Species delimitation integrative approach reveals three new species in the Nemertopsis bivittata complex

The presence of cryptic species is fairly frequent in many invertebrate groups and even more so among invertebrates with simple morphology, such as nemerteans. Consequently, the use of molecular methods for species delimitation has become a needed tool to complement morphological analyses to better recognise such species. Nemertopsis bivittata is one example of species with subtle morphological variation, but ample geographic distribution, being a good candidate for a species complex study. Here we applied two mitochondrial genes, and 2903 single nucleotide polymorphism (SNP) variants in addition to morphological characters to investigate the presence of cryptic species among specimens previously identified as N. bivittata along the Brazilian Coast. To do so, specimens were collected at 15 different sites in the north-east, south-east and southern regions. Three new species of Nemertopsis are described based on morphological and molecular analyses: Nemertopsis caete sp. nov., Nemertopsis pamelaroeae sp. nov. and Nemertopsis berthalutzae sp. nov. The species N. pamelaroeae and N. berthalutzae present broad distributions from north-east to south-east; N. caete, however, is restricted to the north-east coast. This is the first study to use this combined approach in nemerteans and shows the advantages of integrating genomic markers with classical taxonomy, and applying objective approaches to delimiting species as independently evolving entities.