How many native Cerithium species in the Mediterranean Sea? An integrative taxonomic approach

Species are fundamental units in evolutionary biology. However, defining them in taxonomically problematic groups requires integration of independent sources of information in order to develop robust hypotheses for taxonomic classification. Here, we propose an integrative framework for species delimitation in the Mediterranean species of the genus Cerithium (Caenogastropoda: Cerithiidae), whose shells show a wide variety of forms resulting in problematic morphological identification at the species level. Combined sequence data of two mitochondrial genes (COIþ12S) for 55 individuals from the central Mediterranean Sea were used to test the species status of six identified morphotypes. Phylogenetic analyses, as well as DNA-based methods of species delimitation (automatic barcode gap discovery, species delimitation plugin and genealogical sorting index), support the species status of three morphotypes (C. vulgatum ¼ MOTU-A, C. alucastrum ¼ MOTU-B and C. protractum ¼ MOTU-C), sometimes considered as ecotypes. Molecular operational taxonomic unit (MOTU)-D includes large individuals morphologically assigned to C. vulgatum (from Brindisi and Oristano) and C. repandum (from Tunisia), but all probably represent C. repandum which, if valid, would then not be endemic to its type locality in the Gulf of Gabe` s. All individuals identified as C. lividulum grouped in MOTU-E, except those from Tunisia which cluster in MOTU-F with some C. renovatum. Cerithium renovatum from Crete forms an endemic group (MOTU-G), suggesting a possible cryptic species. Our results show only partial concordance between traditional morphology and sequence data, indicating that the former is not always sufficient for recognizing species level taxa within Mediterranean Cerithium, although protoconch morphology is a key feature for distinguishing between species with superficially similar teleoconchs. Further analyses based on more comprehensive geographic sampling and more mitochondrial markers, and including a number of nuclear loci, are needed to clarify Mediterranean Cerithium diversity more fully.