Comparative analysis of complete chloroplast genomes reveals the phylogenetic relationships of Lygodium Sw. (Lygodiaceae) species in China

The family Lygodiaceae occupies a key position in leptosporangiate fern phylogeny. However, its intrafamilial relationships and chloroplast (cp.) genome evolution remain insufficiently explored. The lack of comprehensive cp. genomic data from Chinese species, which represent significant diversity in the family, hinders the development of robust molecular markers and precise phylogenetic resolution. The complete cp. genomes of seven Chinese Lygodium species were assembled and characterized. Comparative analyses across these and one published genome (L. merrillii) showed highly conserved structure and gene order, but total length varied (157,001-164,439 bp). This size variation was primarily driven by large-fragment insertions in intergenic spacers of L. microphyllum and L. longifolium. All genomes shared a common preference for A/U-ending codons and contained 31–65 simple sequence repeats (SSRs) and 31–91 tandem/dispersed repeats. Critically, nucleotide diversity analysis identified nine highly variable regions, including four coding (ycf1, ndhF, matK, and rpoC2) and five non-coding hotspots (e.g., rpl32-trnP-GGG, rpoB-trnC-GCA, trnI-CAU-trnR-ACG), which are proposed as promising novel molecular markers for species delimitation. Phylogenomic reconstruction based on cp. genomes strongly supported the monophyly of Lygodiaceae and clarified interspecific relationships, thereby consolidating previous molecular findings from a genomic perspective. This study provides a comprehensive cp. genomic resource for major Chinese Lygodiaceae species. The identified hypervariable regions offer powerful tools for future phylogenetic and population studies. Furthermore, the resolved phylogeny enhances understanding of the family’s evolutionary history and provides a solid framework for its global systematics.