Genetic analysis for conservation of Austropotamobius italicus populations in Marches Region (Central Italy)

The genetic characteristics of white-clawed crayfish populations in Marches Region (Central Italy) was investigated to plan conservation strategies. In order to clarify their taxonomic status, 50 specimens from ten populations were collected in six different hydrographic drainages of the Umbro-Marchigiano Appennine. The genetic relationships of the Austropotamobius italicus specimens were assessed using DNA sequences of the mitochondrial DNA 16S rRNA gene.The 3 distinct haplotypes M1, M2, M3 detected have been compared to those available in GenBank corresponding to four subspecies described for A. italicus. Our results confirmed the presence of both subspecies A. i. carsicus (M1 & M2) and A. i. meridionalis (M3) in Marches region. Two mixed populations with both subspecies have been characterized. These new data of the genetic structure of population will be used to plan management and recovery programs. Key-words: Austropotamobius italicus, subspecies, taxonomy, conservation. ANALYSES GÉNÉTIQUES POUR LA CONSERVATION DES POPULATIONS D’AUSTROPOTAMOBIUS ITALICUS DANS LA RÉGION DES MARCHES (ITALIE CENTRALE) RÉSUMÉ Les caractéristiques génétiques des populations d’écrevisses à pattes blanches de la région des Marches (Italie centrale) ont été étudiées dans le but de définir une stratégie de conservation. 50 individus provenant de 10 populations appartenant à six bassins hydrographiques des Apennins, de l’Ombrie et des Marches ont été prélevés dans le but de déterminer leurs statuts taxonomiques. Les relations génétiques entre individus d’Austropotamobius italicus ont été appréhendées par sequençage d’une portion du gène d’ADN mitochondrial codant pour l’ARN ribosomique 16S. Les trois haplotypes obtenus, M1, M2 et M3, ont été comparés à ceux mis en ligne dans GenBank correspondant aux quatre sous-espèces décrites chez A. italicus. Nos résultats confirment la présence dans la région des deux sous-espèces A. i. carsicus (M1 & M2) et A. i. meridionalis (M3). Deux populations mixtes ont été caractérisées. Ces nouvelles données sur la structure ha l-0 01 10 85 6, v er si on 1 9 Ja n 20 07 Author manuscript, published in "Bulletin Francais De La Peche Et De La Pisciculture (2006) 991-1000" Bull. Fr. Pêche Piscic. (2006) 380-381 : 991-1000 — 992 — génétique des populations seront utilisées pour définir des plans de gestion et pour mettre en œuvre des programmes de repeuplement. Mots-clés: Austropotamobius italicus, sous-espèces, taxonomie, conservation. INTRODUCTION The white-clawed crayfish, Austropotamobius pallipes complex, is widespread throughout Europe and its range has been drastically restricted (HOLDICH, 2002) due to a combination of several factors affecting the habitat quality or the health of populations (FÜREDER et al., 2003). To protect the existing populations, numerous conservation plans thoughout Europe have emerged during the last 10 years, often including genetic analysis of populations to help management decisions. These studies have revealed a high genetic structure among European populations and more particularly among Italian ones (SANTUCCI et al, 1997; LÖRTSCHER, 1998; GRANDJEAN et al., 2000; LARGIADER et al., 2000, FRATINI et al., 2005, TRONTELJ et al., 2005; ZACCARA et al., 2004 and 2005). These authors reported the presence of two well differentiated groups in Europe corresponding to pallipes and italicus specimens. Although discussions on the status of these two groups are still in progress, separate species status for pallipes and italicus was originally proposed by GRANDJEAN et al. (2002) and accumulating evidence favours that interpretation. Within italicus species, these studies showed a strong genetic structure with the presence of four differentiated groups (FRATINI et al., 2005): A. i. italicus in the TuscanEmilian Apennine (i.e. central Italy); A. i. carsicus in North-eastern Italy; A. i. carinthiacus in Central and and North-western Italy and A. i. meridionalis corresponding to specimens from Latium, Abruzzi, and Southern Italy and also including Slovenian specimens. However, if we take in account the extremely close genetic relationship between A. i. italicus and carinthiacus, it seems to be more reliable to cluster these two subspecies into one A. p. italicus as proposed by Machino in HOLDICH (2002). Actually Austropotamobius italicus is not an official recognised taxon, but the complex phylogenetic structure of the Austropotamobius pallipes supports the necessity of a systematic revision. The aim of the present study was to investigate the genetic relationships among appennine populations by the analysis of sequences of 16 S RNA gene to determine the subspecific status of specimens. This preliminary study on the genetic characteristics of populations in Marches Region will be used to plan management and recovery programs. MATERIALS AND METHODS We collected 50 specimens (five specimens from each population) by hand from ten different rivers of the Umbria-Marches Appennine in six hydrographic drainages of the Adriatic sea (Figure 1). One pereopod was taken per individual and was put in absolute ethanol. Tissues were dehydrated (2 min) in sterile water and ground with plastic pestles in microcentrifuge tubes containing 100 mM Tris, 10 mM EDTA, 100 mM NaCl, 0.1% SDS, 50 mM DTT and 10 μl/ml proteinase K (pH = 8). Samples were incubated at 37°C for 4 hours and DNA extracted twice with phenol/chloroform/isoamyl alcohol (25: 24: 1) followed by an additional extraction with chloroform. The DNA was then precipitated with one volume of isopropanol and 1/10 volume of 3 M (pH = 5.2) sodium acetate. It was then dried and resuspended in sterile water. DNA was collected by centrifugation, dried and diluted in water to a final concentration of 20 ng/μl. Polymerase chain reaction (PCR) amplification of of a rDNA 16S portion about 500 base pairs (bp) long was carried out in a Progene thermocycler using primers available in GRANDJEAN et al. (2002). ha l-0 01 10 85 6, v er si on 1 9 Ja n 20 07 Bull. Fr. Pêche Piscic. (2006) 380-381 : 991-1000 — 993 — Figure 1 Geographical distribution of haplotypes of Austropotamobius italicus subspecies in Marches region. Figure 1 Distribution géographique des haplotypes des sous-espèces de Austropotamobius italicus dans la région des Marches. The optimal cycling program was 2 min at 94°C, 1 min at 35°C, 2 min at 72°C followed by 1 min at 94°C, 1 min at 35°C and 2 min at 72°C for 44 cycles and a final extension of 5 min at 72°C, using a Trio-Thermoblock (BIOMETRA GmBH, Göttingen, Germany).DNA Alignment-The nucleotide sequences were aligned manually with Se-Al v1.0al (Sequence Alignment Editor Version 1.0 alpha 1; Andrew Rambaut, 1996). The sequences obtained from this study were combined with other sequences deposited in GenBank and corresponding to Italian, French and Swiss populations; a sequence of A. torrentium from Genbank was also used as outgroup (AF237599; GRANDJEAN et al., 2000a). Neighbour-Joining (NJ), and Maximum Parsimony (MP) methods were used for phylogenetic reconstructions. The NJ and MP and analyses were conducted on MEGA version 3.0 (KUMAR et al., 2004). MP trees were found by heuristic search using ha l-0 01 10 85 6, v er si on 1 9 Ja n 20 07 Bull. Fr. Pêche Piscic. (2006) 380-381 : 991-1000 — 994 — 101 replicates of random stepwise addition. Bootstrap Percentages (BPMP) were computed after 1,000 replicates. The NJ tree was found using the “p” distance. The pairwise sequence divergence values among haplotypes were used to assess the mean sequence divergence between the two main clusters revealed in phylogeographic analyses. RESULTS All the analyses were based on a 301 bp alignment of mitochondrial 16 S sequences. The sequences imported from GenBank were 199 bp shorter than those of 500 bp obtained in this study. The sequence alignments consisted of 301 pb enclosing 62 variable sites, of which 26 are parsimony informative. Among Marches samples three distinct haplotypes M1, M2, M3 were detected (Table I). The geographical distribution of Marches’ haplotypes is presented in Figure 1. Among these three haplotypes, M1 and M2 were closely related with a nucleotide distance, pi, between the two haplotypes of 0.6%. These two haplotypes differ from M3 by a mean of 2.3%. These haplotypes show a highly structured geographical pattern of distribution (Figure 1 and Table 1). Haplotype M1 was found in northern locations whereas haplotype M3 was found in southern locations. M2 was specific to one location: Rio Freddo. Two centrally located populations harbour both M1 and M3. Both phylogenetic analyses from NJ and MP revealed the presence of two subspecies in hydrographic basins of Marches region: A. i. carsicus (M1 & M2 haplotypes) that has been detected up to now only in the North-eastern Italy and in Alpi Orobie and A. i. meridionalis (M3) in the south. Two mixed populations with both subspecies A. i. carsicus and A. i. meridionalis were present in Amandole and Sanguerone from the hydrographic basin of Esino (the middle of Marches region) (Table I). Pairwise mt16S sequence divergence was reported in Table II. The level of genetic variation within A. italicus subspecies is represented by the pairwise p-distance extimation (expressed as percentage) in Table II. DISCUSSION Our results showed that two subspecies overlap in Marches Region of Italy, A. i. carsicus and A. i. meridionalis. The distribution of A. i. carsicus in the central-northern part of the region was in accordance with the Italian ichthyogeographic Padan-Venetian (PV) district identified by BIANCO (1993). It could be explained by the extension of the Po River basin as far as the border of the meso-adriatic ditch, capturing water from a large number of rivers on both sides of the Adriatic Sea. In fact during glaciations the lowering of the sea level determined confluences of water between rivers flowing into the epicontinental area of the Mediterranean sea (CATTAUTO et al., 1988). A. i. meridionalis, recently described by FRATINI et al. (2005) from molecular data, and to the Apennine cluster defined by TRONTELJ et al., 2005 is located in Central-Southern Italy from both side of the Apennines chain. According to these authors, its presence in Slovenia could result from recolonization events from glacial refugial area during the Pleistocene due to the lowering of the sea level and the consequent confluence of some Adriatic rivers (BIANCO, 1995). In this study, we showed that its northern distribution reaches the central Marches Region where this subspecies is in contact with A. i. carsicus in the hydrographic basin of Esino. Two hypotheses could be drawn to explain the existence of mixed populations in central Marches region. They could be the result of a natural secondary contact between the two species during their spreading from different refugia after the last glacial period of the Pleistocene. According to the distribution of A. i. meridionalis, the refugial area during the last glaciation period could be located in the south of Italy, and from that area postglacial recolonisation events had taken place towards the north. Translocations by humans ha l-0 01 10 85 6, v er si on 1 9 Ja n 20 07 Bull. Fr. Pêche Piscic. (2006) 380-381 : 991-1000 — 995 — Ta b le I Fo r ea ch s it e, d at a ar e re p o rt ed o f: w at er b o d y an d it s hy d ro g ra p hi c d ra in ag e; re g io n o r co un tr y; s am p le s iz e; m it o ch o nd ri al h ap lo ty p es (in p ar en th es es , t he n um b er o f in d iv id ua ls f o r ea ch h ap lo ty p e) . S o m e se q ue nc es a re d o w nl o ad ed f ro m G en eB an k. Ta b le au I P o ur c ha q ue s it e, le s d o nn ée s su iv an te s so nt r ep o rt ée s: c o ur s d ’e au , b as si n hy d ro g ra p hi q ue , ré g io n o u p ay s, t ai lle d e l’é ch an ti llo n, ha p lo ty p es m it o ch o nd ri au x (e nt re p ar en th ès es , le n o m b re d ’in d iv id us p o ur c ha q ue h ap lo ty p es ). Q ue lq ue s sé q ue nc es o nt é té té lé ch ar g ée s à p ar ti r d e G en eB an k. C o d e W at er b o d y H yd ro g ra p hi c d ra in ag e S am p lin g lo ca ti o n S am p le si ze H ap lo ty p e 16 S (n ) G en eb an k ac ce ss io n nu m b er , o ri g in al s o ur ce a nd t ax o no m ic