Amorphophallus is an economically and ornamentally important genus in the arum family, Araceae. Traditional identification is challenging because members of the genus show pronounced morphological plasticity, and the timing of leaf and flower emergence can also be separated. The study therefore used chloroplast-genome comparisons to investigate genome structure, codon usage bias and phylogenetic relationships, including the placement of Amorphophallus among other sampled Araceae.

Researchers sequenced, assembled and annotated chloroplast genomes from A. pendulus, A. glaucophyllus, A. fornicatus, A. macrophyllus, A. titanum and A. hewittii using the PacBio Revio platform. When combined with data from 10 additional species, the dataset covered 16 Amorphophallus taxa. Their chloroplast genomes ranged from 152,492 to 185,810 base pairs and displayed the characteristic quadripartite structure, with the study treating differences in genome size, inverted-repeat regions and gene regions as part of the comparative analysis.

Structural variation was detected in the inverted-repeat regions of several genomes. Expansion of this region occurred in A. macrophyllus and A. glaucophyllus, while contraction was found in A. fornicatus. The comparison also identified the chloroplast gene accD as a highly variable region, with a reported nucleotide diversity value of Pi = 0.1217. These results show that the sampled genomes share a broad structural pattern while differing in specific regions relevant to comparative analysis.

The researchers also assessed codon usage across the 16 chloroplast genomes. Codons showed a bias toward sequences ending in A or U rather than G or C, and the analysis identified natural selection as the predominant driving force behind this pattern. Despite variation among species, six codons were classified as optimal in all 16 Amorphophallus species, indicating a common set of preferred codons within the sampled group. The analysis therefore characterized both shared codon-usage features and differences among individual taxa.

Only C-to-U base-editing events were detected in the chloroplast genomes of the 16 species. In other words, the reported editing pattern was directional within this dataset: cytosine-to-uracil changes were observed, but the analysis did not identify the corresponding U-to-C events. The findings add another layer to the comparison of chloroplast genomes, alongside genome-size variation, inverted-repeat changes and codon-usage bias.

One of the study’s most direct identification results came from its search for an informative DNA barcoding region. The researchers identified the genomic segment spanning 80,401–81,200 base pairs as an optimal barcoding window for the sampled species. Five diagnostic loci within that window could discriminate all 16 Amorphophallus species examined. This finding is specific to the comparative sample used in the study and provides a molecular marker for distinguishing the included taxa.

Phylogenetic reconstruction recovered all sampled Amorphophallus species as a monophyletic group, which the researchers divided into three major clades: Continental Asia I, Continental Asia II and Southeast Asia. The Southeast Asia clade contained A. fornicatus, A. pendulus, A. hewittii and A. titanum. The Continental Asia II clade included A. glaucophyllus, A. macrophyllus, A. konjac, A. krausei, A. albus and A. kachinensis. Continental Asia I comprised A. coaetaneus, A. tonkinensis, A. yunnanensis, A. kiusianus, A. muelleri and one accession that was not identified to species.

Within the wider Araceae sample, Symplocarpus was sister to the remaining sampled members of the family. Amorphophallus was most closely related to Syngonium, Xanthosoma, Zomicarpella and Caladium. Together, the chloroplast analyses provide a comparative account of structural evolution, codon usage and lineage relationships in 16 Amorphophallus species, while the five-locus barcoding window offers a sequence-based means of discriminating the taxa included in the study. The authors identify species identification, germplasm conservation and molecular breeding as areas supported by these findings.

Subject of Research: Comparative chloroplast genomics of structure, codon usage and phylogeny in 16 Amorphophallus species

Article Title: Comparative chloroplast genomics in Amorphophallus: revealing codon usage patterns and phylogenetic clades across 16 species

Article References: Shi, H., Lu, Q., Qi, Y., Yang, M., Zhao, M., Gao, P., Huang, F., Zhao, J., Li, L., & Yu, L. (2026). Comparative chloroplast genomics in Amorphophallus: revealing codon usage patterns and phylogenetic clades across 16 species. BMC Plant Biology. https://doi.org/10.1186/s12870-026-09945-1

Image Credits: AI Generated

DOI: 10.1186/s12870-026-09945-1

Keywords: Amorphophallus, chloroplast genomes, comparative genomics, codon usage bias, plant phylogenetics, DNA barcoding, genome structure, Araceae, base editing, species identification, Comparative, chloroplast

The new study, published in the journal Ecology and Evolution, reports the first complete mitochondrial genome for any species in the genus Rhodobryum and compares RNA editing and codon usage bias between its mitochondrial and chloroplast genomes. The research team collected whole moss individuals from Haba Snow Mountain in Shangri-La, Yunnan, China, at coordinates 100.117 degrees E and 27.313 degrees N, depositing the voucher specimen in the Herbarium of Hebei Normal University. Approximately 5 milligrams of green vegetative tissue from silica gel-dried samples was used for DNA extraction, and sequencing libraries were prepared from 300 to 500 base pair DNA fragments. The sequencing was performed on an Illumina HiSeq X Ten platform using 150-base pair paired-end reads, generating over 5.4 gigabases of raw data. After quality assessment with FastQC and adapter trimming with fastp, the mitochondrial genome was assembled using GetOrganelle with SPAdes as the core assembler and the embplant_mt seed reference database, achieving an average coverage depth of 307.8 times.

The assembled mitochondrial genome of R. laxelimbatum measures 103,071 base pairs in length with a GC content of 39.8 percent, closely resembling the mitochondrial genomes of the model moss Physcomitrella patens, which spans 105,340 base pairs with a GC content of 40.6 percent, and Anomodon rugelii, which spans 104,239 base pairs with a GC content of 41.2 percent. The genome contains 40 protein-coding genes, among which nad7 is identified as a pseudogene, along with 24 transfer RNA genes and three ribosomal RNA genes. The gene sequence accounts for 70,419 base pairs, representing 68.32 percent of the total genome, with exons comprising 39,107 base pairs, introns 31,312 base pairs, and intergenic spacers 32,668 base pairs. The GC content of the genes, at 40.99 percent, is slightly higher than the overall genome value, while the intergenic regions exhibit a lower GC content of 37.24 percent. Three pairs of overlapping genes were identified, with atp4 and atp8 overlapping by 8 base pairs, cox1 and atp4 by 4 base pairs, and ribosomal protein S12 and S7 by 4 base pairs.

The mitochondrial genome harbors 3 Group I introns and 22 Group II introns, a composition largely consistent with that reported for other mosses. Two open reading frames of at least 300 base pairs were detected within the introns of cox1 and nad5, measuring 1,869 and 1,602 base pairs respectively. Notably, no open reading frames were found within the 38 intergenic spacer regions that exceeded 300 base pairs, a result significantly different from observations in other mosses. Local BLASTN searches against the chloroplast and nuclear genomes of the model organism revealed no chloroplast-derived exogenous sequences in the mitochondrial intergenic regions, but 26 nuclear-derived sequences were identified, encompassing 8,625 base pairs and representing 8.37 percent of the complete mitochondrial genome. This proportion is significantly lower than those reported for angiosperms and other moss species, likely because nuclear genomic data from Bryales species were not used as references.

In total, 76 simple sequence repeats were identified in the mitochondrial genome, with mononucleotide repeats being the most abundant at 34 instances, or 44.74 percent, followed by dinucleotide repeats at 23 instances, or 30.26 percent, tetranucleotide repeats at 11 instances, complex repeats at 5 instances, trinucleotide repeats at 2 instances, and pentanucleotide repeats at a single instance. The mononucleotide repeats were predominantly composed of A and T bases, while dinucleotide repeats were primarily composed of AT motifs, a pattern consistent with SSR distributions in numerous plant species. Based on established criteria for reliable SSR primer design, seven feasible SSR loci were identified in R. laxelimbatum, providing a practical resource for SSR mining in mosses under limited conditions.

One of the most striking findings concerns RNA editing, the post-transcriptional process that alters RNA sequences after they are transcribed from DNA. The mitochondrial genome contained a total of 189 predicted RNA editing sites, with the majority attributed to C-to-U transitions at 128 sites, while the chloroplast genome contained a significantly higher number at 608 sites, split between 318 C-to-U and 290 U-to-C editing events. This result contradicts the conventional understanding that mitochondrial editing sites are generally more numerous than chloroplast ones in terrestrial plants. For the model bryophyte Physcomitrella patens, only 11 mitochondrial editing sites and a single chloroplast editing site have been annotated in previous studies. The cytochrome c biogenesis-related genes ccmFc and ccmFN exhibited relatively high numbers of editing sites in the mitochondrion, followed by nad5 and sdh3, while in the chloroplast genome, the hypothetical genes ycf2 and ycf1 showed the highest frequencies, followed by the RNA polymerase beta subunit 2 and maturase K genes. In both organellar genomes, 28 types of amino acid transitions were identified, with the conversion from hydrophilic to hydrophobic amino acids being predominant, aligning with previous findings in other plant species.

The codon usage bias analysis, conducted on 28 mitochondrial and 52 chloroplast protein-coding sequences, revealed that both organellar genomes exhibited the highest GC content at the first codon position, followed by the second and third positions, with an overall preference for A and T. Neutrality plot analysis showed no significant correlation between GC12 and GC3 for mitochondrial protein-coding genes, indicating strong constraints from natural selection on mitochondrial codon composition. In contrast, a significant correlation was observed in the chloroplast genome, suggesting that mutation pressure significantly contributes to shaping chloroplast codon usage bias. The ENC-plot analysis demonstrated that the distribution range of ENC values was wider in the mitochondrial genome, with nearly all values above 35, while the chloroplast genome exhibited a narrower range but contained more genes with ENC values below 35, indicating stronger codon usage bias in a subset of chloroplast genes. The PR2-plot revealed distinct base-composition imbalances at synonymous third codon positions, with mitochondria showing T exceeding A and comparable G and C frequencies, while chloroplasts showed both T exceeding A and G exceeding C.

Five optimal codons were identified in the mitochondrial genome, namely UUG, CCC, AAA, AGC, and GGC, while nine optimal codons were found in the chloroplast genome, all ending in A or U, consistent with the general pattern in land plant chloroplasts. Correspondence analysis revealed that mitochondrial codon variation is primarily driven by the magnitude of codon bias and synonymous third-position base composition, as the second principal axis was significantly correlated with ENC and GC3s. In chloroplasts, the first axis was significantly correlated with CAI, Fop, GC3s, and GC content, and the second axis exhibited a significant negative correlation with the Gravy value, indicating that chloroplast codon variation is affected by translational efficiency, base composition, and protein hydrophobicity. Collectively, the primary determinants of codon usage differ between the mitochondria and chloroplasts, suggesting that these organelles possess distinct replication-repair machinery, tRNA pools, and translational systems shaped by different evolutionary constraints.

Perhaps the most evolutionarily significant finding is the confirmation of nad7 pseudogenization in R. laxelimbatum, a non-basal moss species belonging to the Bryales family. Previous studies had documented nad7 pseudogeneization in the mitochondrial genome of Tetraphis pellucida and proposed that such events evolved independently in liverworts, mosses, and hornworts. Because nad7 pseudogeneization or gene loss was absent in non-basal species, its presence in R. laxelimbatum was unexpected, but it is consistent with a report documenting the same phenomenon in another non-basal moss species, Mielichhoferia elongata. Given that the affected species are phylogenetically distantly related and that nad7 functional loss also occurs in liverworts and hornworts, researchers have hypothesized that the functional loss of this gene evolved at least twice during moss evolution. The identification of nad7 pseudogenization in R. laxelimbatum further supports this hypothesis. Only two introns, nad7i140g2 and nad7i209g2, were absent from the R. laxelimbatum mitochondrial genome, closely associated with the functional impairment of the nad7 gene, while the intronic elements present in basal moss lineages and liverworts were gradually lost during the evolutionary diversification of mosses.

This study presents the entire mitochondrial genome of R. laxelimbatum, elucidating its genome size, GC content, gene composition, intron profile, repetitive sequences, and SSR loci, while providing a dual-organellar comparative perspective on RNA editing and codon usage bias. The predicted RNA editing sites in the chloroplast genome far exceeded those in the mitochondrion, contradicting conventional understanding and exhibiting significant differences from the editing characteristics of most moss species. The overall codon usage bias among the organelles was relatively weak and was regulated by multiple evolutionary factors, with mitochondria subjected to stronger constraints from natural selection and chloroplasts more influenced by mutation pressure alongside translational selection, base composition, and protein physicochemical properties. These findings provide crucial theoretical support for further elucidating the post-transcriptional regulatory differences between the chloroplasts and mitochondria of this species and for understanding the evolutionary mechanisms of organellar genomes, while also serving as a reference for optimizing expression systems and producing metabolites with pharmacological implications.

Subject of Research: Mitochondrial genome characteristics and comparative organelle genome analysis of the medicinal moss Rhodobryum laxelimbatum

Article Title: Mitochondrial Genome Characteristics of Rhodobryum laxelimbatum and Comparative Analysis of RNA Editing and Codon Usage Bias With the Chloroplast Genome

Article References: Niu, J., Shi, S., Li, S., Zhao, J., & Li, L. (2026). Mitochondrial Genome Characteristics of Rhodobryum laxelimbatum and Comparative Analysis of RNA Editing and Codon Usage Bias With the Chloroplast Genome. Ecology and Evolution, 16(10), Article e74421. https://doi.org/10.1002/ece3.74421

Image Credits: AI Generated

DOI: 10.1002/ece3.74421

Keywords: Rhodobryum laxelimbatum, mitochondrial genome, chloroplast genome, RNA editing, codon usage bias, moss genomics, organelle evolution, Mitochondrial, Genome, Characteristics, Rhodobryum, laxelimbatum

The research, published in IMA Fungus, analyzed the genome sequences of 150 strains representing 97 species across 15 species complexes and four singletons. The team integrated newly assembled genomes from 20 species preserved at the Microbial Library of the Germplasm Bank of Wild Species from Southwest China with publicly available data from the National Center for Biotechnology Information. By combining phylogenomic reconstruction, codon usage analysis, functional gene annotation, and gene family evolution modeling, the investigators built a multi-dimensional framework for understanding how Colletotrichum species have diversified across different host plants and ecological niches.

One of the most significant findings concerns taxonomy itself. The phylogenomic analysis, based on 160 single-copy orthologous genes, clarified the relationships among species complexes and resolved several misidentifications in public databases. The researchers confirmed that C. menglaense and C. parvisporum should be synonymized with C. siamense, while C. dimorphum and C. nanhuaense belong within C. gloeosporioides. They also reassigned several strains previously mislabeled in genomic repositories, including two strains known for producing the pharmaceutical compound huperzine that had been widely used as C. gloeosporioides but actually represent distinct lineages likely constituting two novel species. These corrections underscore how pervasive taxonomic misannotation in public databases can mislead comparative genomic analyses.

The study revealed substantial variation in genome architecture across the genus. Genome sizes ranged from 43.97 megabases in C. graminicola to 144.24 megabases in C. ledebouriae, a 3.3-fold difference. Repeat sequences, particularly long terminal repeat retrotransposons, emerged as the primary driver of this variation. Genomes with higher repeat content showed elevated frequencies of repeat-induced point mutation (RIP) signatures, which introduce C-to-T mutations in duplicated sequences and shift GC content downward. The researchers found that inefficiency in the RIP defense mechanism against transposable elements likely permitted significant genome expansion in certain lineages, particularly those associated with monocotyledonous herbaceous hosts and pathogenic strains.

Gene number, by contrast, remained relatively stable across the genus, ranging from 10,279 to 18,313 predicted genes with no correlation to genome size. Instead, gene number was positively correlated with non-repetitive genome size and negatively correlated with both gene length and intergenic spacing. The intergenic distance distributions suggested that some species, including C. cuscutae and C. gigasporum, may possess two-speed genome architectures with distinct gene-dense and repeat-rich compartments, a feature previously associated with adaptive evolution in fungal pathogens.

A particularly striking discovery involved codon usage bias. The researchers found that Colletotrichum exhibits pronounced preference for cytosine at third codon positions and avoidance of adenine at those positions, patterns that closely followed phylogenetic relationships rather than host type or lifestyle. Principal component analysis showed that codon usage profiles formed distinct clusters corresponding to species complexes, and a Mantel test confirmed that non-random usage bias tracked the phylogeny of the genus. Neutrality plot and effective number of codons analyses indicated that natural selection, rather than mutation pressure, was the dominant force shaping codon usage patterns.

The team uncovered a bistable co-evolutionary relationship between codon usage bias and tRNA gene abundance. For twofold-degenerate codon families, tRNA gene copy number generally matched the magnitude of codon bias, consistent with theoretical predictions that codon usage and tRNA content co-evolve into mutually adaptive states. However, for codon families with multiple synonymous members, the relationship was more complex, with some codons showing extremely low or absent tRNA gene counts despite strong usage bias. The researchers explained these discrepancies through the wobble hypothesis, noting that the pattern of inconsistencies aligned with the typical number of inosine-modified tRNAs in eukaryotes, suggesting a dual strategy of strict codon-anticodon pairing and flexible wobble pairing.

Functional gene repertoires displayed coordinated shifts across ecological contexts. The secretome, comprising proteins with signal peptides but lacking transmembrane helices, varied from 549 to 1,867 proteins across strains. Broad host-range lineages such as the C. gloeosporioides and C. acutatum species complexes possessed significantly larger secretomes than host-specialized lineages infecting herbaceous monocots or dicots. Strains associated with woody or dicotyledonous hosts had larger secretomes, transporter repertoires, transcription factor families, and secondary metabolite biosynthetic gene clusters compared with those from herbaceous or monocotyledonous hosts. These coordinated expansions suggest system-level genomic adaptation to the more complex cell walls of woody dicot plants, which contain higher hemicellulose and lignin content.

Gene family evolution analysis revealed divergent ecological strategies at key evolutionary nodes. The woody host-dominant lineage showed expansion of GH39 glycoside hydrolases and alkaline proteases, alongside oxidative detoxification enzymes targeting aromatic compounds and alkaloids, including cytochrome P450 monooxygenases and FAD-dependent monooxygenases. In contrast, herbaceous-associated lineages preferentially expanded laccases, aldo/keto reductases, and glutathione-dependent enzymes, forming a rapid-response system for neutralizing reactive phenolics and aldehydes. Within herbaceous lineages, dicot-infecting clades expanded glucan- and pectin-degrading enzymes along with nitrile and aldehyde detoxification genes, while monocot-infecting clades underwent pronounced contraction of these same families, consistent with the lower pectin content in monocot cell walls.

The C. gloeosporioides species complex emerged with the most comprehensive functional expansion, combining oxidative and rapid-response detoxification systems, hemicellulose- and pectin-degrading enzymes, adhesion proteins, and broad transcription factor repertoires. This genomic versatility likely underpins its exceptionally broad host range across angiosperms. Meanwhile, endophytic lineages exhibited convergent contraction of gene families related to cell adhesion, cellulose degradation, and cell wall remodeling, indicating functional streamlining toward reduced invasiveness and improved host compatibility. Together, these findings establish a robust phylogenomic framework for Colletotrichum and illuminate the evolutionary mechanisms by which fungal pathogens navigate the complex interface between phylogenetic constraint and ecological adaptation.

Subject of Research: Comparative genomics and ecological adaptation of the fungal plant pathogen genus Colletotrichum

Article Title: Genus-wide comparative genomics of Colletotrichum reveals evolutionary conservation and divergent ecological adaptations

Article References: Shen, J.-X., Qiao, M., Hong, J.-N., Dai, X.-W., Li, W.-J., Liu, S.-Q., & Yu, Z.-F. (2026). Genus-wide comparative genomics of Colletotrichum reveals evolutionary conservation and divergent ecological adaptations. IMA Fungus, 17, Article e195303. https://doi.org/10.3897/imafungus.17.195303

Image Credits: AI Generated

DOI: 10.3897/imafungus.17.195303

Keywords: Colletotrichum, comparative genomics, anthracnose, plant pathology, codon usage bias, transposable elements, gene family evolution, host adaptation, secretome, endophytes, phylogenomics, fungal evolution

The research team, led by Ying Xue and Yunpeng Gai of Beijing Forestry University together with colleagues including Guy Smagghe of the Vrije Universiteit Brussel, focused on a natural experiment that has fascinated evolutionary biologists for decades. Within M. sativa subsp. falcata, both diploid individuals with 16 chromosomes and tetraploid individuals with 32 chromosomes occur in nature. Whole-genome duplication, or polyploidization, is one of the most dramatic events that can happen to a plant lineage, reshaping nuclear gene dosage, silencing duplicate genes, and often triggering sweeping changes in cell size and physiology. Whether and how these nuclear upheavals ripple outward into the chloroplast genome, however, has remained poorly resolved, particularly within a single subspecies where cytotypes can be directly compared.

Chloroplasts carry their own small circular genomes, typically around 120,000 to 160,000 base pairs in flowering plants, descended from free-living cyanobacteria that were engulfed by an ancestral eukaryotic cell more than a billion years ago. Because plastid genomes are maternally inherited in most legumes, relatively compact, and present in thousands of copies per cell, they are workhorses of plant evolutionary biology, used to trace lineages, identify species, and reconstruct geographic histories. The team assembled the complete chloroplast genome of the diploid falcata cytotype at 125,934 base pairs and the tetraploid cytotype at 125,770 base pairs, a difference of just 164 base pairs. Both genomes showed nearly identical guanine-cytosine content of approximately 33.80 percent, and gene counts differed only marginally between the two ploidy levels.

That near-identity is itself a finding. If polyploidization imposed strong selective pressure on the plastid compartment, one might expect shifts in genome size, gene content, or structural organization between cytotypes. Instead, the comparison revealed that the chloroplast genomes of diploid and tetraploid falcata are essentially interchangeable in size, composition, and architecture. The authors conclude that polyploidization exerts limited influence on chloroplast genome size, gene content, and structural organization, and that strong functional constraints on the plastid genome are preserved across ploidy levels. In other words, whatever turbulence whole-genome duplication stirs up in the nucleus, the chloroplast keeps its house in order.

Beyond the ploidy comparison, the study dug into the fine-grained mechanics of chloroplast protein evolution through codon usage bias, the phenomenon by which genes preferentially use certain synonymous codons over others. The genetic code is degenerate, meaning most amino acids are specified by multiple three-letter codons, and organisms are far from random in choosing among them. In the Medicago chloroplast genomes, the analysis revealed a dominant preference for codons ending in adenine or uracil, the A/U-ending codons characteristic of AT-rich plastid genomes. Crucially, the statistical signatures indicated that this bias is shaped primarily by natural selection rather than by mutation pressure or random drift alone, suggesting that translational efficiency and accuracy continue to exert measurable selective forces on chloroplast coding sequences.

The team also examined the evolutionary forces acting on individual protein-coding genes by calculating the ratio of nonsynonymous substitution rates to synonymous substitution rates, abbreviated Ka/Ks. A ratio below one indicates purifying selection, the process by which natural selection removes harmful mutations and preserves protein function; a ratio near or above one would suggest neutral evolution or positive selection. Across the Medicago chloroplast gene set, 77.1 percent of genes showed Ka/Ks values below 0.5, a hallmark of strong purifying selection. The constraint was especially pronounced in genes involved in photosynthesis and energy metabolism, the core functions that the chloroplast exists to perform. This deep conservation underscores how little tolerance the photosynthetic machinery has for change, even across a genus spanning dozens of species and millions of years of divergence.

Not every region of the chloroplast genome is locked down, however, and that variability is precisely what makes the plastome useful for breeding and identification. The researchers mapped simple sequence repeats, short tandemly repeated DNA motifs that mutate rapidly and are abundant in the falcata chloroplast genomes. Most of these SSR loci were mononucleotide repeats, runs of a single base such as poly-A or poly-T tracts, and the vast majority sat in non-coding regions rather than within genes. Because these hypervariable stretches evolve quickly while flanking sequences remain conserved, they are ideal raw material for developing chloroplast-derived molecular markers that can distinguish accessions, assess genetic diversity in germplasm collections, and fingerprint cultivars without the complications of nuclear heterozygosity.

To place falcata in its evolutionary context, the team reconstructed phylogenetic relationships across 30 Medicago species and performed collinearity analysis, which compares the order and orientation of genes along the chloroplast genome. Both approaches demonstrated high structural conservation among closely related species, with gene order rearrangements rare and localized. In the resulting trees, the diploid and tetraploid falcata chloroplast genomes clustered together with other M. sativa accessions, confirming the subspecies-level relationships inferred from morphology and nuclear data and reinforcing the view of falcata as the wild gene pool most closely allied to cultivated alfalfa. The tight clustering also validates chloroplast genomes as reliable barcodes for untangling relationships within a genus famous for taxonomic complexity, driven in part by autopolyploidy and hybridization.

The practical implications extend well beyond evolutionary theory. Yellow-flowered alfalfa is regarded as an essential genetic resource for forage improvement, biofuel production, and sustainable agriculture, and its superior cold tolerance and drought resistance make it suitable for cultivation in marginal and climate-stressed environments where common alfalfa fails. As global agriculture confronts hotter summers, colder snaps, and increasingly unreliable rainfall, the wild cytotypes of falcata represent a reservoir of adaptive alleles that breeders are eager to tap. The chloroplast genomic resources and candidate SSR loci delivered by this study can accelerate germplasm evaluation, phylogenetic inference, and marker-assisted breeding of Medicago forage cultivars, allowing breeders to track valuable cytoplasms through crossing programs and to certify the maternal lineage of elite hybrids.

The study also contributes to a broader scientific conversation about how organellar genomes respond to nuclear genome doubling. Because chloroplasts and mitochondria encode proteins that must interface with thousands of nuclear-encoded partners, polyploidization could in principle disrupt these co-adapted systems and force compensatory evolution in the organelle. The falcata comparison suggests that, at least for the chloroplast, such disruption is minimal: the plastome’s size, GC content, gene inventory, and codon preferences ride out whole-genome duplication essentially unchanged, buffered by intense purifying selection. For a crop genus that feeds livestock across the globe and anchors grassland agriculture from China to North America, that stability is good news. It means the chloroplast markers developed here should remain robust across ploidy levels, and it means the wild, cold-hardy cytoplasms of falcata can be introduced into breeding lines without unpredictable plastid side effects. As sequencing costs continue to fall, comparative plastomics of this kind is poised to become a routine tool in the effort to future-proof the world’s forage crops, one chloroplast genome at a time.

Subject of Research: Comparative chloroplast genome evolution and polyploidy in Medicago sativa subsp. falcata

Article Title: Comparative chloroplast genomics of Medicago: codon usage bias, purifying selection, and phylogenomics

Article References: Xue, Y., Liang, X., Zhao, W., Smagghe, G., Shen, L., Li, S., Wang, Z., Gao, F., Li, D., Zhang, T., & Gai, Y. (2026). Comparative chloroplast genomics of Medicago: codon usage bias, purifying selection, and phylogenomics. BMC Plant Biology. https://doi.org/10.1186/s12870-026-10073-z

Image Credits: AI Generated

DOI: 10.1186/s12870-026-10073-z

Keywords: Medicago, chloroplast genome, polyploidy, codon usage bias, purifying selection, simple sequence repeats, phylogenomics, alfalfa, forage breeding, plant molecular biology, genomics, Comparative

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

Codons are the three-letter DNA words that specify amino acids during protein synthesis. Because the genetic code is degenerate, most amino acids can be encoded by several synonymous codons, yet organisms rarely use these synonyms equally. This unevenness, known as codon usage bias, is far from trivial. It influences how efficiently genes are translated into proteins, how quickly chloroplasts can respond to environmental stress, and even how successfully a foreign gene can be expressed if it is engineered into a plant. Understanding which codons a species prefers, and why, therefore has both evolutionary and very practical implications.

The eight species examined span the diversity of cultivated and related millets: barnyard millet (Echinochloa esculenta), finger millet (Eleusine coracana), proso millet (Panicum miliaceum), little millet (Panicum sumatrense), kodo millet (Paspalum scrobiculatum), pearl millet (Pennisetum glaucum), foxtail millet (Setaria italica) and sorghum (Sorghum bicolor). All belong to the grass family Poaceae, and all are prized for their nutritional value and resilience to drought, making them increasingly important crops in a warming world. Yet, as the authors note, comprehensive analyses of their chloroplast genomic architecture, and of codon usage in particular, had been lacking.

To fill that gap, the team deployed a battery of standard but powerful bioinformatic measures. The relative synonymous codon usage (RSCU) quantifies how often each codon appears compared with what would be expected if all synonyms were used equally. The effective number of codons (ENC) summarizes how many distinct codons a gene actually draws upon, ranging from 20, meaning extreme bias with only one codon per amino acid, to 61, meaning no bias at all. GC content and GC3, the fraction of guanine and cytosine at the third, wobble position of codons, reveal compositional tendencies. Neutrality plots and parity rule 2 (PR2) analysis then help distinguish between two competing forces: mutational pressure, which pushes base composition in a direction dictated by the chemistry of DNA repair and replication, and natural selection, which favors codons that are translated more efficiently or accurately.

The results were strikingly consistent. Across all eight species, the chloroplast genomes showed a strong preference for codons ending in adenine or thymine. GC3 values hovered around 29 to 30 percent, a clear signature of compositional bias toward AT-richness. Thirty high-frequency codons, defined as those with an RSCU greater than 1, were identified in each genome, and every one of them ended in A or T. Some of these favored codons, such as TGT, CCT and TTA, were shared across all eight species, hinting at a common ancestral or functional logic. But many optimal codons differed from species to species, suggesting that each lineage has fine-tuned its translational machinery in its own way.

The neutrality plot analysis delivered the study’s central evolutionary verdict. If mutation were the dominant force, GC3 would track the overall GC content of genes closely, and the neutrality plot would show a strong diagonal relationship. Instead, the ENC values and neutrality results indicated that natural selection has played the decisive role in shaping codon usage bias in these chloroplast genomes, outweighing mutational pressure. This makes biological sense for an organelle whose genes encode the photosynthetic machinery on which the entire plant depends. Efficient, accurate translation of photosystem proteins, rubisco subunits and ATP synthase components confers a direct fitness advantage, particularly in the harsh, water-limited environments where millets excel.

The PR2, or parity rule 2, analysis added another layer of nuance. Under purely neutral conditions, adenine should equal thymine and guanine should equal cytosine within a DNA strand. Deviations from this parity reveal directional forces at work. In the millet chloroplast genomes, the PR2 plots showed a preference for thymine over adenine and for guanine over cytosine at the third codon position, a pattern consistent with selection acting on translational efficiency rather than with random drift. Correspondence analysis, a multivariate technique that clusters genes according to their codon usage signatures, revealed that most genes share a similar codon usage profile, but a subset of genes deviated noticeably, possibly reflecting differences in gene function, expression level or adaptation to environmental conditions.

Why does this matter beyond evolutionary theory? Chloroplasts have become a favored target for plant genetic engineering. They are maternally inherited in many crops, which reduces gene flow through pollen, they support very high levels of foreign protein expression, and they offer a natural containment strategy. But chloroplast expression depends on the translational preferences of the plastid itself. Codon optimization, rewriting a transgene to match the host’s preferred codons, has been shown in prior work to dramatically enhance expression levels and yield insights into chloroplast translation. The codon preference data assembled for these eight millets provide exactly the reference table that synthetic biologists would need when designing transgenes for millet improvement, whether the goal is stress tolerance, enhanced photosynthesis or nutritional fortification.

The study also contributes to phylogenetics. Codon usage patterns can carry historical signal, and comparative chloroplast genome analyses across the Poaceae have already been used to resolve relationships among grass lineages. By documenting shared and species-specific optimal codons, the researchers have generated a dataset that can inform both evolutionary reconstructions and the identification of conserved regulatory features in millet chloroplasts. Given that millets are increasingly promoted as climate-resilient staples for future food security, any tool that accelerates their improvement is welcome.

For a crop group often described as orphan grains, overlooked by mainstream breeding programs despite their nutritional virtues, this work represents a meaningful step into the genomic era. It confirms that the chloroplast genomes of millets are not passive repositories of sequence but actively curated libraries, their codon choices sculpted by selection to keep the photosynthetic engine running smoothly under stress. As gene editing and chloroplast transformation mature, the humble codon table may turn out to be one of the most valuable things these ancient grains can teach us.

Subject of Research: Codon usage bias in the chloroplast genomes of eight millet species

Article Title: Comprehensive Analysis of Codon Usage Bias Pattern in Chloroplast Genomes of Eight Millet Species

Article References: Nishanth, J. B., Balaji, R., Govindhan, G., & Somasundaram, S. (2026). Comprehensive Analysis of Codon Usage Bias Pattern in Chloroplast Genomes of Eight Millet Species. Indian Journal of Genetics and Plant Breeding, 86(2), 177-189. https://doi.org/10.1007/s44489-026-00018-z

Image Credits: AI Generated

DOI: 10.1007/s44489-026-00018-z

Keywords: codon usage bias, chloroplast genome, millets, RSCU, effective number of codons, GC3 content, neutrality plot, natural selection, translational selection, codon optimization, Poaceae, plant biotechnology

The research team, led by Xing-Xing Shen of Zhejiang University together with co-first authors Xianfeng Mi and Guo-Zheng Ou and colleague Yixiao Zhu, leveraged a large-scale dataset originally assembled by Tao and colleagues comprising 472 insect species spanning 19 orders. From each species they extracted amino acid sequences of 13 mitochondrial protein-coding genes and 1,367 single-copy nuclear protein-coding genes. Using concatenation-based maximum likelihood inference with the IQ-TREE software and 1,000 ultrafast bootstrap replicates, they reconstructed two genome-scale phylogenies, one from the mitochondrial data under the best-fitting mtInv+F+G4 model and one from the nuclear data under the LG+G4 model. Both trees were exceptionally well supported: 98.30 percent of nodes in the nuclear phylogeny and 88.11 percent of nodes in the mitochondrial phylogeny carried bootstrap values of 90 percent or higher, and within the five largest insect orders, Coleoptera, Diptera, Hemiptera, Hymenoptera, and Lepidoptera, highly supported nodes remained above 95 percent in nuclear trees and above 80 percent in mitochondrial trees.

That high support made the conflict all the more striking. When the researchers compared the two topologies using the normalized Robinson-Foulds distance, a metric ranging from 0, indicating identical trees, to 1, indicating maximal disagreement, they obtained a value of 0.306 for the full dataset, a considerable level of topological discordance that could not be dismissed as an artifact of weak phylogenetic signal. The degree of conflict also varied substantially among the major orders. Lepidoptera, the order containing butterflies and moths, showed the greatest incongruence with an nRF distance of 0.446, while Hymenoptera, the bees, wasps, and ants, showed the least at 0.158. Because both trees were statistically robust, the discordance had to reflect genuine differences in the evolutionary histories or compositional properties of the two genomes rather than simple failure to resolve branching order.

One obvious suspect was model choice. Mitochondrial genomes evolve faster than nuclear genomes and display strong compositional heterogeneity, so critics might argue that standard models simply fail to capture the true substitution process in mitochondrial sequences, generating misleading trees. To test this, the team rebuilt the mitochondrial phylogeny from concatenated amino acid sequences using a site-heterogeneous mixture model, mtInv+F+G4+C60, which allows substitution patterns to vary across sites in far more flexible ways. The result was unambiguous: the more complex model did not eliminate the mito-nuclear discordance. This finding aligns with a growing body of work suggesting that increasing evolutionary model complexity improves fit to the data but does not necessarily resolve gene tree conflict, meaning the source of the disagreement had to lie elsewhere.

The researchers then cast a wide net, systematically quantifying nine gene properties for every mitochondrial and nuclear gene: alignment length, GC content, amino acid substitution saturation, effective number of amino acids, proportion of constant sites, proportion of parsimony-informative sites, external branch length, average bootstrap support value, and treeness, defined as the proportion of internal branch lengths over all branch lengths. Their central analytical innovation was a property-matching strategy. For each property, they selected 13 nuclear genes whose values closely matched those of the 13 mitochondrial genes, for example nuclear genes whose alignment lengths fell within plus or minus 5 percent of the corresponding mitochondrial genes, concatenated these mito-like nuclear genes, built a phylogeny, and measured its topological distance to the mitochondrial reference tree. This sampling and tree-building procedure was repeated 20 times for each property and compared against a background in which 13 nuclear genes were chosen at random, which yielded a median nRF of 0.334.

Against that background, only one property made a difference. When nuclear genes were selected to match the GC content of mitochondrial genes, the resulting trees became significantly more similar to the mitochondrial phylogeny, while matching on alignment length, saturation, amino acid diversity, or any of the other properties failed to reduce the discordance. The compositional gulf between the two genomes is dramatic: mitochondrial genes exhibited significantly lower GC content than nuclear genes, with mitochondrial codons showing a strong bias against codons ending in G or C and a marked preference for A/T-ending codons, whereas nuclear codons displayed a more balanced pattern across the first, second, and third codon positions. This compositional bias cascades into protein composition, since the mitochondrial genome favors amino acids encoded by low-GC codons such as leucine, isoleucine, phenylalanine, methionine, asparagine, and tyrosine, while the nuclear genome favors amino acids encoded by high-GC codons such as alanine, arginine, glutamate, and aspartate.

The GC effect extends beyond the boundary between the two genomes and into the structure of the nuclear genome itself. When the researchers compared pairwise topological differences among mitochondrial genes, they found that mitochondrial genes were more topologically similar to one another than nuclear genes were to each other, consistent with their shared maternal inheritance. Yet even within the mitochondrial genome, low-GC genes produced trees more similar to each other than high-GC genes did. To probe the nuclear side, the team divided 1,367 nuclear genes into three non-overlapping groups by GC content, a low-GC group at 38 to 40 percent, a medium-GC group at 42 to 44 percent, and a high-GC group at 46 to 48 percent, with each group spanning exactly a 2 percent range to ensure comparable variation. Pairwise comparisons of gene trees within each group revealed a clear trend: topological differences increased steadily with GC content, meaning low-GC nuclear genes are more topologically consistent with one another than high-GC nuclear genes are.

Intriguingly, GC content also tracks functional differentiation within the nuclear genome. Using Gene Ontology analysis, in which detailed GO terms were consolidated into 18 broader biological process categories, the researchers found that high-GC nuclear genes were disproportionately involved in complex biological processes including biological regulation, developmental processes, response to stimulus, multicellular organismal processes, growth, and homeostatic processes, while low-GC nuclear genes represented the greatest proportion of genes involved in metabolic processes. This functional stratification suggests that GC content is not merely a technical nuisance for phylogenetic inference but a biologically meaningful axis of genome organization, one that correlates with gene expression patterns and evolutionary dynamics documented in prior studies of codon usage bias and compositional evolution across insect genomes.

What mechanism might link nucleotide composition to tree topology? The authors point to GC-biased gene conversion, a process in which recombination preferentially fixes G and C alleles over A and T during DNA repair, as a plausible driver. GC-rich genomic regions are thought to experience higher recombination rates, and biased conversion can distort substitution patterns in ways that mislead tree-building algorithms, potentially generating greater topological discordance among high-GC genes. The study’s conclusions are notably conservative in what they rule out: gene alignment length, inheritance pattern, and evolutionary model complexity all failed to explain the observed conflicts, overturning several common assumptions in the field. Because mitochondrial genomes are shorter than nuclear genomes, differences in alignment length have long been considered a contributing factor, yet matching on length had no effect, and the persistence of discordance among mitochondrial genes grouped by GC content shows that maternal inheritance alone cannot guarantee topological agreement.

The authors are careful to acknowledge the limits of their analysis. Multiple biological processes are known to generate nuclear phylogenomic incongruence, including incomplete lineage sorting, introgression, hybridization, sex-biased dispersal, and horizontal gene transfer, and future work integrating these factors will be essential to test whether they also contribute to mito-nuclear discordance. The team emphasizes that their aim was to elucidate the cause of discordance rather than to declare which genome tells the truer evolutionary story. Even so, the practical implications are immediate for anyone reconstructing insect phylogenies: sampling nuclear genes whose GC content resembles that of mitochondrial genes, so-called mito-like nuclear genes, can measurably reduce topological conflict, and researchers interpreting mitochondrial barcodes or mitogenomic trees should now weigh compositional bias as a first-order consideration. By bridging molecular composition and evolutionary inference, the study transforms GC content from a footnote in methods sections into a central character in one of phylogenetics’ most persistent mysteries.

Subject of Research: Mito-nuclear phylogenetic discordance and the role of GC content in insect genome evolution

Article Title: Dissecting discordance of mitochondrial and nuclear phylogenetic trees in insects

Article References: Mi, X., Ou, G.-Z., Zhu, Y., & Shen, X.-X. (2025). Dissecting discordance of mitochondrial and nuclear phylogenetic trees in insects. Crop Health, 3(1), Article 23. https://doi.org/10.1007/s44297-025-00062-3

Image Credits: AI Generated

DOI: 10.1007/s44297-025-00062-3

Keywords: phylogenetics, mito-nuclear discordance, insects, GC content, mitochondrial genome, nuclear genome, insect genomics, GC-biased gene conversion, Gene Ontology, Robinson-Foulds distance, codon usage bias, evolutionary models

Chloroplasts, the organelles that convert sunlight into the chemical energy sustaining nearly all life on Earth, descend from a cyanobacterial ancestor engulfed roughly one to one and a half billion years ago. Their genomes retain a semi-autonomous, circular architecture with a large single-copy region, a small single-copy region, and two inverted repeats that help stabilize the molecule through homologous recombination. Within these genomes, codon usage bias, the nonrandom use of synonymous codons, reflects the interplay of natural selection, mutation pressure, and genetic drift. Because synonymous codons encode the same amino acid, their frequencies offer a sensitive record of the evolutionary forces operating on a genome, from translational efficiency demands to directional mutational biases. Studying codon usage in a basal lycophyte like Isoetes therefore provides a rare window into how these forces have balanced each other across hundreds of millions of years of vascular plant evolution.

The research team assembled complete chloroplast genome sequences from 60 Isoetes species drawn from public databases, reannotating each sequence with reference-guided pipelines and manually verifying gene structures to ensure comparability. Despite the genus spanning continents and ecological niches, the plastomes proved remarkably uniform in size, ranging from 142,880 base pairs in Isoetes harleyi to 145,535 base pairs in Isoetes malinverniana. Overall GC content hovered near 37.95 percent with minimal interspecific variation, confirming the strongly AT-rich character of these genomes. Positional analysis of codons sharpened the picture: GC content was highest at first codon positions, intermediate at second positions, and dramatically lowest at third positions, the sites most sensitive to synonymous substitution. The effective number of codons averaged 47.67, approaching the theoretical maximum of 61 and indicating that codon usage bias across the genus is, in genome-wide terms, relatively weak.

The relative synonymous codon usage analysis added a strikingly consistent signature. Across all 60 species, every codon with an elevated frequency, indicating preferential use, terminated in either A or U, mirroring the AT-rich genomic background. The leucine codon UUA stood out as strongly enriched, while the alanine codon GCU showed elevated use despite containing guanine and cytosine at its first two positions, hinting that factors beyond simple third-position nucleotide bias, such as translational considerations or functional constraints, may influence preference for certain amino acids. The fact that these high-frequency codons were conserved across the entire genus suggests that synonymous codon choice is evolutionarily stable in Isoetes, a stability that underscores how deeply conserved the compositional fabric of these plastomes has remained since the lineage’s ancient origin.

Simple sequence repeats, the short tandemly repeated motifs scattered through genomes, told a complementary story. The team identified 5,084 SSR loci across the 60 plastomes, with mononucleotide repeats dominating at nearly 45 percent of all loci and dinucleotide repeats adding another fifth. This prevalence of short, A/T-rich motifs aligns with the AT-biased mutational character of the genome and points to replication slippage as a plausible engine of fine-scale variation. Crucially, most SSRs clustered in noncoding regions rather than within protein-coding sequences, a distribution consistent with weaker functional tolerance for repeat length changes in coding regions, where an insertion or deletion could disrupt a reading frame. The coexistence of broadly shared repeat motifs with species-specific ones offers a practical dividend: these variable loci can serve as molecular markers for distinguishing closely related quillwort species, tracing population structure, and guiding conservation of endangered taxa.

The inverted repeat boundaries, long recognized as dynamic elements of plastome evolution, proved strikingly conserved within Isoetes. Pseudogenes ycf1 and ycf2 were consistently retained near the small single-copy junction, with only minor boundary shifts observed in a handful of species such as Isoetes yunguiensis and Isoetes japonica. By contrast, the closely related spike-moss Selaginella showed far more dramatic boundary dynamics, with substantial expansions and gene duplications. This contrast suggests fundamentally different strategies of chloroplast genome organization between the two lycophyte lineages, with Isoetes adhering to an exceptionally conservative architectural template even as its relatives restructured their plastomes more freely.

The most analytically revealing results came from comparing observed codon usage against theoretical expectations. Using the effective number of codons plotted against GC content at synonymous third positions, the researchers evaluated 4,523 gene records across the genus. More than 61 percent fell below Wright’s expected curve, and 22.91 percent deviated markedly, meaning their codon usage was more biased than mutational composition alone could explain. These marked deviations were not randomly distributed. Photosystem II genes accounted for nearly half of all marked deviations, followed by large ribosomal protein genes, photosystem I genes, and small ribosomal protein genes. In contrast, NADH dehydrogenase genes, RNA polymerase genes, and the RuBisCO large subunit showed no marked deviations. Parity rule 2 analysis reinforced the mutational signal, revealing systematic T-over-A and G-over-C preferences at third positions, while neutrality plots showed regression slopes near zero, indicating that third-position composition varies almost independently of the functionally constrained first and second positions.

Selection pressure analysis at the protein level confirmed pervasive purifying selection across the plastome. Nearly all chloroplast genes displayed ratios of nonsynonymous to synonymous substitution rates well below one, demonstrating that deleterious mutations are being efficiently weeded out. Core photosynthetic genes such as psaB, psbC, and psbD showed extremely low ratios, generally between 0.08 and 0.09, reflecting the intense functional conservation of the photosynthetic machinery, while rbcL, encoding the carbon-fixing enzyme RuBisCO, showed a median ratio of just 0.15. Yet a handful of NADH dehydrogenase complex genes, most notably ndhC, displayed elevated ratios, with a mean of 1.81 and more than half of comparisons exceeding one. The authors interpret these signals cautiously, noting that elevated values may reflect relaxed selective constraints, lineage-specific rate acceleration, or functional divergence rather than definitive adaptive evolution, but the pattern marks the NDH complex as the most dynamic corner of an otherwise deeply conservative genome.

The broader implications reach beyond evolutionary theory into urgent conservation practice. Many Isoetes species are threatened worldwide by habitat destruction, eutrophication, and pollution; in China, every known species enjoys first-grade national protection. The hypervariable loci identified here, including atpF, ycf1, ccsA, matK, and ndhF, together with species-specific SSR markers, provide a toolkit for phylogeographic reconstruction and population monitoring of these critically endangered plants. The study’s hypothesis-driven framework also clarifies a conceptual model for basal vascular plants: genome-wide mutational bias sets the compositional stage, gene-specific constraints concentrate in essential functional modules such as the photosystems and ribosomes, and localized sequence divergence accumulates in noncoding hotspots without disturbing the overall architecture. Whether these codon usage patterns carry adaptive significance in the fluctuating wetland environments quillworts inhabit, some of which demand remarkable physiological feats like the crassulacean acid metabolism evolved by Isoetes howellii, remains an open question. Testing it will require integrating ecological data, transcriptomics, and plastid translational profiles with the genomic baseline this study has now established for one of Earth’s oldest living plant lineages.

Subject of Research: Codon usage bias and chloroplast genome evolution in the ancient aquatic lycophyte genus Isoetes

Article Title: Mutation Pressure and Gene‐Specific Selection Shape Codon Usage Bias in the Chloroplast Genomes of Isoetes, an Ancient Aquatic Vascular Plant Lineage

Article References: Li, B., Xue, Y., Zhang, D., Smagghe, G., Liang, S., Wang, S., Ge, Y., Aimaitijiang, Z., & Gai, Y. (2026). Mutation Pressure and Gene‐Specific Selection Shape Codon Usage Bias in the Chloroplast Genomes of Isoetes , an Ancient Aquatic Vascular Plant Lineage. Ecology and Evolution, 16(9), Article e74216. https://doi.org/10.1002/ece3.74216

Image Credits: AI Generated

DOI: 10.1002/ece3.74216

Keywords: Isoetes, chloroplast genome, codon usage bias, mutation pressure, lycophyte, purifying selection, plastome evolution, simple sequence repeats, photosystem genes, conservation genomics, living fossil, wetland plants

Triozidae belongs to the superfamily Psylloidea, the jumping plant lice, a lineage of small sap-sucking hemipterans that feed on an enormous range of host plants. Several psyllids are notorious agricultural pests, and even species that are not direct crop pests can act as vectors of plant pathogens or cause damage through the galls they induce. Despite their ecological and economic significance, Triozidae has remained poorly represented in mitochondrial genomic databases, and the evolutionary relationships among its genera have never been fully resolved. Mitochondrial genomes, compact and maternally inherited, are among the most widely used tools for untangling insect phylogeny, which made the scarcity of Triozidae mitogenomes a conspicuous gap in insect genomics.

The newly sequenced Bactericera gobica mitogenome is 14,865 base pairs long and contains the standard set of 37 mitochondrial genes: 13 protein-coding genes, 22 transfer RNA genes, and 2 ribosomal RNA genes. Its gene arrangement follows the typical insect pattern, and the genome displays a strong adenine-thymine bias, with AT content reaching 72.1 percent, a hallmark of insect mitochondrial DNA. By integrating this genome with nearly all previously published Triozidae mitogenomes, the researchers constructed a dataset that allows comparative analyses at a scale previously impossible for the family, covering genome architecture, nucleotide composition, codon usage, and gene order.

The comparative analysis revealed a strikingly conservative genomic architecture across the family. Gene order is highly conserved among Triozidae species, and the lengths of the protein-coding genes show little variation from one species to another. Where genomes do differ in size, the variation is concentrated almost entirely in the hypervariable control region, the non-coding stretch of the mitochondrial genome that contains the origin of replication and transcription. This pattern, in which a single labile region accounts for most of the length differences among otherwise stable genomes, mirrors findings from many other insect groups and underscores the control region’s role as the principal arena of mitochondrial genome size evolution.

Codon usage within the protein-coding genes also yielded a clear signal. The team found that codon usage bias was significantly correlated with nucleotide composition across the mitogenomes, indicating that mutational pressure, rather than natural selection acting on translational efficiency, is the primary driver of codon preference in Triozidae mitochondria. In other words, the skewed AT-rich composition of the genome shapes which synonymous codons are favored, a conclusion consistent with the compositional forces known to dominate mitochondrial evolution in insects. Such results matter for downstream phylogenetic work, because compositional bias can mislead tree-building methods if it is not modeled appropriately.

The phylogenetic analyses, based on the 13 protein-coding genes and the 2 ribosomal RNA genes, produced a well-resolved picture of relationships within the family. The analyses strongly supported the monophyly of Triozidae as a whole, confirming that the family represents a single evolutionary lineage. Within it, three genera emerged as robust, coherent units: Bactericera, Egeirotrioza, and Pariaconus were each recovered as monophyletic with strong statistical support. These results provide reassurance that much of the family’s generic framework rests on genuine evolutionary boundaries rather than superficial morphological resemblance.

The exception is the genus Trioza itself. Rather than forming a single clade, species assigned to Trioza were distributed across multiple distinct lineages scattered through the family tree, a textbook case of polyphyly. This means that the morphological characters traditionally used to define Trioza have converged or been retained across unrelated lineages, and that the genus as currently circumscribed mixes species that are not each other’s closest relatives. The authors argue that this confirmed polyphyly highlights the urgent need for taxonomic revision within Triozidae, a process that will likely involve splitting the genus and redefining its boundaries using both molecular and morphological evidence.

Beyond relationships, the study reached back in time. Divergence time estimation, calibrated within the phylogenetic framework, suggested that Triozidae originated in the later Paleogene, the epoch that followed the extinction of the non-avian dinosaurs and saw the rise of modern plant lineages. The family’s major diversification, however, came much later, during the early to mid-Miocene, a period characterized by significant global cooling, the expansion of grasslands, and the diversification of many flowering plant lineages. That timing hints at a possible link between the evolutionary radiation of these plant lice and the ecological transformations of the Miocene, although testing such associations will require denser sampling and richer host-plant data.

The practical implications extend beyond pure systematics. A robust molecular framework for Triozidae is a prerequisite for identifying pest species accurately, tracing the spread of psyllid-borne plant pathogens, and designing targeted management strategies. Because psyllids are often intercepted in trade and quarantine, reliable DNA-based identification grounded in a sound phylogeny can directly support biosecurity. The expanded mitogenomic resource assembled in this study gives researchers a reference set against which new specimens and new species can be rapidly placed, and it establishes a baseline for future studies incorporating whole mitochondrial genomes, nuclear markers, or even full genome-scale datasets.

The work was carried out by Jiangtao Feng, Yanting Yang, Jie Fan, Jia He, Luochong Wang, Fan Song, and Weidong Huang, with contributions from institutions including China Agricultural University in Beijing and the Ningxia Academy of Agriculture and Forestry Science, and was supported by the Key Research and Development Program Project of Ningxia Hui Autonomous Region. As the authors conclude, the study offers comprehensive insights into the mitochondrial genomic architecture and evolutionary history of Triozidae, confirms that one of its flagship genera is an artificial assemblage, and lays the groundwork for a deeper understanding of psyllid evolution. For a family of insects that has flown under the genomic radar for so long, the message is clear: their family tree is due for a thorough rewrite.

Subject of Research: Mitochondrial genomics and phylogenetics of the jumping plant louse family Triozidae

Article Title: Exploring the mitogenomics of Triozidae (Hemiptera: Psylloidea): comparative analysis and phylogenomics

Article References: Feng, J., Yang, Y., Fan, J., He, J., Wang, L., Song, F., & Huang, W. (2026). Exploring the mitogenomics of Triozidae (Hemiptera: Psylloidea): comparative analysis and phylogenomics. BMC Genomics. https://doi.org/10.1186/s12864-026-13337-y

Image Credits: AI Generated

DOI: 10.1186/s12864-026-13337-y

Keywords: Triozidae, mitochondrial genome, phylogenetics, Bactericera gobica, Trioza polyphyly, Psylloidea, divergence time estimation, codon usage bias, BMC Genomics, insect taxonomy, Miocene diversification, plant lice