Phyllidiella zeylanica, Kelaart, 1858

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 : 610

Phyllidiella zeylanica auctt.

Twenty-four specimens are grouped in a clade tentatively named Phyllidiella zeylanica auctt. These specimens (Fig. 10.1a–f) vary in their background colour from grey to pink. Many specimens have the tubercles arranged into longitudinal series; however, some specimens display single conical tubercles which can vary in size. A few have small, rounded, compound tubercles. The black lines can be arranged very differently, forming a reticulate pattern, but the pattern is basically a linear one surrounding larger central tubercular fields (Fig. 10.1a–f). The pink or white margin is followed by a black submarginal line. The rhinophores are black with white base. In contrast to that of many other species, the anal opening is large in all specimens, placed on grey to black dorsum, and surrounded by a ring of tubercles; it has a pink outer surface and black inner surface (Fig. 10.1e). By this unique character, it can be easily distinguished from Phyllidiella sp. a, which is characterised by a grey to black anal opening on a black area of the notum. The oral tentacles are white or pale pink with faded grey on the lateral grooves. Their outer appearance matches the description of Phyllidiella zeylanica of Brunckhorst (1993), but not the original description of P. zeylanica by Kelaart (1858: type locality Sri Lanka, figured by Eliot, 1906: pl. 52, fig. 10). The original description clearly states that there are two black rings and one central black stripe. The two rings are narrower than the central stripe with the outermost ring being the narrowest one. Burn (1970) subsequently described and illustrated specimens of P. zeylanica (sensu Kelaart) from Gulf of Kutch ( India) with three black circular rings on the notum. Phyllidiella zeylanica (sensu Kelaart) was also described and illustrated by Yonow (2012 and references therein) based on specimens from the Maldives, Seychelles, Thailand, and Chagos with two or three circular black lines. Therefore, P. zeylanica was certainly misidentified in several publications, including Brunckhorst (1993), Domínguez et al. (2007), and Gosliner et al. (2015, 2018).

Unfortunately, several species were synonymised with P. zeylanica by Brunckhorst (1993) based on a pattern of black lines that coalesce in the posterior part of the body and pink compound tubercles which form longitudinal ridges and these will now need careful re-examination in light of current results: P. catena ( Pruvot-Fol, 1956) , P. seriata ( Pruvot-Fol, 1957) , and P. empelia ( Yonow, 1984) . Phyllidiella catena (type locality Mayotte, Mauritius), and P. empelia (type locality Sri Lanka) are species only recorded from the Indian Ocean, while the type locality of P. seriata is not known. The animals in our clade are not like Phyllidiella empelia , varying in details such as the numbers of black rings around the lateral mantle (which may be an ontogenetic variation), whether or not the black lines are united around the anal opening, the position of the black line surrounding the rhinophores, the presence of a central black line or band, and the anterior tubercles directly surrounding the rhinophores which may form an angular rectangle-like line instead of a curved line. They also appear to differ significantly from Pruvot-Fol’s species and P. rosans ( Bergh, 1873) . Thus, we cannot assign this clade to any of the above species without molecular evidence of these nominal Indian Ocean species. We urgently need more investigations involving these species to clarify the difficult taxonomy of this group. Our molecular analyses render Phyllidiella zeylanica auctt. as sister taxon to Phyllidiella sp. a with a very low support value of 51 (concatenated data set; Figs. 13 , S 1 ). This result reflects the contradicting results of the single-gene analyses. A joined network analysis of Phyllidiella sp. a and P. zeylanica auctt. ( Fig. 19 ) also clearly shows the distinctiveness of these two species with 50 mutational steps separating them, despite their similar colourations.

Crude extracts of seven P. zeylanica auctt. specimens were analysed with LCMS. The chemotypes found showed a high degree of variation (see Fig. S 9g) with dominant peaks attributable to sesquiterpene isonitriles by mass spectrometry. Careful inspection of the feature-based molecular network pointed out a few features that were detected in all investigated P. zeylanica auctt. specimens, e.g., ions with m/z 262.16 (M + H) and retention times of 14.6 min and 15.8 min match the accurate masses of bicyclic unsaturated isothiocyanate sesquiterpenes axisothiocyanate-4 and 10-isothiocyanato-4,6-amorphadiene (Fig. S4a) reported from various sponges ( Adinolfi et al., 1977; Alvi et al., 1991). These features are very rare among the 70 chemically analysed phyllidiids and were detected in trace amounts in only five other specimens belonging to three genera, Phyllidia varicosa , Phyllidiopsis krempfi , Phyllidiopsis burni , and Phyllidiella sp. c subclade 2. Small specimen size and the resulting low extract amounts hindered us from isolation and unambiguous structure elucidation of the extract constituents.

Figures

Fig.13 Maximum likelihood tree of Phyllidiidae based on the concatenated data set of partial CO1 and 16S sequences. Terminal taxa on species level collapsed. Numbers at nodes are bootstrap values (1000 replicates). The genera are marked with the same colours as in Fig. S1. Numbers behind the species names indicate results of the

Fig. 1 Sampling locations in Sulawesi, Indonesia

Fig.19 Haplotype network of Phyllidiella sp. a (29 sequences, white circles) and Phyllidiella zeylanica auctt. (24 sequences, grey circles) based on the CO1 gene. Note the closer relationship of specimens with similar chemotypes, coloured orange and blue, respectively