Comparative chloroplast genomics of Hibiscus (Malvaceae) and its phylogenetic implications.

Hibiscus L. (Hibisceae, Malvoideae, Malvaceae) is globally distributed and valued for its ornamental, agricultural, and medicinal significance, yet its taxonomic problems remain unresolved. Here, we de novo assembled 17 complete chloroplast (cp.) genomes representing 16 Hibiscus species, as one species was represented by two varieties, and compared them with 13 previously published genomes of this genus. These genomes ranged from 159,125 to 163,360 base pairs and exhibited a conserved quadripartite structure, encoding 112 unique genes (78 protein-coding genes, 30 transfer RNAs, and 4 ribosomal RNAs). While codon usage, amino acid frequency, substitution patterns, and the expansion and contraction of inverted repeats revealed high similarity, the number of simple sequence repeats (SSRs) varied among species. Comparative analyses identified five non-coding polymorphic loci (trnH-psbA, ndhF-rpl32, rps19-rpl22, trnR-atpA, and ndhD-ccsA) and six coding loci (rpl22, ycf1, matK, rpl32, rbcL, and ndhF) as suitable candidates for DNA barcoding and phylogenetic reconstruction. Phylogenomic analyses based on the cp. genome confirmed the polyphyly of Hibiscus, with Urena Dill. ex L., Malvaviscus Fabr., Kosteletzkya C. Presl, and Kydia Roxb. nested within it. Three major clades were recovered (/Phylloglandula, /Trionum, and /Euhibiscus). Hibiscus volkensii Gürke showed a long branch, suggesting a distinct evolutionary trajectory. Thepparatia scandens (Roxb. ex G.Don) Phuph. appeared as a sister to Hibiscus verdcourtii Craven. Congruence was observed between the cp. genome phylogeny and recent sectional classifications, although Hibiscus sect. Bombicella was confirmed to be polyphyletic. Overall, this study provides insights into the cp. genome evolution of Hibiscus and its implications for the phylogeny of this genus. These findings highlight the need for nuclear genomic data to resolve remaining uncertainties and clarify the taxonomy of this complex genus and other genera within the tribe Hibisceae.