Introduction The Zingiberaceae family, which includes numerous economically and medicinally important species, exhibits considerable phylogenetic and genetic diversity. Chloroplast genomes are valuable resources for studying evolutionary relationships, genetic diversity, and adaptive evolution in plants. While many Zingiberaceae chloroplast genomes have been sequenced, the evolutionary mechanisms-including structural variation, codon usage bias, selection pressures, and divergence history-remain to be comprehensively investigated. Methods we performed a comparative analysis of 11 newly identified species (Aframomum alboviolaceum, Amomum longipetiolatum, Amomum petaloideum, Amomum velutinum, Cautleya spicata, Cornukaempferia larsenii, Globba atrosanguinea, Globba variabilis, Hedychium aureum, Riedelia arfakensis, and Zingiber citriodorum) and 110 published data from the Zingiberaceae family, including their structure, codon usage, nucleotide polymorphisms, divergence time, and selection pressures. Results The chloroplast genomes of Zingiberaceae species exhibited a highly conserved structure with no significant expansion or contraction during diversification. Analysis revealed four hypervariable protein-coding genes (atpH, rpl32, ndhA, and ycf1) and one intergenic region (psac-ndhE), which are proposed as potential molecular markers for future phylogeographic and population genetic studies. Codon usage bias was found to be predominantly shaped by natural selection. Phylogenetic analysis strongly supported the division of Zingiberaceae into two primary subfamilies (Alpinioideae and Zingiberoideae) and clarified key relationships, revealing that Globba is more closely related to Curcuma than to Hedychium, and Hedychium is more closely related to the Pommereschea-Rhynchanthus clade than to Cautleya. Divergence time estimation indicated two rapid diversification events within Zingiberoideae, coinciding with the rapid uplift of the Tibetan Plateau and a Late Miocene cooling event linked to declining CO₂ levels. Ancestral range reconstruction suggested an African origin during the Cretaceous period, followed by dispersal to Southeast Asia and India. Selection pressure analysis showed that most protein-coding genes are under negative selection. In contrast, the ycf2 gene was found to be under relaxed selection. Furthermore, two genes (matK and ndhB) were identified to be under positive selection in high-altitude species of Roscoea, suggesting a role in adaptation to alpine environments. Discussion This study provides a comprehensive genomic analysis of the Zingiberaceae family, highlighting the conserved nature of chloroplast genome structure despite extensive diversification. The identified mutation hotspots present valuable tools for developing high-resolution markers for species identification and biogeographic studies. The phylogenetic results resolve longstanding uncertainties in the relationships among key genera. The inferred divergence times and ancestral range suggest that the evolutionary history of Zingiberaceae was significantly influenced by major geological and climatic events, notably the uplift of the Tibetan Plateau and global cooling in the Late Miocene. The prevalence of negative/purifying selection across most genes indicates strong evolutionary constraints to maintain core photosynthetic functions. The discovery of positively selected genes in high-altitude Roscoea species provides insights into adaptive evolution to environmental stressors. These findings offer foundational knowledge for future efforts in crop improvement, species identification, and the conservation of genetic diversity within the Zingiberaceae family.

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Zingiberales includes eight families and more than 2600 species, with many species having important economic and ecological value. However, the backbone phylogenetic relationships of Zingiberales still remain controversial, as demonstrated in previous studies, and molecular dating based on chloroplast genomes has not been comprehensively studied for the whole order. Herein, 22 complete chloroplast genomes from 21 species in Zingiberales were sequenced, assembled, and analyzed. These 22 genomes displayed typical quadripartite structures, which ranged from 161,303 bp to 163,979 bp in length and contained 111-112 different genes. The genome structures, gene contents, simple sequence repeats, long repeats, and codon usage were highly conserved, with slight differences among these genomes. Further comparative analysis of the 111 complete chloroplast genomes of Zingiberales, including 22 newly sequenced ones and the remaining ones from the national center for biotechnology information (NCBI) database, identified three highly divergent regions comprising ccsA , psaC , and psaC - ndhE . Maximum likelihood and Bayesian inference phylogenetic analyses based on chloroplast genome sequences found identical topological structures and identified a strongly supported backbone of phylogenetic relationships. Cannaceae was sister to Marantaceae, forming a clade that was collectively sister to the clade of (Costaceae, Zingiberaceae) with strong support (bootstrap (BS) = 100%, and posterior probability (PP) = 0.99-1.0); Heliconiaceae was sister to the clade of (Lowiaceae, Strelitziaceae), then collectively sister to Musaceae with strong support (BS = 94-100%, and PP = 0.93-1.0); the clade of ((Cannaceae, Marantaceae), (Costaceae, Zingiberaceae)) was sister to the clade of (Musaceae, (Heliconiaceae, (Lowiaceae, Strelitziaceae))) with robust support (BS = 100%, and PP = 1.0). The results of divergence time estimation of Zingiberales indicated that the crown node of Zingiberales occurred approximately 85.0 Mya (95% highest posterior density (HPD) = 81.6-89.3 million years ago (Mya)), with major family-level lineages becoming from 46.8 to 80.5 Mya. These findings proved that chloroplast genomes could contribute to the study of phylogenetic relationships and molecular dating in Zingiberales, as well as provide potential molecular markers for further taxonomic and phylogenetic studies of Zingiberales.

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In order to reveal the constituents and their biological activities, we carried out a phytochemical study on Hedychium ellipticum Buch.-Ham. ex Sm. (Zingiberaceae). Ten labdane diterpenoids (1-10) were isolated from the rhizomes of H. ellipticum for the first time. Their structures were identified on the basis of spectroscopic analyses including two-dimensional NMR and comparison with literature data. All of these compounds were evaluated for their antimycobacterial activity against Mycobacterium tuberculosis and cytotoxicity against KB, MCF7, NCI-H187 and Vero cells. The result showed that compounds 1 and 7 exhibited moderate activity against Mycobacterium tuberculosis and compounds 4, 6 and 7 displayed remarkable cytotoxic activity. This is the first report on the presence of all compounds in H. ellipticum and the first time that their structure activity relationship has been discussed.

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Seven labdane-type diterpenes, coronarin E, coronarin A, yunnancoronarin A, yunnancoronarin B, hedyforrestin B, villosin, and hedyforrestin C were isolated from the rhizome of Hedychium gardnerianum and evaluated for cytotoxic activity against human small cell lung cancer (NCI-H187) and non-cancerous Vero cells. The results showed that villosin exhibited potent cytotoxic activity with IC(50) of 0.40 microM, which was higher than that of the drug ellipticine (IC(50) 1.79 microM). Moreover, ellipticine was very toxic to Vero cells (IC(50) 7.47 microM) whereas the toxicity of villosin was undetectable at concentration lower than 166.42 microM. The results have indicated that the lactone ring is essential for high cytotoxic activity and that the presence of a hydroxyl group at the 6 or 7 position causes decrease in activity. The very high cytotoxicity against the NCI-H187 cells and the exceptionally high selectivity index (>416) of villosin suggested that this compound may be used as a potential lead molecule for antitumor therapeutic development.

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The stems of Hedychium villosum yielded the new diterpenoid 1, named villosin, along with coronarin E and beta-sitosterol. Their structures were elucidated by spectroscopic methods including mono- and bi-dimensional NMR.

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