The DNA RFLP analysis of nine Greek and two central European populations of terrestrial isopods belonging to Ligidium species revealed a strikingly high level of variation between populations, coupled with low levels of variation within populations. Each of the seventeen haplotypes found is unique in one population only. Divergence values between populations are comparable to those between species in other, phylogenetically related, animal groups. In particular, Marcadé et al. (1999) consider the 13% divergence among populations of the species complex Porcellionides pruinosus from France and Tunisia-Greece-Réunion island as indicative of the presence of separate species. A similar value (12.3%) has been found between different species of the crustacean genus Artemia using RFLP analysis of the 16S rDNA gene segment (Gajardo et al., 2004). At the same time, genetic distance between different species of the decapod Emerita, based on nucleotide data, lies also between 13-15% (Tam et al., 1996), while the genetic distance, according to RFLP analysis of COI and COII segments, among allopatric populations of the marine isopod Saduria entomon from northeastern Europe are lower than 2% (Sell, 2003). In comparison, looking also at non-related taxa, divergence values around 10% have been documented for marine and estuarine populations of Atherina boyeri in Greece (Klossa-Kilia et al., 2002), which in fact are considered to be different species. Papasotiropoulos et al. (2002) have found divergence values ranging from 1.61 to 15.12 between different species of Grey Mullets (Mugilidae).
Even though there are no other data concerning genetic distance among terrestrial isopods on the basis of RFLP analysis, it is reasonable to consider that the distance values found between Ligidium populations are very high considering the geographical scale of the present analysis. These results are compatible with the biology of these animals, since they live within a narrow range of habitat types (see Introduction), forming local populations with very restricted gene ﬂow between them.
It is remarkable that population divergence is incongruent with geographical distance among populations. Additionally, the well-established species L. hypnorum (Stuttgart) and L. germanicum (Slovenia) show a divergence of only 5% between them, lower than that of almost all other pairs of populations from Greece, even of populations inhabiting the same river system (Lousios and Vasilaki). Only the two populations from southern Evvoia Island (Karystos 1,2) show a lower nucleotide divergence, but they have been collected within a distance of less than 300 m and from the same river system. These populations, though, show a very high genetic distance from the population of central Evvoia (Dirfys), which belongs to the same species (L. euboicum); nevertheless they are placed in the same clades in both the population tree and the Minimum Spanning Network (MSN) of haplotypes.
The relationships among populations, as revealed by the trees of haplotypes (Fig. 3.populations (Fig. 4.and the MSN of haplotypes (Fig. 2, reveal two pertinent groups, one consisting of the insular populations from Evvoia Island (Dirfys, Karystos 1, 2), and one consisting of the three monomorphic populations from central Peloponnisos (Planiterou, Lousios, Vasilaki). Another relatively well-supported relationship is the one between the population from Mt. Pilio (L. beieri) and the one from northern Peloponnisos (Zarouchla). We should note that whereas Zarouchla and Planiterou lie close to each other from a geographical perspective, the respective Ligidium populations have been collected from rivers that ﬂow towards different directions. The one at Zarouchla (Krathis river) ﬂows to the north (Korinthiakos Gulf), whereas the one at Planiterou to the southwest (Ionian Sea). It seems that the populations from central Peloponnisos belong to the same stock, while those from its northern and southern part (Nedonas river) are more variable and may have a different history. Also, it is evident that it is not possible to draw a clear line between the Greek species and the European ones, even though the haplotype and the population trees suggest that European populations may belong to a distinct clade. Nevertheless, there are certain haplotypes in Greek populations that are more closely related to haplotypes of the European species. Comparing the restriction site patterns (Tables 1, 2) with haplotype distribution on the MSN and the trees, we can see that most variation is due to the repeated and independent gain and/or loss of restriction sites. This means that there are increased levels of homoplasy in the data, probably resulting in low bootstrap values supporting the clades in these trees.
Another important ﬁnding is that the patterns of genetic divergence are not consistent with phenotypic divergence. As already stated, most morphological features of these animals are not subject to differentiation among species, while most of the characters traditionally used in the taxonomy of the genus exhibit overlapping variation (Sfenthourakis, 1993). The only dependable taxonomic character found so far, the male pleopod-endopodite 2, has the same structure in all specimens from Peloponnisos and Evvoia examined so far, whereas it differs in the specimens from Mt. Pilio (see ﬁgures in Sfenthourakis, 1993). Furthermore, this same character, as well as several other morphological features (e.g. coloration, relative length of uropod exo- and endopodite), differ strikingly between the two European populations. Genetic divergence, though, does not agree with these phenotypic patterns. We should note that, even though the present RFLP analysis has not lead to a resolution of phylogenetic relationships among populations, its results are suggestive for some discrepancy among patterns exhibited by molecular and morphological data.
The overall observations indicate an extremely high level of isolation among populations coupled with conservatism in phenotypic characters. It is not safe to translate genetic divergence in statements about phylogenetic relationships and taxonomy, since a large part of the variation is due to homoplasic changes. Further work involving more populations as well as sequence data are needed before we can resolve the phylogeny of Greek Ligidium species. On the other hand, we have shown that morphological features alone seem inadequate to reveal the standing diversity and to resolve the phylogenetic relationships inside this taxon.