Comparative pan-genome analysis of Huperzia and Phlegmariurus and transcriptomics reveals thermal adaptation in Huperzia.

Huperzia and Phlegmariurus are ancient genera within the Lycopodiaceae family with significant medicinal value and ecological adaptability, yet the evolutionary dynamics and genetic diversity of their chloroplast genomes remain poorly characterized. Specifically, critical aspects such as intergeneric differences, phylogenetic relationships, and adaptive evolution within their chloroplast genomes remain insufficiently explored. This study analyzed the chloroplast genomes of 66 species from these two genera through comparative genomics to elucidate their structural dynamics and adaptive mechanisms. Results revealed that Huperzia chloroplast genomes (153-155 kb, GC content 36.25-36.39%) are significantly larger than those of Phlegmariurus (148-151 kb, GC content 33.78-34.26%), with pronounced differences in IR boundary dynamics, repetitive sequence distribution, nucleotide diversity, and codon usage bias. Phylogenetic and population structure analyses confirmed the monophyly of both genera and demonstrated significantly higher genetic diversity in Phlegmariurus, likely linked to adaptive radiation driven by humid tropical environments. Transcriptomic data revealed a temporally coordinated chloroplast response to heat stress in Huperzia serrata. Photosynthetic core genes (such as psaB and rrna16) were downregulated, leading to sustained functional impairment. In contrast, early stress-response genes (such as rbcL and trnI-GAU) peaked at 4 h to enhance carbon fixation and transport. Mid-phase repair genes (such as ndhG and rps8) exhibited inverted U-shaped expression patterns to activate electron transport and protein synthesis, whereas late-stage overexpression of atpI restored energy homeostasis. This coordinated regulatory mechanism illustrates a survival strategy of "photosynthetic inhibition-stress compensation-energy reorganization" for thermal adaptation. Future studies should integrate nuclear genome and epigenetic modification data to further unravel the synergistic nucleo-cytoplasmic interactions underlying environmental adaptation.