Trying to resolve the taxonomic confusion of Paracalanus parvus species complex (Copepoda, Calanoida) in the Mediterranean and Black Seas through a combined analysis of morphology, molecular taxonomy and DNA metabarcoding

Paracalanus parvus is reported as the most abundant representative of the genus and one of the main components of the coastal zooplankton in the Mediterranean and Black Seas. However, the subtle taxonomic differences between P. parvus and the congeneric species P. indicus and P. quasimodo, in combination with the ample morphological variation found in Mediterranean specimens, render problematic the correct identification. A recent molecular study by Cornils and Held (2014) provided evidence of cryptic speciation in the P. parvus complex and indicated that P. parvus s.s. does not have global distribution, but may be restricted to the northeastern Atlantic. In order to clarify the taxonomic status and distribution of this species complex in the Mediterranean and Black Seas, a study was conducted on Paracalanus specimens collected from different locations across the aforementioned marine basins and sequenced for portion of the COI mitochondrial gene. An accurate taxonomic analysis was also carried out to correlate morphological characteristics with the molecular species' assignation. The phylogenetic analysis of the specimens together with the publicly available sequences of P. parvus complex revealed the presence of four molecular operational taxonomic units (MOTUs) in the Mediterranean, which differed in abundance and geographic distribution. The combination of morphological and molecular data revealed great inconsistencies between morphospecies and MOTUs. Moreover, several bulk zooplankton samples were analyzed through DNA metabarcoding in the frame of the “MetaCopepod” project to provide more extensive information on the spatiotemporal distribution and abundance of the target specie

The “MetaCopepod” project: Designing an integrated DNA metabarcoding and image analysis approach to study and monitor the diversity of zooplanktonic copepods and cladocerans in the Mediterranean Sea

The timely and accurate analysis of marine zooplankton diversity is a challenge in ecological and monitoring studies. Morphology-based identification of taxa, which requires taxonomy experts, is time consuming and cannot provide accurate resolution at species level in several cases (e.g. immature stages, cryptic species, broken specimens). The “MetaCopepod” project is aimed at overcoming these limitations by developing a high-throughput and cost effective methodology that integrates DNA metabarcoding and image analysis. Utilizing the accuracy of DNA metabarcoding in species recognition and the quantitative results of image analysis, zooplankton diversity (mainly of copepods and cladocerans) is assessed both qualitatively (species' composition) and quantitatively (abundance, biomass and size-distribution). To achieve this goal, bulk zooplankton samples are first scanned and analyzed with ZooImage and then massively sequenced for a selected fragment of the mitochondrial 16S rRNA gene. Through a bioinformatic pipeline, sequences are compared to a reference genetic database, constructed within the project, and identified at species- level. The methodology was calibrated by using both mock and taxonomically identified samples and demonstrated on samples collected monthly from monitoring stations across the Mediterranean Sea. It is currently optimized for higher integration and accuracy and is expected to become a powerful tool for monitoring zooplankton in the long term and for early warning of bioinvasions and other ecosystem change