A NEW SPECIES OF THE GENUS SQUALIUS BONAPARTE, 1837 (ACTINOPTERYGII, CYPRINIDAE) FROM THE TAGUS RIVER BASIN (CENTRAL SPAIN)

A new species of Squalius is described on the basis of morphological and genetic characters from the Gallo River and its tributaries (Tagus Basin) in central Spain. The new species is distinguished from other Iberian Squalius species by a combination of morphometric, meristic and genetic characters, such as wide infraorbital bones that do not cover the adductor mandibulae muscle; seven scales on the upper transversal line; small number of lateral line scales, mean = 39.8 (39-42); a short and wide head, 4.3-4.9 (mean = 4.5) times the standard length; Short pectoral-ventral length, reaching 2.2-2.4 (mean = 2.3) times the standard length. The new species differs from its closer taxa in terms of genetic GTR distances, S. carolitertii and S. pyrenaicus at 20 fixed nucleotide positions (molecular autapomorphies), in the mitochondrial cytochrome b gene. Genetic divergence values for this new species and S. carolitertii and S. pyrenaicus are 3.8% and 6.1% respectively. The new species should be considered Critically Endangered (CR) according to the IUCN Red List Categories.


Introduction
The genus Squalius Bonaparte, 1837 is widely distributed throughout European freshwater lakes and rivers, and shows a high level of diversity in the Mediterranean area (Zardoya & Doadrio, 1999;Durand et al., 2000;Doadrio & Carmona, 2003).This genus includes approximately 15 species (Kottelat 1997;Doadrio & Carmona 1998) distributed along the Mediterranean basin; two species are widely reported in Central and North Europe (Sanjur et al., 2003).
Nevertheless, the taxonomy of Squalius in the Iberian Peninsula has been controversial because of inconsistencies between analyses based on genetic and morphological characters.Phenotypic plasticity is high among the species and diagnostic characters are difficult to find, but genetic approaches have demonstrated a high degree of differentiation between Iberian Squalius populations based on allozymes (Coelho et al., 1995;Alves et al., 1997;Carmona et al., 1997) and mitochondrial (mt) DNA sequences variation (Brito et al., 1997;Sanjur et al., 2003).These studies have focused mainly on populations of S. pyrenaicus and the high levels of genetic variation between these populations have resulted in the diagnoses and identification of four new species from the southern and eastern Iberian Peninsula, S. aradensis and S. torgalensis from Portugal (Coelho et al., 1998) and S malacitanus and S. valentinus from Spain (Doadrio & Carmona, 2006).Squalius carolitertii, which is also widely distributed throughout the north-western Iberian Peninsula, has not been as thoroughly studied, and no comparable levels of genetic variation have been found in initial studies of this species (Sanjur et al., 2003).
In this paper we reanalysed between-population genetic variation in Squalius carolitertii and S. pyrenaicus including a population from the upper Tagus Basin, which preliminary phenotypic examinations suggested as being distinct from both S. caroliterii and S. pyrenaicus.The purpose of this paper is to examine the phylogenetic relationships of this Tagus Basin population based on mitochondrial cytochrome b sequence data, and to provide diagnostic morphological characters and a description of the new species of Squalius.

MOLECULAR ANALYSIS
Fifty-two samples from seven species of Squalius from the Iberian Peninsula were analysed.Squalius alburnoides and S. palaciosi were not examined because these species are part of a hybridogenetic complex (Carmona et al., 1997;Alves et al., 1997Alves et al., , 1999Alves et al., , 2001;;Cunha et al., 2004).Squalius lucumonis and S. keadicus from the "Mediterranean lineage" (Sanjur et al., 2003) were used as outgroup taxa.New samples examined were collected in the wild from multiple locations (Table 1).DNA was extracted from dorsal muscle, which was preserved in liquid nitrogen or 70% ethanol.Because in the last ten years only one specimen of Squalius from the Gallo River was captured (1999), we only included one specimen from this population in the molecular analysis.Posterior exhaustive samplings in this area have not reported Total cellular DNA was extracted from tissues by a standard proteinase K and phenol/chloroform extraction method (Sambrook et al., 1989) and ethanol purification (Towner, 1991).Two overlapping fragments of the mitochondrial cytochrome b gene (total of 1140 bp) were amplified via polymerase chain reaction (PCR) from each individual DNA sample.The primers used for amplification of the cytochrome b were from Machordom & Doadrio (2001).Thermal cycle parameters were as follows: initial denaturation at 92ºC (2 min), 35 cycles of denaturation at 94ºC (1 min), annealing at 48ºC (1 min), and extension at 72ºC (5 min), and a final extension at 72ºC for 10 min.PCR mixtures were prepared in 25 ml volumes with a final concentration of 0.5 ml each primer, 0.2 mM each dNTP, 1.5 mM MgCl 2 , and 1 unit of Taq DNA polymerase (Biotools).After checking PCR products on 1.5 % agarose gels, they were cloned using the pGEM-T vector (Promega) into E. coli JM109, and were sequenced using the Big Dye Deoxy Ter-minator cyclesequencing kit (Applied Biosystems Inc.).DNA sequences of both strands were obtained using M13 universal (forward and reverse) sequencing primers.All samples were sequenced on an Applied Biosystems 3700 DNA sequencer following the manufacturer's instructions.
Chromatograms and alignments were visually checked and verified and there were no gaps in the resulting DNA sequences.Nucleotide saturation was analysed by plotting absolute number of tran-sitions and transversions against patristic distance values (not shown).The aligned data were analysed by Bayesian inference performed with MrBayes 3.1 (Huelsenbeck & Ronquist, 2001) by simulating a Markov chain for 1.000.000cycles.Model Test vers.3.7 (Posada & Crandall, 1998) indicated that the best-fit model was the GTR (Rodríguez et al., 1990) + I model of evolution.Not burnin parameters were necessary.

Results and discussion
The high degree of genetic and morphological divergence of specimens from the Gallo River (Tagus Basin) to other species of Squalius of the Iberian Peninsula warrants consideration of the Gallo River population as a different species.No available name could be applied to this population, and therefore, a new species was described.
DESCRIPTION: D II-III 8, A II-III 7-8 (mean = 7.5), P I 13, V II 7, C 18, LLS 39-42 (mean = 39.8),A medium sized species that rarely reaches a standard length of 200 mm.Morphometric characters are given in Tables 2 and 3. Maximum body depth is 3.8-4.2(mean = 4) times the standard length.Head short and is 4.3-4.9(mean = 4.5) times the standard length.Head length is lesser than maximum body depth.Preorbital distance is longer than eye diameter, reaching 1.2-1.6 (mean = 1.4) times the eye diameter.There is a long interorbital distance, reaching 1.6-2 (mean = 1.8) times the eye diameter.Ventral fin is inserted slightly before origin of dorsal fin.Predorsal length 1.1-1.2(mean = 1.1) times preventral length.High caudal peduncle.The ventral-anal distance is long, with an anal index (VAL/PRAD) of 2.4-2.9 (mean = 2.7).Minimum body depth is 2.9-3.2(mean = 3) times length of caudal peduncle and 1.7-1.9(mean = 1.8) times length of anal peduncle.The pectoral-ventral distance is short, reaching 2.2-2.4 (mean =2.3).Fin size is moderately large.PIGMENTATION PATTERN: Silver body, darker dorsally.There is a black spot at the base of the scales that appears to be divided by the lateral line pore into two separate spots.The peritoneum is silver with small black spots.DISTRIBUTION: This new species is endemic to the Gallo River and its tributaries in the upper Tagus Basin (Central Spain) (Fig. 2).ETYMOLOGY: The species name "castellanus" derives from the name of the Spanish region -Castille-inhabited by this species.An adjective.COMMON NAMES: We propose using the common name "bordallo del Gallo" in Spanish, and "Gallo chub" in English.
CONSERVATION: Squalius castellanus is only known from small populations along the Gallo River (Tagus basin) and its main tributaries, both Bullones and Arandilla Rivers.In the last ten years only one individual has been collected in the Gallo River.The main threat to the species is pollution from the city of Molina de Aragon.Consequently, the population is currently declining and should be considered Critically Endangered (CR B1 + 2ce) according to the IUCN Red List Categories.
INTERESPECIFIC COMPARISONS: Specimens of Squalius from the Gallo River (Tagus basin) show many differences in both genetic and morphological characters with respect to S. carolitertii and S. pyrenaicus.Thus, this population deserves recognition as a new species, Squalius castellanus.On Table 5 there is a summary of the main differences between the new species S. castellanus and both S.     carolitertii (mean GTR =3.8, 3.7-4.2) than to S. pyrenaicus (mean GTR =6.1, 5.7-6.6).In respect of S. aradensis and S. torgalensis, the genetic distances found between these species and S. castellanus were the highest (mean GTR =11.8, 11.7-11.9and mean GTR =12.9 respectively).If a molecular clock with a 1% rate of mutation per 1 My is assumed for the mt cytochrome b gene (Dowling et al., 2002;Doadrio & Carmona, 2004), the Iberian Squalius species belonging to the "Mediterranean lineage" diverged from other European species in the upper Miocene period (13 MYA).This divergence was possible when Asia Minor, Greece, the Balkans, Italy and the Iberian Peninsula were geographically connected.The break up of this geographical connection may have provided the conditions for the origin of Iberian Squalius species that subsequently became isolated populations that experienced further allopatric speciation (Sanjur et al., 2003).The complexity of the Iberian hydrographic basins in the Tertiary period contributed to allopatric speciation within the Iberian Peninsula, and led to the current diversity of Iberian endemic species.According to the molecular clock for cytochrome b (Dowling et al., 2002;Doadrio & Carmona, 2004), the separation between the new species described, S. castellanus, and the species genetically closer to it, S. carolitertii, is coincident with the lower Pliocene (3.8 MYA).Not geological events during the Pliocene could be claimed to explain the colonization and posterior speciation of S. castellanus.

Fig. 5 .
Fig. 5.-Phylogenetic tree of 52 analysed specimens of genus Squalius recovered from cytochrome b sequences according to the bayesian method.Branch lengths are proportional to the estimated mean number of substitutions per site.Numbers are posterior probabilities of bayesian analysis.Fig. 5.-Árbol filogenético de 52 especímenes del género Squalius obtenido a partir de secuencias de citocromo b analizadas mediante inferencia Bayesiana.Las longitudes de las ramas son proporcionales al número medio de sustituciones por sitio.Los números indican las probabilidades posteriores del análisis bayesiano.
new samples.Voucher specimens for this species were deposited in the collections of the Museo Nacional de Ciencias Naturales, Madrid, Spain.