Phylogenetic species delimitation of the earthworms Eisenia fetida (Savigny, 1826) and Eisenia andrei Bouché, 1972 (Oligochaeta, Lumbricidae) based on mitochondrial and nuclear DNA sequences

Three samples (11 individuals) of Eisenia fetida (Savigny, 1826) and four samples (20 individuals) of Eisenia andrei Bouché, 1972 (focal species), and two samples (six individuals) of Eisenia eiseni (Levinsen, 1884) (nonfocal species; outgroup) were tested for species delimitation using the phylogeny-based method of Wiens and Penkrot (2002). Thirty-seven and 17 DNA sequences of the nuclear 28S (2031 bp) and mitochondrial COI (658 bp) genes, respectively, were generated. Phylogenetic hypotheses for each gene and both combined were built using maximum parsimony, maximum likelihood, and Bayesian approaches. Fully resolved and well-supported trees (>90% bootstrap proportions and 1.0 posterior probabilities) showed both E. fetida and E. andrei samples as monophyletic. Application of Wiens and Penkrot's protocol to those trees resulted in the recognition of these two taxa as different phylogenetic species. This result complements previous studies of crossability and allozyme genetics addressing their different biological status (biological species concept). Finally, our analyses also show that E. fetida is composed of reproductively isolated populations and suggest the presence of a hitherto unrecognized species or subspecies within this taxon.