Feldhoff, R. C. | Search bioRxiv

bioRxiv2022

Recurrent co-option and recombination of cytokine and three finger proteins in multiple reproductive tissues throughout salamander evolution

The proteomic composition of amphibian gametes is largely a molecular mystery, particularly for Urodeles (salamanders and newts) which have few genomic-scale resources. Lungless salamanders (family Plethodontidae) include approximately two thirds of all extant salamander species and are classic models of vertebrate mating behavior. As part of an extended, multi-stage courtship ritual, male plethodontid salamanders deliver rapidly evolving protein pheromones that modify female behavior and improve male reproductive success. Despite great interest in this set of pre-mating reproductive barriers, limited characterization of plethodontid gametes has prohibited investigation of post-mating pre-zygotic barriers such as sperm-egg recognition. In this study, we performed transcriptomic analyses of testis and ovary using long-read PacBio sequencing and proteomic analyses of sperm using mass spectrometry for two evolutionary divergent plethodontid species, Plethodon shermani and Desmognathus ocoee. In both species, many of the most abundant sperm proteins were paralogs of the courtship pheromones Plethodontid Receptivity Factor (PRF), Plethodontid Modulating Factor (PMF), and Sodefrin Precursor-like Factor (SPF). Sperm-specific paralogs of PMF and SPF are likely the most abundant secreted proteins in P. shermani and D. ocoee, respectively. In contrast, sperm PRF lacks a signal peptide and may be expressed in cytoplasm. PRF pheromone genes evolved independently multiple times through repeated gene duplication of sperm PRF genes and signal peptides recovered by recombination with PMF genes. Phylogenetic analysis of courtship pheromones and their sperm paralogs support that each protein family evolved for these two reproductive contexts at distinct evolutionary time points between 17 and 360 million years ago. As the first molecular characterization of salamander gametes, this study expands our knowledge of amphibian fertilization beyond frogs and provides novel insight into the evolutionary processes by which new, rapidly evolving reproductive proteins may evolve.

evolutionary biology↗

bioRxiv2018

An annual cycle of gene regulation in the red-legged salamander mental gland: from hypertrophy to expression of rapidly evolving pheromones

Cell differentiation is mediated by synchronized waves of coordinated expression for hundreds to thousands of genes, and must be an exquisitely regulated process to produce complex tissues and phenotypes. For many animal species, sexual selection has driven the development of elaborate male ornaments, requiring sex-specific differentiation pathways. One such male ornament is the pheromone-producing mental gland of the red-legged salamander (Plethodon shermani). Mental gland development follows an annual cycle of extreme hypertrophy, production of pheromones for the ~2 month mating season, and then complete resorption before repeating the process in the following year. At the peak of the mating season, the transcriptional and translational machinery of the mental gland are almost exclusively redirected to synthesis of many rapidly evolving pheromones. Of these pheromones, Plethodontid Modulating Factor (PMF) has experienced an unusual history of disjunctive evolution: following gene duplication, positive sexual selection has diversified the protein coding region while the untranslated regions have been conserved by purifying selection. However, the molecular underpinnings that bridge the processes of gland hypertrophy, pheromone synthesis, and disjunctive evolution remain to be determined and are the focus of the present investigation. Using Illumina sequencing, we prepared a de novo transcriptome of the mental gland at six stages of development. Differential expression analysis and immunohistochemistry revealed that the mental gland initially adopts a highly proliferative, almost tumor-like phenotype, followed by a rapid increase in pheromone mRNA and protein levels. One likely player in this transition is Cold Inducible RNA Binding Protein (CIRBP), which selectively and cooperatively binds the highly conserved PMF 3' UTR. CIRBP, along with other stress response proteins, have seemingly been co-opted to aid in mental gland development by helping to regulate pheromone synthesis. The P. shermani mental gland utilizes a complex system of transcriptional and post-transcriptional gene regulation to facilitate its hypertrophication and pheromone synthesis. The data support the evolutionary interplay of both coding and noncoding segments in rapid gene evolution, and necessitate study of the co-evolution of pheromone gene products along with their transcriptional and translational regulators. Additionally, the mental gland could be a powerful emerging model of regulated proliferation and subsequent resorption of a tissue, within the dermis, thus having potential links to skin cancer biology.

molecular biology↗