Comparative chloroplast genome of six species in Hypoxidaceae from China: insights into phylogenetic relationships and molecular marker development.

Abstract

Background: The family Hypoxidaceae (order Asparagales) is a predominantly Southern Hemisphere lineage comprising approximately 11 genera and 200 species, many of which possess significant medicinal and ornamental value. Despite their economic importance, Hypoxidaceae has received limited research attention, leading to problematic identification of species and misuse of wild resources in traditional medicine markets. Taxonomically, the phylogenetic position of Hypoxidaceae and the intergeneric relationships within this family remain controversial and unresolved, particularly concerning the delimitation of Curculigo and Molineria. Previous studies based on morphological traits and molecular markers have yielded inconsistent results, highlighting the need for more robust genomic evidence. In angiosperms, complete chloroplast genomes have proven highly effective in resolving systematic uncertainties considering their conserved structure and high informational content. However, such genomic data remain scarce for Hypoxidaceae, limiting phylogenetic clarity. In this research, the complete chloroplast genomes of six species representing three key genera (Curculigo, Molineria, and Hypoxis) were sequenced and characterized for a comparative and phylogenetic analysis.

Results: The chloroplast genomes of six species exhibited conserved quadripartite structures, measured 157,472 bp to 158,550 bp in length. The overall GC content of these genomes ranged between 37.3 and 37.5%. Gene annotations identified 132 genes, 19 duplicated in the inverted repeat regions, and had complete ndh gene. Comparative analysis of six complete chloroplast genomes revealed highly similarity, but they were varied in repeats sequence, codon usage bias, contractions and expansions in the IR region. Five molecular markers showed the highest degree of variability between the six cp genomes. Phylgenetic analysis based on cp genomic data confirmed that Hypoxidaceae was a monophyly, being a sister to Asteliaceae with higher supports than the previous research. Three main clades were recognized in Hypoxidaceae, including Curculigo clade, Hypoxis clade, and Pauridia-Empodium clade. And what's more, Curculigo clade could be divided into three subclades, containing Molineria subclade, Curculigo subclade, and Seychellean subclade, indicating significantly phylogenetic insights.

Conclusions: The complete cp genomes of six species of three representative genera from Hypoxidaceae were sequenced and analyzed in detail, including the general data on the genome length, repeat sequence, codon usage, IR expansion and contraction, structural comparison and divergence hotspot identification analyses, and phylogenetic analysis. A comparative analysis revealed that the cp genome was highly consistent of four Molineria species, but varied greatly at the generic level between Hypoxis, Curculigo, and Molineria, which could be used for generic delimitation. Five DNA barcodes (psbK-psbI, rpoB-trnC, ndhF-rpl32, ycf1, and trnE-trnT) were selected for authentication of Hypoxidaceae medicinal materials. Hypoxidaceae was a monophyletic lineage, containing three major clades, being a sister to Asteliaceae with stronger supports than before. The three main clades in Hypboxidaceae were re-confirmed as the three stable lineages for this family. In the Curculigo Clade, three subclades were identified with significant phylogenetic insights. The phylogenetic evidence presented here, combined with distinct chloroplast genome features, supports Molineria Subclade separated from Curuculigo Subclade, being a monophyletic group by transferring Sinocurculigo taishanica and two Borneo Curculigo species into Molineria. Further research should provide a better understanding of the intergeneric relationships among Hypoxidaceae, adding more genomic data with extensive samplings across the center distribution of Southern Hemisphere.