Molecular confirmation of species status for the rare cephalaspidean Melanochlamys lorrainae (Rudman, 1968), and comparison with its sister species M. cylindrica Cheeseman, 1881

Colour polymorphisms, and phenotypic plasticity of traits such as shells and radulae, complicate the taxonomy of many invertebrate taxa. Recent field surveys in New Zealand yielded specimens of the white cephalaspidean opisthobranch Melanochlamys lorrainae (Rudman, 1968), not reported since the 1960s. We used an integrative approach to test whether M. lorrainae was a distinct species or an unpigmented morph of the more common black congener M. cylindrica Cheeseman, 1881. Molecular data were combined with analysis of shell morphology and observations of relative abundance and habitat preference for the two nominal species. Differences in the shape of the internal shell were con- gruent with body colour, but less extreme than previously reported. In field surveys, M. lorrainae was found on sand- or mudflats while M. cylindrica was primarily associated with rocky habitat and red algal turf. Molecular phylogenetic analyses were performed on three gene regions, the mitochondrial cytochrome c oxidase subunit I (COI) and large ribosomal subunit (16S) rRNA loci, and the nuclear large ribosomal subunit (28S) rRNA locus. In all analyses M. lorrainae and M. cylindrica were reciprocally monophyletic, with net genetic distances of 16.5% for COI, 2.4% for 16S and 0.9% for 28S, all comparable to distances between sister species of other cephalaspideans. The northeastern Pacific M. diomedea and an undescribed Australian species formed a clade with modest support in parsimony analysis of mtDNA, and strong support in Bayesian analysis of the nuclear 28S gene despite an unusually long branch for the Australian species. No strong phylogenetic affinity was detected between the New Zealand species and the other two Melanochlamys spp., which also differ in some morphological respects. Group mating via unilateral insemination is described for M. cylindrica.