First Integrative Morphological and Genetic Characterization of Tremoctopus violaceus sensu stricto in the Mediterranean Sea

SIMPLE SUMMARY: Four rare species are recognized within the genus Tremoctopus (Cephalopoda: Octopoda), i.e., T. gelatus, T. gracilis, T. robsoni, and T. violaceus. The accurate identification of organisms is a fundamental prerequisite to deepen our knowledge of the biology and ecology of a species. In this study, for the first time, an integrative morphological and genetic approach was undertaken to confirm the identity of specimens of the genus Tremoctopus collected in Mediterranean waters. Sequences of two mitochondrial genes were generated and analyzed from three Mediterranean females, allowing ascribing all the samples to the species T. violaceus sensu stricto. For the first time, barcoding sequences have been obtained from the presumed type locality of the species. This information is of particular importance for this rare species; it has been complemented with the detailed descriptions of morphometric and biological features, as well as beaks analyses for the age estimation of the samples. ABSTRACT: An integrative approach based on morphological and genetic analyses was undertaken for the first time to confirm the species identification of Mediterranean samples belonging to the genus Tremoctopus. Sequences of two mtDNA genes (cytochrome c oxidase subunit (COI) and 16S) were generated for the first time from Mediterranean samples. Both the similarity-based identifications and tree-based methods indicated that three females can be identified as Tremoctopus violaceus sensu stricto in agreement with their morphological classifications. All Mediterranean sequences clustered with the sequences of Tremoctopus violaceus from the Gulf of Mexico and were clearly differentiated from the sequences attributed to T. gracilis and T. robsoni. The chromatic pattern of the web and some features of gill filaments, arms formula, stylets, radulae, beaks, and stomach contents were given for all the samples; 105,758, 20,140, and 11,237 oocytes were estimated in the mature, immature, and developing samples, respectively. The presence of four spermatangia inside the cavity of the maturing female suggested the ability of this species to mate before reaching full maturity with more partners. Age investigation using beaks, performed for the first time in T. violaceus and within the genus gave results consistent with the different sizes and maturity conditions of the samples.