After alignment, 380 base pairs were obtained for the 12S gene: 63 invariable, 187 variable uninformative and 130 parsimony informative. The percentage of genetic divergence at the species level ranges from 6% (T. sp. from Zat River vs T. sp. from Ourika River) to 36% (S. v. virei vs Saharolana seurati Monod, 1930). For the 16S gene, we examined 444 aligned base pairs: 99 invariable, 200 variable and 145 parsimony informative. For this gene, the divergence between species ranges from 7% (T. haouzensis vs M. delamarei) to 42% (S. seurati vs Antrolana lira Bowman, 1964).
All the sequences are A-T rich (Table 1), in agreement with the observation that arthropod mitochondrial genomes generally tend to be highly A+T biased, even though the A-T content is lower than in other arthropods such as insects (Simon et al., 1994; Muraji and Nakahara, 2001). The results of the chi-square homogeneity test show homogeneous base composition within the ingroup taxa for the 12S (χ2= 125.65, p<0.0001) and 16S (χ2= 125.80, p<0.0001) rRNAs.
The results of the likelihood mapping method carried out with TREE-PUZZLE suggest the presence of a strong phylogenetic signal in the two mitochondrial gene portions. Sequence congruence between the 16S and 12S gene fragments is not rejected according to the Homogeneity Partition test (p=0.45) for those taxa sequenced for both genes. The heuristic search produced two most parsimonious trees at 1878 steps (CI=0.58, RI=0.54, HI=0.41), from which a strict consensus tree is presented (Fig. 2).
The Sphaeromides group (SPH-G, sensu Botosaneanu, Table 2) appears to be conserved in all trees (Figs 2, 3). It includes the species belonging to the genera Typhlocirolana, Marocolana, Antrolana, Speocirolana and Sphaeromides, as previously suggested on the basis of morphological investigations. The only exception is Turcolana sp., which appears in the 12S tree with an unsolved position and very divergent from all the other Mediterranean stygobitic cirolanid species analysed (Fig. 3). However, the divergence of the 12S sequence in Turcolana sp. could also be due to pseudogene amplification (Song et al., 2008). 16S amplification performed on six individuals of Turcolana sp. yielded unsuccessful products and requires other samples and analyses.
The Sphaeromides group presents two major lineages: the clade containing the Typhlocirolana species and M. delamarei, and the clade containing S. virei and the two American taxa, A. lira and S. bolivari (Rioja, 1953). Within the Typhlocirolana group, T. leptura Botosaneanu, Boutin and Henry, 1985 and Typhlocirolana sp. from Errachidia (ERR) (central-eastern Morocco) show high affinity and are grouped with the taxa (T. spp., GIN, TIZ, SOU, OUR, ZAT) inhabiting the central and southern part of Morocco (Fig. 2). On the other hand, T. cf. fontis from Algeria (ALG) seems to be closely related to the species living in the northern part of Morocco (GAF, GAY), which together with MOR and SIC constitute the M group (a group of species present along or near to Mediterranean coasts), well supported in all trees (Figs 2, 3).
Fig. 2. Consensus of proposed phylogenetic relationships obtained with the combined 16S and 12S data. The consensus diagrams summarize the results of the three reconstruction methods, with their support values for single nodes, Bayesian posterior probability, MP and NJ bootstrap values at the nodes. M: Mediterranean group; SPH-G: Sphaeromides group, explanations in the text. Marine species in bold; taxa with light troglomorphology underlined; taxa with troglomorphic characters only in italic. Abbreviations as in Table 1.
Fig. 3. Bayesian phylogenetic analysis obtained with 12S data. The consensus diagrams summarize the results of the three reconstruction methods, with their support values for single nodes, Bayesian posterior probability, MP and NJ bootstrap values at the nodes. M: Mediterranean group; SPH-G: Sphaeromides group, explanations in the text. Marine species in bold; taxa with light troglomorphology underlined; taxa with troglomorphic characters only in italic. Abbreviations as in Table 1.
The 12S tree confirms the groups detected by the 16S and 12S+16S phylogenies: S. virei and the two American taxa; GUE, GAF, ALG, SIC and MOR; HAU AND MAR (Fig. 3). In contrast, some differences were detected when the 12S and 16S sequence data were analysed separately: S. seurati and F. faucheri cluster together as the sister clade of the Sphaeromides group (SPH-G), as expected on the basis of the previous studies (Botosaneanu, 1986, Waegele, 1989). In the 12S tree (Fig. 3) the Typhlocirolana species from central and southern Morocco (LEP, ERR, ZAT, OUR, SOU, GIN, TIZ), poorly supported in Fig. 2, are split into two groups but with unsolved positions.
The position of M. delamarei, in the same clade as T. haouzensis (‘central group’, Fig. 2), is not clear and its generic status is not supported by the low genetic divergence from T. haouzensis, as also calculated by the divergence sequence data of the COXI gene. The molecular divergence investigated by COXI reveals a genetic distance of 14% between the two taxa, more similar to the distance between close species of Sphaeromides (S. s. virei and S. v. mediodalmatina = 12%) than to the divergence value of 29% recorded among different genera, such as T. haouzensis or M. delamarei and S. virei.
Application of a molecular clock provides a time framework for the evolutionary events that characterized the stygobitic cirolanids analysed here over a time scale of 180 myr up to present. The separation of the different species of Cirolanidae from a common ancestor appears to date back to the late Jurassic/lower Cretaceous 180-165 mya (Fig. 4). In particular, cirolanid species belonging to the different genera (S. v. virei vs T. haouzensis/M. delamarei) could have separated from each other 180 mya (350-150 my 95% HPD). The time of divergence for S. seurati/F. faucheri vs the Sphaeromides group should date back to 165 mya (350-240 95% HPD) (Fig. 4). In the Mediterranean basin, the Typhlocirolana taxa belonging to the M group (Fig. 2) probably separated from the species of central and northern Morocco 90 mya (140-45 my 95% HPD).