Delimiting species boundaries is a fundamental yet challenging task that has been especially problematic in morphologically conserved lineages. The use of genetic markers and phylogenetic approaches has proven of paramount importance to resolve these cases. Using the genus Unio as a case study, we have analysed two mitochondrial markers, cytochrome c oxidase subunit I (COI) and 16S rRNA (16S), phylogenetically and phylogeographically to explore species limits and evolutionary lineages within the group. We followed two different approaches to define species boundaries: the generalized mixed Yule coalescence model in a Bayesian framework and the Poisson tree process model. Phylogenetic relationships among species and divergence times were also assessed using relaxed molecular clock analyses. Taken together, we provide a sound systematic framework for Western Palaearctic Unio species, addressing phylogenetic relationships, and the taxonomy, biogeographic patterns and evolutionary history of this group in this region. The Unio clade from the studied area had a clear phylogenetic structure with four robust lineages that include the following species: (1) Unio durieui + Unio gibbus; (2) Unio tumidus; (3) Unio pictorum, Unio delphinus + Unio foucauldianus, Unio elongatulus, Unio mancus, Unio ravoisieri, Unio tigridis; and (4) Unio tumidiformis + (Unio bruguierianus + (Unio crassus + Unio ionicus)). Deeper analyses, including filling gaps in distributions, should be conducted to disentangle the U. crassus, U. pictorum and U. tigridis species complexes.

Using an approach based on molecular, reproductive and morphological characters, four independent evolutionary lineages represented by the following species were identified in Tunisia: Unio ravoisieri Deshayes, U. gibbus Spengler, U. durieui Deshayes and Potomida littoralis (Cuvier). The species U. ravoisieri and U. durieui are redescribed, including details of their distribution, biology and glochidium morphology. The puzzling distribution of the former (Algeria, Tunisia and two localities in Northeast Spain) could reflect introduction or natural dispersal. In Tunisia, the release of the glochidia of U. ravoisieri starts in March, but in Spain this is delayed until August or September. The entire interlamellar space of the outer demibranchs acts as a marsupium (ectobranchy). Glochidia are rounded-triangular, globose, and either hooked or hookless. Unio durieui is distributed across Algeria and Tunisia, where it releases glochidia in December and in March. Glochidia are always hooked, but the elaboration of the hook is variable. The molecular phylogenetic analysis supports two clusters within Unio, each including both European and North African species. Strong bootstrap support was obtained for the two clades within U. ravoisieri, of which one includes specimens from the Iberian Peninsula as well as Tunisia. Unio durieui appeared as the sister species of U. gibbus. The Tunisian populations of P. littoralis were not segregated from those in Europe, and U. fellmanni should be considered a synonym.

Within the Unionoida the family Margaritiferidae is a small but widely distributed group, whose number of genera and species is under discussion. Using molecular and morphological characters, the present paper redescribes several Moroccan specimens of Margaritifera, previously classified as M. auricularia marocana. The shell and anatomical features of the taxon are similar to those observed in European specimens of M. auricularia auricularia. Notwithstanding, the two subspecies can be separated by certain hinge characters. Partial sequences of the mitochondrial genes COI and 16S rRNA support recognition of the Moroccan populations as a different species: Margaritifera marocana. We examined the phylogenetic relationships of the Margaritiferidae. The COI data indicated two monophyletic clades: one including M. margaritifera, M. dahurica, M. falcata and M. laevis, and the second comprising M. auricularia from the Iberian Peninsula and the Moroccan specimens of M. marocana as an independent lineage. Cumberlandia monodonta was recovered as the basal margaritiferid, sister to Margaritifera.