Living Fossil Quillworts Reveal How Mutation and Selection Sculpt Ancient Chloroplast Genomes

The quillwort genus Isoetes is one of the strangest survivors in the plant kingdom. Resembling a tuft of leek-like blades rising from wetland mud, this spore-producing lycophyte has been persisting since the Devonian period, roughly 360 million years ago, and stands today as the sole surviving genus of its entire family. Its lineage branched from the vascular plant tree long before seed plants diversified, which means that every quillwort genome carries molecular echoes of the evolutionary transitions that shaped the first land plants. Now, a sweeping comparative study of chloroplast genomes from 60 Isoetes species has decoded how these ancient photosynthetic genomes are organized, revealing that AT-biased mutation pressure establishes the genome-wide compositional background while gene-specific selective constraints sculpt the essential functional modules within it.

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