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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.

Animal sex pheromone systems often exist as multicomponent signals [1–11] to which chemical cues have been added over evolutionary time. Little is known on why and how additional molecules become recruited and conserved in an already functional pheromone system. Here, we investigated the evolutionary trajectory of a series of 15 kDa proteins—termed persuasins—that were co-opted more recently alongside the ancient sodefrin precursor-like factor (SPF) courtship pheromone system in salamanders [9, 12]. Expression, genomic, and molecular phylogenetic analyses show that persuasins originated from a gene that is expressed as a multi-domain protein in internal organs where it has no pheromone function but underwent gene duplication and neofunctionalization. The subsequent evolution combined domain loss and the introduction of a proteolytic cleavage site in the duplicated gene to give rise to two-domain cysteine rich proteins with structural properties similar to SPF pheromones [12]. An expression shift to the pheromone-producing glands, where expression of persuasins was immediately spatiotemporally synchronized with the already available pheromone system, completed the birth of a new pheromone. Electrostatic forces between members of both protein families likely enhance co-localization and simultaneous activation of different female olfactory neurons, explaining why persuasins immediately had a selective advantage. In line with this, behavioral assays show that persuasins increase female receptivity on their own but also exert a cumulative or synergistic effect in combination with SPF, clearly reinforcing the pheromone system as a whole. Our study reveals molecular remodeling of an existing protein architecture as an evolutionary mechanism for functional reinforcement of animal pheromone systems. Little is known about how additional molecules are recruited to functional pheromone systems. Maex et al. show how a new salamander courtship pheromone, persuasin, was co-opted alongside an ancient pheromone after a gene duplication and shift in expression. Subsequent evolution led the molecule along a pathway that reinforced the existing pheromone.

In animals that use chemical communication during courtship and reproduction, speciation is often associated with divergence of their sex pheromones. In multicomponent pheromone systems, divergence can be obtained either by adding or deleting components, or by altering the relative contribution of individual components to the mixture. Protein pheromone systems can additionally evolve by amino acid sequence divergence to produce pheromones with a species-specific effect. The sodefrin precursor-like factor (SPF) pheromone system, a blend of proteins that essentially enhances receptivity in salamanders, has had a long and dynamic evolution of gene duplications, but the mechanisms that govern interspecific divergence and the role they play in reproductive isolation remain elusive. Here, we use transcriptomics and proteomics to characterize the SPF protein repertoire of the alpine newt (Ichthyosaura alpestris), and compare it to the previously identified repertoire of SPF proteins of the palmate newt (Lissotriton helveticus), a related but nonhybridizing species. Subsequent phylogenetic analyses indicate that, despite the availability of multiple SPF gene copies, both species predominantly express the same subset of orthologs. Our study demonstrates that species specificity in the SPF protein pheromone system can be established by gradual sequence divergence of the same set of proteins alone.

Sodefrin, a decapeptide isolated from the male dorsal gland of the Japanese fire belly newt Cynops pyrrhogaster, was the first peptide pheromone identified from a vertebrate. The fire belly salamander and sodefrin have become a model for sex pheromone investigation in aquatically courting salamanders ever since. Subsequent studies in other salamanders identified SPF protein courtship pheromones of around 20 kDa belonging to the same gene-family. Although transcripts of these proteins could be PCR-amplified in Cynops, it is currently unknown whether they effectively use full-length SPF pheromones next to sodefrin. Here we combined transcriptomics, proteomics and phylogenetics to investigate SPF pheromone use in Cynops pyrrhogaster. Our data show that not sodefrin transcripts, but multiple SPF transcripts make up the majority of the expression profile in the dorsal gland of this newt. Proteome analyses of water in which a male has been courting confirm that this protein blend is effectively secreted and tail-fanned to the female. By combining phylogenetics and expression data, we show that independent evolutionary lineages of these SPF’s were already expressed in ancestral Cynops species before the origin of sodefrin. Extant Cynops species continue to use this multi-component pheromone system, consisting of various proteins in addition to a lineage-specific peptide.

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.

Throughout the animal kingdom, internal fertilization - the merging of sperm and egg inside the female body - nearly invariably relies on the use of a copulatory organ. In contrast, males of advanced salamanders (Salamandroidea) attain internal fertilization 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, since the majority does not display physical contact between the sexes and largely rely on females following the male track at spermatophore deposition. Although the use of pheromones has been widely assumed during their courtship, molecules able to induce the female following behaviour that culminates in insemination have not been identified. Here we show that uncleaved glycosylated SPF protein pheromones, secreted during courtship, are sufficient to elicit such behaviour in palmate newts (Lissotriton h. helveticus), indicating that these molecules obviate the need for copulation in these salamanders. Surprisingly, our finding of side-by-side secretion of Late Palaeozoic diverged proteins in a single species suggests that these molecules already had a courtship function in stem salamanders about 300 million years ago, rendering them one of the oldest vertebrate pheromone systems.

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

Crna čovječja ribica (Proteus anguinus parkelj Sket & Arntzen, 1994), ili „črni močeril“ na slovenskom, podvrsta je čovječje ribice čiji su predstavnici nađeni samo na dva susjedna izvora u području Bele Krajine, u jugoistočnoj Sloveniji, zapadno od Žumberka (Bela Krajina dobila je ime po brezama kojih je mnogo u tom kraju). Ime podvrste – parkelj – na slovenskom znači krampus.

Prevedeno iz djela: Gerhard J. (1988): Welche Libelle ist das? Die arten Mittel - und Sudeuropas. Kosmos, Stuttgart, pp. 191

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.