Mutation Pressure and Gene-Specific Selection Shape Codon Usage Bias in the Chloroplast Genomes of Isoetes, an Ancient Aquatic Vascular Plant Lineage.

Isoetes is one of the most ancient extant vascular plant genera and represents a distinct evolutionary lineage within lycophytes. Its chloroplast genome preserves molecular signatures relevant to early land plant evolution. However, the evolutionary forces shaping codon usage bias (CUB) in Isoetes chloroplast genomes remain poorly characterized. Here, we analyzed the chloroplast genomes of 60 Isoetes species to characterize CUB patterns and disentangle the contributions of mutation pressure and natural selection to chloroplast genome evolution in this basal vascular plant lineage. Chloroplast genome structure was highly conserved across species, with genome sizes of 142,880 bp to 145,535 bp and GC contents ranging from 37.56% to 38.18%. Parity rule 2 (PR2) plot analysis revealed a pronounced T/G bias at the third codon position, whereas neutrality plot analysis revealed no significant correlation between GC3 and GC12. All preferred codons (RSCU > 1) terminated in A or U, consistent with an AT-ending preference. Simple sequence repeat (SSR) analysis revealed that SSRs are primarily distributed in intergenic regions, with mononucleotide repeats predominating, suggesting valuable molecular markers for phylogenetic studies. Our findings indicate that Isoetes chloroplast genomes exhibit weak but structured codon usage bias. Mutation pressure appears to be the dominant force establishing the AT-rich compositional background, whereas natural selection acts in a gene-specific manner, mainly through purifying selection and functional constraints on particular chloroplast genes. These results provide new insights into chloroplast genome evolution in basal vascular plants and highlight the combined effects of mutational bias and selective constraints on codon usage evolution. Unlike most previous chloroplast CUB studies focused on angiosperms, this genus-wide analysis of Isoetes provides insight into codon usage evolution in a basal lycophyte lineage and suggests potential associations among CUB patterns, plastome conservation, functional constraints, and gene-level evolutionary dynamics.