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All issues No 423 (2022) Knowl. Manag. Aquat. Ecosyst., 423 (2022) 24 Full HTML
Conservation genetics
Open Access
Issue
Knowl. Manag. Aquat. Ecosyst.
Number 423, 2022
Conservation genetics
Article Number 24
Number of page(s) 20
DOI https://doi.org/10.1051/kmae/2022022
Published online 25 November 2022

© P. Berrebi et al., Published by EDP Sciences 2022

Licence Creative CommonsThis is an Open Access article distributed under the terms of the Creative Commons Attribution License CC-BY-ND (https://creativecommons.org/licenses/by-nd/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. If you remix, transform, or build upon the material, you may not distribute the modified material.

1 Introduction

The brown trout Salmo trutta complex is, in its general acceptation, a diversified assemblage of salmonids whose native area covers Europe, Western Asia and North Africa. It has been studied for the last two centuries using multiple methods but reaching no clear agreement about its taxonomic organization (Guinand et al., 2021; Tougard 2022). These taxonomic studies (1758–2007) were compiled by Kottelat and Freyhof (2007), resulting in the proposal of 28 species. After subsequent publications, there are more than 50 species (Delling, 2010; Turan et al., 2009, 2011, 2012, 2014a, 2014b, 2017, 2020, 2021, 2022; Doadrio et al., 2015; Segherloo et al., 2021). The reasons of this taxonomic disorder and “inflation” (Isaac et al., 2004; Zachos et al., 2013; Guinand et al., 2021) are first the diversity of ecological and geographic morphs within this group, the consideration of evolutionary lineages as distinct species and the use of multiple species concepts (see Kottelat, 1997; Guinand et al., 2021).

Brown trout populations frequently show distinct sedentary and anadromous behaviors in sympatry in north western Atlantic rivers of their native area (Behnke 1972; Dorefeyeva et al., 1981; Charles et al., 2005). Similarly, an allacustrine formis is observed, migrating to lakes. The migratory form differs from the resident ecotype by the life cycle, their morphological (especially the large body size), demographic and ecological characteristics (Frost and Brown, 1967; Campbell, 1977; Baglinière et al., 2001). Some taxa from several European locations were first described as species according to their migration behavior, starting with Linnaeus (1758), who described two resident species (S. trutta Linnaeus, 1758 and S. fario Linnaeus, 1758 according to the color patterns and the mandible length), one anadromous (S. eriox Linnaeus, 1758) and one lacustrine species (S. lacustris Linnaeus, 1758).These forms were therefore considered distinct species until recently (e.g. Mills, 1971; Elliott, 1994) despite the early opposite opinion of Berg (1948), who considered these taxa to be the same species: S. trutta. Many ichthyologists later recognized them as subspecies: S. trutta fario, S. trutta trutta and S. trutta lacustris (e.g., Whitehead et al., 1984; Lelek, 1987). However, several studies demonstrated that they are in fact ecotypes of the same population, thus of the same species. Indeed, genetic analyses revealed no difference between anadromous and resident trout of the same river (Fleming, 1983; Skaala and Naevdal, 1989), so they belong to the same population in a given watershed. Moreover, a population of one ecotype can produce progeny developing the other form (Skrochowska, 1969; Ombredane et al., 1996; Dębowski et al., 1999). This has been especially obvious in Kerguelen Islands where domestic sedentary Atlantic trout were introduced in troutless rivers, and anadromous forms appeared (Guyomard et al., 1984, Lecomte et al., 2013). It has also been demonstrated that distinct ecotypes in a given river do not develop heritable behavioral differentiation (Charles et al., 2005; Ferguson et al., 2017, 2019) and that these ecotypes are flexible with a continuum of life cycles, from purely resident to anadromous, with intermediate tactics along a continuum in time (age to reproduce) and space (distance of migration within the watershed up to the sea) (Cucherousset et al., 2005).

The most consensual genetic structure description for the whole brown trout complex is based on mitochondrial DNA (mtDNA) sequences (Bernatchez et al., 1992) and especially that of the Dloop, in the Control Region (CR). This marker discriminates seven main clusters: Danubian (DA), Atlantic (AT), Duero, (DU), Dadès, North Adriatic (MA; the marble trout), Adriatic (AD), Mediterranean (ME) and the North African (NA) lineages (Bernatchez et al., 1992; Giuffra et al., 1994; Weiss et al., 2000; Bernatchez, 2001; Cortey and Garcia-Marin, 2002; Cortey et al., 2004, 2009; Snoj et al., 2011; Tougard et al., 2018; Sanz, 2018). The first molecular phylogenies grouped all the western Mediterranean lineages (MA+AD+ME), however they disagreed on the next closer lineage (DA in Bernatchez et al., 1992 and in Cortey et al., 2004; AT in Bernatchez, 2001).

The Rhône River is a French river drainage, flowing southward to the Mediterranean and known to harbor mainly the ME lineage (Bernatchez et al., 1992; Bernatchez, 2001; Reynaud et al., 2011). It is composed of dozens of large sub basins, mainly characterized by Mediterranean flow regimes (overflow in spring and autumn, low waters in summer and winter) and of mountainous flow regime (overflow in late spring when snow melts). Trout populations living there are highly diversified (Berrebi and Cherbonnel, 2009; Berrebi and Schikorski, 2016). The Mediterranean trout is deeply sedentary: some studies determined its range of movement to be less than 50 km (Berrebi and Shao, 2009).

With the successive dam buildings near the Rhône delta (Fig. 1) at Donzère-Mondragon (1952) and Vallabrègues (1970), several anadromous species such as the river lamprey Lampetra fluviatilis (Linnaeus, 1758) or the European sturgeon Acipenser sturio Linnaeus, 1758 have disappeared upstream (Keith et al., 1992; Chassaing et al., 2016). Anadromous trout are observed, but very rarely, in the Rhône drainage (Audouin and Maurin, 1958; Le Gurun et al., 2012). A few specimens are sometimes caught near the Rhône delta (Camargue wetland) and in other coastal Mediterranean catchments, like the Var River, or captured by trawl nets at sea (Didry, 1953; Audouin and Maurin, 1958; Spillmann, 1961; local fishermen personal communications). Iglésias et al. (2021) recorded a similar capture of trout at sea, but this is probably a resident specimen washed out to the sea (recognizable by its general punctuation including the dorsal fin and indented caudal fin). Didry (1953) affirmed that Mediterranean trout can easily migrate from a coastal drainage to another one through the sea but provided no supporting data. On the other hands Spillmann (1961) claimed that restocking in headwaters of these coastal Mediterranean drainages from Danish eggs explained the presence of sea trout in this area. As anadromous species are the most threatened taxa (UICN Comité Français et al., 2019), essentially due to the presence of dams preventing their migrations, degrading local environmental conditions and fragmentation of the hydrographic networks (Merg et al., 2020), all diadromous fish species are attentively monitored by riverine managers. Thus, as a diadromous species, sea trout management is driven by the French code of the environment, establishing rules for its fishing (i.e. open fishing season, minimum catch size, catch quota per year and fisherman; art. R436-44 of the 5 August 2005).

The aim of this study is to assign sea trout caught in French Mediterranean rivers using molecular data to their closest genetic lineage, in order to clarify the mysterious identity of these large-bodied trout going up the Rhône River and often blocked downstream of the dams. Two hypotheses will be tested: (i) the sea trout are genetically similar to some natural Mediterranean populations of the Rhône basin (if Mediterranean trout can spontaneously develop an anadromous strategy) or (ii) they are similar to hatchery stocks and come from restocking with Atlantic domestic trout. This determination will provide key information to protect this Mediterranean population of sea trout.

thumbnail Fig. 1

Geographic localization of the samples. Red letters = sea trout individuals (Tab. 2), blue numbers = comparative samples (Tab. 1). Trout 1d is missing because of unknown origin. D1 and D2 = large downstream dams (Donzère-Mondragon and Vallabrègues dams, respectively). Hatcheries are not represented.

2 Materials and methods

2.1 Sampling rare specimens

Sea trout are reported from time to time along the main Rhône River bed, in some of its tributaries like the Sorgues, Veuze and Gardon Rivers, in the delta formed by its estuary, the Camargue wetland, and at sea, in trawl nets, off the coast of this Mediterranean zone. They have also been observed in another Mediterranean river of southern France: the Var River. The rarity of this category of trout is such that it cannot reliably be captured using scientific methods (e.g., programmed electric fishing). In our study, we considered 14 trout specimens caught by anglers or in rare occurrences by electrofishing between 2004 and 2007, in the Rhône and Var rivers. Among them, as an example, the 1b specimen, featured in Figure 2, was fished with a spinning spoon (i.e. artificial metallic bait) in the Gardon River, a tributary of the Rhône River. The Migrateurs-Rhône-Méditerranée association (MRM), a nongovernmental organization for the protection of the migratory fish of the Rhône River, has for several years collected and conserved some organs (eggs, digestive tracts, fin-clips or scales) from these sporadic captures. These tissues were conserved in more or less concentrated ethanol, in small or big jars, without guarantee as to the good preservation of the tissues or their DNA.

Specimens were identified morphologically according to several external criteria. Sea trout is differentiated from the resident trout by (i) a larger size in total length reaching easily 50 cm (vs. not exceeding 35 cm), (ii) a silver coat (vs. greenish to brownish skin coloration), even if in large rivers this typical coloration pattern tends to disappear in favor of being brownish, (iii) the presence of star-shaped black spots (vs. round red and black spots as well as 4 or 5 blackish vertical bands on the flanks) and (iv) the straight margin of caudal fin (vs. lightly forked) (Fig.2: Spillmann, 1961; Baglinière et al., 2000; Jonsson and Jonsson, 2007). As a last parameter for identification, the stations of capture of all sea trout (with the exception of specimen 1h) were outside the distribution of resident trout, strictly distributed in salmonid-favorable rivers whose mapping is well known in France.

In order to genetically characterize the putative sea trout of our sampling, other reference samples, already genotyped by the same team (grey literature), were added for comparison. These reference samples, belong to the Institut des Sciences de l'Evolution (Montpellier University, France) tissues collection and include numerous resident populations living in tributaries all along the Rhône River basin as well as commercial domestic strains bred in several French hatcheries for stocking (details in Tab. 1).

thumbnail Fig. 2

(A) Sea trout 1b (52 cm TL for 1745 g) fished with spinning spoon in the Gardon River in 2007. White arrows highlight morphological characters allowing the identification as sea trout: straight margin of caudal fin and the presence of star-shaped black spots. (B) Resident Mediterranean trout (28 cm TL, 460 g) electro-fished in the Sorgue River, left tributary of the Rhône River. This relatively large river trout shows a typical forked tail, black and red spots on the flanks and the dorsal fin, a yellow-brown skin color.
Table 1

Composition of the 27 samples considered in the present study.

2.2 DNA extraction, mitochondrial DNA and microsatellites amplifications

Total DNA was isolated from fin tissue preserved in 96% ethanol following the protocol of Medrano et al. (1990) for mtDNA sequencing and using Chelex 100 according to Walsh et al. (1991) method.

The complete CR (ca. 1100bp) was amplified following the protocol in Marić et al. (2012) using primers LRBT-25 (5‵-AGA GCG CCG GTG TTG TAA TC-3‵) and LRBT-1195 (5‵-GCT AGC GGG ACT TTC TAG GGT C-3‵; Uiblein et al., 2001). Purified PCR products were Sanger sequenced in one direction using primer LRBT-1195 (3‵-end) and BigDye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems, Inc.).

Primers of the seven microsatellite markers used in this study were obtained from the literature (Oneµ9, Ss0SL-311, Omy21DIAS, Mst543, Sfo1, Ssasl197 and OMM1105, see Tab. 2 for details). Microsatellite repeated sequences are dinucleotide except Ssa197 which is a tetranucleotide microsatellite (O'Reilly et al., 1996). For each marker, one of the 5‵ ends of the two primers was end-labeled with a fluorescent dye. Polymerase chain reactions (PCR) were performed using the Qiagen multiplex PCR kit in a final volume of10 μl, containing 3 μl of genomic DNA diluted at 10 ng/μl, 5 μl of Qiagen PCR Master Mix, 1 μl of Qiagen Q-solution, and 1 μl of primer mix at 2 μM each (Eurofins MWG Operon).

Amplifications and genotyping were carried out in a Gene Amp PCR System 2700 thermal cycler (Applied Biosystems), according to the supplier's instructions (Qiagen multiplex PCR kit): initial denaturation step (95 °C, 15 min), followed by 35 cycles of denaturation (94 °C, 30 s), annealing (55 °C for all loci, 90 s), and extension (72 °C, 60 s), with a final extension step (60 °C, 30 min). Amplified PCR fragments were then diluted and separated on an ABIPRISM 3130/xl/sequencer (Applied Biosystems) with Gene Scan 500 Rox dye size standards. Allele sizes were determined using the Gene Mapper v4.1 software system (Applied Biosystems, Life Technologies). A genotype matrix was then constructed and used as a basis for all the following statistical analyses.

Table 2

Characteristics of the seven microsatellite loci. The second column indicates the gathering of each locus to one of the three multiplexes developed.

2.3 Data processing

MtDNA partial CR sequences were aligned with haplotypes previously identified in the literature using the Muscle package (Edgar, 2004) in Mega X (Kumar et al., 2018). CR sequences of S. trutta haplotypes available in the literature (Cortey and García-Marín, 2002; Duftner et al., 2003; Cortey et al., 2004, 2009; Meraner et al., 2007; Snoj et al., 2011) as well as those of S. ohridanus and S. salar used as outgroups are reported in Appendix A with their GenBank accession numbers.

Partial CR diagnostic sites for each lineage were identified with the Quiddich package (Kühn and Haase, 2019) for R (R Core Team, 2013). Lineage affiliation using the entire reference sequences (1125 bp) was made with a phylogenetic tree by Bayesian inference (MrBayes 3.2, Ronquist et al., 2012), with the HKY+I+G model selected by JModelTest 2.1.1 (Darriba et al., 2012) according to Bayesian criteria. Bayesian analysis was performed launching two runs with 5 million generations and sampling every 100 generations. The subsequent tree files were summarized and 10% of trees eliminated as burn' in after checking for convergence. The analysis was reiterated twice cutting our alignment to our partial CR sequences lengths (532 bp and 325 bp) using respectively HKY+G and HKY+I models.

For microsatellites, standard parameters were calculated using the Genetix 4.05 software (Belkhir et al., 2004): the expected heterozygosity (He), corrected for sample sizes (Nei, 1978), the observed heterozygosity (Ho) and the mean number of alleles by locus (A). While the sea trout sample st01 is far from constituting a functional population (sampled over several years, in several locations, during migration stage), it is interesting to compare its global diversity with the diversity of the other river and hatchery samples. In order to estimate the sea trout genetic diversity, only Ho and A were calculated because these parameters do not require a Hardy-Weinberg equilibrium. For this, the Genetix software was used for the 26 comparative samples calculations. The allelic richness of a population, Ar, is the expected number of distinct alleles in a sample and can provide strict comparisons if the sample sized are equalized to the smaller one (rarefaction method, Kalinowski, 2004). The HP-Rare 1.0 software (Kalinowski, 2005) was used, reducing all samples to eight individuals.

A general picture of the trout genetic structure was first researched through multidimensional analyses on microsatellites data. Here, Factorial Correspondence Analyses (FCA: Benzécri, 1973) were carried out as implemented in Genetix, allowing the overall structure of the sampling to be explored. The clusters (or clouds) observed in the diagrams correspond to nuclear genetic homogeneous lineages. The mathematical method is detailed in She et al. (1987).

In order to detect differentiated subgroups, assignment tests using the Bayesian Structure 2.1 program (Pritchard et al., 2000), subdivided the whole sample into K subgroups (K is the number of biological subgroups tested within the entire sample), characterized by their best genetic equilibrium in terms of best panmixia and lower linkage disequilibrium. The admixture ancestry model and correlated allele frequencies option were chosen. A burn-in of 50,000 iterations followed by 100,000 additional Markov Chain Monte Carlo iterations were applied on 5 runs for each K value. Here K has been tested between 1 and 8. The DeltaK method (Evanno et al., 2005) was applied through Structure Harvester (Earl and von Holdt, 2012) in order to suggest the best K value in terms of likelihood. The objective of the test is to link the sea trout to one of the lineages included in the sampling: the domestic Atlantic lineage or one of the natural Mediterranean lineages present in the detailed sampling of the tributaries of the Rhône River.

3 Results

3.1 Degraded DNA amplification

Among the 14 sea trout tissues, nine provided amplifiable DNA: one sample of eggs and digestive tract, two samples of fin-clip and six samples of scales (Tab. 3). One trout (specimen 1c) was not used because of too much missing data.

The mtDNA CR marker was amplified on only three out of the nine specimens (sea trout 1f, 1g and 1i). All sequences were short, between 270 and 489 bp, and at the 3‵ end of the CR. All three DNA samples came from scales (Tab. 3).

Concerning microsatellites, we obtained complete genotypes from only five out of the nine specimens. For the following analyses on microsatellite data, only eight specimens were considered, showing at most two missing locus genotypes (Tab. 3).

Table 3

Genotypes and mtDNA sequences lengths (bp) of the sea trout samples with details on tissues used for DNA extraction (Ѡ = eggs, DT = digestive tract, F = fin, S = scales), locality, date of capture and total length. na = no amplification. Trout 1c, with 4 missing loci, has been removed.

3.2 MtDNA affiliation

Three short CR sequences (270 and 489 bp) were obtained and aligned with other reference sequences of the main lineages of S. trutta. The phylogenetic tree obtained by Bayesian inference (Fig. 3) follows mainly the one of Snoj et al. (2011) with an irresolution grouping AT, DU, DA and Dadès lineages, and a second one clustering AD, MA and ME lineages but distinguishing well this last one (Fig. 3). Our three partial sequences are placed in the AT+DU clade, and do not share diagnostic sites with the ME lineage (T in positions 878 and 961 vs. C and A in position 901 vs. G. See Appendix B). The two specimens 1g and 1i are clustered with the haplotype ATcs4 sharing the A in positions 908 and 937. The specimen 1f differs from the ATcs1, ATcs3, ATcs14, ATcs15, ATcs25, ATcs45, ATM2 haplotypes by only the T in position 548 (vs. C). The specimens 1f and 1h seem to correspond to unknown AT haplotypes. The three new sequences were deposited in GenBank with the accession numbers OP719777 to OP719779. The two phylogenetic trees on shorter sequences (532 bp and 325 bp) gave the same results (Appendix C).

thumbnail Fig. 3

Phylogenetic tree by Bayesian inference with the CR marker (1125 bp) on 124 sequences of Salmo trutta haplotypes. Colored blocks represent S. trutta lineages. White arrows designate the three Mediterranean sea trout 1f, 1g and 1i. Posterior probability values are indicated above the nodes.

3.3 Microsatellite genotyping information

Observed heterozygosity (Tab. 4) was 0.74 for the sea trout assemblage, 0.60 for the river populations (0.41 < Ho < 0.68) and 0.73 for the hatchery strains (0.55 < Ho < 0.82). The calculation of the A parameter gave 6.6 for the sea trout, 7.8 (5.4 < A < 10.9) for the river populations and 8.3 (5.5 < A < 12.14) for the hatchery ones. The eight sea trout samples are as diverse as hatchery samples and about 20% more diverse than the resident Mediterranean lineage.

In order to overcome the differences in samples sizes, rarefaction calculations allowed the homogenization of all samples to eight individuals. The expected number of alleles in the sea trout sample was 4.82; it was 4.36 in average for hatcheries and 3.91 in average for river samples. Among rivers, only one sample was more diverse than the sea trout sample: the population 8 of the Drac River flowing to the Isère River, an upstream left tributary of the Rhône River. Also, two hatcheries were shown more diverse than sea trout.

The numerous reference samples used (18 river samples from the Rhône watershed and 8 hatchery strains from France) allowed the multidimensional construction of a background map (Fig. 4), clustering Atlantic domestic strains at the left of the diagram and wild Mediterranean populations at its right. All eight sea trout are grouped in the domestic Atlantic zone of the hyperspace.

The DeltaK method (Evanno et al., 2005) suggested a partition into two groups (K = 2, obviously Atlantic and Mediterranean lineages). The separation between wild Mediterranean populations and domestic Atlantic strains permitted to clearly place all the sea trout in the domestic lineage (Fig. 5). Exploration of higher partitions (here up to K = 5) only allowed the distinction of several Mediterranean sub-lineages. For the Atlantic lineage, for K = 5, it remained a single unit and grouped sea trout with domestic Atlantic strains (st20 to st27) in Figure 5.

Table 4

Genetic diversity of the analyzed samples measured with: Expected heterozygosity (He, unbiased parameter of Nei 1978), Observed heterozygosity (Ho), mean number of alleles by locus (A) and Allele richness (Ar after rarefaction calculation). For the sea trout sample, He cannot be calculated because, composed of several independent captures, it does not represent a population. For each parameter, high values are indicated in grey.

thumbnail Fig. 4

The FCA (axis 1 horizontal and 2 vertical) presents a very clear classification of the genotypes of the 27 analyzed samples into two clusters: domestic Atlantic at the left and wild Mediterranean at the right. Legend at the right uses the numbers from the first column of Table 1: R samples with small circles = river sample; H with small triangles = hatchery sample; large circles represent sea trout (st01), with Rhône River in red, Var River in green; unknown origin (X = Rhône or Var River) in grey. In circles are given the number of missing locus genotypes among 7.
thumbnail Fig. 5

Different histograms produced by the STRUCTURE software after assignment analysis of the 649 sampled specimens (individual 1c has been removed). A = Assignment of the whole sampling to two expected subgroups (K = 2). B = STRUCTURE histogram when K = 5 showing the wild Mediterranean populations diversity (domestic Atlantic strains, in red, stay homogeneous).

4 Discussion

The present study addresses a category of trout that is hard − if not impossible − to sample using classical sampling approaches, making inferences complicated. Yet, the distinction between resident and anadromous forms is essential for conservation. The method used and the confidence that can be given to this determination condition the strength of the conclusions. The first element for determination was the overall morphology of the trout, as reported by the fishermen who brought the specimens to the MRM association: generally large, silvery skin with only a few cross-shaped spots, end of the tail straight (Spillmann, 1961; Baglinière et al., 2000; Jonsson and Jonsson, 2007). This morphology is only reported, not observed and measured by researchers. However, the MRM association has worked on diadromous fish species since nearby 30 years with collaboration networks all over the country with the National Federation of Angling in France (FNPF) and the French Biodiversity Agency (OFB) as well as several research labs. So, it has good knowledge in fish identification and is therefore a trustful source. The other important element is location. French rivers are classified as first category (environments favorable to salmonids, generally upstream of the rivers) and as second category (downstream of the rivers, water temperature in summer incompatible with the life of salmonids, cyprinids dominant). This is particularly true for the Rhône drainage (Changeux, 1995). All trout analyzed, except specimen 1 h, were caught in second category rivers, or even at sea. These characteristics as well as the molecular results obtained all point toward anadromous trout. In addition, the behavior observed by anglers (large trout “waiting” downstream from major dams) corresponds to anadromy.

4.1 Degraded DNA analysis

Among the 14 sea trout tissues, nine provided amplifiable DNA, three gave partial mtDNA CR sequences and eight gave microsatellites data, five completely genotyped at seven loci.

The incomplete molecular data is a sign that the DNA was probably not well preserved, coherent with an apparent state of degradation of several samples. It is known that dried tissues give better results than other materials stored in ethanol in uncontrolled conditions (Rowe et al., 2011). Dried DNA on scales can be preserved over 60 years for molecular studies (e.g., Nielsen et al., 1997, 1999) giving some perspectives of research including historical data (Nielsen and Hansen 2008; Levin et al., 2018). However, in the samples of the present study, DNA is degraded probably due to low ethanol concentration. Obtaining microsatellite data appears to be more efficient than obtaining short mtDNA sequences in this case.

4.2 Nature and origin of the Rhône River anadromous trout

Mitochondrial data on the three sequenced specimens affiliates them clearly, despite the short lengths of sequences, to the Atlantic lineage and not to the Mediterranean cluster.

Among the nine genotyped sea trout, two had more than one missing locus: individual 1i (2 missing locus genotypes) but also the individual 1d with 4 missing loci. This last individual has been discarded from the last analyses. The eight remaining sea trout were assigned to the Atlantic domestic trout lineage, according to microsatellites multidimensional (Fig. 4) and assignment (Fig. 5) analyses.

The genetic composition of the Rhône River anadromous trout contrasts with that of the resident populations sampled in tributaries all around the Rhône watershed that are mostly composed of wild Mediterranean trout (among the 425 trout belonging to the 18 Rhône River samples, 7 are stocked individuals and 30 are hybrids between Atlantic and resident trout, according to microsatellite genotypes).

All sea trout analyzed showed microsatellite genotypes and mitochondrial sequences of Atlantic origin. Because the Atlantic trout is not natural in the Rhône basin or in any Mediterranean river, it is most likely that these eight sea trout come from restocking. These results are similar to other molecular studies realized on sea trout from the Adriatic Sea; sea trout from this area were also assigned to stocked Atlantic strains from hatcheries (Snoj et al., 2002; Splendiani et al., 2020).

While not constituting a functional population, the assembled sea trout samples showed a high level of diversity, as generally observed in the commercial strains found everywhere in France and in other countries (Bohling et al., 2016; Berrebi et al., 2019). This provides another line of evidence for a domestic origin of sea trout. In hatcheries, this high diversity derives from the stock constitution more than thirty years ago by crossing several north European natural reproducers (Bohling et al., 2016; Berrebi et al., 2019).

As a first result, the Rhône sea trout is likely originating from stocking using the national (and international) commercial Atlantic strain, far from what can be seen in natural populations of the Rhône watershed. Because of its North European origins, the Atlantic domestic strain seems to produce migrating individuals first swimming to sea, crossing obstacles thanks to their small size at the smolt stage, then stopped by dams when returning for reproduction, due to the large size of the adults. According to Jorgensen and Berg (1991), Atlantic domestic trout are known to produce post-stocking downstream movements. Domestic stocked populations have showed such migratory behavior in other circumstances, as when introduced in Kerguelen Islands' troutless streams (Davaine and Beall, 1997; Lecomte et al., 2013). Similarly, after introduction in Newfoundland, Canada, of brown trout fertilized eggs shipped in 1883 from Stirling, Scotland and from Germany (Westley and Fleming, 2011), brown trout developed an invasive behavior. The dispersal of these trout was facilitated principally by anadromy along the north and south coasts of the Avalon Peninsula, expressing both anadromous and resident ecotypes (O'Toole et al., 2021).

4.3 Ecology of Rhône River anadromous vs resident trout

Neither Risso (1810, 1826) nor Crespon (1844) mentioned the sea trout in their inventories of southern France fishes. According to fossil data, this ecotype was probably absent from the Mediterranean catchments since the last ice age events between the Holocene and Pleistocene (Durante, 1978; Hamilton et al., 1989; Bouza et al., 1999; Splendiani et al., 2016). As an ancestral character (since also present in Atlantic salmon Salmo salar), anadromy should have disappeared in modern Mediterranean trout. Several explanations have been suggested. (i) This absence could be attributed to some physicochemical properties of the sea water, such as surface salinity around 38‰ and surface temperature peaks over 25 °C, which correspond to upper critical values for the trout (Tortonese, 1970). (ii) Possibly, the anadromous behavior has been counter-selected in Mediterranean trout lineage since the Pleistocene and Holocene (Splendiani et al., 2016). (iii) Another possibility could be that the migratory capacity of Mediterranean trout is silent during hot interglacial periods but triggered by glacial ecological conditions, a hypothesis invoked to explain the Mediterranean trout invasion of Corsica during or after the last glaciations (Gauthier and Berrebi, 2007). (iv) The lower Rhône River is characterized by disproportionately large floods after particular Cevenol-type storm events, with discharges reaching 1720 m3 s−1 at Beaucaire, corresponding to the discharge of the Loire and Seine rivers added together (Pardé, 1925). These conditions are clearly inadequate for the establishment of the sea trout in the Mediterranean catchments (Snoj et al., 2002; Splendiani et al., 2020).

Anadromous trout reappeared in the Mediterranean (at least along French Mediterranean coasts and in north Adriatic Sea) due to Atlantic strains introductions. One of the first trout restocking in the Rhône River took place in 1851 from an Alsatian fish farm breeding a strain coming from the Rhine catchment (Vivier, 1956). After 1950, because of the construction of important dams few kilometers upstream to the delta (Donzère-Mondragon and Vallabrègues dams), several anadromous fish species disappeared upstream. For sea trout, nowadays, according to local anglers, this big form is in drastic regression, but growing stocking reduction is a part of the cause.

4.4 Consequences for the conservation and the management

Our results clarified the status of Mediterranean sea trout as an allochthonous lineage of S. trutta, which confirms and supports previous findings in the Adriatic Sea (Snoj et al., 2002; Splendiani et al., 2020).The justification of protective measures for an introduced taxon may be questioned, like for the European bitterling Rhodeus amarus (Linnaeus, 1758). This species, considered as threatened in most of Europe, have been evidenced as recently invasive in west and central Europe, following carp culture, and threatening freshwater mussels (Van Damme et al., 2007). The rare (and regressing) presence of the sea trout in the French Mediterranean basin has no incidence on the management of the anadromous species already established (Lebel et al., 2007). However, autochthonous resident Rhône trout populations, called Salmo rhodanensis Fowler, 1974 by some authors (e.g., Kottelat and Freyhof, 2007), belonging mainly to the Mediterranean lineage (sensu Bernatchez et al., 1992), are threatened by human activities. Threat comes especially from hydropower reservoir building (Grimardias et al., 2017) or non-native Atlantic brown trout stockings (Caudron and Champigneulle, 2011), leading to natural/domestic introgression (Poteaux et al., 1999). Thus, there is no interest in protecting an allochthonous lineage which could also be a threat for the autochthonous one. Managers can then focus their efforts on the three remaining threatened diadromous species: the Mediterranean shad Alosa agone (Scopoli, 1786), the sea lamprey Petromyzon marinus Linnaeus, 1758 and the European eel Anguilla Anguilla (Linnaeus, 1758).

Acknowledgements

The authors thank the Migrateurs-Rhône-Méditerranée association and especially Yann Abdallah, the Fédération de la Pêche des Alpes-Maritimes and the anglers Bernard Marécaux, Christophe Marcellino and Guillaume Deth for providing the sea trout tissues and photo used in this study. David Schikorski (Labofarm, private laboratory at Loudéac, France, sub-contractor for amplifications and genotyping), Aleš Snoj and Simona Sušnik Bajec (University of Ljubljana) were of great help in mtDNA molecular analyses. Thanks to a reviewer and to editors of the journal for their very beneficial help. Finally we warmly thank Mélyne Hautecoeur and Agnès Dettai for their help to check the English.

Appendix A: GenBank accession numbers of CR sequences used in this study

Lineage Haplotype GenBank Source
Atlantic ATcs1 AF273086 Cortey and García-Marín, 2002
Atlantic ATcs2 AF273087 Cortey and García-Marín, 2002
Atlantic ATcs3 AF274574 Cortey and García-Marín, 2002
Atlantic ATcs4 AF274575 Cortey and García-Marín, 2002
Atlantic ATcs5 AF274576 Cortey and García-Marín, 2002
Atlantic ATcs6 AF274577 Cortey and García-Marín, 2002
Atlantic ATcs11 AY836327 Cortey et al., 2004
Atlantic ATcs12 AY836328 Cortey et al., 2004
Atlantic ATcs13 AY836329 Cortey et al., 2004
Atlantic ATcs14 EF530476 Cortey et al., 2009
Atlantic ATcs15 EF530477 Cortey et al., 2009
Atlantic ATcs16 EF530478 Cortey et al., 2009
Atlantic ATcs17 EF530479 Cortey et al., 2009
Atlantic ATcs18 EF530480 Cortey et al., 2009
Atlantic ATcs19 EF530481 Cortey et al., 2009
Atlantic ATcs20 EF530482 Cortey et al., 2009
Atlantic ATcs21 EF530483 Cortey et al., 2009
Atlantic ATcs22 EF530484 Cortey et al., 2009
Atlantic ATcs23 EF530485 Cortey et al., 2009
Atlantic ATcs24 EF530486 Cortey et al., 2009
Atlantic ATcs25 EF530487 Cortey et al., 2009
Atlantic ATcs26 EF530488 Cortey et al., 2009
Atlantic ATcs27 EF530489 Cortey et al., 2009
Atlantic ATcs28 EF530490 Cortey et al., 2009
Atlantic ATcs29 EF530491 Cortey et al., 2009
Atlantic ATcs30 EF530492 Cortey et al., 2009
Atlantic ATcs31 EF530493 Cortey et al., 2009
Atlantic ATcs32 EF530494 Cortey et al., 2009
Atlantic ATcs33 EF530495 Cortey et al., 2009
Atlantic ATcs34 EF530496 Cortey et al., 2009
Atlantic ATcs35 EF530497 Cortey et al., 2009
Atlantic ATcs36 EF530498 Cortey et al., 2009
Atlantic ATcs37 EF530499 Cortey et al., 2009
Atlantic ATcs38 EF530500 Cortey et al., 2009
Atlantic ATcs39 EF530501 Cortey et al., 2009
Atlantic ATcs41 EF530502 Cortey et al., 2009
Atlantic ATcs42 EF530503 Cortey et al., 2009
Atlantic ATcs43 EF530504 Cortey et al., 2009
Atlantic ATcs45 EF530505 Cortey et al., 2009
Atlantic ATcs46 EF530506 Cortey et al., 2009
Atlantic ATcs47 EF530507 Cortey et al., 2009
Atlantic ATcs48 EF530508 Cortey et al., 2009
Atlantic ATcs49 EF530509 Cortey et al., 2009
Atlantic ATcs50 EF530510 Cortey et al., 2009
Atlantic ATcs51 EF530511 Cortey et al., 2009
Atlantic ATcs52 EF530512 Cortey et al., 2009
Atlantic ATM1 JF297978 Snoj et al., 2011
Atlantic ATM2 JF297979 Snoj et al., 2011
Atlantic ATM3 JF297980 Snoj et al., 2011
Atlantic ATM4 JF297975 Snoj et al., 2011
Atlantic ATM5 JF297977 Snoj et al., 2011
Atlantic ATSic JF297974 Snoj et al., 2011
Mediterranean MEcs1 AY836350 Cortey et al., 2004
Mediterranean MEcs2 AY836351 Cortey et al., 2004
Mediterranean MEcs3 AY836352 Cortey et al., 2004
Mediterranean MEcs4 AY836353 Cortey et al., 2004
Mediterranean MEcs5 AY836354 Cortey et al., 2004
Mediterranean MEcs6 AY836355 Cortey et al., 2004
Mediterranean MEcs7 AY836356 Cortey et al., 2004
Mediterranean MEcs8 AY836357 Cortey et al., 2004
Mediterranean MEcs9 AY836358 Cortey et al., 2004
Mediterranean MEcs10 AY836359 Cortey et al., 2004
Mediterranean MEcs11 AY836360 Cortey et al., 2004
Mediterranean MEcs12 AY836361 Cortey et al., 2004
Mediterranean MEcs13 AY836362 Cortey et al., 2004
Mediterranean MEcs14 AY836363 Cortey et al., 2004
Mediterranean MEcs15 AY836364 Cortey et al., 2004
Adriatic ADcs1 AY836330 Cortey et al., 2004
Adriatic ADcs2 AY836331 Cortey et al., 2004
Adriatic ADcs3 AY836332 Cortey et al., 2004
Adriatic ADcs4 AY836333 Cortey et al., 2004
Adriatic ADcs5 AY836334 Cortey et al., 2004
Adriatic ADcs6 AY836335 Cortey et al., 2004
Adriatic ADcs7 AY836336 Cortey et al., 2004
Adriatic ADcs8 AY836337 Cortey et al., 2004
Adriatic ADcs9 AY836338 Cortey et al., 2004
Adriatic ADcs10 AY836339 Cortey et al., 2004
Adriatic ADcs11 AY836340 Cortey et al., 2004
Adriatic ADcs12 AY836341 Cortey et al., 2004
Adriatic ADcs13 AY836342 Cortey et al., 2004
Adriatic ADcs14 AY836343 Cortey et al., 2004
Adriatic ADcs15 AY836344 Cortey et al., 2004
Adriatic ADcs16 AY836345 Cortey et al., 2004
Adriatic ADcs17 AY836346 Cortey et al., 2004
Adriatic ADcs18 AY836347 Cortey et al., 2004
Adriatic ADcs19 AY836348 Cortey et al., 2004
Adriatic ADcs20 AY836349 Cortey et al., 2004
Dadès Dades JF297981 Snoj et al., 2011
Danubian Da1a AY185568 Duftner et al., 2003
Danubian Da3 AY185571 Duftner et al., 2003
Danubian Da9 AY185572 Duftner et al., 2003
Danubian Da22 AY185573 Duftner et al., 2003
Danubian Da24 AY185576 Duftner et al., 2003
Duero DUcs1 EF530513 Cortey et al., 2009
Duero DUcs2 EF530514 Cortey et al., 2009
Duero DUcs3 EF530515 Cortey et al., 2009
Duero DUcs4 EF530516 Cortey et al., 2009
Duero DUcs5 EF530517 Cortey et al., 2009
Duero DUcs6 EF530518 Cortey et al., 2009
Duero DUcs7 EF530519 Cortey et al., 2009
Duero DUcs8 EF530520 Cortey et al., 2009
Duero DUcs9 EF530521 Cortey et al., 2009
Duero DUcs10 EF530522 Cortey et al., 2009
Duero DUcs11 EF530523 Cortey et al., 2009
Duero DUcs12 EF530524 Cortey et al., 2009
Duero DUcs13 EF530525 Cortey et al., 2009
Duero DUcs14 EF530526 Cortey et al., 2009
Duero DUcs15 EF530527 Cortey et al., 2009
Duero DUcs16 EF530528 Cortey et al., 2009
Duero DUcs17 EF530529 Cortey et al., 2009
Duero DUcs18 EF530530 Cortey et al., 2009
Duero DUcs19 EF530531 Cortey et al., 2009
Duero DUcs20 EF530532 Cortey et al., 2009
Duero DUcs21 EF530533 Cortey et al., 2009
Duero DUcs22 EF530534 Cortey et al., 2009
Duero DUcs23 EF530535 Cortey et al., 2009
North Adriatic MAcs1 AY836365 Cortey et al., 2004
North Adriatic MA2a DQ841189 Meraner et al., 2007
North Adriatic MA2b DQ841190 Meraner et al., 2007
Salmo ohridanus   AY926564 Sušnik et al., 2006
Salmo salar   AF133701 Arnason et al., unpublished data

Appendix B. Diagnostic sites on the partial CR mtDNA haplotypes characterizing the main S. trutta lineages (Atlantic (AT), Mediterranean (ME), Adriatic (AD), Dadès, Danubian (DA), Duero (DU), Marbled (MA)) with the three partial sequences obtained from the sea trout specimens. GenBank accession numbers are listed in Appendix A.

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Appendix C. Phylogenetic trees by Bayesian inference with the partial CR marker (532 bp (A) and 325 bp (B)) on respectively 123 and 124 sequences of Salmo trutta haplotypes. Colored blocks represent S. trutta lineages. White arrows designate the three Mediterranean sea trout 1f, 1g and 1i. Posterior probability values are indicated above the nodes.

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Cite this article as: Berrebi P, Campton P, Denys GPJ. 2022. Molecular characterization of rare anadromous Rhône River brown trout. Knowl. Manag. Aquat. Ecosyst., 423, 24.

All Tables

Table 1

Composition of the 27 samples considered in the present study.

In the text
Table 2

Characteristics of the seven microsatellite loci. The second column indicates the gathering of each locus to one of the three multiplexes developed.

In the text
Table 3

Genotypes and mtDNA sequences lengths (bp) of the sea trout samples with details on tissues used for DNA extraction (Ѡ = eggs, DT = digestive tract, F = fin, S = scales), locality, date of capture and total length. na = no amplification. Trout 1c, with 4 missing loci, has been removed.

In the text
Table 4

Genetic diversity of the analyzed samples measured with: Expected heterozygosity (He, unbiased parameter of Nei 1978), Observed heterozygosity (Ho), mean number of alleles by locus (A) and Allele richness (Ar after rarefaction calculation). For the sea trout sample, He cannot be calculated because, composed of several independent captures, it does not represent a population. For each parameter, high values are indicated in grey.

In the text

All Figures

thumbnail Fig. 1

Geographic localization of the samples. Red letters = sea trout individuals (Tab. 2), blue numbers = comparative samples (Tab. 1). Trout 1d is missing because of unknown origin. D1 and D2 = large downstream dams (Donzère-Mondragon and Vallabrègues dams, respectively). Hatcheries are not represented.
In the text
thumbnail Fig. 2

(A) Sea trout 1b (52 cm TL for 1745 g) fished with spinning spoon in the Gardon River in 2007. White arrows highlight morphological characters allowing the identification as sea trout: straight margin of caudal fin and the presence of star-shaped black spots. (B) Resident Mediterranean trout (28 cm TL, 460 g) electro-fished in the Sorgue River, left tributary of the Rhône River. This relatively large river trout shows a typical forked tail, black and red spots on the flanks and the dorsal fin, a yellow-brown skin color.
In the text
thumbnail Fig. 3

Phylogenetic tree by Bayesian inference with the CR marker (1125 bp) on 124 sequences of Salmo trutta haplotypes. Colored blocks represent S. trutta lineages. White arrows designate the three Mediterranean sea trout 1f, 1g and 1i. Posterior probability values are indicated above the nodes.
In the text
thumbnail Fig. 4

The FCA (axis 1 horizontal and 2 vertical) presents a very clear classification of the genotypes of the 27 analyzed samples into two clusters: domestic Atlantic at the left and wild Mediterranean at the right. Legend at the right uses the numbers from the first column of Table 1: R samples with small circles = river sample; H with small triangles = hatchery sample; large circles represent sea trout (st01), with Rhône River in red, Var River in green; unknown origin (X = Rhône or Var River) in grey. In circles are given the number of missing locus genotypes among 7.
In the text
thumbnail Fig. 5

Different histograms produced by the STRUCTURE software after assignment analysis of the 649 sampled specimens (individual 1c has been removed). A = Assignment of the whole sampling to two expected subgroups (K = 2). B = STRUCTURE histogram when K = 5 showing the wild Mediterranean populations diversity (domestic Atlantic strains, in red, stay homogeneous).
In the text
thumbnail
In the text
thumbnail
In the text
thumbnail
In the text

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