Comparing four mitochondrial genes in earthworms – Implications for identification, phylogenetics, and discovery of cryptic species

Earthworms play a key role in soil ecology as they can reach high densities, are well known as soil engineers, and occupy a central position in soil food-webs. The identification of earthworms, however, is notoriously difficult and morphologically only possible for well-preserved adult specimens. Molecular markers could facilitate earthworm identification and would be a huge advantage in studies where it is important to species-specifically identify juveniles or where badly preserved specimens and remains of earthworms need to be identified. The aim of this study was to compare four mitochondrial genes (12S rRNA, 16S rRNA, COI, and COII) with respect to their value for identifying earthworms, calculating earthworm phylogenies, and discovering cryptic species. Our results indicate that all four genes are suitable for species identification. However, the genetic distances were approximately twice as high for the protein coding genes than for RNA coding ones. High genetic distances and deep genetic lineages, e.g. for Octolasion lacteum , Lumbricus rubellus and Aporrectodea rosea , indicate the possible presence of cryptic species and hamper molecular identification. The Bayesian analysis based on concatenated sequence data resulted in a phylogenetic tree with high posterior probabilities. The con-specific relationship of Aporrectodea spp. and Allolobophora spp. was not confirmed, underlining the ongoing discussion about the revision of these two genera. In conclusion, our findings suggest using 12S and 16S rRNA sequences as molecular markers for species identification whereas the COI gene is better suited to address genetic lineages and to explore possible cryptic species. Taxonomy and the identification of species are essential for most ecological studies. This study provides the needed molecular sequence information to develop molecular tools that can overcome many hurdles in studies on earthworms, their relationship and their ecology.