The six newly sequenced species—B. repanda, B. axillaris, B. sinuata, B. martiniana, B. megacephala, and B. formosana—were collected from Yunnan Province in southwestern China, where more than half of the country’s Blumea species have documented medicinal uses. Among them, B. balsamifera, known locally as Dafeng’ai, has been used for thousands of years across China and Southeast Asia to treat eczema, dermatitis, beriberi, menorrhagia, and skin injuries. Modern pharmacological studies have identified L-borneol as its primary bioactive constituent, and the species holds a special place in traditional Chinese medicine as the only botanical source of Aipian, a substance used to clear orifices and invigorate the spirit. Other species such as B. megacephala, B. riparia, B. aromatica, B. formosana, and B. densiflora have likewise demonstrated considerable pharmacological potential.

Chloroplasts are the photosynthetic powerhouses of plant cells, and their genomes typically measure between 115 and 165 kilobases in a conserved quadripartite structure: a large single-copy region, a small single-copy region, and two inverted repeats. Because chloroplast DNA is maternally inherited, lacks recombination, and has a reduced effective population size, it is a stable and reliable tool for reconstructing plant evolutionary relationships. Advances in next-generation sequencing have made plastid genome assembly cheaper and faster, accelerating phylogenomic studies across diverse taxonomic scales. The research team used the Illumina HiSeq 2500 platform to generate 150-base-pair paired-end reads, filtered low-quality data with Fastp, and assembled complete plastomes with GetOrganelle, verifying each sequence by alignment against the reference genome of B. balsamifera.

The resulting plastomes ranged from 150,783 base pairs in B. sinuata to 151,281 base pairs in B. formosana, all falling within the typical size range for most angiosperms. Each contained the canonical quadripartite architecture, with inverted repeats measuring 24,924 to 24,957 base pairs, a large single-copy region of 82,524 to 82,894 base pairs, and a small single-copy region of 18,355 to 18,509 base pairs. Overall GC content was nearly constant across species at 37.5 to 37.6 percent, with the inverted repeats showing the highest GC content at 43 percent, followed by the large single-copy region at 35.6 to 35.7 percent and the small single-copy region at 30.8 to 31.0 percent. Each plastome contained 131 genes, including 8 ribosomal RNAs, 37 transfer RNAs, and 86 protein-coding genes, 18 of which were duplicated within the inverted repeats.

Whole-genome alignments with Mauve revealed no detectable gene rearrangements or inversions across all ten plastomes, confirming that Blumea’s chloroplast genome evolution has been relatively conservative. Sequence identity analysis using mVISTA confirmed that inverted repeats are more conserved than single-copy regions and that coding regions are more conserved than non-coding ones. Nucleotide diversity estimates yielded the same pattern: in non-coding regions, variation spanned 0.19 to 8.36 percent with a mean of 3.82 percent, averaging 4.21 percent in the large single-copy region, 0.87 percent in the inverted repeats, and 6.25 percent in the small single-copy region. In coding regions, variation ranged from 0 to 3.70 percent with a mean of 1.25 percent. Non-coding regions diverged 3.06-fold more than coding regions overall.

Six non-coding regions showed over 6 percent variability—trnH-psbA, rps16-trnQ-UUG, atpI-atpH, ycf4-cemA, ndhG-ndhI, and rps15-ycf1—making them candidate DNA barcodes for genus-level phylogenetic studies. Four genes—ycf1, rps8, ndhF, and matK—showed the highest variability among coding regions. Comparison of the inverted repeat and single-copy boundary positions across the ten plastomes revealed generally conserved inverted repeat regions along with limited structural variation at specific junctions. At the large single-copy and inverted repeat b boundary, rps19 sits in the large single-copy region 10 to 13 base pairs from the junction, while rpl2 resides in the inverted repeat b region 52 to 60 base pairs away. A truncated copy of ycf1 straddles the inverted repeat b and small single-copy junction, extending 0 to 21 base pairs into the small single-copy region in nine species; in B. balsamifera, this copy is pseudogenized. The intact ycf1 copy in the small single-copy region extends 564 to 581 base pairs into the inverted repeat a region across the small single-copy and inverted repeat a junction.

The research team also catalogued repetitive sequences across the ten plastomes, identifying 395 dispersed repeats comprising 181 forward, 202 palindromic, 11 reverse, and one complementary repeat. B. axillaris exhibited the highest number at 49, while B. martiniana had the fewest at 34. Forward and palindromic repeats were present in all species, reverse repeats were detected in six species, and complementary repeats were exclusive to B. tenella. Most dispersed repeats were located in the large single-copy and inverted repeat regions, with the majority residing in intergenic spacers. Repeats of 30 to 39 base pairs were the most abundant, followed by those of 40 to 49 base pairs. In addition, 79 tandem repeats and 547 simple sequence repeats were identified across the ten plastomes. Mononucleotide repeats were the most abundant at 425, accounting for 77.70 percent of all SSRs, and among mononucleotide SSRs, A/T motifs overwhelmingly dominated over G/C motifs at 417 versus 8. B. formosana exhibited the highest number of SSRs at 63, and B. martiniana the lowest at 50.

Codon usage analysis of 52 filtered coding sequences revealed identical patterns across all ten plastomes, with 29 codons showing frequent usage and 28 of those ending in A or U, while 30 codons were used less frequently and 28 of those ended in C or G. The codon UUA encoding leucine exhibited the highest relative synonymous codon usage value at 1.89 to 1.94, whereas AGC encoding serine showed the lowest at 0.32 to 0.33. Leucine was the most abundant amino acid at 10.97 to 11.07 percent of all codons, and cysteine was the least abundant at 1.12 to 1.16 percent. This strong preference for A/U-ending codons is consistent with numerous angiosperm plastomes and provides critical insight into the molecular evolutionary mechanisms underlying Blumea’s plastid genomes.

Phylogenetic trees reconstructed from 79 shared protein-coding genes across 23 taxa, with two Helianthus species as outgroups, divided the 21 Inuleae species into four major clades. All ten Blumea species formed a highly supported monophyletic group, internally divided into three subclades. Subclade I comprised six species—B. axillaris, B. oxyodonta, B. megacephala, B. repanda, B. sinuata, and B. tenella—within which B. sinuata was sister to B. tenella and B. megacephala was sister to B. repanda. Subclade II consisted of a single species, B. balsamifera, which formed a sister lineage to Subclade I. Subclade III comprised three species—B. aromatica, B. formosana, and B. martiniana—with B. martiniana as the basal taxon and B. aromatica sister to B. formosana. Notably, all three species of Subclade III belong to Section Macrophylla, and the Bayesian inference tree topology was entirely congruent with the maximum likelihood tree for the ten Blumea species.

Positive selection analysis using the MEME method identified 21 genes containing significant episodic positively selected sites along the Blumea lineage, while the FUBAR method detected no pervasive positive selection, indicating that the signals represent lineage-specific events. Among the 21 candidate genes, ycf1 contained the highest number of positively selected sites at 75, followed by ycf2 at 17 and rpoB at 7. These genes are primarily involved in photosynthesis, energy metabolism, gene transcription, and protein synthesis. Photosystem I genes psaA and psbB, photosystem II gene psbA, ATP synthase genes atpA and atpF, and cytochrome b6f complex gene petA support the overall optimization of photosynthetic electron transport and energy metabolism. Multiple NADH dehydrogenase complex genes—ndhA, ndhB, ndhD, ndhF, and ndhG—and the acetyl-CoA carboxylase gene accD are involved in cyclic electron transport and fatty acid synthesis, respectively. Blumea species are predominantly distributed in tropical and subtropical open habitats, exhibiting strong adaptability to varying light environments, and their relatively high photosynthetic efficiency and broad light adaptation range are consistent with the positive selection observed in these photosynthesis-related genes. The ycf1 and ycf2 genes encode key components of the plastid inner envelope protein transport complex, with Ycf1 potentially serving as the protein translocation channel and Ycf2 providing ATPase activity to energize the transport process. These two genes harbored the highest number of positively selected sites in Blumea, suggesting they may play important roles in the adaptive evolution of the genus and even the Asteraceae family. The mixed life forms within subclades—annuals such as B. tenella and B. axillaris alongside perennials such as B. megacephala—suggest that the evolution of life-history strategies in this genus may be more complex than previously recognized, and the unique phylogenetic position of B. balsamifera may indicate it has undergone a different evolutionary history from other Blumea species, possibly retaining ancestral traits. The identified SSRs and candidate barcodes may facilitate molecular marker development and population genetics, providing a genomic foundation for future studies on the phylogeny and adaptive evolution of this medicinally important genus.

Subject of Research: Comparative chloroplast genome analysis and phylogenetic relationships of ten Blumea species in the Asteraceae family

Article Title: Plastomes of Ten Blumea Species (Asteraceae): Comparative Analysis and Phylogenetic Relationships

Article References: Zhao, Y., Yang, W., Qian, C., Zhou, T., Zhang, X., Chen, X., Zhang, R., & Liu, X. (2026). Plastomes of Ten Blumea Species (Asteraceae): Comparative Analysis and Phylogenetic Relationships. Ecology and Evolution, 16(10), Article e74361. https://doi.org/10.1002/ece3.74361

Image Credits: AI Generated

DOI: 10.1002/ece3.74361

Keywords: Blumea, Asteraceae, chloroplast genome, plastome, phylogenetics, positive selection, Inuleae, SSRs, codon usage, DNA barcodes, Balsamifera, Yunnan China