Ginger Family Tree Rewritten: Chloroplast Genes Reveal Hidden Splits in Malaysian Amomum

Deep in the rainforests of Peninsular Malaysia, a group of aromatic gingers has been quietly defying the labels botanists have pinned on them for two centuries. Now, a new phylogenomic study drawing on tens of thousands of base pairs of chloroplast DNA has mapped the evolutionary relationships of the genus Amomum in unprecedented detail, confirming what many taxonomists suspected: the group is not a single natural lineage at all. The research, published in the journal PhytoKeys, used RNA sequencing to reconstruct the family tree of seven Amomum species from across the peninsula, and the results are already reshaping how scientists think about one of tropical Asia’s most economically important plant families.

The Zingiberaceae, the ginger family, is the largest lineage in the order Zingiberales, with more than 57 genera and over 1,600 species spread across tropical and subtropical Asia. It includes some of humanity’s most familiar spices and medicines: ginger, turmeric, cardamom, galangal, and white ginger. Within this sprawling family, Amomum has long stood out for its value as a spice and its extensive use in traditional remedies across Southeast Asia, where its species have served as substitutes for true cardamom. But despite this importance, the genus has been a taxonomic headache. Species identification relies heavily on inflorescence characteristics that bloom only once or twice a year, and morphological convergence has repeatedly blurred the boundaries between Amomum and its close relatives.

Previous molecular studies had already confirmed that Amomum, as traditionally circumscribed, was paraphyletic, meaning it excluded some of its own descendants. A comprehensive taxonomic revision by de Boer and colleagues in 2018 resurrected three old genera, Conamomum, Meistera, and Wurfbainia, and described three new ones, Epiamomum, Lanxangia, and Sundamomum, within the broader Amomum complex. That revision updated the list of Amomum species in Peninsular Malaysia to 28 species spread across five genera. Yet even with this framework in place, species-level relationships among Peninsular Malaysian taxa remained poorly resolved, particularly where morphological convergence is common and herbarium specimens with both vegetative and reproductive material are scarce.

To break through this impasse, researchers turned to chloroplast phylogenomics, a powerful approach that exploits the relatively conserved structure and slower mutation rates of chloroplast genes to resolve evolutionary relationships. The team collected fresh flowering material of seven Amomum species from type localities across Peninsular Malaysia, including sites at Batu Caves in Selangor, the Forest Research Institute Malaysia, Penang Hill, and the Penang Botanic Gardens. Each species was represented by two biological replicates, and tissues were harvested at equivalent developmental stages during the flowering season between March and April 2022, always between 8 and 10 in the morning, to ensure comparability.

The sequencing effort generated approximately 170 gigabytes of raw RNA data. From the transcriptome assemblies, the researchers identified 85 chloroplast protein-coding genes, of which 43 complete genes were retained for analysis after excluding short or incomplete sequences. The concatenated length of these genes ranged from 27,414 to 27,500 base pairs across the seven species, with GC content between 37.4 and 38.0 percent, a figure characteristic of the AT-biased plastid genomes of monocots. Codon position analyses revealed purifying selection at the first codon position, stronger evolutionary constraint at the second, and relaxed selection at the third, where synonymous substitutions accumulate more freely and contribute to evolutionary plasticity.

Beyond raw sequence data, the study also examined chloroplast gene expression profiles across species. A hierarchical clustering heatmap of normalized expression values revealed distinct species-specific transcriptional signatures. Most strikingly, the species Amomum elan formed a separate cluster entirely, with genes such as ycf68, ycf4, rpl33, and rps14 showing their highest expression levels in this species, while Amomum aculeatum and Amomum trilobum generally displayed lower expression across the same genes. Meanwhile, Amomum trilobum and Amomum testaceum clustered closely together, suggesting similar plastid gene regulation. These patterns hint at lineage-specific functional divergence in chloroplast biology that may reflect adaptation to different ecological niches or regulatory changes accumulated over evolutionary time.

Sliding-window analysis of nucleotide diversity identified clear hotspots of sequence polymorphism. The ndhJ gene region exhibited the highest nucleotide diversity, followed by elevated values near psbT, psbZ, and rps14. These loci could serve as molecular markers for species differentiation and evolutionary research, filling a critical gap in a group where traditional DNA barcoding has struggled. Conversely, genes such as rbcL, psbA, and atpI, which perform essential photosynthetic or housekeeping functions, remained highly conserved with minimal variability, reducing their effectiveness in distinguishing species-level variation. The contrast between hypervariable and conserved regions highlights the dynamic evolutionary forces shaping the Amomum chloroplast genome.

For the phylogenetic reconstruction, the team combined their transcriptome-derived sequences with 93 related taxa downloaded from NCBI GenBank and two outgroups, Hedychium coronarium and Roscoea humeana. Bayesian inference and maximum likelihood analyses, both based on 43 concatenated chloroplast genes totaling 28,794 aligned positions, recovered two major well-supported clades. Clade A primarily comprised species from the genera Zingiber and Kaempferia, while Clade B contained the Amomum-Alpinia complex, including representatives of Amomum, Wurfbainia, Lanxangia, and Alpinia. All seven Peninsular Malaysian species fell within Clade B, though their positions within it varied considerably.

The most consequential finding concerns Amomum trilobum. In both the Bayesian and maximum likelihood trees, this species showed a closer association with the Wurfbainia lineage than with the core Amomum-Alpinia cluster, suggesting that its transfer to Wurfbainia may be warranted pending further taxonomic evaluation. Similarly, Amomum uliginosum and Amomum testaceum, previously transferred to Wurfbainia, resolved within the Wurfbainia lineage, supporting the 2018 taxonomic revision. Meanwhile, Amomum curtisii, Amomum elan, and Amomum smithiae clustered together with strong support, forming a sister lineage to two Alpinia species. These placements collectively affirm the paraphyletic nature of Amomum as traditionally circumscribed and reinforce the recognition of segregate genera within the broader complex.

The study provides the first transcriptome-derived chloroplast phylogenomic framework for Peninsular Malaysian Amomum, demonstrating that even partial plastome data recovered from RNA sequencing can recover robust phylogenetic patterns where complete plastomes are unavailable. The identification of hypervariable loci such as ndhJ, psbT, and rps14 offers promising candidates for future barcoding and phylogeographic studies, while the species-specific expression profiles of genes like rps14, rpl33, and ycf68 suggest potential functional shifts that merit further investigation. As genomic datasets expand and taxon sampling broadens, the evolutionary history of this charismatic ginger lineage is finally coming into focus, one chloroplast gene at a time.

Subject of Research: Chloroplast phylogenomics of Amomum s.l. in Peninsular Malaysia reveals paraphyly and species-level evolutionary relationships within Zingiberaceae.

Article Title: Comprehensive plastid phylogenomics reveal the phylogeny of Amomum s.l. ( Zingiberaceae ) from Peninsular Malaysia

Article References: Fareed, F. S., Singaram, N., Abdul Malek, A. Z., Hashim, A. M., Sam, Y. Y., & Hin, O. I. (2026). Comprehensive plastid phylogenomics reveal the phylogeny of Amomum s.l. (Zingiberaceae) from Peninsular Malaysia. PhytoKeys, 276, 151-174. https://doi.org/10.3897/phytokeys.276.192768

Image Credits: AI Generated

DOI: 10.3897/phytokeys.276.192768

Keywords: phylogenomics, chloroplast, Zingiberaceae, Amomum, Peninsular Malaysia, RNA sequencing, taxonomy, Wurfbainia, nucleotide diversity, ginger family, Comprehensive, plastid