Deeply divergent sympatric mitochondrial lineages of the earthworm Lumbricus rubellus are not reproductively isolated

Background: The accurate delimitation of species is essential to numerous areas of biological research. An unbiased assessment of the diversity, including the cryptic diversity, is of particular importance for the below ground fauna, a major component of global biodiversity. On the British Isles, the epigeic earthworm Lumbricus rubellus, which is a sentinel species in soil ecotoxicology, consists of two cryptic taxa that are differentiated in both the nuclear and the mitochondrial (mtDNA) genomes. Recently, several deeply divergent mtDNA lineages were detected in mainland Europe, but whether these earthworms also constitute cryptic species remains unclear. This information is important from an evolutionary perspective, but it is also essential for the interpretation and the design of ecotoxicological projects. In this study, we used genome-wide RADseq data to assess the reproductive isolation of the divergent mitochondrial lineages of L. rubellus that occur in sympatry in multiple localities in Central Europe. Results: We identified five divergent (up to 16 % net p-distance) mitochondrial lineages of L. rubellus in sympatry. Because the clustering of the RADseq data was according to the population of origin and not the mtDNA lineage, reproductive isolation among the mtDNA lineages was not likely. Although each population contained multiple mtDNA lineages, subdivisions within the populations were not observed for the nuclear genome. The lack of fixed differences and sharing of the overwhelming majority of nuclear polymorphisms between localities, indicated that the populations did not constitute allopatric species. The nucleotide diversity within the populations was high, 0.7-0.8 %. Conclusions: The deeply divergent mtDNA sympatric lineages of L. rubellus in Central Europe were not reproductively isolated groups. The earthworm L. rubellus, which is represented by several mtDNA lineages in continental Europe, apparently is a single highly polymorphic species rather than a complex of several cryptic species. This study demonstrated the critical importance of the use of multilocus nuclear data for the unbiased assessment of cryptic diversity and for the delimitation of species in soil invertebrates.