Phyllidiella albonigra Quoy & Gaimard, 1832

Papu, Adelfia, Bogdanov, AleXander, Bara, Robert, Kehraus, Stefan, König, Gabriele M., YonoW, Nathalie & Wägele, Heike, 2022, Phyllidiidae (Nudibranchia, Heterobranchia, Gastropoda): an integrative taxonomic approach including chemical analyses, Organisms Diversity & Evolution (New York, N. Y.) 22 (3), pp. 585-629 : 615

Phyllidiella albonigra Quoy & Gaimard, 1832

All nine specimens of Phyllidiella albonigra (Fig. 11.2a–f) are more elongated in shape than the other Phyllidiella clades, with rather pointed ends and a greater proportion of black background to the mantle than all other species except P. nigra , and a narrow pink, green, or white band along the mantle margin. The dense complex tubercles are small but compound, arranged in small clusters on the notum, and their colour varies from pink to green or grey. The rhinophores are black with a white base. The black anal opening arises on black background. This pattern most resembles the external appearance of the figured type material of P. albonigra (pl. 21, figs. 26, 27; type locality Tonga islands): the original description and drawing depict a specimen that is also rather black with irregular clusters of small tubercles. We therefore remove P. albonigra from synonymy with P. pustulosa (see Brunckhorst, 1993) and reinstate it here. Phyllidiella albonigra is clearly separate from other Phyllidiella species (bootstrap value of the clade is 100). Minimum genetic differences varied from 10.52 (from Phyllidiella sp. c, subclade 1) up to nearly 18% (from P. rudmani ; Table S6). Its sister-taxa relationship with Phyllidiella sp. c is also supported in the tree reconstruction based on the concatenated data set and CO1 (bootstrap value 99), but is not resolved in the 16S analysis. Haplotype analysis of this species, together with Phyllidiella sp. c, provides further evidence of its distinctiveness ( Fig. 20 ).

The chemical analysis of P. albonigra was described in detail in a recent publication (see Bogdanov et al., 2020 as Phyllidiella pustulosa clade 6). We were able to isolate the very rare dichloroimidic sesquiterpenes ( Figs. 16 , S4a) and their derivatives from one specimen (Phpu15Bu21, Fig. 11.2d) of this clade. These dichloroimids were identified as the major extract constituents in three out of four analysed specimens of Phyllidiella albonigra . Importantly, all chemically studied specimens were collected at different localities around North Sulawesi during the course of three years. Whereas the chemical profiles (UV chromatograms obtained during LCMS analysis) of two specimens (Phpu15Bu21, Fig. 11.2d; Phpu16Sa32, Fig. 11.2e) were identical, the chromatogram of a third specimen (Phpu17Ba4, Fig. 11.2f) was very different at first glance (see Fig. S9l). However, after obtaining pure compounds from the in-depth analysis, the different metabolomes could be explained by the presence of degradation products of the chemically unstable dichloroimids. Intriguingly, no dichloroimids or derivatives thereof could be detected in the crude extract of the fourth analysed specimen Phpu16Bu8 (Fig. 11.2b), which was smaller (30 mm) compared to the other three specimens (38–80 mm). Chemical analyses thus provide further evidence that P. albonigra is a separate species and can be distinguished from its closest relative Phyllidiella sp. c. ( Fig. 20 ).

Figures

Fig. 20 Haplotype network from concatenated sequences of Phyllidiella sp. c with subclade 1 (32 sequences, white circles), subclade 2 (24 sequences, light grey circles), and Phyllidiella albonigra (9

Fig.16 Phylogeny of Phyllidiidae and chemical analysis of 70 preserved specimens from three genera (Phyllidia, Phyllidiopsis, and Phyllidiella) and 19 species. a Coloured clusters were obtained with classical or feature-based molecular networking and depict families of secondary metabolites characteristic for certain clades, e.g., brominated alkaloids for the genus Phyllidia, amphilectene-type diterpenes for P. coelestis and P. elegans, kalihinol-type diterpenes for P. rudmani, as well less specific secondary metabolites (sesquiterpene isonitriles and derivatives) that were detected throughout the Phyllidiidae. The chemical structures were established either with NMR after compound isolation, or were assigned based on HR-LCMS/MS