Phyllidia coelestis Bergh, 1905

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 : 595-596

Phyllidia coelestis Bergh, 1905

Fifty specimens of Phyllidia coelestis (Fig. 4.2a–e) collected around Sulawesi exhibit the typical colouration as described by Brunckhorst (1993) and Yonow (2012). The animals all have three longitudinal black lines on the notum and a general bluish background that is granulated. The middle line is usually interrupted by yellow-capped tubercles, but these central tubercles are encircled by the median black line in only two of our 50 specimens, rendering the usually interrupted line into a continuous line (Phco18Po1, Fig. 4.2b). The median line is divided by an anterior tubercle, thus forming the species-specific Y-shaped black marking in front of the rhinophores. The lateral black lines do not unite in either the anterior or posterior parts of the body. Other large and usually single rounded tubercles, capped by yellow, lie on blue ridges between the black lines. The tubercles lying laterally are smaller and can be yellow-capped or bluish; additionally, there are tiny black flecks in this lateral blue area. The species always lacks a black line on the foot. One pale specimen has yellow tubercles protruding only from the central black line (Phco15Bu16, Fig. 4.2d). There was only one interesting and different specimen within the P. coelestis clade: one with the identifier Phpic16Sa2 (Fig. 4.2e) differs in its black background colouration, resembling Phyllidia madangensis Brunckhorst, 1993 . A dark form of P. coelestis was described and illustrated from Australia in Yonow (2011): externally they differed in having a completely black central area with three rows of isolated, orange-tipped tubercles, and differences in the internal anatomy from the typical P. coelestis . Some individuals of P. coelestis with darker colouration are illustrated on Sea Slug Forum ( Cobb & Rudman, 2008) and GBIF (2019); however, it should be emphasised that these refer only to externally identified individuals. Nonetheless, 50 specimens of P. coelestis from Sulawesi are included in our molecular analyses with further eight sequences retrieved from NCBI from Lizard Island, Australia ( Cheney et al., 2014), and New Caledonia ( Valdés, 2003) which clearly group with our sequences with a bootstrap value of 100 (concatenated tree, Figs. 13 , S 1 ). Intraspecific variability lies within 2%. Phyllidia coelestis and P. elegans form a monophyletic clade supported by a bootstrap value of 99.

Metabolomic analysis of P. coelestis and P. elegans specimens collected in Bunaken National Park in 2015 demonstrated very similar characteristic chemotypes predominant in these two species (Fig. S7a, b), thus supporting their sister-taxa relationship (bootstrap in the concatenated data set is 99). HR-LCMS analysis of crude extracts revealed a series of major peaks with m/z values that can be assigned to amphilectene-type diterpene isonitriles (m/z [M + H] + 298.250), diisonitriles (m/z [M + H] + 325.259), and formamides (m/z [M + H] + 316.261) (Fig. S4a) that were recently reported from two Hainan P. coelestis populations ( Carbone et al., 2019). The amphilectene diterpenes detected in P. elegans and P. coelestis form a separate cluster in molecular networks ( Figs. 16 , S2, S3). Metabolomic analysis using GNPS tools also facilitated an intriguing detection of dichloroimidic sesquiterpenes in P. elegans that were characterised by us previously ( Bogdanov et al., 2020). Even though present in trace amounts, dichloroimidic sesquiterpenes can provide a chemotaxonomic separation between the two closely related species P. coelestis and P. elegans . Two specimens (Phco15Bu6, Phel15Bu8) among the chemically analysed specimens of both species possess an additional unknown metabolite with an m/z 404.187 [M + H] + and a retention time of 13.9 min. Interestingly, the same metabolite is detected in only one specimen of Phyllidiella sp. c subclade 2 (Phpu15Bu27). Minute extract amounts prevented us from isolating and characterising this compound.

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.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