Titel Deelnemers "Korte inhoud" "Exaptation as a Mechanism for Functional Reinforcement of an Animal Pheromone System" "Margo Maex, Dag Treer, Henri De Greve, Paul Proost, Ines Van Bocxlaer, Franky Bossuyt" "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." "Origin and diversification of a salamander sex pheromone system" "Sunita Leentje Janssenswillen, Wim Vandebergh, Dag Treer, Bert Willaert, Margo Maex, Ines Van Bocxlaer, Franky Bossuyt" "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." "Beyond sodefrin: evidence for a multi-component pheromone system in the model newt Cynops pyrrhogaster (Salamandridae)." "Ines Van Bocxlaer, Margo Maex, Dag Treer, Sunita Leentje Janssenswillen, Rik Janssens, Wim Vandebergh, Franky Bossuyt" "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." "Chemistry Between Salamanders: Evolution of the SPF Courtship Pheromone System in Salamandridae" "Margo Maex, Lisa Maria Schulte, Ines Van Bocxlaer, Sunita Leentje Janssenswillen" "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." "Love Is Blind: Indiscriminate Female Mating Responses to Male Courtship Pheromones in Newts (Salamandridae)" "Dag Treer, Ines Van Bocxlaer, Severine Matthijs, Dimitri Du Four, Sunita Leentje Janssenswillen, Bert Willaert, Franky Bossuyt" "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." "Side-by-side secretion of Late Palaeozoic diverged courtship pheromones in an aquatic salamander" "Ines Van Bocxlaer, Dag Treer, Margo Maex, Wim Vandebergh, Sunita Leentje Janssenswillen, Gwij Stegen, Philippe Kok, Bert Willaert, Severine Matthijs, Anneleen Mortier, Henri De Greve, Franky Bossuyt" "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." "Love bites: male frogs (Plectrohyla, Hylidae) use teeth scratching to deliver sodefrin precursor-like factors to females during amplexus" "Lisa Maria Schulte, Geen naam beschikbaar, Raciel Cruz-Elizalde, Aurelio Ramirez-Bautista, Franky Bossuyt" "BACKGROUND: Efficient transfer of chemical signals is important for successful mating in many animal species. Multiple evolutionary lineages of animals evolved direct sex pheromone transmission during traumatic mating-the wounding of the partner with specialized devices-which helps to avoid signal loss to the environment. Although such direct transmission modes of so-called allohormone pheromones are well-documented in invertebrates, they are considered rare in vertebrates. Males of several species of the frog genus Plectrohyla (Hylidae, Anura) have elongated teeth and develop swollen lips during the breeding season. Here we investigated the possibility that these structures are used to scratch the females' skin and apply allohormone pheromones during traumatic mating in several Plectrohyla species.RESULTS: Our behavioural observations revealed that males press their upper jaw onto the females' dorsum during amplexus, leaving small skin scratches with their teeth. Histological examinations of the males' lips identified specialized mucus glands, resembling known amphibian pheromone glands. Whole-transcriptome sequencing of these breeding glands showed high expression of sodefrin precursor-like factor (SPF) proteins, which are known to have a pheromone function in multiple amphibian species.CONCLUSIONS: Our study suggests SPF delivery via traumatic mating in several anuran species: the males have specialized breeding glands in the lips for production and secretion and use their elongated teeth as wounding devices for application. We hypothesize that these SPF proteins end up in the females' circulatory system, where understanding their exact function will require further molecular, physiological and behavioural testing." "Side-by-side secretion of Late Palaeozoic diverged courtship pheromones in an aquatic salamander" "Paul Proost" "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."