TY - JOUR
AU - , Van Bocxlaer, Ines
AB - Abstract Chemical signaling in animals often plays a central role in eliciting a variety of responses during reproductive interactions between males and females. One of the best-known vertebrate courtship pheromone systems is sodefrin precursor-like factors (SPFs), a family of two-domain three-finger proteins with a female-receptivity enhancing function, currently only known from salamanders. The oldest divergence between active components in a single salamander species dates back to the Late Paleozoic, indicating that these proteins potentially gained a pheromone function earlier in amphibian evolution. Here, we combined whole transcriptome sequencing, proteomics, histology, and molecular phylogenetics in a comparative approach to investigate SPF occurrence in male breeding glands across the evolutionary tree of anurans (frogs and toads). Our study shows that multiple families of both terrestrially and aquatically reproducing frogs have substantially increased expression levels of SPFs in male breeding glands. This suggests that multiple anuran lineages make use of SPFs to complement acoustic and visual sexual signaling during courtship. Comparative analyses show that anurans independently recruited these proteins each time the gland location on the male’s body allowed efficient transmission of the secretion to the female’s nares. chemical communication, Amphibia, behavioral evolution, sodefrin precursor-like factor Introduction Throughout the animal kingdom, sex pheromones play an essential role in eliciting a variety of responses during reproductive interactions between males and females. Being involved in mate location, evaluation, and copulatory synchronization, chemical communication can be one of the key criteria for sexual selection (Wyatt 2014). While long-range signals facilitate the encounter between potential mates, short-range signals (courtship pheromones) alter the mating behavior after initial contact (Costanzo and Monteiro 2007). In amphibians, the latter are particularly involved in courtship of salamanders, where several male protein pheromones have been characterized. For example, various protein pheromones (plethodontid receptivity factor, PRF; plethodontid modulating factor, PMF; sodefrin precursor-like factor, SPF) have been isolated from the mental gland of terrestrially mating lungless salamanders (Plethodontidae) (Rollmann et al. 1999; Houck et al. 2007, 2008). The chemicals secreted from these glands are transmitted from male to female either by direct contact with the nares or via skin abrasions (Arnold 1977; Houck et al. 1998; Rollmann et al. 2003). In Salamandridae, several related decapeptides (sodefrin and its variants) and two unrelated longer proteins (SPFs and persuasins) were demonstrated to have a pheromone function during courtship (Kikuyama et al. 1995; Van Bocxlaer et al. 2015; Maex et al. 2018). Males of aquatically courting species transmit these proteins, which are released from internal glands via the cloaca by tail-fanning (Janssenswillen et al. 2015). Interestingly, SPF proteins are shared by all salamander families in which pheromone communication was studied so far (Houck 2009; Janssenswillen et al. 2015; Van Bocxlaer et al. 2015; Maex et al. 2016). These proteins generally belong to two ancient clades of two-domain three-finger proteins (2 D-TFPs), termed alpha and beta, that originated through a Late Palaeozoic gene duplication (Van Bocxlaer et al. 2015). Behavioral tests measuring various aspects of female response behavior to proteins from both clades have shown that they essentially enhance the female’s receptivity, resulting in a reduction of the duration and/or latency of courtship (Park and Park 2002; Houck 2009; Janssenswillen et al. 2015; Van Bocxlaer et al. 2015; Janssenswillen and Bossuyt 2016; Maex et al. 2018). However, it is currently unknown whether this is the only effect, or whether females also can make choices by evaluating quantitative or qualitative properties of the pheromone mix. In addition to elevated expression in pheromone glands (where they are known as SPFs), 2 D-TFPs are also expressed in the eyes and regenerative tissues of salamanders (Maki et al. 2010; Campbell et al. 2011), and in various tissues of other vertebrates, where their roles are often unknown. Several 2 D-TFPs have been characterized as phospholipase inhibitors (PLIs) in snake blood, where they were postulated to function as a self-protecting agent against phospholipase A2 from their own venom, although they also occur in nonvenomous species (Dunn and Broady 2001; Fortes-Dias et al. 2017). Analysis of the Anolis carolinensis lizard genome revealed 19 2 D-TFP sequences in a single cluster (Janssenswillen et al. 2015), and several of these are expressed in male dewlap and testes, as well as in female ovaries, implying a function in reproduction (Eckalbar et al. 2013). The frog Silurana tropicalis has at least 26 2 D-TFP genes in its genome, some of which are expressed in the gastrula embryo stage (Amaya et al. 2006; Clark et al. 2016). However, despite the fact that 2 D-TFPs are expressed in a variety of tissues and several vertebrates have many copies of these genes, the functions of most of these remain unknown (Janssenswillen et al. 2015). The ancient divergence of the alpha and beta SPF clades that fulfill a pheromone function in extant salamanders opens the possibility for a similar role in other amphibians, or even other vertebrates, but involvement of these proteins in chemical signaling so far has not been detected. In contrast to salamanders, which are basically mute, the sister group of nearly 7,000 species of frogs and toads (Anura) are thought to predominantly use acoustic communication for sexual interactions (Starnberger et al. 2013). Evidence for sexual chemical signaling in this group is limited, and consists of molecules that assist in localization or attraction of potential mates (Wabnitz et al. 1999; Byrne and Keogh 2007) or compounds that may play a role in distinguishing conspecifics (Poth et al. 2012, 2013; Starnberger et al. 2013). Still, anurans display a wide variety of glands, many of which are sexually dimorphic and have been hypothesized to be involved in chemical communication (Thomas et al. 1993; Duellman et al. 1997; Brizzi et al. 2002, 2003; Brunetti et al. 2014), but the secretions from these glands remain largely uncharacterized. Here, we combined proteomics, histology, molecular phylogenetics, and whole transcriptome sequencing (RNAseq) across anurans to search for a potential wider utilization of SPFs in sexual chemical signaling. Since male frogs use different courtship or amplexus (i.e., the embrace of the female by the male) strategies to deliver the secretion of these glands to the female (table 1), we investigated sexually dimorphic breeding glands and microgland-containing skins from different male body parts (chin, fingers and thumbs, armpits, thighs, and multiple locations on the trunk) (supplementary fig. 1, Supplementary Material online). Table 1. Taxonomic Sampling, Sexually Dimorphic Glands, and Associated Courtship Behavior. Family Species Gland Position On Male Delivery to Female Associated Courtship Behavior Nyctibatrachidae Nyctibatrachus petraeus Thumbs Nares Nuptial pads are held near female nostrils, during straddle amplexus N. petraeus Thighs Back Unknown N. humayuni Thumbs Nares Nuptial pads are held near female nostrils, during straddle amplexus N. humayuni Thighs Back Unknown N. sanctipalustris Thumbs Armpits Axillary amplexus N. sanctipalustris Thighs Back Unknown N. grandis Thumbs Armpits Axillary amplexus Dicroglossidae Fejervarya goemchi Thumbs and fingers Armpits Axillary amplexus Bufonidae Bufo bufo Thumbs and fingers Armpits Axillary amplexus Hylidae Boana cinerascens Chin Nares Male rubs mental gland over female nares during courtship and amplexus Hyloscirtus phyllognatus Chin Nares Unknown, related species H. mashpi rubs mental gland over female nares Dendrosophus sarayacuensis Breast Back Axillary amplexus Ranidae Pelophylax esculentus Thumbs and fingers Armpits Axillary amplexus Rhacophoridae Pseudophilautus amboli Ventral skin Back Unknown Pipidae Hymenochirus boettgeri Axilla Nares Subaquatic waving with hind limbs to the female Bombinatoridae Bombina orientalis Thumbs and fingers Groin Inguinal amplexus Ranixalidae Indirana chiravasi Thighs Back Unknown I. chiravasi Thumbs Groin Inguinal amplexus Micrixalidae Micrixalus mallani Thumbs and fingers Armpits Axillary amplexus Microhylidae Phrynomantis microps Dorsal skin Unknown Unknown Family Species Gland Position On Male Delivery to Female Associated Courtship Behavior Nyctibatrachidae Nyctibatrachus petraeus Thumbs Nares Nuptial pads are held near female nostrils, during straddle amplexus N. petraeus Thighs Back Unknown N. humayuni Thumbs Nares Nuptial pads are held near female nostrils, during straddle amplexus N. humayuni Thighs Back Unknown N. sanctipalustris Thumbs Armpits Axillary amplexus N. sanctipalustris Thighs Back Unknown N. grandis Thumbs Armpits Axillary amplexus Dicroglossidae Fejervarya goemchi Thumbs and fingers Armpits Axillary amplexus Bufonidae Bufo bufo Thumbs and fingers Armpits Axillary amplexus Hylidae Boana cinerascens Chin Nares Male rubs mental gland over female nares during courtship and amplexus Hyloscirtus phyllognatus Chin Nares Unknown, related species H. mashpi rubs mental gland over female nares Dendrosophus sarayacuensis Breast Back Axillary amplexus Ranidae Pelophylax esculentus Thumbs and fingers Armpits Axillary amplexus Rhacophoridae Pseudophilautus amboli Ventral skin Back Unknown Pipidae Hymenochirus boettgeri Axilla Nares Subaquatic waving with hind limbs to the female Bombinatoridae Bombina orientalis Thumbs and fingers Groin Inguinal amplexus Ranixalidae Indirana chiravasi Thighs Back Unknown I. chiravasi Thumbs Groin Inguinal amplexus Micrixalidae Micrixalus mallani Thumbs and fingers Armpits Axillary amplexus Microhylidae Phrynomantis microps Dorsal skin Unknown Unknown Note.—Male gland position and delivery to the female are indicated. In absence of knowledge on behavior associated with transmission of the gland secretion, delivery to the female is indicated as the zone of contact between gland and female. Open in new tab Table 1. Taxonomic Sampling, Sexually Dimorphic Glands, and Associated Courtship Behavior. Family Species Gland Position On Male Delivery to Female Associated Courtship Behavior Nyctibatrachidae Nyctibatrachus petraeus Thumbs Nares Nuptial pads are held near female nostrils, during straddle amplexus N. petraeus Thighs Back Unknown N. humayuni Thumbs Nares Nuptial pads are held near female nostrils, during straddle amplexus N. humayuni Thighs Back Unknown N. sanctipalustris Thumbs Armpits Axillary amplexus N. sanctipalustris Thighs Back Unknown N. grandis Thumbs Armpits Axillary amplexus Dicroglossidae Fejervarya goemchi Thumbs and fingers Armpits Axillary amplexus Bufonidae Bufo bufo Thumbs and fingers Armpits Axillary amplexus Hylidae Boana cinerascens Chin Nares Male rubs mental gland over female nares during courtship and amplexus Hyloscirtus phyllognatus Chin Nares Unknown, related species H. mashpi rubs mental gland over female nares Dendrosophus sarayacuensis Breast Back Axillary amplexus Ranidae Pelophylax esculentus Thumbs and fingers Armpits Axillary amplexus Rhacophoridae Pseudophilautus amboli Ventral skin Back Unknown Pipidae Hymenochirus boettgeri Axilla Nares Subaquatic waving with hind limbs to the female Bombinatoridae Bombina orientalis Thumbs and fingers Groin Inguinal amplexus Ranixalidae Indirana chiravasi Thighs Back Unknown I. chiravasi Thumbs Groin Inguinal amplexus Micrixalidae Micrixalus mallani Thumbs and fingers Armpits Axillary amplexus Microhylidae Phrynomantis microps Dorsal skin Unknown Unknown Family Species Gland Position On Male Delivery to Female Associated Courtship Behavior Nyctibatrachidae Nyctibatrachus petraeus Thumbs Nares Nuptial pads are held near female nostrils, during straddle amplexus N. petraeus Thighs Back Unknown N. humayuni Thumbs Nares Nuptial pads are held near female nostrils, during straddle amplexus N. humayuni Thighs Back Unknown N. sanctipalustris Thumbs Armpits Axillary amplexus N. sanctipalustris Thighs Back Unknown N. grandis Thumbs Armpits Axillary amplexus Dicroglossidae Fejervarya goemchi Thumbs and fingers Armpits Axillary amplexus Bufonidae Bufo bufo Thumbs and fingers Armpits Axillary amplexus Hylidae Boana cinerascens Chin Nares Male rubs mental gland over female nares during courtship and amplexus Hyloscirtus phyllognatus Chin Nares Unknown, related species H. mashpi rubs mental gland over female nares Dendrosophus sarayacuensis Breast Back Axillary amplexus Ranidae Pelophylax esculentus Thumbs and fingers Armpits Axillary amplexus Rhacophoridae Pseudophilautus amboli Ventral skin Back Unknown Pipidae Hymenochirus boettgeri Axilla Nares Subaquatic waving with hind limbs to the female Bombinatoridae Bombina orientalis Thumbs and fingers Groin Inguinal amplexus Ranixalidae Indirana chiravasi Thighs Back Unknown I. chiravasi Thumbs Groin Inguinal amplexus Micrixalidae Micrixalus mallani Thumbs and fingers Armpits Axillary amplexus Microhylidae Phrynomantis microps Dorsal skin Unknown Unknown Note.—Male gland position and delivery to the female are indicated. In absence of knowledge on behavior associated with transmission of the gland secretion, delivery to the female is indicated as the zone of contact between gland and female. Open in new tab Results and Discussion De Novo Transcriptome Assembly To gain insight into overall gene expression in anuran male sexually dimorphic glands, we conducted RNAseq for a wide variety of tissues (table 1). We generated over 3.0 billion reads in total (50–150 bp each) from males of 16 species, belonging to 11 families that represent a broad anuran phylogenetic diversity with divergence times up to over 200 Ma (Kumar et al. 2017). After quality-filtering and retaining transcripts with expression >2 transcripts per million (TPM), our assemblies contained between 10,368 and 55,055 transcripts (supplementary table 1, Supplementary Material online). Calculation of Metazoa Benchmarking Universal Single-Copy Orthologs (BUSCO) scores (Simão et al. 2015) using dammit v0.3.2 (Scott 2016) showed a high completeness of these assemblies (supplementary table 1, Supplementary Material online). RNAseq expression analyses indicate that anuran sexually dimorphic glands are often highly specialized organs, which express a relatively small set of genes, with 22–170 transcripts constituting 36–83% of the cumulative expression (see Materials and Methods and supplementary table 1, Supplementary Material online, for details). Comparison across Anuran Glands Housekeeping and ubiquitous (e.g., structural) genes, which are expressed in all tissues, can make up a significant proportion of the transcriptome and do not contribute to our search for molecules involved in chemical communication. To perform a principal component analysis (PCA) across frog families and glands, we excluded these transcripts and focused on genes with expression >0.1% (see Materials and Methods and supplementary table 1, Supplementary Material online, for details). These analyses reveal that the mRNA expression levels show remarkable similarities and contrasts across different taxa and gland types (fig. 1 and supplementary fig. 2 and table 2, Supplementary Material online). For example, glands on the thighs (femoral glands) share conserved expression patterns of two unrelated proteins without N-terminal signal peptide (fig. 1, N-methyltransferase and Steroid 17-alpha-hydroxylase; supplementary table 1, Supplementary Material online) across different anuran families (Nyctibatrachidae and Ranixalidae), despite their divergence ∼94 Ma (Kumar et al. 2017) (supplementary fig. 2, Supplementary Material online). Other glands mainly express proteins that have an N-terminal signal peptide (many of which thus likely are secreted) and several of them cluster by gland type (such as most nuptial pads on the thumbs and fingers), suggesting conserved functions (fig. 1 and supplementary fig. 2, Supplementary Material online). Intriguingly, four glands located on different body parts (chin, axilla, and thumbs), sampled from highly divergent evolutionary lineages (Pipidae, Hylidae, and Nyctibatrachidae, divergence ∼204 and 155 Ma, respectively, Kumar et al. 2017) show similar expression patterns (fig. 1 and supplementary fig. 2, oval, Supplementary Material online) that include highly elevated SPF expression, exceeding that of the highest expressed housekeeping gene (Elongation factor EF-1α 1) 9.3–45.1 times in these glands (supplementary table 1, Supplementary Material online). Mass spectrometry-based (MS) proteomics on gland secretion confirms that these proteins are abundantly secreted. In total, 72 (7-38 AA long) peptides aligning with the identified SPF precursor sequences were found, 36 of which were unique. The sequence coverage varied between 9% and 50%, being covered by one to ten peptides per sequence, some of which aligned with several sequences (supplementary fig. 3 and table 3, Supplementary Material online). Both anuran and salamander SPFs share a high number of cysteines in an evolutionary conserved pattern (fig. 2A), and most of them have similar theoretical masses of ∼20 kDa. Fig. 1. Open in new tabDownload slide Heatmap of gene expression in male sexually dimorphic glands. The analysis is based on the data shown in supplementary table S1, Supplementary Material online. Rows are centered; unit variance scaling is applied to rows. Both rows and columns are clustered using correlation distance and average linkage. The heatmap shows 13 gene families that were clustered based on their expression in the 18 tissues. Overall, similar glands show comparable expression patterns despite the deep divergence of the respective species. However, glands on different body parts, belonging to frog families (Hylidae, Nyctibatrachidae, and Pipidae) that diverged ∼204 and 155 Ma, respectively (Kumar et al. 2017) (supplementary fig. S2, Supplementary Material online, indicated with *), share an enhanced SPF expression (vertical column indicated with black line). Fig. 1. Open in new tabDownload slide Heatmap of gene expression in male sexually dimorphic glands. The analysis is based on the data shown in supplementary table S1, Supplementary Material online. Rows are centered; unit variance scaling is applied to rows. Both rows and columns are clustered using correlation distance and average linkage. The heatmap shows 13 gene families that were clustered based on their expression in the 18 tissues. Overall, similar glands show comparable expression patterns despite the deep divergence of the respective species. However, glands on different body parts, belonging to frog families (Hylidae, Nyctibatrachidae, and Pipidae) that diverged ∼204 and 155 Ma, respectively (Kumar et al. 2017) (supplementary fig. S2, Supplementary Material online, indicated with *), share an enhanced SPF expression (vertical column indicated with black line). Fig. 2. Open in new tabDownload slide Evolutionary relationships and structural similarities of SPF in frogs and salamanders. (A) Alignment of frog and salamander SPF transcripts shows a conserved cysteine pattern, suggesting a conserved tertiary structure and protein folding. Note that the typical terminal asparagine in the first TFP domain is missing in the precursor of Hymenochirus. (B) Bayesian consensus phylogram (−Ln L = 28,313.352; 249 aligned amino acids) of 2 D-TFP evolution in vertebrates. Supported nodes (Bayesian posterior probabilities >95 and/or bootstrap values >70) are indicated with a black square. Top-expressed transcripts from sexually dimorphic frog glands are in orange, well-established salamander alpha and beta pheromones are in blue. The sequences of the frog Silurana tropicalis are genomic sequences with largely unknown function or expression patterns. The transmission of secretion during frog courtship shows striking similarities with that salamanders, both in water (waving behavior creating a water current from the glands toward the female’s nares) and on land (rubbing of glands directly on or near female nares). Fig. 2. Open in new tabDownload slide Evolutionary relationships and structural similarities of SPF in frogs and salamanders. (A) Alignment of frog and salamander SPF transcripts shows a conserved cysteine pattern, suggesting a conserved tertiary structure and protein folding. Note that the typical terminal asparagine in the first TFP domain is missing in the precursor of Hymenochirus. (B) Bayesian consensus phylogram (−Ln L = 28,313.352; 249 aligned amino acids) of 2 D-TFP evolution in vertebrates. Supported nodes (Bayesian posterior probabilities >95 and/or bootstrap values >70) are indicated with a black square. Top-expressed transcripts from sexually dimorphic frog glands are in orange, well-established salamander alpha and beta pheromones are in blue. The sequences of the frog Silurana tropicalis are genomic sequences with largely unknown function or expression patterns. The transmission of secretion during frog courtship shows striking similarities with that salamanders, both in water (waving behavior creating a water current from the glands toward the female’s nares) and on land (rubbing of glands directly on or near female nares). Anuran SPFs Are Homologous with Salamander Pheromones Maximum likelihood and Bayesian phylogenetic analyses on vertebrate 2 D-TFPs (fig. 2B and supplementary table 4, Supplementary Material online) congruently imply multiple gene duplications before the last common ancestor of anurans and urodelans (∼297 Ma, Kumar et al. 2017). All analyses show that the top-expressed 2 D-TFPs of two hylid and two nyctibatrachid species (fig. 2B, orange) are most closely related to the salamander beta SPF clade and are nested among the whole of well-established salamander SPF pheromones (fig. 2B, blue). For a fifth species (a pipid), the highest expressed transcript belongs to a clade of proteins in which no urodelan pheromones have been detected so far, but show signs of abundant duplications in the S. tropicalis genome. Since anuran SPF transcripts are highly expressed in male sexually dimorphic breeding glands and for four of the five species are orthologous with salamander pheromones of the beta SPF clade, we propose similar roles for anuran and urodelan SPFs in chemical communication. Validation of this hypothesis will require behavioral or physiological evidence and will be a challenging task in future research. Independent Recruitment in Sexual Communication Because SPF proteins typically show enhanced expression in glands where they have a pheromone function (Van Bocxlaer et al. 2015; Maex et al. 2018), we used transcript expression levels as a proxy for estimating the probability that anuran SPFs will be able to induce female courtship responses across the anuran evolutionary tree (fig. 3A). Mapping of the SPF transcript expression relative to the most abundant housekeeping gene EF-1α 1 shows that elevated expression originated at least three times independently in different breeding glands of various frog lineages, and evolved in concert with modified courtship behavior that allows delivery or transmission to the female nares (fig. 3A and table 1). In hylid treefrogs, SPF expression was highly elevated in the mental glands. Males of the Boana punctatus group (B. cinerascens) and the Hyloscirtus bogotensis group (H. phyllognathus) apply these proteins by rubbing their mental gland onto the female’s nares, shortly before and during amplexus (Telles et al. 2013; Brunetti et al. 2014) (table 1, fig. 2B, andsupplementary movie S1, Supplementary Material online). In night frogs (Nyctibatrachidae), elevated expression was observed in the nuptial pads of two species (Nyctibatrachus petraeus and N. humayuni) that hold their fingers in the proximity of the female’s nares (table 1 and fig. 2B), but was not found in the sister group that retained axillary amplexus in which the nuptial pads remain firmly pressed under the female axilla (N. grandis and N. sanctipalustris) (table 1) (Biju et al. 2011; Willaert et al. 2016). Finally, elevated SPF expression was also observed in the postaxillary glands of the aquatically courting frog Hymenochirus boettgeri. These glands do not come into direct contact with the female during amplexus, but the males evolved a courtship dance (Pearl et al. 2000) in which they quickly shuffle with their hind limbs (Rabb and Rabb 1963), a behavior that may serve to wave molecules secreted from their postaxillary glands to the female’s nares (fig. 2B andsupplementary movie S2, Supplementary Material online). Overall, the terrestrial and aquatic delivery modes in anurans represent remarkable parallelisms to those observed in salamanders (fig. 2B). Fig. 3. Open in new tabDownload slide Recruitment of SPFs in male breeding glands across the anuran evolutionary tree. (A) Reconstruction of SPF expression in anuran breeding glands. For the three species for which data on two gland types were available, the femoral glands were omitted (lowest SPF expression, thus conservative in light of this study). The log2 scale visualizes transcript expression relative to the housekeeping gene EF-1α 1, with each color change on the scale representing a 2-fold change in expression. The inferred recruitment in each of the breeding glands (i.e., expression exceeds 9.3- to 45.1-fold that of the housekeeping gene, vs. expression is lower) is indicated in red on the phylogenetic tree. (B) The histological sections of three male frog breeding glands indicate that carbohydrates, such as glycoproteins, are produced in specialized mucous glands (SMGs; PAS-positive reaction) but not in specialized serous glands (SSGs). The density of SMGs correlates with the RNA-expression of SPFs, which in salamanders are known to be glycosylated. Fig. 3. Open in new tabDownload slide Recruitment of SPFs in male breeding glands across the anuran evolutionary tree. (A) Reconstruction of SPF expression in anuran breeding glands. For the three species for which data on two gland types were available, the femoral glands were omitted (lowest SPF expression, thus conservative in light of this study). The log2 scale visualizes transcript expression relative to the housekeeping gene EF-1α 1, with each color change on the scale representing a 2-fold change in expression. The inferred recruitment in each of the breeding glands (i.e., expression exceeds 9.3- to 45.1-fold that of the housekeeping gene, vs. expression is lower) is indicated in red on the phylogenetic tree. (B) The histological sections of three male frog breeding glands indicate that carbohydrates, such as glycoproteins, are produced in specialized mucous glands (SMGs; PAS-positive reaction) but not in specialized serous glands (SSGs). The density of SMGs correlates with the RNA-expression of SPFs, which in salamanders are known to be glycosylated. To understand the potential mechanism behind independent recruitment of SPFs in male macroglands, we compared their expression level with the relative abundance of sexually dimorphic microglands in the tissues. Such microglands can occur dispersed in the male skin (Thomas et al. 1993; Brizzi et al. 2002), but breeding glands are often a congregate of two types of sexually dimorphic microglands, specialized mucous glands (SMGs) and specialized serous glands (SSGs) (Thomas et al. 1993; Brunetti et al. 2012). We used histology to visualize microglands in glandular and adjacent tissues of three hylid species that show distinct SPF RNA-expression levels. Because SPF protein pheromones in salamanders have been shown to be glycosylated (Van Bocxlaer et al. 2015; Maex et al. 2016), we examined the gland content for carbohydrate macromolecules, such as glycoproteins, using the Periodic Acid Schiff (PAS) reaction. Our histological slides reveal that the presence of SMGs, which show morphological and histochemical similarities to the pheromone producing glands of salamanders by being densely packed with PAS positive secretory content (Sever 2017), is highly associated with SPF transcript expression. SMGs are present as sole microglands (H. phyllognathus) or in combination with SSGs (B. cinerascens) in breeding glands that show elevated expression, but are absent in adjacent skin and breeding glands lacking such expression (Dendrosophus sarayacuensis) (fig. 3B andsupplementary fig. 4, Supplementary Material online). This suggests that SPFs are produced and stored in SMGs, and when showing elevated expression, clustering of these microglands can result in SPF-producing breeding glands that may become effective in sexual communication each time courtship behavior allows efficient transmission to the female’s nares. Our study reveals that SPF proteins have the ability to dynamically gain substantially enhanced expression levels in male courtship glands. If an associated role in chemical communication would be confirmed, this would greatly expand the potential scale of courtship pheromone use in amphibians, or potentially even in other vertebrates. For example, these proteins may have been equally recruited for chemical sexual signaling in animals with reduced vision and/or vocal communication (e.g., caecilians: Wilkinson 2012), or in evolutionary lineages showing enhanced expression of their 2 D-TFP protein repertoire in sexually dimorphic structures (e.g., squamates: Garcia-Roa et al. 2017). Because 2 D-TFP proteins serve various biological functions (e.g., Campbell et al. 2011), both their corresponding genes and receptors could easily be retained during evolutionary periods where chemical sexual communication is less essential. When behavioral or morphological evolution creates the opportunity to exploit chemical signaling during courtship, the molecules do not have to originate de novo, but the available protein repertoire could be recruited again. SPF proteins thus may be surprisingly dynamic in their involvement in chemical sexual signaling. Materials and Methods Animal Collection and Sampling Animals were selected to represent a wide phylogenetic diversity as well as a broad diversity in sexually dimorphic breeding glands and associated behaviors. Data on reproduction and behavior (summarized in table 1) were taken from various sources in the literature (Duellman and Trueb 1986; Thomas et al. 1993; Pearl et al. 2000; Brizzi et al. 2002; Rabb and Rabb 1963; Biju et al. 2011; Brunetti et al. 2012, 2014; Willaert et al. 2013, 2016; Gaitonde and Giri 2014; AmphibiaWeb 2018; Frost 2018). Because pheromones often only show elevated expression during the breeding season, all frogs were either collected during this period in their natural habitats while calling, or kept under natural conditions in the laboratory and sampled during reproductive activity. The animals were killed with a 2% Lidocaine (Xilonest) spray and were either preserved in 10% formalin for histological examinations, or their glands were surgically removed and placed into RNAlater or acetylcholine chloride (for transcriptome sequencing or shotgun proteomics, respectively). After surgical removal, glands were placed into 1 ml RNAlater (Life Technologies) each, stored for 24 h at 4 °C, and subsequently at −20 °C until RNA isolation. For proteomic analyses, the surgically removed tissues/glands where placed for 1 h at 4 °C into an 1.48-mM acetylcholine chloride solution (Alfa Aegar; in 1 ml phosphate-buffered saline [PBS] with protease inhibitor [one cOmplete Mini Protease Inhibitor Cocktail tablet per 10 ml PBS]). After discarding the remaining tissue, the gland extract solutions were filtered through Minisart RC4 syringe filters and subsequently mixed with 9 ml 2% (v/v) acetonitrile (ACN) containing 0.1% (v/v) trifluoroacetic acid (TFA) and loaded with a syringe onto solid phase extraction cartridges (Sep-Pak C8 plus cartridge, 400 mg sorbent, Waters). Cartridges were wet beforehand with 8 ml 90% (v/v) ACN with 0.1% (v/v) TFA and equilibrated with 8 ml 2% (v/v) ACN with 0.1% (v/v) TFA. After loading the gland extracts, cartridges were washed using the equilibration buffer and wrapped into parafilm for storage at 4 °C. Whole Transcriptome Sequencing (RNAseq) and Assembly For each tissue, multiple specimens were pooled to minimize individual or temporal variation (supplementary table S1, Supplementary Material online). Total RNA was extracted using TRI Reagent (Sigma-Aldrich) and the RNeasy Plus Universal Mini Kit (Qiagen). De novo transcriptome sequencing was performed at DNAvision (Belgium), Xcelris (India), and Macrogen (Korea). A paired-end cDNA sequencing library (either 100 or 150 bp) was constructed using the Illumina TruSeq RNA sample preparation kit for sequencing on a Nextseq500, HiSeq2000, or HiSeq2500 platform (Illumina, San Diego, California). Raw reads have been deposited in the NCBI Sequence Read Archive (SRA) database under BioProject PRJNA534223. Adapter sequences and low-quality bases were trimmed using trim-galore v0.4.4. Both read pairs containing reads with an average quality score <5 (Phred33) and read pairs for which forward or reverse reads were trimmed to smaller than 25 nucleotides were discarded. Per sample, three paired de novo assemblies were constructed using Trinity v2.5.1 (Grabherr et al. 2011; Haas et al. 2013), indicating respectively no strand-specificity, RF strand-specificity, and FR strand-specificity. Although our RNA-sequencing reads are not strand-specific, the De Bruyn graphs in Trinity are less complicated when indicating strand-specificity (http://groups.google.com/forum/#! msg/trinityrnaseq-users/2SY_L7tKx88/Qj7hqBVTAQAJ), and often transcripts with longer open reading frames are built (Maex et al. 2018). By subsequently combining the three assemblies using the EvidentialGene “transcript to amino acid coding sequence” (tr2aacds) script (Gilbert 2010,, 2013), we reduced the over-assembly to a more accurate and concise assembly (Mamrot et al. 2017; Maex et al. 2018). This meta-assembly was further assessed by calculating Metazoa Benchmarking Universal Single-Copy Orthologs (BUSCO) scores (Simão et al. 2015) using dammit v0.3.2 (Scott, online ref.). For two samples, we also used data from cDNA libraries to confirm the full-length sequences (Hym. boettgeri postaxillary glands) and calculate approximate expression values of 2 D-TFPs (N. humayuni nuptial pads). For those species, total RNA was extracted using the TRI-reagent protocol (Sigma-Aldrich). The RNA extracts of the glands were sent to Advanced BiotekServices (San Diego, CA) for construction of a standard cDNA library with mammalian expression vector pCMVEXP. The Escherichia coli culture transformed with cDNA library plasmids was diluted with SOB medium and plated on LB/ampicillin plates. Colonies were randomly selected the next day. The CMV forward primer and TK pA reverse primer were used to amplify the inserts. The polymerase chain reaction (PCR) was performed using Taq DNA Polymerase (Fermentas) under the following conditions: 94 °C for 4 min, followed by 25 cycles of 94 °C for 40 s, 63 °C for 1 min, and 72 °C for 1 min, and a final extension step of 2 min at 72 °C. A total of 500 PCR products were purified (Wizard SV 96 PCR Clean-Up System, Promega), amplified with the CMV forward primer using the BigDye Terminator Sequencing Kit v.3.1 and sequenced on an ABI Prism 3100 automated sequencer (Applied Biosystems). Expression Analyses and PCA Sequences and motifs were identified using ExPASy (Gasteiger et al. 2003), SignalP (Petersen et al. 2011), InterProScan 5 (Jones et al. 2014), and RAPSearch2 (Zhao et al. 2012). Gene expression levels were calculated using RSEM 1.3.0. All transcript with a TPM value >1,000 were identified and catalogued using Blastx and Blastn against the vertebrate RefSeq protein database (www.ncbi.nlm.nih.gov/), PFAM domains were identified using dammit v0.3.2 (Scott, online ref.). Unidentified sequences with no obvious open reading frame were regarded as untranslated regions and were discarded from further analyses. PCA and visualization of heatmaps were done with ClustVis (Metsalu and Vilo 2015). To reduce the number of variables in the PCA, sequences belonging to ubiquitous (e.g., ribosomal proteins), structural (e.g., keratin), or housekeeping (e.g., EF-1α 1) genes were not included in the analysis (see supplementary table S1, Supplementary Material online, for overview). The cumulative TPM of transcripts with TPM >1,000 represents 36.2–82.8% of the total expression, and is thus a good representation of the major expression pattern per gland. RAPSearch2 was used to identify the housekeeping gene EF-1α 1 transcripts in each data set. The complete repertoire of 2 D-TFPs was re-evaluated by using tblastn and tblastx in Blast2Go (Götz et al. 2008) and by checking all transcripts (i.e., including lowly expressed) against a database of 2 D-TFPs (containing all vertebrate sequences used in the phylogeny). Total 2 D-TFP expression values were then calculated by adding up the TPM values of all identified 2 D-TFP transcripts and dividing them by the TPM value of the housekeeping gene EF-1α 1, resulting in the fold increase in expression relative to the housekeeping gene and allowing better comparison across different glands. Proteomics Gland extracts were eluted from the cartridges with 7.5 ml 90% (v/v) ACN containing 0.1% (v/v) TFA. About 400 µl aliquots of each individual were pooled and MS proteomics were conducted by the Proteomics Core Facilities of the VIB Medical Biotechnology Center, in order to identify proteins by their peptide homologies. Samples were diluted with 20 mM HEPES pH 8.0 to a final urea concentration of 4 M and proteins were predigested with Lysyl Endopeptidase (Wako) (1/250, w/w) for 4 h at 37 °C. Samples were then further diluted to 2 M urea and digested with Trypsin (Promega) (1/100, w/w) overnight at 37 °C. Peptides were purified on C18 Omix tips (Agilent) and injected for LC-MS/MS analysis on Q Exactive HF mass spectrometer. The MaxQuant software (Cox and Mann 2008; Tynova et al. 2016) was used to construct comprehensive lists of confident protein identifications. As a protein reference, a FASTA database of B. hypsiboas-specific proteins (containing 3,690 entries) obtained via RNA sequencing of the mental glands was used. MaxQuant (version 1.6.0.16) was used with default search settings including a false discovery rate set at 1% on both the peptide and protein levels. The mass tolerance for precursor and fragment ions was set to 4.5 and 20 ppm, respectively, during the main search. Enzyme specificity was set as C-terminal to arginine and lysine (trypsin), also allowing cleavage at arginine/lysine-proline bonds with a maximum of two missed cleavages. Carbamidomethylation of cysteine residues was set as a fixed modification and variable modifications were set to oxidation of methionine residues (to sulfoxides) and acetylation of protein N-termini. Only proteins with at least one unique or razor peptide were retained. iBAQ quantification done by MaxQuant was used to calculate the relative abundance of the different proteins. Histology Glands were fixed in formaldehyde and dehydrated in a graded series of ethanol (50–100%). Dehydration began in 70% ethanol for samples derived from ethanol-preserved specimens. After dehydration, samples were paraffin-embedded, cut at 5 μm using a rotary microtome, and mounted onto microscope slides. Sections were stained with Ehrlich’s haematoxylin eosin (H/E) stain for general cytology and histology, and with Periodic acid–Schiff (PAS) for the detection of mucins, glycogens, and neutral glycoproteins. Samples were examined with a Leica DMLB2microscope and images were captured with a Leica DFC320 camera and processed with LAS4.8.0. Molecular Phylogenetics For each of the frog tissues that showed enhanced SPF expression relative to the housekeeping gene EF-1α 1, we assembled the cumulative 90% of highest expressed amino acid sequences (GenBank accession numbers MK457705–MK457719). We combined this data set with the highest expressed pheromones of available salamander species of plethodontids, ambystomatids, salamandrids, and a representative set of genomic sequences of 2 D-TFPs from fish (outgroup), coelacanth, amniotes, and amphibians (supplementary table S4, Supplementary Material online). One domain TFPs are not closely related to 2 D-TFPs and were therefore not included in the analyses. Alignment of 2 D-TFPs was done in MAFFT version 7 using the L-INS-I method (Katoh and Standley 2013), and the part with excess of missing data in the posterior part of the alignment was trimmed. This resulted in a data matrix of 249 amino acids, for which Mr Bayes assigned the WAG model as best fitting the data. Likelihood analyses were run in PAUP* 4.0a (Swofford 1998) and Bayesian analyses were performed in MrBayes 3.2.232 (Ronquist et al. 2012) at the CIPRES Science Gateway v3.333 (Miller et al. 2010). Two parallel runs of four Markov chain Monte Carlo were executed for 10,000,000 generations, with trees sampled every 1,000th generation and the first 5,000 generations discarded as burn-in. Convergence of parallel runs was confirmed by split frequency SDs (<0.01) and potential scale reduction factors (approximating 1.0) for all model parameters. Adequate posterior sampling for each run was verified using Tracer 1.634, by examining the effective sampling sizes of all model parameters. Supplementary Material Supplementary data are available at Molecular Biology and Evolution online. Acknowledgments Research permits were obtained from the Servicio Nacional Forestal y de Fauna Silvestre Peru (SERFOR) in Lima (No. 069-2016-SERFOR/DGGSPFFS, export authorization No. 003051-SERFOR), ANB (No. ANB/BL-FF/V15-00104), and the State Forest Departments of Kerala and Maharashtra (No. NWL10-25421/2014, MSBB/A-27/232/12-13/D-22 (8)/Research/4543/2012-13, D.WL.CR-5/2009-10), and research was authorized by the ethical committee of the VUB (permit no. 15-AAA-2). Special thanks to Andy C. Barboza and Pablo J. Venegas (CORBIDI), Evan Twomey and Sonali Garg for help in the field, Francis Impens, Delphi Van Haver, and An Staes for help with the proteomics analyses, Tristram Wyatt for commenting on an earlier draft, Evan Twomey for proofreading the article, and Alejandro Arteaga for sharing the courtship video of H. mashpi. This work was supported by the European Research Council (ERC 204509 StG grant to F.B.), Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (FWO research project G020318N to F.B. and A.M.), the Strategic Research Program of the Vrije Universiteit Brussel (SRP30 to F.B., K.R., and I.V.B.), a Research Fellowship of the Deutsche Forschungsgemeinschaft (to L.M.S.), and a doctoral fellowship from FWO-Vlaanderen (11Q0616N to M.M.). Author Contributions F.B., L.M.S., and I.V.B. conceived the study. F.B., I.V.B., L.M.S., S.J.W., K.R., S.M., and S.D.B. sampled frogs and tissues. S.D.B. provided RNAseq data for Indian species. F.B., L.M.S., I.V.B., S.J.W., M.M., Y.V.D.P., and P.Y.N. performed RNAseq analyses. L.M.S. performed protein analyses. A.M. prepared histological slides. F.B. and I.V.B. performed phylogenetic analyses. F.B., L.M.S., and I.V.B. wrote, and all authors read and approved the article. 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TI - Multiple Independent Recruitment of Sodefrin Precursor-Like Factors in Anuran Sexually Dimorphic Glands
JF - Molecular Biology and Evolution
DO - 10.1093/molbev/msz115
DA - 2019-09-01
UR - https://www.deepdyve.com/lp/oxford-university-press/multiple-independent-recruitment-of-sodefrin-precursor-like-factors-in-bdqtGfcUbT
SP - 1921
VL - 36
IS - 9
DP - DeepDyve
ER -