Multiple Independent Recruitment of Sodefrin Precursor-Like Factors in Anuran Sexually Dimorphic Glands

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.

Love bites: male frogs (Plectrohyla, Hylidae) use teeth scratching to deliver sodefrin precursor-like factors to females during amplexus

“…The strong positive staining of the SMGs with Coomassie Brilliant Blue R-250 indicates a proteinaceous content of the glands, and the PAS-positive staining suggests that these proteins may contain carbohydrate chains. Combined, these results hint towards a potential glycoprotein allohormone pheromone content, as previously shown for other breeding glands [ 8 , 41 – 43 ].…”

“…In the lips of all three examined species, we identified specialized mucus glands (SMGs), a type of sexually dimorphic skin gland, that has been described in various amphibian species (including another Plectrohyla species [ 33 ]). In several anurans which bring their SMGs in direct contact with the female´s nares during mating [ 8 ] as well as in plethodontid salamanders known for transdermal transmission [ 40 ] these glands have been shown to produce proteins that are involved in chemical communication. In Plectrohyla the ducts of the SMGs exit through the epidermis onto the inner and outer lips (also see [ 33 ]).…”

“…Furthermore, a recent study showed that multiple families of both terrestrially and aquatically reproducing anurans also independently recruited SPF proteins, most likely for chemical sexual communication. The recruitment of this potential pheromone happened when the evolution of the male breeding gland location and courtship behavior allowed transmission of its secretion to the female’s nares [ 8 ]. In our study we compared the SPF proteins found in the Plectrohyla lip glands with those nasally delivering anurans as well as the same set of urodelan SPF sequences used before [ 8 ], in order to further estimate the evolution of chemical communication roles for SPF.…”

“…These kind of pheromones can be transferred directly to the nose of the receiver (e.g. during copulation in anurans [ 8 ]) or by wounding the mating partner with specialized devices (i.e. traumatic mating, traumatic secretion transfer [ 9 ]).…”

“…The chemical signals used during salamander courtship are typically protein pheromones, used for short-range communication (for an overview of the different types of amphibian pheromones see [ 22 – 24 ]). Although some of these proteins have been shown to have originated early in amphibian evolution and can be found among several urodele and anuran families [ 8 , 25 , 26 ], their transmission via traumatic mating using extra-genital wounding structures [ 9 ] is currently only known from these salamanders.…”