RESEARCH ARTICLE

Origins and genetic diversity among Atlantic salmon recolonizingupstream areas of a large South European river followingrestoration of connectivity and stocking

Charles Perrier • Jerome Le Gentil •

Virginie Ravigne • Philippe Gaudin •

Jean-Claude Salvado

Received: 8 December 2013 / Accepted: 25 March 2014! Springer Science+Business Media Dordrecht 2014

Abstract The restoration and maintenance of habitatconnectivity are major challenges in conservation biology.

These aims are especially critical for migratory species

using corridors that can be obstructed by anthropogenicbarriers. Here, we explored the origins and genetic diver-

sity of Atlantic salmon (Salmo salar) recolonizing

upstream areas of the largest South European Atlanticsalmon population (Adour drainage, France) following

restoration of connectivity and stocking. We genotyped

1,009 juvenile individuals, sampled either in continuouslyinhabited downstream sites or in recently reconnected and

recolonized upstream locations, at 12 microsatellite loci.

We found significant fine scale genetic structure, with threemain genetic clusters corresponding to the Nive, Nivelle

and Gaves rivers. Within each of these clusters, samples

collected in continuously inhabited and recently

recolonized sites had comparable allelic richness andeffective population sizes and were only weakly differen-

tiated. Genetic structure among basins was also similar

among continuously inhabited and recently recolonizedsites. The majority of the individuals sampled from

recently recolonized sites were assigned to neighboring

continuously inhabited downstream sites, but noticeableproportions of fish were assigned to samples collected in

more distant sites or identified as putative hybrids. Overall,

this study suggests that the restoration of accessibility toupstream areas can allow for the recolonization and

effective reproduction of Atlantic salmon from proximate

downstream refugia, which does not decrease local diver-sity or disrupt existing genetic structure.

Keywords Recolonization ! Genetic diversity ! Dam !Connectivity ! Assignment ! Salmo salar

Introduction

The fragmentation of habitat is one of the major humanthreats to wild populations (Vitousek et al. 1997; Ewers

and Didham 2006; Fahrig 2003) and is often due to the

construction of artificial barriers, which can result in patchsize reduction and patch isolation (Fahrig 2003). For a wide

range of species, habitat fragmentation can also modify

dispersal and gene flow (Coulon et al. 2010; Van Oort et al.2011; Pepino et al. 2012). Moreover, population size and

effective population sizes as well as genetic diversity can

be affected (Couvet 2002; Blanchet et al. 2010; Dixo et al.2009; Tsuboi et al. 2013; Whiteley et al. 2013). Eventually,

fragmentation may affect the evolutionary trajectories of

populations and lead up to local extinction. Therefore, tomitigate habitat fragmentation and its impacts on local

Charles Perrier and Jerome Le Gentil have contributed equally to thisstudy.

C. PerrierDepartement de Biologie, Universite Laval, Quebec G1V 0A6,Canada

C. Perrier (&) ! J. Le GentilUMR 0985 ESE, INRA, 35042 Rennes, Francee-mail: charles5perrier@gmail.com

J. Le Gentil ! P. Gaudin ! J.-C. SalvadoUMR 1224 Ecobiop, INRA, 64310 St Pee sur Nivelle, France

V. RavigneUMR BGPI, CIRAD, TA A 54/K, Campus International deBaillarguet, 34398 Montpellier Cedex 05, France

J.-C. SalvadoUMR 1224 Ecobiop, Universite de Pau Et des Pays de l’Adour,Campus de Montaury, 64600 Anglet, France

123

Conserv Genet

DOI 10.1007/s10592-014-0602-3

populations, numerous conservation actions have been

undertaken to conserve, restore (Clewell and Aronson2006; Aronson 2011; De Groot et al. 2013), and increase

habitat connectivity (Brown et al. 2013). Therefore,

effective conservation and restoration of habitat connec-tivity requires knowledge of how fragmentation and

reconnection impacts local species.

Freshwater ecosystems are particularly subject to frag-mentation due to anthropogenic activities (Nilsson et al.

2005; Hall et al. 2011). One of the main causes of frag-mentation of freshwater habitats has been the widespread

construction of dams for irrigation purposes, drinking

water retention, watermills, hydroelectric power plants, andrecreational activities. Their impacts on freshwater eco-

systems are well recognized, and range from modifications

to population genetic diversity (Horreo et al. 2011; Neraasand Spruell 2001; Wofford et al. 2005) to changes in

species assemblages (Poulet 2007; Boet et al. 1999; Brown

et al. 2013; Grenouillet et al. 2008). Barriers sizes andpermeability are important parameters affecting the

movement of fish species (Raeymaekers et al. 2009) and

thus their demographic and genetic characteristics. Inaddition, swimming ability of the fish and their capacity to

disperse through barriers are often species specific (Haro

et al. 2004). Accordingly, recent studies demonstrated thatthere are species-specific modifications of gene flow and

genetic structure due to weirs (Blanchet et al. 2010) and

waterfalls (Gomez-Uchida et al. 2009). In particular,anadromous species like Atlantic salmon, which migrate

between spawning grounds located in rivers and feeding

zones at sea (Jonsson and Jonsson 2011), can be highlyimpacted by reduced connectivity as a result of dams

(Brown et al. 2013; Hall et al. 2012). Therefore, many

conservation studies and programs aimed at documentingthe impact of artificial barriers on the demography and

evolutionary trajectories of anadromous fish populations

have highlighted the importance of the restoration of hab-itat connectivity.

In many regions of the world, there are regulations to

re-establish connectivity among freshwater habitats thathave been disconnected by weirs and dams to restore fish

movement within watersheds. Migratory fish species have

been the main targets of these restoration regulations sincethey are highly impacted by habitat fragmentation and

because they are of high biological, economic and societal

interest. Partial restoration of river connectivity can beprovided by the addition of fishways (Coutant and Whitney

2000; Brown et al. 2013; Larinier and Boyer-Bernard

1991). Even though fishways have proven their effective-ness, dam removal remains the best option to effectively

improve fish movement (Brown et al. 2013; Oldani and

Baigun 2002; Mallen-Cooper and Brand 2007). In additionto restoring fish movement, dam removal can also allow

for the restoration of spawning grounds that were previ-

ously buried under sediment (Bednarek 2001). Geneticanalyses of these recolonizing individuals together with

baseline information from samples collected in geograph-

ically close and continuously inhabited sites may help toidentify the origin and genetic diversity of these colonists

and help us to better understand the recolonization process

(Kiffney et al. 2009; Perrier et al. 2010; Griffiths et al.2011; Winans et al. 2010; Ikediashi et al. 2012). Several

recent studies have found that the Atlantic salmon recol-onizing rivers mainly came from nearby rivers but that

some fish also came from more distant source populations

(Perrier et al. 2010; Griffiths et al. 2011; Ikediashi et al.2012). However, there have not yet been any studies

aiming at identifying the origin, among productive down-

stream areas, of salmon recolonizing recently reconnectedupstream parts of rivers. In addition to determining the

origin of the immigrants, such studies within a single river

system will provide insight into the diversity and effectivepopulation size in recently recolonized river sections, two

population genetic parameters predicted to influence the

success of recolonization (Naish et al. 2013; Oakley 2013;Fraser et al. 2007).

As an alternative approach to sustain declining or re-

establish extinct populations of migratory salmonids, thestocking of wild or captive hatchery-reared individuals is

often used. However, although stocking may help rees-

tablish populations, it has been highly criticized because ofits potential negative effect on the fitness of wild popula-

tions in the long term (Aprahamian et al. 2003; Araki and

Schmid 2010; Fraser 2008). Indeed, the fitness of stockedfish may be reduced due to the effects of unintentional

selection and domestication during early life in hatcheries

that affects survival, migration and reproductive success ofstocked fish (Araki et al. 2008; Milot et al. 2013; Theriault

et al. 2011). Moreover, stocking with individuals origi-

nating from highly differentiated stocks has resulted in theloss of neutral genetic integrity in salmonids (Marie et al.

2010; Bourret et al. 2011; Perrier et al. 2013a, b; Hansen

et al. 2009). Since fine scale genetic structure may existwithin large basins (Dionne et al. 2009; Ensing et al. 2011;

Vaha et al. 2007; Primmer et al. 2006), even the use of

local wild fish to produce stocked fry may ultimately resultin the disruption of local genetic structure and local

adaptation if subpopulations are not carefully considered

(Eldridge et al. 2009; Pearse et al. 2011). Therefore,genetic analyses can be used not only to document the

origin of fish recolonizing recently reconnected sites but

also to estimate the relative contributions of ‘‘natural’’recolonization versus stocking (Beaudou et al. 1994;

Griffiths et al. 2011).

In this study, we explore the distribution of geneticdiversity within the largest Atlantic salmon catchment in

Conserv Genet

123

Southern Europe (Adour, France), to determine the origin

of Atlantic salmon recolonizing upstream areas followingrecent restoration of connectivity and stocking. The Adour

hydro-geographic basin has a surface of 16,880 km2 and

harbours a large wild Atlantic salmon population, which isan important target of both recreational and commercial

fishing (Vauclin 2007). On the basis of relatively few

samples, Perrier et al. (2011b) revealed that Atlantic sal-mon samples collected in Nive, Nivelle and Adour rivers

(see the map Fig. 1) clustered together and were geneti-cally differentiated from other French stocks. Nevertheless,

given the size of the Adour basin and the distance among

spawning grounds where Atlantic salmon have beenobserved, a fine scale population genetic structure may

exist. Of principal interests the connectivity of several

upstream areas previously inaccessible to migratory fishdue to the presence of impassable dams have been restored

since 1986 (see Fig. 1; Table 1 and methods to locate

impassable and passable dams). In parallel, salmon fry andparr, mainly the offspring of wild caught parents, have

been released since the 1970s into various tributaries of the

Adour catchment in order to sustain populations and to aidin the reestablishment of new populations in recently

reconnected sites (Marty 1984; Beall et al. 1995; Davaine

et al. 1996). How this history of human intervention hasimpacted the Salmon populations in this area is unknown.

Therefore, the specific aims of the present study were to:

(1) investigate the fine scale genetic population diversityand structure within the Adour catchment and identify

conservation units, (2) determine whether Atlantic salmon

recolonizing recently restored habitats originated from

proximate downstream sites in the basin or from the other

basins, (3) test whether the genetic diversity, effectivepopulation size, and genetic structure within recently

recolonized areas are reduced compared to continuously

inhabited sites, (4) test whether stocking impacted thedistribution of genetic diversity within the basin, and (5)

discuss the relevance of our results for the sustainable

management and conservation of this important Southern-European Atlantic salmon stock and generally discuss the

effects of the restoration of connectivity in freshwaterfishes.

Materials and methods

Study site and Atlantic salmon population

The Adour catchment is a 16,880 km2 river-system.

Atlantic salmon (Salmo salar) spawning sites can be foundin several major sub-drainages including: the Nive

(993 km2), the Saison (631 km2), the Gave d’Oloron

(2,000 km2), the Gave d’Aspe (598 km2), the Gave d’Os-sau (493 km2) and the Gave de Pau (2,600 km2). The

Nivelle River (244 km2) estuary is twenty kilometres away

from the Adour estuary and harbours a small Atlanticsalmon population. On average over the last decade, 6,500

adults annually return to the Adour Drainage, and 300,000

parr 0? are produced per year (Barracou 2008; Le Gentilet al. in prep). Thus, the Adour River is currently the most

productive drainage in France as well as in Southern Eur-

ope. The Adour population is large when compared to mostNorthern-European populations, although much larger

populations do exist (Tonteri et al. 2009).

Numerous dams have been built on the Adour River sincethe beginning of the eighteenth century, and habitat frag-

mentation reached its maximum during the 1940’s due to

hydro-electric power plant construction. Dam constructiondecreased or prevented the migratory fish from reaching

upstream spawning sites (Fig. 1; Table 1). As a result, the

abundance and catch of Atlantic salmon dramaticallydeclined in the 1960’s in the Adour basin (Barracou 2008).

For example, more than 10,000 fish were caught annually at

the beginning of the twentieth century but \500 fish werecaught in 1976 (Marty and Bousquet 2001). The intensifi-

cation of agriculture, urbanization, industrialization and

overexploitation at sea may have also contributed to thisdecline in Atlantic salmon. Since 1986, in response to

changes in regulations, much work has been done to

improve the accessibility of upstream spawning sites tomigratory fish, principally Atlantic salmon (Barracou 2008).

Connectivity restoration mainly consisted of building fish-

ways on dams isolating sectors 2, 3, 5, 7, 8, 11, 12, 13 and 16(Fig. 1). The dam that isolated sector 6 was destructed. The

N

1

23

4

56

7

8

9

10

11

12

13

15

14 16

Nive

Nivelle

Lurgorieta

Béhérobie Arnéguy

Gaves Adour

Pau Ossau

Aspe

Lourdios Vert

Saison

Oloron

Fig. 1 Location of the sampling sites that were continuouslyinhabited (blue circles), or which have been recently recolonized(green circles). Dams which were passable (green stars) or impass-able (red stars) at the time of sampling are also noted. For site numbercorrespondence, see Table 1. (Color figure online)

Conserv Genet

123

Tab

le1

Ch

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stic

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fsa

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san

dg

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sity

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sam

ple

sin

clu

din

gF

IS,

exp

ecte

dh

eter

ozy

go

sity

(He)

,al

leli

cri

chn

ess

(Ar)

,p

riv

ate

alle

lic

rich

nes

s(P

Ar)

,ef

fect

ive

size

(Ne)

,an

dce

nsu

ssi

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erb

asin

(Nc

corr

esp

on

ds

toth

eav

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en

um

ber

of

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rom

ou

sfi

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ng

the

bas

ins,

Lan

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01

1)

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inR

iver

Tri

bu

tary

Co

ord

inat

esL

oca

tio

nD

ista

nce

tori

ver

mo

uth

(km

)

Yea

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rati

on

of

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ssib

ilit

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N gen

oty

ped

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llsi

bs

%o

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ter

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ing

full

sib

s

FIS

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PA

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Niv

elle

Niv

elle

Niv

elle

43

"200

5200 N

/0

1"3

30 0

600 W

11

7–

30

82

72

20

.08

0.8

48

.59

0.3

43

3.8

10

5

Niv

elle

43

"180

3300 N

/0

1"3

10 4

900 W

2*

22

19

92

45

51

14

00

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0.8

48

.61

0.3

11

24

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Lu

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3"1

802

800 N

/0

1"3

30 5

800 W

3*

22

19

92

19

94

71

00

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0.8

58

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0.4

13

02

.6

Ad

ou

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Niv

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3"2

004

400 N

/0

1"2

60 5

300 W

45

9–

25

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25

0.0

40

.80

7.2

40

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59

5.8

56

0

Lau

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43

"150

4000 N

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67

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63

63

18

00

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0.8

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71

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3"1

208

900 N

/0

1"2

60 7

400 W

6*

70

19

92

17

45

02

91

24

0.0

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5.5

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3"3

102

700 N

/0

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70 1

900 W

7*

88

19

86

27

00

27

0.0

50

.85

8.4

30

.28

14

2.6

9,2

00

Sai

son

43

"190

5100 N

/0

0"9

10 3

300 W

8*

10

51

99

59

87

79

10

.02

0.8

48

.45

0.3

0.0

40

0.5

Gav

ed

’Olo

ron

43

"260

8500 N

/0

0"6

80 4

100 W

91

06

–6

53

56

20

.02

0.8

38

.21

0.1

92

38

.4

Gav

ed

’Olo

ron

43

"230

1100 N

/0

0"6

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10

11

5–

47

71

54

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0.8

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64

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t4

3"1

603

100 N

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0"6

80 1

500 W

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20

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31

29

0.0

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.83

8.3

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ed

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3"0

308

800 N

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7.9

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43

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6900 N

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0"6

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9.0

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ed

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6

Conserv Genet

123

Dam that isolated sector 15 was equipped with a fish lift.

Thanks to these improvements in habitat connectivity sec-tors 7, 2–3–6–12–13–16, 15, 8 and 5–11 became accessible

in 1986, 1992, 1993, 1995 and 2001, respectively.

To sustain the Atlantic salmon populations inhabitingthe Adour drainage as well as facilitate the recolonization

of upstream habitats, stocking programs were implemented

in the 1970s (Marty 1984; Beall et al. 1995; Davaine et al.1996; Marty and Bousquet 2001). Eggs, young of the year

and smolts were stocked in several tributaries. Until 1990,eggs, fry and smolts produced from local and non-local

parents (mainly originating from Scotland) were stocked in

the Gaves, Nive and Nivelle. Since 1990, local parentswere used in the Nive, Nivelle and Gaves Rivers (Marty

and Bousquet 2001). At present, stocking is sustained only

in the Gave de Pau. As presented in (Perrier et al. 2013b),little introgression by non-local strains occurred in the

Adour populations and this introgression has remained

stable over the last decades.

Sampling

During September and October 2005, 0? and 1? year old

juvenile Atlantic salmon were sampled by cutting off a

small part of their pectoral fins (non-lethal sampling). Finswere stored in 95 % ethanol. A total of 1,009 individuals

were sampled in 16 locations distributed in the Adour

drainage (Fig. 1; Table 1). To increase the number ofindividuals analysed per location, but to limit biases

resulting from sampling individuals from the same family,

each sampling site consisted in a section of 10–30 km long.Individuals sampled were wild fish for all but site #14,

where a restocking operation occurred in 2004.

DNA extraction and genotyping

DNA extraction, amplification and genotyping were com-pleted according to the procedures described in (Horreo

et al. 2008). A total of twelve microsatellites were selected

on the basis of consistency of amplification, ease of scoringand variability: Ssa197, Ssa202, Ssa171 (O’Reilly et al.

1996), SSsp1605, SSsp2210, SSspG7, SSsp2201 (Paterson

et al. 2004), Ssosl417, Ssosl85 (Slettan et al. 1995),SsaD144b, SSa157a (King et al. 2005), Ssa289 (McConnell

et al. 1995). A thirteenth microsatellite was used to dis-

tinguish S. salar from Salmo trutta and detect possiblehybrids between these species (Perrier et al. 2011a).

Data quality

ML-relate (Kalinowski et al. 2006) was used to detect full-

sibs. Full-sibs were subsequently removed from the dataset

to avoid bias caused by the overrepresentation of individ-

uals from the same family (Hansen et al. 1997). We usedthe software Micro-Checker to test for the presence of null

alleles (Van Oosterhout et al. 2004). Linkage disequilib-

rium was estimated using Genepop 3.4 (Raymond andRousset 1995b), with sequential Bonferroni correction

(Raymond and Rousset 1995a). FIS and tests for Hardy–

Weinberg disequilibrium were conducted withFSTAT2.9.3.2 based on 1,000 permutations.

Analysis of genetic diversity within samples

Observed (Ho) and expected heterozygoties (He) wereestimated using Genepop 3.4. The number of alleles per

locus and population, allelic richness (Ar) and private

allelic richness (PAr) were estimated using Hp-rare 1.0(Kalinowski 2005). Effective population size (Ne) was

estimated for each location using the LDNe method (Wa-

ples and Do 2008) implemented in NeEstimator V2.0 (Doet al. 2013). We used an allele frequency threshold of 0.05.

We tested for data normality using a Shapiro test, and then

compared average Ar and Ne among recently recolonizedand continuously inhabited sites using student’s t tests for

normally distributed data and Mann–Withney tests for non-

normal distributed data. Census size (Nc) were obtainedfrom Lange et al.’s (2011) and corresponded to the average

number of anadromous fish entering in the Nive, Nivelle

and Gaves rivers.

Analysis of genetic structure among sites

We used Genepop 3.4 to estimate FST between sites. Sig-

nificance of FST was estimated using 1,000 permutations.

We compared average FST among: (A) recently recolon-ized sites and sites that were continuously inhabited within

each basins, (B) recently recolonized sites from different

basins and (C) sites from different basins that remainedinhabited by Atlantic salmon. We tested for sstatistical

significance using student’s t tests. We performed three

AMOVAs using Arlequin v3.5 (Excoffier and Lischer2010). The first AMOVA was conducted using Nivelle,

Nive and Gaves as groups to test for hierarchical structure.

The second AMOVA was conducted using the samegrouping but on continuously inhabited sites only. The last

AMOVA was conducted using the same grouping but on

recently recolonized sites only. These last two AMOVAswere conducted to investigate whether genetic differenti-

ation was lower among recently recolonized sites com-

pared to continuously inhabited ones at both hierarchicallevels. A neighbor-joining dendrogram based on pairwise

Nei (Da) genetic distances (Nei et al. 1983) was con-

structed with Populations 1.2.30 (http://bioinformatics.org/*tryphon/populations/). Confidence estimates of tree

Conserv Genet

123

topology were calculated by 1,000 bootstraps of loci.

Dendrograms were visualized using TreeView (Page1996).

Bayesian clustering and assignment of individuals

We examined the clustering of populations and individuals

using Baps v2.0 (Corander et al. 2004) and Structure(Pritchard et al. 2000). While Baps rapidly and accurately

finds main clusters, the estimation of individual admixturemay be less accurate. Alternatively, Structure accurately

estimates population and individual admixture but is rela-

tively slow and may have difficulty finding main clusterswhen some populations are under or over-represented

(Kalinowski 2010). First, we used the population clustering

option implemented in BAPS to delineate main geneticclusters in the dataset, with a maximum number of

potential clusters set to 10. Population and individual

admixture was subsequently tested on the basis of thesepopulation-clustering results. Second, the Bayesian clus-

tering method implemented in the software Structure was

used to delineate K genetic clusters. The best K valueswere defined according to the DK procedure as described

by (Evanno et al. 2005), using STRUCTURE HAR-

VESTER (Earl and vonHoldt 2012). A total of 10 runswere computed for each value of K tested, from 1 to 10.

Each run started with a burn-in period of 50,000 steps

followed by 300,000 Markov Chain Monte Carlo (MCMC)replicates. We used an admixture model, without prior

information regarding population clustering.

We used the software Geneclass2 (Piry et al. 2004)following the methods of (Baudouin and Lebrun 2000) to

assign individuals sampled in either recently recolonized

sites or continuously inhabited ones by Atlantic salmon to abaseline constituted of samples from continuously inhab-

ited sites (sites 1, 4, 9, 10 and 14). This was done to

identify the source populations for the newly foundedpopulations. Individuals that had scores \70 % were con-

sidered as potential hybrids or migrants from un-sampled

populations.

Results

Data quality

Of the 1,009 sampled individuals, 960 individuals were

successfully genotyped with a minimum of 66 % of indi-

vidual amplification success. The total amplification suc-cess was 99.68 %. Using the SSAD486 marker to identify

species, we found that 5 individuals were S. trutta, 26

individuals were hybrids between S. trutta and S. salar, and5 could not be identified (amplification failed at this locus).

We discarded these 36 individuals and conducted the

subsequent analyses on the 924 remaining Atlantic salmon.Following the results from ML-relate, we removed a total

of 187 (20 %) individuals that were found to have full-sibs

in our dataset. These 187 removed individuals represented0–47 % of the total number of individuals at each site

(median value of 15 %). We thus conducted all the sub-

sequent analyses on a total of 737 individuals (Table 1).Micro-Checker detected no null alleles. No linkage dis-

equilibrium was detected by Genepop among loci(p [ 0.05 for all them after Bonferroni corrections), thus,

all loci were considered to be genetically independent.

Only eight out of 192 FIS computed in Fstat were signif-icant. No locus presented significant deviation from Hardy

to Weinberg Equilibrium over all populations. At the

population level, locations 6, 11 and 15 yielded signifi-cantly smaller observed than expected heterozygosities

(Table 1).

Analysis of genetic diversity within samples

Loci had 6 (Ssa289) to 49 (Ssa157a) alleles over the entiredataset, with a median value of 24 and a total of 322 alleles

over all loci. Average He (Expected heterozygosity) over

all loci per population varied from 0.80 to 0.87 (Table 1),0.84 on average in the Nivelle basin, 0.81 in the Nive basin

and 0.84 in the Gaves basin. He was on average of 0.83 in

continuously inhabited locations and of 0.84 in recentlyrecolonized sites. Average Ar (Allelic richness) over all

loci varied from 7.28 to 8.93 depending on the location. Ar

was on average 8.62 in the Nivelle basin, 7.45 in the Nivebasin and 8.27 in the Gaves basin. Ar was on average 8.09

in continuously inhabited locations and 8.23 in recently

recolonized sites (Fig. 2). Ar was not significantly differentbetween continuously inhabited locations and recently

recolonized ones (Table 2, t test, t = -0.50, df = 7.26,

p value = 0.63). Average PAr (Private allelic richness)varied from 0.15 to 0.41 depending on the location. PAr

was on average 0.35 in the Nivelle basin, 0.18 in the Nive

basin and 0.25 in the Gaves basin. PAr was on average 0.23in continuously inhabited locations and 0.27 in recently

recolonized sites. Effective size (Ne) varied from 33.8 to

3,583.8 depending on the location (Table 1). Overall, themedian value of Ne was 238.4 in continuously inhabited

locations and 124.0 in recently recolonized sites (Fig. 2).

These two medians of Ne estimates were not significantlydifferent (Table 2, Mann–Whitney test, W = 30,

p value = 0.83).

Analysis of genetic structure among sites

FST among sites ranged from 0.000 to 0.054 (Table 2).Table 2 and Fig. 3 show small FST among continuously

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inhabited sites and recently recolonized ones within each

basin (FSTA = 0.012 on average), but relatively high FST

among sites located in different basins, either recently

recolonized ones (FSTB 0.035 on average) or continuously

inhabited sites (FSTC 0.042 on average). These three FST

were significantly different (FSTB vs FSTC t = -2.54,

df = 10.99, p value = 0.03; FSTA vs FSTB t test, t =

-10.30, df = 27.75, p value = 5.6e -11; FSTA vs FSTCt test, t = -10.36, df = 12.87, p value = 1.3e-07).

Considering all the sites, AMOVAs revealed that2.53 % of the genetic variance was found among basins

and 1.08 % among populations within basins (Table 3).

When considering only continuously inhabited sites,AMOVAs revealed that 3.75 % of the variance was found

among basins and 0.47 % among populations within

basins. When considering only recently recolonized sites,AMOVAs revealed that 2.15 % of the variance was found

among basins and 1.22 % among populations within

basins. These two last AMOVAs showed that geneticstructure was higher among continuously inhabited sites

than among recently recolonized ones, according to the

5–95 % confidence intervals.A neighbor-joining dendrogram of Nei’s genetic dis-

tance revealed three genetically distinct populations cor-

responding to the Nivelle, Nive and Gaves basins (Fig. 4).This dendrogram also reveals relatively little differentia-

tion among sites within each basin.

Bayesian clustering and assignment of individuals

While the best k value found by BAPS was k = 3 (Fig. 5),the first delta k pick found in STRUCTURE result corre-

sponded to k = 4, followed by a smaller pick at k = 8.

However, the existence of several clusters in Gaves fork = 4 and in Gaves and Nive for k = 8 did not clearly

corresponded to any geographic grouping and individuals

were highly admixed. We therefore proposed that the mostrealistic number of genetic clusters was three, corre-

sponding to the three main basins.

According to the assignment conducted using Geneclass,an average of 86 % of the individuals sampled in continu-

ously inhabited sites were assigned to their site of origin, 95,

100 and 78 % on average in the Nivelle, Nive and Gavesrivers, respectively (Table 4), suggesting a relatively high

power to assign fish to the three different basins. In contrast,

an average of 54 % of putatively local individuals was foundin recently recolonized sites (48, 71 and 50 % in the Nivelle,

Nive and Gaves rivers, respectively). Within the continu-

ously inhabited sites, 0 % of the individuals that could not beassigned to a basin were putative migrants from other basins

and 12 % of individuals were hybrids or migrants from other

unsampled populations. Within the recently recolonizedsites, the individuals non-assigned to the basin were 4 %

putative migrants from other basins and 38 % hybrids or

migrants from other unsampled populations.

Table 2 Table of FST among sites. (Color table online)

Significant values are indicated in bold and non-significant ones in italic. A color gradient help to visualize the hierarchical differentiation amongpopulations

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Discussion

Fine scale genetic diversity within continuously

inhabited sites

The significant genetic differentiation (FST) observed

among continuously inhabited sites within the Adourcatchment was comparable to the FST observed among

other Atlantic salmon populations at the within river scale

using markers with similar level of polymorphism (Elliset al. 2011; Dionne et al. 2009; Vaha et al. 2007; Primmer

et al. 2006). This significant differentiation among popu-

lations from these three rivers contrasted with the results ofPerrier et al. (2011b), who only found one major genetic

cluster within the Adour River. However, Perrier et al.

(2011b) analyzed a much smaller number of individuals.The clear clustering of individuals in three genetically and

geographically distinct groups suggests limited dispersal

and gene flow among the Nive, Nivelle and Gaves rivers.

This is in line with the relatively strict homing behaviour of

Atlantic salmon (Stabell 1984) that has been suggested tobe accurate to the tributary level (Vaha et al. 2008; Dillane

et al. 2008). This existing fine scale genetic structure may

also be linked to fine scale local adaptation (Vaha et al.2008, 2007) and should be taken into account for man-

agement (Fraser and Bernatchez 2001). Hence, these

results support the local management strategy that has been

Fig. 2 Boxplots of allelic richness and effective population size incontinuously inhabited and recently recolonized sites

Fig. 3 Boxplots of FST: A between continuously inhabited andrecently recolonized sites in each basin; B between recentlyrecolonized sites among each basins; C between continuouslyinhabited sites among each basins

Table 3 Analysis of molecular variance partitioning genetic struc-ture among and within the 3 main drainages (Gaves, Nivelle, Nive)

Populationconsidered

Source ofvariation

% ofvariation

U-Statistic mean(5–95 %)

All, n = 16 Among groups 2.534 0.031 (0.021–0.041)

Amongpopulationswithin groups

1.076 0.011 (0.009–0.013)

Continuouslyinhabited,n = 5

Among groups 3.749 0.037 (0.027–0.049)

Amongpopulationswithin groups

0.469 0.005 (0.001–0.010)

Recentlyrecolonized,n = 11

Among groups 2.145 0.021 (0.017–0.027)

Amongpopulationswithin groups

1.222 0.012 (0.011–0.014)

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applied since the 1990’s, which considers the Nive, Nivelleand Gaves rivers as three distinct conservation units.

Effective population sizes estimated for the continu-

ously inhabited sites in these three rivers were relativelyhigh compared to other rivers located in Southern Europe

(Perrier et al. 2013b; Nikolic et al. 2009). In particular, the

populations located at the edge of this species’ range (likethe study population) have dramatically declined (Boylan

and Adams 2006; Parrish et al. 1998; Dumas and Prouzet

2003; Prouzet 1990). While the size of Atlantic salmonpopulations inhabiting the Adour basin has declined during

the past decades, the effective population sizes we detected

appear may be high enough for maintaining geneticdiversity on the short term. However, these effective pop-

ulation sizes are relatively low compared to what is needed

for the long-term conservation of wild populations(Frankham 2002, 2005; Traill et al. 2010). Nevertheless,

the role of migration among populations within the Adour

basin or even from distant rivers should not be neglected insustaining long-term effective population sizes and genetic

diversity (Gomez-Uchida et al. 2013; Palstra and Ruzzante2011; Kuparinen et al. 2010). Moreover, high proportions

of mature male parr may also increase effective population

size in these southern populations (Saura et al. 2008;Garcia-Vazquez et al. 2000; Martinez et al. 2000; Moran

et al. 1996).

The low admixture and low proportions of putativemigrants or hybrids within continuously inhabited sites

suggests a relatively low impact of stocking. In particular,

the use of geographically and genetically distant popula-tions to stock the river from the Adour catchment may have

resulted in noticeable admixture (Perrier et al. 2013b;

Finnengan and Stevens 2008; Hansen et al. 2009) and in alack of differentiation among locations due to a local

homogenisation of the genetic diversity (Marie et al. 2010;

Eldridge and Naish 2007). Similarly, since stocked fish canhave a higher dispersal than their wild counterparts (Pe-

dersen et al. 2007; Quinn 1993), even stocking local but

hatchery-reared individuals may have led to admixtureamong local clusters. However, admixture appeared low

within continuously inhabited sites and these locations

were significantly differentiated. This may be due to arelatively low return rate (Perrier et al. 2013a) and fitness

0.05

1

3*

50

2*

50

4

6*

985*

97

77

7*

8*

4713*

9

10

75

14

34

15*

14

16*

3

12*

2

11*Gaves

Nive

Nivelle

Fig. 4 Neighbor joining tree based on Nei 1983 genetic distances.Bootstrap values are indicated upside the node. Recently recolonizedsites are indicated with an asterisk

Fig. 5 Bayesian individual clustering obtained using the softwareBAPS for k = 3 the software STRUCTURE for k = 8 and k = 4.Vertical bars represent proportions of membership of each individual

to each cluster represented, which are represented by different colors.Recently recolonized sites are indicated with an asterisk. (Color figureonline)

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123

of non-native salmon stocked before the 90’s (Milot et al.

2013; Perrier et al. 2013b; Theriault et al. 2011). Relativelylow survival and/or low straying of local fish stocked since

the 90’s using Nive, Nivelle and the Gaves basins as

conservation units may also explain low admixture amongthese populations. Overall, while we cannot rule out the

impacts of historical stocking on present neutral genetic

structure among populations or on their adaptive potential,our results suggests that stocking did not significantly

affect genetic structure within continuously inhabited sites

from the Adour basin.

The recolonization of reconnected sites by Atlantic

salmon

A relatively high production of fry was found in recently

reconnected spawning grounds (Barracou 2008), illustrat-ing that the construction of fishways on dams may aid in

the effective recolonization of Atlantic salmon populations

(i.e. recolonization followed by successful reproduction).Even though noticeable proportions of fish sampled in

recently recolonized sites were assigned to distant sites or

were identified as putative hybrids, the majority of theindividuals were assigned to neighboring downstream sites,

suggesting a higher colonization success of local fish.

Accordingly, the genetic structure among samples from asingle river was small, suggesting a relatively high con-

tribution of fish having local origin, either wild or stocked,

to the establishment of new populations. This was also

supported by our finding that there is a similar genetic

structure among basins for both continuously inhabited andrecently recolonized sites. These results agree with (Perrier

et al. 2010; Ikediashi et al. 2012) data, which found that

large proportions of fish recolonizing depopulated riversoriginated from nearby rivers. However, in contrast with

the previous studies documenting recolonization processes

in Atlantic salmon by inferring the origin of adults’ indi-viduals recolonizing rivers (Griffiths et al. 2011; Perrier

et al. 2010; Ikediashi et al. 2012), here we genotypedjuveniles caught in recolonized parts. Thus, we character-

ized both a recolonization and a successful reestablishment

of Atlantic salmon populations. Nevertheless, it did notallow us to compare reproductive success among adult fish

returning to upstream areas. Hence, the fact that large

proportions of fry caught in recently recolonized habitatswere assigned to downstream sites of the same rivers

(Nive, Nivelle, Gaves, respectively) does not necessarily

indicate that stocked or wild adult fish had high homingrates but instead may suggest that adults with local genetic

characteristics had high reproductive success relative to

potential migrants. This illustrates that even though dis-persal of both wild and stocked fish might have contributed

to the reproductive effort within recently reconnected sites,

the parents with the highest reproductive success, overall,originated from close downstream sites. This result agrees

with studies showing that immigrants often come from

nearby sites (Perrier et al. 2010; Ikediashi et al. 2012) andwith those finding locally adapted fish may have higher

reproductive success (Dionne et al. 2008; McGinnity et al.

2007; Hendry 2004). However, it was not possible to dis-entangle the relative contributions of stocking and coloni-

zation of wild fish since recent stocking has used local

parents for each of the three rivers. Pedigree reconstructionwould have allowed us to address this question (Milot et al.

2013; Araki et al. 2007; Theriault et al. 2011), but this was

not possible due to the prohibitively extensive and costlygenotyping effort needed to address this issue in such a

large population. Given that local fish might be locally

adapted, as widely suggested for Salmonids (Bourret et al.2013; Primmer 2011; Garcia de Leaniz et al. 2007; Taylor

1991), the fact that local fish may have had a higher

reproductive contribution than non-local fish has importantimplications for the long term recolonization of the Adour

basin. In particular, local fish might be more prone to

establish a viable population than exogenous individuals.We expected a relatively low effective population sizes

in recently recolonized locations compared to continuously

inhabited sites, as a result of founder effects. However,effective population sizes in these two groups were rela-

tively similar. Such comparable effective sizes among

recently recolonized and continuously inhabited sites couldbe explained by relatively high contemporary gene flow

Table 4 Assignation of Atlantic salmon among rivers and tributaries(sampling sites grouped by basins)

River Sites 1 (%) 4 (%) 9, 10, 14(%)

Genetically admixedindividuals (%)

Nivelle 1 95 0 5 0

2* 35 5 13 48

3* 60 0 10 30

Nive 4 0 100 0 0

5* 0 65 15 20

6* 1 77 9 13

Gaves 7* 7 7 48 37

8* 4 3 42 51

9 0 0 85 15

10 0 0 73 28

11* 0 14 41 45

12* 0 0 45 55

13* 0 0 65 35

14 0 0 78 22

15* 0 0 60 40

16* 0 0 50 50

* Recently recolonized sites

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123

(Waples and England 2011). Of particular interest, effec-

tive population size tended to be larger for recentlyrecolonized habitats within the Nivelle River. This result

suggests that the new population that recolonized reopened

areas is even larger than the populations located down-stream in continuously inhabited sites. This may occur

because upstream habitats are, in general, more suitable for

Atlantic salmon than downstream sites (Barracou 2008). Inturn, this observation may not be linked to stocking oper-

ations since hatcheries generally use a reduced numbers ofparents harbouring reduced diversity compared to the

population of origin (Araki and Schmid 2010). Indeed,

these results likely suggest a relatively low impact ofstocked Atlantic salmon, which is in line with recent

studies on the impact of stocking on the breeding system in

this species (Milot et al. 2013; Jonsson and Jonsson 2006).In the case of the Nivelle River the effective population

size appeared larger than the census size. Such a result is

difficult to explain without invoking a potentially largecontribution of precocious parr (Johnstone et al. 2013;

Saura et al. 2008; Jones and Hutchings 2001) but could also

be linked to gene flow from the Nive and Gave rivers.Indeed, census size has been estimated through an

exhaustive monitoring of adult anadromous salmon but did

not included precocious parr (Lange et al. 2011). Withinthe Nive and the Gaves rivers, the effective population

sizes tended to be smaller than the census sizes, which is

more in lines with the expectations of a typical Atlanticsalmon population (Palstra and Ruzzante 2011) in which a

large variance among breeders exists (Richard et al. 2013;

Fleming 1996). Overall, the relatively large effectivepopulation sizes estimated for recently recolonized sites

might allow for the conservation of a relatively high level

of genetic diversity in these new populations, which maylimit short-term extinction risks (Traill et al. 2010;

Frankham 2005). Accordingly, within each of the three

rivers, samples collected in continuously inhabited and inrecently recolonized sites had comparable allelic richness,

suggesting no or only a weak loss of genetic diversity

during the recolonization process. This is a critical obser-vation because effective population size and genetic

diversity is positively linked to the effectiveness of selec-

tion relative to drift (Charlesworth 2009; Olson-Manninget al. 2012).

Conclusion

Overall, our results suggest that restoring accessibility toupstream areas can allow for the recolonization of Atlantic

salmon. This recolonization mainly comes from individuals

from proximate downstream sites, with neither a decreaseof local diversity nor disruption of existing genetic

structure. Along with previous studies (Perrier et al. 2010;

Ikediashi et al. 2012; Kiffney et al. 2009; Griffiths et al.2011; Schreiber and Diefenbach 2005), this study shows

connectivity restoration as an effective way to support

recolonization of rivers from which salmonids have beenpreviously extirpated. Nevertheless, the ecological resto-

ration policy should also aim to reconnect several other

upstream tributaries and improve water quality. Indeed, theactual census size of the Atlantic salmon population in the

Adour basin is much smaller than the estimated potentialcapacity (Barracou 2008). While the implementation of

fishways in the Adour drainage allowed an effective

recolonization of Atlantic salmon within several upstreamareas, little is known about the impacts of these fish pas-

sages on the recolonization dynamic of other migratory

species. While Atlantic salmon can cross over relativelychallenging fishways, several other migratory fish having

lower swimming capacities may need more specific and

less challenging fishways to effectively recolonize depop-ulated areas. More broadly, even though the recolonization

of some fish migratory species can be enabled by fishways,

such devices may not compensate the overall ecosystem-wide dramatic impacts of dams (Brown et al. 2013).

Acknowledgments We acknowledge all participants to the collec-tion of samples and of various historical and environmental data, witha special attention to A. Manicki, J. Chat, D. Barracou. We also thankP. Regnacq, JB Torterotot and Anne Dalziel for their help whileanalyzing data and writing the paper. We thank two anonymousreviewers and the associate editor C. Primmer for their very con-structive comments. Authors also thank all French organizations thatprovided their technical assistance for electric fishing: the NationalInstitute for Agricultural Research (INRA), the National Office ofWater and Aquatic Media (ONEMA) and Migradour. This work wasfunded by the European Union INTERREG IIIB program [AtlanticSalmon Arc Project (ASAP)] and the European Union INTERREGIVB program [Atlantic Arc Resource Conservation (AARC)].

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