High pheromone diversity in the male cheek gland of the red-spotted newt Notophthalmus viridescens (Salamandridae)

Sunita Janssenswillen, Bert Willaert, Dag Treer, Wim Vandebergh, Franky Bossuyt, Ines Van Bocxlaer

Male salamanders (Urodela) often make use of pheromones that are produced in sexually dimorphic glands to persuade the female into courtship and mating. The mental gland of lungless salamanders (Plethodontidae) and dorsal cloacal glands (or abdominal glands) of newts (Salamandridae) have been particularly well studied in that respect. In both families, sodefrin precursor-like factor (SPF) proteins have been identified as major components of the courtship pheromone system. However, similar to plethodontids, some newts also make use of subtle head glands during courtship, but few pheromones have been characterized from such structures. Males of red-spotted newts (Notophthalmus viridescens, Salamandridae) have both cloacal and cheek (genial) glands, and are known to apply secretions to the female's nose by both tail-fanning and cheek-rubbing. Here we combined transcriptomic and phylogenetic analyses to investigate the presence, diversity and evolution of SPF proteins in the cloacal and cheek glands of this species. Our analyses indicate that the cheek glands of male N. viridescens produce a similar amount and diversity of SPF isoforms as the cloacal glands in this species. Expression in other tissues was much lower, suggesting that both male-specific courtship glands secrete SPF pheromones during courtship. Our phylogenetic analyses show that N. viridescens expresses a combination of isoforms that stem from four highly diverged evolutionary lineages of SPF variants, that together form a basis for the broad diversity of SPF precursors in the breeding glands. The similar SPF expression of cheek and cloacal glands suggests that this protein family is used for pheromone signalling through cheek rubbing in the red-spotted newt. Since several male salamandrids in other genera have comparable head glands, SPF application via other glands than the cloacal glands may be more widespread than currently appreciated in salamandrids.