Genetic differentiation and evolutionary process of speciation in the Idotea chelipes complex (Crustacea, Isopoda)

Idotea chelipes a ete definie, sur la base des criteres morphologiques et de l'hemocyanine, comme espece polytypique qui groupe trois sous-especes : I. c. bocqueti, I. c. mediterranea et I. c. chelipes. Le statut taxinomique de ces trois sous-especes est reexamine en utilisant les donnees du polymorphisme enzymatique et les resultats des tests d'interfertilite par comparaison avec une autre espece, Idotea balthica. Douze populations d'I. chelipes ont ete analysees par electrophorese sur gel de polyacrylamide mettant en evidence 13 loci. A partir des frequences alleliques, les distances genetiques de Nei ont ete estimees. Celles-ci sont faibles entre les populations mais augmentent entre les sous-especes et deviennent importantes entre les especes. Un dendrogramme est construit et un modele de speciation est propose. Les statuts taxinomiques, specifique et subspecifique sont confirmes.

The majority of the I. chelipes subspecies features, particularly those concerning the secondary sexual male characteristics are identical. However, they are very different from I. b. basteri ones !8!.
Similar observations were reported that concern the hemocyanin electrophoretic mobility and its molecular weight. Hemocyanin, used as a crustacean specific marker, showed the same relative electrophoretic mobility and the similar molecular weight within I. chelipes subspecies. Thus, we observed clear differences between I. chelipes and I. b. basteri [4, 9!. The former results, when combined with some minor morphological characteristics (coxal plates and pleotelson shape) and with biochemical markers !7!, allowed the following: -to confer a subspecific level to I. bocqueti, the endemic species of the eastern Tunisia coasts, which was described as a new species !26!; -to separate the western Mediterranean and the Atlantic I. chelipes populations into two subspecies, I. c. mediterranea [4] and I. c. chelipes [22!. The purpose of the present paper is to verify this viewpoint. Two approaches are used: -breeding tests to determine whether genetic divergence is high enough for the populations or the subspecies to be considered as separate species; -enzymatic polymorphism to estimate the intraspecific and the interspecific genetic divergence.

MATERIALS AND METHODS
Laboratory cross-breedings were tested: intrasubspecific, intersubspecific and interspecific crosses were made.
Electrophoretical study used specimens collected from natural populations (figure 1). Descendants and hybrids obtained from laboratory intra or interspecific cross-breedings were used to determine the genetic control of the different allozymes. Thirteen populations were submitted to enzymatic analysis: . basteri population of Bizerta Lake was used as an outgroup to compare the three geographical 7. chelipes subspecies described above with this I. balthica basteri.
The enzymatic polymorphism was studied on polyacrylamide gels. Specimens at stage C of the moult cycle interecdysis [12] were homogenized in a migration buffer (Tris-glycin pH 8,6) and saccharose 40 % in the same proportions.
Only one or two enzymes were scored per specimen. The hemocyanic fractions and the following enzymes were analysed: amylases (AMY, EC Sims [27], Harris and Hopkinson [13], Legrand-Hamelin et al. [17], Laulier [14], Pasteur et al. [24] and Charfi-Cheikhrouha !5!. According to the population, the number of specimens varied from 14 to 184. Genetic interpretation of the gels was based upon the nomenclature of Pasteur et al. [24]: the most common allele at each locus was named 100.
For the other alleles, numerical values were obtained by adding or subtracting migration distances from 100.
The Biosys-1 program of Swofford and Selander [29] was used: -to calculate the coefficients [20,21] of genetic identity (I) and genetic distance (D); -to construct the phylogenetic tree using the unweighted pair group method (UPGMA) and the single linkage (SL) or nearest-neighbour method !28!.
This last method offers the advantage of requiring no assumptions about equal rates of evolution.

Experimental cross-breedings
Owing to the cannibalism of males against females during ecdysis, only some cross-breedings were realized in the laboratory. The interspecies crossbreedings (I. balthica basteri 7. c. mediterranea and I. c. bocqueti) were unsuccessful. The intersubspecies cross-breedings (I. c. mediterranea and L c. bocqueti) yielded viable and fertile individuals. The intrasubspecies cross-breedings (I. c. mediterranea, I. c. bocqueti and I. c. chelipes) were always successful. The progeny numbers produced by the intra and the inter cross-breedings (table 1) were compared statistically. The F test application indicates no statistical difference between the matings (F = 0.1383, P < 0.05). Enzyme structure, deduced from pedigree analysis, is monomeric (AMY, AO-2) or dimeric (ALP, EST-2). At the esterase locus (EST-2), diagnostic alleles were identified: each subspecies is characterized by its own allele. According to this table, we can observe that the alleles with the highest frequencies are the same in each subspecies.
For some genes, frequencies differ notably between the three subspecies. Such results are observed when L c. bocqueti is compared with the other I. chelipes subspecies (AMY-2). The same results are also observed within 7. chelipes mediterranea when the northern lagoons of Tunisia are compared with the ponds of Roussillon (PGI).
The similarity of hemocyanin fractions was observed. The homology of the fraction, considered as the constitutive monomer, was demonstrated. These observations support the hypothesis of the hemocyanic allele identity.
Only nine loci were scored in the 7. balthica basteri population. Three of them were polymorphic (AMY-1, PGI, LDH) and six were monomorphic withinpopulation (AMY-2, GOT, MDH-2, MDH-1, EST-1, HCY). The monomorphic loci are as important as the polymorphic ones. The three last loci were considered as biochemical markers. They separate 7. b. basteri from I. chelipes samples. Allele frequencies are compiled in table Il.

Genetic divergence
The genetic similarity (I) and distance (D) values were calculated among I. chelipes subspecies on the basis of the allelic frequency data ( The dendrogram reported in figure 2 summarizes genetic relationships among all populations. It shows three levels of genetic differentiation corresponding to three main clusters: -a first subdivision which separates the two species, I. b. basteri and I. chelipes; -a second subdivision which isolates I. c. bocqueti populations from I. c. chelipes and I. c. mediterranea ones; -a third subdivision which separates 1. c. mediterranea and I. c. chelipes populations.
The groupings of the various populations of 1. c. mediterranea, using the UPGMA and the SL methods, are different. These populations are represented on the dendrogram at the same level.

DISCUSSION
Experimental studies showed the similarity of interbreedings between and within I. chelipes subspecies. Negative results and unsuccessful matings were observed when I. b. basteri was involved. According to the biological species concept: the mating of I. bocqueti sensu Rezig with other 7. chelipes populations produces hybrid offspring that interbreed with both parents and with one another !6!. We conclude that I. bocqueti is not a 'good' species like I. b. basteri but a race or a subspecies which belongs to the same species T chelipes. However, these conclusions would be confirmed by further experiments based on the possibility of the choice of partners and the investigation of sympatric areas such as the Siculo Tunisia strait.
Apart from the morphological similarities, there are diagnostic alleles at the esterase 2 locus which constitute the best way to characterize hybrids in potential contact zones. At the locus EST-2 of the hybrids I. c. bocqueti-7. c. mediterranea, three bands at equal distances are evident. This result proves a diallelic locus and a dimeric structure of the EST-2.
Like other Crustaceans, the hemocyanic electrophoregram might be used as a specific taxonomic criterion. Manwell and Baker [19] and Maguire and Fielder [18] reported a similar hemocyanin pattern of various Crustacean species.
Furthermore, in the case of Sphaeroma, the hemocyanin is both specific and subspecific !15!. In the Idotea genus, the comparison of hemocyanic fractions showed the identity of L c. chelipes, I. c. mediterranea and 7. c. bocqueti. This pattern differs from that of I. b. basteri !9!. These results are consistent with the hypothesis of genetic identity of the hemocyanin fraction inside closely related taxa.
An interesting observation should be made: the genetic distinction of the three subspecies is related to the geographic areas based on the enzymatic polymorphism and the allelic frequencies. Many geographically distinct populations of Jaera, Talitrus saltator were separated when allelic frequencies were used [3, 11!. The genetic distance values (table 1T! might be directly compared with the taxonomic categories based on morphological criteria. The interspecific (chelipes-balthica) genetic distance as well as the subspecific one (c. bocqueti, c. mediterranea and c. chelipes) agree with estimates reported for other animal groups [2,23]. The former values confirm that populations of the western Mediterranean and the Atlantic lagoons of I. chelipes would be considered as local populations of the two subspecies, I. chelipes mediterranea and I. chelipes chelipes and suggest that 7. bocqueti cannot be isolated from the 1. chelipes complex and elevated to the rank of species like 7. balthica basteri. This example shows the excellent overall agreement between the genetic data and the taxonomic grouping.
The genetic distances within I. chelipes subspecies were used to draw the dendrogram ( figure 2). The results suggest a relatively recent separation of the three subspecies populations. The first cladogenetic event would isolate the two species, L chelipes and L balthica. The second one would lead to the separation of I. c. bocqueti from 7. c. mediterranea-L c. chelipes and the third one would separate L c. mediterranea and 7. c. chelipes. It would be important to examine, with particular attention, the presumed contact zones considering that the process of geographic differentiation is reversible, whether there is an opportunity of gene exchange. The clustering in the dendrogram agrees with the morphological affinities. The two species, 7. chelipes and I. balthica basteri, are distinguishable by strong features notably the male sexual characteristics such as appendix masculina and pereiopod's two seta. Only minor features corresponding to the pleotelson and the first pereionite shape are observed to differ within 7. chelipes subspecies. Thus, these characteristics are more pronounced in I. c. bocqueti than in the two other I. chelipes subspecies.
The results of the genetic analysis confirm our morphological observations and our tests of tentative hybridization and reinforce our hypothesis of the distinction of three subspecies, I. c. bocqueti, I. c. mediterranea and I. c. chelipes of a single polytypic species I. chelipes.
Contrary to some assertions [30,31] for Idotea balthica [16] for Sphaeroma, the pattern of I. chelipes would suggest colonization from the western Mediterranean sea !10!. These apparently contradictory results must be reconsidered.
The present Mediterranean Isopoda fauna might be related to a conquest by species which originated from the present oriental basin and which did not undergo drying during the salinity crisis as expounded by Charfi-Cheikhrouha and Zaghbib-'llzrki [10] and a reconquest by Atlantic species [25, 16!. To better solve this puzzle, the study must be enlarged to other species of the Idotea genus. The cladogram obtained must be compared with the corresponding geological events as has been carried out for Jaera genus on the basis of morphological characters [32]. ACKNOWLEDGEMENT We are grateful to the referees for their comments and correction.