Comparative and Phylogenetic Analysis of the Complete Chloroplast Genomes of <em>Lithocarpus </em>Species (Fagaceae) in South China

In South China, Lithocarpus species dominate mixed evergreen broadleaf forests, forming symbiotic relationships with ectomycorrhizal fungi and serving as food resources for diverse fauna, including frugivorous birds and mammals. The limited understanding of chloroplast genomes in this genus restricts our insights into its species diversity. This study investigates the chloroplast genome (cp genome) sequences from seven Lithocarpus species, aims to elucidates their structural variation, evolutionary relationships, and functional gene content, providing a foundation for future genetic conservation and breeding efforts. Methods: We isolated total DNA from fresh leaves and sequenced the complete cp genomes of these samples. Comparative cp genome analysis and phylo-genetic analysis were conducted to develop genomic resources and elucidate the intra-specific phylogeny of Lithocarpus. Results: All studied species exhibited a conserved quadripartite chloroplast genome structure, with sizes ranging from 161,495 to 163,880 bp. Genome annotation revealed 130 functional genes and a GC content of 36.72&ndash;37.76%. Codon usage analysis showed a predominance of leucine-encoding codons. Our analysis identified 322 simple sequence repeats (SSRs), predominantly palindromic in structure (82.3%). These SSRs comprised 19 distinct categories, with similar type distributions observed across all examined species. Eight highly variable regions (ndhF, ycf1, ycf1, trnS-trnG-exon1, trnk(exon1)-rps16(exon2), rps16(exon2), rbcL-accD and ccsA-ndh) have been identified, which could serve as potential DNA markers for future population genetics or phylogeographic studies on this genus. The well-resolved plastome phy-logeny tree provided critical insights into the evolutionary trajectory of Fagaceae, sug-gested that Lithocarpus was strongly supported as monophyletic, while Quercus was in-ferred to be polyphyletic, showed a significant cytonuclear discrepancy. Conclusions: We characterized and compared the chloroplast genome features across eight Lithocarpus species, followed by comprehensive phylogenetic analyses. These findings provide critical insights for resolving taxonomic uncertainties and advancing systematic research in this genus.