Comparative analysis of phylogeny and chloroplast genome evolution of Salix

Abstract

Willows play a crucial role in landscape architecture and the restoration of ecosystems. The phylogenetic relationships within Salix remain problematic due to phenotypic plasticity, frequent hybridization, and persistent discordance between nuclear and plastid data, particularly among recently diverged lineages. However, the current molecular markers and genomic resources are inadequate for facilitating accurate enhancement and stewardship of genetic resources. Salix gordejevii and S. microstachya are ecologically and horticulturally important shrub species widely utilized for sand dune fixation and riparian soil stabilization in arid environments. In this study, we assembled and annotated the complete chloroplast genomes of S. gordejevii and S. microstachya, revealing typical angiosperm quadripartite structures with total lengths of 155,643 bp and 155,555 bp, respectively. Both plastomes encoded the same set of functional genes, comprising protein-coding genes, tRNA genes, and rRNA genes, and exhibited highly conserved gene content and structural organization. Codon usage was strongly biased toward A/U-ending synonymous codons, primarily due to functional constraints and natural selection rather than mutational pressure alone. Comparative genomics further revealed several mutational hotspots in non-coding regions, such as psbA-trnH and trnS-trnG, which may serve as potential molecular markers for species identification. The major diversification of Salix occurred during the Miocene, with S. microstachya diverging approximately 14.32 Mya, while S. gordejevii belongs to a more recent cluster that diverged within the last 10 Mya. Overall, these taxa likely originated in East Asian, with their current distribution in Northern China shaped by historical migration and continuous genomic adaptation to intensifying continental climates. Collectively, these findings provided reliable chloroplast molecular markers for species identification and population genetics, and accelerated the breeding of stress-resistant elite cultivars, thereby supporting ecological restoration and landscaping in arid and semi-arid regions.