Genetic differentiation between anadromous and

 2006 The Authors Journal compilation  2006 Blackwell Munksgaard

Ecology of Freshwater Fish 2006: 15: 255–263 Printed in Singapore Æ All rights reserved

ECOLOGY OF FRESHWATER FISH

Genetic differentiation between anadromous and freshwater resident brown trout (Salmo trutta L.): insights obtained from stable isotope analysis Charles K, Roussel J-M, Lebel J-M, Baglinie`re J-L. Genetic differentiation between anadromous and freshwater resident brown trout (Salmo trutta L.): insights obtained from stable isotope analysis. Ecology of Freshwater Fish 2006: 15: 255–263.  2006 The Authors. Journal compilation  2006 Blackwell Munksgaard Abstract – A genetic survey was carried out on recently emerging fry to investigate interbreeding between anadromous and freshwater resident brown trout in La Roche Brook, a major spawning tributary of the Oir River system (Normandy, France). Emerging fry were sampled at three different sites in spring 2002 and 2003. Stable isotope analysis (SIA) allowed the classification of each individual as the progeny of resident (N ¼ 76) or anadromous (N ¼ 58) female trout. Microsatellite DNA analysis showed that genetic diversity between fry samples can be explained by microgeographical (various sampling sites) and temporal (2 years of sampling) variations rather than maternal origin (anadromous vs. freshwater resident). We conclude that a high level of gene flow exists between the two morphs when anadromous adults have access to the spawning grounds of residents. We highlight the relevance of using SIA in conjunction with genetic analysis to study interactions between the two morphs.

Introduction

Brown trout Salmo trutta is a highly polymorphic species that lives in a variety of aquatic systems including lakes, streams and the ocean (Jonsson 1985). In Western Europe, the presence of anadromous (or sea-run) brown trout has been reported in streams from the Bay of Biscay to northern Scandinavia (Frost & Brown 1967; Elliott 1994; Baglinie`re 1999). Studies reveal a great genetic variability both within and between brown trout populations (Ferguson 1989; Guyomard 1989). Genetic divergence has been reported between anadromous and non-anadromous sympatric morphs in salmonids (Salmo salar: Verspoor & Cole 1989; Vuorinen & Berg 1989, Oncorhynchus nerka: Foote et al. 1989; Taylor et al. 1996; Wood & Foote 1996, Salvelinus alpinus: Jonsson & Jonsson 2001, Oncorhynchus mykiss: Narum et al. 2004). However, for brown trout, studies have led to various conclusions ranging doi: 10.1111/j.1600-0633.2006.00149.x

K. Charles1, J.-M. Roussel1, J.-M. Lebel2, J.-L. Baglinie`re1, D. Ombredane1 1

UMR Agrocampus Rennes–INRA Ecobiologie et Qualite´ des Hydrosyste`mes, C´ontinentaux Rennes Cedex, France, 2IBBA Laboratoire de Biologie et Biotechnologie Marines, Universite´ de Caen, Caen, France

Key words: sea trout, emerging fry, isotopic ratio, genetic divergence, microsatellite K. Charles, UMR Agrocampus Rennes–INRA Ecobiologie et Qualite´ des Hydrosyste`mes Continentaux, 65 rue de Saint-Brieuc, 35042 Rennes Cedex, France; e-mail: [email protected], [email protected] Accepted for publication 19 January, 2006

from a lack of genetic divergence between anadromous and non-anadromous morphs (Guyomard et al. 1984; Guyomard 1989; Hindar et al. 1991; Cross et al. 1992; Pettersson et al. 2001) to the existence of some degree of isolation (Jonsson 1982; Krieg & Guyomard 1985; Skaala & Nævdal 1989). Freshwater resident and anadromous brown trout often reach the same spawning grounds in the lower parts of rivers. In some cases, however, freshwater resident brown trout colonise upstream tributaries whereas anadromous brown trout use downstream spawning grounds, for instance when the sea-run fish are stopped by a migratory barrier such as a waterfall, a high elevation or a long migratory distance. It has also been reported that resident females can ovulate later than anadromous females (Jonsson & Jonsson 1999). Therefore, it remains unclear whether genetic divergence between anadromous and freshwater resident trout, whenever observed in the wild, is due to geographical isolation 255

Charles et al. Conversely, the absence of genetic divergence would reflect a high level of gene flow between the two morphs, thus indicating frequent interbreeding on redds.

(Skaala & Nævdal 1989; Knutsen et al. 2001), delay in gonad maturation process (Jonsson 1985) or the existence of behavioural barriers that may prevent mating from occurring. Part of the confusion about the nature of genetic divergence between the two morphs may be due to the technical difficulty of field investigations, meaning that data on interbreeding are scarce. Campbell (1977) observed the presence of resident males together with spawning sea trout females on redds, but there is no evidence that the eggs were actually fertilised. Laboratory experiments have shown that anadromous · freshwater resident crosses are possible and produce fertile descendants in brown trout (Skrochowska 1969; Ombredane et al. 1996). Nevertheless, the existence of hybrids in nature has not yet been reported. In the present study, we investigated interbreeding between anadromous and non-anadromous adult brown trout through a genetic analysis of new cohorts emerging from redds and entering the population after incubation of eggs and embryos into the substrate. The study took place in La Roche Brook, a small spawning tributary of the Oir River (Normandy, France) where both anadromous and non-anadromous spawning grounds largely overlap (Charles et al. 2004). Since anadromous and freshwater resident brown trout are not morphologically distinguishable at the juvenile stage (Baglinie`re et al. 2000), we first used stable isotopes analyses (SIA) to identify the maternal origin of each emerging fry (i.e., anadromous or freshwater resident) following the method described by Charles et al. (2004). We then genotyped the fry at six DNA microsatellites. In cases of low interbreeding level between adults of the two morphs on the redds, we assumed that the anadromous and non-anadromous female progenies would be genetically divergent.

Materials and methods Study site and brown trout population

The study was carried out in La Roche Brook (Fig. 1), a 4.5-km long second-order tributary stream of the Oir River (Normandy, France), which provides suitable spawning grounds for both freshwater resident and anadromous brown trout and for Atlantic salmon (Salmo salar). An impassable dam disrupts the stream so that only the lower 2.2 km of the stream is accessible to anadromous brown trout. Since 1985, brown trout movement and population abundance have been monitored on the Oir River and major tributaries by electrofishing juveniles (October and May) and by counting smolts and adults at a fish trap located on the Oir River. A thorough description of the Oir River biota, fish community and salmonid populations monitoring can be found in Baglinie`re et al. (1993, 2005). Anadromous brown trout generally enter the Oir River and La Roche Brook in November and spawn from late November to January. Young-of-the-year start to emerge from redds in March, while emigrant juveniles leave the brook in early spring (age-1 and age-2 parr, pre-smolt and smolt). Fry sampling

Emerging fry were sampled at three sites in La Roche Brook (Fig. 1): the lower site (S1) was situated 30 m

La Roche Brook Scale S3 S2

0

1 Km

S1 OIR River Emerging fry

Mont Saint-Michel Bay

sample sites Invertebrates sample sites

OIR River

256

Impassable dam

Fig. 1. Study area: sites 1, 2 and 3 on the La Roche Brook (48 38.3¢N, 01 10.6¢W), second order spawning tributary of the Oir River (Normandy, France).

Genetic variability in sea and resident trout fry upstream from the confluence with the Oir River; the middle site (S2) and the upper site (S3) were at 0.8 km and nearly 1.6 km, respectively, from the outlet of the brook. The sites were each 150 m long and, taken together they represented 20% (450 m) of the stream length between the confluence and the dam. Each site was electrofished weekly from 15 March to 10 April 2002 and from 3 March to 24 April 2003, to sample the entire periods of emergence. Equipped with a backpack electrofisher (24 V input; 150–550 V, 400 Hz pulsed DC output; DreamElectronics, Pessac, France), the operator progressed in an upstream direction and performed a minimum of 40-point samples per site along the right and left banks, alternately. Fry collections from each sample point were placed in a separate water-filled container at the end of the site and only recently emerged fry (i.e., with yolk adsorption not completed or just completed) were kept for SIA and genetic analysis. The sampling protocol for collecting emerging fry has been described in detail and discussed in Charles et al. (2004). We established reference isotopic signatures of anadromous and non-anadromous spawners by collecting fin and egg tissue samples from male and female brown trout from the Oir River system during November and December 2002. Moreover, we sampled benthic invertebrates from electrofishing sites (S1, S2 and S3) and three other locations on the Oir River system (Fig. 1) in April 2003 to determine the stable isotope values of potential prey available to freshwater resident fish. Isotopic analysis

The selected fry were over-anaesthetised in a solution of phenoxyethanol (0.1%), according to the regulations for the ethical treatment of wild fish. The digestive tract was removed and discarded, and the remaining tissues were rinsed with distilled water. All samples (fry, eggs, fin clips and benthic invertebrates) were freeze-dried at )40 C and ground to a fine powder using a mixer mill (Retsch MM 200, Fisher Bioblock Scientific, Illkirch, France). Approximately 0.2 mg of sample were weighed (Sartorius M2P, Sartorius, Goettingen, Germany) into a tin capsule and combusted in a Carlo Erba N/C-2500 elemental analyzer interfaced with a Thermo-Finnigan Delta Plus XP continuous flow isotopic ratio mass spectrometer (CF-IRMS, Thermo Finnigan, Bremen, Germany). Isotope ratios are expressed in parts per thousand (&) according to the following equation: X ¼ ½ðRsample  Rstandard Þ=Rstandard   1000 where X is d13C or d15N and R is the corresponding 13 12 C/ C or 15N/14N ratio. Rstandard for d13C was carbon from carbonate rock (Pee Dee Belemnite formation,

Craig 1957) and Rstandard for d15N was atmospheric nitrogen (Mariotti 1983). Positive delta values indicate enrichment of the heavier isotope and negative values indicate depletion. Repeat analyses of IAEA standards (N1, N2, CH6 and CH7), along with three elemental standards (acetalinide, cyclohexanone and nicotinamide) and one internal standard (homogenised bovine liver), showed that d13C and d15N were precise to 0.20&. Sample reproducibility was tested by making duplicate measurements (two or three per sample) on 43 tissue samples. The coefficient of variation ranged between 0.001 and 0.077, and single measurements were carried out on all remaining samples. Genetic analysis

DNA extractions from fry samples were performed according to the Proteinase K/Chelex method (Estoup et al. 1996) and DNA was stored at )20 C. Samples were analysed with six microsatellite markers: Str85INRA, StrBS131INRA, Ssa0064NHV, Ssa103NHV, Str543NHV and Ssa179NHV (PCR conditions and primer sequences are given in Charles et al. 2005). The protocol followed for the PCR reaction as well as the PCR product separation and visualisation was the same as in Launey et al. (2003). Stocking programmes have been discontinued for more than 10 years on the Oir River catchment. However, to check that the genetic analysis was carried out on native fish, we included DNA samples from fish belonging to four hatchery strains (local: ‘Fe´de´ration’ and ‘Farcy’; national: ‘Chauvet’ and ‘Xertigny’). Statistical analysis

Assumptions of normality and homogeneity of variance in stable isotope values were tested using Kolmogorov–Smirnov and F-tests, respectively. Student’s t-tests were used to assess variations in isotopic composition (d13C and d15N) between anadromous and freshwater resident groups. Statistical analyses were performed with S-PLUS (Professional Edition Version 6.1.2, Insightful Corp. S: Copyright Lucent Technologies, Inc., Seattle, WA, USA). Maximum type-I error rates were set at a ¼ 0.05. Fry were grouped by year (2002 and 2003), site (S1, S2 and S3), and, according to the SIA results, by maternal origin (anadromous and freshwater resident: abbreviated as ANA and FHW, respectively). In this way, we defined 12 emerging fry groups (Table 1). Departure from Hardy–Weinberg equilibrium and the null hypothesis of homogeneity in allelic distribution between the groups were assessed with the genepop3.1d program (Raymond & Rousset 1995). The genetic differentiation between groups was 257

Charles et al. Table 1. Total number of alleles per locus (Ta), number of alleles per locus per sample (a), expected heterozygosity (HE) and observed heterozygosity (HO) at six microsatellite loci analysed in 12 groups of emerging fry Group

Str85INRA

Ta 10 02FHWS1 (N ¼ 5) a 3 HE 0.540 HO 0.400 02FHWS2 (N ¼ 22) a 5 HE 0.732 HO 0.727 02FHWS3 (N ¼ 19) a 6 HE 0.760 HO 0.790 02ANAS1 (N ¼ 14) a 4 HE 0.543 HO 0.357 02ANAS2 (N ¼ 3) a 3 HE 0.611 HO 1.000 02ANAS3 (N ¼ 6) a 4 HE 0.736 HO 0.833 03FHWS1 (N ¼ 7) a 5 HE 0.735 HO 0.571 03FHWS2 (N ¼ 17) a 5 HE 0.766 HO 0.882 03FHWS3 (N ¼ 6) a 4 HE 0.667 HO 0.667 03ANAS1 (N ¼ 12) a 5 HE 0.715 HO 1.000 03ANAS2 (N ¼ 11) a 5 HE 0.756 HO 1.000 03ANAS3 (N ¼ 12) a 5 HE 0.684 HO 0.917 Fe´de´ration (N ¼ 35) a 8 HE 0.813 HO 0.857 Farcy (N ¼ 35) a 7 HE 0.763 HO 0.857 Chauvet (N ¼ 15) a 7 HE 0.804 HO 0.857 Xertigny (N ¼ 15) a 7 HE 0.780 HO 0.933

StrBS131INRA

Ssa0064NHV

Ssa103NHV

Str543INRA

Ssa179NHV

Mean

11

27

29

17

29

21

4 0.580 0.800

3 0.620 0.600

5 0.780 0.600

4 0.580 0.800

6 0.800 0.800

4 0.650 0.667

6 0.803 0.909

10 0.690 0.773

11 0.819 0.773

7 0.636 0.682

10 0.791 0.727

8 0.745 0.765

5 0.726 0.789

9 0.799 0.842

12 0.896 0.947

7 0.697 0.737

9 0.852 0.947

8 0.788 0.842

6 0.786 0.929

5 0.722 0.923

7 0.814 0.429

6 0.648 0.857

6 0.747 1.000

6 0.710 0.749

3 0.611 1.000

3 0.611 1.000

4 0.722 1.000

2 0.444 0.667

4 0.722 1.000

3 0.620 0.944

5 0.778 0.667

5 0.694 1.000

4 0.708 0.833

4 0.653 1.000

7 0.833 1.000

5 0.734 0.889

5 0.674 0.857

7 0.786 0.857

7 0.796 1.000

3 0.541 0.714

5 0.611 0.667

5 0.690 0.778

5 0.704 0.765

13 0.867 0.940

12 0.887 1.000

4 0.394 0.471

8 0.822 0.875

8 0.740 0.763

5 0.764 1.000

8 0.861 0.833

6 0.778 0.667

7 0.820 1.000

7 0.820 1.000

6 0.785 0.862

4 0.715 0.750

6 0.802 1.000

6 0.792 0.833

6 0.765 1.000

8 0.826 1.000

6 0.769 0.931

5 0.744 0.727

8 0.820 0.900

6 0.765 0.889

4 0.566 0.454

8 0.833 0.778

6 0.747 0.791

5 0.722 0.833

7 0.640 0.818

9 0.822 0.909

6 0.793 1.000

9 0.876 0.818

7 0.756 0.883

8 0.618 0.629

11 0.860 0.882

16 0.852 0.971

12 0.847 0.743

15 0.885 0.970

12 0.813 0.825

8 0.748 0.857

14 0.861 0.971

15 0.824 0.857

9 0.812 0.857

14 0.890 0.943

11 0.816 0.890

7 0.749 0.600

10 0.842 1.000

13 0.856 0.733

11 0.822 0.867

13 0.864 0.867

10 0.823 0.852

6 0.784 0.733

10 0.784 1.000

8 0.820 0.800

9 0.822 0.800

11 0.889 0.800

9 0.813 0.844

Emerging fry were collected in the lower (S1), middle (S2) or upstream (S3) site of La Roche Brook, in 2002 (02) and 2003 (03), and identified as progeny of anadromous (ANA) or freshwater resident (FHW) female brown trout. Results of the four hatchery strains (Fe´de´ration, Farcy, Chauvet and Xertigny) are also reported.

258

Genetic variability in sea and resident trout fry quantified with the genetix 4.02 program (Belkhir 2000) by calculating unbiased pairwise Fst values (Weir & Cockerham 1984). To account for temporal and microgeographical genetic variations when analysing differentiation between morphs, we performed a hierarchical analysis of molecular variance (amova) using the software arlequin version 2.000 (Schneider et al. 2000). Fry were grouped first by year and site, then by morph and site and, lastly, by morph and year. We calculated the probability of each fish belonging to each group using assignment tests performed with the geneclass2 program (Piry et al. 2004). We used the populations program (O. Langella, http://www.cnrsgif.fr/pge/bioinfo/populations) to assess relationships between groups by means of a Neighbour-joining analysis performed with the Cavalli-Sforza & Edwards (1967) chord distance, which assumes pure genetic drift. A bootstrap was carried out on loci (3000 permutations) to assess the robustness of the clustering. The population phenogram was visualised using the program treeview 1.6.6. (Page 1996). Results Isotope analysis

We collected 69 and 65 emerging brown trout fry in La Roche Brook in 2002 and 2003, respectively. The combined d13C–d15N scatter plot shows that emergent fry are split into two distinct groups (Fig. 2). The first group (d13C ¼ )18.92& ± 0.69 SD, d15N ¼ 15.82 & ± 1.34 SD) is significantly 13C-enriched (t-test: d.f. ¼ 132, t ¼ 55.54, P ¼ 0) and 15N-en-

19 17

13

15

δ N (%)

15

11 9 7 5 –31

–29

–27

–25

–23

–21

–19

–17

–15

13

δ C (%)

Fig. 2. Combined d13C and d15N of emerging fry collected in La Roche Brook in March to April 2002 (d) and 2003 ( ). Vertical and horizontal lines show the range of isotopic values (mean, minimum and maximum) for anadromous (h) and freshwater resident ( ) adult trout and benthic invertebrates ( ) collected in the Oir River Basin in winter 2002 and spring 2003.

riched (t-test: d.f. ¼ 132, t ¼ )15.22, P ¼ 0) compared with the second group (d13C ¼ 15 )25.91 & ± 0.75 SD, d N ¼ 12.10 & ± 1.45 SD). Using the collection of samples with known origin (anadromous and resident brown trout and benthic invertebrate tissues, Fig. 2), we conclude that fry of the first group (N ¼ 58) have a marine maternal origin, whereas fry from the second group (N ¼ 76) have a freshwater maternal origin. Genetic analysis

Table 1 reports the results obtained on the genetic diversity (number of alleles and heterozygosities) among groups (12 emerging fry groups and four hatchery groups). Multitests (over all groups and loci) for deviations from Hardy–Weinberg expectations yield no global heterozygote deficit (P ¼ 0.71) or excess (P ¼ 0.243). When fry are grouped by year, the test for global heterozygote deficit is only significant for the 2002 group (P ¼ 0.04) but not for the 2003 group (P ¼ 0.68). When fry are grouped by morph, the FHW group show a heterozygote deficit (P ¼ 0.04), but not the ANA group (P ¼ 0.55). Departure from Hardy–Weinberg expectations is only significant for the ANA group in 2003, which shows a global heterozygote excess (P ¼ 0.0216). The quantification of genetic differentiation by Fst yields a high level of genetic divergence between the four hatchery groups and the 12 emerging fry groups (0.04 < Fst < 0.11), confirming that fry collected in La Roche Brook were native fish. Most of the multiple comparison tests between groups listed in Table 1 reveal significant genetic differences (59 of 66 comparisons). The exact test for genetic differentiation shows no significant differentiation for the 02ANAS2/02FHWS2 (P ¼ 0.17) and 03ANAS3/03FHWS3 (P ¼ 0.21) pairwise comparisons. We find a significant differentiation with the other comparisons between morphs of the same year and the same site: 02ANAS1/02FHWS1 P < 0.001, 02ANAS3/02FHWS3 P < 0.001, 03ANAS1/ 03FHWS1 P ¼ 0.024, 03ANAS2/03FHWS2 P < 0.001. Between-year (for the same site and same morph) comparisons show that differentiation is always significant except for 03ANAS3/02ANAS3 (P ¼ 0.093). Genetic differentiation is significant for all intersite comparisons (for the same year and morph). The exact test for genetic differentiation between ANA and FHW groups is statistically significant (P < 0.01) for each sampling year, but its magnitude is small since the Fst varies from 0.03 in 2002 to 0.02 in 2003. Genetic divergences between sampling dates are higher for ANA (Fst ¼ 0.04, P < 0.0001) than FHW (Fst ¼ 0.01, P < 0.05) groups. There is a closer genetic relationship between ANA 259

Charles et al. Variance component

F-statistics

5

0.076

0.035

3.50

0.014

6 122 5

0.086 2.018 0.002

0.040 0.074 0.001

4.00 92.60 0.08

0.000 0.000 0.495

6 122 3

0.155 2.018 0.002

0.071 0.072 0.001

7.13 92.79 0.07

0.000 0.000 0.506

8 122 133

0.155 2.018 2.180

0.072 0.072

7.14 92.79

0.000 0.000

d.f. Between emerging fry grouped by year and site Between maternal morph Within emerging fry groups Between emerging fry grouped by morph and site Between year Within emerging fry groups Between emerging fry grouped by morph and year Between site Within emerging fry groups Total

and FHW samples from the same year than between ANA samples from two consecutive years (2002 and 2003). The results of the hierarchical genetic diversity analysis (amova; Table 2) show that 4% of the total genetic variance is explained by morph (Fsc ¼ 0.041, P < 0.001), whereas 7% is explained by site (Fsc ¼ 0.071, P < 0.001) and 7% by year (Fsc ¼ 0.071, P < 0.001). The majority of the genetic variance (varying from 92.6 % to 92.8 %) is due to variability within emerging fry groups. Moreover, the results of the assignment tests show that most of the individuals (89%) are assigned to the group they actually belong to, and no fry is assigned to a hatchery group. Among the misassigned individuals, 40% are assigned to the correct site, 53% to the correct morph phenotype and 73% to the correct year. The neighbour-joining dendrogram based on the Cavalli–Sforza genetic distance confirms this spatiotemporal pattern, since clusters do not correspond to the morph but tend to group samples together from the same year and the same site (Fig. 3). Hatchery strains clearly form a separate cluster. Discussion

Genetic diversity among emerging fry collected in La Roche Brook is better explained by microgeographical (between-site samples) and temporal (between-year samples) effects rather than the morph (anadromous or resident) of their maternal ancestry. Such results corroborate those found by Hindar et al. (1991) for adult brown trout in River Voss (Norway), support the hypothesis that interbreeding frequently occurs on redds when anadromous and freshwater resident trout reach the same spawning grounds and indicates a high level of gene flow between the two morphs via reproduction. Gene flow between freshwater resident and anadromous trout in La Roche Brook is probably promoted by extensive overlapping of their spawning grounds as previously observed 260

% of the total variance

P-values

Table 2. Results of analysis of molecular variance on the number of different alleles at six microsatellites for emerging fry collected from La Roche Brook in 2002–2003, grouped by year and site, by morph and site, and by morph and year

(Charles et al. 2004), together with the absence of behavioural barriers on the redds (Campbell 1977). Moreover, there appear to be no temporal barriers either, as fish of both morph phenotypes enter La Roche Brook to spawn at the same time. In a companion study, Charles et al. (2005) collected tissue samples on adults en route to the spawning grounds at different locations in the Oir river catchment, i.e., without information on where they were going to spawn. Microsatellite analysis revealed no genetic differentiation between the two morphs. Therefore, gene flow between the two morphs appears to be a general trend occurring over the entire population of the Oir river catchment. The gene flow may be regarded as a mechanism to reduce genetic divergence between morphs, which is promoted by divergent selection, thus ensuring a high level of plasticity for the species. It is not surprising that the geographical factor has an influence on the genetic structuring of brown trout populations, since this species has already been found in genetically subdivided populations over different geographical areas (Ferguson 1989; Skaala & Nævdal 1989; Hansen & Loeschcke 1996; Estoup et al. 1998). In La Roche Brook, the genetic differentiation between sampling sites located a few hundred metres apart may reflect the fine-scale genetic structure of brown trout within a single stream, as already observed elsewhere (see Carlsson et al. 1999; Carlsson & Nilsson 2000). When sampling short stretches of stream however, a potential problem is that the catch may be biased towards a few families (Allendorf & Phelps 1981; Hansen et al. 1997), and in La Roche Brook, between-site genetic differences could be reinforced by the small size of the population of spawners at each site. A positive correlation between geographical distance and genetic differentiation, i.e., ‘isolation by distance’, has been reported in several salmonid populations (e.g., Mo´ran et al. 1995; Hansen & Mensberg 1998; Knutsen et al. 2001). Carlsson &

Genetic variability in sea and resident trout fry 02ANAS3

03FHWS3

02ANAS1

02FHWS1

03FHWS1 89 03ANAS1 19 Fédération

52

Chauvet 11

Farcy

14 Xertigny

03FHWS2 35 03ANAS2

02FHWS2 40 02ANAS2

03ANAS3 65 02FHWS3 0.1 units Fig. 3. Neighbour-joining dendrogram with pairwise Cavalli– Sforza and Edwards’ chord distance units for 12 emerging fry groups collected in La Roche Brook and four hatchery samples (see Table 1 for details). Nodes values are given when >10% (3000 bootstrap replicates).

Nilsson (2000) provided an example of isolation by distance in brown trout on a microgeographical scale (seven sections of 70–90 m along a 15.7-km stream)

in the absence of impassable geomorphological structures. In La Roche Brook, the lack of isolation by distance may result from the relatively homogeneous habitat in this tributary, where water depth ranges from 20 to 60 cm and the gradient (1.1%) is almost constant from the confluence with Oir River up to the dam. Thus, the entry of adults into La Roche Brook in early winter for spawning is largely facilitated, and they can quickly reach the dam located 2.2 km upstream from the confluence. If the dispersion of adults on redds is not actually constrained by any habitat feature, then we may reasonably assume that adults are randomly distributed on redds. This accounts for the absence of a correlation between the genetic structure of the emerging fry population and the distance between the three sampling sites S1, S2 and S3. Several studies on salmonids have highlighted the temporal stability of the genetic structure of populations over several decades (Nielsen et al. 1999; Tessier & Bernatchez 1999; Hansen et al. 2002). For brown trout, Carlsson & Nilsson (2000) reported temporal stability in the genetic structure within sites in a Swedish stream. However, Østergaard et al. (2003) observed that temporal differentiation vastly exceeded geographical differentiation in Danish rivers with periodically extreme low water levels. In the present study, the observed temporal instability (i.e., the genetic differences) between sampling dates for the same site may directly reflect the genetic variation between cohorts. Again, this variation may be due to a genetic drift related to the small size of the brown trout population (Jorde & Ryman 1996). This last feature could produce an artefact in the study of genetic differentiation between the anadromous and freshwater resident trout in our study, and the question should be further investigated on larger brown trout populations. Finally, our study represents the first attempt to analyse genetic differentiation between the progenies of freshwater resident and anadromous female brown trout, thanks to SIA. The results strongly support the hypothesis that interbreeding frequently occurs on redds when anadromous and freshwater resident trout reach the same spawning ground and maturate at the same time, thus indicating a high level of gene flow between the two morphs via reproduction. It is therefore suggested that genetic divergence between anadromous and freshwater resident morphs, whenever observed in brown trout populations, may be due to geographical isolation and/or delay in gonad maturation process rather than behavioural barriers that prevent anadromous trout from mating with resident trout. The present work further illustrates that SIA – in conjunction with other appropriate tools such as genetic analysis — can improve our understanding

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Charles et al. of some complex aspects of animal ecology (Cunjak et al. 2005). Acknowledgements We are very grateful to R. Guyomard and P. Riera who made helpful suggestions during this work, to R.A. Cunjak and two anonymous referees for their valuable comments on earlier versions of the manuscript, to R. Delanoe¨, D. Huteau, F. Marchand, M. Roucaute, J. Tremblay and M. Andriamanga for laboratory and field assistance, and to the Stable Isotopes in Nature Laboratory (Canadian Rivers Institute, University of New Brunswick) for stable isotope analyses. M.S.N. Carpenter and D. Morris postedited the English style. The present study was enabled thanks to the financial support of the Conseil Re´gional de Basse-Normandie.

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