Comparative plastome analyses of Lewinskya (Orthotrichaceae): insights into genome structure, molecular evolution, and phylogenetic relationships.

Mosses are key components of terrestrial ecosystems and provide important systems for studying plant diversity, adaptation, and genome evolution. Lewinskya is a species-rich moss genus in Orthotrichaceae, but species delimitation and phylogenetic reconstruction within the genus remain difficult because diagnostic characters are often subtle or convergent. Chloroplast genomes can provide useful genomic resources and complementary evidence for comparative and systematic studies. This study aimed to generate new Lewinskya plastome resources and evaluate plastome structure, sequence variation, codon usage, and plastid-based phylogenetic relationships in the genus. Five newly sampled Lewinskya chloroplast genomes were assembled from genome-skimming data, including three circular plastome assemblies and two high-quality single-scaffold assemblies. Together with the published plastome of L. incana, the six Lewinskya plastomes ranged from 122,258 to 123,526 bp and showed conserved genome organization, gene content, GC composition, and inverted repeat boundaries. Each plastome encoded 128 genes, including 83 protein-coding genes, 37 transfer RNA genes, and eight ribosomal RNA genes. A total of 520-542 simple sequence repeats were detected per plastome, with mononucleotide repeats being dominant and most repeats located in the large single-copy region. Comparative analyses revealed no large-scale rearrangements, but several localized divergence regions were detected. Nucleotide diversity analysis identified 11 highly variable regions, including five genic regions (rps18, rpl22, infA, rpl32 and rps3) and six intergenic spacers, most of which were located in the large single-copy region. Codon usage patterns were highly similar among species and showed a preference for A/T-ending codons. Phylogenetic analyses based on 78 plastid protein-coding genes from 26 Orthotrichaceae plastomes strongly supported the sampled Lewinskya species as a clade, although some deeper relationships within the genus remained weakly resolved. The newly assembled Lewinskya plastomes expand genomic resources for Orthotrichaceae and show that chloroplast genome evolution in the sampled species is structurally conservative but contains informative localized variation. The identified repeat loci and highly variable regions provide candidate markers for future species identification and population-level studies. Plastome-scale data offer useful evidence for Lewinskya systematics, but broader taxon sampling and integration with nuclear genomic and morphological evidence will be needed to resolve difficult interspecific relationships.