Phylogenetic insights and chloroplast genome structure of Bothriochloa ischaemum reinforce the unified classification of the BCD clade

Bothriochloa ischaemum belongs to the Bothriochloa genus within the Poaceae family and exhibits close morphological and distributional similarities with species from the Capillipedium and Dichanthium genera, collectively termed the BCD clade. However, the phylogenetic relationships within this clade have been the subject of ongoing debate. To address these controversies, we uti-lized the chloroplast genome, a resource known for its evolutionary conservation, as an invaluable tool for clarifying species delineations and exploring evolutionary dynamics. In this study, we sequenced and assembled the chloroplast genome of B. ischaemum, achieving a complete sequence of 138,316 bp with a GC content of 38.53%. Annotation of the genome identified 138 genes, comprising 89 protein-coding genes, 41 tRNA genes, and 8 rRNA genes. In a comparative genomic analysis of six chloroplast genomes from the BCD clade, (A/T) mononucleotide repeats were the most abundant, and forward (F) repeats predominated among the longer repeat sequences. Structural variations associated with specific genes were detected in the IR/SC boundary regions, and four mutation hotspots (rps16/trn-UUG, trnG-UCC/trnE-UUC, rps12/trnL-UAG, ccsA/ndhD) were pinpointed using sliding window analysis, which could serve as potential molecular markers for BCD clade identification. Phylogenetic tree construction indicated that Bothriochloa, Capillipedium, and Dichanthium form a monophyletic group, suggesting that the BCD clade may be recognized as a single genus from an evolutionary perspective. This research provides new insights into the intergeneric relationships within the BCD clade through chloroplast phylogenomics.