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Genetic structure of Spanish white-clawed crayfish Austropotamobius pallipes populations as determined by RAPD analysisreasons for optimism.

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AQUATIC CONSERVATION: MARINE AND FRESHWATER ECOSYSTEMS
Aquatic Conserv: Mar. Freshw. Ecosyst. 18: 190–201 (2008)
Published online 17 July 2007 in Wiley InterScience
(www.interscience.wiley.com) DOI: 10.1002/aqc.829
Genetic structure of Spanish white-clawed crayfish
(Austropotamobius pallipes) populations as determined by
RAPD analysis: reasons for optimism
B. BEROIZa,1, C. CALLEJASa, F. ALONSOb and M.D. OCHANDOa,*
a
b
Departamento de Gene´tica, Facultad de CC. Biológicas, Universidad Complutense, 28040 Madrid, Spain
Centro de Investigación Agraria de Albaladejito, Junta de Comunidades de Castilla-La Mancha, 16194 Cuenca, Spain
ABSTRACT
1. Spanish populations of the white-clawed crayfish have declined sharply over the last three
decades. Although Austropotamobius pallipes was once widely distributed and very abundant in most
of the limestone basins of the country, outbreaks of crayfish plague since 1978 have reduced its
populations, and now only some 500–600 small populations are left.
2. Consequently, the species now enjoys protection under national legislation. Management
decisions regarding the conservation of a threatened species require an understanding of the genetic
structure of its populations.
3. Using random amplified polymorphic DNA (RAPD) fingerprinting the genetic variability of 11
populations of A. pallipes was assessed over the species’ range in Spain, and their phylogenetic
relationships determined.
4. Substantial genetic differentiation was detected among the populations tested; no clear
relationship was found between patterns of genetic variability and hydrological basin. The RAPD
markers showed the degree of genetic variability of these populations to be similar to, and in some
cases slightly higher than, that reported in previous studies on other Spanish and European
populations of A. pallipes.
5. The results offer hope for the recovery of this species in Spain, and provide information that
might be useful in the management of crayfish reintroduction programmes.
Copyright # 2007 John Wiley & Sons, Ltd.
Received 26 June 2006; Accepted 26 December 2006
KEY WORDS: Austropotamobius pallipes; RAPD; genetic variability; genetic structure; bottleneck; genetic drift;
fragmentation; conservation
*Correspondence to: M.D. Ochando, Departamento de Genética, Facultad de CC. Biológicas, Universidad Complutense, Ciudad
Universitaria, 28040 Madrid, Spain. E-mail: dochando@bio.ucm.es
1
Present address: Departamento de Biologı́a de Plantas, CIB-CSIC, 28040 Madrid, Spain.
Copyright # 2007 John Wiley & Sons, Ltd.
GENETIC STRUCTURE OF SPANISH FRESHWATER CRAYFISH
191
INTRODUCTION
Austropotamobius pallipes (Lereboullet, 1858) – the white-clawed crayfish – is a freshwater species
indigenous to Western Europe. Its current distribution extends from Ireland and the British Isles to the
Adriatic, and from southern Spain and Italy to Belgium. However, its original distribution (as well as some
features of its taxonomy) remains unclear.
The species was once a cornerstone of Iberian freshwater ecosystems with large populations widely
distributed throughout most of the country’s limestone basins; indeed, it was absent only from the highest
mountain ranges and most arid areas. The dramatic decline in its numbers all over its European range is the
result of a combination of factors such as the introduction of exotic crayfish species, the related spread of
crayfish plague (caused by the oomycetous fungus Aphanomyces astaci), and human-activity-linked
problems such as habitat destruction and pollution. The Iberian Peninsula is probably one of the most
affected areas; indeed, some of the current populations have experienced rates of decline of 30–50% per
5-year period (Alonso et al., 2000). As a result, the species now has a very patchy distribution, with the
surviving populations occupying marginal areas or short stretches of watercourses usually isolated from the
main river systems (Martı́nez et al., 2003). As a consequence the species is now protected by national
legislation (Ministry of Agriculture, Boletı́n Oficial del Estado, 149: 24098, 2003). It is listed as vulnerable in
the Red List of Threatened Animals of the International Union for the Conservation of Nature and
Natural Resources (IUCN) (Baillie and Groombridge, 1996), and is included in Annexes II and IV of the
European Community Directives for the Conservation of natural habitats and of wild flora and fauna (92/
43/EEC and 94/62/EU) as requiring special conservation measures.
Plans for the restoration of A. pallipes in Spain and several conservation actions have been proposed and
indeed implemented (Diéguez-Uribeondo et al., 1997; Alonso et al., 2000). However, the development of
strategies for the conservation of a threatened species requires an understanding of its biology and the
possession of data on the genetic variability and structure of its populations. The latter is critical in
decision-making since one of the major goals of conservation is to preserve genetic variability; the
importance of such data has recently been highlighted in two workshops (IAA European meetings:
Innsbruck, 2004, Bulletin Franc-ais de la Peˆche et de la Pisciculture, vol. 3, and Florence, 2005, Bulletin
Franc-ais de la Peˆche et de la Pisciculture, vol. 4).
The ecological importance of A. pallipes and the conservation interest surrounding it have led
researchers to study the genetic variability of its populations. The first papers in this area focused
on allozyme variation (Attard and Vianet, 1985; Zarazaga, 1993; Lörtscher et al., 1997, 1998; Santucci
et al., 1997) and the analysis of mtDNA by RFLP fingerprinting (Grandjean et al., 1997, 2000, 2001;
Souty-Grosset et al., 1997, 1999). More recently, analysis has centred on mtDNA sequences (Grandjean
et al., 2002; Zaccara et al., 2004; Baric et al., 2005a, b; Fratini et al., 2005; Trontelj et al., 2005;
Diéguez-Uribeondo et al., in press), microsatellites (Gouin et al., 2000; Baric et al., 2005a) and RAPD
markers (Gouin et al., 2001, 2003). All have made contributions to the present understanding of the
species’ genetic structure (see Schulz and Grandjean (2005), and references therein). However, few
of these studies included Spanish crayfish populations, and all but two (Grandjean et al., 2001; DiéguezUribeondo et al., in press) were based on small samples from limited areas. In addition, some
major hydrographic basins formerly sustaining large crayfish populations were not represented in those
studies.
The aim of the present study was to assess the genetic structure of populations of A. pallipes and their
phylogenetic relationships throughout the range of the species in Spain. Random amplified polymorphic
DNA (RAPD) fingerprinting (Williams et al., 1990; Welsh and McClelland, 1990) was employed for this
purpose. This technique involves the amplification of random segments of genomic DNA using a single
primer of arbitrary sequence to amplify polymorphic DNA. Like any other genetic marker, RAPD markers
have some limitations: limited reproducibility across laboratories and marker dominance. Among the DNA
Copyright # 2007 John Wiley & Sons, Ltd.
Aquatic Conserv: Mar. Freshw. Ecosyst. 18: 190–201 (2008)
DOI: 10.1002/aqc
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B. BEROIZ ET AL.
fingerprinting techniques available, however, RAPD requires the least equipment and is the cheapest in
terms of labour and other costs, and its usefulness in population studies has been widely shown.
The data presented may well be of use in the development of conservation strategies for this endangered
species and in the management of restocking programmes.
MATERIALS AND METHODS
Sampling
Ten populations (25 individuals per population) of A. pallipes from the major hydrographic basins within
the species’ Spanish range were sampled (Table 1; Figure 1). As far as possible, these were healthy
populations from places not known to have been recently stocked.
An additional sample was obtained from the main Spanish crayfish farm at Rillo de Gallo (Guadalajara),
which is devoted to providing individuals for restocking. This government-owned facility keeps a stock of
some 6000 reproducing animals originally from the Ebro, Tagus and Guadiana basins. This stock has
received no new imports for at least 15 years.
Crayfish were captured in the field with baited traps or by hand, and transported live to the laboratory.
One chela was removed from each animal and stored at –808C until DNA extraction. This avoids killing the
animals. However, most animals were killed by freezing and kept by the Complutense University’s
Department of Genetics for further studies.
DNA extraction
Genomic DNA was extracted from claw muscle tissue, adjusting the protocol of Benito et al. (1993) to the
present material. Samples were treated with SDS and the genomic DNA phenol:chloroform-extracted and
precipitated with isopropanol. The resulting pellets were washed in 70% ethanol, dried, and resuspended in
Tris-EDTA (10 mM Tris pH 8.0, 1 mM EDTA).
RAPD-PCR analysis
Ten different oligodecamers from two sets of primers (kits A and C, Operon Technologies) were used
(OPA-01, OPA-06, OPA-11, OPA-12, OPC-04, OPC-06, OPC-07, OPC-14, OPC-15, OPC-16). An in-house
protocol for DNA amplification was followed, modifying the conditions reported by Williams et al. (1990).
Table 1. Code, location, year of capture and habitat altitude of populations of Austropotamobius pallipes analysed (MED:
Mediterranean basin; ATL: Atlantic basin; H: hatchery)
Population
Code
Collection sites
Year
Altitude (m)
Cuende Stream
Santa Margarida Stream
Ermitas Stream
Guztar Stream
Valsemana Pool
Magdalena Spring
River Ega
Pozuelo Stream
River Nervión
Crayfish hatchery
River Guadazaón
CUE
GIR
GRA
GUZ
LEO
MAD
NAV
POZ
PVS
RIL
VIL
Huerta Obispalia, Cuenca (Guadiana Basin, ATL)
Olot, Gerona (Costero Catalana Basin, MED)
Albuñuelas, Granada (Guadalquivir Basin, ATL)
Padrones de Bureba, Burgos (Ebro Basin, MED)
Lugán, Leon (Duero Basin, ATL)
Rebolledo de Traspeña, Burgos (Duero Basin, ATL)
Estella, Navarra (Ebro Basin, MED)
El Pozuelo, Cuenca (Tagus Basin, ATL)
Altuve, Alava (North Basin, ATL)
Rillo de Gallo, Guadalajara (H)
Valdemoro, Cuenca (Júcar Basin, MED)
2000
2000
1999
1999
1999
1999
1998
1998
2000
1998
1998
900
435
1050
800
990
1180
480
1200
209
1100
830
Copyright # 2007 John Wiley & Sons, Ltd.
Aquatic Conserv: Mar. Freshw. Ecosyst. 18: 190–201 (2008)
DOI: 10.1002/aqc
GENETIC STRUCTURE OF SPANISH FRESHWATER CRAYFISH
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Figure 1. Map showing the location of the 11 Spanish white-clawed crayfish populations sampled in this study.
PCR reactions were performed in a total volume of 12.5 mL containing: 12.5 ng template DNA, 0.4mM of
primer, 100 mM of each dNTP, 4 mM MgCl2, 1.25 mL Stoffel buffer 10 (Applied Biosystems), and 1.25
units of Stoffel Fragment DNA polymerase (Applied Biosystems).
An M.J. Research PT-100 thermal cycler was used for all amplifications. The optimal cycling programme
was 6 min at 948C followed by 55 cycles of amplification (1 min at 948C, 1 min at 368C, and 4 min at 728C),
and a final step at 728C for 6 min. Each amplification reaction was performed at least twice: the results were
consistently reproducible.
A negative control (no DNA template) was added to each amplification run. Amplified products were
separated in 2% agarose gels with TAE buffer (40 mM Tris-Acetate, 1 mM EDTA pH 8.0) containing
ethidium bromide and visualized using a UV transilluminator. A 100 bp ladder marker (MBI Fermentas)
was used as a molecular size standard.
Data analysis
Locus phenotype was determined according to the presence (1) or absence (0) of the corresponding band.
The polymorphism for each population was then estimated. Mean bandsharing similarity indices were
calculated using the simple matching coefficient, Sxy ¼ nxy =nx þ ny (Sokal and Michener, 1958), where nxy is
the number of bands shared by two individuals, and nx and ny the total number of bands present in
individuals x and y respectively.
Genetic differentiation among samples was examined by clustering and multivariate analysis. Corrections
for dominant markers were made as described by Lynch and Milligan (1994).
Analysis of molecular variance (AMOVA; Excoffier et al., 1992) was performed to determine the
variance components for the RAPD profiles, the aim being to assess the partitioning of genetic variation
among the populations sampled, within these populations, and among the basins sampled. The levels of
significance for these variance component estimates were calculated using permutational procedures.
Copyright # 2007 John Wiley & Sons, Ltd.
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DOI: 10.1002/aqc
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B. BEROIZ ET AL.
Variance components were tested to determine whether they were different from zero (Arlequin 2000
software package) (Schneider et al., 2000).
The FST coefficient (Wright, 1951) and y index (Weir and Cockerham, 1984) were then calculated. Gene
flow (Nm) was estimated from FST and y using the RAPDFST programme (part of the RAPD-PCR
software package, Black IV 1997).
Nei’s (1972) genetic distances were calculated from the marker frequencies using the RAPDdist
programme (RAPD-PCR software package Black IV 1997). The distances and interpopulation similarity
matrices were used to construct dendrograms using the unweighted pair-group (UPGMA) (Sneath and
Sokal, 1973) and neighbour-joining (NJ) (Saitou and Nei, 1987) methods employing NTSYSpc software v.
2.01b (Rohlf, 1997). The reliability of the trees was evaluated using 1000 bootstrap replicates (RAPDdist
software). Correlations between the genetic and geographic distances among populations were calculated
using the Mantel test, a randomized test for matrix correspondence (Mantel, 1967). Principal components
analysis (PCA) (Sneath and Sokal, 1973) was performed (using NTSYS software) to visualize the grouping
of populations.
RESULTS
A total of 133 bands were scored; 40 were monomorphic for all 275 crayfish from the 11 sample
populations. Representative DNA profile of the species with the primer OPA-06 is shown in Figure 2.
Polymorphism ranged between 7–8% (populations CUE and GRA) and 22–24% (LEO and NAV)
(Table 2). The mean polymorphism for the 11 populations was 15%. The intrapopulational similarity
indices ranged from 78% to 92%; interpopulation similarity ranged from 70% to 86% (Table 2). The mean
similarity interpopulation value for all A. pallipes samples was 78%.
All the AMOVA analyses (Table 3) showed that most of the total genetic variation was expressed among
populations (67.55–52.09%, depending on the analysis), whereas about one-third (32.45%) existed within
populations. Only 15.74% of the total variation was found among river basins. A random permutational
test revealed that all but two of the variance components were significant (p50.001). According to the
Figure 2. RAPD profiles of Austropotamobius pallipes with primer OPA-06. First and last lanes contain a 100 bp ladder molecular
weight marker.
Copyright # 2007 John Wiley & Sons, Ltd.
Aquatic Conserv: Mar. Freshw. Ecosyst. 18: 190–201 (2008)
DOI: 10.1002/aqc
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GENETIC STRUCTURE OF SPANISH FRESHWATER CRAYFISH
Table 2. Nei’s genetic distances (below diagonal), interpopulation (above diagonal) and intrapopulation simple matching similarity
indices (on diagonal) for the studied populations. Polymorphism values (P) are provided in the last row
CUE
GIR
GRA
GUZ
LEO
MAD
NAV
POZ
PVS
RIL
VIL
CUE
GIR
GRA
GUZ
LEO
MAD
NAV
POZ
PVS
RIL
VIL
0.8390
0.1907
0.1645
0.2048
0.1419
0.1906
0.1947
0.1561
0.1260
0.1980
0.2083
0.7824
0.9181
0.2107
0.2497
0.2313
0.2723
0.2667
0.2202
0.1896
0.2651
0.2746
0.8127
0.7580
0.8089
0.2383
0.1042
0.1955
0.1763
0.1535
0.1999
0.2334
0.2164
0.7781
0.7268
0.7445
0.7900
0.1769
0.0715
0.1301
0.1763
0.1782
0.2089
0.1792
0.8036
0.7352
0.8391
0.7713
0.8020
0.1425
0.1557
0.1691
0.1709
0.1856
0.1811
0.7865
0.7115
0.7837
0.8703
0.7942
0.7858
0.1109
0.1470
0.1892
0.2018
0.1465
0.7616
0.7020
0.7467
0.7980
0.7534
0.8118
0.7850
0.0828
0.1883
0.1387
0.0969
0.8020
0.7453
0.8035
0.7846
0.7607
0.8116
0.8294
0.8019
0.1667
0.1108
0.1148
0.8049
0.7777
0.7831
0.7823
0.7859
0.7775
0.7676
0.7950
0.8348
0.1740
0.2044
0.7790
0.7124
0.7458
0.7494
0.7596
0.7583
0.7845
0.8206
0.7908
0.7793
0.0795
0.7786
0.7134
0.7663
0.7871
0.7739
0.8167
0.8287
0.8344
0.7715
0.8625
0.7837
P
0.07
0.15
0.08
0.18
0.22
0.15
0.24
0.15
0.13
0.17
0.11
Table 3. Analysis of the molecular variance (AMOVA) of the 275 individuals from all 11 populations of Austropotamobius pallipes
using RAPD bands. The data show the degrees of freedom (d.f.), variance component estimates, percentage of total variance
contributed by each component, and the probability (p) of obtaining a more extreme component estimate by chance alone. 1000
permutations were used for each analysis
Source of variation
d.f.
Variance component
Percentage total variance
p-value
All populations: 1 group
Among populations
Within populations
10
264
10.05
4.82
67.55
32.45
50.001
50.001
All basins: 9 groups
Among basins
Among populations within basins
Within populations
8
2
264
2.34
7.79
4.82
15.74
52.09
32.27
0.1070
50.001
50.001
All populations: 3 ocean basinsa
Among ocean basins
Among populations within ocean basins
Within populations
8
2
264
0.22
9.91
4.82
1.51
66.22
32.27
0.3326
50.001
50.001
a
Atlantic, Mediterranean and hatchery.
FST and y indices, the genetic differentiation among the populations accounted for 60.9% and 75.7% of the
total variation, respectively (FST ¼ 0:609 0:030; y ¼ 0:757 0:030). Gene flow (Nm) among the
populations was indirectly estimated from these indices. Depending on the index used, the calculated
Nm value was either 0.2 or 0.1.
The genetic distance values between all pairwise comparisons, obtained from the marker frequencies
(Nei, 1972), ranged from 0.07 to 0.27 with a mean genetic distance of 0.18 (Table 2, below diagonal). The
highest distance values were obtained between the most north-easterly (GIR) and southerly (GRA)
populations and the rest of the populations. The Mantel test revealed no significant correlation (r ¼ 0:47;
p[random Z4 observed Z]¼ 0:01) between the genetic and geographic distance matrices.
The UPGMA and NJ clusterings based on Nei genetic distances and similarity indices showed a
similar topology; Figure 3 therefore only shows the UPGMA dendrogram from Nei genetic distances.
The bootstrap values were high in most cases. The dendrogram revealed that the GIR population (from
Copyright # 2007 John Wiley & Sons, Ltd.
Aquatic Conserv: Mar. Freshw. Ecosyst. 18: 190–201 (2008)
DOI: 10.1002/aqc
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B. BEROIZ ET AL.
Figure 3. Dendrogram of populations of Austropotamobius pallipes produced by UPGMA clustering based on Nei’s genetic distances.
Bootstrap values, based on 1000 replications, are shown near the corresponding branches.
Figure 4. Results of the PCA of RAPD bands. Eigenvalues for each principal component are listed beside each axis.
Copyright # 2007 John Wiley & Sons, Ltd.
Aquatic Conserv: Mar. Freshw. Ecosyst. 18: 190–201 (2008)
DOI: 10.1002/aqc
GENETIC STRUCTURE OF SPANISH FRESHWATER CRAYFISH
197
north-east Spain) formed a separate group. Two main branches can be seen, one contains the populations
CUE, GRA, LEO and PVS (Atlantic basins), the other includes the remaining samples. The graphical
representation of the PCA is congruent with the distribution of the groups as revealed by the dendrogram
(Figure 4). The first PCA axis explains 40.12% of the variance and reveals two well-separated groups: the
GIR population and the remaining populations. The second PCA axis explains 21.09% of the variance and
separates the RIL population (from the crayfish farm) from the rest. The third axis explains somewhat less
(18.72 %) of the variance and separates the GRA population.
DISCUSSION
Levels of genetic variability
The results show a mean polymorphism of 15% for the 11 populations of A. pallipes tested. This value is
consistent with the low polymorphism levels already detected by isozyme analysis in crustacean decapods
(p ¼ 0:15; Tracey et al., 1975; p ¼ 0:22; Hedgecock et al., 1976; p ¼ 0:32; Nevo et al., 1984), in other
freshwater crayfish species (e.g. p ¼ 0:1020:25 in Astacus and p ¼ 0:05 in Orconectes, Attard and Pasteur,
1984; p ¼ 0:12; Crandall, 1997; p ¼ 0:0120:12 in Procambarus, Busack, 1988) and in Spanish (p ¼ 0:13;
Zarazaga, 1993) and European populations of A. pallipes (p ¼ 0:10; Attard and Pasteur 1984; p ¼ 020:05;
Attard and Vianet 1985; p ¼ 0:15; Lörtscher et al., 1997; p ¼ 0:056; Santucci et al., 1997; p ¼ 020:10;
Lörtscher et al., 1998).
The polymorphism detected in the present work ranged between 7–8% (CUE and GRA) and 22–24%
(LEO and NAV). It is interesting to note that the GRA population, one of the most southerly of all Europe,
had the lowest polymorphism value. This population was once large but, owing to disease caused by the
fungus Saprolenia parasitica (amongst other factors), its numbers crashed during the 1990s (Gil and AlbaTercedor, 1998, 2000; Galindo et al., 2003). The NAV and LEO populations showed the highest
polymorphism values of all. These two populations are located in protected areas where diseases have been,
in part, avoided, and their effective population numbers maintained at high levels. The moderate variability
observed in the farmed crayfish population (RIL, 0.17) may be explained by the fact that it was established
more than 20 years ago, and since then has been kept under favourable conditions which have helped
maintain a high population density.
The present results show that RAPD-PCR is a sensitive technique for detecting genetic variability in
Spanish populations of the threatened white-clawed crayfish. In fact, the only two other works in A. pallipes
using RAPD markers (however, without Spanish samples) also found a higher genetic variability
(Ho ¼ 0:159; Gouin et al., 2001; Ho ¼ 0:299; Gouin et al., 2003). Three population studies based on
mtDNA analysis revealed no genetic variability in Spanish populations (Grandjean et al., 2001; Trontelj
et al., 2005) or only two haplotypes (Diéguez-Uribeondo et al., in press). This is probably due to the
different nature of genetic markers used (most RAPD markers target non-coding regions). The single
haplotype found by two of the above authors, and one of the haplotypes found by the third, is also seen
in northern Italian populations, which generates debate over whether the Iberian Peninsula has been
artificially stocked (Albrecht, 1982; Laurent, 1988; Royo et al., 2003). Human translocations have been
speculated also for other geographic areas (Grandjean et al., 2002; Demers et al., 2005; Trontelj et al.,
2005). However, Santucci et al. (1997) ruled out artificial introduction since, in the two Iberian populations
they studied (from Granada and Teruel), they found genetic variability, including a number of alleles that
had not been detected elsewhere (private alleles).
Genetic drift and inbreeding in small effective-size populations (in this case a consequence of
fragmentation, pollution, overfishing, crayfish plague transmitted by introduced species, and habitat loss)
lead to the loss of genetic variability. However, this survey showed levels of genetic variability to be similar
Copyright # 2007 John Wiley & Sons, Ltd.
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to – and in some cases slightly higher than – those reported for Spanish and European populations of
A. pallipes by other authors using different markers. Regardless of the origin of this species and given
its current risk status over its entire range, this variability in certain populations offers some hope for
the species from a management point of view. A level up to 24% in natural populations, and 17% in the
farm-reared populations, could be sufficient for restoration plans and conservation measures.
Genetic variability in crayfish distribution
RAPD marker analysis showed there to be a significant degree of genetic differentiation among the 11
populations tested. Intrapopulational genetic similarity estimates were greater than their interpopulational
counterparts (Table 2, above the diagonal). This implies that individuals within each population are
genetically more similar to each other than to individuals from any other population. The analyses of
molecular variance (Table 3) showed that most of this genetic variation was among populations. No
important levels of genetic differentiation were seen among hydrological or ocean (i.e. Mediterranean and
Atlantic) basins.
The high FST (60.9%) and y (75.7%) values and the Nei genetic distances (0.08 to 0.27) also indicate
differentiation among populations. This agrees with data reported by other authors for both Spanish
(Zarazaga, 1993; Diéguez-Uribeondo et al., in press) and European (geographically well-differentiated)
white-clawed crayfish populations (Attard and Vianet, 1985; Santucci et al., 1997; Gouin et al., 2001).
The existence of small and isolated populations leads to divergence between populations and
homogeneity within them. Conversely, the presence of large, connected populations results in less
differentiation among them and higher diversity within them (Lin et al., 1999). The gene flow estimated for
the Spanish populations of crayfish was very low, 0.2 or 0.1 depending on the use of the FST or y index.
Thus, the current structure and recent history of the Spanish populations of A. pallipes was probably
shaped by the joint action of bottlenecks and genetic drift in fragmented populations. However, ancient
historical events such as population fragmentation, recolonizations from refugia during the ice ages
(Grandjean et al., 2001; Gouin et al., 2003; Trontelj et al., 2005; Diéguez-Urebiondo et al., in press), or the
formation of fluvial basins, may also have influenced their present structure. Such influences have been seen
to operate on many animal and plant species (Taberlet et al., 1998; Callejas and Ochando, 2002).
The dendrogram (Figure 3) shows the phylogenetic relationships among the 11 populations analysed.
The robustness of the tree is shown by the generally high bootstrap values, which agree with the existence of
genetically differentiated populations. Although the dendrogram and PCA (Figure 4) reflect no clear
phylogeographic structure, some interesting features can be seen. In a very general way, the samples appear
clustered by ocean basin; populations from the Mediterranean basin are separated from their Atlantic
counterparts, with the exception of the POZ and MAD populations; the latter group with the
Mediterranean samples probably because of their geographical proximity.
The most north-easterly population of the Iberian Peninsula (GIR) is somewhat different from the
others, showing the largest genetic distances from the other populations. The introduction of this
population (the origin of which remains unknown) in the 1930s has been documented (Pardo, 1942). Even
though no significant correlation was found between genetic and geographic distances (Mantel test
r ¼ 0:47), the largest genetic distances were seen between the GIR population (the most north-easterly) and
the southern GRA population when compared with the rest of the populations. In addition, the smallest
genetic distance was found between the GUZ (Ebro basin) and MAD (Duero basin) populations, which
belong to different river basins but in the same province and only 51 km apart.
Unlike some other freshwater crayfish species, A. pallipes has very limited land dispersal ability. At
present their populations are confined to headwaters or closed water bodies isolated from the main fluvial
systems. In Spain, human translocations of crayfish have been common practice since the 19th century
(Pardo, 1942; Torre and Rodrı́guez, 1964) and this very probably influenced the present distribution of the
Copyright # 2007 John Wiley & Sons, Ltd.
Aquatic Conserv: Mar. Freshw. Ecosyst. 18: 190–201 (2008)
DOI: 10.1002/aqc
GENETIC STRUCTURE OF SPANISH FRESHWATER CRAYFISH
199
species. This may explain why the Atlantic populations POZ and MAD appear clustered with
Mediterranean populations to which they are geographically close. These findings agree with previous
surveys in Switzerland (Lörtscher et al., 1998) and Ireland (Gouin et al., 2003) where human influence is
identified as one of the main factors in the genetic variability distribution patterns.
In conclusion, the use of RAPD markers is a useful tool – and more sensitive than others – for assessing
the genetic structure of Spanish populations of A. pallipes. This study shows a certain degree of
polymorphism in some populations and a substantial amount of genetic differentiation among them. No
clear relationship was found between genetic variability and the hydrological basins inhabited. Human
activities may have influenced this. Future conservation programmes should take heart from the fact that
the present data offer hope for the recovery of this species in Spain.
ACKNOWLEDGEMENTS
This work was funded by the Project INIA SC-96-006. We would like to thank Dr J. Reynolds and the editor, Dr P.J.
Boon, whose constructive comments greatly improved the manuscript, and Dr Diéguez-Uribeondo for permitting us
the use of their manuscript. Thanks are due to the following people for help in sampling the populations: Mari Cruz
Cano (Castilla-La Mancha populations), Consuelo Temiño (Burgos populations), Javier Diéguez-Uribeondo and José
Luis Múzquiz (Navarra population), José Marı́a Gil (Granada population), Javier Pinedo (Paı́s Vasco population),
Emili Bassols (Gerona population) and Javier Sancho (León population). We also thank Susana Ramı́rez for her help
with the distribution map.
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