DNA barcoding of earthworms (Eisenia fetida/andrei complex) from 28 ecotoxicological test laboratories

Plenty of evidence indicates that the earthworms Eisenia fetida (Savigny, 1826) and Eisenia andrei Bouché, 1972 (Lumbricidae) can be distinguished by morphological, physiological and molecular traits. However, the morphological differences alone do not allow to correctly identify these taxa. This may be a serious problem for the reliability of the standard ecotoxicological tests for which these worms are used. Recently, DNA barcoding (i.e., sequencing of a standard mtDNA COI gene fragment for identification purposes) has been proposed as a quick and simple method to identify species, including earthworms. In order to assess the practicability and reliability of this method, an international ring test was organized by the “Eisenia barcoding Initiative (EBI)”, a group of scientists from four public institutions and two contract laboratories. Coded samples of E. fetida, E. andrei, and Eisenia sp. were provided by 28 ecotoxicological laboratories from 15 countries on four continents. Five laboratories in Belgium, Canada, Germany, and Spain identified the specimen through DNA barcoding. All steps of the sample preparation were described by Standard Operating Procedures (SOP). The COI sequences (581 bp) obtained were used to construct a neighbor-joining tree based on the uncorrected pairwise p-distance. This analysis revealed three distinct haplotype clusters: one including only E. andrei sequences (mean within-group p-distance 0.026 ± 0.002) and two with only E. fetida sequences, referred to as E. fetida 1 and E. fetida 2. Each of the latter two in fact represented one single haplotype. The mean p-distance between E. fetida 1 and E. fetida 2 was 0.112, whereas the mean p-distances between these two taxa and E. andrei were 0.142 and 0.143, respectively. Such COI divergence levels are usually indicative of species level differentiation. Hence it is hypothesized that E. fetida 1 and E. fetida 2 refer to different cryptic species. Since the attribution of the individual worms to these three clusters was completely consistent among the five DNA barcoding laboratories the good applicability of DNA barcoding for the identification of these ecotoxicological test species is proven. Remarkably, specimens of the molecular E. fetida clusters were always identified morphologically as E. fetida. However, this was not true the other way round, i.e., some specimens of the molecular E. andrei cluster were identified morphologically as E. fetida. The results of this ring test are presented to standardization organizations (OECD, ISO) in order to improve the standardization and thus quality of ecotoxicological routine testing by using DNA barcoding.