Dos nuevas especies de truchas atlánticas (Actinopterygii, Salmonidae) de Marruecos ; Two new species of atlantic trout (Actinopterygii, Salmonidae) from Morocco

Four trout species of the genus Salmo Linnaeus, 1758 have been traditionally recognized in Morocco: S. macrostigma Duméril, 1858; S. pallaryi Pellegrin, 1924; S. pellegrini Werner, 1931 and S. akairos Delling & Doadrio, 2005. Two of these species are mainly distributed in Mediterranean (S. macrostigma) and Atlantic (S. pellegrini) river basins while the other two species are restricted to isolated lakes in the Atlas Mountains, one of them to Ifni Lake (S. akairos) and the other, probably extinct, to Sidi Ali Lake (S. pallaryi). Preliminary phylogenetic and regional studies based on molecular data have found high structuration of the populations of this genus in Morocco. These studies focused on allozymes, microsatellites and mitochondrial markers found genetic differences in populations from Isli Lake (Atlas Mountains) and the Draa Basin in southern Morocco. In this work we provide different morphological and genetic traits to distinguish these populations from Isli Lake and Draa Basin as two different species. urn:lsid:zoobank.org:pub:131DCCC9-5D2D-40B9-8F53-A2DAFA1AAB45


Introduction
Salmonid fishes constitute a monophyletic clade that is composed of three main lineages, namely coregonins, thymallins and salmonins.These three lineages are widely spread in cold waters of the northern hemisphere (Shedko et al., 2013).Within salmonids, the taxonomy of trouts (Salmo spp.) is controversial because of their high phenotypic variation in contrast to the scarce genotypic differentiation between populations, occurring in an extensive original distribution in the mountains of Eurasia and North Africa.This disagreement between morphological and genetic studies has led to different taxonomic approaches.Molecular studies usually prefer the use of lineages within Salmo trutta Linnaeus, 1758 to refer to different geographical populations (Machordom et al., 2000;Bernatchez, 2001;Suárez et al., 2001;Cortey et al., 2004).In contrast, in recent years, morphological studies prefer to fragment S. trutta into different species, using the traditional morphological species concept with a phylogenetic argument based on the data provided by molecular studies (Delling, 2003;Kottelat & Freyhof, 2007).
In contrast to other North African countries, Morocco preserves natural populations of trout and three of the four currently recognized species in North Africa are present in the country: S. macrostigma, S. pellegrini and S. akairos.The species S. pallaryi was described from Sidi Ali Lake, in the Middle Atlas Mountains (Morocco), but the autochthonous population of the lake seem to have disappeared due to the introduction of exotic species.Nevertheless, some authors consider that the population from Isli Lake could belong to S. pallaryi (Schöffmann, 1993;Delling, 2003).
The current taxonomy of Moroccan trout recognizes one species distributed in the Atlantic basins (S. pellegrini); one species from the Mediterranean basins (S. macrostigma); another species confined to Ifni Lake in the Souss basin (S. akairos) and the uncertain taxonomic position of the Isli Lake population.The main aims of this study are to clarify the taxonomic position of the Isli trout population and to characterize morphologically, for the first time, the populations from the Draa basin (located in southern Moroccan Atlas).Consequently we describe two new species of genus Salmo Linnaeus, 1758.
Twenty-six morphometric measurements and ten meristic variables were recorded for all specimens.All morphometric measurements were in millimetres.Measurements and counts were taken following Delling (2002).The following abbreviations were used for morphometric and meristic characters: AFH, anal fin height; AFL, anal fin length; AP, anal fin pterygiophores; APL, anal peduncle length; AR, anal fin rays; BA, body depth, at level of origin of anal fin; BD, body depth, at level of origin of dorsal fin; BLD, body least depth; Bq, left side branquiostegals; C, central caudal fin rays; CFL, length of lower caudal fin lobe; CPA, caudal peduncle length at level of end of adipose fin; CPL, caudal peduncle length at level of end of dorsal fin; Cpr, number of procurrent rays upper caudal lobe.DFL, dorsal fin length; DFH, dorsal fin height; DP, dorsal fin pterygiophores; DR, dorsal fin rays; EDH, horizontal eye diameter; EDV, vertical eye diameter; HL, head length; LL, lateral line scales; ML, length of maxilla, from premaxilla end to posterior end of maxilla; PFL, pectoral fin length; PrAD, preanal distance; PrDD, predorsal distance; PrOL, preorbital length; PrPD, prepectoral distance, PrVD, preventral distance; PsOL, postorbital length, RSA, scales from base of adipose fin to lateral line; SL, standard length; UJD, upper jaw depth; UJL, upper jaw length, from symphysis of premaxilla to posterior end of maxilla; VFL, ventral fin length; Ve, total number of vertebrae.Number of vertebrae was obtained by direct counting on X-rays images of individuals from all studied populations.
After constructing the measurement matrix, Burnaby's method was used to correct size effect (Burnaby, 1966;Rohlf & Bookstein, 1987).All analyses were conducted with the corrected matrix.Morphometric and meristic characters were analysed independently.A two-way analysis of variance (ANOVA) comparing morphometric characters was conducted to test for sexual dimorphism and variation among populations.To identify the variables that contributed most to the variation among populations, a principal components analysis (PCA) was performed using the covariance matrix for morphometric characters.Statistical analyses were carried out using PAST software (Hammer et al., 2001).
For the molecular approach, we analysed trout samples from Atlantic basins in Morocco corresponding to individuals of the genus Salmo from Ifni Lake (Salmo akairos), Isli Lake (Salmo sp.), Melloul R. (Salmo pellegrini), Miaami Lagoon (Salmo pellegrini), Dades R. (Salmo sp.), Lakhdar R. (Salmo pellegrini), Imlil R. (Salmo pellegrini) and Ziz R. (Salmo pellegrini) (Table 1).Likewise, we included one population from the Ikiss R. (Moulouya Basin) from the Mediterranean slope that has been associated to Atlantic trout populations (Tensift Basin) in previous molecular studies (Snoj et al., 2011).The species Salmo salar and Salmo orhidanus were selected as outgroups on the basis of previous phylogenetic analyses (Snoj et al., 2011).Analyses included mitochondrial cytochrome b gene (MT-CYTB) and control region (CR) for all data sets.Total genomic DNA was extracted from fin-clip tissue using the commercial kit Biosprint15 for tissue and blood (Qiagen).For each specimen, the complete region (1140bp) of MT-CYTB and a fragment (947bp including gaps) of CR were amplified.Primers and protocols used for PCR followed Machordom & Doadrio (2001) for MT-CYTB and Bernatchez (2001) for CR.After checking PCR products on 1% agarose gels, they were purified by ExoSAP-IT TM (USB) and directly sequenced on  Phylogenetic analyses were performed using Bayesian inference (BI) implemented in MrBayes v. 3.2 (Ronquist et al., 2012).The best-fitting models of sequence evolution for MT-CYTB and the CR under the AIC criterion (Akaike, 1973) were selected using PartitionFinder v1.1.1. (Lanfear et al., 2012).The best model scheme for running phylogenetic analysis was constituted by four partitions: CR (HKY+I), MT-CYTB 1 st aminoacidic position (K80), MT-CYTB 2 nd aminoacidic position (F81) and MT-CYTB 3 rd aminoacidic position (TrN).This partitioning scheme was used in phylogenetic performance unlinking evolutionary models among partitions.Bayesian analysis was performed using two independent runs of four Markov Montecarlo coupled chains (MCMC) of 10 6 generations each, to estimate the posterior probability distribution.Topologies were sampled every 100 generations, and majority-rule consensus tree was estimated after discarding the first 10% of generations.Robustness of clades was assessed using Bayesian posterior probabilities.The average genetic distances between Salmo populations were calculated for each gene using MEGA package v.6.0 (Tamura et al., 2013) according to the uncorrected-p distances.

MORPHOLOGICAL COMPARISON BETWEEN POPULATIONS
Analysis of variance (ANOVA) for sexual dimorphism showed significant differences between sexes (p<0.05) for variables associated with preorbital length, anal fin and body depth (Table 2).Orbital size was also different in males in relation to females but probably as a consequence of the large difference in eye size in the Ifni lake population (in comparison to the other populations).When we removed the Ifni Lake population from the analysis no significant differences were found between males and females.In general, all variables showing significant dimorphism were greater in females than in males (Table 3).Due to the presence of sexual dimorphism and because we collected few female samples (n=38), we removed females for subsequent morphological analyses.
On the other hand, most of the morphometric variables showed significant differences between populations in the two-way ANOVA analysis (Table 2).An analysis of body proportions based on non-parametric Kruskal-Wallis and Mann-Whitney post hoc comparisons were used, to find differences in body shape between populations that can remain masked if only linear untransformed measurements are considered.According to these analyses, morphological differences in body shape were found in almost all morphometric measurements (Appendix 1).The population from Isli Lake showed the largest body size compared to the remaining populations.Otherwise, the maxilla in the Isli population was long but not significantly larger than in the other populations (Table 4).In Isli Lake trout, the size of the eye was smallest of all populations studied, but since mature individuals from this population exhibited the largest body size of all studied populations, the smaller size eye found in this population could reflect allometric growth (Table 4).The Isli Lake individuals showed the smallest pectoral fin in comparison to other populations.The population of Miaami Lagoon in the upstream Chbouka River also had a long preorbital distance, which is in agreement with a well-developed maxilla in length and height.Trout of Miaami Lagoon were shorter, which is revealed in body depth and caudal
The PCA divided the Salmo populations into three groups that correspond to those of Ifni Lake, those of Miaami Lagoon and a third large group containing the remaining Salmo populations (Fig. 2).The eigenvalues for the two first Principal Components, with the Burnaby-corrected matrix for removing size effect, mostly explained the variance (Table 5).The variables that contributed most to the ordination in the PCA were orbital and preorbital lengths, caudal peduncle lengths, size and height of the maxilla and length of the dorsal fin (Table 5).
With regard to meristic characters, we found no significant differences according to Kruskal-Wallis and Mann-Whitney non parametric analyses for the following traits: number of vertebrae, number of scales in lateral line, number of scales in transversal on adipose fin, number of rays in dorsal and anal fins, number of procurrent rays on the caudal fin and number of branchyostegal rays.Only two traits showed significant differences, dorsal and anal pterygophores (Table 6).The number of pterygophores in dorsal fin had its highest value in Salmo akairos and its lowest value in the Isli Lake population (Fig. 3).Likewise, the number of pterygophores in the anal fin presented its highest value in Salmo pallaryi from Sidi Ali Lake comparing with the remaining populations studied (Fig. 3).The species Salmo akairos (Ifni population) also showed significant differences in relation to the other populations in anal fin pterygophores.

MOLECULAR APPROACHES
The concatenated matrix including both mitochondrial genes rendered a total of 2088bp (MT-CYTB: 1140bp+CR: 947bp including gaps).Bayesian tree reconstruction revealed well-supported phylogenetic relationships among trout populations from Morocco and demonstrated the early divergence of the Dades population (Draa basin) from the others (Fig. 4).In fact, the genetic distances between Dades and the remaining populations, including the recognized species Salmo akairos (Ifni), were the highest recorded for both genes and for all comparisons (Table 7).The Dades populations also showed nine autapomorphies in the MT-CYTB, all of them transitions (Table 9).Genetic distances among the rest of trout populations from Morocco ranged from 0.2-0.6% for MT-CYTB and 0.2-0.5% for CR (Table 7).Autapomorphies in the MT-CYTB gene, although not so numerous as in the Dades population, were found in the Ifni, Isli, Lakhdar and Tensift populations (Table 8).

PIGMENTATION PATTERNS
The Isli population showed a pigmentation pattern without spots and parr marks, as also occurred in Salmo pallaryi.The Dades population showed numerous irregular parr marks, usually greater to or equal than 14, which usually are retained in adult specimens.In the Dades population, the body is covered by small and numerous red and black spots that sometimes extend to the caudal fin and opercular area.Adult specimens from the Moulouya, Ziz and Oum er Rbia basins usually retained large and scarce parr marks.Adults from the Tensift population did not retain parr marks and showed a dark coloration (Fig. 5).

OSTEOLOGICAL REMARKS
Vomer teeth in Moroccan trout were arranged in zigzag with the exception of the Isli population, whose teeth were placed in one row within a narrow vomer.The vomer length was lesser than half of the parasphenoid length in all the studied populations except for the Ifni population.In this last population vomer length was greater than half of the parasphenoid length (Appendix 2).
For Dades individuals, the lateral processes of the ethmoid bone were extended, conferring a T shape to the bone, and the upper process of the premaxilla bone was small and laterally inclined.In comparison to other populations studied, where the upper maxillary   The anterior maxillary process in the Isli population was thinner than in the remaining Moroccan populations and the inflection of this process, in relation to the  Table 7.-Uncorrected-p genetic distances, in percentage, among Atlantic Moroccan trout populations based on MT-CYTB gene (below diagonal) and the CR (above diagonal).In bold on diagonal uncorrected-p genetic distances within populations on the left MT-CYTB and on the right CR.

DESCRIPTION OF POPULATIONS OF GENUS SALMO FROM MOROCCO
Our morphological and molecular analyses revealed that the population from Ifni Lake, recently described as a different species (Salmo akairos), had a high degree of distinct morphological features and moderate genetic differentiation in mitochondrial genes.The populations from the Oum er Rbia Basin showed scarce morphological and genetic differentiation relative to the Tensift Basin, where the species Salmo pellegrini was described (Delling & Doadrio, 2005;Geiger et al., 2014); therefore we provisionally assigned populations from Oum er Rbia basins to Salmo pellegrini.However, one of the populations of this latter basin (Miaami Lagoon) showed differential morphological traits, particularly in the length and height of the maxilla and premaxilla bones, but it was nested within the Oum er Rbia Basin mitochondrial clade and therefore this population should be also provisionally assigned to S. pellegrini.Isli no presenta las "rayas de pinto" ni manchas en el cuerpo.Dades presenta "rayas de pinto" irregulares y estrechas en los adultos y multiples puntos en el cuerpo.Poblaciones de Ziz y Moulouya tienen "rayas de pinto" grandes en los adultos.Los ejemplares adultos del Tendift no presentan "rayas de pinto".parr=parr marks.
The populations of the Ziz and Moulouya basins showed moderate genetic differentiation for the MT-CYTB gene; genetic differences in allozyme markers had also been previously reported for the Ziz population (Lbadaoui et al., 2011).Nevertheless, no morphological differentiation was detected when compared to other populations from the Oum er Rbia Basin, and so in the absence of the inclusion of other Mediterranean populations in the morphological and genetic analyses, we named these two populations as Salmo sp.The populations from Isli Lake and the Dades Basin showed a high degree of morphological and genetic differentiation that justifies their consideration as distinct species.No available name for these populations exists at present, and therefore the two new species are described in this study.The head is large in relation to the body, in comparison to all trout populations except those of the Ifni and Miaami populations, with head length 22.6-24.9%SL in females and 23.7-25.8%SL in males.The anterior part of the head shows a flat snout.The preorbital distance is the shortest of the populations studied and the proportion with respect to the head length is 18.6-20.7%HL in females and 18.1-21.3%HL in males.The fourth and fifth infraorbitals are narrow and sparsely extend over the hyomandibular bone (Fig. 7).The eye size in Salmo multipunctata and Salmo akairos is the largest in relation to other populations studied, and in Salmo multipunctata the horizontal diameter of the eye is 29.4-31.8%HL in females and 28.5-33.5% HL in males.

Salmo multipunctata
The supramaxilla is large and slender; its length is more than half of the maxilla.The maxilla is short and the proportion, with respect to the head length, is 46.3-53.2%SL in females and 48.9-52.3%SL in males.The upper jaw is deep as in the populations of the Ziz and Oum er Rbia river basins, except for the   The coloration is slightly yellow, lighter in the ventral area, and brownish in the dorsal region.The fins are slightly yellow with orange marginal borders of caudal and adipose fins.Dorsal, pelvic and anal fins in adults have white anterior edges.The dorsal fin presents a black line below the white mark.The dorsal fin has small black and red spots.Body with numerous small spots, which are black and slightly ocellated in the dorsal region and red spots (not ocellated) in the middle of the body.The iris has two black, conspicuous lateral spots and two less conspicuous above and below the iris.Parr marks are narrow, irregular and numerous, placed in the middle of body, sometimes divided into two marks.The species has a characteristic opercular black spot, and another spot just above it (usually ocellated).There are usually numerous black spots in the opercular area and the caudal fin.There are eight or nine black spots on the dorsal body, very conspicuous in juveniles, which tend to disappear in adults.

ETYMOLOGY:
The species name "multipunctata" refers to its diagnostic pigmentation pattern, which is constituted by numerous spots and marks along the body.

COMMON NAME:
We propose the English common name "Draa trout" for this species.
HABITAT AND BIOLOGY: Similar to other riverine trout of Morocco, this species inhabits small and shallow streams with clear and cold waters.No information about spawning season is available.

CONSERVATION:
The habitat of this species suffers catastrophic seasonal floods that influence dramatically the demography of the population.The continuous deforestation of the Atlas Mountains and the disappearance of the riparian vegetation, increase the adverse effect of the overflows over the small Atlas rivers.Overfishing is also a problem for a small population such as the one from the Dades River.For this reason, we suggest that this species should be included in the IUCN category of Endangered (EN).

GENETICS: Uncorrected genetic distances between
Salmo multipunctata and other studied populations of trout for the MT-CYTB gene ranged from 0.9 to 1.4%.Salmo multipunctata is phylogenetically basal to S. trutta from Europe and other Salmo populations from Morocco assigned to S. akairos, S. pellegrini and S. macrostigma.DIAGNOSIS: Differs from other known species of the genus Salmo from Morocco (except Salmo pallaryi) by the absence of spots or marks on the body.The species Salmo viridis has a lesser number of anal pterygophores (AP 10-11; Median=10) than Salmo pallaryi (AP 12-14; Median=13) and shorter orbital length.Horizontal orbital length with respect to the head length is 16-23% HL in Salmo viridis vs 25-31% HL in Salmo pallaryi.The following combination of characters are also diagnostic to Salmo viridis: Vomer teeth in one row, premaxilla extended laterally, supramaxilla short and deep, dorsal fin pterygophores from 11 to 13 (Median=12).One molecular autapomorphy in the MT-CYTB gene in the nucleotide position 508.
The supramaxilla is short and deep.The length of the maxilla is variable in this species and usually reaches the edge of the posterior eye orbit (Fig. 10).The length of the maxilla, in proportion and with respect to head length is 44.4-56.3%SL in females and 46.5-52.8%SL in males.The upper jaw is narrower than in other populations except that of Ifni Lake, which belongs to

S. akairos.
The maximum height of the upper jaw, in proportion to its length, is 28.4-33.1% UJL in females and 24.6-32.2%UJL in males.The eye orbit is small, in proportion to head length, the horizontal diameter being 17.5-19.4%HL in females and 15.6-22.8%HL in males and the vertical diameter being 16.4-19.8%HL in females and 13.8-20% HL in males (Fig. 10).The vomer teeth are placed in one row and are small and thin.
The coloration is uniformly grayish with some greenish iridizations, paler in the ventral region.The pectoral fins are slightly yellow and caudal, adipose and anal fins are greenish.No spot or marks are found, only some traces in caudal fin and dorsal skull.

ETYMOLOGY:
The species name "viridis" refers to its diagnostic greenish coloration in some parts of body and fins.
DISTRIBUTION: This new species is endemic to Isli Lake in the Atlas Mountains of Morocco.It is the only fish species inhabiting this lake.
COMMON NAME: We propose the English common name "green trout" for this species.
HABITAT AND BIOLOGY: This species inhabits the Isli Lake (Fig. 11).Isli is situated on a high plateau, 2270 meters above sea level.It is a large and deep (≈2 km of diameter, 92 m deep) oligotrophic lake.The geological origin of this Lake is under debate.Some authors point to its origin as a consequence of a meteorite impact that occurred approximately 40 000 years ago (Ibhi et al., 2013;Nachit et al., 2013), while others consider a tectonic origin during the Pleistocene period, similar to the one proposed for other Moroccan lakes (Chaabout et al., 2013).The presence of a natural population of Salmo viridis in Isli Lake is in agreement with the latter hypothesis.The spawning of this species takes place at the end of October and November in the sandy beaches of the lake.

CONSERVATION:
The habitat of this species is threatened due to eutrophication, related to intensive overpasture.Overfishing is also a problem due to the low protection of the lake.No juveniles have been observed in the last years and several malformations in fins and skull were observed in this population.For this reason, we suggest that this species should be included in the IUCN category of Endangered (EN).

GENETICS: Uncorrected genetic distances between
Salmo viridis and other studied populations of trout for the MT-CYTB gene ranged from 0.2 to 1 %.The

Fig. 4 .
Fig. 4.-Phylogenetic tree rendered by Bayesian inference based on the concatenated matrix of both mitochondrial genes (MT-CYTB+CR).Numbers on branches indicate posterior probability values.

Table 1 .
-Sampling localities for Salmo spp from Moroccan Atlantic basins and GenBank Accession numbers.Tabla 1.-Localidades de muestreo de las especies de Salmo spp de las cuencas atlánticas de Marruecos y números de acceso de GenBank.

Table 4 .
-Values of the means and between brackets maximum and minimum, to different ratios of the morphometric variables and scales count.Abbreviations are described in the Material and Methods epigraph.Tabla 4.-Valores de la media, y entre paréntesis valores máximos y mínimos, para diferentes proporciones de las variables morfométricas y número escamas.Las abreviaturas están descritas en el epígrafe de Material y Métodos.

Table 5 .
-Eigenvalues and eigenvectors for the first three principal components (PC1-PC3) from 26 morphometric variables for Salmo populations.Abbreviations are described in the Material and Methods epigraph.In bold, variables with the highest eigenvectors for each PC.

Table 6 .
-Non-parametric Kruskal-Wallis test and Mann-Whitney's post hoc comparisons for testing significant differences in pterygophores in dorsal fin (above diagonal) and anal fin (below diagonal) among Atlantic Moroccan trout populations.Significant values are presented in bold.Tabla 6.-Test no paramétrico de Kruskal-Wallis y comparaciones post hoc de Mann-Whitney para testar diferencias significativas en los pterigóforos de la aleta dorsal (sobre la diagonal) y de la aleta anal (debajo de la diagonal) entre las poblaciones atlánticas de truchas de Marruecos.Valores significativos se representan en negrita.

234 447 474 504 508 616 732 849 915 990 1090 Ifni
Numerous black, small spots placed at the upper side of the body and several red ocellated and non ocellated spots placed in middle of the body.Black spots sometimes extended to the opercular region and the caudal fin.Ethmoid bone with a characteristic T shape.Premaxilla bone with small and inclined upper process.
Paratypes: Table 9. MNCN 290751-290752, MNCN 290754-290785: Thirty-four specimens from the Dades River, Draa Basin, Aït Aatou O'Moussa (31.871776, -5.738205), Morocco; 21/10/2014.Collected by Doadrio, I; Yahyaoui, A; Garzón P; Perea, S. Holotype and a series of paratypes (35 specimens) have been deposited at the Fish Collection of the Museo Nacional de Ciencias Naturales, (Madrid, Spain).DIAGNOSIS: Differs from other known species of the genus Salmo in the numerous irregular narrow parr marks, usually ≥14, in the lateral line.Upper row of parr marks ≥10.Lower row of parr marks variable, usually ≥5.Two conspicuous black spots placed at the right and left sides of the iris and two less visible spots at the upper and lower sides of the iris.dard length.The body is slightly more elongated in comparison to other trout populations, with maximum body depth, at the level of the anal origin, ranging from 16.1-18.7%SL in females and 15.5-18.5% SL in males.