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.

Recurrent Duplication and Diversification of Acrosomal Fertilization Proteins in Abalone

Reproductive proteins mediating fertilization commonly exhibit rapid sequence diversification driven by positive selection. This pattern has been observed among nearly all taxonomic groups, including mammals, invertebrates, and plants, and is remarkable given the essential nature of the molecular interactions mediating fertilization. Gene duplication is another important mechanism that facilitates the generation of molecular novelty. Following duplication, paralogs may parse ancestral gene function (subfunctionalization) or acquire new roles (neofunctionalization). However, the contributions of duplication followed by sequence diversification to the molecular diversity of gamete recognition genes has been understudied in many models of fertilization. The marine gastropod mollusk abalone is a classic model for fertilization. Its two acrosomal proteins (lysin and sp18) are ancient gene duplicates with unique gamete recognition functions. Through detailed genomic and bioinformatic analyses we show how duplication events followed by sequence diversification has played an ongoing role in the evolution of abalone acrosomal proteins. The common ancestor of abalone had four members of its acrosomal protein family in a tandem gene array that repeatedly experienced positive selection. We find that both sp18 paralogs contain positively selected sites located in different regions of the paralogs, consistent with a subfunctionalization model where selection acted upon distinct binding interfaces in each paralog. Further, a more recent species-specific duplication of both lysin and sp18 in the European abalone H. tuberculata is described. Despite clade-specific acrosomal protein paralogs, there are no concomitant duplications of egg coat proteins in H. tuberculata, indicating that duplication of egg proteins per se is not responsible for retention of duplicated acrosomal proteins. We hypothesize that, in a manner analogous to host/pathogen evolution, sperm proteins are selected for increased diversity through extensive sequence divergence and recurrent duplication driven by conflict mechanisms.

Evolutionary rates of testes-expressed genes differ between monogamous and promiscuous Peromyscus species

Reproductive proteins, including those expressed in the testes, are among the fastest evolving proteins across the tree of life. Sexual selection on traits involved in sperm competition is thought to be a primary driver of testes gene evolution and is expected to differ between promiscuous and monogamous species due to intense competition between males to fertilize females in promiscuous lineages and lack thereof in monogamous ones. Here, we employ the rodent genus Peromyscus as a model to explore differences in evolutionary rates between testis-expressed genes of monogamous and promiscuous species. We find candidate genes that may be associated with increased sperm production in promiscuous species and gene ontology categories that show patterns of molecular convergence associated with phenotypic convergence in independently evolved monogamous species. Overall, our results highlight possible molecular correlates of differences in mating system, which can be contextualized in light of expected selective pressures.

Reproductive Proteins Evolve Faster Than Non-reproductive Proteins Among Solanum Species

Elevated rates of evolution in reproductive proteins are commonly observed in animal species, and are thought to be driven by the action of sexual selection and sexual conflict acting specifically on reproductive traits. Whether similar patterns are broadly observed in other biological groups is equivocal. Here, we examine patterns of protein divergence among wild tomato species (Solanum section Lycopersicon), to understand forces shaping the evolution of reproductive genes in this diverse, rapidly evolving plant clade. By comparing rates of molecular evolution among loci expressed in reproductive and non-reproductive tissues, our aims were to test if: (a) reproductive-specific loci evolve more rapidly, on average, than non-reproductive loci; (b) ‘male’-specific loci evolve at different rates than ‘female’-specific loci; (c) genes expressed exclusively in gametophytic (haploid) tissue evolve differently from genes expressed in sporophytic (diploid) tissue or in both tissue types; and (d) mating system variation (a potential proxy for the expected strength of sexual selection and/or sexual conflict) affects patterns of protein evolution. We observed elevated evolutionary rates in reproductive proteins. However, this pattern was most evident for female- rather than male-specific loci, both broadly and for individual loci inferred to be positively selected. These elevated rates might be facilitated by greater tissue-specificity of reproductive proteins, as faster rates were also associated with more narrow expression domains. In contrast, we found little evidence that evolutionary rates are consistently different in loci experiencing haploid selection (gametophytic-exclusive loci), or in lineages with quantitatively different mating systems. Overall while reproductive protein evolution is generally elevated in this diverse plant group, some specific patterns of evolution are more complex than those reported in other (largely animal) systems, and include a more prominent role for female-specific loci among adaptively evolving genes.

Rapid divergence of the male reproductive proteins in theDrosophila dunnigroup and implications for postmating incompatibilities between species

Proteins involved in post-copulatory interactions between males and females are among the fastest evolving genes in many species, usually attributed to their involvement in reproductive conflict. As a result, these proteins are thought to often be involved in the formation of postmating-prezygotic incompatibilities between species. The Drosophila dunni subgroup consists of a dozen recently diverged species found across the Caribbean islands with varying levels of hybrid incompatibility. We performed experimental crosses between species in the dunni group and see some evidence of hybrid incompatibilities. We also find evidence of reduced survival following hybrid mating, likely due to postmating-prezygotic incompatibilities. We assessed rates of evolution between these species genomes and find evidence of rapid evolution and divergence of some reproductive proteins, specifically the seminal fluid proteins. This work suggests the rapid evolution of seminal fluid proteins may be associated with postmating-prezygotic isolation, which acts as a barrier for gene flow between even the most closely related species.

Reproductive proteins evolve faster than non-reproductive proteins among Solanum species

Elevated rates of evolution in reproductive proteins are commonly observed in animal species, and are thought to be driven by the action of sexual selection and sexual conflict acting specifically on reproductive traits. Whether similar patterns are broadly observed in other biological groups is equivocal. Here we examine patterns of protein divergence among wild tomato species (Solanum section Lycopersicon), to understand forces shaping the evolution of reproductive genes in this diverse, rapidly evolving plant clade. By comparing rates of molecular evolution among loci expressed in reproductive and non-reproductive tissues, our aims were to test if: a) reproductive-specific loci evolve more rapidly, on average, than non-reproductive loci; b) ‘male’-specific loci evolve at different rates than ‘female’-specific loci; c) genes expressed exclusively in gametophytic (haploid) tissue evolve differently from genes expressed in sporophytic (diploid) tissue or in both tissue types; and d) mating system variation (a potential proxy for the expected strength of sexual selection and/or sexual conflict) affects patterns of protein evolution. We observed elevated evolutionary rates in reproductive proteins; however this pattern was most evident for female-rather than male-specific loci, both broadly and for individual loci inferred to be positively selected. These elevated rates might be facilitated by greater tissue-specificity of reproductive proteins, as faster rates were associated with more narrow expression domains. In contrast we found little evidence that evolutionary rates are consistently different in loci experiencing haploid selection (gametophytic-exclusive loci), or in lineages with quantitatively different mating systems. Overall while reproductive protein evolution is generally elevated in this diverse plant group, specific patterns of protein evolution are more complex than those reported in other (largely animal) systems, and include a more prominent role for female-specific loci among adaptively evolving genes.

Differences in Postmating Transcriptional Responses between Conspecific and Heterospecific Matings in Drosophila

In many animal species, females undergo physiological and behavioral changes after mating. Some of these changes are driven by male-derived seminal fluid proteins and are critical for fertilization success. Unfortunately, our understanding of the molecular interplay between female and male reproductive proteins remains inadequate. Here, we analyze the postmating response in a Drosophila species that has evolved strong gametic incompatibility with its sister species; Drosophila novamexicana females produce only ∼1% fertilized eggs in crosses with Drosophila americana males, compared to ∼98% produced in within-species crosses. This incompatibility is likely caused by mismatched male and female reproductive molecules. In this study, we use short-read RNA sequencing to examine the evolutionary dynamics of female reproductive genes and the postmating transcriptome response in crosses within and between species. First, we found that most female reproductive tract genes are slow-evolving compared to the genome average. Second, postmating responses in con- and heterospecific matings are largely congruent, but heterospecific matings induce expression of additional stress-response genes. Some of those are immunity genes that are activated by the Imd pathway. We also identify several genes in the JAK/STAT signaling pathway that are induced in heterospecific, but not conspecific mating. While this immune response was most pronounced in the female reproductive tract, we also detect it in the female head and ovaries. These results show that the female’s postmating transcriptome-level response is determined in part by the genotype of the male, and that divergence in male reproductive genes and/or traits can have immunogenic effects on females.

Rapid Divergence Of The Copulation Proteins In The Drosophila Dunni Group Is Associated With Hybrid Post-Mating-Prezygotic Incompatibilities

Background: Proteins involved in post-copulatory interactions between males and females are among the fastest evolving genes in many species and this has been attributed to reproductive conflict. Likely as a result, these proteins are frequently involved in cases of post-mating-prezygotic isolation between species. The Drosophila dunni subgroup consists of a dozen recently diverged species found across the Caribbean islands with varying levels of hybrid incompatibility.Results: We performed experimental crosses between species in the dunni group and find evidence of hybrid inviability likely due to post-mating-prezygotic incompatibilities. We next assessed rates of evolution between these species genomes and find evidence of rapid evolution and divergence of some reproductive proteins, specifically the seminal fluid proteins.Conclusions: This work suggests the rapid evolution of seminal fluid proteins can lead to post-mating-prezygotic isolation, which acts as a barrier for gene flow between even the most closely related species.

The Female Snark Is Still a Boojum: Looking toward the Future of Studying Female Reproductive Biology

Synopsis Philosophical truths are hidden in Lewis Carroll’s nonsense poems, such as “The hunting of the snark.” When the poem is used as a scientific allegory, a snark stands for the pursuit of scientific truth, while a boojum is a spurious discovery. In the study of female biology, boojums have been the result of the use of cultural stereotypes to frame hypotheses and methodologies. Although female reproduction is key for the continuation of sexually reproducing species, not only have females been understudied in many regards, but also data have commonly been interpreted in the context of now-outdated social mores. Spurious discoveries, boojums, are the result. In this article, we highlight specific gaps in our knowledge of female reproductive biology and provide a jumping-off point for future research. We discuss the promise of emerging methodologies (e.g., micro-CT scanning, high-throughput sequencing, proteomics, big-data analysis, CRISPR-Cas9, and viral vector technology) that can yield insights into previously cryptic processes and features. For example, in mice, deoxyribonucleic acid sequencing via chromatin immunoprecipitation followed by sequencing is already unveiling how epigenetics lead to sex differences in brain development. Similarly, new explorations, including microbiome research, are rapidly debunking dogmas such as the notion of the “sterile womb.” Finally, we highlight how understanding female reproductive biology is well suited to the National Science Foundation’s big idea, “Predicting Rules of Life.” Studies of female reproductive biology will enable scholars to (1) traverse levels of biological organization from reproductive proteins at the molecular level, through anatomical details of the ovum and female reproductive tract, into physiological aspects of whole-organism performance, leading to behaviors associated with mating and maternal care, and eventually reaching population structure and ecology; (2) discover generalizable rules such as the co-evolution of maternal-offspring phenotypes in gestation and lactation; and (3) predict the impacts of changes to reproductive timing when the reliability of environmental cues becomes unpredictable. Studies in these key areas relative to female reproduction are sure to further our understanding across a range of diverse taxa.

Quantitative proteomics reveals rapid divergence in the postmating response of female reproductive tracts among sibling species

Fertility depends, in part, on interactions between male and female reproductive proteins inside the female reproductive tract (FRT) that mediate postmating changes in female behaviour, morphology, and physiology. Coevolution between interacting proteins within species may drive reproductive incompatibilities between species, yet the mechanisms underlying postmating–prezygotic (PMPZ) isolating barriers remain poorly resolved. Here, we used quantitative proteomics in sibling Drosophila species to investigate the molecular composition of the FRT environment and its role in mediating species-specific postmating responses. We found that (i) FRT proteomes in D. simulans and D. mauritiana virgin females express unique combinations of secreted proteins and are enriched for distinct functional categories, (ii) mating induces substantial changes to the FRT proteome in D. mauritiana but not in D. simulans , and (iii) the D. simulans FRT proteome exhibits limited postmating changes irrespective of whether females mate with conspecific or heterospecific males, suggesting an active female role in mediating reproductive interactions. Comparisons with similar data in the closely related outgroup species D. melanogaster suggest that divergence is concentrated on the D. simulans lineage. Our study suggests that divergence in the FRT extracellular environment and postmating response contribute to previously described patterns of PMPZ isolation and the maintenance of species boundaries.