Hybridization relics complicate barcode-based identification of species in earthworms.

Introgressive hybridization results in mito-nuclear discordance which could obscure the delimitation of closely related taxa. Although such events are increasingly reported, they have been poorly studied in earthworms. Here, we propose a method for investigating the degree of introgressive hybridization between three taxa of the Allolobophora chlorotica aggregate within two field populations (N = 67 and N = 105) using a reference data set including published DNA barcoding and microsatellite data of all known A. chlorotica lineages (N = 85). For this, we used both molecular phylogenetic and population genetic approaches. The test of correspondence between mitochondrial cytochrome c oxidase I (COI) lineages and clusters of nuclear microsatellite genotypes allowed individuals to be sorted in three categories (matching, admixed and nonmatching) and additional markers (mitochondrial NADH dehydrogenase subunit 1, nuclear Histone 3 and Internal transcribed Spacer Region 2) were used for phylogenetic reconstructions in order to check assignments. Although 15 admixed individuals were observed, no early-generation hybrids were detected within the two populations. Interestingly, 14 nonmatching individuals (i.e. with a mtDNA haplotype that did not correspond to their nuclear cluster) were detected, a pattern that would result after multiple generations of unidirectional hybridization of female from one taxon to male of the other taxon. Because earthworms are simultaneous hermaphrodites, these events of unidirectional hybridization suggest sterility of the male function in several crosses and highlight that some individuals can be misidentified if reliance is placed on COI barcodes alone. These findings could improve the use of these barcodes in earthworms for species delineation.

Re-integrating earthworm juveniles into soil biodiversity studies: species identification through DNA barcoding.

Species identification of earthworms is usually achieved by careful observation of morphological features, often sexual characters only present in adult specimens. Consequently, juveniles or cocoons are often impossible to identify, creating a possible bias in studies that aim to document species richness and abundance. DNA barcoding, the use of a short standardized DNA fragment for species identification, is a promising approach for species discrimination. When a reference library is available, DNA-based identification is possible for all life stages. In this study, we show that DNA barcoding is an unrivaled tool for high volume identification of juvenile earthworms. To illustrate this advance, we generated DNA barcodes for specimens of Lumbricus collected from three temperate grasslands in western France. The analysis of genetic distances between individuals shows that juvenile sequences unequivocally match DNA barcode clusters of previously identified adult specimens, demonstrating the potential of DNA barcoding to provide exhaustive specimen identification for soil ecological research.