Patrick Berrebi
email: patrick.berrebi@laposte.net

V Caputo Barucchi

A Splendiani

S Muracciole

A Sabatini

F Palmas

Christelle Tougard

M Arculeo

S Marić

Keywords:

Brown trout (Salmo trutta L.) high genetic diversity around the Tyrrhenian Sea as revealed by nuclear and mitochondrial markers

Italy. 10 (c) Office National des Forêts, Pont de l 'Orta, 20250 Corte, France. 11 (d) Dipartimento di Scienze della Vita e dell'Ambiente, Università di Cagliari, Via Fiorelli 1,Cagliari,[START_REF] Pujolar | Restricted gene flow at the micro-and 1052 macro-geographical scale in marble trout based on mtDNA and microsatellite polymorphism[END_REF] Italy. 13 (e) Dipartimento di STEBICEF, Università di Palermo, via Archirafi 18,Palermo 90123,Italy. 14 (f) University of Belgrade, Faculty of Biology, Institute of Zoology, Studentski Trg 16, 11001, Belgrade, 15 Serbia. [START_REF] Zaccara | Morphological and 1064 genetic characterization of Sardinian trout Salmo cettii Rafinesque, 1810 and their conservation implications[END_REF] 17

Abstract 18

The brown trout (Salmo trutta L.) is widely distributed all around Europe but its natural diversity is 19 threatened by massive stocking with Atlantic domestic strains. Describing the remaining natural genetic 20 diversity and the proportion of domestic hatchery strains in rivers is a prerequisite for smart conservation. 21

The high genetic diversity of brown trout populations around the Tyrrhenian Sea is well known. Use of 22 twelve microsatellites has allowed description of the natural genetic structure of populations and detection 23 of the consequences of stocking. Mitochondrial DNA control region sequences and the LDH-C1* gene 24 enabled placement of each population into one of the six mitochondrial and two allozymic known

Introduction 42 43

The brown trout is widespread all around Europe, western Asia and North Africa at medium and high 44 elevation [START_REF] Behnke | The systematics of salmonid fishes of recently glaciated lakes[END_REF][START_REF] Behnke | Brown trout[END_REF]. It is one of the most extensively managed freshwater fish species 45 worldwide because of its high economical value, mainly for sport fishing. As a consequence, brown trout

Materials and methods 93 94

Sampling distribution 95 96

The present survey concentrates sampling to the freshwater basins distributed all around the Tyrrhenian 97

Sea with some Ionian Sea tributaries (Fig. 1). In order to introduce comparative samples, a river entering 98

the Ligurian Sea at the French-Italian boundary and three commercial Atlantic domestic samples from 99 northern France and central Italy have been included (Table 1). The French hatchery is representative of 100 the international commercial Atlantic strain and the Italian ones of local domestic strains, both derived 101 from genitors from several countries [START_REF] Bohling | Genetic diversity and population structure of domestic brown 631 trout (Salmo trutta) in France[END_REF]. 102

Electrofishing was conducted between 2004 and 2014. A total of 365 river trout (sub-adults and adults) 103 was anaesthetized and a small fin clip taken (preserved in 96% ethanol) before each fish was returned to 104 its river. In the three hatcheries sampled, the method was similar but the fish were caught with dip nets. 105 Some of the samples contained few fish (Table 1): lower Tiber in Lazio region (7 to 9 specimens), 106

Calabria (3 to 7) and Sicily (6 to 11), except for the Anapo River. These samples were very difficult to 107 constitute because of the low trout density. In Sicily and Calabria, the species is protected and special 108 permits are needed with a given maximum number of trout to be caught. Fortunately, most often, this 109 concerned nearby small rivers which could be concatenated for population parameter calculations. 110 111 DNA extraction 112 113 DNA was extracted using the Chelex/proteinase K based method described by [START_REF] Walsh | Chelex 100 as a medium for simple extraction of DNA 860 for PCR-based typing from forensic material[END_REF] and 114 [START_REF] Estoup | Rapid one-tube DNA extraction for reliable 664 PCR detection of fish polymorphic markers and transgenes[END_REF] then improved by [START_REF] Yue | A simple and affordable method for high-throughput DNA extraction from 867 animal tissues for polymerase chain reaction[END_REF]. A small piece of fin was incubated overnight 115 at 56°C in 195 µL of 5% Chelex 100 Resin (Biorad) solution containing 50 mM of Tris-HCL (pH 7) and 116 500 µg/mL of proteinase K. Samples were then incubated at 95°C for 10 min before centrifugation at 3500 117 g for 5 min. Supernatants were recovered and frozen at -20°C until required for use.

119

Mitochondrial marker 120 121

The mtDNA CR was amplified by PCR using the PST and FST primers (Cortey & García-Marín, 2002). 122

Each 50 µL reaction included 0.4 µM of each primer (Eurofins MWG Operon), dNTP (2 mM each), 2 mM 123 of MgCl2, 10 µL of 5× PCR buffer, 1 U of Taq polymerase (GoTaq® Promega) and about 50 ng of 124 genomic DNA. The PCR conditions included initial denaturation (95°C, 5 min) followed by 30 strand 125 denaturation (94°C, 1 min), primer annealing (52°C, 1 min) and DNA extension (72°C, 1 min) cycles, 126 followed by a final extension (72°C, 5 min). All PCR amplifications were performed in Eppendorf 127

Mastercycler thermocyclers. The amplified DNA fragments were run on a 0.8% agarose gel to verify the 128 amplification efficiency. The amplified products were purified and sequenced in both directions to 129 confirm the polymorphic sites in an ABIPRISM 3130/xl/ sequencer (Applied Biosystems). 130

The mtDNA CR sequences were aligned using the computer program Clustal X (Thompson et al., 131 1997) implemented in MEGA version 6 [START_REF] Tamura | MEGA6: Molecular Evolutionary 834 Genetics Analysis Version 6.0[END_REF]. In order to assign Tyrrhenian trout to a 132 lineage, data were aligned and compared with reference S. trutta CR sequences from GenBank (AT, DU, 133 ME, MA, AD and DA) (see Table S1 for haplotype details). The genealogical relation of haplotypes was 134 depicted using a 95% statistical parsimony network constructed using TCS 1.3 [START_REF] Clement | TCS: a computer program to estimate gene genealogies[END_REF]. 135 136 LDH-C1* genotypes 137 138 Some of the sampled specimens were genotyped at the LDH-C1* gene coding the LDH enzyme (Table 1). 139

This marker, largely used in the past through allozyme electrophoreses [START_REF] Hamilton | Post-glacial 694 colonisation of brown trout, Salmo trutta L.: Ldh-5 as a phylogeographic marker locus[END_REF]Almodóvar 140 et al., 2006;[START_REF] Berrebi | Three brown trout Salmo trutta lineages in Corsica described through allozyme 622 variation[END_REF] was analyzed by the PCR-RFLP method of [START_REF] Mcmeel | Partial nucleotide sequences, and routine typing by 721 polymerase chain reaction-restriction fragment length polymorphism, of the brown trout (Salmo 722 trutta) lactate dehydrogenase, LDH-C1*90 and *100 alleles[END_REF]. This popular 141 marker separates the ancestral allele *100 found in Atlantic salmon and southern brown trout lineages and 142

Atlantic domestic strains carrying the derived *90 allele [START_REF] Hamilton | Post-glacial 694 colonisation of brown trout, Salmo trutta L.: Ldh-5 as a phylogeographic marker locus[END_REF][START_REF] García-Marín | Postglacial colonization of brown trout in Europe based 677 on distribution of allozyme variants[END_REF]143 Berrebi, 2015). Native Mediterranean trout populations belong to the mitochondrial trout lineages ME, 144 AD, MA and AT. This last lineage, is represented by the so-called southern Atlantic clade, sensu Cortey et 145 al. (2004) and also exhibits the LDH-C1*100 allele [START_REF] Aurelle | Natural and artificial secondary contact in brown trout (Salmo trutta, L.) 600 in the French western Pyrenees assessed by allozymes and microsatellites[END_REF]. Therefore, this marker is 146 used, here, mainly to distinguish between the AT haplotypes belonging to the southern Atlantic clade and 147 the AT haplotypes of northern origin (marked with LDH-C1*90) that invaded the Mediterranean region 148 subsequent to stocking activities. According to [START_REF] Hamilton | Post-glacial 694 colonisation of brown trout, Salmo trutta L.: Ldh-5 as a phylogeographic marker locus[END_REF], all Mediterranean trout populations 149 are characterized by the allele *100, except the marble trout (allele LDH-C1*120). On the contrary, most 150 north Atlantic populations carry the allele *90 with several exceptions [START_REF] García-Marín | Postglacial colonization of brown trout in Europe based 677 on distribution of allozyme variants[END_REF]. This 151 marker can be used to distinguish natural trout, including natural Atlantic populations in Sicily carrying 152 the *100 allele [START_REF] Schöffmann | Phylogenetic origin of Salmo trutta L 1758 from Sicily, based 790 on mitochondrial and nuclear DNA analyses[END_REF] from introduced hatchery Atlantic trout. 153 154

Microsatellite loci 155 156

The primers of the twelve microsatellite markers used in this study were obtained from the literature. 157

Repeated sequences are all dinucleotide except Ssa197 which is a tetranucleotide microsatellite (O'Reilly 158 et al., 1996). Details about each locus and multiplexes are given in Table 2. 159

For each marker, one of the 5' ends of the two primers was end-labelled with a fluorescent dye, either 160 6-FAM, HEX or NED. Polymerase chain reactions (PCR) were performed using the Qiagen multiplex 161 PCR kit in a final volume of 10 µL, containing 3 µL of genomic DNA diluted at 10 ng/µL, 5 µL of Qiagen 162 PCR Master Mix, 1 µL of Qiagen Q-solution, and 1 µL of primer mix at 2 µM each (Eurofins MWG 163 Operon). Amplifications were carried out in a GeneAmp PCR System 2700 thermal cycler (Applied 164 Biosystems), according to the supplier's instructions (Qiagen multiplex PCR kit): initial denaturation step 165 (95°C, 15 min;) followed by 35 cycles of denaturation (94°C, 30 s), annealing (55°C for the three 166 multiplexes, 90 s) and extension (72°C, 60 s); with a final extension step (60°C, 30 min). Amplified PCR 167 fragments were then diluted and separated on an ABIPRISM 3130/xl/ sequencer (Applied Biosystems) 168

with GeneScan 500 Rox dye size standards. Allele sizes were determined using the GeneMapper v4.1 169 software system (Applied Biosystems, Life Technologies).

8

A genotype matrix was then constructed and used as a basis for all the following statistical analyses 171 mainly carried out using GENETIX 4.05 [START_REF] Belkhir | GENETIX 4.05: logiciel sous Windows pour la 610 génétique des populations[END_REF]. 172 173

Microsatellites statistics 174 175

For estimation of genetic polymorphism, expected heterozygosity (He), unbiased expected 176 heterozygosity (Hnb: [START_REF] Nei | Estimation of average heterozygosity and genetic distance from a small number of 735 individuals[END_REF]) and observed heterozygosity (Ho) were calculated for each sample or 177 river system. The mean number of alleles by locus (A) is another way to estimate the diversity in a 178 population. These calculations were limited to samples of ten or more specimens. For this reason, nearby 179 locations characterized by very low fish number were grouped in order to increase the sample size. 180

Inter-sample or inter-river system differentiations (Fst) and the intra-river panmixia (Fis) were 181 estimated (ɵ and f estimators of Weir & Cockerham, 1984, respectively). The significance of the Fst and 182

Fis values was tested by random permutation procedures: 5000 individual permutations between samples 183 for Fst and 5000 allele permutations within samples for Fis, processed using GENETIX. 184

The sequential Bonferroni correction was applied for multiple tests [START_REF] Rice | Analyzing tables of statistical tests[END_REF]. Micro-Checker 185 software (van Oosterhout et al., 2004) was run in order to detect null alleles, drop-out or stuttering 186

perturbations. 187

A general picture of the trout genetic diversity was first obtained through multidimensional analyses. 188

Here Factorial Correspondence Analyses (FCA: [START_REF] Benzécri | L'analyse des données[END_REF], allowing the overall structure of the 189 sampling to be explored, were carried out as implemented in GENETIX. Three focuses were chosen: all 190 the samples, the Sicilian two lineages and the Atlantic diversity of the sampling (Fig. 3a, b andc). The 191 clusters (or clouds) observed in the diagrams correspond to nuclear genetic homogeneous lineages. The 192 mathematical method is clearly detailed in She et al. (1987). 193 In order to detect differentiated subgroups, hierarchical STRUCTURE assignment analyses were 194 performed for the whole sample set [START_REF] Vähä | Life-history and habitat features influence the 855 within-river genetic structure of Atlantic salmon[END_REF]Marić et al., 2017). The program STRUCTURE 195 2.3.2 (Pritchard et al. 2000) runs Markov chain Monte Carlo simulations to partition individuals into K 196 clusters (here, K was run between 1 and 24). Basic assignment criteria are the minimization of Weinberg and of linkage disequilibria [START_REF] Pritchard | Inference of population structure using multilocus 770 genotype data[END_REF]. Different run lengths were used at each step 198 (from 20,000 to 100,000 burn-in and 100,000 to 500,000 total lengths, repeated seven times for each K) 199 depending on convergence (proportion of identical structure among the seven repeated runs at each step). 200

The admixture ancestry model and correlated allele frequency options were chosen. Sampling location 201

was not used as prior information. 202

The ΔK method [START_REF] Evanno | Detecting the number of clusters of individuals using the 667 software STRUCTURE: a simulation study[END_REF] was applied to estimate the most probable K at each step (see 203

Table S2). The first step determines the first hierarchical level, the whole sample set being split into K 204 subgroups. Then each subgroup was analyzed separately, allowing for more precise clustering of 205 individuals without eliminating admixed individuals. This hierarchical method was applied until no further 206 substructure was observed. A comparison with the simple assignment test with the best K (here K=19) is 207 provided in Fig. S5 andS6). 208

Finally, the parentage software COLONY 2.0.6.4. [START_REF] Jones | COLONY: a program for parentage and sibship inference from 703 multilocus genotype data[END_REF] was applied especially on 209

Corsican and Sardinian samples. We have chosen the Pair-Likelihood-Score (PLS)/Full-Likelihood (FL) 210 combined (=FPLS) algorithm in order to establish only full-sibs listing. The objective is to understand the 211 family structure of these very low polymorphic and very differentiated/isolated populations. 212 The new haplotype AD-Tyrrh1 is the most common, widespread in all regions except Sicily, and in a 227 central position in the AD haplogroup (Fig. 2). AD-Tyrrh5 and 6 are exclusive to Nera River (i.e. upper 228 Tiber). With nine different haplotypes detected, the AD haplogroup was the main non-domestic mtDNA 229 haplogroup observed in the Tyrrhenian region (56%, Table 3). Note that the sequence of the AD-Tyrrh4 230 haplotype, newly discovered in the present study, contains an 82 bp repeat towards the 3′-end of the CR. 231

As the elongation model of this repeat is generally thought to be the result of intra-molecular processes 232 [START_REF] Buroker | Length heteroplasmy of sturgeon mitochondrial DNA, an illegitimate elongation model[END_REF], only the first copy was kept in the analysis. The native AD lineage was present in 233 96% of the Corsican Tyrrhenian trout, 100% of the upper Tiber River (Nera), 67% of the lower Tiber 234 River (Simbrivio and Aniene), 50% of the Sardinian trout, 46% of Calabrian trout and 0% of Sicilian 235 ones, representing a north-south cline partly linked to stocking activities. In the present survey, six AD 236 haplotypes are new, suggesting their endemism (Table S1). 237

Corsica was mainly characterized by the new haplotype AD-Tyrrh1, except for the Acqua d'Acelli 238 population that had only the ADcs15 haplotype. AD-Tyrrh1 accounted for 88% of the Corsican AD 239 haplotypes, 31% of the Sardinian ones, 8% in the Tiber basin in central Italy, 50% of the Calabrian 240 diversity (but with a sample size of 6) and was not present in Sicily. Here again, a cline affected the 241 distribution of this variant, exhibiting a decrease from north to south. The other AD haplotypes were 242 distributed without clear structure in Sardinia, the Tiber River and Calabria (Table 3). 243

Among the AT lineage, the AT-s6 haplotype should represent a natural migration of Atlantic trout into 244 the Mediterranean [START_REF] Schöffmann | Phylogenetic origin of Salmo trutta L 1758 from Sicily, based 790 on mitochondrial and nuclear DNA analyses[END_REF]. This haplotype of 380 bp is included in the 985 bp haplotype 245 ATSic detected here. This haplotype was the only one present in the Anapo River (Sicily). "Clone JE1", 246 the other Sicilian AT haplotype, was the only one found in the Manghisi River, a tributary of the River 247

Cassibile. On the contrary, haplotypes 1 to 4 (Table 3) are well known to characterize north Atlantic 248 populations and the domestic commercial Atlantic hatchery strain (Cortey & García-Marín, 2002;Cortey 249 et al., 2004). These haplotypes, synonymous with ATcs1 to 4 (Cortey et al., 2004), are well represented in 250 both Italian and French hatcheries, and in rivers where they had been introduced by stocking. They were 251 observed in Corsica (globally 4%), Sardinia (50%), Calabria (54%) and in the lower Tiber basin (33%). 252

The new and rare haplotype AT-Tyrrh1 was found only in the Cantiano hatchery (sample 15) but not in 253 any river. In Sicily, only the Atlantic haplotype was observed. Their natural/domestic origin is discussed 254 below. 255 256

LDH-C1* genotypes 257 258

Genotyping this gene is especially useful to distinguish between natural and domestic origins of the AT 259 haplotypes of Sicily. Some of the samples were tested, especially those with AT mtDNA haplotypes 260 (Table 1) Ermolinus and Sadali populations are composed of 7 to 9 families of generally 1 or 2 individuals (except 325 two families of 3 and 5 trouts, see Table S3). 326 327

Comparing different marker systems 328 329

Comparing data provided by the three categories of markers allowed the general genetic structure of trout 330 around the Tyrrhenian Sea to be described. The LDH-C1* marker is monomorphic for the considered 331 natural populations and was used as a diagnostic marker between natural and domestic trout. 332

The Roya trout population belongs to the ME lineage, according to mtDNA CR sequences 333 (unpublished data) constituting the ME lineage reference for microsatellite diversity and assignment 334 (Table 4, Fig. 4). In the sequence data constituting the present study, this ME lineage was not observed all 335 around the Tyrrhenian Sea. 336

Corsican and Sardinian natural populations were mostly characterized by the AD-Tyrrh1 haplotype 337 (with a few AD-Tyrrh2 and AD-Tyrrh4). Among this mitochondrial homogeneity, there were two 338 exceptions: the Corsican population of Acqua d'Acelli had only the ADsc15 haplotype and the Sardinian 339 population Is Abius was characterized by the A_2 Adriatic haplotype. The microsatellite markers divided 340

Corsican and Sardinian natural populations into nine clusters in Fig. 3a and at the third step of the 341 hierarchical assignment analysis (Fig. 4). 342

Along the Tyrrhenian-Ionian Italian Peninsula the upper Tiber River trout (Nera River, sample 17) 343 appear as a separate cluster in the third step of the hierarchical analysis, which is congruent with the 344 mtDNA haplotype composition. Here, two private haplotypes were observed (AD-Tyrr5 and 6) that 345 probably have a trans-Apennine origin due to their genetic similarity with haplotype ADcs11 (see Fig. 2), 346 this latter being very common in the Adriatic region (Sušnik et al., 2007;Berrebi et al., 2013). 347

On the contrary, the lower Tiber River trout (samples 18 and 19) showed wide and distinct haplotype 348 diversity of more western origin (AD-Tyrrh1, 2 and 4). 349

Further south, the Calabrian samples were separated into two lineages at the second step of the 350 hierarchical analysis (B1 and B2): the Ancinale River system (samples 20 to 22) and lower Calabria 351 (samples 23 to 26). The Ancinale cluster was characterized by the common haplotype AD-Tyrrh1 and the 352 lower Calabrian by two private haplotypes: AD-Tyrrh3 and ADcs1. 353

Sicilian trout populations exhibited two AT haplotypes (ATSic and "clone JE1"). The clone JE1 354 haplotype, fixed in the Manghisi River population, can be considered to be of native origin on the basis of 355 the associated fixation of the LDH-C1*( 100) allele (Table 3). Microsatellite Sicilian clusters separated 356 lately (at the third step) the Manghisi and San Marco populations (south Sicily -samples 28 and 29) from 357 the Anapo population ( 27) in correlation with the two haplotypes (Table 3). With only one exception, all 358 analyzed Sicilian trouts showed the LDH-C1*100 wild genotype. 359

All around the Tyrrhenian Sea, AT haplotypes AT-Tyrrh1 and "haplotypes 1 to 4" marked the 360 domestic lineage assigned to the B2 cluster (Fig. 4) based on microsatellites and recognizable with the 361 LDH-C1*90 allele, while clusters B1 and B3 dominantly represented composite natural lineages. 362 363

Population parameters 364 365

Population parameters add some biological information. Heterozygosity, as measured with various H 366 parameters and with the mean number of alleles A (Table 4), was very low in Corsica (Hnb=0.17), but 367 very high in Sardinia (0.58). Parameter A, the mean number of alleles by locus, was also very distinctive 368 (respectively 1.9 and 4.7). Hatchery populations are considered to be highly polymorphic (Berrebi et al., 369 2000;[START_REF] Bohling | Genetic diversity and population structure of domestic brown 631 trout (Salmo trutta) in France[END_REF] and displayed large values: 0.67<Hnb<0.78 and 5.7<A<8. 3. 370 Inter-sample differentiation can be determined using the estimations of Fst (Table 5). Most sample 371 pairs was highly significantly differentiated except for a few cases which became moderately significant 372 after Bonferroni correction. Considering only feral trout, in Corsica the mean inter-sample Fst was 0.74, 373 which is a very high value among neighboring rivers (0.47>Fst>0.90). The mean Fst was lower in Sicily 374 (0.34) and among the Tiber tributaries (0.29). In Sardinia, the Ermolinus and Sadali trout populations were 375 deeply introgressed by domestic forms. Only Marroccu, Is Abius and Camboni populations are native, 376 with very low differentiation (0.03<Fst<0.08). In Calabria, the small sample sizes limited the estimations 377 between groupings to an opposition between the Ancinale and Alaro/Assi/Stilaro/Precariti basins (Fst was 378 0.17 Corsican trout can be considered as the best conserved natural stock around the Tyrrhenian Sea, since 392 all samples (except number 2) were in the A cluster, which characterizes mostly AD individuals (Fig. 4). 393

As a possible explanation, the riverine ecosystems have been relatively well preserved in the island and 394 the upstream part of the watersheds is generally free of pollution. Hydrogeological characteristics are also 395 favorable to salmonids. According to [START_REF] Gauthier | La colonisation de l'île par différentes souches de truite[END_REF], the ancestral lineage (AD) has been 396 isolated upstream by impassable waterfalls since the last glacial maximum. Below these waterfalls, 397 postglacial Mediterranean invader trout (ME, not sampled here) hybridized with resident AD populations. 398

The numerous upstream isolated ancestral populations are free of ME invader or of domestic introduction 399 (Fig. 4), confirming the [START_REF] Berrebi | Three brown trout Salmo trutta lineages in Corsica described through allozyme 622 variation[END_REF] survey of 38 samples, some of which were re-sampled for this 400 study (stations 2, 6 and 7). Traces of stocking (AT) are rare there (imperceptible with nuclear markers and 401 4% according to the mitochondrial sequences). This favorable situation explains the preserved high 402 differentiation between populations (0.47>Fst>0.90; mean value 0.74, the highest value among all the 403 samples). Moreover, the small size of streams together with possible bottlenecks could explain the very 404 low intra-population diversity (0.08<Hnb<0.26, the lowest values among all the samples) compared to 405 native grouped Tiber populations (0.51<Hnb<0.75), native Sardinian trout (0.45<Hnb<0.51), and even the 406 grouped Calabrian (0.62<Hnb<0.78) and Sicilian populations (0.49<Hnb<0.59). Table 3 shows that these 407 populations exhibit only one haplotype each (except for sample 9 from the Lataga River), mostly the 408 newly described AD-Tyrrh1. 409

In order to better describe these island isolated small populations, COLONY software (Jones & Wang 410 2010) was used to research family structure in Corsican populations (not shown). In Corsica, each 411 population shows only one to three families (Table S3). In fact, the polymorphism is so low 412 (0.08<Hnb<0.26) that we can deduce that these small populations, totally isolated, have suffered several 413 drastic recent bottlenecks. We can also suppose that one or two pairs of parents have recently re-funded 414 each population making naturally each sample 1 to 3 families. The other interpretation is that such low 415 polymorphism does not allow families detection. In Sardinia, the landscape is totally different: (i) in north-416 east populations (Flumendosa basin), mostly composed of Atlantic domestic lineage, the Ermolinus and 417

Sadali populations are composed of 7 to 9 families of generally 1 or 2 individuals. These highly 418 polymorphic populations (here 0.74<Hnb<0.79) are composed of various origins. (ii) In the south, the 419 same local lineage has been observed in three samples of the same watershed (Cixerri basin) with possible 420

exchanges. The medium level of polymorphism (0.41<Hnb<0.51) corresponds to rather large populations 421 and possibly exchanges between them. The whole Cixerri sampling forms 12 families of full-sibs 422 frequently composed of individuals of the three sampled locations (Table S3). 423

If we compare these values with populations analyzed in the literature with similar markers (9 to 11 424 microsatellite loci), the first difficulty is the diversity of cases mainly in terms of population size. 425

However, with the filter of case similarity, we observed very high diversity in northern populations 426 (generally of AT mtDNA lineage) as in Switzerland (Stelkens et al, 2012: 0.73<He<0.81) or in Romania 427 (Popa et al. 2016: 0.79<He<0.82), but lower values in Catalonia/Spain, as around the Tyrrhenian Sea 428 (Araguas et al., 2017: 0.36<He<0.66). 429

The Sardinian populations seem to be far more degraded than the Corsican ones. Currently trout 430 populations can be found in a few basins in Sardinia, where they are confined to areas of medium 431 elevation. In this island, known pure natural populations are limited to one southern basin located within 432 the Regional Natural Reserve (Foresta di Monte Arcosu): the Cixerri basins [START_REF] Sabatini | Conservation and management of Salmo 781 (trutta) macrostigma in Sardinian freshwaters: first results of genetic characterization[END_REF][START_REF] Duchi | Fecundity, egg and alevin size in the River Irminio population of the threatened Salmo 659 cettii Rafinesque-Schmaltz, 1810 (Sicily, Italy)[END_REF]433 Zaccara et al., 2015;[START_REF] Sabatini | Restoration 784 of native Mediterranean trout Salmo cetti Rafinesque, 1810 (Actinopterygii, Salmonidae) 785 populations using an electric barrier as mitigation tool[END_REF]. Cixerri basin samples (numbers 12 to 14) were grouped at the 434 second step of hierarchical assignment (Fig. 4). Flumendosa basin (samples 10 and 11), located east-435 central of the island, is inhabited by hybrid trout with domestic dominance, assigned to the B2 cluster 436 gathering the domestic Atlantic lineage (Fig. 4), with a majority (76%) of hatchery haplotypes (Table 3). 437 Tiber River trout are probably influenced by the numerous upstream hatcheries settled in the Marche 438 (samples 15 and 16) and Umbria regions. However, this influence seems limited in the wild according to 439 the mitochondrial marker (no AT haplotype upstream in the Nera River and 33% downstream). Nuclear 440 markers clearly isolate a first natural group, the upstream Nera River sample alone, visible outside the 441 central part of the multidimensional diagram (Fig. 3a) in the B1 lineage (Fig. 4) and with haplotypes AD-442 Tyrrh5 and 6, not observed elsewhere (Table 3). According to microsatellites (Fig. 3c and the B2 cluster 443 in Fig 4 .), another lower Tiber group is genetically closer to the domestic samples (haplotypes AD-Tyrrh 444 1, 2 and 4, all three not endemic to the Tiber River). 445

Calabria is a dry area cut at the south by small parallel coastal rivers flowing into the Ionian Sea. 446 Domestic Atlantic strains were introduced into this region, and totally replaced the Allaro population 447 (Table 3, Fig. 3c and B2 cluster in Fig. 4). The Diga Giulia population is hybridized and shows 70% of 448 domestic LDH alleles (*90) and 30% of Atlantic haplotypes (Table 3). Natural Calabrian trout are limited 449 to Ancinale and partly in the Diga Giulia Rivers with haplotypes AD-Tyrrh1 and 3, and ADcs1. 450

According to [START_REF] Schöffmann | Phylogenetic origin of Salmo trutta L 1758 from Sicily, based 790 on mitochondrial and nuclear DNA analyses[END_REF] and the present study, Sicily is inhabited by trout 451 belonging only to the AT mitochondrial lineage. [START_REF] Schöffmann | Phylogenetic origin of Salmo trutta L 1758 from Sicily, based 790 on mitochondrial and nuclear DNA analyses[END_REF] described haplotype AT-s6 in a 452 sample of 26 specimens from three rivers including the River Anapo. The AT-s6 haplotype is a short 453 synonym (380 bp reported by [START_REF] Bernatchez | The evolutionary history of brown trout (Salmo trutta L.) inferred from 614 phylogeographic, nested clade, and mismatch analyses of mitochondrial DNA variation[END_REF] of three published longer sequences: the ATSic 454 haplotype (endemic to Sicily, Snoj et al., 2011), and the ATM1 and ATM6 haplotypes, known in Morocco 455 (Snoj et al., 2011). These natural AT haplotypes are phylogenetically far from "haplotypes 1 to 4" marking 456 domestic AT trout (Snoj et al., 2011). Using one enzymatic marker, [START_REF] Schöffmann | Phylogenetic origin of Salmo trutta L 1758 from Sicily, based 790 on mitochondrial and nuclear DNA analyses[END_REF] indicated 457 that the LDH-C1*100/100 genotype is dominant in Sicily (24/26). This last genotype is absent from north 458 Atlantic domestic strains [START_REF] Berrebi | Stocking impact and allozyme diversity 624 in brown trout from Mediterranean southern France[END_REF]Cortey et al., 2004). It was thus deduced that the Sicilian 459 lineage should be a natural immigration of the south Atlantic lineage, characterized by the LDH-C1*100 460 allele. [START_REF] Schöffmann | Phylogenetic origin of Salmo trutta L 1758 from Sicily, based 790 on mitochondrial and nuclear DNA analyses[END_REF] also indicated that the AT-s6 haplotype sequence is close to those found 461 in Atlantic basins in the Iberian Peninsula [START_REF] Weiss | Mitochondrial haplotype diversity among 864 Portuguese trout Salmo trutta L. populations: relevance of the post-Pleistocene recolonization of 865 northern Europe[END_REF]Suárez et al., 2001) and southern France 462 [START_REF] Aurelle | Genetic structure of brown trout (Salmo trutta, L.) populations from 597 south-western France: data from mitochondrial control region variability[END_REF]. The other haplotype found in Sicily in the Manghisi River (tributary of the 463 River Cassibile), "clone JE1", was first described in the Jerte River (tributary of the River Tajo) in Spain 464 (Suárez et al., 2001). This haplotype is phylogenetically close to the ATSic natural haplotype but also to 465 several domestic haplotypes like "haplotype 3" (Fig. 2). Therefore, the presence of the sole allele LDH-466 C1*100 in a sub-sample from the Manghisi River (Table 3) demonstrates the natural origin of the 467 haplotype clone JE1. Taking these new results into account confirm the delimitations of two Atlantic 468

sublineages: the natural one mainly in Sicily and the domestic ones mainly in hatchery samples but 469 dispatched over the whole zone. This is justified by (i) the two exclusive groups of haplotypes, haplotypes 470 1 to 4 vs ATsic and clone JE1 for domestic and natural AT and (ii) by Fig. 4, where the Atlantic lineage is 471 in black at step 1 and in green and black (B1 and B2) at step 2 for natural and domestic Atlantic 472

sublineages. 473

Recently, Splendiani et al. (2016) detected the ATsc33 haplotype in sub-fossil remains of brown 474 trout from a Tyrrhenian locality of northern Calabria dating about 13,000-8,000 years BP. This haplotype 475 is considered to belong to the southern Atlantic haplogroup together with ATSic and clone JE1 476 haplotypes, and was already detected in Sicily [START_REF] Fruciano | Phylogeographical relationships of Sicilian 672 brown trout and the effects of genetic introgression on morphospace occupation[END_REF], Spain (Suárez et al., 2001;Cortey et 477 al., 2009) and Morocco (Snoj et al., 2011). This observation is coherent with the recent expansion of this 478 haplogroup related to the last glacial maximum as proposed by Snoj et al. (2011). 479

In the samples from Calabria analyzed in the present study, no native Atlantic haplotypes were 480 found. However, due to the very low size of the Calabrian samples analyzed here (13 sequenced 481 specimens), we cannot make any assumptions about a possible natural presence of the Atlantic lineage in 482 current brown trout populations of the southern Italian peninsula. 483 484

Conservation purposes 485 486

The brown trout is generally accepted as a diversified and complex assemblage of salmonids of the 487 genus Salmo. It has been investigated for a long time by several methods, but without a clear description 488 of its taxonomy. According to [START_REF] Sanz | Phylogeographic history of brown trout: a review[END_REF], who reviewed the entire S. trutta complex data, the origin of 489 this difficulty is due to the multiple episodes of colonization and secondary contacts. This high 490 phylogenetic complexity with large genetic differences within lineages is accompanied by morphological 491 and life-history diversity that has led to the description of dozens of nominal species (Kottelat & Freyhof, 492 2007). 493

The increase in the number of new trout species, sometimes elevating known subspecies to the species 494 rank [START_REF] Isaac | Taxonomic inflation: its influence on macroecology and 701 conservation[END_REF][START_REF] Zachos | Species inflation and taxonomic artefacts -A critical 874 comment on recent trends in mammalian classification[END_REF], sometimes accepting ancient morphological species as true 495 species [START_REF] Ninua | Phylogeography and taxonomic status of trout and 737 salmon from the Ponto-Caspian drainages, with inferences on European brown trout evolution and 738 taxonomy[END_REF], is a strategy to be considered as a threatened taxon in the IUCN Red List 496 criteria. This should be a help for conservation (Garnet & Christidis, 2007) but such a strategy can lead to 497 mismatches with described molecular lineages [START_REF] Phillimore | Are subspecies useful in evolutionary and conservation biology? 756[END_REF]. Considering the complex 498 evolutionary history of the brown trout, Sanz (2018) recommended defining conservation units based on 499 genetic assessments at the population level. 500 Thirty years ago, molecular investigations proposed a first global description of the S. trutta complex. 501

The most popular description of genetic structure is based on the mtDNA CR sequences, showing five 502 main clusters closely linked to geographic distribution [START_REF] Bernatchez | DNA sequence variation of the mitochondrial 617 control region among geographically and morphologically remote European brown trout Salmo 618 trutta populations[END_REF][START_REF] Bernatchez | The evolutionary history of brown trout (Salmo trutta L.) inferred from 614 phylogeographic, nested clade, and mismatch analyses of mitochondrial DNA variation[END_REF]. This 503 organization has been confirmed with rDNA ITS markers [START_REF] Presa | Phylogeographic congruence between mtDNA 768 and rDNA ITS markers in brown trout[END_REF]. 504

Numerous publications used the CR marker, increasing the number of sequences or the sequence 505 length [START_REF] Weiss | Mitochondrial haplotype diversity among 864 Portuguese trout Salmo trutta L. populations: relevance of the post-Pleistocene recolonization of 865 northern Europe[END_REF]Cortey & García-Marín, 2002) until sequencing the 1250 bp that cover the 506 whole CR in [START_REF] Giuffra | Mitochondrial control region and protein coding genes 683 sequence variation among phenotypic forms of brown trout Salmo trutta from northern Italy[END_REF] or 1013 bp in Cortey et al. (2004). This marker is perhaps not the best 507 for phylogenetic reconstruction, but it is the most used and so the most practical for general phylogenies 508 with numerous GenBank sequences involved. 509 Beside the S. trutta complex, few taxa have been demonstrated as being outside the species complex 510 according to mtDNA phylogenies. S. ohridanus and S. obtusirostris are rare Salmo taxa considered as 511 distinct species [START_REF] Snoj | DNA phylogeny supports revised 812 classification of Salmothymus obtusirostris[END_REF][START_REF] Sušnik | Genetic and morphological characterization of a Lake 825 Ohrid endemic, Salmo (Acantholingua) ohridanus with a comparison to sympatric Salmo trutta[END_REF][START_REF] Snoj | Genetic architecture of trout 807 from Albania as revealed by mtDNA control region variation[END_REF] as is S. salar. At the same time, 512 other morphological species have been returned to the S. trutta complex (S. platycephalus: Sušnik et al., 513 2004;S. dentex: Snoj et al., 2010). 514

Most molecular publications apply an exclusivity criterion which is the reciprocal monophyly, the 515 basis of the phylogenetic species concept [START_REF] Hebert | Biological identifications through DNA 697 barcodes[END_REF][START_REF] Sites | Empirical criteria for delimiting species[END_REF], so that taxonomy 516 is as close as possible to the phylogeny. However, discrepancies between morphology and genetics 517 (especially for recent species) should be explained by the expected discrepancy between gene history 518 [START_REF] Avise | Phylogeography -The history and formation of species[END_REF] and/or gene phylogeny and species phylogeny [START_REF] Knowles | Delimiting species without monophyletic gene trees[END_REF]. 519

According to [START_REF] Moritz | Defining evolutionarily significant units for conservation[END_REF] and to the arguments given here, conservation units should be management 520 units (MUs), i.e. conservation units based on molecular assessments at the population level, without 521 phylogenetic prerequisites, considering population assemblages forming units that deserve distinct 522 management for conservation. 523

In the Tyrrhenian region, several attempts at nomenclature have been made. The proposition of 524 [START_REF] Kottelat | Handbook of European freshwater fishes[END_REF] to retain the name S. cettii for the whole Tyrrhenian region ignores the local 525 high diversity of trout populations and numerous genetic sub-groups observed with two molecular markers 526 (Fig. 2, 3 and4). Trout in the Tyrrhenian Sea region were rarely investigated with molecular methods and 527 never as a whole in order to distinguish the different taxa and conservation units, although these data are 528 necessary for intelligent management and prioritization of stock preservation. The present survey 529 demonstrated that several categories of molecular markers are necessary for this. Crossing the well visible 530 clusters in Fig. 2, 3a and 3b and the distinct lineages obtained by assignment (Fig. 4), the following 531 significantly differentiated MUs, can be proposed: 532 The northern islands host precious MUs (not seen elsewhere) with special mention of Corsica, where 551 the relative good health of very small populations suggests that dozens of MUs need protection. Sardinia 552 is similar except that very few trout populations remain for investigation with molecular markers: two 553

MUs could be defined in the Cixerri basin. Sicily is similar to Sardinia since only the south of the island is 554 populated by small populations, but at least two MUs were detected. We do not know if the limited trout 555 range in Sicily is natural or due to human activity. 556

Populations from continental Italy appear less structured, probably as a consequence of both fewer 557 sampling efforts and a strong influence of genetic introgression with the Atlantic genome of domestic 558 origin. Here, therefore, on one hand, two MUs are still recognizable (in central and south Tyrrhenian 559 Italy). On the other hand, caution should be taken for estimation of the number of MUs detected. In future, 560 more effort should be made to include samples from other parts of Tyrrhenian Italy where the historical 561 presence of native brown trout genetic diversity has been highlighted (e.g, [START_REF] Splendiani | Museum samples could help to reconstruct the original 818 distribution of Salmo trutta complex in Italy[END_REF]Fabiani 562 et al., 2018). 563

The maintenance of these natural lineages, some without domestic introgression, allows optimism 564 regarding conservation of Tyrrhenian trout, which is indispensable for their role in freshwater biodiversity. 565

In view of the great genetic diversity that characterizes Mediterranean trout and the risks of undesirable 566 genetic homogenization by domestic forms, an intelligent conservation strategy should never be carried 567 out without preliminary genetic description of the populations involved in conservation actions. 568

Unfortunately, the number of conservation programs carried out without any kind of control is becoming 569 more and more frequent. but weighted depending on the sample size [START_REF] Nei | Estimation of average heterozygosity and genetic distance from a small number of 735 individuals[END_REF], Ho = observed heterozygosity, A = mean 890 number of allele by locus. Values of Fis between parentheses have no real sense as several samples 891 were grouped to obtain a sample size over 10. 892 Table 5: Double matrix indicating the Fst (Ɵ, [START_REF] Weir | Estimating F-statistics for the analysis of population structure[END_REF] values (upper triangle) and their 893 significance (lower triangle). All Fst were very highly significant ***. After Bonferroni correction, 894 only the level of significance was reduced, indicated between parentheses, for some comparisons 895 simply significant (*) or highly significant (**). Med. = Roya River flowing to the Ligurian Sea, Dom. 896 = domestic Atlantic commercial strain. Numbers refer to the first column of Table 1 andFig shown). The station numbers refer to the first column of Table 1. 903 River ME and the Anapo River AT samples. Black ellipse = true domestic Atlantic trout (samples 10, 918 11, 15, 16, 18, 23 and 30). Red ellipse = Calabrian sample containing both wild and domestic trout 919 (samples 20 to 26). Lower Tiber = populations close to Rome. The upper Tiber gathers the two 920 domestic strains from the hatcheries Cantiano and Visso (samples 15 and 16). South Sicily samples are 921

Manghisi and San Marco (28 and 29). 922 White lines separate the sampling sites. Cluster names correspond to river names or to regions. In order 924 to link each cluster to the elementary sample(s) involved, the numbers from the first column in Table 1 925 are indicated at the left of the figure -first histogram. The most probable K for the analyzed samples 926 shown in the arrows is based on the ΔK method; no further structures were detected in subsequent 927 rounds (after the sixth step) and within the excluded clusters (K = 1). Arrows delineate the progress of 928 the hierarchical approach, where subsets of the data were subsequently analyzed. Corresponding 929 mtDNA (control region haplotypes) and LDH-C1* alleles are indicated at the right of the figure. 930 931 Table S2: Hierarchical steps in estimating K (the number of genetic clusters) from STRUCTURE runs 935 using the ΔK method. L(K) -posterior probability of K; stdev -standard deviation of L(K) from seven 936 independent runs; ΔK -an ad hoc quantity, predictor of the real number of clusters (Evanno et al., 937 2005), best ΔK are highlighted. 938 Table S3: Detection of fullsibs in Corsican and Sardinian samples. 939 DNA data. White lines separate sampling sites, the most probable K = 19 is based on maximizing the 947 mean estimated ln probability of data [START_REF] Pritchard | Inference of population structure using multilocus 770 genotype data[END_REF]. Names and codes of sampling 948 sites/clusters are reported in Table 1 and4 andFigure 3 Ind. 4 Ind. 5 Ind. 6 Ind. 7 Ind. 8 Ind. 9 Ind. 10 Ind. 11 Ind. 12 Ind. 13 Ind. 14 Ind. 15 Ind. 16 Ind. 17 Ind. 18 Ind. 19 Ind. 20 Ind. 21 1 1.0000 1.0000 FUR01 FUR02 FUR03 FUR04 FUR05 FUR06 FUR07 FUR08 FUR09 FUR10 FUR11 FUR12 FUR14 FUR15 FUR16 FUR17 FUR19 FUR20 2 1.0000 0.1236 FUR13 FUR18 3 1.0000 1.0000 TAS01 TAS02 TAS03 TAS04 TAS05 TAS06 TAS07 TAS08 TAS09 TAS10 TAS11 TAS12 TAS13 TAS14 TAS15 TAS16 TAS17 TAS18 TAS19

  (a), V. Caputo Barucchi (b), A. Splendiani (b), S. Muracciole (c), A. Sabatini (d), F. Palmas (d), 5 C. Tougard (a), M. Arculeo (e), S. Marić (f). 6 7 (a) ISEM, Université de Montpellier, CNRS, IRD, EPHE, Montpellier, France. 8 (b) Dipartimento di Scienze della Vita e dell'Ambiente, Universita Politecnica delle Marche, Ancona, 9

  144 mtDNA CR sequences were obtained in this survey corresponding to sixteen different 219 haplotypes, seven of which are undescribed so far (AD-Tyrrh1 to 6 and AT-Tyrrh1: Tables3 and S1for 220 distribution and accession numbers). The alignment length was 987 bp providing 34 parsimony 221 informative sites. Haplotype classification allows the relationships between Tyrrhenian populations and 222 the five main known lineages widespread around Europe plus the DU junior lineage to be understood. The 223 network (Fig. 2) represents the haplotype organization within the Tyrrhenian region, with GenBank 224 published haplotypes used as references. Tyrrhenian haplotypes all belong to AD or AT lineages 225 exclusively. 226

  . Hatchery samples were nearly all characterized by the LDH-C1*90 allele except for one 261 *90/100 heterozygote genotype among the 20 Cantiano hatchery trouts (sample 15). This hatchery *90 262 allele was also found in 8/8 in the Allaro River and 7/10 in the Diga Giulia River, both in Calabria, and 263 just 1/10 in the Anapo River in Sicily. The domestic allele LDH-C1*90 was not found in Corsica (but only 264 four fishes analyzed) nor in the Manghisi River in Sicily (five trouts). over ten specimens (20 of the 30 considered), the Micro-Checker software 269 made no drop-out detection, two tests among 240 had suspect stuttering and 17 among 240 suggested the 270 occurrence of null alleles. The 17 possible null allele detections were spread among 8 of the 12 loci, with 271 0 to 3 cases each, showing that there was no systematic presence of null alleles in a given marker, 272 probably not disturbing the calculations. 273 Parentage analyses performed with COLONY software detected one to three families in each 323 autochthonous Corsican or Sardinian population. However, in north-east Sardinian populations, the 324

  and mitochondrial markers provided a rather clear structure of the Tyrrhenian (and 383 Ionian) trout populations. Very high inter-population diversity was observed in Corsica. A double north-384 south cline was detected: (i) high inter-population differentiation in the north (mainly Corsica) and lower 385 in the south (ii) predominance of the Adriatic lineage in the north, which decreased towards the south due 386 to stocking in Sardinia, the Tiber basin and Calabria and because of the natural Atlantic lineage settlement 387 in Sicily. 388 389 Origin of Tyrrhenian trout diversity 390 391

  The eight Corsican samples (U Furcone, A Tassineta, Aqua d'Acelli, E Maghjine, Speloncellu, Pozzi, 533 Val d'Ese and Lataga Rivers) and the three Sardinian samples of the Cixerri basin divided into two MUs 534 (Marroccu and Is Abius-Camboni) constitute ten MUs, genetically clearly different, mostly grouped in 535 cluster A of Fig. 4. They constitute the island part of the Tyrrhenian trout diversity. The detection of these 536 MUs allows much more isolated and differentiated populations to be described in Corsica. These island 537 MUs are almost not subject to domestic admixture. 538 Secondly, continental Italy revealed only two MUs positioned in cluster B1 of Fig. 4, i.e. dominated by 539 natural trout: i) the Nera River (sample 17, central Italy) and ii) the Ancinale River (sample 22, Calabria), 540 which seems to be the only local population dominated by a natural lineage. 541Third, the Sicilian trouts, confirmed in their natural AT lineage, are nevertheless separated into two 542 MUs: i) the well-known Anapo River[START_REF] Schöffmann | Phylogenetic origin of Salmo trutta L 1758 from Sicily, based 790 on mitochondrial and nuclear DNA analyses[END_REF] characterized by haplotype ATSic and ii) 543 the pure wild populations of Manghisi and San Marco with the "clone JE1" haplotype, although the San 544 Marco River specimens were not haplotyped. whole for the Tyrrhenian Sea region, trout have revealed their geographical genetic 549 organization. 550

Table 1 :Table 2 :Table 3 :Table 4 :

 1234 Bernard Lasserre, Sophie Dubois, Yuki Minegishi and Zhaojun Shao for laboratory 574 technical help, Pietro Armenia, Giuseppe Pisani et Andrea Belluscio for field help and David Schikorski 575 (of the private company Labofarm-Genindexe -France) for genotyping most of the considered trouts. 576 Thank you to Martin Laporte and Séverine Roques who participated to statistical solving. Some of the 577 samples were constituted by the French anglers' Federations of Alpes Maritimes and Corsica and the 578 Genesalm project (CIPA). They are warmly thanked. Sicilian samples were collected with permission 579 from the Regional Province of Syracuse and the professional assistance of Pietro Armena. In Calabria and 580 Lazio (Rome) corresponding managers (Giuseppe Pisani in Calabria and Andrea Belluscio in Lazio) 581 linked us with professional fishermen for sampling. We thank them for their irreplaceable assistance. The 582 authors would also like to acknowledge OSU-OREME and Juliette Fabre for their help in database 583 construction and mapping. Saša Marić was supported by the Ministry of Education, Science and 584 Technological Development of the Republic of Serbia (Grant No. 173045). Details about the trout samples. Italian hatcheries were linked to the host river because a possible 879 exchange was expected. The French hatchery has a national dispatch and no link with a river. Map = 880 see Fig. 1, N = number of analyzed individuals, MS = microsatellites, mtDNA = control region 881 haplotype, LDH-C1* = enzyme gene RFLP. FR=France, IT=Italy, AM=Alpes-Maritimes, a southern 882 France region/area, W. Med=Western Mediterranean basin. Very small samples are due to low density 883 of natural populations or limited capture authorization. In the calculations, they are grouped by region. 884 List of the twelve microsatellites genotyped. Their grouping into three multiplexes and their 885 original publication are indicated. 886 Control region haplotypes and LDH-C1* allele distribution. Map = see Fig. 1. 887 Parameters estimating the genetic diversity and the panmixia. Map = see Fig. 1, He = calculated 888 heterozygosity under the hypothesis of Hardy-Weinberg Equilibrium, Hnb = similar to He parameter 889

Fig. 1 :

 1 Figure captions 900

Fig. 2 :Fig. 3 :

 23 Fig. 2: Network presenting the relationships between Tyrrhenian control region sequences (colored 904

Fig. 4 :

 4 Fig. 4: Population structure as inferred by hierarchical STRUCTURE analysis of microsatellite data. 923

Fig. S4 :

 S4 Fig. S4:New projection of the FCA analysis presented in Fig.3aaccording to axes 1 and 3. In this new 940

  Figure 1

  0.29 0.26 0.46 0.35 0.37 A (18 to 19) (**) *** *** *** *** *** *** *** *** *** *** *** (**) *** (**) ***

Table 1 :

 1 Details on the trout samples. Italian hatcheries were linked to the host river because a possible exchange is 978 expected. The French hatchery has a national dispatch and no link with a river. Map = see Fig. 1, N = number of 979 analyzed individuals, MS = microsatellites, mtDNA = control region haplotype, LDH-C1* = Enzyme gene 980 RFLP. FR=France, IT=Italy, AM=Alpes-Maritimes, a southern France region/area, W. Med=Western 981 Mediterranean basin. Very small samples are due to low density of natural populations or limited capture 982 authorization. In the calculations, they are grouped by region.

	983
	Map River or hatchery	Watershed	Sea	Region (country)	Date	N MS	N mtDNA	N LDH-C1*
	1	Roya	Roya	Ligurian	AM (FR)	2007	20	0	-
	2	U Furcone	Luri	Tyrrenian	Corsica (FR)	2011	20	6	-
	3	A Tassineta	Golo	Tyrrenian	Corsica (FR)	2012	19	7	-
	4	E Maghjine	Fango	W. Med.	Corsica (FR)	2012	21	7	4
	5	Speloncellu	Tavignanu	Tyrrenian	Corsica (FR)	2013	20	7	-
	6	Pozzi	Fium'Orbu	Tyrrenian	Corsica (FR)	2004	20	11	-
	7	Val d'Ese	Prunelli	W. Med.	Corsica (FR)	2004	20	5	-
	8	Acqua d'Acelli	Travo	Tyrrenian	Corsica (FR)	2013	20	5	-
	9	Lataga	Ortolo	W. Med.	Corsica (FR)	2005	20	4	-
	10	Ermolinus	Flumendosa	Tyrrenian	Sardinia (IT)	2007	17	7	-
	11	Sadali	Flumendosa	Tyrrenian	Sardinia (IT)	2009	14	10	-
	12	Marroccu	Cixerri	Tyrrenian	Sardinia (IT)	2007	20	4	-
	13	Is Abius	Cixerri	Tyrrenian	Sardinia (IT)	2009	12	5	-
	14	Camboni	Cixerri	Tyrrenian	Sardinia (IT)	2009	19	0	-
	15	Cantiano hatchery	Tiber	Tyrrenian	Marche (IT)	2006	19	8	20
	16	Visso hatchery	Tiber	Tyrrenian	Marche (IT)	2000	20	16	40
	17	Nera	Tiber	Tyrrenian	Marche (IT)	2006	20	8	-
	18	Simbrivio	Tiber	Tyrrenian	Lazio (IT)	2014	9	3	-
	19	Aniene	Tiber	Tyrrenian	Lazio (IT)	2014	7	3	-
	20	Rotta	Ancinale	Ionian	Calabria (IT)	2013	3	0	-
	21	Carusi	Ancinale	Ionian	Calabria (IT)	2013	5	0	-
	22	Ancinale	Ancinale	Ionian	Calabria (IT)	2013	4	3	-
	23	Allaro	Alaro	Ionian	Calabria (IT)	2013	5	3	4
	24	Mula	Assi	Ionian	Calabria (IT)	2013	4	0	-
	25	Diga Giulia	Stilaro	Ionian	Calabria (IT)	2013	7	5	5
	26	Precariti	Precariti	Ionian	Calabria (IT)	2013	5	0	-
	27	Anapo	Anapo	Ionian	Sicily (IT)	2013	17	7	5
	28	Manghisi	Casibile	Ionian	Sicily (IT)	2013	11	5	5
	29	San Marco	Asinaro	Ionian	Sicily (IT)	2013	6	0	-
	30	Isère hatchery	-	-	(FR)	2008	20	3	-
	984
	985

Table 2 .

 2 List of the 12 microsatellites genotyped, their grouping into three multiplexes and their original publication.

	986
	Locus	multiplex	Reference
	MST543	C	Presa et al., 1994
	MST85	B	Presa & Guyomard 1996
	Omm1105	A	Rexroad et al., 2002
	OMY21DIAS	B	Holm & Bendixen 2000
	Oneµ9	C	Scribner et al., 1996
	Sfo1	A	Angers et al., 1995
	Ssa197	A	O'Reilly et al., 1996
	SsoSL311	C	Slettan et al., 1995
	SSOSL417	A	Slettan et al., 1995
	SSOSL438	C	Slettan et al., 1996
	STR591	B	Presa & Guyomard 1996
	STRBS131	C	Charles et al., 2005
	987
	988

Table 3 .

 3 Control region haplotypes and LDH-C1* allele distribution. Map = see Fig. 1.

	989

Table 4 .

 4 Parameters estimating the genetic diversity and the panmixia. Map = see Fig.1, He = calculated 992 heterozygosity under the hypothesis of Hardy-Weinberg Equilibrium, Hnb = similar to He parameter but 993 weighted depending on the sample size[START_REF] Nei | Estimation of average heterozygosity and genetic distance from a small number of 735 individuals[END_REF], Ho = observed heterozygosity, A = mean number of allele by 994 locus. Values of Fis between parentheses have no real sense since several samples have been grouped for a

	995
	996	sample size over 10.
		Map	River or hatchery	N	He	Hnb	Ho	A	Fis	Fis signif.
		1	Roya	20	0.63	0.65	0.55	7.75	0.164	***
		2	U Furcone	20	0.21	0.22	0.20	2.92	0.075	ns
		3	A Tassineta	19	0.15	0.15	0.18	1.42	-0.148	ns
		4	E Maghjine	21	0.01	0.10	0.09	1.50	0.144	ns
		5	Speloncellu	20	0.20	0.20	0.18	1.75	0.113	ns
		6	Pozzi	20	0.23	0.23	0.21	2.08	0.104	ns
		7	Val d'Ese	20	0.25	0.26	0.28	2.17	-0.094	ns
		8	Acqua d'Acelli	20	0.08	0.08	0.08	1.75	-0.041	ns
		9	Lataga	20	0.08	0.08	0.07	1.58	0.083	ns
		10	Ermolinus	17	0.72	0.74	0.67	6.25	0.102	ns
		11	Sadali	14	0.76	0.79	0.78	7.17	0.008	ns
		12	Marroccu	20	0.44	0.45	0.40	3.25	0.118	**
		13	Is Abius	12	0.40	0.41	0.47	2.58	-0.133	ns
		14	Camboni	19	0.49	0.51	0.51	4.42	-0.009	ns
		15	Cantiano hatchery	19	0.73	0.75	0.73	7.42	0.031	ns
		16	Visso hatchery	20	0.76	0.78	0.75	8.83	0.029	ns
		17	Nera	20	0.49	0.51	0.47	5.83	0.063	ns
		18&19	lower Tiber	16	0.72	0.75	0.62	6.42	(0.177)	ns
		20to22	Ancinale	12	0.59	0.62	0.57	5.00	(0.082)	ns
		23to26	lower Calabria	21	0.76	0.78	0.55	8.33	(0.310)	(***)
		27	Anapo	17	0.48	0.49	0.47	4.67	0.046	ns
		28&29	south Sicily	17	0.57	0.59	0.49	6.33	(0.182)	(***)
		30	Isere hatchery	20	0.65	0.67	0.67	5.75	0.007	ns
	997
	998

Table 5 .

 5 Double matrix indicating the Fst (Ɵ,[START_REF] Weir | Estimating F-statistics for the analysis of population structure[END_REF] values (upper triangle) and their 999 significance (lower triangle). All Fst were very highly significant ***. After Bonferroni correction, only the level 1000 of significance was reduced, indicated between parentheses, for some comparisons simply significant (*) or 1001 highly significant (**). Med. = Roya River flowing to the Ligurian Sea, Dom. = domestic Atlantic commercial 1002 strain. Numbers refer to the first column of Table1 and Fig. 1. A, B, C and D are grouped samples in order to 1003 reach sample sizes of 10 and more.

	1004

Table S1

 S1

	Haplotype	Accession number		Distribution -country/drainages
			3 Denmark (Skals), Norway (Bjornes Lake, Sima), Spain (hatchery
	haplotype 1 (ATcs1) AF273086	stocks), (Skorradalsvatn), British Isles (Coquet, Wear, Lune, Melvin), 5 Spain (Garona), France (Gulf of Biscay), Iceland
			21 continental Italy (IT), Sardinia, Corsica
			3 Denmark (Skals, Karup), Norway (Guddal, Sima), Spain (hatchery
	haplotype 2 (ATcs2) AF273087	stocks), Fowey, Teifi, Conwy, Loch Romoch), Russia (Nilima, Vorobiev), 5 France (Gulf of Biscay), British Isles (Coquet, Stour, Rother,
			21 Sardinia, Corsica
			3 Denmark (Skals), Norway (Bjornes Lake, Guddal, Sima), Spain
	haplotype 3 (ATcs3) AF274574	(hatchery stocks), Isles (Coquet, Wear, Rother, Teifi, Conwy, Melvin), 5 Spain (Garona), France (Gulf of Biscay), British 21 continental
			Italy, Sardinia, Calabria,
			3 Denmark (Skals, Karup), Norway (Bjornes Lake, Guddal, Sima),
	haplotype 4 (ATcs4) AF274575	Spain (hatchery stocks),	5 France (Gulf of Biscay), British Isles
			(Lune),	21 Sardinia, Calabria.
	ATSic	JF297974	13 Sicily (Ánapo)
	clone JE1	AF253557	14 Iberian (South European Atlantic region),	21 Sicily (Casibile)
	*AT-Tyrrh1	KX450263	21 Italy hatchery stock (Cantino, Marche region)
	ATcs14	EF530476	5 Iceland (Skorradalsvatn)
	ATcs33	EF530495	5 Spain (Minho, Atlantic Ocean, Cantabric Sea)
	ATcs35	EF530497	5 Spain (Atlantic Ocean)
	ATcs37	EF530499	5 Spain (Atlantic Ocean)
	ATcs38	EF530500	5 Spain (Atlantic Ocean)
	ATcs47	EF530507	5 Iceland (Skorradalsvatn)
	ATcs50	EF530510	5 British Isles (Stour)
	ATcs51	EF530511	5 British Isles (Teifi)
	ATcs52	EF530512	5 British Isles (Teifi)
	ADcs1	AY836330	4 Spain (Ter, Ebre, Túria, Segura), Maritza), 2,9 Macedonia (Prespa Lake, Vardar) 8 Bulgaria (Struma, Mesta,
	ADcs15	AY836344	4 France -Corsica (Corsica stream)
	A_2	KM216129	16 Sardinia (Pula)
	*AD-Tyrrh1	KX450257 21 Corsica, Sardinia, Calabria, continental Italy (Aniene River)
	*AD-Tyrrh2	KX450258 21 Corsica, continental Italy (Aniene River)
	*AD-Tyrrh3	KX450259	21 Calabria (Diga Giulia River)
	*AD-Tyrrh4	KX450260	21 Sardinia, continental Italy (Aniene River)
	*AD-Tyrrh5	KX450261	21 continental Italy (Nera River)

Table S2

 S2 Hierarchical steps in estimating K (the number of genetic clusters) from STRUCTURE runs 1083 using the ΔK method. L(K) -posterior probability of K; stdev -standard deviation of L(K) from seven 1084 independent runs; ΔK -an ad hoc quantity, predictor of the real number of clusters[START_REF] Evanno | Detecting the number of clusters of individuals using the 667 software STRUCTURE: a simulation study[END_REF], 1085 best ΔK are highlighted. In the second step of analysis on the Corse-Sardinian dataset (A Tassineta, E 1086 Maghjine, Speloncellu, Pozzi, Val d'Ese, Acqua d'Acelli, Lataga, Marroccu, Is Abius & Camboni), ΔK 1087value was strongly affected by two outlier runs for K = 7, which are discarded from the analysis.TableS3Parentage analyses performed with COLONY for Corsican and Sardinian samples. We have 1094 chosen the Pair-Likelihood-Score (PLS)/Full-Likelihood (FL) combined (=FPLS) algorithm and used only 1095 full-sibs listing to give the results below. In Corsica, each population shows only one to three families. In fact, the polymorphism is so low 1099 (0.08<Hnb<0.26) that we can deduce that these small populations, totally isolated, have suffered several 1100 drastic recent bottlenecks. We can also suppose that one or two pairs of parents have recently re-funded 1101 each population making naturally each sample 1 to 3 families. The other interpretation is that such low 1102 polymorphism does not allow families detection.

	1096
	1097
	1088	FullSibs families	Prob(Inc.) Prob(Exc.) Ind. 1 Ind. 2 Ind.
	1089
			K L(K)	stdev ΔK
	1098
	A. 1103
	1104
	1105

Acknowledgements

573

Supporting information 932

Table S1: Accession numbers of all haplotypes positioned in Fig. 2 (* = new), completed with their 933 geographic distribution according to twenty main publications. Da1a (Iran 3) AY185568 1,6 Austria (Drau, Inn, Kamp, Danube), 8 Bulgaria (Cerny Iskar), Serbia (Džepska River), 19 Iran (Tonekabon) Da2a (Da2) GQ284834 1,6 Austria (Inn, Mur, Drau), 10 Italy (Adige) Da3 AY185571 6,20 Austria (Kamp) Da22 AY185573 1,6,20 Austria (Inn, Kamp, Enns, Drau, Danube), 9 Macedonia (Vardar), 10 Italy (Adige), 18 Bosnia and Herzegovina (Una) Da23a AY185574 6,20 Austria (Kamp)