The study, published in BMC Plant Biology, assembled the complete mitochondrial genomes of four species in the genus: Rehmannia glutinosa, the source of the famous herbal medicine dihuang, along with R. henryi, R. piasezkii, and R. chingii. All four belong to the broomrape family, Orobanchaceae, a group better known for its parasitic members but which also contains these therapeutic and horticulturally valuable plants. Despite decades of research into the bioactive compounds and metabolic pathways that make Rehmannia medicinally important, its mitochondrial genomes had remained essentially unexplored territory until now.

What the team found is a striking demonstration of structural plasticity. The mitochondrial genome of R. glutinosa spans 547,002 base pairs and exists as a typical single circular molecule, as does that of R. piasezkii, which is considerably larger at 742,657 base pairs. But the other two species broke the mold entirely. R. chingii, at 662,985 base pairs, is organized as one linear molecule accompanied by two circular structures, while R. henryi, at 663,966 base pairs, is split across four separate circular molecules. In other words, four species from the same genus carry mitochondrial genomes that differ not just in size but in their fundamental molecular architecture.

This kind of multipartite organization is not merely a curiosity. Plant mitochondrial genomes are known to exist as dynamic populations of molecules whose shapes depend on recombination between repeated sequences scattered throughout the DNA. When repeat-mediated recombination occurs at certain sites, a single master circle can be resolved into multiple smaller circles or linear fragments, and different configurations can coexist within the same cell. The assembly approach used in this study relied on long-read PacBio HiFi sequencing, which allowed the researchers to resolve the complex assembly graphs and confirm which alternative connections between contigs were genuinely supported by the read data, a critical step when the genome refuses to conform to a single circular model.

Beneath the architectural drama, the researchers documented a detailed landscape of repetitive DNA. Simple sequence repeats, short tandem motifs that are useful as molecular markers, showed a broadly conserved composition across the four species. However, the numbers of dispersed repeats, longer duplicated sequences that can mediate recombination, and tandem repeats varied quantitatively among the species. This variation in repeat content is exactly what would be expected to drive the differences in genome structure, since each pair of sufficiently large repeats represents a potential recombination point that can reshape the molecule into alternative conformations. The findings thus connect the fine-scale repeat inventory directly to the large-scale structural diversity observed among the four genomes.

Perhaps the most surprising result is how little the functional content of these genomes varies despite their structural chaos. The team predicted 394 C-to-U RNA editing sites across 31 protein-coding genes in each of the four species, a profile that was highly conserved throughout the genus. RNA editing of this kind is a hallmark of plant mitochondria, where specific cytidines in the transcribed RNA are converted to uridines, often restoring codons that would otherwise be nonfunctional. The consistency of this editing landscape suggests that while the physical packaging of the genome is fluid, the information it encodes and the post-transcriptional processing it requires are under strong stabilizing selection.

The study also traced the ongoing conversation between two organelles. Mitochondrial genomes in plants frequently absorb DNA from the chloroplast, and these mitochondrial plastid DNA sequences, or MTPTs, were abundant in all four Rehmannia species, with 21 to 28 such transfers identified in each genome. Among them was a conserved region of approximately 4.5 kilobases containing complete sequences of ndhB, a photosynthesis-related gene, along with two plastid ribosomal protein genes, rps7 and rps12. These fragments are essentially molecular fossils of past DNA traffic from chloroplast to mitochondrion, and their conservation across the genus points to a shared history of inter-organelle gene transfer predating the divergence of these species.

To place these genomes in an evolutionary context, the researchers conducted phylogenetic analysis that strongly supported the monophyly of Rehmannia, confirming that the genus forms a single coherent lineage within Orobanchaceae. They then measured nucleotide diversity and calculated the ratio of nonsynonymous to synonymous substitution rates, known as Ka/Ks, across the shared protein-coding genes. The overall picture was one of conservation: most mitochondrial genes are evolving slowly and under purifying selection, as expected for genes encoding essential components of the respiratory chain. Yet a handful of genes stood out, with sdh3, atp6, and matR showing relatively higher sequence variation among the species, hinting at localized hotspots of divergence within an otherwise conservative genome.

The contrast between these two layers of evolution, structural and sequence-based, is what makes the study compelling. On one hand, the mitochondrial genomes of Rehmannia have diversified dramatically in size and organization over what is presumably a modest evolutionary timescale, with one species carrying its genome in four circles and another in a hybrid of linear and circular molecules. On the other hand, the gene content, RNA editing patterns, and rates of sequence evolution remain remarkably stable. This coexistence of pronounced structural plasticity with conserved genomic features appears to be a defining characteristic of plant mitochondrial genomes, and the Rehmannia data provide a particularly clean example because the four species are close relatives, minimizing confounding effects from deep evolutionary divergence.

For the broader research community, the work delivers a set of complete, well-annotated mitochondrial genomes for a medicinally significant genus, along with a framework for understanding how repeat-mediated recombination, chloroplast DNA influx, and selective constraint interact to shape mitochondrial genome evolution in flowering plants. It also underscores a practical lesson for genome assembly: assuming a single circular mitochondrial chromosome can lead researchers astray, and long-read sequencing with careful validation of alternative assembly paths is increasingly essential. As more species across Orobanchaceae and beyond receive the same treatment, comparisons like this one will help reveal whether the structural exuberance seen in Rehmannia is the rule or the exception in plant mitochondrial evolution, and whether the genes that vary most, such as sdh3 and atp6, hold clues to adaptation in these remarkable medicinal plants.

Subject of Research: Comparative assembly and evolutionary analysis of mitochondrial genomes in four Rehmannia species

Article Title: Assembly and comparative analysis of the four complete mitochondrial genomes in Rehmannia (Orobanchaceae): structural diversity and evolutionary insights

Article References: Jiang, X., Yan, C., Qiu, A., Hou, R., Wu, Q., Huang, S., Wang, J., Ren, M., Wang, S., Li, J., & Liu, Z.-L. (2026). Assembly and comparative analysis of the four complete mitochondrial genomes in Rehmannia (Orobanchaceae): structural diversity and evolutionary insights. BMC Plant Biology. https://doi.org/10.1186/s12870-026-10054-2

Image Credits: AI Generated

DOI: 10.1186/s12870-026-10054-2

Keywords: Rehmannia, mitochondrial genome, Orobanchaceae, genome assembly, RNA editing, MTPTs, chloroplast gene transfer, repeat-mediated recombination, plant evolution, PacBio HiFi, Ka/Ks selection, medicinal plants

The research focused on four sympatric species: Ajania sericea, Aster brachytrichus, Anaphalis pannosa, and Leontopodium dedekensii. Although all belong to the vast sunflower family, they sit on different branches of the Asteraceae tree, which makes them an ideal natural experiment. If species from distinct lineages living in the same alpine setting show shared chloroplast genomic features, those features could plausibly relate to environmental response rather than shared ancestry. The team sequenced, assembled, and annotated the complete chloroplast genomes of all four species, then layered on analyses of codon usage, repeat composition, variable regions, selection pressure, and phylogenetic relationships to build a comprehensive comparative picture.

Structurally, the four plastomes turned out to be remarkably conservative. Each exhibited the typical quadripartite architecture found across most flowering plants, consisting of a pair of inverted repeat regions separated by a large single-copy and a small single-copy region. Guanine-cytosine content peaked in the inverted repeats, ranging from 42.99 to 43.07 percent, a pattern consistent with the elevated GC levels typically observed in these conserved segments. Gene content was similarly stable, with between 142 and 145 gene copies annotated in each genome. The differences that did emerge were subtle but informative: Anaphalis pannosa and Leontopodium dedekensii had both lost the ycf15 gene, and Anaphalis pannosa additionally lacked the transfer RNA gene trnT-GGU. These small-scale gene losses, along with variation at the boundaries of the inverted repeats and differences in repeat sequence composition, provide potential markers for distinguishing closely related species within the family.

Beyond gross structure, the researchers probed how the coding sequences themselves are used. Codon usage bias across the four plastomes was generally weak, and the preferred codons predominantly ended in adenine or thymine, reflecting the AT-rich character of chloroplast genomes. Two complementary approaches, the neutrality plot and the effective number of codons plot, suggested that mutational pressure alone could not fully explain the observed patterns. Factors beyond mutation, potentially including natural selection, appear to contribute to shaping codon preferences in these genomes. That conclusion matters because it hints that even the fine-grained mechanics of translation in alpine chloroplasts may not be entirely neutral, opening the door to functional interpretations of codon-level patterns in extreme environments.

One of the most practically valuable findings concerns DNA barcoding. By scanning the alignment of the four plastomes for regions of elevated sequence divergence, the team identified five hypervariable loci: petN, trnY-GUA, pafI-trnS-GGA, ndhC-trnV-UAC, and rpl32. These segments, which mutate faster than the surrounding genome, are prime candidates for developing DNA barcodes capable of discriminating Asteraceae species, a family so large and morphologically diverse that traditional identification is often difficult. Hypervariable chloroplast regions have long been sought as barcodes because they combine the advantages of plastid inheritance, including haploidy and lack of recombination, with enough variability to separate closely related taxa. The five regions flagged here could feed directly into future studies of interspecific differentiation and species delimitation across the daisy family.

To place the four species in evolutionary context, the researchers reconstructed phylogenetic trees using both maximum likelihood and Bayesian inference, two methods that rely on different statistical philosophies. The resulting topologies were fully consistent, a reassuring sign of robustness. Within this framework, Ajania sericea was most closely related to Ajania nematoloba, confirming its generic placement and providing a phylogenetic anchor for interpreting its genomic features. Reliable species trees are essential groundwork for any claim of convergent molecular evolution, because shared features must be distinguished from those simply inherited from a common ancestor.

The heart of the adaptation story lies in the analysis of selection. By calculating the ratio of nonsynonymous to synonymous substitution rates, known as Ka/Ks, across all protein-coding genes, the team assessed which genes might be evolving under relaxed constraint or positive selection. The verdict was clear for most of the genome: the vast majority of chloroplast protein-coding genes are under purifying selection, meaning that harmful mutations are being steadily weeded out, a hallmark of genes essential to survival. Only a handful of genes, including rpl14, rpl33, and rps2, showed relatively elevated Ka/Ks values or candidate signals of selection. These genes encode components of the chloroplast ribosome, the molecular machine that translates plastid proteins, and their elevated divergence in alpine species is intriguing given the demands that cold and high light place on plastid protein synthesis.

When the researchers integrated sequence variation, selection signals, and functional annotation into a single framework, a coherent picture emerged. Five genes stood out as candidates associated with alpine response: rpl14 and rpl33, both ribosomal protein genes tied to chloroplast translation; petN and ndhC, components of the photosynthetic electron transport chain; and ndhF, part of the chloroplast NADH dehydrogenase-like complex involved in redox regulation. Together, these genes point toward three interlinked processes, namely translation, photosynthetic electron transport, and redox homeostasis, as the chloroplast functions most plausibly shaped by life at high elevation. The logic is biologically appealing: alpine plants must keep photosynthesis running under intense light and cold while avoiding oxidative damage, and the plastid NADH dehydrogenase-like complex participates in cyclic electron flow and stress-related redox balancing, making its subunits plausible targets of environmental selection.

Not every result pointed toward adaptation, and the negative findings are just as instructive. The team compared chloroplast genomes from three Ajania sericea samples collected at different elevations and found them highly similar, with no clear elevation-associated plastome differentiation within the range examined. This suggests that, at least for this species and this elevational span, the plastid genome does not track altitude in a simple, linear fashion, and that any adaptive variation may be shared across populations or reside elsewhere in the genome. The authors are careful to frame their candidate genes as shared molecular response features rather than proven adaptations, a scientifically appropriate level of caution for a comparative study without reciprocal experiments or functional validation.

The broader significance of the work is twofold. For evolutionary biologists, it provides new chloroplast genomic evidence that sympatric Asteraceae species from different lineages may harbor convergent candidate features linked to alpine environments, enriching the growing literature on how plants respond genetically to mountain life. For taxonomists and conservationists, the five hypervariable regions identified here offer ready-made tools for species discrimination and population studies in one of the world’s largest plant families. As sequencing costs continue to fall, comparative plastomics of co-occurring species in other extreme habitats, from deserts to Arctic tundra, could reveal whether the same translation, electron transport, and redox genes recur as molecular common denominators of environmental stress response, or whether each habitat writes its own signature into the chloroplast genome.

Subject of Research: Comparative chloroplast genomics of sympatric alpine Asteraceae species to identify molecular features associated with alpine adaptation

Article Title: Comparative chloroplast genomics of Ajania sericea and sympatric Asteraceae species identifies shared candidate molecular features associated with alpine adaptation

Article References: Yang, H., Wu, S., He, Q., Xin, H., Li, W., & Chen, X. (2026). Comparative chloroplast genomics of Ajania sericea and sympatric Asteraceae species identifies shared candidate molecular features associated with alpine adaptation. BMC Genomics. https://doi.org/10.1186/s12864-026-13312-7

Image Credits: AI Generated

DOI: 10.1186/s12864-026-13312-7

Keywords: chloroplast genome, Ajania sericea, Asteraceae, alpine adaptation, molecular evolution, codon usage bias, Ka/Ks selection, DNA barcoding, phylogenetics, Yunnan, photosynthetic electron transport, redox regulation