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

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.

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“…As shown in Figure 7 , the phylogenetic tree constituted three main branches: one included the reference sequence of T. robsoni ; the second included one reference sequence deposited as T. gracilis and four reference sequences deposited in GenBank as T. violaceus , but reclassified by Agus et al [ 41 ] as T. gracilis ; a third branch included two sub-branches, one with the compiled sequences obtained in this study for TV006 and TV008, highly similar to the T. violaceus homologue sequence from the Mediterranean Sea (OM025233), and one with the sequences from our individuals T. gracilis TG002 and TG003 clustering with the two homologue sequences from the Gulf of Mexico (MW351786 and MW351787) ( Table S4 ). To the best of our knowledge, these are among the first haplotypes of cytochrome c oxidase subunits I (COI) of Tremoctopus sp.…”

“…To the best of our knowledge, these are among the first haplotypes of cytochrome c oxidase subunits I (COI) of Tremoctopus sp. from the Mediterranean Sea publicly available in GenBank, following the study of Agus et al [ 41 ].…”

“…In this study, some Mediterranean individuals of holopelagic octopuses were genetically characterized using the mitochondrial fragment COI genetic marker, mostly used for the identification of cephalopod species [ 41 , 72 , 73 , 74 ].…”

“…The presence of T. gracilis in the Mediterranean basin was discovered by analysing a photo of an adult female in the Tyrrhenian Sea in August 2002, as discussed by Orsi Relini et al [ 3 , 6 ]. After this event, a deeper analysis of previous records has shown that other individuals of T. gracilis had been erroneously ascribed to T. violaceus [ 3 , 5 , 6 , 41 ]. The study of Agus et al [ 41 ] supported the hypothesis that errors/misidentifications are common in public repositories, probably because there are not many and easily applicable taxonomic characters to distinguish T. violaceus and T. gracilis .…”

“…The total COI region sequences were submitted to GenBank using Basic Local Alignment Search Tool (BLAST) [ 39 ] and were then aligned with highly similar sequences using the Clustal W algorithm implemented in MEGA v7 ( , accessed on 29 January 2021) [ 40 ]. COI sequences were also compared to available barcode records in BOLD, using the identification engine BOLD-IDS, with the option “COI Full Database”, as described by Agus et al [ 41 ]. Sequences derived from a complete mitochondrial genome (AB158363), attributed to the species Octopus vulgaris , were used as an outgroup for both COI.…”