SRA

Here, we present the results of an integrative study that leverages naturally segregating variation and a recent, adaptive divergence event affecting seasonal timing to identify developmental mechanisms underlying diapause progression in a tephritid fly, Rhagoletis pomonella. Also called the apple maggot fly, R. pomonella is native to North America, where it infests fruits of native Crataegus (hawthorn) species throughout its range. Derived populations of the fly infest apples (Malus domesticus), and thus have evolved in the last ~250 years since apples were introduced. Many molecular studies in conjunction with mark-recapture experiments document that the populations, or host races, remain genetically distinct despite ongoing gene flow, making R. pomonella a textbook example of incipient speciation with gene flow and host associated divergence. Strong natural selection on two primary traits, host finding behavior and seasonal timing, maintain genetic divergence. Both populations (hereafter apple and haw flies) have one generation per year, with a functionally obligate pupal diapause, overwintering in the soil. Adults must emerge coincident with host fruit availability, typically a period of only a few weeks, in order to successfully oviposit into fruits. Apple flies have evolved an earlier (~3 week) emergence timing to synchronize with apples, which fruit about 3 weeks earlier than hawthorn at a typical, sympatric site in the Midwest (e.g., Michigan or Illinois, USA). We combined RNA sequencing (RNAseq), phenotyping of emergence timing and brain morphology, and whole genome pooled resequencing (Poolseq) to infer mechanisms underlying segregating variation for diapause phenology in each Rhagoletic pomonella host race, and the evolved difference in phenology between host races. Overall design: Infested apple and hawthorn fruits were collected from Urbana, IL in the late summer and fall of 2014. Infested fruit were returned to the laboratory at the University of Florida, emerging larvae were collected and held at ~24C 14:10 LD for 10 days to complete pupation and enter diapause, then pupae were transferred to 3.5C at 95% Relative Humidity (RH). At monthly intervals from 2 to 6 months we dissected out the Central Nervous System (CNS), homogenized pools of 10 CNS, and extracted RNA (5 samples per time point per host). RNA sequencing libraries were prepared using Illumina's tru-seq stranded mRNA kit and sequenced using 100bp single end Illumina HiSeq 2500. Cleaned reads were mapped to the R. zephyria genome using STAR, and read counts were generated using RSEM.