The formation of continental Europe in the Neogene was due to the regression of the Tethys Ocean and of the Paratethys Sea. The dynamic geology of the area and repetitious transitions between marine and freshwater conditions presented opportunities for the colonization of newly emerging hydrological networks and diversification of aquatic biota. Implementing mitochondrial and nuclear markers in conjunction with a large-scale sampling strategy, we investigated the impact of this spatiotemporal framework on the evolutionary history of a freshwater crustacean morphospecies. The Gammarus balcanicus species complex is widely distributed in the area previously occupied by the Paratethys Sea. Our results revealed its high diversification and polyphyly in relation to a number of other morphospecies. The distribution of the studied amphipod is generally characterized by very high local endemism and divergence. The Bayesian time-calibrated reconstruction of phylogeny and geographical distribution of ancestral nodes indicates that this species complex started to diversify in the Early Miocene in the central Balkans, partially in the shallow epicontinental sea. It is possible that there were several episodes of inland water colonization by local brackish water lineages. Subsequent diversification within clades and spread to new areas could have been induced by Alpine orogeny in the Miocene/Pliocene and, finally, by Pleistocene glaciations. The present distribution of clades, in many cases, still reflects Miocene palaeogeography of the area. Our results point out that investigations of the historical aspect of cryptic diversity in other taxa may help in a general understanding of the origins of freshwater invertebrate fauna of Europe.

A number of studies have claimed that recombination occurs in animal mtDNA, although this evidence is controversial. Ladoukakis and Zouros (2001) provided strong evidence for mtDNA recombination in the COIII gene in gonadal tissue in the marine mussel Mytilus galloprovincialis from the Black Sea. The recombinant molecules they reported had not however become established in the population from which experimental animals were sampled. In the present study, we provide further evidence of the generality of mtDNA recombination in Mytilus by reporting recombinant mtDNA molecules in a related mussel species, Mytilus trossulus, from the Baltic. The mtDNA region studied begins in the 16S rRNA gene and terminates in the cytochrome b gene and includes a major noncoding region that may be analogous to the D-loop region observed in other animals. Many bivalve species, including some Mytilus species, are unusual in that they have two mtDNA genomes, one of which is inherited maternally (F genome) the other inherited paternally (M genome). Two recombinant variants reported in the present study have population frequencies of 5% and 36% and appear to be mosaic for F-like and M-like sequences. However, both variants have the noncoding region from the M genome, and both are transmitted to sperm like the M genome. We speculate that acquisition of the noncoding region by the recombinant molecules has conferred a paternal role on mtDNA genomes that otherwise resemble the F genome in sequence.

Many bivalve species, including mussels of the genus Mytilus, are unusual in having two mtDNA genomes, one inherited maternally (the F genome) and the other inherited paternally (the M genome). The sequence differences between the genomes are usually great, indicating ancient divergence predating speciation events. However, in Mytilus trossulus from the Baltic, both genomes are similar to the F genome from the closely related M. edulis. This study analyzed the mtDNA control region structure in male and female Baltic M. trossulus mussels. We show that a great diversity of structural rearrangements is present in both sexes. Sperm samples are dominated by recombinant haplotypes with M. edulis M-like control region segments, some having large duplications. By contrast, the rearranged haplotypes that dominate in eggs lack segments from this M genome. The rearrangements can be explained by a combination of tandem duplication, deletion, and intermolecular recombination. An evolutionary pathway leading to the recombinant haplotypes is suggested. The data are also considered in relation to the hypothesis that the M. edulis M-like control region sequence is necessary to confer the paternal role on genomes that are otherwise F-like. S TRICTLY uniparental inheritance of organelle genomes is a rule in nearly all anisogamic organisms. One of the most prominent exceptions is the mitochondrial inheritance system of mussels of the family Mytilidae in which separate maternal (the F genome) and paternal (the M genome) routes of mtDNA inheritance occur (for review, see Skibinski et al.1994a,b; Zouros et al. 1994a,b;Zouros 2000). This system, called doubly uniparental inheritance (DUI) (Zouros et al. 1994a), has also been observed in freshwater mussels of the family Unionidae (Hoeh et al. 1996;Liu et al. 1996) and clams of the family Veneridae (Passamonti and Scali 2001). Phylogenetic analysis indicates that divergence of the F and M genomes can be great, predating speciation events, and that role reversal or masculinization events, whereby the F genome takes on the role of the previous M genome, has occurred repeatedly in the evolution of marine mussels (Hoeh et al. 1996(Hoeh et al. , 1997, but is absent or less frequent in freshwater mussels (Hoeh et al. 2002).A hybrid zone separates Baltic Mytilus trossulus from North Sea M. edulis populations (see Riginos and Cunningham 2005 for review). Although there is little introgression of mtDNA between American M. trossulus and M. edulis (Saavedra et al. 1996;Comesana et al. 1999) in Baltic M. trossulus, the mtDNA in heteroplasmic male individuals is similar to that in the F genome from M. edulis (Quesada et al. 1995(Quesada et al. , 2003Wenne and Skibinski 1995; Zbawicka et al. 2003a,b). It appears that there has been complete asymmetric introgression of M. edulis F mtDNA into Baltic M. trossulus, accompanied by role reversal and masculinization (Rawson and Hilbish 1998;Quesada et al. 1999). These processes might be coupled and associated with cytonuclear incompatibilities that ...