Scripts and detailed command lines supporting: Ecological selection as drivers during early speciation: Insights from two allopatric cypress species in the Himalaya

Understanding the factors driving speciation is fundamental to evolutionary biology. While geographic isolation has long been considered a primary factor, the role of ecological selection during early speciation stages is poorly understood. The Himalaya, a biodiversity hotspot, offers a unique opportunity to study these processes. We investigated two closely related conifer species, Cupressus torulosa and C. cashmeriana, which are geographically separated yet exhibit distinct ecological adaptations. Using target-capture sequencing of 52 C. cashmeriana samples from Bhutan and 46 C. torulosa samples from Nepal, we surveyed their genetic diversity, demographic history, and adaptive evolution. Genomic analyses revealed moderate genetic differentiation between them, yet confirmed they are genetically distinct sister species. Demographic modeling indicated their divergence began around 6.57 million years ago, with strong gene flow persisting until approximately 2.08 million years ago, shortly after the initiation of Quaternary climate oscillations. Ecological niche modeling showed low niche overlap, reflecting adaptations to different climatic conditions—C. cashmeriana to warmer, wetter habitats and C. torulosa to colder, drier habitats. We identified positively selected genes related to environmental stress responses, highlighting the crucial role of ecological selection. Genotype–environment association and partial redundancy analysis emphasized that ecological factors significantly drive genetic variation. Under future climate scenarios, both species show increased genomic vulnerability, particularly C. torulosa. Our findings underscore the importance of ecological selection in initiating speciation despite strong gene flow and have significant implications for managing mountain biodiversity in the face of climate change.