Comparative genomics of plastid genome within Curculigo orchioides subclade (Hypoxidaceae) reveals evolutionary relationships and document the shortest ndhA intron in seed plants.

Main conclusion: This study presents the first comprehensive plastome analysis of Curculigo, filling a genomic gap in Hypoxidaceae, reveals its evolutionary relationships, and identifies the shortest known ndhA intron among seed plants. Comparative genomics is an effective tool for exploring plastome evolution and gene content variation across species. In the genus Curculigo (Hypoxidaceae), plastome research has been restricted to C. orchioides and C. capitulata. To enhance our understanding of evolutionary relationships within this genus, we sequenced and assembled complete plastomes of the five Indian species: C. brevifolia, C. janarthanamii, C. konkanensis, C. orchioides, and C. sabui, all belonging to the C. orchioides subclade. The plastomes (158,114-158,205 bp) contained 113 unique genes, including 79 protein coding, with codon usage ranging from 23,139 to 24,434. We identified 49 long repeats (30-66 bp), primarily in intergenic spacers and eight genes and 42-47 SSRs per genome. LSC/IR/SSC junction analyses revealed conserved positioning across species. Six hotspot regions were detected, with trnL-trnF and rpl32-trnL-UAG previously reported in Asparagales. A unique 508-nt deletion in the ndhA intron of C. orchioides 2 resulted in the shortest known intron among seed plants. Complete ribosomal DNA sequences (7015-8702 bp) were also assembled and utilized for phylogenetic analysis. Phylogenies based on whole plastomes, 79 coding sequences, and rDNA data resolved the relationships among six Curculigo taxa. Further multigene analysis with additional taxa supported earlier studies while offering greater resolution within the C. orchioides group. Our results reveal a largely conserved plastome architecture punctuated by lineage-specific indels that constitute useful synapomorphies within Hypoxidaceae. The new genomic resources and clarified phylogeny provide a robust framework to support future research in species delimitation, adaptive evolution, and conservation genetics of this medicinally valuable genus.