Speciation mechanisms in hawaiian Drosophila: Hybrid male sterility, mating behavior, and differential gene expression in D. planitibia and D. silvestris

One of the grand challenges in evolutionary biology is determining the phenotypic and genetic mechanisms that create and maintain species. Island systems such as the Hawaiian archipelago offer excellent opportunities to examine genetic and phenotypic differences between lineages that are at different stages of the speciation process. Divergences in courtship behavior and hybrid male sterility are recognized as important reproductive isolating mechanisms that can limit hybridization between species that are currently undergoing divergence or have recently speciated. Behavioral reproductive isolation is a pre-zygotic barrier that is expected to be stronger in sympatry due to reinforcement selection and generally weaker in allopatry. Hybrid male sterility (HMS) is an important post-zygotic reproductive isolating mechanism in many eukaryotic organisms as it restricts gene flow even in the presence of hybridization and more often occurs in males in species where males are the heterogametic sex. Only recently has genome-wide gene expression analysis been used to investigate the full suite of genes involved in the expression of this complex trait. Here we analyze the courtship behavior, HMS, and differential gene expression of testis-level fertility and allele-specific expression in backcrossed (BC) males of two closely related endemic Hawaiian Drosophila species, D. silvestris and D. planitibia. These two species evolved in allopatry with D. silvestris endemic to Hawai`i Island and D. planitibia endemic to the island of Maui. Previous studies have shown that D. planitibia can hybridize with D. silvestris, indicating little pre-zygotic reproductive and behavioral isolation barriers. These two species also produce viable F1 offspring and fertile F1 females but sterile hybrid males, a classic post-zygotic reproductive barrier. Backcross (BC) males, produced by crossing hybrid F1 females with parental males, exhibit a range in fertility, providing potential introgression should the two species ever come back into contact. The behavioral studies reported here show that mating success and sperm phenotype in BC males are uncorrelated, indicating that as expected these allopatric species exhibit little reinforcement of behavioral reproductive isolation. The lack of correlation between courtship behaviors and male fertility in the BC males also suggests that these two systems are controlled by different sets of genes. Using the RNA-Seq platform and a de novo transcriptome assembly, we identified over 100 differentially expressed genes across three BC fertility phenotypes, showing a clear down-expression of key functional genes in BC individuals who lack sperm (sterile) compared with those individuals who are effectively fertile. These results demonstrate a clear directional gene expression change regarding HMS, providing insight to the mechanisms of speciation. This research indicates that the genetic basis of male fertility has diverged to a greater extent than courtship behavior between these two species and suggests that the evolution of male reproductive systems are under strong divergent selection leading to the evolution of reproductive isolating mechanisms when species are in allopatry.