Phyllidiella sp.

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 : 611-615

Phyllidiella sp. c

Phyllidiella sp. c consists of two different subclades (subclades 1 and 2) which are not considered separate species according to the molecular phylogenetic analyses or the ABGD test based on the CO1 and 16S data sets. Nevertheless, these two clades are considered as two different species when applying bPTP tests (both maximum likelihood and Bayesian analyses). Moreover, they are morphologically different and therefore described below as two separate entities.

Phyllidiella sp. c subclade 1 comprises 32 specimens. The black pattern of the specimens consists of two longitudinal lines and one outer ring (Fig. 10.4a–d). The obvious margin is pink. The inner median lines unite with the outer one anteriorly in front of the rhinophores and posteriorly and are connected to each other by several black X-shaped marks. There is no black band between the rhinophores, and the tubercular clusters in front of and behind the rhinophores are continuous. Several pink compound tubercles lie between these black lines forming short ridges arranged in clusters in the central part of the notum and in the lateral parts. The

◂ Fig. 11 Phyllidiella species and specimens with identifiers. Scale bars: 10 mm. 1a–e Phyllidiella sp. c subclade 2: a Phpu18Ba12; b Phan15Bu3; c Phli16Sa5; d Phli18Ba1; e Phpu16Sa11. 2a–f Phyllidiella albonigra : a Phpu15Bu5; b Phpu16Bu8; c Phpu16Bu3; d Phpu15Bu21; e Phpu16Sa32; f Phpu17Ba4. 3a–d Phyllidiella sp. d: a Phpu18Po1; b Phpu18Ba1; c Phpu18Po3; d. Phpu18Bl5. 4a–d Phyllidiella species complex e: a Phpu15Bu28; b Phpu16Sa35; c Phpu15Bu39; d Phpu18Bl2

long pointed rhinophores are black with white to pink or red bases. The anal opening is on the black part of the notum. The hyponotum is grey with pink-white patches corresponding to the dorsal tubercles and ridges, the foot sole is white to grey and often with two distinct darker longitudinal lines (e.g., Phpu18Po4, Fig. 10.4d), and the oral tentacles are whitish pink with black along the lateral grooves. Some of these characters are similar to parts of the original description of P. granulata Brunckhorst, 1993 (type locality Guam), but future molecular analyses including specimens from Guam are needed .

All 24 specimens of Phyllidiella sp. c subclade 2 have one outer black ring and two lateral black lines (Fig. 11.1a–e), similar to subclade 1. The outer ring and the inner longitudinal lines unite anteriorly and posteriorly, similar to subclade 1. The inner lines also connect to each other transversely, forming two central tubercular areas, but these are H-shaped in subclade 2, and there is a black mark in the middle of these regions. The pink anal opening lies in a pink field, not in a black area as in subclade 1. There is a very obvious white–pink margin. In the central part of the notum, the compound pink tubercles are usually arranged in a circular form, with only four specimens exhibiting tubercles arranged in elongate, but short ridges interrupted by smooth black areas (Fig. 11.1e). The compound lateral tubercles are arranged in curved or straight series and separated by black lines. The rhinophores are predominantly black with a white base, but in Phpu18Ba12 (Fig. 11.1a), the rhinophores have a red base. The hyponotum is pink with grey patches caused by the dorsal colouration showing through, the gills are grey to black, the foot sole is white to pinkish grey, and the outer sides of the oral tentacles are black.

The specimens somewhat match the original description of P. melanocer a Yonow, 1986 from the Red Sea, but our specimens have a distinct black submarginal line. There are also vague similarities with P. annulata ( Gray, 1853; type locality French Polynesia), but this species was described with the absence of a white margin and an overall black colouration with a high number of pink rings (4–14) on the notum ( Brunckhorst, 1993). Re-investigated type material of P. annulata ( Fig. 3 a-e) indicates similarities to the description of the holotype and paratype (only) of P. melanocera (Fig. 2f) in all other respects. In Phyllidiella sp. c, all specimens from both subclades 1 and 2 lack the black stripe between the rhinophores that is characteristic for most Phyllidiella species, and therefore, the pale pink tubercular field is continuous. The anal opening is not in a black area as usual, but lies between two compound tubercles in a pink to grey field.

Molecular evidence, including haplotype network analyses ( Fig. 20 ), does not strongly separate P. sp. c subclade 2 from P. sp. c subclade 1. Both clades are united with a bootstrap support of 100. Species delimitation tests also reject subclade 2 as a separate species from subclade 1, except when applying bPTP algorithms. Within the network analysis based on CO1, only seven mutational steps are present between the two clades.

Four specimens of Phyllidiella sp. c subclade 1 (Phpu15Bu35, Fig. 10.4b; Phpu16Sa14, Phu16Sa37, Phpu16Sa92) and two members of subclade 2 (Phli16Sa5, Fig. 11.1c; Phpu15Bu27) were chemically investigated. Metabolomes found in both subclades exhibited a high degree of variation rendering P. sp. c “difficult” from a chemical point of view, thus reflecting the discrepancy when comparing morphological and molecular data (see Fig. S9j, k). The crude extracts contained, like many other phyllidiids, various sesquiterpenes with usual functionalities, i.e., isonitriles and formamides. We consider two major sesquiterpene isonitriles with retention times of 18.2 min and 18.4 min as a unifying chemical feature of subclade 1. Even though these metabolites are found in varying amounts in all four subclade 1 specimens, they could not be detected in the two specimens of subclade 2. Crude extracts of both specimens of P. sp. c subclade 2 had different chemical profiles (see Fig. S9k) which also differed from those of subclade 1 (Fig. S9j). Interestingly, the chemical profile of Phpu15Bu27 (subclade 2) was identical with those found in one specimen of Phyllidia coelestis (Phco15Bu6) and one specimen of Phyllidia elegans (Phel15Bu8) (see Fig. S10). All three specimens did not contain the usually observed sesquiterpenes, but several amphilectene-type diterpenes with an unknown major metabolite with an m/z 404.187 [M+ H] +. In contrast, the extract of the other Phyllidiella sp. c subclade 2 specimen (Phli16Sa5) is composed of sesquiterpene isonitriles and corresponding formamides with a major compound eluting at 18.1 min in LCMS analysis. This compound is probably 7-isocyano- 7,8-dihydrobisabolene, also isolated from a P. krempfi specimen (Phpu16Sa66). Both Phyllidiella sp. c subclades seem to lack obvious chemotaxonomic markers other than individual isonitrile compositions, and thus, they are difficult to clearly separate from other phyllidiids. This high degree of variability makes chemical characterisation of Phyllidiella sp. c on various species delimitation tests. One number indicates that all species delimitation tests showed the same result on species numbers; four numbers indicate the species calculated according to (1) ABGD test based on CO1, (2) ABGD test based on 16S, (3) ML bPTP, and (4) Bayesian bPTP tests based on the concatenated data sets Phyllidiidae ( Nudibranchia,Heterobranchia , Gastropoda):an integrative taxonomic approach… 615

subclade level, as well as on species level, inconclusive and does not contribute to a taxonomic solution.

Figures

Fig. 3 Type and museum voucher material of Phyllidiella and Phyllidiopsis: a Phyllidiella annulata non-type C159519; b Phyllidiella annulata non-type C159520; c Phyllidiella annulata non-type C159520b; d Phyllidiella annulata non-type C159522; e Phylli-

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