Comparative chloroplast genomics of 22 species of Sida (Malvaceae) reveals structural conservation, adaptive evolution, and preliminary phylogenetic structure.

The genus Sida L. (Malvoideae, Malvaceae) comprises 275 species with a near-cosmopolitan distribution. Persistent taxonomic uncertainties, driven by morphological complexity and low resolution of traditional molecular markers, hinder systematic understanding of the genus. Chloroplast (cp) genomes provide genome-scale data capable of resolving these taxonomic uncertainties, but genomic resources for Sida remain limited. Here, we substantially expand cp genome sampling, perform comparative and selection analyses, identify candidate loci for species discrimination and phylogenetic inference, and conduct phylogenomic inference to clarify infrageneric relationships. We sequenced and de novo assembled the complete cp genomes of Sida cordata (Burm.f.) Borss.Waalk. and Sida rhombifolia L., and assembled cp genomes of 18 additional species de novo from publicly available raw sequencing reads. Together with two previously reported cp genomes, 22 cp genomes were subjected to comparative and phylogenetic analyses. Genome sizes ranged from 159,706 to 160,513 base pairs (bp), comprising a large single-copy region (88,771-89,551 bp), inverted repeats (IRa/IRb; 25,288-25,640 bp), and a small single-copy region (19,863-20,294 bp). Each genome contained 113 unique genes (79 protein-coding, 30 tRNAs, 4 rRNAs). Comparative analyses revealed conserved gene order, guanine-cytosine content, codon usage, and simple sequence repeat composition, as well as a consistent transition bias in substitution patterns across all Sida species. Gene-level selection analyses identified episodic positive selection in psaB, psbB, rbcL, and rps8, while site-level analyses detected positive selection at specific codons in rbcL and ycf1. Ten highly polymorphic loci with elevated nucleotide diversity and low missing data were identified as candidates for species identification and phylogenetic inference, including psbT-psbN, ndhD-psaC, psaC-ndhE, rps15-ycf1, and psaI. Phylogenetic analysis of the 22 species resolved two strongly supported clades: a monophyletic endemic Australian lineage and a broadly distributed Asian/pantropical lineage. Our findings reveal conserved cp genome structure across 22 Sida cp genomes, identify polymorphic loci as potential barcodes, and resolve two clades (an endemic Australian lineage and a widespread Asian/pantropical lineage), demonstrating the value of whole-cp genome data. However, due to sampling limitations and sole reliance on the maternally inherited cp genome, we recommend that future studies refine biogeographic inferences using denser taxon sampling and nuclear genomic data.