibians to predation of rotan decreases in the row: B. bufo (R. temporaria, R. arvalis and R. lessonae), T. vulgaris, T. cristatus. The Crested newt (T. cristatus) is the most endangered species and could extinct in next years.     

Occurrence of Monogenea in Italian freshwater fish (Po river basin)

Sixteen Monogenean species were found on the skin and gills of 10 different fish species collected from north Italian water bodies. Cleidodiscus pricei from Ictalurus melas; Dactylogyrus alatus from Alburnus alburnus; D. anchoratus, D. formosus and D. vastator from Carassius carassius; D. difformis from Scardinius erythrophthalmus; D. rutili from Rutilus aula; D. tincae and Gyrodactylus gasterostei from Tinca tinca; D. ergensi from Leuciscus cephalus and Telestes muticellus; D. vistulae and Paradiplozoon rutili from Leuciscus cephalus; D. extensus and G. katharineri from Cyprinus carpio; G. lucii and Tetraonchus monenteron from Esox lucius. All species are new records for Italy with the exception of D. difformis, D. ergensi, D. vistulae and T. monenteron.