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Chemical communication is an important aspect of courtship and reproduction in salamanders ( Urodela ), where males secrete several protein pheromones from various sexually dimorphic glands. The most widely used sex pheromone system consists of proteins of the Sodefrin Precursor-like Factor (SPF) family. This protein family already started diversifying through gene duplications in the Late Palaeozoic and continued to do so in various urodelan lineages. As a result, males of multiple extant salamander species secrete different sets or ratios of SPF protein pheromones which, as shown in behavioral tests, can evoke various female responses during the courtship process, such as following behavior, cloacal gaping, or an acceleration of courtship in general. Still, all observable effects essentially are a consequence of female receptivity enhancement, indicating a preserved role for SPF pheromones. Salamandridae is a large family with currently 119 described species that show a considerable variety in courtship behaviour. In this family, different lineages evolved various modes of pheromone transfer, ranging from direct application during amplexus on land, to an indirect transfer in which males abandoned physical contact and instead tail-fan pheromones underwater toward a nearby female. In one genus, an additional decapeptide pheromone, sodefrin, originated by cleavage from a precursor of the SPF family and was co-opted alongside uncleaved SPF protein pheromones. Here we discuss our current understanding and knowledge gaps on the use of the SPF pheromone system in salamandrids, and we define some testable hypotheses on its evolution in relation to changes in mating habitat and courtship modes.

Background 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. Results 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. Conclusions 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. Electronic supplementary material The online version of this article (doi:10.1186/s12862-015-0333-1) contains supplementary material, which is available to authorized users.

Males of the advanced salamanders (Salamandroidea) attain internal fertilization without a copulatory organ by depositing a spermatophore on the substrate in the environment, which females subsequently take up with their cloaca. The aquatically reproducing modern Eurasian newts (Salamandridae) have taken this to extremes, because most species do not display close physical contact during courtship, but instead largely rely on females following the male track at spermatophore deposition. Although pheromones have been widely assumed to represent an important aspect of male courtship, molecules able to induce the female following behaviour that is the prelude for successful insemination have not yet been identified. Here, we show that uncleaved sodefrin precursor-like factor (SPF) protein pheromones are sufficient to elicit such behaviour in female palmate newts ( Lissotriton helveticus ). Combined transcriptomic and proteomic evidence shows that males simultaneously tail-fan multiple ca 20 kDa glycosylated SPF proteins during courtship. Notably, molecular dating estimates show that the diversification of these proteins already started in the late Palaeozoic, about 300 million years ago. Our study thus not only extends the use of uncleaved SPF proteins outside terrestrially reproducing plethodontid salamanders, but also reveals one of the oldest vertebrate pheromone systems.

Sex pheromones form an important facet of reproductive strategies in many organisms throughout the Animal Kingdom. One of the oldest known sex pheromones in vertebrates are proteins of the Sodefrin Precursor-like Factor (SPF) system, which already had a courtship function in early salamanders. The subsequent evolution of salamanders is characterized by a diversification in courtship and reproduction, but little is known on how the SPF pheromone system diversified in relation to changing courtship strategies. Here we combined transcriptomic, genomic and phylogenetic analyses to investigate the evolution of the SPF pheromone system in nine salamandrid species with distinct courtship displays. First we show that SPF originated from vertebrate three-finger proteins and diversified through multiple gene duplications in salamanders, while remaining a single copy in frogs. Next, we demonstrate that tail-fanning newts have retained a high phylogenetic diversity of SPFs, whereas loss of tail-fanning has been associated with a reduced importance or loss of SPF expression in the cloacal region. Finally, we show that the attractant decapeptide sodefrin is cleaved from larger SPF precursors that originated by a 62 base pair insertion and consequent frameshift in an ancestral Cynops lineage. This led to the birth of a new decapeptide that rapidly evolved a pheromone function independently from uncleaved proteins.

Males of many frog species develop spiny nuptial pads with underlying glands on their thumbs during the mating period. We used 3D visualization on the European common frog Rana temporaria to show that the morphology of these glands allows the channelling of secreted molecules to the pad’s surface during amplexus. Combined transcriptome and proteome analyses show that proteins of the Ly-6/uPAR family, here termed amplexins, are highly expressed in the nuptial glands during the mating season, but are totally absent outside that period. The function of amplexins remains unknown, but it is interesting to note that they share structural similarities with plethodontid modulating factors, proteins that influence courtship duration in salamanders. Willaert, Bert Bossuyt, Franky Janssenswillen, Sunita Adriaens, Dominique Baggerman, Geert Matthijs, Severine Pauwels, Elin Proost, Paul Raepsaet, Arent Schoofs, Liliane Stegen, Gwij Treer, Dag Van Hoorebeke, Luc Vandebergh, Wim Van Bocxlaer, Ines eng Research Support, Non-U.S. Gov’t England 2013/08/21 06:00 J Exp Biol. 2013 Nov 15;216(Pt 22):4139-43. doi: 10.1242/jeb.086363. Epub 2013 Aug 15. keywords: *Animal Communication Animals Anura/metabolism/*physiology Chromatography, High Pressure Liquid Forelimb/*secretion Gene Library Histological Techniques Mass Spectrometry Polymerase Chain Reaction Sex Attractants/genetics/*secretion *Sex Characteristics Species Specificity Urodela/metabolism X-Ray Microtomography ispartof: Journal of Experimental Biology vol:216 issue:22 pages:4139-4143 ispartof: location:England status: published

Internal fertilization without copulation or prolonged physical contact is a rare reproductive mode among vertebrates. In many newts (Salamandridae), the male deposits a spermatophore on the substrate in the water, which the female subsequently takes up with her cloaca. Because such an insemination requires intense coordination of both sexes, male newts have evolved a courtship display, essentially consisting of sending pheromones under water by tail-fanning towards their potential partner. Behavioral experiments until now mostly focused on an attractant function, i.e. showing that olfactory cues are able to bring both sexes together. However, since males start their display only after an initial contact phase, courtship pheromones are expected to have an alternative function. Here we developed a series of intraspecific and interspecific two-female experiments with alpine newt (Ichthyosaura alpestris) and palmate newt (Lissotriton helveticus) females, comparing behavior in male courtship water and control water. We show that male olfactory cues emitted during tail-fanning are pheromones that can induce all typical features of natural female mating behavior. Interestingly, females exposed to male pheromones of their own species show indiscriminate mating responses to conspecific and heterospecific females, indicating that visual cues are subordinate to olfactory cues during courtship.