Untangling a mess of worms: Species delimitations reveal morphological crypsis and variability in Southeast Asian semi-aquatic earthworms (Almidae, Glyphidrilus).

Semi-aquatic freshwater earthworms in the genus Glyphidrilus from Southeast Asia are characterized by both an extreme morphological crypsis among divergent phylogenetic lineages and a high morphological variability within the same phylogenetic lineages. The present study provides a new taxonomic framework for this problematic genus in SE Asia by integrating DNA sequence and morphological data. When single-locus and multilocus multispecies coalescent-based (MSC) species delimitation methods were applied to DNA sequence data, they usually yielded highly incongruent results compared to morphology-based species identifications. This suggested the presence of several cryptic species and high levels of intraspecific morphological variation. Applying reciprocal monophyly to the cytochrome c oxidase subunit 1 (COI) gene tree allowed us to propose the existence of 33 monophyletic species. Yet, often substantially more molecular operational taxonomic units (MOTUs) were obtained when species delimitation was based on COI and 16S rRNA sequences. In contrast, the ITS1 and ITS2 sequences suggested fewer MOTUs and did not recover most of the monophyletic species from the Mekong basin. However, several of these latter taxa were better supported when MSC species delimitation methods were applied to the combined mtDNA and ITS datasets. The ITS2 secondary structure retrieved one unnamed Mekong basin species that was not uncovered by the other methods when applied to ITS2 sequences. In conclusion, based on an integrative taxonomic workflow, 26 Glyphidrilus candidate species were retained and two remained to be confirmed. As such, this study provides evidence to suggest nine species new to science and to synonymize 12 nominal morphospecies. It also illustrates that the uncritical use of COI as a universal DNA barcode may overestimate species diversity because COI may be unable to distinguish between divergent conspecific lineages and different candidate species.