Metabolome of the Phyllidiella pustulosa Species Complex (Nudibranchia, Heterobranchia, Gastropoda) Reveals Rare Dichloroimidic Sesquiterpene Derivatives from a Phylogenetically Distinct and Undescribed Clade

Phyllidiid nudibranchs are brightly colored gastropod mollusks, frequently encountered in coral reefs of the tropical Indo-Pacific. The lack of a protective shell is suggested to be compensated by toxic secondary metabolites that are sequestered from specific prey sponges. Our ongoing reconstruction of phyllidiid phylogeny using molecular data of more than 700 specimens, based on published data and newly collected specimens in various seasons and localities around North Sulawesi (Indonesia), demonstrates that Phyllidiella pustulosa is a species complex with at least seven well-supported clades. A metabolomic analysis of 52 specimens from all seven clades of P. pustulosa was performed. Secondary metabolite profiles were found to correlate with the phylogenetic study and not the prevailing food sponges as expected. GNPS molecular networking revealed a unique chemotype in clade 6. Detailed chemical analysis of a specimen from this chemically and genetically distinct P. pustulosa clade led to the identification of seven new sesquiterpenoids with a rare dichloroimidic moiety (1 and 4) and derivatives thereof (2, 3, 5-7). Our findings suggest that P. pustulosa clades should be raised to the species level.