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Genetic and morphological variation in the endangered crayfish species, Austropotamobius pallipes (Lereboullet) (Crustacea, A.... Article  in  Aquatic Sciences · January 2000 DOI: 10.1007/PL00001327

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Aquatic Sciences

Genetic and morphological variation in the endangered crayfish species, Austropotamobius pallipes (Lereboullet) (Crustacea, Astacidae) from the Poitou-Charentes region (France) Frédéric Grandjean* and Catherine Souty-Grosset Laboratoire de Biologie Animale, URM 6556, Université de Poitiers, 40 Avenue du Recteur Pineau, 86022 Poitiers Cedex, France

Key words: Austropotamobius pallipes, endangered species, mtDNA, multivariate analysis, genetic and morphological population structure, conservation genetics. ABSTRACT The white-clawed crayfish, Austropotamobius pallipes, an endangered species in Europe, was surveyed for genetic and morphological variation as part of a larger project aimed at clarifying its French stock structure. Analysis from four morphological characters discriminated two groups, one including the 8 stream populations and the other comprising a single pond population. Total mitochondrial DNA variation was examined by RFLP analysis using 11 restriction enzymes for 120 animals sampled from these 9 populations located in Poitou-Charentes region. Among the three haplotypes revealed, two were found in animals sampled from brook populations and the third haplotype was only found in individuals from the pond population. Mitochondrial DNA nucleotide diversity values within species ranged from 0.57 to 1.31 %. The analysis of genetic variance showed no structuring of genetic variation by hydrographic basins and could reflect the impact of translocations by man although other explanations are possible.

Introduction The white-clawed crayfish Austropotamobius pallipes, native to northern Europe, has been in decline for 100 years (Vigneux et al., 1993) primarily due to habitat destruction, pollution, the introduction of exotic crayfish and the crayfish plague Aphanomyces astaci Shikora (Westman, 1985). It is listed as vulnerable and rare by the I.U.C.N (International Union for the Conservation of Nature and Natural Resources) (Groombridge, 1994). Throughout its range, this species exhibits geographically fragmented populations mainly due to degradation of water quality. Population genetics theory predicts that small isolated populations with low levels of gene flow, characteristically show a low genetic diversity within populations and * Corresponding author, e-mail: [email protected].

2

Grandjean and Souty-Grosset

a high genetic differentiation among populations. These phenomena are often responsible for a genetic fragmentation of species into discrete stocks which could reflect local adaptation to their environment. Information on the degree and distribution of genetic variability in natural endangered species stocks is important for conservation strategies. Conservationists working to reintroduce crayfish to native waters from which they have disappeared need to identify genetic stocks of crayfish in order to avoid the mixing of genetically highly differentiated populations within a hydrographic basin. Moreover, in case of population reinforcement, introduction of genetically different individuals could adversely alter the gene pool of locally adapted populations. Thus the application of genetics to conservation issues is a practical endeavour and has produced concrete recommendations for management of animal species (Echelle et al., 1989; Ashley et al., 1990; Welsh and McClelland, 1990; Vogler et al., 1993a, b; Williams et al., 1990; Gray, 1995). Allozymes have been widely used as markers to study the genetic variability of crayfish populations. However, most allozyme studies have revealed very low levels of genetic variation in crustaceans (Hedgecock et al., 1982) and in crayfish species in particular (Nemeth and Tracey, 1979; Brown, 1980; Albrecht and Von Hagen, 1981; Attard and Vianet, 1985; Austin, 1986; Austin and Knott, 1996; Busack, 1988, 1989; Fevolden and Hessen, 1989; Agerberg, 1990). Recently, two studies have revealed the suitability of allozymes to assess the genetic structure of A. pallipes in a survey of Italian and Swiss populations (Santucci et al., 1997; Lörtscher et al., 1997). However, a low level of genetic variation within populations is reported in both studies. These results could be related to the increased fragmentation of habitat which has allowed genetic variation to develop between subpopulations and to decrease within subpopulations as a result of genetic drift. In regard to these results, it seemed interesting to obtain additional data from a more variable genetic marker such as mitochondrial DNA. Within the last ten years, techniques using mitochondrial DNA variability have become widely used in genetic studies of population differentiation (Avise et al., 1987). Restriction fragment length polymorphism analysis (RFLP) from total mtDNA provides a sensitive method to reveal genetic differentiation that may exist among populations within a species (Avise et al., 1987; Avise, 1991). In marine Decapods, several studies have revealed the suitability of this genetic marker in stock identifications (Komm et al., 1982; Mc Lean et al., 1983; Brasher et al., 1992 a, b; Ovenden et al., 1992). Recently, Grandjean et al. (1997 a, 1998) have shown the suitability of mitochondrial DNA to assess the genetic variation between four European populations of the white-clawed crayfish, A. pallipes. We present here a preliminary attempt to describe the degree of morphological and genetic variation among nine French populations of the endangered crayfish species A. pallipes sampled from Poitou-Charentes region. The purpose of this study was to define a coherent plan for the management of this species on a regional scale.

Materials and methods Adult crayfish were collected by drop-nets, hand and electric fishing from nine locations in the region of Poitou-Charentes (France) (Fig. 1). One population (Pinail)

Genetic variability in white-clawed crayfish

3

Figure 1. Geographical location of the population samples studied in Austropotamobius pallipes from Poitou-Charentes with main hydrographic systems. The numbers: 16, 79 and 89 are the French numbers for the departments of Charentes, Deux-Sèvres and Vienne, respectively

4

Grandjean and Souty-Grosset

Table 1. Distribution of nine populations according to their hydrographic basins. Number of animals collected by different fishing techniques for morphometric analysis Departments

Basins

Sub-basins

Deux-Sèvres

La Vienne La Viette La Martinière L’Auxances Le Magot La Sèvre Niortaise

Vienne

Charente

La Vienne

La Charente

Populations

number of males

number of females

fishing techniques

34 24

41 25

Drops-nets Drops-nets

Le Magnerolle 37 Le Puits 44 d’Enfer Le Gatineau 20

33 88

Drops-nets Drops-nets

25

Drops-nets/ Hand fishing

La Crochatière 26

27

Drops-nets

Pinail

11

15

Drops-nets

29 13

20 21

Drops-nets Drops-nets Electric fishing

La Tude La Gace La Tardoire La Fontaine St Pierre

was collected from small ponds; the remainder were stream (brook) populations which classified according to their hydrographic basins (Table 1).

Morphometric analysis Measurements were made to the nearest 0.1 mm using an eyepiece micrometer. The following morphometric data were collected (Fig. 2): post-orbital length (POL) (from the posterior orbital edge to the dorsal extremity of the cephalothorax), abdominal width (AW) (at the 2nd abdominal segment), and the width (CW) and length (CL) of the claws. Measurements have been selected from the work of Agerberg (1990). After measurements, most individuals were returned to their sampling site except those used in the genetic study. Individuals with apparent skeletal damage or signs of regeneration were omitted from morphological analysis. The data from males and females were analysed separately because these traits are known to be sexually dimorphic in crayfish (Stein et al., 1977, Grandjean et al., 1997b, c). To investigate differences in morphology, several analyses were performed: Mean Comparison of POL to check sample bias, Analysis of Covariance (ANCOVA) taking POL as reference value and Canonical Variate Analysis (CVA). ANCOVA and CVA were run using logarithmically transformed data grouped by populations. The CVA maximises the separation between groups relative to the within-group variance, taking into account the within-group correlation between characters (Thorpe, 1976, 1980), and is the most commonly used method for the investigation of morphological differentiation between populations.

Genetic variability in white-clawed crayfish

5

Figure 2. Morphometric measurements of crayfish collected in the nine populations of PoitouCharentes

Genetic analysis The number of animals per population used for genetic analysis is given in Table 8. Heart, green glands and testes obtained from fresh animals were used for the mtDNA extraction. The extraction of total mtDNA was performed according to the adjusted method of Grandjean and Souty-Grosset (1996). MtDNA samples were cleaved with eleven restriction endonucleases: five 6base cutters (Bgl II, Eco RI, Hind III, Pst I, Xho I); one 5-base cutter (Hinf I) and five 4-base cutters: Acc II, Hae III, Hpa II, Nde II, Taq I. Digestions were performed according to the manufacturer’s instructions (Gibco BRL). The restriction fragments obtained were separated in 1.2% agarose gels in TE-buffer for 15 h at 30 volts. Gels were stained with SYBRTM Green I (FMC Bioproducts) and visualized with a UV light transluminator. The restriction fragment patterns from each of the eleven endonucleases were identified by a letter, each individual being characterized by a composite haplotype of eleven letters in the order presented in Table 3. The total proportion of shared fragments (S-value) between two individuals was calculated from the following equation (Nei and Li, 1979).

6

Grandjean and Souty-Grosset

2mij Sij = 04 mi + mj where mi and mj are the numbers of restriction fragments in DNA sequences i and j, respectively, whereas mij is the number of fragments shared by the two sequences. The number of nucleotide substitutions per site d can be estimated by – ln Sij dˆ ij = 03 r where r is the number of bases per restriction site (Nei and Li, 1979). When different kinds of enzymes with different r values are used, the mean number of nucleotide substitutions can be estimated by the formula given by Nei and Tajima (1981): ∑ mkrkdij (k) k dˆ ij = 039 ∑ mkrk k

mi (k) + mj (k) where mk = 00 and k refers to the kth class of restriction enzymes. 2 The data were tested for genetic subdivision using analysis of molecular variance (AMOVA) from a program developed by Excoffier et al. (1992). This method was used to analyze the genetic structure within and among brook populations using variance component estimates in a hierarchical analysis. Three hierarchical levels were recognized : (1) within populations (within each local sampling), (2) among populations within hydrographic basins, (3) among hydrographic basins. A Mantel test was performed to determine if positive covariation exists among morphological and genetic distance matrices. This non parametric test analyses similarity between two distance matrices (Mantel, 1967; Douglas and Elder, 1982).

Results Morphometric data The mean comparisons of POL are given in Table 2. Significant differences for both sexes are revealed among many populations. On average, the smallest individuals were collected in Fontaine Saint-Pierre, Gatineau and Pinail and the largest in Crochatière, Magnerolle and Martinière populations. ANCOVA results for CL, CW and AW are given in Tables 3, 4 and 5 respectively. For CL, there were significant differences between the regression slope values of Pinail and most other populations except for Gatineau in both sexes. In

– 0,78 (1; 31)

– 6,73 (1; 48)

– 6,28 (1; 58)

0,19 (1; 40)

– 5,84 (1; 49)

– 3,36 (1; 35)

– 5,49 (1; 37)

– 1,95 (1; 22)

Gatineau

Magnerolle

Puits d’Enfer

Gace

Martinière

Magot

Crochatière

Pinail

Fontaine St-Pierre

Fontaine St. Pierre

– 1,44 (1; 29)

– 5,86 (1; 44)

– 3,43 (1; 42)

– 6,05 (1; 56)

0,925 (1; 47)

– 6,62 (1; 65)

– 7,19 (1; 55)

– 1,7 (1; 44)

Gatineau

4,62 (1; 46)

0,15 (1; 61)

1,83 (1; 59)

0,09 (1; 73)

7,23 (1; 64)

– 0,52 (1; 82)

– 11,09 (1; 56)

– 9,18 (1; 52)

Magnerolle

4,13 (1; 56)

0,58 (1;71)

2,11 (1; 69)

0,57 (1; 82)

7,33 (1; 74)

5,07 (1; 119)

– 6,58 (1; 111)

– 7,51 (1; 107)

Puits d’Enfer

– 1,65 (1; 38)

– 5,93 (1; 53)

– 3,97 (1; 51)

– 6,53 (1; 65)

2,04 (1; 106)

6,55 (1; 51)

– 3,87 (1; 43)

– 3,94 (1; 39)

Gace

3,74 (1; 47)

0,05 (1; 62)

1,58 (1; 60)

– 5,69 (1; 59)

– 4,61 (1; 127)

0,71 (1; 72)

– 10,26 (1; 64)

– 9,13 (1; 60)

Martinière

– 1,83 (1; 33)

– 1,41 (1; 48)

2,85 (1; 64)

– 0,93 (1; 43)

0,33 (1; 111)

3,1 (1; 56)

– 3,45 (1; 48)

– 3,94 (1; 44)

Magot

3,74 (1;35)

– 3,13 (1; 50)

– 1,3 (1; 66)

– 6,05 (1; 45)

– 5,2 (1; 113)

– 0,69 (1; 58)

– 9,96 (1; 50)

– 8,41 (1; 46)

Crochatière

8,16 (1; 40)

2,32 (1; 38)

8,12 (1; 54)

2,86 (1; 33)

4,47 (1; 101)

9,3 (1; 46)

– 0,99 (1; 38)

–2 (1; 34)

Pinail

Table 2. Mean comparisons of POL between the nine samples using t-test. Values for males are given above the diagonal. Values in bold characters showed significant differences between samples (P < 0.05). The degrees of freedom are given in parentheses

Genetic variability in white-clawed crayfish 7

20.32 (1; 30)

0.028 (1; 47)

7.39 (1; 57)

0.24 (1; 39)

2.95 (1; 48)

2.35 (1; 34)

20.93 (1; 36)

10.41 (1; 21)

Gatineau

Magnerolle

Puits d’Enfer

Gace

Martinière

Magot

Crochatière

Pinail

Fontaine St-Pierre

Fontaine St-Pierre

128.87 (1; 28)

2.18 (1; 43)

1.48 (1; 41)

0.85 (1; 55)

15.72 (1; 46)

0,15 (1; 64)

7.68 (1; 54)

4.27 (1; 43)

Gatineau

3.48 (1; 45)

2.74 (1; 60)

0.42 (1; 58)

0.41 (1; 72)

5.54 (1; 63)

1.06 (1; 81)

0.34 (1; 55)

0.38 (1; 51)

Magnerolle

21.21 (1; 55)

0.29 (1; 70)

0.62 (1; 68)

0.19 (1; 82)

21.26 (1; 73)

1.03 (1; 118)

0.04 (1; 110)

5.59 (1; 106)

Puits d’Enfer

0.91 (1; 37)

35.17 (1; 52)

9.55 (1; 50)

13.98 (1; 64)

23.37 (1; 105)

15.79 (1; 50)

4.37 (1; 42)

4.89 (1; 38)

Gace

15.18 (1; 46)

1.21 (1; 61)

0.04 (1; 59)

25.21 (1; 58)

4.18 (1; 126)

0.9 (1; 71)

0.19 (1; 63)

1.54 (1; 59)

Martinière

14.14 (1; 32)

2.26 (1; 47)

2.74 (1; 63)

9.31 (1; 42)

0.02 (1; 101)

1.58 (1; 55)

0.55 (1; 47)

4.6 (1; 43)

Magot

61.29 (1; 34)

0.33 (1; 49)

6.46 (1; 65)

16.34 (1; 44)

0.14 (1; 112)

1.48 (1; 57)

0.02 (1; 49)

0 (1; 45)

Crochatière

61.86 (1; 39)

44.4 (1; 37)

67.89 (1; 53)

55.9 (1; 32)

113.29 (1; 100)

39.65 (1; 45)

36.32 (1; 37)

83.6 (1; 33)

Pinail

Table 3. ANCOVA of CL with POL as covariate. F-value are given above and below the diagonal for females and males, respectively. F-values in bold characters show significant differences between samples (P < 0.05). The degrees of freedom are given in parentheses

8 Grandjean and Souty-Grosset

4.05 (1; 30)

0.22 (1; 47)

10.57 (1; 57)

0.03 (1; 39)

13.37 (1; 48)

0.043 (1; 34)

13.85 (1; 36)

2.25 (1; 21)

Gatineau

Magnerolle

Puits d’Enfer

Gace

Martinière

Magot

Crochatière

Pinail

Fontaine St-Pierre

Fontaine St-Pierre

7.34 (1; 28)

15.3 (1; 43)

0.21 (1; 41)

12.53 (1; 55)

0.63 (1; 46)

9.52 (1; 64)

0.03 (1; 54)

5.19 (1; 43)

Gatineau

1 (1; 45)

2.47 (1; 60)

1.21 (1; 58)

2.56 (1; 72)

6.32 (1; 63)

0 (1; 81)

0.93 (1; 55)

2.68 (1; 51)

Magnerolle

10.18 (1; 55)

2.13 (1; 70)

2.6 (1; 68)

2.45 (1; 82)

16.16 (1; 73)

0.77 (1; 118)

4.36 (1; 110)

19.47 (1; 106)

Puits d’Enfer

0.1 (1; 37)

18.71 (1; 52)

1.52 (1; 50)

16.37 (1; 64)

0.08 (1; 105)

0.3 (1; 50)

1.94 (1; 42)

9.4 (1; 38)

Gace

16.41 (1; 46)

0.18 (1; 61)

9.38 (1; 59)

0.45 (1; 58)

5.69 (1; 126)

2.19 (1; 71)

4.02 (1; 63)

12.62 (1; 59)

Martinière

0.44 (1; 32)

7.75 (1; 47)

0.35 (1; 63)

1.94 (1; 42)

6.34 (1; 101)

1.4 (1; 55)

7.36 (1; 47)

17.86 (1; 43)

Magot

15.55 (1; 34)

0.94 (1; 49)

1.6 (1; 65)

0.37 (1; 44)

1.33 (1; 112)

0.08 (1; 57)

0.75 (1; 49)

4.26 (1; 45)

Crochatière

0.37 (1; 39)

8.5 (1; 37)

6.31 (1; 53)

2.79 (1; 32)

5.32 (1; 100)

0.32 (1; 45)

0.04 (1; 37)

9.31 (1; 33)

Pinail

Table 4. ANCOVA of CW with POL as covariate. F-value are given above and below the diagonal for females and males, respectively. F-values in bold characters show significant differences between samples (P < 0.05). The degrees of freedom are given in parentheses

Genetic variability in white-clawed crayfish 9

15.5 (1; 30)

0.21 (1; 47)

6.24 (1; 57)

1.43 (1; 39)

1.79 (1; 48)

1.02 (1; 34)

17.11 (1; 36)

0.15 (1; 21)

Gatineau

Magnerolle

Puits d’Enfer

Gace

Martinière

Magot

Crochatière

Pinail

Fontaine St-Pierre

Fontaine St-Pierre

6.56 (1; 28)

5.1 (1; 43)

2.49 (1; 41)

2.15 (1; 55)

1.47 (1; 46)

1.13 (1; 64)

6.21 (1; 54)

2.01 (1; 43)

Gatineau

0.17 (1; 45)

0.28 (1; 60)

1.17 (1; 58)

1.22 (1; 72)

1.83 (1; 63)

0.22 (1; 81)

0.83 (1; 50)

0.2 (1; 51)

Magnerolle

4.24 (1; 55)

0.02 (1; 70)

0.61 (1; 68)

0.17 (1; 82)

9.56 (1; 73)

0.61 (1; 118)

0.21 (1; 110)

0.72 (1; 106)

Puits d’Enfer

0.05 (1; 37)

17.86 (1; 52)

1.53 (1; 50)

5.36 (1; 64)

0.01 (1; 105)

0.19 (1; 50)

0.17 (1; 42)

0.43 (1; 38)

Gace

1.44 (1; 46)

0.13 (1; 61)

0.68 (1; 59)

0.4 (1; 58)

1.34 (1; 126)

0.38 (1; 71)

0.73 (1; 63)

0.57 (1; 59)

Martinière

0.6 (1; 32)

0.99 (1; 47)

0.74 (1; 63)

1.49 (1; 42)

0.62 (1; 101)

0.64 (1; 55)

5.3 (1; 47)

5.69 (1; 43)

Magot

12.88 (1; 34)

0.05 (1; 49)

4.45 (1; 65)

0.04 (1; 44)

0 (1; 112)

1,61 (1; 57)

0.36 (1; 49)

0.06 (1; 45)

Crochatière

6.58 (1; 39)

10.52 (1; 37)

6.1 (1; 53)

10.66 (1; 32)

3.09 (1; 100)

4.68 (1; 45)

6.7 (1; 37)

3.92 (1; 33)

Pinail

Table 5. ANCOVA of AW with POL as covariate. F-value are given above and below the diagonal for females and males, respectively. F-values in bold characters show significant differences between samples (P < 0.05). The degrees of freedom are given in parentheses

10 Grandjean and Souty-Grosset

Genetic variability in white-clawed crayfish

11

most cases, no significant differences are shown between all other populations. Similar results are observed for CW. However, the regression slope values of females from Pinail showed significant differences from those of the other populations except those of Fontaine Saint-Pierre and Puits d’Enfer. In males, the Pinail population seemed to be less differentiated than in females because only the differences between the regression slope values with Gatineau, Puits d’Enfer and Crochatière populations were significant. For AW, the regression slope values are more homogenous than those for CW and CL. In females, the regression slope values of Fontaine Saint-Pierre showed significant differences from those of all the other populations excluding Magnerolle.

Multivariate analysis In males, canonical discriminant analysis using all mature animals indicated significant differences in the centroid positions among the nine populations (Wilk’s l = 0.29, F (32, 919) = 11.37, P < 0.0001). In females, this analysis showed significant differences in the centroid positions among the nine populations (Wilk’s l = 0.23, F (32, 1090) = 16.54, P < 0.0001). Position of centroïds on canonical variates one and two were plotted (Fig. 3). For both sexes, two distinct clusters could be defined: one represented by the Pinail population, the second by all stream populations. In males (Fig. 3a), the first and second canonical variates account for 78.9% and 16.9% of total variance, respectively. In females (Fig. 3b), the first and second canonical variates express 69.38% and 25.37% of total variance, respectively. The first component represents overall size, since all characters are positively correlated with this component in both sexes (Table 6). From the loadings given in Table 6, it can be seen that AW and CW contributed most strongly to the second canonical variable for both sexes. Reclassification of animals into the population of origin based on morphological features is highly ineffective in separating individuals from random unknown samples. The average of reclassification was less than 40% in both sexes. However, animals from Pinail population were correctly reclassified in 93% and 82% of cases in males and in females, respectively. Table 6. Relationship between the original variables and the canonical axes in males (Table a) and in females (Table b) a

Variables POL AW CL CW

CV I 0.734 0.856 0.743 0.847

CV II – 0.464 – 0.491 – 0.415 – 0.108

b

Variables POL AW CL CW

CV I 0.818 0.871 0.796 0.972

CV II 0.390 0.449 0.284 – 0.107

12

Grandjean and Souty-Grosset

a

b

Figure 3. Centroïd position of the nine population sampled in Poitou-Charentes along canonical variables 1 and 2 based on an analysis of residual morphometric characteristics for males (a) and females (b)

Genetic data 120 animals obtained from the nine sampled populations were used for mtDNA extraction. For nine of the eleven tested enzymes, no difference in the restriction pattern between individuals could be detected (Table 7). Two patterns (A and B) were observed for the enzymes Hpa II and Hind III (Table 7). For these enzymes, different genotypes were separated by a single loss or gain of a restriction site. In total, three different composite haplotypes detected among the 120 crayfish from

Genetic variability in white-clawed crayfish

13

Table 7. Estimated sizes (in base pairs) of mtDNA fragments resulting from digestion with eleven restriction endonucleases in nine populations of Austropotamobius pallipes in Poitou-Charentes (France). For each enzyme, letters refer to the different obtained profiles Hind III Hind III

Hpa II

Hpa II

Hae III

Acc II

Nde II

Hinf I

Taq I

Bgl II

Pst I

Xho I

Eco RI

A

B

A

B

A

A

A

A

A

A

A

A

A

5520 2710 1425 1050 935 845 615 530

5520 1590 1425 1130 1050 935 845 615 530

4330 1730 1360 1250 1160 ¥ 2 885 630 560

4330 1460 1250 ¥ 2 1160 ¥ 2 950 630 560

2730 1380 1310 1230 770 710 680 610 530

10 475 2160 1395 1330 870

1770 1450 1250 950 790 740 690 630 530

1930 1600 1170 1100 980 790 700 650 570 500 460

2340 2070 1120 950 860 770 660 590 570 490 460

4710 3070 2520

8235 9200 ¥ 2 7460 935 ¥ 2

Total 13630

13630

9930

16 230

8800

10 450

13 165

12 895

10880 10300

17565

18400

13545 1865 935 ¥ 2

17280

nine populations and the distribution of haplotypes are given in Table 8. No intraspecific variability of the mitochondrial genome was apparent in La Martinière, La Gace and La Fontaine Saint-Pierre (haplotype 1: AAAAAAAAAAA), in Le Magot (haplotype 2: BAAAAAAAAAA) and in Le Pinail (haplotype 3: ABAAAAAAAAA). Haplotype 3 was only found in Le Pinail. The population of Crochatière showed intraspecific variability for the restriction enzyme Hind III. Haplotype 1 was more frequent in this population than haplotype 2, with frequencies of 75% and 25%, respectively (Table 8). Intraspecific variation was also found in the three populations within the Basin of La Sèvre Niortaise (Magnerolle, Puits Table 8. Percentage of the different composite haplotypes revealed in the nine populations sampled in three departments of Poitou-Charentes (France) and sample sizes Department

Basin

Sub-basin

Populations

number of animals

Haplotypes 1

Deux-Sèvres

La Vienne

La Viette L’Auxances

La Sèvre Niortaise Vienne

Charente

La Vienne

La Charente

La Tude La Tardoire

2

3

La Martinière Le Magot

10 20

Le Magnerolle Le Puits d’Enfer Le Gatineau

20 14 16

65 86 12,5

35 14 87,5

La Crochatière

20

75

25

Pinail

5

La Gace La Fontaine St Pierre

7 8

100 100

100 100 100

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Grandjean and Souty-Grosset

Table 9. Genetic variance components and F-statistics for the white-clawed crayfish populations estimated using AMOVA program (Excoffier et al., 1992) Genetic variance

F statisticts

Among hydrographic basins Among populations within basins Within populations

F ct = – 0.02 F sc = 0.631 F st = 0.624

P = 0.38 P < 0.002 P < 0.002

d’Enfer, Gatineau) with variable proportion of both haplotypes 1 and 2. Mitochondrial DNA nucleotide diversity values within the species ranged from 0.57 to 1.31%. Estimates of the F-values for mtDNA of A. pallipes are given in Table 9. The substantial amount of intrapopulational variation was evident in the AMOVA analysis, in which 59% of the total variation was found among populations within hydrographic system. 33% of the total variation was found among individuals within populations and 8% of the variation was found among the hydrographic systems. The correlation of Nei’s genetic distance and morphological distance between samples was not significant (P = 0.68).

Discussion This study revealed relatively low levels of morphological and genetic variation among the nine geographically close populations of A. pallipes. In both sexes, bivariate and multivariate analyses showed a low morphometrical differentiation between the Pinail population collected from artificial ponds and a homogeneous group including all populations sampled in streams. These results could be explained by two hypotheses. The first is based on the habitat variation existing between the populations sampled. The habitat of the crayfish, A. pallipes, correponds generally to brooks with cool running waters, rich in calcium and in dissolved oxygen and with hiding places or refuges (Lachat and Laurent, 1987). The Pinail could be considered as an atypical habitat for this species because it is constituted by very small artificial ponds located in a nature reserve. Physical and chemical measurements carried out by Grimaldi and Roskam (1994), have revealed very low rates of dissolved oxygen and calcium in these ponds. Futhermore, the temperature reading gave values greater than 26°C at the bottom of the ponds during summer which was unusual for this species (Fenouil, 1987). The impact of habitat variation on morphology has been illustrated in several animals and particularly in crayfish (Horwitz, 1990; Horwitz et al., 1990; Austin and Knott, 1996). The second hypothesis is based on human introduction which could be responsible for the morphological differentiation, either by a founder effect or by the introduction of animals from another French region. The results of the mtDNA survey revealed a limited amount of genetic diversity in these A. pallipes populations. Among the three haplotypes seen, two were only found in the populations sampled within the hydrographic basin while the third haplotype was exclusively recovered from an artificial pond (Pinail population).

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Furthermore, the nucleotide divergence between haplotype pairs showed that the two haplotypes found in streams are closely related. This result could be explained by the small sampling area where environmental conditions were similar for all populations except for the Pinail. According to Hewitt (1996), the lack of genetic variability between and within populations located in the northern part of the range was a general phenomenon in European species following their recolonization from southern refugial areas after the last ice ages. A post-glacial recolonization of Poitou-Charentes region from an unique refugial zone located in southern France could explain the low level of genetic divergence within and between populations. Among populations sampled in brooks, the F-values showed no structure of genetic variability according to hydrographic basins. Our results are not in accordance with those obtained from other freshwater species where the fragmentation due to hydrography plays an important role in structuring populations (Ward et al., 1994). Indeed, hydrography limits the gene flow between populations confined to different drainages such that it could permit their genetic divergence. Thus in most population genetic studies of freshwater species, particularly in fish, high levels of genetic structure have been shown among populations in different river systems (Ward et al., 1994). In our study, the chaotic pattern of genetic variation distribution between hydrographic basins could be explained by two non-exclusive hypotheses. It could reflect the occurrence of many translocations by man. Crayfish transplantation was a common practice as early the Middle Ages and isolated populations exist as a result of such activities (Lowery and Holdich, 1988). Thus, transplantations of crayfish from populations located in different hydrographic basins may have altered the natural genetic pattern. However, given that the degree of genetic differences found between sites and haplotypes are not extensive, this genetic pattern within and between populations could be accounted by a period of population expansion and migration or gene flow followed by contraction, isolation and fragmentation of populations leading to genetic divergence mainly under the process of genetic drift which resulted in fixation of the alternative variants in a stochastic manner. Recently, similar patterns of genetic variability have been shown in Australian crayfish species by allozyme studies (Horwitz, 1990; Campbell et al., 1994; Austin and Knott, 1996). Concerning the haplotype found in Pinail, it seems that the best explanation is the introduction of animals from another French region rather than founder effect. A more exhaustive survey of French populations could allow this possibility to be examined. Genetic distances between A. pallipes populations fall within the range of those calculated for several other crustaceans, which reveal moderate levels of genetic differentiation. In Panulirus argus, Silberman et al. (1994) have found a mean interpopulation nucleotide sequence diversity of 1.44%. Bouchon et al. (1994) have showed a significant genetic differentiation (divergence = 1.7%) between three populations of Penaeus japonicus. The levels of intraspecific nucleotide divergence displayed between populations of Poitou-Charentes region are nevertheless smaller than mtDNA nucleotide divergence estimated for European populations of A. pallipes by Grandjean et al. (1997a, 1998). They reported nucleotide divergence of more than 12% between Spanish, British and Slovenian populations of subspecies A. p. lusitanicus, A. p. pallipes and A. p. italicus respectively. However, the low levels

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of genetic variability found in this study are in accordance with the lack of genetic differentiation observed from allozymes between populations sampled from England, southern France and north-western Italy (Santucci et al., 1997). Similarly, Grandjean et al. (1997 a) showed no genetic variation between four English populations from analysis based on mtDNA. This genetic homogeneity between English populations could be explained by a post-glacial colonization of Britain of animals of French stock either naturally through a post-glacial stream connection with France or by an human introduction (Albrecht and Von Hagen, 1981). Insufficient time for the accumulation of mutations due to their recent establishment may explain the absence of genetic variability found in A. pallipes between these two countries. If management of A. pallipes is to be based on the identification of stocks, then efficient management including both England and Poitou-Charentes should be possible. The nonsignificant relationship between morphological and genetic distances could be explained by the fact that morphological characters are unsuitable to reveal a clear discrimination between samples. Thus, the genetic heterogeneity between populations revealed by a high F-st value created a nonsignificant relationship between the genetic and morphological data matrices. All the morphological variables used in this study are highly correlated with the total size of animals contributing strongly to the first canonical variable which explained approximately 80% of total variance. Thus, the morphological distances are mainly explained by the heterogeneity of total size mean of samples. The second canonical variate explained by CW and AW variables allows information to be obtained about population discrimination. It seems to differentiate the Pinail population from a group comprising all stream populations. According to this axis, a congruence between morphological and genetic data appeared, which could justify a particular management of populations from ponds or closed water. A search for more discriminant morphological characters will be required. Further analysis involving several regions must be done in order to obtain a complete picture of the population structure of this species in France. In the light of our results and those obtained by Santucci et al. (1997) and Lörtscher et al. (1997), a combination of both genetic markers, (i.e. mtDNA and allozymes) should be used to understand the genetic structure in A. pallipes. ACKNOWLEDGEMENTS Thanks are due to Michel Bramard, member of the Délégation Régionale du Conseil Supérieur de la Pêche du Poitou-Charentes (C.S.P) for field assistance during collecting the crayfish samples. This work was supported by financial grant from the C. S. P.

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