Phylogenetic assessment of the earthworm Aporrectodea caliginosa species complex (Oligochaeta: Lumbricidae) based on mitochondrial and nuclear DNA sequences.

The Aporrectodea caliginosa species complex includes the most abundant earthworms in grasslands and agricultural ecosystems of the Paleartic region. Historically this complex consisted of the following taxa: A. caliginosa s.s.Savigny, 1826, A. trapezoides Dugés (1828), A. tuberculata (Eisen, 1874), and A. nocturna Evans (1946). These four taxa are morphologically very similar and difficult to differentiate because of their morphological variability. Consequently, their taxonomic status and their phylogenetic relationships have been a matter of discussion for more than a century. To study these questions, we sequenced the COII (686 bp), 12S (362 bp), 16S (1200 bp), ND1 (917 bp), and tRNAs(Asn-Asp-Val-Leu-Ala-Ser-Leu) (402 bp) mitochondrial and 28S (809 bp) nuclear gene regions for 85 European earthworms from 27 different localities belonging to the A. caliginosa species complex and four outgroup taxa. DNA sequences were analyzed using maximum parsimony, maximum likelihood, and Bayesian approaches of phylogenetic inference. The resulting trees were combined with morphological, ecological, and genomic evidence to test species boundaries (i.e., integrative approach). Our molecular analyses showed that A. caliginosa s.s. and A. tuberculata form a sister clade to A. trapezoides, A. longa, and A. nocturna, which indicates that A. longa is part of the A. caliginosa species complex. We confirm the species status of all these taxa and identify two hitherto unrecognized Aporrectodea species in Corsica (France). Moreover our analyses also showed the presence of highly divergent lineages within A. caliginosa, A. trapezoides, and A. longa, suggesting the existence of cryptic diversity within these taxa.