Molecular Ecology (2004) 13, 1035– 1045

doi: 10.1111/j.1365-294X.2004.02117.x

Phylogeography of a game species: the red-crested pochard (Netta rufina) and consequences for its management

Blackwell Publishing, Ltd.

L . G A Y ,* P . D E F O S D U R A U ,† J . - Y . M O N D A I N - M O N V A L † and P . - A . C R O C H E T ‡ *CEFE-CNRS, 1919 route de Mende, F-34293 Montpellier cedex 5, France, †Office National de la Chasse et de la Faune Sauvage, CNERA Avifaune Migratrice, Le Sambuc, 13200 Arles, France, ‡Laboratoire de Biogéographie et Ecologie des Vertébrés, EPHE, box 94, Université Montpellier II, 34095 Montpellier cedex, France

Abstract Western European populations of red-crested pochard (Netta rufina) are characterized by low size and high fragmentation, which accentuate their sensitivity to hunting. Uncertainties regarding the demographic trends of these populations highlight the need for pertinent hunting regulations. This requires identification of the limits of the populations under exploitation, i.e. delimiting a management unit. We used the left domain of the mitochondrial control region and seven nuclear loci (four microsatellites and three introns) to assess the level of genetic structure and demographic independence between the fragmented Western European and the large Central Asian populations. The second objective was to investigate the colonization history of the Western European populations. This study demonstrated that the Western European populations of red-crested pochard constitute a separate demographic conservation unit relative to the Asian population as a result of very low female dispersal (mitochondrial DNA: ΦST = 0.152). A morphometric analysis further suggested that Central Asian and Western European specimens of both sexes originate from different pools of individuals. Male dispersal seems higher than female dispersal, as suggested by the lack of clear genetic structure for the nuclear markers at this continental scale. Genetic data, in conjunction with historical demographic data, indicate that the current Western European populations probably originate from a recent colonization from Central Asia. As numbers of red-crested pochards in Western Europe cannot be efficiently supplemented by immigration from the larger Asian populations, a management plan regulating the harvest in Western Europe is required. Keywords: birds, control region, management unit, microsatellites, nuclear introns, phylogeography Received 16 October 2003; revision received 8 December 2003; accepted 8 December 2003

Introduction Securing the local persistence of a species requires testing for demographic isolation, which identifies sets of populations whose dynamics are not significantly affected by influences from adjacent populations. These units are often called management units (MUs; Moritz 1994; Fraser & Bernatchez 2001). This concept is crucial in the case of exploited populations for which we have to identify those sets of populations that will be affected by human harvest and thus evaluate the impact of exploitation (e.g. Ruzzante et al. 2000; Koljonen 2001). The degree of demographic isolation is determined by the occurrence of dispersal Correspondence: Laurène Gay. E-mail: [email protected] © 2004 Blackwell Publishing Ltd

among populations. Genetic methods can provide an indirect estimate of dispersal, even if an absence of genetic differentiation does not necessarily imply a high level of dispersal (for example in the case of recently isolated populations, see Whitlock & McCauley 1999). Therefore, Moritz (1994) advocated recognizing as MUs ‘populations with significant divergence of allele frequencies at nuclear or mitochondrial loci’. The red-crested pochard (Netta rufina, Aves, Anseriforme) is a diving duck with a vast Palearctic range extending from Western Europe to Central Asia (Scott & Rose 1996) (Fig. 1). It is one of the least abundant of the Western Palearctic waterfowl species (Anatidae) (Scott & Rose 1996; Dehorter & Rocamora 1999). Hunting is nevertheless allowed in Spain, Portugal and France, resulting in an annual

1036 L . G A Y E T A L .

Fig. 1 Distribution of the red-crested pochard, Netta rufina, in Eurasia (after Scott & Rose 1996). Shaded areas indicates the breeding distribution. Filled circles indicate sampling sites: 1, Donana; 2, Ebrodelta; 3, Camargue; 4, Dombes; 5, Constance Lake; 6, Volga delta; 7, Kazakhstan. Shaded lines indicate population boundaries (broken line: uncertain) (outlining the breeding, wintering and migration range).

harvest estimated at 8000 birds (Shedden 1986). The uncertainties regarding the evolution of the population size and the fragmentation of the distribution range in Western Europe highlight the need for an international action plan, including proposals for sustainable hunting (Defos du Rau 2002). This requires that the impact of hunting be estimated, which necessitates identification of the limits of the exploited populations. Based on census data and ringing recoveries, three distinct population groups have been postulated (Fig. 1) (Monval & Pirot 1989; Scott & Rose 1996). The first group (hereafter described as ‘Western European populations’) occupies the western Mediterranean region and western and central Europe, with an estimated wintering population size of 50 000 birds (Delany & Scott 2002). The second group (‘Eastern European populations’) inhabits the area of the Black Sea and eastern Mediterranean basins, with a wintering population size of 20 000 – 43 500 birds (Delany & Scott 2002). The third population group (‘Central Asian populations’) occupies the steppe areas from the Caspian Sea to Mongolia and western China and is estimated at 250 000 individuals (Delany & Scott 2002). The Western European breeding range is highly fragmented and comprises apparently isolated small-sized subpopulations (Hagemeijer & Blair 1997), while the distribution in the eastern part of the global range appears more continuous. The Western European populations are sedentary or short-distance migrants (within the Mediterranean basin), while the eastern populations migrate longer distances. Wintering areas for the three populations are clearly separated from each other (Cramps & Simons 1977; Saez-Royuela 1997). As for other birds presenting the same type of distribution (dense populations in Asia and fragmented subpopulations

in Western Europe; Cramps & Simons 1977), the distribution of the red-crested pochard may result from: (i) recent colonization of Western Europe by individuals from further east, or (ii) fragmentation of a previously continuous distribution range. Historical data suggest that the species colonized Western Europe in the late nineteenth century (Mayaud 1966; Cramps & Simons 1977; Hagemeijer & Blair 1997). The genetic study of Western European and Central Asian populations would allow us to evaluate this hypothesis. The first objective of this work was to investigate the amount of gene flow between Western European and Central Asian populations of red-crested pochard. A significantly reduced gene flow would indicate a currently essentially independent demographic functioning of the Western European populations relative to the much larger Asian populations. The second objective was to understand the biogeographical history of the red-crested pochard. Using genetic data, it is not possible to distinguish ancient fragmentation from colonization. Thus the analysis is restricted to the case of recent events. The observed patterns of genetic diversity were compared with the pattern expected for two possible biogeographical scenarios: recent colonization of Europe or recent range fragmentation. Recent colonization should result in a loss of genetic diversity along the colonization axis and marked genetic structure (Austerlitz et al. 1997). Positive exponential growth rate should also be detected. Under the recent rangefragmentation hypothesis, little difference would be expected in the diversity pattern in both populations and imprints of a negative exponential growth rate. The genetic analyses included two classes of molecular markers, the left domain of the mitochondrial DNA (mtDNA) control region (hypervariable control region I, CRI) and seven nuclear markers with biparental inheritance (four microsatellites, three nuclear introns). Two populations were included: Western Europe and Central Asia. Genetic analyses were supplemented by a morphometric analysis because morphology sometimes diverges quicker than neutral markers as a result of local selection (see Podolsky & Holtsford 1995; Nice & Shapiro 1999; Chan & Arcese 2003 for bird, insect and plant examples respectively).

Materials and methods Morphological analyses The 100 specimens (Western Europe n = 20; Central Europe n = 13; Central Asia n = 67) held at the Muséum National d’Histoire Naturelle, Paris, France and the British Museum of Natural History, Tring, UK were measured by one of the authors (P.D.D.R.). Each specimen was referenced according to the locality of collection. The following variables were measured with a calliper to the nearest mm: wing length, © 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 1035–1045

C O N S E R V A T I O N G E N E T I C S O F A D U C K S P E C I E S 1037 Table 1 Sample localities (population and subpopulation) and sample size for mitochondrial sequences, microsatellites (APH11, APH10, APH1 and SFI4) and nuclear introns (GAPD, OD and RP40) Microsatellites

Nuclear introns

Population

Sub-population

mtDNA

APH11

APH10

APH1

SFI4

GAPD

OD

RP40

Western Europe

Camargue Dombes Ebro delta Doñana Constance Total WE

10 9 2 10 2 33

24 30 7 21 3 85

23 30 4 22 3 82

23 30 5 21 3 82

17 24 0 0 2 43

13 27 6 19 2 67

5 25 5 2 2 39

1 28 6 0 2 37

Central Asia

Kazakhstan Volga delta Total CA

27 4 31

58 3 61

54 3 57

56 3 59

54 3 57

57 4 61

53 4 57

43 4 47

tarsus length, culmen length, bill width at base of bill, bill depth at base of bill, length between bill base and inner edge of nostril, length between bill tip and outer edge of nostril. The number of lamellae on the bill was also noted for each specimen. All characters were standardized (to zero mean and unit variance) prior to principal components analysis (PCA). Both sexes were pooled for the PCA. Differences between populations or sexes (a potentially confounding effect) in the multivariate morphometric space were investigated by mean of successive analyses of variance (anova) on the principal components (PCs).

Samples for the genetic analyses and DNA extraction Samples for the genetic analyses were either muscles in alcohol or dried legs (kept at room temperature) collected from red-crested pochards shot by hunters in different localities of the distribution range (Table 1, Fig. 1). All these localities are breeding sites but most samples might include migrating or wintering birds. Only samples from Doñana most likely represent breeding birds. Since the migration pathways and wintering areas seem to be distinct for Western European and Central Asian populations (see above), birds caught in Western Europe or Central Asia would be expected to belong to their respective population. Total genomic DNA was extracted using mini column extraction kits (DNeasy Tissue Kit, Qiagen) following the manufacturer’s instructions.

Mitochondrial DNA sequencing A 450 base pair (bp) fragment of the first (left) domain of the mtDNA control region (CR1) was amplified by the polymerase chain reaction (PCR) with the following primers: NDLF, 5′-AAA-TAA-GTC-ATT-ATT-CCT-GC-3′ (3′ end at position 253 in the Anas platyrhynchos sequence, GenBank accession number L22477) and NDLIR, 5′-AACCAG-AGG-CGC-AAA-AAT-GTG-3′ (3′ end at position 821 © 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 1035–1045

in the same sequence). Primers were designed by aligning the sequences of waterfowl species found in GenBank. PCR amplification was performed in a 50-µL reaction volume containing 2 µL DNA solution (variable concentration), 5 µL 10× buffer (Tris–HCl 100 mm + KCl 500 mm), 6.25 µm MgCl2, 3.33 µm dNTP, 1.67 µm of each primer and 0.2 units Taq DNA polymerase (Eurogentec). The annealing temperature was 52 °C and annealing duration was 45 s. Sequencing reactions were conducted with ABI dye terminator chemistry (Applied Biosystem) following the standard ABI cycle sequencing protocol and were electrophoresed on an ABI Prism 310 Genetic Analyser following recommended procedures, using NDLIR as the sequencing primer.

Microsatellites Between 100 and 146 individuals were genotyped at four microsatellite loci (see Table 1 for the sample size of each marker). Primer sequences were found in the literature (APH11, APH10 and APH1 in the Peking duck Anas platyrhynchos: Maak et al. 2000; SFI4 in the spectacled eider Somateria fisheri: Fields & Scribner 1997). PCRs were performed in a total volume of 12 µL containing 2 µL DNA solution (concentration variable), 1.2 µL 10× buffer (Tris– HCl 100 mm + KCl 500 mm), 2.08 µm MgCl2, 0.5 µm dNTP, 0.83 µm of forward primer, 0.17 µm of γ[32P]ATP-labelled reverse primer and 0.1 units Taq DNA polymerase. The annealing temperature was 52 °C for all marker except APH1 (for which the annealing temperature was 54 °C) and annealing duration was 30 s. PCR products were resolved by electrophoresis on 5% denaturing polyacrylamide gels, exposed for 12–72 h.

Nuclear introns Three nuclear introns were amplified on 130 individuals for GAPD, 96 for OD and 84 for RP40 (Table 1) using PCR

1038 L . G A Y E T A L . (same protocol as for the control region). Primer sequences were found in the literature (Friesen et al. 1997, 1999): GAPD (glyceraldehyde-3-phosphate dehydrogenase gene), RP40 (ribosomal protein 40 gene) and OD (ornithine decarboxylase gene). The annealing temperature was 56 °C for 45 s. Sequence polymorphism was revealed by single-strand conformation polymorphism (SSCP; Lessa 1992; Palumbi & Baker 1994) performed using an ABI Prism 310 Genetic analyser, following the manufacturer’s instructions (Applied Biosystems).

Data analysis Within-population analysis. The number of haplotypes (na), number of polymorphic sites (S), haplotype diversity (H ± SD) and mean number of pairwise differences (π ± SD) were estimated on mtDNA data using arlequin version 2.0 (Schneider et al. 2000). The hypothesis of selective neutrality of the control region fragment sequenced was tested using the D* and F* tests (Tajima 1989a; Fu 1997) with the program arlequin. Maximum likelihood estimates of the exponential growth rate ( g, scaled to the per sequence mutation rate) were obtained using a coalescence-based method (fluctuate, Kuhner et al. 1998). For nuclear data, the mean number of alleles (na), Nei’s unbiased estimates of expected heterozygosity (HE) and observed heterozygosity (HO) were calculated using genetix version 4.02 (Belkhir et al. 2001). FIS was calculated for each locus separately and for all loci together and significance was tested by permutation of individuals among populations (1000 permutations). genetix was used to test for linkage disequilibrium between pairs of loci in each population (1000 permutations). Levels of significance were adjusted using sequential Bonferroni corrections (Rice 1989). Among-population analyses. modeltest version 3.0 (Posada & Crandall 1998) was used to determine the appropriate model of substitution for the control region sequences. The selected model was Tamura–Nei with a gamma distribution of the substitution rates and a proportion of invariable sites. mega version 2.1 (Kumar et al. 2001) was then used to generate a phylogenetic tree of the mtDNA haplotypes by the neighbour-joining method based on a Tamura–Nei distance matrix with the shape parameter of the gamma distribution determined by modeltest. The analysis of population structure was based on variance partitioning. Mitochondrial DNA data were analysed by analysis of molecular variance (amova, Excoffier et al. 1992) with arlequin using a Kimura two-parameter distance for estimating ΦST values. For nuclear markers, the estimator θ of FST (Weir & Cockerham 1984) was estimated using genetix. RST was not used for microsatellites because both sample size and number of loci were too small for RST to give a better estimation of population structure than

FST (Gaggiotti et al. 1999). The significance of ΦST and FST estimates was tested by permutations of individuals among populations (1000 permutations). FST and ΦST estimates were used to estimate gene flow between the two populations, assuming a finite-island model, with the following equation at migration/drift equilibrium: FST = 1/(1 + 4Nmα) and Φ ST = 1/(1 + N fm fα), Nm being the number of migrants entering a population per generation, Nfmf is the number of female migrants and α = r/(r − 1) with r being the number of populations. In this study, r = 2 and α = 2. Estimates of gene flow based on FST and its analogues rely on the island model assumptions requiring equal population sizes and symmetric migration rates (Rousset 2001). In the case of the red-crested pochard, both assumptions probably do not hold (see Introduction). Therefore, the gene flow was also estimated using a maximumlikelihood method based on coalescence (Beerli & Felsenstein 1999) implemented in migrate Version 1.5.1. (Beerli 2002), using mtDNA sequences, microsatellites and nuclear introns separately, because these markers follow different mutational models (mtDNA sequences, Felsenstein’s mutation model; nuclear introns, infinite allele model; microsatellites, stepwise-mutation model). For all analyses, the default settings of migrate were used except that the number of short and long Markov chains and the number of trees sampled were increased in some runs (20 short chains with 5000 recorded genealogies and four long chains with 50 000 genealogies). Because convergence problems are common with Markov chain estimations, the convergence of the program was tested by using the options ‘heating’ and ‘replication’ and by reiterating the estimations with different starting values (for example the estimations from previous runs).

Results Morphometric analysis The two first axes of the PCA explain 50% of the total variance. The first axis of the PCA (PC1) indicates an obvious morphological differentiation between individuals from Western Europe and Central Asia, with the Eastern European population having an intermediate position (Fig. 2). This is confirmed by the analysis of variance: there is a strong population effect on PC1 (one-way anova; F2,97 = 59.28; P < 10−6) but no effect of sex (one-way anova; F2,97 = 0.37; P = 0.54). This first axis is mainly a size axis, with most biometric variables having a large contribution, except the number of lamellae (see Table 2 for the contribution of each variable to the PCs and percentages of explained variance for PC1 and PC2), Western and Central European specimens having, on average, smaller biometrics than individuals from Central Asia. © 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 1035–1045

C O N S E R V A T I O N G E N E T I C S O F A D U C K S P E C I E S 1039 Table 2 Eigenvalues, per cent of explained variance and contribution of each variable to the principal components

Eigenvalue Per cent of explained variance Wing Tarsus Culmen Width at bill base Depth at bill base Bill base to nostril Bill tip to nostril No. of lamellae

Fig. 2 Bivariate plot of PC1 and PC2 scores generated by a PCA on all specimens (sexes pooled) using the eight morphological variables (see Materials and methods).
, Western European population; +, Eastern European population; , Central Asian population.

Population genetic diversity The control region was sequenced in 64 individuals ( Table 1; GenBank accession numbers AY465764 to AY465827). The 450-bp analysed fragment starts around position 367 of the Anas plathyrhyncos sequence (GenBank accession number L22477) and ends around position 821. This segment proved highly variable, with one haplotype per individual in Central Asia (31 individuals) and 13 haplotypes in Western Europe (33 individuals). The sequence exhibited 74 polymorphic sites in Central Asia and 41 in Western

Locus

Allele

Central Asia

APH11

1 2 3 4

0.2869 0.6557 0.0410 0.0164

APH10

1 2 3 4 5

SFI4

1 2 3 4 5 6 7 6

Western Europe

Locus

Allele

0.2118 0.6706 0.1176 0.0000

APH1

1 2 3 4

0.6949 0.2288 0.0763 0.0000

0.6585 0.2256 0.1098 0.0061

0.7018 0.1228 0.0439 0.0000 0.0263

0.7195 0.2012 0.0000 0.0183 0.0488

GAPD

1 2 3 4 5

0.2951 0.1475 0.1639 0.0902 0.2213

0.2239 0.2090 0.0597 0.0672 0.2985

0.2105 0.5000 0.0965 0.0702 0.0088 0.0526 0.0614 0.1053

0.2558 0.4651 0.0233 0.0698 0.0000 0.0465 0.1395 0.0122

6 1 2 3 1 2 3

0.0820 0.3830 0.6064 0.0106 0.7544 0.2281 0.0175

0.1418 0.4595 0.5270 0.0135 0.6667 0.3205 0.0128

OD

© 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 1035–1045

PC2

2.83 35.37

1.14 14.29

0.40 0.64 0.65 0.62 0.78 0.71 0.56 0.07

0.55 − 0.31 − 0.35 − 0.19 − 0.05 0.17 0.35 0.65

Europe. Haplotype diversity was 1.000 ± 0.008 (± SD) and 0.723 ± 0.080 for Central Asia and Western Europe, respectively. The mean number of pairwise differences ranged from 0.027 ± 0.0125 in Central Asia to 0.012 ± 0.006 in Western Europe. Mitochondrial DNA diversity was thus significantly lower in Western Europe than in Central Asia (Mann–Whitney test, retaining only independent pairwise comparisons: NCentral Asia = 15, NWestern Europe = 16; U = 54.5; Z = 2.589; P = 0.009). There were between three and seven alleles per locus for the four microsatellites and the three nuclear introns (Tables 3 and 4). Unlike the sequence data, there was no significant difference of diversity between the two populations at the nuclear loci. The mean number of alleles per locus was 4.43 in Central Asia and 4.28 in Western Europe. Expected heterozygosities were similarly high (0.539 ± 0.148 and 0.554 ± 0.133 for Central Asia and Western Europe,

Central Asia

RP40

PC1

Western Europe

Table 3 Allele frequencies for seven nuclear loci (microsatellites: APH11, APH10, APH1 and SFI4; introns: GAPD, RP40 and OD) for Central Asian and Western European populations

1040 L . G A Y E T A L . Table 4 Diversity at seven nuclear loci (microsatellites: APH11, APH10, APH1 and SFI4; introns: GAPD, RP40 and OD) for Central Asian and Western European populations Locus name

Population

APH11 Central Asia Western Europe APH10 Central Asia Western Europe APH1 Central Asia Western Europe SFI4 Central Asia Western Europe GAPD Central Asia Western Europe RP40 Central Asia Western Europe OD Central Asia Western Europe

n

nall HE

HO

61 85 57 82 59 82 57 43 61 67 47 37 57 39

4 3 5 5 3 4 7 6 6 6 3 3 3 3

0.475 0.029 0.317 0.541 − 0.095 0.839 0.421 0.129 0.052 0.463 − 0.050 0.699 0.373 0.196 0.020 0.329 0.351 0.000 0.691 − 0.067 0.789 0.698 0.002 0.410 0.770 0.046 0.170 0.731 0.080 0.075 0.425 0.134 0.127 0.378 0.272 0.031 0.316 0.174 0.056 0.410 0.106 0.192

0.486 0.492 0.479 0.439 0.459 0.503 0.685 0.737 0.800 0.789 0.485 0.511 0.379 0.453

FIS

P

n is the sample size; nall the number of alleles per locus; HE the gene diversity; HO the observed heterozygosity; FIS Wright’s inbreeding coefficient and P the probability associated with the test of Hardy–Weinberg equilibrium.

respectively; Mann–Whitney test on expected heterozygosities for each locus: NCentral Asia = 7; NWestern Europe = 7; U = 20; Z = −0.575; P = 0.565). There was a significant deviation from Hardy–Weinberg equilibrium when considering all loci together (Central Asia: FIS = 0.076, P = 0.019; Western Europe: FIS = 0.093, P = 0.006). However, this result was predominantly caused by a significant heterozygote deficiency at locus APH1 (Central Asia: FIS = 0.196, P = 0.02; Western Europe: FIS = 0.351, P < 10−3). The FIS calculated without APH1 is much lower and only marginally significant (Central Asia: FIS = 0.060, P = 0.05; Western Europe: FIS = 0.055, P = 0.09). APH1 was not discarded from further analyses because this heterozygote deficiency should have limited effect on the estimates of interpopulation differentiation, as FST is especially intended to separate deviations from Hardy–Weinberg equilibrium due to geographical structuring, as opposed to other causes. After Bonferroni correction, no pair of loci was in significant linkage disequilibrium.

Population genetic structure The phylogenetic relationships between the control region haplotypes based on the neighbour-joining method are presented in Fig. 3. The topology of the tree is poorly supported, as shown by low bootstrap values. Haplotypes found in the two populations do not form any distinct clade. However, a nonrandom distribution of haplotypes is apparent: the Western European haplotypes are mainly

Fig. 3 Phylogenetic tree obtained by the neighbour-joining method, based on Tamura–Nei distances with gamma correction for 64 individuals. Each symbol corresponds to one individual, filled symbol indicate Western European populations while open symbols indicate Central Asian populations. The symbol corresponds to the subpopulation: , Dombes; , Camargue;
, Ebro Delta; , Doñana; , Constance; , Volga Delta; , Kazakhstan. Bootstrap values based on 1000 permutations.

grouped into two clades and several haplotypes are shared by more than one individual, while some haplotype groups are not detected in Western Europe. On the contrary, all haplotypes from Central Asia are present in only one individual, and all haplotypic groups are present in Central Asia. In addition, the branches of the tree are much shorter on average for the Western Europe haplotypes. This nonrandom distribution of haplotypes was confirmed by the amova, which indicates significant differentiation between Central Asian and Western European populations (ΦST = 0.152, P < 0.01) explaining 15% of the total genetic variance. On the contrary, estimates of population structure based on nuclear genes are very low and not significantly different from zero ( FST = 0.004; P = 0.14). © 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 1035–1045

C O N S E R V A T I O N G E N E T I C S O F A D U C K S P E C I E S 1041 Table 5 Number of migrants estimated using F-statistics assuming a finite-island model for the three different markers

Mitochondrial DNA Multilocus nuclear markers Microsatellites only (four loci) Nuclear introns only (three loci)

FST or ΦST

Nm or Nfmf estimation

ΦST = 0.152 0.004 (ns) 0.003 (ns) 0.006 (ns)

Nfmf = 1.40 31.20 49.48 21.43

ns, not significantly different form zero.

The corresponding Nm values are about 30 migrants per generation for nuclear markers and fewer than two female migrants per generation for mtDNA between Western Europe and Central Asia (Table 5). No significant structure was detected among European populations for mtDNA (amova, P = 0.312) or for nuclear markers (FST not significantly different from zero). When using migrate on mtDNA sequences, some runs produced biologically unrealistic values (of population size for example) suggesting convergence towards local likelihood maxima, and were excluded. Regarding microsatellites, gene flow and population size estimates were convergent but some runs gave extremely wide confidence intervals. For nuclear introns, all estimations were convergent. Estimates of gene flow with migrate provided more similar results between the different markers than estimation based on the finite-island model. Gene flow from Central Asia to Western Europe was estimated as 0.64 female migrants (95% confidence interval: 0.249–1.145) for mtDNA control region sequences (or 1.280 migrants per generation assuming a balanced sex ratio and no sex-biased dispersal), 1.706 (1.098 –2811.914) for microsatellites [0.969 (0.944–1.004) if runs with very large confidence intervals are excluded] and 4.095 (2.680 – 4.976) for nuclear introns (Fig. 4). The numbers of migrants from Western Europe to Central Asia were similar (symmetrical gene flow) for nuclear markers. For mtDNA sequence data though, migrate estimated significantly higher female gene flow from Western Europe to Central Asia [51.468 (16.550 – 75.247)] than from Central Asia to Western Europe [0.640 (0.249 –1.145)]. This could however, be a consequence of the hierarchy of diversities among the two populations: all haplotypes found in Europe are also present in Asia, so each European individual could be a potential Asian migrant.

Demographic inferences Neutrality tests for mtDNA control region sequences rejected the hypothesis of the neutral equilibrium model for the Western Europe population with Tajima’s test © 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 1035–1045

Fig. 4 Mean effective size (2Nf µ for mitochondrial sequences, with Nf = females effective size = 0.5 Ne for a balanced sex ratio; Neµ for nuclear introns and microsatellites) and mean number of migrants (2Nfmf for mitochondrial sequences, with mf = females migration rate; Nem for nuclear introns and microsatellites) estimated by the software migrate on mitochondrial sequences (a), nuclear introns (b) and microsatellites (c). Values in bold type indicate the maximum likelihood estimator, and those in italic indicate 95% confidence intervals.

(D = −1.637; P = 0.0425) and for Central Asia with Fu’s test (Fs = −24.101; P < 10− 4). In the two other tests, the neutral hypothesis was not rejected. Growth rates estimated by fluctuate were positive for both populations, with a larger growth rate for Central Asia (g = 409.18; SD = 32.79 for Central Asia and g = 64.03; SD = 22.70 for Western Europe). Considering a per-site mutation rate µ for the mtDNA control region comprised between 10−5 and 10−7 (Quinn 1992; Freeland 1997; Kidd & Friesen 1998), an absolute growth rate (λ = exp g *µ) and the time until the populations double were evaluated, as presented in Table 6. Even if a mutation rate as high as 10 −5 is considered, λ is higher than one for both populations.

1042 L . G A Y E T A L . Table 6 Estimates of θ (effective population size × mutation rate) and of g (exponential growth rate) for Central Asian and Western European populations using fluctuate

g θ λ µ = 10−5 µ = 10−7 td µ = 10−5 µ = 10−7

Central Asia

Western Europe

409.184 (SD 32.791) 3.177 (SD 0.931)

64.032 (SD 22.701) 0.065 (SD 0.010)

1.004100 (1.004429 –1.003771) 1.000041 (1.000044 –1.000038)

1.000640 (1.000413 –1.000413) 1.000006 (1.000009 –1.000004)

170 16.906

1.083 115.525

The ratio of transitions to transversions was 7.5; θ = 2Nf µ with Nf being the female effective population size and µ being the mutation rate. λ is the absolute growth rate [λ = exp(g × µ)] and td is the time until the population doubles in years. Confidence intervals are shown in parenthese and SD are indicated.

Thus, neither population shows any sign of demographic decline.

summary statistics and should thus be more powerful. However, it is computationally very demanding (Emerson et al. 2001) and it is practically difficult to find the appropriate chain length for reaching convergence. As a consequence, the robustness of these estimations to violations of the underlying assumptions has still to be tested (Neigel 2002) and it seems difficult to conclude on the comparison of FST-based and coalescence-based estimates of gene flow. The existence of morphological differences between the Western European and Central Asian populations indicates that they constitute distinct pools of individuals. Morphological differences are not necessarily linked to genetic structure, as they could result from phenotypic plasticity (James 1983). However, if dispersal between Western Europe and Central Asia was completely random, no morphological differences would be expected, not even as a result of plasticity. This morphological analysis thus further confirms that individuals do not move randomly between these two distant areas. Moreover, banding data do not mention any bird banded in Central Asia being recaptured in Western Europe. Of course, the validity of that observation depends on the ‘banding effort’ in Central Asia, but it argues for restricted migration from Central Asia to Western Europe.

Discussion

Comparison of mitochondrial and nuclear genetic structure

Population structure

Whereas ΦST indicates a substantial level of mitochondrial genetic structure, its nuclear analogue F ST is very low (0.004) and not significantly different from zero. Because of differences in effective population size of the markers, structure indices estimated with nuclear or mitochondrial markers are not directly comparable (see Crochet 2000). If the red-crested pochard populations were at equilibrium and without sex-biased dispersal, the observed mitochondrial ΦST of 0.152 would be equivalent to a theoretical nuclear FST of 0.043, which is approximately 10 times higher than the observed value for nuclear markers. Two hypotheses could explain the discrepancy between mitochondrial and nuclear structure: (i) return time to equilibrium and (ii) sex-biased dispersal. In the first of these hypotheses, because the effective population size of mitochondrial markers is four times lower than for nuclear DNA in gonochoric species, mtDNA returns faster to equilibrium than nuclear DNA. In humans, it has been shown that demographic events disturbing population equilibrium resulted in different genetic diversity patterns for mitochondrial and nuclear DNA (Fay & Wu 1999; Hey & Harris 1999). In the red-crested pochard, recent demographic events (founding effect or bottlenecks) could have affected genetic equilibrium (see below), leading to different nuclear and mitochondrial genetic structure.

The lack of significant genetic structure among Western European subpopulations for all markers fits well with the ringing data (Defos du Rau 2002), which shows numerous exchanges of individuals between these various subpopulations. A significant genetic structure exists between Western Europe and Central Asia for the mtDNA (ΦST = 0.152, P < 0.01) but not for nuclear loci. Estimates of female dispersal rates from Central Asia to Western Europe using mtDNA are consistently low (1.4 females per generation for FST-derived estimates and 0.64 females per generation for coalescence-based methods). Considering the recent history of the species (see below), these results are probably based on a nonequilibrium situation and actual levels of dispersal between these populations are probably even lower (see Whitlock & McCauley 1999 for details on the implications of deviation from the assumptions of the island model). Estimates of the amount of nuclear gene flow are more difficult to interpret. On the one hand, the nonsignificant value of nuclear FST precludes making any precise estimates of gene flow, but suggests a high level of gene flow. On the other hand, migrate estimates suggest a low level of nuclear gene flow, even if the very large confidence intervals for microsatellites make this conclusion questionable. The approach employed in migrate makes full use of data rather than relying on

© 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 1035–1045

C O N S E R V A T I O N G E N E T I C S O F A D U C K S P E C I E S 1043 Alternatively, the difference of structuring patterns between mtDNA and nuclear markers could be the consequence of sex-biased dispersal. In Anseriformes (geese and ducks, Greenwood & Harvey 1982), unlike the prevalent pattern of male-biased philopatry in avian species, females often display strong natal- and breeding-site fidelity while males can migrate long distances (see Avise et al. 1992 for the snow goose Anser caerulescens and Blums et al. 2002 for three duck species). As a result, a recent study using nuclear and mitochondrial markers in the spectacled eider duck Somateria fisheri found the same pattern of strong mitochondrial structure and low nuclear differentiation as obtained here (Scribner et al. 2001). A comparison between male and female relatedness within populations, as has been performed in the red grouse Lagopus lagopus scoticus (Piertney et al. 1998) could allow discrimination between these two hypotheses. If natal philopatry reduces female-mediated gene flow, females are expected to be more closely related than males (Luikart & England 1999, see Prugnolle & De Meeus 2002 for a review).

Neutral evolution of mtDNA and demographic inferences The failure of red-crested pochard control region sequences to pass Tajima’s test (Tajima 1989a) (for Western Europe) and Fu’s test (Fu 1997) (for Central Asia) does not necessarily indicate a non-neutral molecular evolution. Deviations from the assumption of demographic equilibrium (after demographic expansions or bottlenecks) can lead to the rejection of neutrality in absence of selection (Tajima 1989b). Indeed, Tajima’s test has been used before to make demographic inferences (e.g. Fry & Zink 1998 in the song sparrow Melospiza melodia; Fay & Wu 1999 in humans). fluctuate also estimates a positive growth rate for each population, but could be affected by selective processes as well. However, various studies have indicated that mtDNA control region polymorphism is usually selectively neutral (Fry & Zink 1998; Milot et al. 2000; Griswold & Baker 2002), even if hitchhiking cannot be excluded because of the complete genetic linkage of mitochondrial genes. The results of Tajima’s and Fu’s tests and fluctuate are thus likely to reflect demographic fluctuations of red-crested pochard populations (demographic expansion in Central Asia, founding effect in Western Europe). To reject the selection hypothesis, other neutrality tests discriminating the effect of demography and selection would be necessary (see Nielsen 2001 for a review).

Phylogeography of the red-crested pochard Despite low mitochondrial gene flow, the absence of original mitochondrial lineages in Western Europe and the lack of reciprocal monophyly between Western Europe © 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 1035–1045

and Central Asia indicate a recent isolation of Western European from Central Asian populations. The high FST-estimates for mitochondrial sequences are compatible with an event of colonization (Austerlitz et al. 1997). The ‘star-like’ phylogeny of Western Europe haplotypes contrasts with the deeper branches among Central Asian lineages, and haplotype diversity is low in Europe compared to central Asia. All lineages present in Western Europe are also present in Central Asia. The haplotype diversity in Western Europe is thus a limited sample of the Central Asian diversity. This loss of diversity is the expected outcome of a colonization event by a low number of founders, with the source population containing the most divergent haplotypes (Austerlitz et al. 1997; see also Ingman et al. 2000 for an example in humans). Mitochondrial data are thus compatible with a recent colonization scenario for Western Europe. Nevertheless, a similar genetic signal would result from a strong decrease in population size in Europe following fragmentation of a formerly continuous distribution with little or no isolation by distance. The demographic inferences obtained with fluctuate and confirmed by the results of Tajima’s test, however, indicate an increase in population size both in Central Asia and Western Europe, which does not support the fragmentation scenario. Moreover, historical data date the first breeding attempts by the red-crested pochard in Western and Central Europe from the late 1800s (Mayaud 1966; Cramps & Simons 1977; Hagemeijer & Blair 1997). A recent colonization of Western Europe by the red-crested pochard is therefore the most likely scenario.

Conservation of the red-crested pochard in Western Europe Analyses of mtDNA and nuclear markers clearly demonstrate that populations of red-crested pochard are not structured in reciprocally monophyletic clusters for any marker and that the Western European populations do not contain any original genetic variants. Western European populations thus do not constitute an Evolutionarily Significant Unit (Moritz 1994). However, mitochondrial allele frequencies are significantly different between Western Europe and Central Asia, which corresponds to the definition of the Management Unit sensu Moritz (1994). Given the low amount of female-mediated gene flow estimated from FST or migrate, the demographic contribution of migrant females from Central Asia to Western Europe can be confidently considered as negligible. Morphological differences between these populations regardless of the sex show that males do not disperse freely among these areas either. In terms of demography, the low number of female migrants means that numbers of red-crested pochards in Western Europe cannot be efficiently supplemented by

1044 L . G A Y E T A L . immigration from the larger Asian populations. The Western European populations of red-crested pochard thus need to be managed independently from the large Central Asian populations. Defining precisely the limits of this management unit will require more sampling in Eastern Europe and Turkey, to determine whether Eastern European populations belong to the same management unit as the Western European populations or are part of the Central Asian populations, or even constitute a distinct unit.

Acknowledgements We thank all the duck hunters from Camargue and Dombes (France) and Ebro Delta (Catalonia) as well as from the Chassorbis, D.H.D. Laïka and Seladang hunting companies who provided us with the materials used in this work. In particular, we thank Cati Gerique (Generalitat Valenciana), Christophe Buquet, Fransesc Vidal Esquerre (Natural Park of Ebro Delta), Jean-Yves Fournier (ONCFS), Andy Green and Jordi Figuerola (Estación Biológica de Doñana), Alain Méric Grossi, Dr Botond Kiss (Danube Delta National Institute), Eric Wacheux (Chassorbis). We are also grateful to Patricia Sourrouille and Chantal Debain for technical assistance with the molecular work and to Mark Adams, Frédéric Jiguet and Jean-Marc Pons for their assistance in the British Museum of Natural History and the Muséum National d’Histoire Naturelle, respectively. We thank Philippe Jarne, Nicolas Galtier and two anonymous referees for helpful comments on the manuscript.

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This work formed part of the Master of Science degree for L. Gay who is currently pursuing a doctorate at the CEFE-CNRS in Montpellier on the impact of selection and hybridization on the speciation process in large white-headed gulls. P-A. Crochet is a post-doctorate researcher at the CEFE–CNRS. His main interest is in the application of molecular methods to the study of natural populations with an emphasis on questions ranging from dispersal to systematics, phylogeny and biogeography. P. Defos du Rau and J-Y. Mondain-Monval work for ONCFS (the French Game and Wildlife Service) and are involved in monitoring surveys and management plans for various animal species and wetland sites.