Bo Delling

Andrea Sabatini

Stéphane Muracciole

Christelle Tougard

Patrick Berrebi
email: patrick.berrebi@laposte.net

Stephane Muracciole

Stéphane Muracciole

Stephane Muracciole

Morphologic and genetic characterisation of Corsican and Sardinian trout with comments on Salmo taxonomy

Keywords: brown trout, Tyrrhenian Sea, morphology, meristics, mtDNA Caractérisation morphologique et génétique de la truite

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Résumé -La présente étude détaille et discute les données morphologiques et moléculaires des truites indigènes, Salmo sp. de Corse et de Sardaigne, ici appelée truites tyrrhéniennes. À titre de comparaison, les données morphologiques obtenues à partir de spécimens de musée, y compris S. macrostigma d'Algérie, sont discutées à la lumière des découvertes moléculaires récentes et nouvelles. Au total, 29 mesures et 20 caractères méristiques ont été considérés pour chaque spécimen. Parmi ces caractères méristiques, 12 ont été obtenus au moyen de rayons X. Un caractère morphométrique important dans la présente étude est la taille de la tête mesurée du prémaxillaire à la marge postérieure du préopercule. Ce caractère est particulièrement stable chez toutes les truites tyrrhéniennes, qui ont montré une tête relativement grande par rapport celle de la truite de l'Atlantique et de S. macrostigma. Au contraire, d'autres caractères comme les ponctuations du corps, les franges noires et blanches des nageoires, la profondeur du corps ou le nombre d'hypuraux dans le squelette caudal sont assez polymorphes. Pour certains caractères méristiques, la gamme de variation de la truite tyrrhénienne dépasse celle de tous les taxons comparés. Chaque truite a été génétiquement caractérisée et de nouveaux haplotypes de truite tyrrhénienne ont été découverts, appartenant à trois lignées mitochondriales à savoir les lignées adriatique, marbrée et méditerranéenne, les haplotypes adriatiques étant dominants. En combinant les données morphologiques et génétiques, il est montré que la morphologie n'a aucune corrélation évidente avec les lignées mitochondriales. D'autre part, la truite tyrrhénienne n'a aucune affinité particulière avec S. macrostigma d'Algérie.

Mots-clés: truite commune / mer Tyrrhénienne / morphologie / caractères méristique / ADNmt

Introduction

The taxonomic status of Eurasian trouts, i.e., all Salmo spp. except Atlantic salmon, Salmo salar, is revised continuously. Among molecular oriented ichthyologists, this part of Salmo diversity is most often referred to as brown trout Salmo trutta or "brown trout complex", whereas taxonomic oriented scientists, mainly focusing on morphology, continue to describe new species and recognizes at least 50 distinct taxa [START_REF] Delling | Systematics of the trouts endemic to Moroccan lakes, with description of a new species (Teleostei: Salmonidae)[END_REF]Sušnik et al., 2006Sušnik et al., , 2007;;Kottelat and Freyhof, 2007;[START_REF] Delling | Diversity of western and southern Balkan trouts, with the description of a new species from the Louros River, Greece (Teleostei: Salmonidae)[END_REF]Turan et al., 2009Turan et al., , 2011Turan et al., , 2012Turan et al., , 2014aTuran et al., , 2014bTuran et al., , 2017Turan et al., , 2020;;[START_REF] Doadrio | Two new species of Atlantic trout (Actinopterygii, Salmonidae) from Morocco[END_REF]Froese and Pauly, 2019).

The extensive molecular studies of the genus started mainly with allozymes (Ferguson and Mason, 1981;Karakousis and Triantaphyllidis, 1990;García-Marín et al., 1999;Berrebi et al., 2000a;Berrebi, 2015), later on shifting focus towards DNA sequencing mainly of mitochondrial origin (Giuffra et al., 1994;Apostolidis et al., 1997;Aurelle and Berrebi, 2001;Snoj et al., 2011). These studies led to numerous publications describing genetic variation in the genus. One of the most important findings within the taxonomic context is probably the proposed five main mitochondrial DNA (mtDNA) lineages within the brown trout complex, Atlantic (AT), Mediterranean (ME), Adriatic (AD), marble (MA) and Danubian (DA) (Bernatchez et al., 1992;Bernatchez, 2001). These lineages are augmented by geographically more limited lineages such as the Duero (DU) lineage (Suarez et al., 2001) and the Dades trout (Snoj et al., 2011), both close to AT, and the Tigris (TI) lineage (Bardakçi et al., 2006) close to DA. A recent analysis of a larger portion of the mtDNA allowed for a division of the AT lineage into two sister clades: a North African lineage (NA, in Morocco, Algeria and Sicily) and a well-known European AT lineage (Tougard et al., 2018).

In several cases, morphological and molecular data are correlated, strengthening hypotheses on taxa delimitation. In several cases, morphological and molecular data are correlated, strengthening hypotheses on taxa delimitation (Sanz, 2018). However, they sometimes disagree: for example, Salmo marmoratus, considered as very distinct in morphology [START_REF] Delling | Morphological distinction of marble trout, Salmo marmoratus, in comparison to marbled Salmo trutta from River Otra, Norway[END_REF], is also characterized by the MA mtDNA and the LDH-C1*(120) allozyme allele. However, MA haplotypes are also found in low frequencies outside the taxon (Bernatchez et al., 1992;Snoj et al., 2009;Pustovrh et al., 2011;Tougard et al., 2018) and the 120-allele is rare in Trebuscica River (Slovenia) otherwise pure and isolated S. marmoratus population (Berrebi et al., 2000b). Another example of marker disagreement is illustrated by S. obtusirostris. This species, while fixed for a unique and specific mtDNA haplotype in the Neretva River (Snoj et al., 2002), is fixed for the AD mtDNA lineage in Jadro River population. Other frequent kinds of contradictions have been observed, especially in the Balkans with numerous taxa sharing similar AD haplotypes (Sušnik et al., 2004(Sušnik et al., , 2006;;Snoj et al., 2010). These kinds of discrepancies may be explained by ancient introgression (Sušnik et al., 2007). Another explanation is the Dobzhansky-Muller model which accounts for cytonuclear incompatibilities (Burton and Barreto, 2012).

Despite the high number of more or less distinguishable taxa within the genus Salmo, large portions of its populations are not easily referred with accuracy to any existing taxon (Splendiani et al., 2019). This is partly due to lack of morphological data, lack of studies including both kinds of data and the fact that several tentatively valid taxa are poorly described lacking clear diagnoses (Kottelat and Freyhof, 2007). Within the native distribution of Salmo, a large part of its diversity is found in basins of the Tyrrhenian islands, Corsica, Sardinia and Sicily (Berrebi et al., 2019), and especially in Corsica where numerous differentiated indigenous populations still survive. Trout from Corsica and Sardinia, together with several other Mediterranean trouts, are often referred to as Salmo macrostigma [START_REF] Duméril | Note sur une truite d'Algérie (Salar macrostigma, A. Dum.)[END_REF])a species originally described from Algeria. The name macrostigma refers to the parr marks retained in adults [START_REF] Duméril | Note sur une truite d'Algérie (Salar macrostigma, A. Dum.)[END_REF]. This is a common feature in many Salmo spp. and may explain the broadened usage of this name, as applied to Corsican trout by Roule (1933) and to Sardinian trout at first by Boulenger (1901) also confirmed by Pomini (1941). Since that, Corsican trout have been characterized both for allozymes (Guyomard and Krieg, 1986;Berrebi, 1995), mtDNA (Bernatchez et al., 1992;Berrebi et al., 2019) and microsatellites (Berrebi et al., 2007(Berrebi et al., , 2019)), showing that they mainly belong to the AD lineage and possess the highly diagnostic allozyme allele LDH-4*(040). Morphological data on Corsican trout is so far restricted to pyloric caeca counts (Olivari and Brun, 1988;Guyomard, 1989) and the description of variation in color pattern among populations (Lascaux et al., 2010). In the same way, the non-introgressed Sardinian populations were characterized by only the AD lineage and allele LDH-C1 100/100 (Sabatini et al. 2018). Some authors describe, for the Sardinian populations, different haplotypes (Ad1, Ad2, Tyrrh1) with highly polymorphic characteristics accompanied by different phenotypes (Sabatini et al., 2011;Zaccara et al., 2015) Regarding distinctiveness of S. macrostigma sensu stricto, Tougard et al. (2018) analyzed complete mtDNA sequences from one syntype and one topotypic specimen and concluded they belonged to the NA lineage. In the same study, samples from Corsica and Sardinia were associated to AD, ME, MA or AT lineages. The present study is deliberately "cross-disciplinary", the main focus being to describe and discuss the Salmo diversity irrespective of different views on classification and taxonomy. Consequently, the use of different names, e.g. S. marmoratus or S. lourosensis only serve the purpose of pointing out a certain subset of trouts. Both molecular and morphological data are presented and discussed for indigenous Salmo sp. from Corsica and Sardinia, here called Tyrrhenian trout. Regarding comparison to Algerian S. macrostigma, morphological data obtained from museum specimens are also included and discussed in the light of recent molecular findings (Tougard et al., 2018).

Material and methods

Sampling

Contemporary specimens (N=38) from one Sardinian and six Corsican localities were captured in the wild by electrofishing, anesthetized with clove essence to death, sampled for tissue (fin clip in 95% ethanol), fixed in formalin 5%, and later transferred to ethanol prior to morphological analyses. The geographic positions of sampling stations are given in Fig. 1.

Tyrrhenian Salmo is referred to with an abbreviation of the stream, e.g. CAM for Camboni River in text, certain graphs and tables (Table 1). As detailed in Fig. 1, samples for genetic and morphological analyses are not exactly the same.

Comparative material includes different sets of Salmo spp. depending on analyses and the question of interest: distinction towards i) Salmo macrostigma from Algeria, ii) Atlantic basin Salmo trutta, iii) Salmo sp. from Spain. Comparative material for morphometry is restricted to specimens within standard length (SL) -range (116-208 mm), i.e., within the SL-range of Tyrrhenian trout samples. A description of contemporary and comparative material is given in (Delling, unpublished) is included for a broader comparison of head length within Salmo. A complete list of studied material is provided as supplementary information (Table S1).

Molecular methods

DNA was extracted from fin clips using the Chelex/proteinase K protocol described by Estoup et al. (1996). Three to six individuals have been considered by locality in the Tyrrhenian region, being or not the exact individuals included in morphological analyses.

Partial mtDNA control region (CR) was amplified by PCR using the PST and FST primers (Cortey and García-Marín, 2002). Each 50 μl reaction included 0.4 µM of each primer (Eurofins MWG Operon), 0.2 mM of dNTP (2 mM each), 2 mM of MgCl2 (25 mM), 10 μl of 5x PCR buffer, 1 U of Taq polymerase (GoTaq® Promega) and about 50 ng of genomic DNA. The conditions for PCR were an initial denaturation (95°C, 5 min) followed by 30 cycles of strand denaturation (94°C, 1 min), primer annealing (52°C, 1 min) and DNA extension (72°C, 1 min), then followed by a final extension (72°C, 5 min). All PCR amplifications were performed in Eppendorf Mastercycler thermocyclers. The amplified DNA fragments were run on a 0.8% agarose gel to verify the efficiency of amplification. The PCR products were purified and sequenced in both directions to confirm polymorphic sites by the Macrogen Company, Seoul, South Korea (https://dna.macrogen.com/) and the platform GenSeq of the Institut des Sciences de l'Evolution de Montpellier (Montpellier, France).

The sequences of CR were aligned together with reference haplotypes retrieved from GenBank, using MEGA v5.05 (Tamura et al., 2011). Haplotypes for the new sequences were generated with DnaSP v5.10.1b (Librado and Rozas, 2009). Haplotype relationships and distribution among populations were evaluated with a median-joining network (Bandelt et al., 1999) constructed with PopART (Leigh and Bryant, 2015). In order to assign a phylogenetic position to the seven contemporary samples (CAM, SPE, POZ, ESE, CAR, CHA and NIN), the network included published GenBank sequences of the lineages AT, ME, AD, MA, DA and NA, all belonging to the brown trout complex. [START_REF] Delling | Morphological variation in hybrids between Salmo marmoratus and alien Salmo species in the Volarja stream, Soca River basin, Slovenia[END_REF] and [START_REF] Delling | Morphological distinction of marble trout, Salmo marmoratus, in comparison to marbled Salmo trutta from River Otra, Norway[END_REF]. The length of the uppermost gill raker on the lower limb of the first gill arch (right side) was measured in situ using a pair of dividers. All other measurements were taken on the left side of the specimen with a digital calliper and rounded to the nearest 0.1 mm (Fig. 2). One important morphometric character in the present study is head length (HL) measured from tip of the snout to posterior margin of the operculum. However, the measurement that quantifies the size of the head more accurately is the distance from the premaxilla to the posterior margin of the preoperculum (No. 24 in Fig. 2). Below, the abbreviation HLpp is applied for that measurement.

Morphology methods

Methodology follows

The number of i) pored scales along the lateral line to the end of the caudal peduncle (left side), ii) scales in an oblique row from the base of the adipose fin backwards down to the lateral line including lateral line scales (left side), iii) gill rakers, including rudimentary elements, on lower and upper limbs of the first gill arch separately (right side), and iv) branchiostegal rays on both sides, were counted under a binocular dissection microscope.

The number of abdominal vertebrae, caudal vertebrae, pterygiophores supporting anal and dorsal fins, caudal fin upper and lower procurrent rays, and interneurals were taken from radiographs (Fig. 3). Rudimentary vertebrae in the caudal skeleton in addition to the three upturned vertebrae were not included in the counts. In cases of fused centra, the number of neural arches or spines was counted. The last abdominal vertebra is herein defined as the last one having ribs (sometimes rudimentary or missing) and/or having the haemal spine much shorter than in the consecutive first caudal vertebra. The positions of the dorsal and anal fins were estimated in relation to the vertebral column. The most strongly developed anterior pterygiophore was used as a marker of dorsal and anal fin position, respectively. Dorsal and anal fin pterygiophores do not articulate with neural and haemal spines, respectively, and in uncertain cases the lower value was chosen. The dorsal and anal fin positions are treated as meristic characters in statistical analyses. A membranous triangular bone sometimes present, located above the neural spine of the first vertebrae (Fig. 3), was not included in interneural counts.

Principal component analyses (PCA) on log transformed measurements and square rooted counts were used as an ordination method (Bookstein et al., 1985). Some informative meristic characters are summarised in frequency tables. The inclusion of 'soft' measurements, e.g. body width and body depth in analyses, depends on the state of preservation of specimens. PCAs were performed using SYSTAT 13. Colour pattern descriptions are most often restricted to contrasting markings: size, density and distribution of spots; presence of black and white leading edges of dorsal and anal fins; any other markings such as dark bars were also considered. In preserved material, light spots are interpreted as red spots based on personal observations: after transfer to ethanol, red spots disappear transformed into pale spots. Spots described as ocellated refer to spots enclosed by a light ring.

Results

Molecular results

Among the 38 contemporary specimens from Corsica and Sardinia, 34 CR sequences were obtained, corresponding to eight new haplotypes (Table 2). The alignment of CR sequences are 998 nucleotides long with 47 phylogenetically informative sites. These haplotypes are genetically very close (distant from each other by up to two mutations, Fig. 4) to 37 published GenBank haplotype sequences used as reference and illustrating the diversity on all the range of the S. trutta complex. A comprehensive network of these new and published haplotypes is presented Fig. 4. The sequences of the contemporary analyzed populations were all clustered into ME, AD and MA lineages, according to the reference sequences. AT, DA and NA lineages were represented only by GenBank sequences.

New haplotypes from Tyrrhenian trout were called ADcr2 to 6, MAcr1 and 2, and MEcr1 (Table 2). Three populations are characterized by one private haplotype each: NIN, (Corsica) with MEcr1; CAM (Sardinia) with ADcr2 and POZ (Corsica) with ADcr4. CAR, (Corsica) is characterized by a majority of MA private haplotypes (MAcr1 and 2) and one AD haplotype (ADcr5), while CHA (Corsica) is characterized by two AD haplotypes (ADcr3 and ADcr6). SPE and ESE shared ADcr3 with CHA.

Morphology

Morphometric data are given in Table 3, and meristic data are summarized in Tables 4 to 6 including extensive comparative material (Tables 1 andS1). Selected results from ordination by means of PCA are given for analyses focusing on variation between Tyrrhenian trout as a whole towards Atlantic basin S. trutta (Fig. 5). Corresponding character loadings are given in Supplementary Tables S2 andS3. The distinction of Tyrrhenian trout towards S. macrostigma is illustrated with a biplot (Fig. 6) focusing on the major morphological trait of the Tyrrhenian trout, viz. the longer head (HLpp) and slightly shorter caudal peduncle. The two Spanish specimens are included in all analyses and graphs. For discussion related to the comparatively large head in Tyrrhenian trout, HLpp is also presented as box plots in comparison to an extended number of Salmo samples (Fig. 7).

Variation among Tyrrhenian trout samples

At first glance (Figs. 8A to 8G), the Tyrrhenian trout resembles Atlantic basin S. trutta, i.e. rather strong jaws, numerous red and black spots, black and white edges of fins, most prominent in CAM, CAR, CHJ and SPE. Black spots are sometimes irregularly distributed, more or less aggregated along the flanks of the body (SPE, NIN) in contrast to, e.g. CAM having its spots more evenly distributed (Fig. 8A). There are large variations in meristic characters between different populations and the range of variation sometimes exceeds that of the extensive comparative material (Tables 4 to 6). The NIN-sample and the two Spanish specimens were not markedly different in multivariate statistics in comparison to the six remaining samples.

Four specimens (1 POZ, 3 ESE) were different in the number of epurals in the caudal skeleton, having three instead of two. All Salmo except S. salar have two, sometimes fused (anomaly) to one. Salmo salar is polymorphic but two is more common (see below).

Distinction of Tyrrhenian trout samples towards North Atlantic basin S. trutta

The rather strong jaws and a colour pattern with prominent black and white leading edges on the fins are shared between several populations of Atlantic basin S. trutta and the Tyrrhenian trouts. Dark, more or less ocellated, spots on flanks of the body are also common in both. However, some Tyrrhenian trout have their spots aggregated (Figs. 8F, 8G and p. 415 in Kottelat and Freyhof, 2007). The sample from Spain also possesses this uncommon pattern and was therefore especially highlighted in the comparative material. Sparsely or densely distributed, dark spots on caudal fin are also common in Tyrrhenian trout (Figs. 8B and 8G,CAM and SPE,respectively). Spots on caudal fins are rarely found in Atlantic basin S. trutta.

Multivariate statistics (Fig. 5) indicate distinction but not complete separation of Tyrrhenian trout from Atlantic S. trutta due to differences in vertebral counts and head size.

Distinction of Tyrrhenian trout samples towards S. macrostigma

The extensive variation in meristic characters in Tyrrhenian trout as a whole (Tables 4 to 6) covers the range of variation in S. macrostigma and limits the analyses to morphometric data. PCA (not shown) reveals that HLpp and caudal peduncle length are the two morphometric characters that distinguish them best (Fig. 6). The Tyrrhenian trout has longer head (HLpp) and slightly shorter caudal peduncle compared to S. macrostigma (Table 3).

Discussion

Genetic diversity: a strong differentiation pattern

According to results from previous (Tougard et al., 2018;Berrebi et al., 2019) and present studies, the Tyrrhenian trout is mainly characterized by an island specific mtDNAradiation within the AD lineage, as well as, to a lesser degree, by other lineages (AT, ME and MA). The recently described NA lineage is also naturally present in Sicily. Thus, within a rather limited and nowadays isolated region in the south center of Salmo distribution, a comparatively high number of mtDNA lineages (four of the five recognizable major lineages) occur naturally. It is also striking that a majority of the haplotypes recovered in the present study were new (Table 2) despite more than two decades of CR sequencing in Salmo. The presence of the ME lineage in the NIN sample, also observed in Corsica by Tougard et al. (2018) and in other Tyrrhenian samples not included in the morphological analyses, is explained by ancient introgressions evidenced elsewhere using nuclear markers (Berrebi et al., 2007;Berrebi, 2015). It shows that possible secondary contacts must have occurred, according to the post-glacial invasion of Corsica hypothesis (Gauthier and Berrebi, 2007).

Moreover, the presence of MA lineage in some isolated Corsican rivers including CAR and the range of distribution of the NA lineage (Morocco, Algeria, Sicily: Tougard et al., 2018;Berrebi et al., 2019) demonstrates the multiple unknown events of migrations, invasions and hybridizations which complicate the trout genetic pattern in the Tyrrhenian region. Finally, the presence of several northern AT haplotypes recorded in the Tyrrhenian trout (Tougard et al., 2018;Berrebi et al., 2019) is due to stocking with commercial AT hatchery strains.

Nuclear markers (microsatellites) have also shown that the Tyrrhenian trouts exhibit exceptionally differentiated genotypes, at a continental-like level, within the two small sampled islands, but especially in Corsica where numerous autochthonous isolated small populations still survive (Berrebi et al., 2019). This strong differentiation among neighboring rivers is typical of dry Mediterranean mountainous regions, never frozen by glaciation and providing way for migration [START_REF] Apostolidis | Genetic structure and demographic history of brown trout (Salmo trutta) populations from the southern Balkans[END_REF]Berrebi et al., 2019).

Morphological diversity in light of genetic diversity

Regarding the strong morphological diversification between studied populations of Tyrrhenian trout, one explanation may involve random effects. Berrebi et al. (2019) showed very low levels of genetic variation within populations in Corsican streams based on microsatellites, suggesting small population sizes and repeated bottleneck events.

Hypothetically, the frequent (c. 10 %), occurrence of three epurals in Tyrrhenian trout compared to c. 0.1 % in comparative material might be a result of genetic drift accelerated by bottlenecks. Three epurals in the caudal skeleton are typical for, e.g. most Pacific trouts and salmons (Oncorhynchus), graylings (Thymallus) and whitefishes (Coregonus) (Norden, 1961;Stearley and Smith, 1993) and appear to be the ancestral state also retained as a polymorphism in S. salar with 12 out of 40 studied specimens having three. [START_REF] Delling | Systematics of the trouts endemic to Moroccan lakes, with description of a new species (Teleostei: Salmonidae)[END_REF] also described a situation with a seemingly plesiomorphic condition in rostrodermethmodid bone in the lake endemic S. pallaryi from Lake Sidi Ali, Morocco, not recorded elsewhere in Salmo. The genetic characteristics of this extinct trout is unknown but it is likely that these kinds of reversals approaching morphological anomalies may occur under certain conditions involving random processes in temporarily small populations.

Comparing Tyrrhenian samples to other Salmo spp., it is tempting to search for a pattern connecting certain characters to certain mtDNA lineages. However, ancient introgression in certain populations without strong impacts on morphology seems rather to be the "rule" in many salmonids and other taxa (Martinez et al., 2009;Gratton et al., 2013;Lerceteau-Köhler et al., 2013;Berrebi et al., 2017). The CAR sample also possessing the MA mtDNA lineage shows no typical S. marmoratus characters, e.g. marbled color pattern, high vertebral counts or a hypethmoid bone embedded in the rostral cartilage. In contrast, more recent hybrids involving S. marmoratus show a variable but, overall, intermediate phenotype [START_REF] Delling | Morphological variation in hybrids between Salmo marmoratus and alien Salmo species in the Volarja stream, Soca River basin, Slovenia[END_REF]. Prominent black and white leading edges on fins in several Tyrrhenian populations are similar to Atlantic basin S. trutta and could tentatively be regarded as ancient traces of the AT lineage. This pattern is also present in some North African trout, e.g. S. akairos and S. macrostigma belonging to the NA lineage, close to the AT one (Tougard et al., 2018). However, neither NA nor AT lineages have so far been reported from Corsica and Sardinia, except AT lineages of hatchery origin.

Despite the complex pattern of migrations, hybridizations and genetic radiation in the

Tyrrhenian trout history, also giving rise to rather morphologically distinct populations in Corsica and Sardinia (Figs. 8A to 8G), they all share a comparatively large head (Fig. 6 and7). This contradictory pattern of morphological homogeneity for certain characters opposed to strong differentiation in others take probably account of the genetic data in the search for possible explanations. Turning focus to the overall large head in Tyrrhenian trout, it is noticeable that this character varies extensively within and between different kinds of trouts (Fig. 7). It should be noted that sample sizes vary considerably (Fig. 7) but several of them can be characterized as typically small-headed, e.g. S. salar, S. obtusirostris and S. lourosensis. Next after the Tyrrhenian trouts, the two North African lake trouts endemic from Morocco, S. akairos and S. pallaryi, together with S. marmoratus and S. dentex inhabiting Adriatic basin drainages, possess comparatively large heads. Thus, there is no obvious correlation between size of head and habitat and/or lifestyle, e.g. rapid streams vs. lakes, and it is fully possible that the large head is a result of a founder effect during ancient establishment on the islands. However, body proportions can be affected by, e.g. growth rate. Barlow (1961) refers to studies where it was shown that head length was smaller in faster growing rainbow trout, Oncorhynchus mykiss. Thus, the comparatively large head in Tyrrhenian trout can be a consequence of slow growth under harsh condition, or a consequence of earlier sexual maturity. However, Pankhurst and Montgomery (1994) showed, also for O. mykiss that retarded growth results in larger eyes. The Tyrrhenian trout possesses on the average slightly smaller eyes compared to Atlantic S. trutta (Table 3) and consequently retarded growth seems not to be a likely explanation for the large head.

The two Spanish specimens, referred to as Salmo sp. MNHN 1920 228-229, possess a color pattern with irregularly distributed spots, also found in some Tyrrhenian trouts, and are comparatively large headed (Fig. 6). They provide an example of what a hypothetical ancestor could have looked like and, if the interpretation of locality information (Ebro basin) is correct, it makes sense as the basin is dominated by AD haplotypes (Cortey et al., 2004). Also, the haplotype ADcs13 found in Ebro is very close to the Tyrrhenian AD haplotypes (Fig. 4).

Conclusions

Data presented herein suggest that within the rather unresolved Salmo complex in the Mediterranean region, we may start to perceive a kind of large headed trout. However, more populations from the islands and surrounding mainland (France, Spain, and Italy) need to be studied to survey the distribution of this morphology. It would be a large step forward if this kind of trout could get an identity, i.e. a scientific name to balance a perhaps too broad or erroneous usage of names such as S. trutta and S. macrostigma in the Mediterranean region. This long-term work already began with several recent molecular papers (Sanz, 2018;Tougard et al., 2018;Berrebi et al., 2019) and the present study. The Tyrrhenian trout studied here are left without a taxonomic identity but it is clearly demonstrated, in line with molecular data (Tougard et al., 2018), that they show no particular affinity to S. macrostigma once described from Algeria.

Acknowledgements

We thank Douglas Jones at the Institute of Freshwater Research, Drottningholm, for improving the English. This work was realized with the support of LabEx CeMEB, an ANR "Investissements d'avenir" program (ANR-10-LABX-04-01).

Apostolidis AP, Madeira M-J, Hansen MM, Machordom A. 2008. Genetic structure and demographic history of brown trout (Salmo trutta) populations from the southern Balkans. Freshw Biol 53: 1555-1566. Fig. 2. Measurements taken on Salmo specimens; 1, upper jaw depth, as largest depth of the maxilla and supramaxilla; 2, snout length, from symphysis of premaxilla to osseous orbit margin; 3, orbital horizontal diameter, between osseous orbital margin; 4, head depth, just posterior to orbit; 5, orbital vertical diameter, between osseous orbital margin; 6, length of maxilla, from premaxilla end to posterior end of maxilla; 7, upper jaw length, from symphysis of premaxilla to posterior end of maxilla; 8, lower jaw length, from symphysis of dentary to retroarticular; 9, pectoral fin length, from base of first ray to tip of longest ray; 10, body depth, at level of origin of dorsal fin; 11, dorsal fin length, from base to tip of longest ray; 12, pelvic fin length, from base of first ray to tip of longest ray; 13, body depth, at level of origin of anal fin; 14, adipose fin length, from origin to tip; 15, anal fin length, from base of first ray to tip of longest ray; 16, caudal peduncle length, from end of anal fin to middle base of caudal fin; 17, least depth of caudal peduncle; 18, length of upper caudal fin lobe, from base to tip of longest ray; 19, length of middle caudal fin ray, from base to tip of shortest ray; 20, length of lower caudal fin lobe, from base to tip of longest ray; 21 standard length (SL), from upper jaw symphysis to middle base of caudal fin; 22, predorsal length from upper jaw symphysis to origin of dorsal fin; 23, head length, from upper jaw symphysis to posterior tip of operculum; 24, premaxilla to preoperculum length, from premaxilla end of maxilla to posterior margin of preoperculum (HLpp); 25, prepelvic length, from upper jaw symphysis to origin of pelvic fin; 26, preanal length, from upper jaw symphysis to origin of anal fin; 27, interorbital width, transverse at narrowest part of skull; 28, body width, transverse at widest part of body at level of dorsal fin origin, above abdominal cavity. Haplotypes are indicated by numbers as given in Table 2. Black circles are for nodes, and hatch marks are for mutation steps. 1. Additional samples given in capital letters (Delling, unpublished) refer to rivers or streams in the given regions (Table S1). Ezenam is a lake in Daghestan and labrax represents trout from Black Sea basin with an anadromous silvery and slender appearance. The taxonomic status of Eurasian trouts, i.e., all Salmo spp. except Atlantic salmon, Salmo salar, is revised continuously. Among molecular oriented ichthyologists, this part of Salmo diversity is most often referred to as brown trout Salmo trutta or "brown trout complex", whereas taxonomic oriented scientists, mainly focusing on morphology, continue to describe new species and recognizes at least 50 distinct taxa [START_REF] Delling | Systematics of the trouts endemic to Moroccan lakes, with description of a new species (Teleostei: Salmonidae)[END_REF]Sušnik et al., 2006Sušnik et al., , 2007;;Kottelat and Freyhof, 2007;[START_REF] Delling | Diversity of western and southern Balkan trouts, with the description of a new species from the Louros River, Greece (Teleostei: Salmonidae)[END_REF]Turan et al., 2009Turan et al., , 2011Turan et al., , 2012Turan et al., , 2014aTuran et al., , 2014bTuran et al., , 2017Turan et al., , 2020;;[START_REF] Doadrio | Two new species of Atlantic trout (Actinopterygii, Salmonidae) from Morocco[END_REF]Froese and Pauly, 2019).

Figures captions

The extensive molecular studies of the genus started mainly with allozymes (Ferguson and Mason, 1981;Karakousis and Triantaphyllidis, 1990;García-Marín et al., 1999;Berrebi et al., 2000a;Berrebi, 2015), later on shifting focus towards DNA sequencing mainly of mitochondrial origin (Giuffra et al., 1994;Apostolidis et al., 1997;Aurelle and Berrebi, 2001;Snoj et al., 2011). These studies led to numerous publications describing genetic variation in the genus. One of the most important findings within the taxonomic context is probably the proposed five main mitochondrial DNA (mtDNA) lineages within the brown trout complex, Atlantic (AT), Mediterranean (ME), Adriatic (AD), marble (MA) and Danubian (DA) (Bernatchez et al., 1992;Bernatchez, 2001). These lineages are augmented by geographically more limited lineages such as the Duero (DU) lineage (Suarez et al., 2001) and the Dades trout (Snoj et al., 2011), both close to AT, and the Tigris (TI) lineage (Bardakçi et al., 2006) close to DA. A recent analysis of a larger portion of the mtDNA allowed for a division of the AT lineage into two sister clades: a North African lineage (NA, in Morocco, Algeria and Sicily) and a well-known European AT lineage (Tougard et al., 2018).

In several cases, morphological and molecular data are correlated, strengthening hypotheses on taxa delimitation. In several cases, morphological and molecular data are correlated, strengthening hypotheses on taxa delimitation (Sanz, 2018). However, they sometimes disagree: for example, Salmo marmoratus, considered as very distinct in morphology [START_REF] Delling | Morphological distinction of marble trout, Salmo marmoratus, in comparison to marbled Salmo trutta from River Otra, Norway[END_REF], is also characterized by the MA mtDNA and the LDH-C1*(120) allozyme allele. However, MA haplotypes are also found in low frequencies outside the taxon (Bernatchez et al., 1992;Snoj et al., 2009;Pustovrh et al., 2011;Tougard et al., 2018) and the 120-allele is rare in Trebuscica River (Slovenia) otherwise pure and isolated S. marmoratus population (Berrebi et al., 2000b). Another example of marker disagreement is illustrated by S. obtusirostris. This species, while fixed for a unique and specific mtDNA haplotype in the Neretva River (Snoj et al., 2002), is fixed for the AD mtDNA lineage in Jadro River population. Other frequent kinds of contradictions have been observed, especially in the Balkans with numerous taxa sharing similar AD haplotypes (Sušnik et al., 2004(Sušnik et al., , 2006;;Snoj et al., 2010). These kinds of discrepancies may be explained by ancient introgression (Sušnik et al., 2007). Another explanation is the Dobzhansky-Muller model which accounts for cytonuclear incompatibilities (Burton and Barreto, 2012).

Despite the high number of more or less distinguishable taxa within the genus Salmo, large portions of its populations are not easily referred with accuracy to any existing taxon (Splendiani et al., 2019). This is partly due to lack of morphological data, lack of studies including both kinds of data and the fact that several tentatively valid taxa are poorly described lacking clear diagnoses (Kottelat and Freyhof, 2007). Within the native distribution of Salmo, a large part of its diversity is found in basins of the Tyrrhenian islands, Corsica, Sardinia and Sicily (Berrebi et al., 2019), and especially in Corsica where numerous differentiated indigenous populations still survive. Trout from Corsica and Sardinia, together with several other Mediterranean trouts, are often referred to as Salmo macrostigma [START_REF] Duméril | Note sur une truite d'Algérie (Salar macrostigma, A. Dum.)[END_REF])a species originally described from Algeria. The name macrostigma refers to the parr marks retained in adults [START_REF] Duméril | Note sur une truite d'Algérie (Salar macrostigma, A. Dum.)[END_REF]. This is a common feature in many Salmo spp. and may explain the broadened usage of this name, as applied to Corsican trout by Roule (1933) and to Sardinian trout at first by Boulenger (1901) also confirmed by Pomini (1941). Since that, Corsican trout have been characterized both for allozymes (Guyomard and Krieg, 1986;Berrebi, 1995), mtDNA (Bernatchez et al., 1992;Berrebi et al., 2019) and microsatellites (Berrebi et al., 2007(Berrebi et al., , 2019)), showing that they mainly belong to the AD lineage and possess the highly diagnostic allozyme allele LDH-4*(040). Morphological data on Corsican trout is so far restricted to pyloric caeca counts (Olivari and Brun, 1988;Guyomard, 1989) and the description of variation in color pattern among populations (Lascaux et al., 2010). In the same way, the non-introgressed Sardinian populations were characterized by only the AD lineage and allele LDH-C1 100/100 (Sabatini et al. 2018). Some authors describe, for the Sardinian populations, different haplotypes (Ad1, Ad2, Tyrrh1) with highly polymorphic characteristics accompanied by different phenotypes (Sabatini et al., 2011;Zaccara et al., 2015) Regarding distinctiveness of S. macrostigma sensu stricto, Tougard et al. (2018) analyzed complete mtDNA sequences from one syntype and one topotypic specimen and concluded they belonged to the NA lineage. In the same study, samples from Corsica and Sardinia were associated to AD, ME, MA or AT lineages. The present study is deliberately "cross-disciplinary", the main focus being to describe and discuss the Salmo diversity irrespective of different views on classification and taxonomy. Consequently, the use of different names, e.g. S. marmoratus or S. lourosensis only serve the purpose of pointing out a certain subset of trouts. Both molecular and morphological data are presented and discussed for indigenous Salmo sp. from Corsica and Sardinia, here called Tyrrhenian trout. Regarding comparison to Algerian S. macrostigma, morphological data obtained from museum specimens are also included and discussed in the light of recent molecular findings (Tougard et al., 2018).

Comparative material in addition to that in Table 1 (Delling, unpublished) is included for a broader comparison of head length within Salmo. A complete list of studied material is provided as supplementary information (Table S1).

Molecular methods

DNA was extracted from fin clips using the Chelex/proteinase K protocol described by Estoup et al. (1996). Three to six individuals have been considered by locality in the Tyrrhenian region, being or not the exact individuals included in morphological analyses.

Partial mtDNA control region (CR) was amplified by PCR using the PST and FST primers (Cortey and García-Marín, 2002). Each 50 μl reaction included 0.4 µM of each primer (Eurofins MWG Operon), 0.2 mM of dNTP (2 mM each), 2 mM of MgCl2 (25 mM), 10 μl of 5x PCR buffer, 1 U of Taq polymerase (GoTaq® Promega) and about 50 ng of genomic DNA. The conditions for PCR were an initial denaturation (95°C, 5 min) followed by 30 cycles of strand denaturation (94°C, 1 min), primer annealing (52°C, 1 min) and DNA extension (72°C, 1 min), then followed by a final extension (72°C, 5 min). All PCR amplifications were performed in Eppendorf Mastercycler thermocyclers. The amplified DNA fragments were run on a 0.8% agarose gel to verify the efficiency of amplification. The PCR products were purified and sequenced in both directions to confirm polymorphic sites by the Macrogen Company, Seoul, South Korea (https://dna.macrogen.com/) and the platform GenSeq of the Institut des Sciences de l'Evolution de Montpellier (Montpellier, France).

The sequences of CR were aligned together with reference haplotypes retrieved from GenBank, using MEGA v5.05 (Tamura et al., 2011). Haplotypes for the new sequences were generated with DnaSP v5.10.1b (Librado and Rozas, 2009). Haplotype relationships and distribution among populations were evaluated with a median-joining network (Bandelt et al., 1999) constructed with PopART (Leigh and Bryant, 2015). In order to assign a phylogenetic position to the seven contemporary samples (CAM, SPE, POZ, ESE, CAR, CHA and NIN), the network included published GenBank sequences of the lineages AT, ME, AD, MA, DA and NA, all belonging to the brown trout complex. [START_REF] Delling | Morphological variation in hybrids between Salmo marmoratus and alien Salmo species in the Volarja stream, Soca River basin, Slovenia[END_REF] and Delling (2002). The length of the uppermost gill raker on the lower limb of the first gill arch (right side) was measured in situ using a pair of dividers. All other measurements were taken on the left side of the specimen with a digital calliper and rounded to the nearest 0.1 mm (Fig. 2). One important morphometric character in the present study is head length (HL) measured from tip of the snout to posterior margin of the operculum. However, the measurement that quantifies the size of the head more accurately is the distance from the premaxilla to the posterior margin of the preoperculum (No. 24 in Fig. 2). Below, the abbreviation HLpp is applied for that measurement.

Morphology methods

Methodology follows

The number of i) pored scales along the lateral line to the end of the caudal peduncle (left side), ii) scales in an oblique row from the base of the adipose fin backwards down to the lateral line including lateral line scales (left side), iii) gill rakers, including rudimentary elements, on lower and upper limbs of the first gill arch separately (right side), and iv) branchiostegal rays on both sides, were counted under a binocular dissection microscope.

The number of abdominal vertebrae, caudal vertebrae, pterygiophores supporting anal and dorsal fins, caudal fin upper and lower procurrent rays, and interneurals were taken from radiographs (Fig. 3). Rudimentary vertebrae in the caudal skeleton in addition to the three upturned vertebrae were not included in the counts. In cases of fused centra, the number of neural arches or spines was counted. The last abdominal vertebra is herein defined as the last one having ribs (sometimes rudimentary or missing) and/or having the haemal spine much shorter than in the consecutive first caudal vertebra. The positions of the dorsal and anal fins were estimated in relation to the vertebral column. The most strongly developed anterior pterygiophore was used as a marker of dorsal and anal fin position, respectively. Dorsal and anal fin pterygiophores do not articulate with neural and haemal spines, respectively, and in uncertain cases the lower value was chosen. The dorsal and anal fin positions are treated as meristic characters in statistical analyses. A membranous triangular bone sometimes present, located above the neural spine of the first vertebrae (Fig. 3), was not included in interneural counts.

Principal component analyses (PCA) on log transformed measurements and square rooted counts were used as an ordination method (Bookstein et al., 1985). Some informative meristic characters are summarised in frequency tables. The inclusion of 'soft' measurements, e.g. body width and body depth in analyses, depends on the state of preservation of specimens. PCAs were performed using SYSTAT 13. Colour pattern descriptions are most often restricted to contrasting markings: size, density and distribution of spots; presence of black and white leading edges of dorsal and anal fins; any other markings such as dark bars were also considered. In preserved material, light spots are interpreted as red spots based on personal observations: after transfer to ethanol, red spots disappear transformed into pale spots. Spots described as ocellated refer to spots enclosed by a light ring.

Results

Molecular results

Among the 38 contemporary specimens from Corsica and Sardinia, 34 CR sequences were obtained, corresponding to eight new haplotypes (Table 2)

Morphology

Morphometric data are given in Table 3, and meristic data are summarized in Tables 4 to 6 including extensive comparative material (Tables 1 andS1). Selected results from ordination by means of PCA are given for analyses focusing on variation between Tyrrhenian trout as a whole towards Atlantic basin S. trutta (Fig. 5). Corresponding character loadings are given in Supplementary Tables S2 andS3. The distinction of Tyrrhenian trout towards S. macrostigma is illustrated with a biplot (Fig. 6) focusing on the major morphological trait of the Tyrrhenian trout, viz. the longer head (HLpp) and slightly shorter caudal peduncle. The two Spanish specimens are included in all analyses and graphs. For discussion related to the comparatively large head in Tyrrhenian trout, HLpp is also presented as box plots in comparison to an extended number of Salmo samples (Fig. 7).

Variation among Tyrrhenian trout samples

At first glance (Figs. 8A to 8G), the Tyrrhenian trout resembles Atlantic basin S. trutta, i.e. rather strong jaws, numerous red and black spots, black and white edges of fins, most prominent in CAM, CAR, CHJ and SPE. Black spots are sometimes irregularly distributed, more or less aggregated along the flanks of the body (SPE, NIN) in contrast to, e.g. CAM having its spots more evenly distributed (Fig. 8A). There are large variations in meristic characters between different populations and the range of variation sometimes exceeds that of the extensive comparative material (Tables 4 to 6). The NIN-sample and the two Spanish specimens were not markedly different in multivariate statistics in comparison to the six remaining samples.

Four specimens (1 POZ, 3 ESE) were different in the number of epurals in the caudal skeleton, having three instead of two. All Salmo except S. salar have two, sometimes fused (anomaly) to one. Salmo salar is polymorphic but two is more common (see below).

Distinction of Tyrrhenian trout samples towards North Atlantic basin S. trutta

The rather strong jaws and a colour pattern with prominent black and white leading edges on the fins are shared between several populations of Atlantic basin S. trutta and the Tyrrhenian trouts. Dark, more or less ocellated, spots on flanks of the body are also common in both.

However, some Tyrrhenian trout have their spots aggregated (Figs. 8F, 8G and p. 415 in Kottelat and Freyhof, 2007). The sample from Spain also possesses this uncommon pattern and was therefore especially highlighted in the comparative material. Sparsely or densely distributed, dark spots on caudal fin are also common in Tyrrhenian trout (Figs. 8B and8G, CAM and SPE, respectively). Spots on caudal fins are rarely found in Atlantic basin S. trutta.

Multivariate statistics (Fig. 5) indicate distinction but not complete separation of Tyrrhenian trout from Atlantic S. trutta due to differences in vertebral counts and head size.

Distinction of Tyrrhenian trout samples towards S. macrostigma

The extensive variation in meristic characters in Tyrrhenian trout as a whole (Tables 4 to 6) covers the range of variation in S. macrostigma and limits the analyses to morphometric data. PCA (not shown) reveals that HLpp and caudal peduncle length are the two morphometric characters that distinguish them best (Fig. 6). The Tyrrhenian trout has longer head (HLpp) and slightly shorter caudal peduncle compared to S. macrostigma (Table 3).

Discussion

Genetic diversity: a strong differentiation pattern

According to results from previous (Tougard et al., 2018;Berrebi et al., 2019) and present studies, the Tyrrhenian trout is mainly characterized by an island specific mtDNAradiation within the AD lineage, as well as, to a lesser degree, by other lineages (AT, ME and MA). The recently described NA lineage is also naturally present in Sicily. Thus, within a rather limited and nowadays isolated region in the south center of Salmo distribution, a comparatively high number of mtDNA lineages (four of the five recognizable major lineages) occur naturally. It is also striking that a majority of the haplotypes recovered in the present study were new (Table 2) despite more than two decades of CR sequencing in Salmo. The presence of the ME lineage in the NIN sample, also observed in Corsica by Tougard et al. (2018) and in other Tyrrhenian samples not included in the morphological analyses, is explained by ancient introgressions evidenced elsewhere using nuclear markers (Berrebi et al., 2007;Berrebi, 2015). It shows that possible secondary contacts must have occurred, according to the post-glacial invasion of Corsica hypothesis (Gauthier and Berrebi, 2007).

Moreover, the presence of MA lineage in some isolated Corsican rivers including CAR and the range of distribution of the NA lineage (Morocco, Algeria, Sicily: Tougard et al., 2018;Berrebi et al., 2019) demonstrates the multiple unknown events of migrations, invasions and hybridizations which complicate the trout genetic pattern in the Tyrrhenian region. Finally, the presence of several northern AT haplotypes recorded in the Tyrrhenian trout (Tougard et al., 2018;Berrebi et al., 2019) is due to stocking with commercial AT hatchery strains.

Nuclear markers (microsatellites) have also shown that the Tyrrhenian trouts exhibit exceptionally differentiated genotypes, at a continental-like level, within the two small sampled islands, but especially in Corsica where numerous autochthonous isolated small populations still survive (Berrebi et al., 2019). This strong differentiation among neighboring rivers is typical of dry Mediterranean mountainous regions, never frozen by glaciation and providing way for migration [START_REF] Apostolidis | Genetic structure and demographic history of brown trout (Salmo trutta) populations from the southern Balkans[END_REF]Berrebi et al., 2019).

Morphological diversity in light of genetic diversity

Regarding the strong morphological diversification between studied populations of Tyrrhenian trout, one explanation may involve random effects. Berrebi et al. (2019) showed very low levels of genetic variation within populations in Corsican streams based on microsatellites, suggesting small population sizes and repeated bottleneck events.

Hypothetically, the frequent (c. 10 %), occurrence of three epurals in Tyrrhenian trout compared to c. 0.1 % in comparative material might be a result of genetic drift accelerated by bottlenecks. Three epurals in the caudal skeleton are typical for, e.g. most Pacific trouts and salmons (Oncorhynchus), graylings (Thymallus) and whitefishes (Coregonus) (Norden, 1961;Stearley and Smith, 1993) and appear to be the ancestral state also retained as a polymorphism in S. salar with 12 out of 40 studied specimens having three. [START_REF] Delling | Systematics of the trouts endemic to Moroccan lakes, with description of a new species (Teleostei: Salmonidae)[END_REF] also described a situation with a seemingly plesiomorphic condition in rostrodermethmodid bone in the lake endemic S. pallaryi from Lake Sidi Ali, Morocco, not recorded elsewhere in Salmo. The genetic characteristics of this extinct trout is unknown but it is likely that these kinds of reversals approaching morphological anomalies may occur under certain conditions involving random processes in temporarily small populations.

Comparing Tyrrhenian samples to other Salmo spp., it is tempting to search for a pattern connecting certain characters to certain mtDNA lineages. However, ancient introgression in certain populations without strong impacts on morphology seems rather to be the "rule" in many salmonids and other taxa (Martinez et al., 2009;Gratton et al., 2013;Lerceteau-Köhler et al., 2013;Berrebi et al., 2017). The CAR sample also possessing the MA mtDNA lineage shows no typical S. marmoratus characters, e.g. marbled color pattern, high vertebral counts or a hypethmoid bone embedded in the rostral cartilage. In contrast, more recent hybrids involving S. marmoratus show a variable but, overall, intermediate phenotype [START_REF] Delling | Morphological variation in hybrids between Salmo marmoratus and alien Salmo species in the Volarja stream, Soca River basin, Slovenia[END_REF]. Prominent black and white leading edges on fins in several Tyrrhenian populations are similar to Atlantic basin S. trutta and could tentatively be regarded as ancient traces of the AT lineage. This pattern is also present in some North African trout, e.g. S. akairos and S. macrostigma belonging to the NA lineage, close to the AT one (Tougard et al., 2018). However, neither NA nor AT lineages have so far been reported from Corsica and Sardinia, except AT lineages of hatchery origin.

Despite the complex pattern of migrations, hybridizations and genetic radiation in the Tyrrhenian trout history, also giving rise to rather morphologically distinct populations in Corsica and Sardinia (Figs. 8A to 8G), they all share a comparatively large head (Fig. 6 and7). This contradictory pattern of morphological homogeneity for certain characters opposed to strong differentiation in others take probably account of the genetic data in the search for possible explanations. Turning focus to the overall large head in Tyrrhenian trout, it is noticeable that this character varies extensively within and between different kinds of trouts (Fig. 7). It should be noted that sample sizes vary considerably (Fig. 7) but several of them can be characterized as typically small-headed, e.g. S. salar, S. obtusirostris and S. lourosensis. Next after the Tyrrhenian trouts, the two North African lake trouts endemic from Morocco, S. akairos and S. pallaryi, together with S. marmoratus and S. dentex inhabiting Adriatic basin drainages, possess comparatively large heads. Thus, there is no obvious correlation between size of head and habitat and/or lifestyle, e.g. rapid streams vs. lakes, and it is fully possible that the large head is a result of a founder effect during ancient establishment on the islands. However, body proportions can be affected by, e.g. growth rate. Barlow (1961) refers to studies where it was shown that head length was smaller in faster growing rainbow trout, Oncorhynchus mykiss. Thus, the comparatively large head in Tyrrhenian trout can be a consequence of slow growth under harsh condition, or a consequence of earlier sexual maturity. However, Pankhurst and Montgomery (1994) showed, also for O. mykiss that retarded growth results in larger eyes. The Tyrrhenian trout possesses on the average slightly smaller eyes compared to Atlantic S. trutta (Table 3) and consequently retarded growth seems not to be a likely explanation for the large head.

The two Spanish specimens, referred to as Salmo sp. MNHN 1920 228-229, possess a color pattern with irregularly distributed spots, also found in some Tyrrhenian trouts, and are comparatively large headed (Fig. 6). They provide an example of what a hypothetical ancestor could have looked like and, if the interpretation of locality information (Ebro basin) is correct, it makes sense as the basin is dominated by AD haplotypes (Cortey et al., 2004). Also, the haplotype ADcs13 found in Ebro is very close to the Tyrrhenian AD haplotypes (Fig. 4).

Conclusions

Data presented herein suggest that within the rather unresolved Salmo complex in the Mediterranean region, we may start to perceive a kind of large headed trout. However, more populations from the islands and surrounding mainland (France, Spain, and Italy) need to be studied to survey the distribution of this morphology. It would be a large step forward if this kind of trout could get an identity, i.e. a scientific name to balance a perhaps too broad or erroneous usage of names such as S. trutta and S. macrostigma in the Mediterranean region. This long-term work already began with several recent molecular papers (Sanz, 2018;Tougard et al., 2018;Berrebi et al., 2019) and the present study. The Tyrrhenian trout studied here are left without a taxonomic identity but it is clearly demonstrated, in line with molecular data (Tougard et al., 2018), that they show no particular affinity to S. macrostigma once described from Algeria. Fig. 2. Measurements taken on Salmo specimens; 1, upper jaw depth, as largest depth of the maxilla and supramaxilla; 2, snout length, from symphysis of premaxilla to osseous orbit margin; 3, orbital horizontal diameter, between osseous orbital margin; 4, head depth, just posterior to orbit; 5, orbital vertical diameter, between osseous orbital margin; 6, length of maxilla, from premaxilla end to posterior end of maxilla; 7, upper jaw length, from symphysis of premaxilla to posterior end of maxilla; 8, lower jaw length, from symphysis of dentary to retroarticular; 9, pectoral fin length, from base of first ray to tip of longest ray; 10, body depth, at level of origin of dorsal fin; 11, dorsal fin length, from base to tip of longest ray; 12, pelvic fin length, from base of first ray to tip of longest ray; 13, body depth, at level of origin of anal fin; 14, adipose fin length, from origin to tip; 15, anal fin length, from base of first ray to tip of longest ray; 16, caudal peduncle length, from end of anal fin to middle base of caudal fin; 17, least depth of caudal peduncle; 18, length of upper caudal fin lobe, from base to tip of longest ray; 19, length of middle caudal fin ray, from base to tip of shortest ray; 20, length of lower caudal fin lobe, from base to tip of longest ray; 21 standard length (SL), from upper jaw symphysis to middle base of caudal fin; 22, predorsal length from upper jaw symphysis to origin of dorsal fin; 23, head length, from upper jaw symphysis to posterior tip of operculum; 24, premaxilla to preoperculum length, from premaxilla end of maxilla to posterior margin of preoperculum (HLpp); 25, prepelvic length, from upper jaw symphysis to origin of pelvic fin; 26, preanal length, from upper jaw symphysis to origin of anal fin; 27, interorbital width, transverse at narrowest part of skull; 28, body width, transverse at widest part of body at level of dorsal fin origin, above abdominal cavity. Haplotypes are indicated by numbers as given in Table 2. Black circles are for nodes, and hatch marks are for mutation steps. 1. Additional samples given in capital letters (Delling, unpublished) refer to rivers or streams in the given regions (Table S1). Ezenam is a lake in Daghestan and labrax represents trout from Black Sea basin with an anadromous silvery and slender appearance. Measurements taken on Salmo specimens; 1, upper jaw depth, as largest depth of the maxilla and supramaxilla; 2, snout length, from symphysis of premaxilla to osseous orbit margin; 3, orbital horizontal diameter, between osseous orbital margin; 4, head depth, just posterior to orbit; 5, orbital vertical diameter, between osseous orbital margin; 6, length of maxilla, from premaxilla end to posterior end of maxilla; 7, upper jaw length, from symphysis of premaxilla to posterior end of maxilla; 8, lower jaw length, from symphysis of dentary to retroarticular; 9, pectoral fin length, from base of first ray to tip of longest ray; 10, body depth, at level of origin of dorsal fin; 11, dorsal fin length, from base to tip of longest ray; 12, pelvic fin length, from base of first ray to tip of longest ray; 13, body depth, at level of origin of anal fin; 14, adipose fin length, from origin to tip; 15, anal fin length, from base of first ray to tip of longest ray; 16, caudal peduncle length, from end of anal fin to middle base of caudal fin; 17, least depth of caudal peduncle; 18, length of upper caudal fin lobe, from base to tip of longest ray; 19, length of middle caudal fin ray, from base to tip of shortest ray; 20, length of lower caudal fin lobe, from base to tip of longest ray; 21 standard length (SL), from upper jaw symphysis to middle base of caudal fin; 22, predorsal length from upper jaw symphysis to origin of dorsal fin; 23, head length, from upper jaw symphysis to posterior tip of operculum; 24, premaxilla to preoperculum length, from premaxilla end of maxilla to posterior margin of preoperculum (HLpp); 25, prepelvic length, from upper jaw symphysis to origin of pelvic fin; 26, preanal length, from upper jaw symphysis to origin of anal fin; 27, interorbital width, transverse at narrowest part of skull; 28, body width, transverse at widest part of body at level of dorsal fin origin, above abdominal cavity. 2. Black circles are for nodes, and hatch marks are for mutation steps. G. Salmo sp. NIN, Lake Ninu Corsica. © S. Muracciole Table 1. Studied material of Salmo from Sardinia and Corsica and selected comparative material (Tables 3456). Catalog numbers are given for the Tyrrhenian samples only. BMHN 1924.3.14:2-3, 1924.3.14:4-6, 1924.3.14:7-10, 1924.3.14:11-13 MNHN 0000-3635, 0000-3636, 0000-4920, B1139 NMW 65890, 65895 NRM 41516, 41519, 41522, 41523, 41528, 41529, 44701, 44702, 44703 ZISP 48210 Salmo dentex NMW 65864, 65887, 65895 (LECTOTYPE), 95248 NRM 25000 NERETVA NRM 46358, 46359, 46361, 46362, 46370 KRKA NMW 65860, 65915, 65930 Salmo trutta (Atlantic basin, not listed above) BMNH 1885.9.18:11-21, 1908.4.28:4, 1908.4.28:5, 1937.9.15:9-14, 1947.8.15:1-2, 1991.7.12:18-20 MHNG 642.70, 816.8 MNHN 0000-2898, 0000-2909, 0000-3634, 0000-3638, 0000-3639, 0000-3641, 0000-3642, 0000-3649, 0000-3650, 0000-3651, 0000-6321, 1923-0215, 1982-0480, A5532, A5533 NRM 24849, 24866, 24885, 24894, 24895, 24903, 45239, 42543, 42544, 42559 ZMH 1206, 6344, 10718, 10721, 10731, 10736 OUM ER RBIA BMNH 1934.10.25:1-2 MNCN 208127-139 MNHN 1925-0350, 1925-0351, 1977-0282 BETH MNHN 1920-0200, 1926-0013, 1977-0272, 1977-0285 MARTIL BMNH 1887.12.23:4-10 MESSAOUD MNHN 1926-0018, 19260019, 1926-0020, 1926-0021, 1926-0022, 1926-0023, 1926-0024, 1926-0025, 1926-0026, 1926-0027 MOULOYA MNHN 1926-0014, 1926-0015, 1926-0016, 1926-0017, 1947-0018, 1977-0269 Salmo pellegrini NMW 19546 (SYNTYPE) Salmo pallaryi BMHN 1926.5.5:1 BMHN 1926.6.24:1 BMHN 1934.10:3-5 MNHN 1923-0066 (HOLOTYPE), 1925-0341, 1925-0342, 1925-0343, 1925-0344, 1925-0346, 1925-0347, 1925-0348, 1925-0349, 1977-0261, 1977-0273, 1977-0280 NRM41452 Salmo akairos MNCN 115018-029, 115022 (HOLOTYPE) Salmo sp. (Corsica and Sardinia) BMNH 1901.6.4:1-6, 1909.2.25:14 MHCH 730.22 MNHN 1896-0005, 1896-0006, 1896-0007, 1896-0008, 1896-0009, 1896-0010, 1896-0011 NMW66115 ZMH4302 MEDITERRANEAN BMNH 1877.1.6:3-4, 1887.1.6:1-2, 1901.8.6:6-7, 1940.2.10:1-3 MHCH 52.82 MNHN 0000-0002, 0000-2575, 0000-3646, 0000-3652, 0000-3653, 000-3354, 1960-

Figures captions

M O U L O U Y A 5 M A R T I L 5 B E T H 5 O U M E R R B I A 8 M E S S A O U D 7 t r u t t a 1 4 1 K R K A 1 0 N E R E T V A

Fig. 1 .

 1 Fig. 1. Geographic position of the new sampled populations in Corsica and Sardinia.

Fig. 3 .

 3 Fig. 3. Radiograph of Salmo sp. POZ, NRM62572, 197.3 mm SL; Pozzi di Marmanu Corsica

Fig. 4 .

 4 Fig. 4. Median-joining network of control region haplotypes of some trout samples (new

Fig. 5 .

 5 Fig. 5. Morphometric PC II plotted against meristic PC I for Salmo from Sardinia, Corsica

Fig. 6 .

 6 Fig. 6. Premaxilla to preoperculum length plotted against caudal peduncle length for Salmo

Fig. 7 .

 7 Fig. 7. Box plot of premaxilla to preoperculum length as % of SL, totally 518 specimens 116-

  Turkey and Mediterranean are samples scattered in the regions. Boxes represent median value +/-25% of the observations, and whiskers the inner fences. Asterisks are outside or far outside values.

Fig. 8 .

 8 Fig. 8. External aspect of seven of the trouts, Salmo sp. analyzed in the present study. Picture

.

  The alignment of CR sequences are 998 nucleotides long with 47 phylogenetically informative sites. These haplotypes are genetically very close (distant from each other by up to two mutations, Fig. 4) to 37 published GenBank haplotype sequences used as reference and illustrating the diversity on all the range of the S. trutta complex. A comprehensive network of these new and published haplotypes is presented Fig. 4. The sequences of the contemporary analyzed populations were all clustered into ME, AD and MA lineages, according to the reference sequences. AT, DA and NA lineages were represented only by GenBank sequences. New haplotypes from Tyrrhenian trout were called ADcr2 to 6, MAcr1 and 2, and MEcr1 (Table 2). Three populations are characterized by one private haplotype each: NIN, (Corsica) with MEcr1; CAM (Sardinia) with ADcr2 and POZ (Corsica) with ADcr4. CAR, (Corsica) is characterized by a majority of MA private haplotypes (MAcr1 and 2) and one AD haplotype (ADcr5), while CHA (Corsica) is characterized by two AD haplotypes (ADcr3 and ADcr6). SPE and ESE shared ADcr3 with CHA.

Fig. 1 .

 1 Fig. 1. Geographic position of the new sampled populations in Corsica and Sardinia.

Fig. 3 .

 3 Fig. 3. Radiograph of Salmo sp. POZ, NRM62572, 197.3 mm SL; Pozzi di Marmanu Corsica

Fig. 4 .

 4 Fig. 4. Median-joining network of control region haplotypes of some trout samples (new

Fig. 5 .

 5 Fig. 5. Morphometric PC II plotted against meristic PC I for Salmo from Sardinia, Corsica

Fig. 6 .

 6 Fig. 6. Premaxilla to preoperculum length plotted against caudal peduncle length for Salmo

Fig. 7 .

 7 Fig. 7. Box plot of premaxilla to preoperculum length as % of SL, totally 518 specimens 116-

  Turkey and Mediterranean are samples scattered in the regions. Boxes represent median value +/-25% of the observations, and whiskers the inner fences. Asterisks are outside or far outside values.

Fig. 8 .

 8 Fig. 8. External aspect of seven of the trouts, Salmo sp. analyzed in the present study.

Fig

  Fig.2. Measurements taken on Salmo specimens; 1, upper jaw depth, as largest depth of the maxilla and supramaxilla; 2, snout length, from symphysis of premaxilla to osseous orbit margin; 3, orbital horizontal diameter, between osseous orbital margin; 4, head depth, just posterior to orbit; 5, orbital vertical diameter, between osseous orbital margin; 6, length of maxilla, from premaxilla end to posterior end of maxilla; 7, upper jaw length, from symphysis of premaxilla to posterior end of maxilla; 8, lower jaw length, from symphysis of dentary to retroarticular; 9, pectoral fin length, from base of first ray to tip of longest ray; 10, body depth, at level of origin of dorsal fin; 11, dorsal fin length, from base to tip of longest ray; 12, pelvic fin length, from base of first ray to tip of longest ray; 13, body depth, at level of origin of anal fin; 14, adipose fin length, from origin to tip; 15, anal fin length, from base of first ray to tip of longest ray; 16, caudal peduncle length, from end of anal fin to middle base of caudal fin; 17, least depth of caudal peduncle; 18, length of upper caudal fin lobe, from base to tip of longest ray; 19, length of middle caudal fin ray, from base to tip of shortest ray; 20, length of lower caudal fin lobe, from base to tip of longest ray; 21 standard length (SL), from upper jaw symphysis to middle base of caudal fin; 22, predorsal length from upper jaw symphysis to origin of dorsal fin; 23, head length, from upper jaw symphysis to posterior tip of operculum; 24, premaxilla to preoperculum length, from premaxilla end of maxilla to posterior margin of preoperculum (HLpp); 25, prepelvic length, from upper jaw symphysis to origin of pelvic fin; 26, preanal length, from upper jaw symphysis to origin of anal fin; 27, interorbital width, transverse at narrowest part of skull; 28, body width, transverse at widest part of body at level of dorsal fin origin, above abdominal cavity.

Fig. 3 .

 3 Fig. 3. Radiograph of Salmo sp. POZ, NRM62572, 197.3 mm SL; Pozzi di Marmanu Corsica (same specimen as in Fig. 8E). Upper left; a thin membranous bone indicated with dashed white line. Upper right; the uncommon condition with three (i-iii) epurals in the caudal skeleton.

Fig. 4 .

 4 Fig. 4. Median-joining network of control region haplotypes of some trout samples (new Tyrrhenian haplotypes and AT, AD, DA, MA, ME and NA haplotypes from GenBank). Haplotypes are indicated by numbers as given in Table2. Black circles are for nodes, and hatch marks are for mutation steps.

Fig. 5 .

 5 Fig. 5. Morphometric PC II plotted against meristic PC I for Salmo from Sardinia, Corsica and Spain in comparison to Atlantic basin S. trutta.

Fig. 6 .

 6 Fig. 6. Premaxilla to preoperculum length plotted against caudal peduncle length for Salmo from Sardinia, Corsica and Spain in comparison to S. macrostigma. Linear regression lines with 95% confidence bands are shown for each group separately.

Fig. 8 .

 8 Fig. 8. External aspect of seven of the trouts analyzed in the present study. For A to F, the pictures were taken after fixation in formalin prior to transfer to ethanol.

Table 1 ,

 1 obtained from several museum collections: CMK, Collection of

	Maurice Kottelat, Cornol, Switzerland; BMNH, British Museum of Natural History, London,
	UK; MHNG, Museum d´Histoire Naturelle, Geneva, Switzerland; MNCN, Museo Nacional
	de Ciencias Naturales, Madrid, Spain; MNHN, Museum National d'Histoire Naturelle, Paris,
	France; NMW, Naturhistorisches Museum, Wien, Austria; NRM, Swedish Museum of
	Natural History, Stockholm, Sweden; ZISP, Zoological Institute, Russian Academy of
	Sciences, St. Petersburg, Russia; ZMH, Zoologisches Museum für Hamburg, Germany. The
	sample from Spain, MNHN 1920 228-229, consists of two specimens only but is included in

the study because their morphology resembles Tyrrhenian trout (see below). Comparative material in addition to that in Table

1

  Locality data for MNHN 1920 0228-0229: Spain, Lerida, Sarrade, 2052 m.a.s.l. is interpreted as close to the mountain Pic de la Pala Alta de Sarradé (2893 m, 42° 34′ 27.1″ N, 0° 53′ 16.82″ E) in the Lerida/Lleida region in Catalonia, Spain, most probably part of Ebro basin.

						NMW
	Salmo carpio*	Lake Garda	Italy		1971-1998 13 NRM, ZISP,	Delling (2002)	Neotype included
						BMNH
	Taxon Salmo ischchan*	River/Lake Lake Sevan	Armenia	Region	Date of	N 24 NRM, ZISP Museum	Reference	Remarks
	Salmo letnica*	Lakes Ohrid and	Albania, Macedonia and	capture 1890-1995 11 NRM, ZISP, Collection	Delling (2003)	Endemic to Ohrid but stocked into
	Salmo spp. Sardinia	Prespa	Sardinia Greece		1909-1960	9 ZMH, MHNG MHCH, ZMH	Delling and	Incomplete locality data Prespa lakes
	Salmo platycephalus* Seyhan	Turkey		1966-2003	3 NRM, ZMH	Doadrio (2005) Turan et al.(2012) Holotype included
	CAM Salmo obtusirostris Salmo spp. Corsica Salmo ohridanus* SPE Salmo lourosensis*	Camboni Jadro and Neretva Rivers Lakes Ohrid and Speloncellu Prespa Louros Stream	Sardinia Croatia, Bosnia and Herzegovina Corsica Albania, Macedonia and Corsica Greece Greece	2010-2012 1883-2000 19 NRM, ZMH, 8 NRM 61782, 61783 ZISP, MNHN, 65092 1895-1909 10 MNHN, NMW, BMNH MHCH 1924-2002 13 ZMH, MNHN 2011 5 NRM 61812 1977 7 NRM	this study Delling (2003) Delling and Delling & Doadrio Doadrio (2005) (2005) this study Delling (2010)	Incomplete locality data Endemic to Ohrid but stocked into Prespa lakes
	POZ Salmo peristericus*	Pozzi di Marmanu Agios Germanos	Corsica Greece		2012 1977, 1998 9 NRM 5 NRM 62572	this study Delling (2010)
	ESE	Val d'Ese Stream	Corsica		2011	5 NRM 61813	this study
	CAR CHJ Salmo dentex	Carnevale Chjuvone Rivers Neretva and Cetina	Corsica Corsica Bosnia and Herzegovina	2012 2012 1843-	5 NRM 62571 5 NRM 62573 5 NMW, NRM	this study this study Delling (2010)	Morphology only Lectotype included
	CHA	Chjuva	Corsica		2004	5	this study	Genetics only
	NIN	Lake Ninu	Corsica		2013	5 NRM 65092	this study
	Salmo sp. Spain **		Spain		1920	2 MNHN	this study
	Salmo macrostigma * El Abaich River	Algeria		1866-1907	9 MNHN, BMNH,	Delling and	Syntype included
						NMW	Doadrio (2005)
	Salmo pallaryi *	Lake Sidi Ali	Morocco		1927-1936 17 MNHN, BMNH,	Delling and	Syntypes included
						NRM	Doadrio (2005)
	Salmo pellegrini *	Tensift River	Morocco		1931	1 NMW	Delling and	Syntype
							Doadrio (2005)
	Salmo akairos*	Lake Ifni	Morocco		1995	10 NRM, MNCN	Delling and	Holotype and paratypes
							Doadrio (2005)
	Salmo trutta	Details in reference European Atlantic basin	1827-1999 243	Delling (2002)	Numerous samples from several
								countries and collections
	Salmo salar	Details in reference European Atlantic basin	1882-1998 40	Delling (2002)	Numerous samples from several
								countries and collections
	Salmo marmoratus	Po, Adige, Soca and	Italy, Slovenia and Bosnia	1823-1998 77 NRM, ZISP,	Delling (2002)
		Neretva Rivers	and Herzegovina		MNHN, BMNH,

*) Endemic taxa and/or samples restricted to type locality **)

Table 2 .

 2 Distribution of the haplotypes involved in this study

	Haplotypes	Accession Number	References / Samples	Locality of first observation
	ATcs14	EF530476	Cortey et al. (2009)	Iceland (Skorradalsvatn R.)
	ATcs16	EF530478	Cortey et al. (2009)	Spain (Several Cantabric rivers)
	ATcs20	EF530482	Cortey et al. (2009)	Russia (Vorobiex R.)
	ATcs26	EF530488	Cortey et al. (2009)	Spain (Duero R.)
	ATcs28	EF530490	Cortey et al. (2009)	Spain (Tajo R.)
	ATcs30	EF530492	Cortey et al. (2009)	Spain (Tajo R.)
	ATcs43	EF530504	Cortey et al. (2009)	Spain (Duero R.)
	ATcs45	EF530505	Cortey et al. (2009)	Iceland (Skorradalsvatn R.)
	ATcs50	EF530510	Cortey et al. (2009)	UK (Stour R.)
	ADC1	DQ381567	Sušnik et al. (2007)	Montenegro + Serbia + Albania (3 rivers)
	ADM1	DQ381566	Sušnik et al. (2007)	Montenegro + Serbia + Albania (3 rivers)
	ADcs14	AY836343	Cortey et al. (2004)	France (Corsica)
	ADcs15	AY836344	Cortey et al. (2004)	France (Corsica)
	Ma1a	DQ841191	Meraner et al. (2007)	Italy (Po R.)
	Ma2a	DQ841189	Meraner et al.

Table S2 .

 S2 Character loadings on principal component I-V for 24 measurements taken on Salmo from Corsica, Sardinia, Spain and Atlantic basin.

		PC I	PC II	PC III	PC IV	PC V
	Standard length (mm)	0.165	-0.039	-0.003	0.013	0.011
	Preanal length	0.170	-0.036	-0.002	0.014	0.014
	Prepelvic length	0.178	-0.029	0.003	0.010	0.013
	Predorsal length	0.169	-0.027	-0.001	0.006	0.013
	Head length	0.175	0.016	0.005	0.002	0.008
	Premaxilla to preoperculum length	0.188	0.028	0.006	0.003	0.007
	Caudal peduncle length	0.144	-0.065	0.001	0.015	0.023
	Caudal peduncle depth	0.153	-0.016	0.014	-0.026	-0.027
	Length of middle caudal fin ray	0.167	0.005	-0.008	0.013	-0.016
	Pectoral fin length	0.150	0.004	-0.010	0.018	-0.016
	Pelvic fin length	0.165	0.004	-0.006	0.007	-0.015
	Adipose fin length	0.157	-0.056	0.009	0.004	-0.020
	Body depth at origin of dorsal fin	0.161	-0.048	0.011	-0.016	-0.030
	Body depth at origin of anal fin	0.168	0.018	0.016	-0.002	0.003
	Head depth	0.137	0.035	-0.010	0.051	-0.005
	Horizontal orbit diameter	0.144	0.055	-0.012	0.040	-0.002
	Vertical orbit diameter	0.180	-0.029	0.013	-0.006	0.000
	Interorbital width	0.193	0.024	0.028	-0.038	0.027
	Snout length	0.204	0.055	0.022	-0.018	0.019
	Upper jaw length	0.163	0.040	0.000	-0.014	-0.047
	Length of maxilla	0.199	0.035	0.019	-0.012	0.011
	Height of maxilla	0.177	0.003	-0.106	-0.034	0.010
	Lower jaw length	0.165	-0.039	-0.003	0.013	0.011
	Gill raker length	0.170	-0.036	-0.002	0.014	0.014
	Variance explained (%)	87.9	3.78	1.99	1.37	1.06

Acknowledgements

We thank Douglas Jones at the Institute of Freshwater Research, Drottningholm, for improving the English. This work was realized with the support of LabEx CeMEB, an ANR "Investissements d'avenir" program (ANR-10-LABX-04-01).

 NRM 61783, 61950, 61812, 61813, 62571, 62572, 62573, 65092Comparative material (Figures 56, Tables 3, S2 andS3) Primary and comparative material is also included in Fig. 7 and Tables 4-6 together with additional comparative material listed below.

Salmo macrostigma from Algeria BMNH 1866.1.18:1-3 MNHN A7585 (SYNTYPE), 0000-1909, 1899-0242 NMW 67970 Salmo sp. Spain MNHN 19200228-0229 Salmo trutta, Atlantic basin BMNH 1865.7.10:2-12, 1866.1.8:1-4, 1947.9.12:1-3, 1982.11.15:6-9, 1986.5.20:208-213, 1986.5.20:94-95, 1991.7.12:18-20 MHNG 816.8 MNHN 0000-3639, 0000-3640, 0000-3643, 1923-0216, 1939-0017, A5532, A5533, B0722 NMW66013 NRM 20125, 20126, 23404, 23406, 23661, 24343, 24344, 24345, 24357, 24361, 24841, 36995, 41373, 41781, 41785, 41785, 41790, 41791, 41793, 41794, 42540, 42541, 42542, 42548, 42549, 42551, 42558, 43978, 44037, 44475, 44780 ZMH 10712, 10733, 10734, 10738, 1206, 5672, 5735, 6346 Additional comparative material (Figure 7, Tables 4-6) Sequence of listed taxon or geographic region follows Fig. 7 (left to right) and inclusion of a particular specimen in Fig. 7 and/or Tables 4-6 depends on SL, state of preservation and, availability of X-ray data for Table 5. MNHN 0000-1441, 1898-1143, 1894-0004, 1894-0005, 1939-0016 NRM 21142, 24914, 41372, 42545, 42546, 42547, 46136 ZMH 10727, 10714 EZENAM (Salmo ezenami) ZISP 28356, 48317 Salmo cf. labrax BMNH 1913.5.25:1-3, 1962.9.25:1, 1991.7.12:21-22 NMW 65628, two uncatalogued specimens TURKEY NMW 80837, 90952, 50581, 50582, 50583 ZMH 2450, 3578, 4222, 4223

Salmo salar