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

Abstract

Background/Objectives: 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 elucidate their structural variation, evolutionary relationships, and functional gene content to provide effective support for future genetic conservation and breeding efforts. Methods: We isolated total DNA from fresh leaves and sequenced the complete cp genomes of these samples. To develop a genomic resource and clarify the evolutionary relationships within Lithocarpus species, comparative chloroplast genome studies and phylogenetic investigations were performed. 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 36.72-37.76%. Codon usage analysis showed a predominance of leucine-encoding codons. Our analysis identified 322 simple sequence repeats (SSRs), which were predominantly palindromic in structure (82.3%). All eight species exhibited the same 19 SSR categories in similar proportions. Eight highly variable regions (ndhF, ycf1, trnS-trnG-exon1, trnk(exon1)-rps16(exon2), rps16(exon2), rbcL-accD, and ccsA-ndh) have been identified, which could be valuable as molecular markers in future studies on the population genetics and phylogeography of this genus. The phylogeny tree provided critical insights into the evolutionary trajectory of Fagaceae, suggesting that Lithocarpus was strongly supported as monophyletic, while Quercus was inferred to be polyphyletic, showing 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.

Keywords: Lithocarpus; chloroplast genomes; comparative genomics; genetic resources; phylogenetics analysis.

Figure 1

Genome map of L. longipedicellatus presents a typical chloroplast genome structure and content Genes illustrated within the circular diagram exhibit clockwise transcription, while those positioned externally transcribe in the counterclockwise direction. Functional gene classification is visually represented through a color-coded system.

Figure 2

Subsequent sequence identity plots depict the chloroplast genome sequences of 8 Lithocarpus species. Grey directional arrows mark gene orientation. Coding elements are color-coded (purple: exons; blue: introns) with non-coding regions in red. The y axis shows sequence similarity (50–100%).

Figure 3

Nucleotide polymorphisms (Pi) of the 8 Lithocarpus chloroplast genomes. The nucleotide diversity values are plotted on the y axis, with the corresponding genomic positions of the sliding windows displayed along the x axis.

Figure 4

Comparison of the boundaries of a large single copy (LSC), small single copy (SSC), and inverted repeat (IR) regions in the Lithocarpus cpDNAs. Note: Genes are illustrated as horizontal bars in schematic diagrams, and the intervals and boundaries between genes are represented by base pair length. Structural extensions such as exon elongations or regulatory regions should be clearly marked above the corresponding bars.

Figure 5

Quantitative analysis of long repeat sequences in eight Lithocarpus species. (A) Proportion of four types of long repeat sequences in each species: p represents palindromic sequences, F represents forward repeat sequences, R represents reverse repeat sequences, and C represents complementary sequences. (B) Proportion of repeat sequences of different lengths in each species.

Figure 6

Quantitative analysis of SSRs in eight Lithocarpus species. (A) Proportion of SSRs with different repeat unit lengths in each species. (B) Frequency and types of identified SSRs.

Figure 7

Bar chart illustrating codon usage bias across eight Lithocarpus species. The x axis denotes the 20 essential amino acids and a termination codon (*), while the y axis represents the codons employed for each amino acid and their corresponding usage frequencies within the analyzed dataset.

Figure 8

Phylogenetic analysis of chloroplast genomes from 71 Fagaceae species. Blue bold font indicates the eight investigated lithocarpus species.