Many cellular processes and organismal behaviours are time-dependent, and asynchrony of these phenomena can facilitate speciation through reinforcement mechanisms. The Mojave and Sonoran desert tortoises (Gopherus agassizii and G. morafkai respectively) reside in adjoining deserts with distinct seasonal rainfall patterns and they exhibit asynchronous winter brumation and reproductive behaviours. We used whole genome sequencing of 21 individuals from the two tortoise species and an outgroup to understand genes potentially underlying these characteristics. Genes within the most diverged 1% of the genome (FST ≥ 0.63) with putatively functional variation showed extensive divergence in regulatory elements, particularly promoter regions. Such genes related to UV nucleotide excision repair, mitonuclear and homeostasis functions. Genes mediating chronobiological (cell cycle, circadian and circannual) processes were also among the most highly diverged regions (e.g., XPA and ZFHX3). Putative promoter variants had significant enrichment of genes related to regulatory machinery (ARC-Mediator complex), suggesting that transcriptional cascades driven by regulatory divergence may underlie the behavioural differences between these species, leading to asynchrony-based prezygotic isolation. Further investigation revealed extensive expansion of respiratory and intestinal mucins (MUC5B and MUC5AC) within Gopherus, particularly G. morafkai. This expansion could be a xeric-adaptation to water retention and/or contribute to differential Mycoplasma agassizii infection rates between the two species, as mucins help clear inhaled dust and bacterial. Overall, results highlight the diverse array of genetic changes underlying divergence, adaptation and reinforcement during speciation.
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