Frédéric Grandjean

Mitochondrial DNA variation and population genetic structure of the white-clawed crayfish, Austropotamobius pallipes pallipes

Here we report a high level of mtDNA variationfrom RFLP analysis within A. p. pallipes from 21 French and 4British populations. Seventeen haplotypes were recorded among 269individuals. Nucleotide sequence divergence (p) among mtDNAhaplotypes ranged from 0.0019 to 0.0425. Nucleotide diversity (π)ranged from 0.01 to 0.006 (mean = 0.0031) and from 0 to 0.001(mean = 0.00037) for populations located in southern and northernFrance, respectively. Graphical representation from principalcoordinate analysis based on Neis genetic distance values amongpopulations showed two groups, cluster A, including the 16populations sampled in the north-western and north-easternFrance, England and Wales and cluster B containing 9 populationssampled in the central and southern of France. No geneticdifferences were noted among the most northern French and Englishpopulations whereas marked interpopulation genetic diversity wasobserved in southern populations. Such genetic heterogeneityamong populations of A. p. pallipes appears to be related tohabitat fragmentation and subsequent recolonizations fromrefugial areas during the Pleistocene. Implications for theconservation of A. p. pallipes are discussed.

Population genetic structure of the endangered freshwater crayfish Austropotamobius pallipes, assessed using RAPD markers

Random amplified polymorphic DNA (RAPD) analysis was performed to characterize the genetic diversity of Austropotamobius pallipes, a threatened freshwater crayfish native to Europe. Four decamer primers which generated six unambiguous polymorphic bands were used to analyse crayfish from 21 populations sampled in the major part of its range. Genetic diversity within populations of A. pallipes, estimated by Shannon’s diversity index, ranged from 0 to 0.446 with a mean of 0.159. A UPGMA dendrogram constructed from pairwise ΦST values between populations, revealed three clusters corresponding to populations sampled in the southern, northwestern and eastern part of its range. AMOVA analysis revealed a high genetic structure of A. pallipes populations ΦST=0.814, with 73.11% of the genetic variation distributed between these clusters. It suggests a historical geographical separation of these groups into three refugial areas, probably in the Rhine, Mediterranean and Atlantic basins during recent glaciations. The close genetic relationships between English and western French populations are in accordance with a natural postglacial origin of English populations from individuals having survived in an Atlantic refugium. However, the present results suggest that the Irish stock originated from a human translocation of individuals from an Atlantic refugium.

Drastic bottlenecks in the endangered crayfish species Austropotamobius pallipes in Spain and implications for its colonization history

Variation in mitochondrial DNA (mtDNA) was surveyed, using restriction endonucleases, in the white-clawed crayfish, Austropotamobius pallipes lusitanicus, from 14 populations sampled in Spain. Four additional samples from France (1), Slovenia (1) and Italy (2) were also analysed. Among the 11 haplotypes listed, only one was detected from the 154 animals sampled from Spanish populations. This haplotype was also recorded in the Fosso di Ferfereta population (Italy). Estimates of nucleotide sequence divergence among haplotypes ranged from 0.45% to 17.4%. Interpopulational genetic relationships showed that Spanish populations were closely related to those of Fosso di Ferfereta with a small genetic distance (0.0003) found between them. AMOVA revealed that most of the genetic variance (71.97%) was attributed to variation between European regions. These results are in accordance with a drastic bottleneck event during the history of the Spanish populations. Four suggestions, based on human introduction, selection and recent or ancient historical events are discussed in relation to the lack of genetic variation in the Spanish crayfish stock.

Origin and colonization history of the white-clawed crayfish, Austropotamobius pallipes, in Ireland.

The presence of the white-clawed crayfish Austropotamobius pallipes complex in Ireland is suspected to be a result of human translocations. Two hypotheses have been formulated about the origin of the crayfish: from British populations or from western French populations. In order to resolve this question, nine Irish crayfish populations (a total of 124 individuals) were sampled along a south–north cline and investigated by combining two molecular markers: mtDNA and RAPDs. The mtDNA marker, analysed by RFLP on the entire molecule, showed an absence of polymorphism within and among Irish populations. The RFLP haplotype found in Irish populations was only recorded in western French populations and was different from those found in English populations. This result may be explained by a human introduction of crayfish to Ireland from western French populations. RAPD analysis showed a clinal reduction of genetic variability within Irish populations from south to north, associated with an increase in their genetic differentiation. A stepwise model of translocation from the south to the north of Ireland is proposed and discussed.

Prevalence of the Crayfish Plague Pathogen Aphanomyces astaci in Populations of the Signal Crayfish Pacifastacus leniusculus in France: Evaluating the Threat to Native Crayfish

Aphanomyces astaci, the crayfish plague pathogen, first appeared in Europe in the mid-19th century and is still responsible for mass mortalities of native European crayfish. The spread of this parasite across the continent is especially facilitated by invasive North American crayfish species that serve as its reservoir. In France, multiple cases of native crayfish mortalities have been suggested to be connected with the presence of the signal crayfish Pacifastacus leniusculus, which is highly abundant in the country. It shares similar habitats as the native white-clawed crayfish Austropotamobius pallipes and, when infected, the signal crayfish might therefore easily transmit the pathogen to the native species. We investigated the prevalence of A. astaci in French signal crayfish populations to evaluate the danger they represent to local populations of native crayfish. Over 500 individuals of Pacifastacus leniusculus from 45 French populations were analysed, plus several additional individuals of other non-indigenous crayfish species Orconectes limosus, O. immunis and Procambarus clarkii. Altogether, 20% of analysed signal crayfish tested positive for Aphanomyces astaci, and the pathogen was detected in more than half of the studied populations. Local prevalence varied significantly, ranging from 0% up to 80%, but wide confidence intervals suggest that the number of populations infected by A. astaci may be even higher than our results show. Analysis of several individuals of other introduced species revealed infections among two of these, O. immunis and P. clarkii. Our results confirm that the widespread signal crayfish serves as a key reservoir of Aphanomyces astaci in France and therefore represents a serious danger to native crayfish species, especially the white-clawed crayfish. The prevalence in other non-indigenous crayfish should also be investigated as they likely contribute to pathogen transmission in the country.

Cryptic diversity within the invasive virile crayfish Orconectes virilis (Hagen, 1870) species complex: new lineages recorded in both native and introduced ranges

The virile crayfish (Orconectes virilis) represents a cryptic species complex with several lineages known in the USA, and a wide introduced range. In Europe, O. virilis is an emerging invader, established during the last decade in at least two areas—one in the United Kingdom and another in the Netherlands. We assessed the position of both known European populations within the species complex by sequencing part of the mitochondrial gene for cytochrome c oxidase subunit I. Tested UK and Dutch individuals did not belong to any mitochondrial lineage recorded in North America so far but formed a separate clade, the original distribution area of which is unknown. Additionally sequenced virile crayfish from Iowa (USA) also represented a new clade, suggesting that undiscovered lineage variation within O. virilis remains high. This exemplifies that genetic analyses of invading populations may provide new insights into diversity of a taxon in its original range.

Microsatellite markers for direct genotyping of the crayfish plague pathogen Aphanomyces astaci (Oomycetes) from infected host tissues.

Aphanomyces astaci is an invasive pathogenic oomycete responsible for the crayfish plague, a disease that has devastated European freshwater crayfish. So far, five genotype groups of this pathogen have been identified by applying random amplified polymorphic DNA analysis on axenic cultures. To allow genotyping of A. astaci in host tissue samples, we have developed co-dominant microsatellite markers for this pathogen, tested them on pure cultures of all genotype groups, and subsequently evaluated their use on tissues of (1) natural A. astaci carriers, i.e., North American crayfish species, and (2) A. astaci-infected indigenous European species from crayfish plague outbreaks. Out of over 200 potential loci containing simple sequence repeat (SSR) motifs identified by 454 pyrosequencing of SSR-enriched library, we tested 25 loci with highest number of repeats, and finally selected nine that allow unambiguous separation of all known RAPD-defined genotype groups of A. astaci from axenic cultures. Using these markers, we were able to characterize A. astaci strains from DNA isolates from infected crayfish tissues when crayfish had a moderate to high agent level according to quantitative PCR analyses. The results support the hypothesis that different North American crayfish hosts carry different genotype groups of the pathogen, and confirm that multiple genotype groups, including the one originally introduced to Europe in the 19th century, cause crayfish plague outbreaks in Central Europe. So far undocumented A. astaci genotype seems to have caused one of the analysed outbreaks from the Czech Republic. The newly developed culture-independent approach allowing direct genotyping of this pathogen in both axenic cultures and mixed genome samples opens new possibilities in studies of crayfish plague pathogen distribution, diversity and epidemiology.

GENETIC AND MORPHOLOGICAL SYSTEMATIC STUDIES ON THE CRAYFISH AUSTROPOTAMOBIUS PALLIPES (DECAPODA: ASTACIDAE)

Morphological criteria and mitochondrial DNA (mtDNA) were analyzed to examine the taxonomic and systematic relationships of the endangered crayfish species Austropotamobius pallipes. Morphological data confirmed the difficulty of differentiating the 3 subspecies: A. p. pallipes, A. p. italicus, and A. p. lusitanicus. MtDNA analysis by RFLP using 6 endonucleases revealed 5 haplotypes and allowed unequivocal identification of the subspecies. The nucleotide sequence divergence among haplotypes characteristic of each subspecies ranged from 10.93-13.6% and may be explained by an ancient geographical separation of each subspecies. This distribution of genetic variation agrees with the presumption of 3 refugial regions during the glaciation periods, possibly the Iberian Peninsula, southern France, and the Balkans. UPGMA analysis from nucleotide distances placed A. p. italicus and A. p. lusitanicus in a cluster separate from A. p. pallipes, suggesting that

Genetic and morphological variation in the endangered crayfish species, Austropotamobius pallipes (Lereboullet) (Crustacea, Astacidae) from the Poitou-Charentes region (France)

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.

Genetic variability of the threatened crayfish Austropotamobius italicus in Tuscany (Italy): implications for its management

We studied the genetic variability of the indeigenous crayfish Austropotamobius italicus in 14 populations inhabiting five basins in Tuscany (Italy). This species is subject to a drastic decline in abundance across its entire range. Using a fragment of the mitochondrial DNA 16S rRNA gene, we identified eight haplotypes, six corresponding to A.i. italicus and two to A.i. meridionalis; the two clades were found in syntopy in one stream. Eight populations of A. i. italicus analyzed for their microsatellite loci, showed a low intra-population genetic variability and a high inter-population genetic divergence. Populations sampled in the Sieve basin showed no heterozygotes and a high level of inbreeding. The knowledge on the genetic structure of the studied populations, combined with information on their ethology, ecology, and demography, is an essential prerequisite for any action aimed at reintroducing or restocking this threatened species.