Phyllidiella pustulosa ( Cuvier, 1804 )

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 : 604-607

Phyllidiella pustulosa ( Cuvier, 1804)

Our results strengthen the need to formulate adequate diagnostic characters for P. pustulosa , the type species of the genus Phyllidiella , as described and illustrated by Cuvier (1804). Only then can we characterise the other clades to distinguish them from the nominal species and solve the P. pustulosa “species complex” problem (see Stoffels et al., 2016; Bogdanov et al., 2020). All eight synonymies by Brunckhorst (1993) must be reconsidered in light of these results. However, of the type material of P. pustulosa , only one syntype is available that was selected as lectotype by Brunckhorst (1993: 49). Pruvot-Fol (1957) re-described the material of Phyllidiella pustulosa deposited by Cuvier in the Muséum nationale d’Histoire naturelle in Paris, as did Brunckhorst (1993), and our re-analysis of this lectotype did not reveal any further information. The single animal is completely white with no traces of any black pigment (Fig. 2e). Brunckhorst (1993) revised the genus and its species based on his material as well as some museum specimens. He described P. pustulosa as having an elliptical to oval shape, with a black dorsal background, rounded tubercles of various numbers grouped into clusters of two or three, and a pink margin. This description refers to the same set of characters previously described and illustrated by Cuvier (1804) and matches the majority of our specimens in the first Phyllidiella clade in our tree ( Figs. 13 , S 1 ), so we assign the nominal name P. pustulosa to this clade, comprising 74 specimens from our collection. Our animals all have a black background with a narrow white mantle margin (Fig. 9.1a–d). The tubercles are rather small and conical, and they usually cluster in two, three, or more in confined tubercular fields. Their colour can vary from pale pink to dark pink and even green. The rhinophores are black; the black anal opening lies in a black field. The hyponotum is grey, with pale pink patches, and the gills are dark grey-pink. The foot sole is a Phsan18Ba1; b Phsan18Ba2; c Phsan18Ba4. 3a Phyllidiopsis sphingis : a. Phsph15Bu1. 4a–c Phyllidiopsis shireenae : a 18LePhsh1; b Phsh16Sa2; c Phpi15Bu4

pale grey to white, as are the oral tentacles, which have a dark grey line along the lateral grooves. Regarding the structure of the tubercles, most of our specimens also match the type materials of Phyllidia nobilis Bergh, 1869 synonymised with P. pustulosa . Examination of the three

◂ Fig. 9 Phyllidiella species and specimens with identifiers. Scale bars: 10 mm. 1a–d Phyllidiella pustulosa : a Phpu15Bu8; b Phpu15Bu33; c Phpu18Ba12a; d Phpu17Ba2. 2a–d Phyllidiella cf. pustulosa : a Phpu18Ko7; b Phpu18Ba3; c Phpu18Ko8; d Phpu18Sm1. 3a–f Phyllidiella nigra : a Phpu15Bu7; b Phni18Po1; c Phpu18Ko9; d Phpu18Ba19; e Phpu17M3; f Phpu17Bu1. 4a–d Phyllidiella sp. a: a Phpu18Po2; b Phpu18Bu4; c Phpu16Sa9; d Phpu16Sa1

paralectotypes deposited in Copenhagen (NHMD-633656, Fig. 2b; NHMD-633657, Fig. 2c; NHMD-633658, Fig. 2d) revealed that the tubercles cluster in groups of three or more on a black background and the mantle has a white band along the rim. These specimens all appear to have median clusters with a clear separation from the lateral clusters. Examination of the lectotype of P. pustulosa deposited in Paris (MNHN, IM-2000–35147, Fig. 2e) also showed the same tubercular dorsal morphology as P. nobilis , but the black pigmentation is lost. Our material is here identified as the true P. pustulosa and groups with most other sequences retrieved from GenBank under the name P. pustulosa . The 94 concatenated sequences from our collection and GenBank cluster with a support value of 96 and are sister taxon to Phyllidiella cf. pustulosa with a bootstrap support value of 100 ( Figs. 13 , 14 , S 1 ). Intraspecific genetic variability is 9.45%.

Numerous chemical studies of P. pustulosa have been published ( Hirota et al., 1998; Jomori et al., 2015; Kassühlke et al., 1991; Lyakhova et al., 2010; Manzo et al., 2004; Okino et al., 1996; Sim et al., 2020; White et al., 2017; Wright, 2003) as well as our recent publication ( Bogdanov et al., 2020) highlighting the chemical variability that is found in nudibranchs commonly encountered and identified as P. pustulosa . Eight P. pustulosa specimens were utilised in this chemical analysis: LCMS revealed three main chemotype variants within the “true” P. pustulosa (see Fig. S9a–c). As expected from previous reports, all investigated specimens possess sesquiterpene isonitriles. The first chemotype is found in specimens Phpu15Bu14 and Phpu16Sa5 (from Bunaken National Park and Sangihe Island), both lying within the first branch of the clade. The major constituent 3-isocyanotheonellin was isolated from the crude extract of Phpu15Bu14 with its formamide derivative ( Gulavita et al., 1986), and the structures were confirmed with 1 H and 13 C NMR experiments. The second chemotype is characterised by having unidentified isonitriles in addition to 3-isocyanotheonellin and is shared by three specimens (Phpu16Sa3, Phpu16Sa4, Phpu16Sa6) collected in Sangihe Island and grouping in a subclade of P. pustulosa supported by a bootstrap value of 100. The third chemotype is found in specimens Phpu15Bu8 (Fig. 9.1a), Phpu15Bu9, and Phpu16Sa52 from different localities (Bunaken National Park and Sangihe Island) and also occurs in different subclades. Intriguingly, the observed chemotypes represent the subclades within P. pustulosa rather than the collection locality. Figure 18 illustrates the relationship between these clades within P. pustulosa in a network analysis. The clades are separated by a few shared mutations but also confirm the closer relationship of specimens sharing the same chemotype.

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. 14 Maximum likelihood tree of Phyllidiidae based on partial CO1 sequences. Terminal taxa on species level collapsed. Numbers at nodes are bootstrap values. The genera are marked with the same colours as in Fig. S1

Fig. 18 Haplotype network of Phyllidiella pustulosa (74 sequences, white circles) and P. cf. pustulosa (20 sequences, grey circles) based on the CO1 gene, with the four described chemotypes (coloured cir-