Chloroplast genomes provide new insights into the phylogeny and evolution of the genus Hibiscus L

Hibiscus L., the largest genus in the Malvaceae family, comprises numerous species of significant ornamental value. However, it remains a phylogenetically contentious taxon that has been historically neglected in systematic evolutionary studies.

In this study, we sequenced and analyzed the chloroplast genomes of 51 samples from 36 Hibiscus species and related genera. Multiple analyses were performed to reveal their genome structures, GC contents, codon usage, cross-species Ka/Ks ratios, SSR distributions, and nucleotide diversities. Concatenated and coalescent-based approaches were used for phylogenetic analyses. Several methods, including the calculation of concordance factors (gCFs and sCFs), MSCquartets and reticulate networks, were employed to explore the causes of phylogenetic conflicts. Furthermore, using branch-site model, we identified positively selected genes. Our results revealed that the chloroplast genomes of Hibiscus species were highly conserved in structure. Their sizes ranged from 160,068 to 163,579 bp, encoding approximately 130 genes, and their GC content varied only between 36.55% and 37%. Notably, 15 highly polymorphic loci (e.g., trnS-GCU, trnS-UGA, and trnG-GCC) were identified as candidate molecular markers for population genetics and horticultural breeding. Phylogenetic analysis revealed that Hibiscus and related genera are polyphyletic and could be divided into three well-supported groups. Novel species relationships were first detected at the plastome level. We also revealed significant phylogenetic conflicts between Hibiscus and related genera, attributed to incomplete lineage sorting (ILS) and reticulate evolution, which collectively explain the complex and polyphyletic relationships among them. Evolutionary rate analyses revealed a Ka/Ks ratio > 1.0 for the ycf1 gene, while positive selection analysis identified eleven genes (e.g., accD, atpF and ndhA) harboring amino acid sites under selection, primarily involved in photosynthetic pathways.

Our results highlight the complex relationships within Hibiscus species and their close relatives, characterized by blurred intergeneric boundaries. Moreover, Hibiscus species and their close relatives have undergone significant purifying selection, with many photosynthesis-related genes playing pivotal roles. This study provides essential genomic resources and evolutionary insights to guide future taxonomic and phylogenetic studies in Hibiscus.