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Comparative Analysis of Chloroplast Genomes and Phylogenetic Relationships of Eight Quercus Species
Institute of Plant Conservation, Hunan Botanical Garden, Changsha, China
* Corresponding Authors: Xiaoming Tian. Email: ; Hao Lv. Email:
# These authors contributed equally to this work
Phyton-International Journal of Experimental Botany 2026, 95(6), 8 https://doi.org/10.32604/phyton.2026.079853
Received 29 January 2026; Accepted 06 May 2026; Issue published 29 June 2026
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Quercus is widely distributed globally and holds significant ecological and economic value. However, the morphological classification of this genus has long been controversial, with the core issue being whether the Cyclobalanopsis should be treated as an independent genus or as a subgenus within Quercus. In this study, the chloroplast genomes of eight Quercus species were determined and analyzed. Among these, the complete chloroplast genome of Quercus pachyloma is reported for the first time, alongside newly published chloroplast genome data for seven other Quercus species collected from Hunan and Jiangxi provinces in China. All eight species exhibited a typical quadripartite chloroplast genome structure, with genome sizes ranging from 160,716 to 160,842 bp. The gene composition and arrangement were highly conserved, with each species containing 131 genes. Three highly variable regions (psaI, infA, rpl22) were identified as candidate DNA barcodes. The total number of simple sequence repeats (SSRs) was similar across species, with all types distributed among them; however, dispersed repetitive sequences exhibited distinct regional characteristics and interspecific differences, providing potential marker resources for distinguishing closely related species. Phylogenetic analysis revealed that all eight species nested within Quercus, with Quercus shennongii and Cyclobalanopsis edithae clustered together as a distinct branch with 100% bootstrap support. Samples from different geographic sources within the same species did not cluster directly, suggesting the potential for intraspecific genetic variation and geographic population structure differentiation within Quercus. This study provides new chloroplast genomic evidence for the taxonomic status of Cyclobalanopsis. The phylogenetic results are compatible with treating Cyclobalanopsis as a subgenus within Quercus but may not be the sole explanation. Final taxonomic treatment requires careful evaluation integrating nuclear genomic, phytogeographic, and population data.Keywords
Supplementary Material
Supplementary Material FileChloroplasts (cp) are essential matrilineal genetic organelles for green plant cells with independent circular genomes and play an important role in plant photosynthesis [1]. The chloroplast genome of terrestrial plants exhibits a typical quadripartite structure, consisting of a large single-copy region (LSC) and a small single-copy region (SSC) separated by two inverted repeat regions (IR), independent of the nuclear genome [2,3,4]. Chloroplast genome is highly conservative with low recombination and mutation rates, making it an effective tool for studying plant evolution and taxonomy [5,6]. In recent years, chloroplast DNA has played an increasingly important role in systematic geography and phylogenetic studies [7,8]. Under the premise of ensuring the full availability of cp DNA data, it is very efficient to compare sister species or other possible species through cp genome information to create phylogenetic relationships, and can provide data for the study of population dynamics and phylogeographic evolution patterns of late evolved species [9]. Chloroplast genes are also regarded as “DNA barcodes” and are expected to become an efficient tool for species identification, contributing not only to molecular identification and differentiation of multiple species, but also to biodiversity analysis [10].
Fagaceae is a key foundational family in the forest ecosystems of the Northern Hemisphere [11]. This family comprises core groups including Quercus, Fagus, Castanopsis, and Lithocarpus. Among these, Fagus is considered an early-diverging basal lineage within Fagaceae, while Quercus represents a core branch resulting from later radiation and evolution within the family [12,13,14]. Quercus is an important member of middle subtropical evergreen broad-leaved forest, possessing important ecological and economic value, and is widely distributed throughout tropical and subtropical Asia [15,16,17]. Currently, Quercus is generally considered to comprise two main branches consisting of 5–6 subgenera [18,19]. However, its infrageneric classification, particularly the taxonomic status of the Cyclobalanopsis group, remains a subject of long-standing debate. Traditional morphological classification primarily relies on cupule scale morphology: the cupule scales of Cyclobalanopsis are usually connate into concentric rings, whereas those of typical Quercus are imbricately arranged [20,21]. Based on this significant difference, many taxonomic systems have treated the Cyclobalanopsis group as a distinct genus. However, recent molecular phylogenetic studies have consistently shown that this group is stably nested within Quercus, forming a monophyletic clade, which supports its treatment as a subgenus of Quercus [22,23,24]. This conflict between morphological and molecular phylogenetic evidence renders the generic delimitation and infrageneric classification of Quercus an urgent issue requiring clarification.
Currently, phylogenetic studies of Quercus based on chloroplast genomes have largely focused on European and North American taxa, with insufficient attention given to Asian lineages, particularly the rich endemic species in China, which limits our comprehensive understanding of the evolutionary patterns within this genus [25,26]. To address this gap and provide new genomic insights into the aforementioned taxonomic controversy, this study selected eight Cyclobalanopsis species distributed in central-southern China for comparative chloroplast genome analysis. The selection of these species was based on the following rationales: (1) Representativeness: they encompass the morphological diversity of cupule forms within Cyclobalanopsis [27]; (2) Geographical focus: they target the understudied subtropical regions of China; (3) Novelty: in this study, the complete chloroplast genome of Quercus pachyloma is reported for the first time. Although chloroplast genome data for Quercus jenseniana and the other six species have been previously published, this study provides independent genomic resources from new geographical populations (Hunan and Jiangxi Province, China) for each of them. In summary, this study presents, for the first time, a systematic characterization of the complete chloroplast genomes and their annotations for eight Cyclobalanopsis species. Through high-throughput sequencing, comparative genomics, and phylogenetic analysis of these key lineages, this study aims to elucidate their genetic relationships, evaluate the efficacy of chloroplast genomes in resolving taxonomic issues within Cyclobalanopsis, and provide new data to support the systematic classification of Quercus.
2.1 Sample Materials Collection, DNA Extraction, and Sequencing
Fresh leaves of 8 species of Quercus were collected from the native environment (Table 1). The collected leaf samples were immediately dried and preserved in silica gel. All samples were identified by the plant taxonomist Lihong Yan (Hunan Botanical Garden) in the field and at the herbarium based on authoritative taxonomic works, including the the Flora of China [27,28]. Voucher specimens for all samples were prepared and deposited in the Hunan Botanical Garden Herbarium (HNBG); their corresponding collection numbers are listed in Table 1.
Genomic DNA was isolated by the modified method CTAB. Agarose gel electrophoresis and a microspectrophotometer (OD-1000, Shanghai Cytoeasy Biotech Co., Ltd., Shanghai, China) were used to detect DNA integrity and quality. The sequencing of cp genome was conducted by Gene pioneer Biotechnologies Co., Ltd. (Nanjing, China) using the Illumina NovaSeq6000 Sequencing System.
Table 1: Information on the collection of samples from eight species of Quercus.
| Plant Name | Collection Place | Latitude/Longitude | Altitude (m) | Voucher Specimen |
|---|---|---|---|---|
| Quercus pachyloma | Tongdao Dong Nationality Autonomous County, Hunan Province | 26°4′27″ N, 109°30′45″ E | 506 m | HNBG20190419 |
| Quercus jenseniana | Yizhang County, Hunan Province | 25°23′57″ N, 112°57′5″ E | 575 m | HNBG20190512 |
| Quercus fleuryi | Dayu County, Jiangxi Province | 25°33′29″ N, 114°26′05″ E | 700 m | HNBG20200502 |
| Quercus gilva | Jingzhou County, Hunan Province | 26°34′48″ N, 109°40′48″ E | 650 m | HNBG20200505 |
| Quercus glauca | Yongshun County, Hunan Province | 29°0′48″ N, 109°57′53″ E | 780 m | HNBG20200519 |
| Quercus shennongii | Longshan County, Hunan Province | 29°38′4″ N, 109°46′8″ E | 550 m | HNBG20200530 |
| Quercus multinervis | Guzhang County, Hunan Province | 28°37′10″ N, 109°56′53″ E | 750 m | HNBG20200628 |
| Quercus myrsinifolia | Guzhang County, Hunan Province | 28°37′10″ N, 109°56′53″ E | 605 m | HNBG20200630 |
2.2 Chloroplast Genome Assembly and Annotation
The data quality control is carried out by using FASTQ software, and the clean data obtained by quality control is assembled by GetOrganelle software [29]. The original data is map to the assembled sequence to check the coverage, and combined with Gapcloser software v1.12 [30] (https://sourceforge.net/projects/soapdenovo2/files/GapCloser/) to fill the gaps in assembly. The cp genome of Q. stewardiana (MN199023) was uploaded to NCBI (https://www.ncbi.nlm.nih.gov/) as the reference sequences, and annotated by CpGAVAS2 (http://www.herbalgenomics.org/cpgavas/) [31]. The annotated data were manually adjusted and modified by Geneious Prime 2021 (Biomatters Ltd., Auckland, New Zealand). The physical structure maps of chloroplast genomes were drawn by OGDRAW v1.3.132 (https://chlorobox.mpimp-golm.mpg.de/OGDraw.html). Finally, the annotated data were submitted to NCBI. The sequence accession numbers of Q. jenseniana, Q. pachyloma, Q. fleuryi, Q. gilva, Q. glauca, Q. shennongii, Q. multinervis and Q. myrsinifolia are respectively: OP442516.1, OP442517.1, OP450821.1, OP450822.1, OP450818.1, OP450820.1, OP450823.1, OP450819.1.
The relative synonymous codon usage of chloroplast genomes of 8 species of Quercus was determined and analyzed by CodonW1.4.2 (http://mobyle.pasteur.fr/cgi-bin/portal.py?form=codonw), and the heat map was drawn by RSCU. The RSCU value of >1 indicates that the codon is used more frequently; equal to 1 indicates that the codon has no usage preference; <1 indicates that the codon is used less frequently.
2.4 Comparative Analysis of Quercus Chloroplast Genomes
The comparative cp genome includes the sequence of 8 species of Quercus. The using DNA Baser Sequence Assembler v5.152 to quantificat the base content, the 8 chloroplast genomes were aligned using the MAFFT v7.475 [32] with default parameters to identify highly variable regions. The number of polymorphic sites and nucleotide variability (Pi) were evaluated using a sliding window with 200-bp step size and a 600-bp window length implemented in DnaSP v5.10.1 [33]. IRscope (https://irscope.shinyapps.io/irapp/) was used to map the chloroplast genomic IR boundaries [34].
2.5 Repeat Sequences Analysis of Quercus Chloroplast Genomes
Four types of repeat sequences: forward, reverse, complementary, and palindromic, were identified using REPuter with a Hamming distance of 3 and a minimum repeat size of 30 bp [35]. In addition, tandem repeats were identified with Tandem Repeats Finder v4.09 with default parameters [36]. The overlapped repeats of the results were removed manually.
2.6 Phylogenetic Analysis of Quercus Chloroplast Genomes
To construct the phylogenetic tree, we assembled a dataset comprising the complete chloroplast genome sequences of the eight species from this study and 39 closely related species downloaded from the NCBI database, totaling 47 species. To enhance taxonomic representativeness, the analysis included two accessions each for Quercus fleuryi, Quercus myrsinifolia, and Quercus gilva: one newly sequenced in this study (accession numbers OP450821.1, OP450819.1, and OP450822.1, respectively, collected from Jiangxi and Hunan) and one previously published from different geographical regions—Quercus fleuryi (MZ502291.1, Fujian), Quercus myrsinifolia (MN199025.1, Anhui), and Quercus gilva (MK986651.1, Fujian) [37,38,39]. Species from the genera Corylus and Alnus of Betulaceae were selected as the familial outgroup [40,41], while species of Fagus (Fagaceae) were chosen as the intra-familial basal outgroup to jointly root the phylogenetic tree [1,14,23].
The tree reconstruction procedure was as follows: all circular chloroplast genome sequences were linearized by aligning them to a common starting point. Multiple sequence alignments were performed using MAFFT software (v7.427, —auto mode). The resulting alignments were trimmed using trimAl (v1.4.rev15) to remove poorly aligned regions. Finally, a maximum likelihood (ML) phylogenetic tree was constructed using RAxML v8.2.10 under the GTRGAMMA model with 1000 rapid bootstrap replicates. Node support was assessed using bootstrap values (BS).
3.1 Chloroplast Genomes of Eight Species of Quercus
In this study, we obtained the complete chloroplast genomes of eight Quercus species, including Quercus pachyloma and Quercus jenseniana. Among these, the complete chloroplast genome of Q. pachyloma is reported here for the first time. Although chloroplast genome information for Q. jenseniana and the other six Quercus species has been previously published, this study provides independent genomic resources from new geographical populations (Hunan and Jiangxi) for each of them. All genomes exhibited a typical quadripartite structure (Fig. 1; Supplementary Fig. S1). The complete chloroplast genome of Q. pachyloma had a total length of 160,778 bp, comprising a LSC of 90,205 bp, an SSC of 18,889 bp, separated by two 25,842 bp IRs. The total length of chloroplast genomes of Q. jenseniana was 160,811 bp and consisted of one LSC (90,237 bp), one SSC (18,906 bp), and two IRs (25,834 bp). The cp genomes of Q. pachyloma, Q. jenseniana, and other 6 species all contained 131 genes, including 85 protein-coding genes, 37 tRNA genes, 8 rRNA genes, and one pseudogene (Table 2).
In addition, the GC content of the complete cp genomes, as well as those of the LSC, SSC, and IR regions, were similar among the eight species, ranging from 36.89% to 36.90%, 34.74% to 34.75%, 31.06% to 31.11%, and 42.77% to 42.80%, respectively (Table 2). A total of 23 genes contained introns in their complete cp genomes, among which 21 genes (13 protein-coding genes and 8 tRNA genes) harbored a single intron, while two genes (ycf3 and clpP) contained two introns. Furthermore, 15 protein-coding genes, 11 tRNA genes, and 8 rRNA genes were duplicated in the IR regions. The LSC region harbored 62 protein-coding genes and 22 tRNA genes, whereas the SSC region contained 12 protein-coding genes and 1 tRNA gene.
To comprehensively evaluate the genomic characteristics obtained in this study, we conducted comparisons at two levels: First, we performed a direct comparison with previously published cp genomes of the same species. We collected published cp genome annotation data for seven species—Q. jenseniana, Q. fleuryi, Q. gilva, Q. glauca, Q. shennongii, Q. multinervis, and Q. myrsinifolia (accession numbers are provided in Supplementary Table S1) [37,38,39,42,43,44,45], and compared them in detail with the corresponding newly assembled genomes from this study (Supplementary Table S2). The comparison revealed that, for the same species, the newly assembled genomes differed only minimally from the published versions in total length and the length of LSC, SSC, and IR regions (average difference < 0.1%), and the GC content of each region were nearly identical (Table 2; Supplementary Table S2). This suggests the stability of the overall genome structure within species. However, while all species in this study consistently contained 131 genes, the gene counts in previously published genomes of the same species fluctuated between 128 and 134. For instance, Q. glauca (KX852399.1) contained 134 genes, whereas Q. fleuryi (MZ502291.1) contained 129 genes. Second, a comparison of gene numbers with closely related species. Compared to the 131 genes identified in the eight species of this study, previously reported Quercus species generally possessed slightly higher gene numbers. For example, Yang et al. reported 134 genes for five species including Quercus variabilis [10]; Huang et al. reported 133 genes for four species including Q. ningangensis [46]; and Li et al. reported 132 genes for four species including Q. disciformis [47]. Given that this study employed a uniform and rigorous annotation workflow, these discrepancies more likely reflect inconsistencies in annotation standards or assembly completeness across different studies.
In summary, these comparisons indicate that the cp genome data obtained in this study are of high quality and internal consistency. The observed differences in gene counts suggest the importance of adopting uniform standards in comparative genomic research, while also potentially revealing genuine microevolutionary variations among different lineages within Quercus.
Figure 1: (A) Chloroplast genome maps of Q. pachyloma; (B) Chloroplast genome maps of Q. jenseniana. Genes are colored based on different function. The inner circle (dashed gray area) indicates the proportional GC content of the corresponding genes. Regions of the LSC, SSR, IRa, and IRb are indicated.
Table 2: Comparison of chloroplast genomes among eight species of Quercus.
| Species | Quercus jenseniana | Quercus pachyloma | Quercus fleuryi | Quercus gilva | Quercus glauca | Quercus shennongii | Quercus multinervis | Quercus myrsinifolia |
|---|---|---|---|---|---|---|---|---|
| Genome size (bp) | 160,811 | 160,778 | 160,778 | 160,779 | 160,814 | 160,716 | 160,776 | 160,842 |
| Length of LSC (bp) | 90,237 | 90,205 | 90,205 | 90,219 | 90,256 | 90,213 | 90,232 | 90,235 |
| Length of SSC (bp) | 18,906 | 18,889 | 18,889 | 18,880 | 18,900 | 18,881 | 18,882 | 18,939 |
| Length of IRs (bp) | 25,834 | 25,842 | 25,842 | 25,840 | 25,829 | 25,811 | 25,831 | 25,834 |
| Number of protein-coding genes | 85 | 85 | 85 | 85 | 85 | 85 | 85 | 85 |
| Number of tRNA genes | 37 | 37 | 37 | 37 | 37 | 37 | 37 | 37 |
| Number of rRNA genes | 8 | 8 | 8 | 8 | 8 | 8 | 8 | 8 |
| Total number of genes | 131 | 131 | 131 | 131 | 131 | 131 | 131 | 131 |
| GC content of overall (%) | 36.89 | 36.90 | 36.90 | 36.90 | 36.89 | 36.90 | 36.90 | 36.89 |
| GC content of LSC (%) | 34.74 | 34.74 | 34.75 | 34.75 | 34.74 | 34.75 | 34.74 | 34.75 |
| GC content of SSC (%) | 31.10 | 31.10 | 31.11 | 31.10 | 31.08 | 31.07 | 31.11 | 31.06 |
| GC content of IRs (%) | 42.77 | 42.77 | 42.77 | 42.77 | 42.77 | 42.80 | 42.78 | 42.77 |
3.2 Relative Synonymous Codons Usage
For the two species newly reported in this study, 26,400 and 26,402 codons were obtained from Q. jenseniana and Q. pachyloma, respectively. A total of 66 codons were detected across the cp genomes of the eight Quercus species. Excluding the three stop codons, the remaining codons encode 20 amino acids. Serine was the most frequently used amino acid, while cysteine was the least frequent. Among the encoded amino acids, tryptophan is encoded exclusively by the single codon UGG, with a relative synonymous codon usage (RSCU) value of 1.00, indicating no obvious preference. The codon AUG, encoding methionine, exhibited the highest RSCU value of 6.99. Analysis of the cp genomes of the eight species revealed that multiple codons encode a single protein (Fig. 2). Among these, three amino acids (leucine, arginine, and serine) are encoded by six codons, four amino acids (alanine, glycine, threonine, and valine) are encoded by four codons, one amino acid (isoleucine) is encoded by three codons, and the remaining 11 amino acids are each encoded by two codons.
Figure 2: Heat map of the Relative synonymous codons usage of the cp genomes in the eight Quercus species.
Approximately 290 SSR loci were identified in each Quercus species, yielding a total of 2328 SSRs across all species. Each species contained over 160 single-nucleotide repeats, far exceeding the numbers of dinucleotides, trinucleotides, tetranucleotides, and pentanucleotides. The predominant motif types among the eight species were A/T, C/G, AT/AT, ATA/TAT, and TAA/TTA, while tetranucleotides and pentanucleotides were less abundant than other types. Furthermore, all detected SSR types were present in all eight species, with no unique SSR types observed (Fig. 3).
Among the eight species, P repeats (palindromic) were the most common, followed by F repeats (forward). R repeats (reverse) were present in only four species, and C repeats (complement) were found in only three species. In the SSC region, only F and P repeats were present, with Q. myrsinifolia and Q. jenseniana exhibiting the highest numbers of F repeats, while the number of P repeats was generally similar across species. In the LSC region, four types of repeats (P, F, R, C) were present in Q. pachyloma, Q. glauca, and Q. gilva; three types were present in Q. jenseniana (P, F, R); and only F and P repeats were found in the remaining species. Among these, Q. gilva had the highest number of F repeats, while the number of P repeats remained largely consistent across species. In the IR region, only F and P repeats were detected, and with the exception of Q. shennongii, the numbers of F and P repeats were identical across the other species (Fig. 3).
Figure 3: (A) Number of different SSR types in eight Quercus species; (B) Number of SSRs and dispersed repeats in eight Quercus species; (C) Number of different dispersed repeat types in the SSC region; (D) Number of different dispersed repeat types in the LSC region; (E) Number of different dispersed repeat types in the IR region.
3.4 Comparative Analysis of the Cp Genome
In the cp genomes of the eight Quercus species, the boundaries of LSC/IRb, SSC/IRb, SSC/IRa, and LSC/IRa were stably positioned, although the distances to adjacent genes exhibited some variation. For example, the JSB boundary was located 7 bp from the ndhF gene in Q. pachyloma, but only 1 bp away in Q. glauca (Fig. 4). Furthermore, we compared nucleotide variation across all genes and intergenic regions among the eight cp genomes. Sliding window analysis revealed that nucleotide diversity (Pi) values ranged from 0 to 0.004. Three mutational hotspot regions were identified, encompassing the psaI, infA, and rpl22 genes (Fig. 5). These regions represent potential markers for population genetic studies and species identification within Quercus.
Figure 4: Analyses of expansion and contraction of inverted repeats in the eight Quercus chloroplast genomes.
Figure 5: Nucleotide diversity (Pi) values among the eight Quercus species.
In this study, a phylogenetic tree was constructed using the cp genomes of 47 species, including the eight newly sequenced Quercus species from this study (Fig. 6). The overall topological structure shows that Quercus species (including the traditional Cyclobalanopsis group) formed a monophyletic clade together with their closely related genera Lithocarpus, Castanopsis, and Castanea. However, the internal topology of Quercus reveals a complex basal structure, detailed as follows: Quercus rubra and Quercus griffithii formed a clade located at the most basal position of the entire group. Above this clade, Quercus engleriana and Quercus senescens formed a second basal branch. Further above, Quercus tarokoensis, Quercus phillyreoides, and Quercus bawanglingensis formed a third basal branch. Above these basal Quercus lineages, Castanea, Castanopsis, and Lithocarpus formed an independent monophyletic branch, clearly separated from Quercus. The crown Quercus clade, which includes the eight species from this study and other typical Quercus species, was located at the top of the phylogenetic tree and formed a monophyletic group (bootstrap support = 100%).
Within this crown clade, the following topological structure was observed: First, Q. fleuryi (OP450821.1) and Q. pachyloma (OP442517.1) formed a sister group (BS = 100%), while another previously published sample of Q. fleuryi from Fujian (MZ502291.1) was positioned in a neighboring branch within the same major clade [39]. Second, the Q. myrsinifolia sample from this study (OP450819.1) and another previously published sample from Anhui (MN199025.1) did not cluster directly together but were placed in different positions within the same branch [37]; the two samples of Q. gilva exhibited a similar pattern [38]. Third, Q. shennongii (OP450820.1) and Cyclobalanopsis edithae (MZ929422.1) clustered together as a single branch (BS = 100%), and this branch did not group with the other seven species from this study. The remaining species, including Q. jenseniana, Q. gilva, Q. glauca, and Q. multinervis, each formed independent terminal branches. These results provide a preliminary plastome-based phylogenetic framework for investigating the phylogenetic relationships within Quercus.
Figure 6: Phylogenetic relationships of 47 species inferred from maximum likelihood method.
In this study, we compared the chloroplast genomes of eight Quercus species. Among these, the complete chloroplast genome of Quercus pachyloma is reported here for the first time. Although chloroplast genome information for Q. jenseniana and the other six species has been previously published, this study provides independent genomic resources from new geographical populations (Hunan and Jiangxi) for each of them. Comparative analysis revealed that the chloroplast genomes of the eight species were highly similar in size and composition, ranging from 160,716 to 160,842 bp in total length. The LSC regions ranged from 90,205 to 90,237 bp, the SSC regions from 18,880 to 18,939 bp, and the IR regions from 25,811 to 25,842 bp. The GC content of the IR regions was higher than that of the LSC and SSC regions, and all GC content were below 50%. All eight species contained 131 genes. Notably, as shown in Section 3.1, compared to the consistent gene number of 131 obtained in this study using a uniform workflow, previously published data for the same species exhibited some fluctuation in gene numbers (ranging from 128 to 134) (Supplementary Table S2) [10,37,38,39,42,43,44,45,46,47]. This suggests that annotation consistency may be an important factor when conducting cross-study comparisons of chloroplast genomes. The chloroplast genome structure of Quercus species is highly conserved [48]. We detected several genes located at boundary regions including rps19, rpl2, ycf1, ndhF, trnN, and trnH in all eight species, a finding consistent with previous studies on other Quercus species [1,47]. Although the copy numbers and positions of these genes varied slightly among species, their localization at boundary regions showed no significant differences. Clear codon usage bias was observed in all eight species, and the codon usage patterns were highly consistent across species, with the methionine-encoding codon AUG exhibiting the highest RSCU value.
In Quercus, the frequent occurrence of transitional morphologies resulting from hybridization and environmentally driven convergent evolution has substantially compromised the reliability of morphological characteristics for systematic classification [49]. Therefore, the development of efficient and stable molecular markers is crucial for establishing reliable species identification systems [50]. Repetitive sequences play an important role in genetic variation and can serve as molecular markers for phylogenetic analysis and population structure studies [50]. In this study, the eight Quercus species exhibited similar SSR abundances, and all detected SSR types were distributed across all species, with no species-specific SSRs identified. This indicates that overall SSR abundance is highly conserved within Quercus, and species identification based on SSRs should rely on allele frequency differences rather than simple presence/absence variations. Additionally, all species showed the highest proportion of A/T combinations, a bias that may be associated with lower energy consumption during information transfer [51]. In contrast to the conservatism of SSRs, the distribution of dispersed repeats exhibited pronounced regional characteristics and interspecific variation. In the SSC and IR regions, only F and P repeats were present, while the repeat types in the LSC region were more complex. Certain species, such as Q. gilva and Q. pachyloma, possessed all four types of repeats in the LSC region, whereas Q. shennongii showed a markedly different number of repeats in the IR region compared to other species. These differences provide potential marker resources for molecular identification of closely related Quercus species. This study compared the abundance, types, and distribution patterns of chloroplast SSRs and dispersed repeats across eight Quercus species, providing foundational data for molecular marker development, species identification, and conservation genetics research [30].
Mutational hotspots are hypervariable regions identified through comparative analysis of chloroplast genomes across different species, making them valuable candidates for DNA barcoding in species identification [52]. The comparison of cp genes differences among species in this study showed that the cp genes of 8 Quercus species had high similarity and there might be no obvious differentiation among them. However, there are still three hypervariable regions, namely: psaI, infA, rpl22. Among these, rpl22 has been consistently detected as a hypervariable region in both this study and previous investigations, suggesting its potential as a candidate DNA barcode for Quercus [1,10]. These findings could provide reference information for taxonomic classification and genetic resource development in Quercus. However, it should be noted that comparisons based on only eight species may not fully capture the overall characteristics of this diverse genus, and further studies incorporating more taxa and broader population-level sampling are needed to validate these observations.
Phylogenetic analysis plays an essential role in elucidating genetic relationships among species, and understanding these interspecific relationship is of great significance for the rational development, utilization, and conservation of plant resources [53]. Chloroplast genomes provide a molecular perspective for resolving difficult issues in morphological classification and have been widely used in phylogenetic studies [48,54]. However, due to the highly conserved nature of chloroplast genomes (with the highest nucleotide diversity Pi value in this study being only approximately 0.004), their resolving power at the species level is limited. Therefore, although the use of chloroplast genomes to elucidate the genetic relationships of Quercus species can provide clues for taxonomic research, it should be integrated with other lines of evidence for comprehensive assessment.
The chloroplast phylogenetic tree constructed in this study (Fig. 6) reveals a complex basal topology within Quercus. Notably, three basal Quercus lineages appear sequentially at the most basal positions of the phylogenetic tree: Q. rubra with Q. griffithii, Q. engleriana with Q. senescens, and Q. tarokoensis with Q. phillyreoides and Q. bawanglingensis. Above these basal Quercus lineages, the clades of Lithocarpus, Castanopsis, and Castanea appear, while the core Quercus clade (including the eight species from this study) is located at the most crown position of the phylogenetic tree. This topology warrants further investigation. Liu et al. demonstrated that there is strong cytonuclear discordance at deep nodes within Quercoideae and Quercus, wherein Quercus is non-monophyletic in the chloroplast phylogenetic tree, mainly due to frequent historical gene flow. Therefore, the “basal Quercus lineages-closely related genera-core Quercus clade” topology observed in this study may not fully reflect the true species divergence order, but rather more likely reflects extensive chloroplast capture or ancient hybridization events between Quercus and its closely related genera during early diversification [55]. Indeed, Liu et al. confirmed the existence of extensive gene flow between Quercus and major lineages of Quercoideae during early evolution, with these ancient hybridization events dating back to the early-to-middle Eocene [55]. This interpretation is also echoed by another observation in this study: some Quercus species (e.g., Q. tarokoensis and its relatives) are placed below the closely related genera in the chloroplast tree, which may reflect that the chloroplast genomes of these species experienced capture events from the ancestors of closely related genera during their evolutionary history.
Phylogenetic analysis revealed that samples of Q. fleuryi, Q. myrsinifolia, and Q. gilva from different geographical origins did not form monophyletic groups. This phenomenon is relatively common in recently radiated and rapidly diversifying woody genera [56,57]. Considering the characteristics of the chloroplast genomes and the evolutionary background of Quercus, the causes of this pattern may be related to the following two factors. On the one hand, the highly conserved nature of the chloroplast genome (with the highest nucleotide diversity Pi value in this study being only approximately 0.004) results in a slow rate of molecular differentiation. Meanwhile, the core Quercus clade belongs to a recently and rapidly radiated group [56]. The ancestral population’s chloroplast haplotype polymorphism may not have been completely and randomly fixed during species divergence, leading to incomplete lineage sorting. In this scenario, different geographical populations of the same species may carry different ancestral haplotypes, resulting in their dispersal across different branches in the phylogenetic tree and forming a “non-monophyletic” topological pattern. On the other hand, this result may reflect intraspecific genetic variation and geographical population structure differentiation within Quercus [26,58]. Due to the maternal uniparental inheritance of the chloroplast genome, its phylogeographic structure may be more pronounced, with different geographical populations potentially retaining distinct plastid haplotypes [48,59]. This geographically driven haplotype differentiation may lead to substantial differences in the chloroplast genomes of different geographical populations of the same species, resulting in a “same species, different branches” clustering pattern. This may imply the existence of gene flow or complex phylogeographic histories within these widely distributed species [26]. Furthermore, the phenomenon of conspecific samples failing to form monophyletic groups is not rare in chloroplast phylogenetic trees. Ogishima et al. found that different populations of the same species in the genus Isodon often appear paraphyletically distributed in chloroplast trees, suggesting that chloroplast capture may be a cause of this phenomenon [60]. Therefore, the non-monophyly of conspecific samples observed in this study may reflect the influence of evolutionary events commonly found in Quercus, such as chloroplast capture and incomplete lineage sorting. These findings suggest that when using chloroplast genomes for species-level phylogenetic inference, it is crucial to fully consider the geographical representativeness of samples and potential intraspecific variation. Future studies integrating more extensive population-level sampling with nuclear genomic data will be necessary to more comprehensively elucidate the evolutionary dynamics and species boundaries of these taxa.
Within the crown clade, the highly supported clustering of Q. shennongii with Cyclobalanopsis edithae is another topological feature worthy of attention. This clustering pattern suggests that the chloroplast genome of Q. shennongii is highly similar to that of C. edithae, while being differentiated from other species. However, interpretation of this result requires caution. In Quercus, a genus characterized by frequent hybridization and introgression, chloroplast capture is a widely documented phenomenon [48,59,61,62] The strongly supported clustering of Q. shennongii with C. edithae could reflect either close common ancestry or, alternatively, the possibility of historical interspecific hybridization resulting in chloroplast genome introgression from one lineage into another. Chloroplast data alone cannot distinguish between these two hypotheses. This result is compatible with, but does not uniquely support, the treatment of Cyclobalanopsis as a subgenus of Quercus; it represents only one of several possible explanations. Notably, we observed no significant structural differentiation in the chloroplast genomes, further suggesting that phylogenetic inferences based on this uniparentally inherited marker require comprehensive evaluation integrating multiple lines of evidence, including nuclear genomic data.
The phylogenetic results based on chloroplast genomes in this study provide a new molecular perspective on the traditional taxonomic issues within Quercus, while also highlighting conflicts with morphological evidence. First, regarding the discordance between molecular and morphological evidence, the chloroplast phylogeny supports the inclusion of Cyclobalanopsis (characterized by concentric cupule scales) within Quercus, contradicting traditional morphological classifications that recognize it as a distinct genus based on its imbricate cupule scales [20,21]. Second, concerning evidence supporting the nesting of Cyclobalanopsis within Quercus, the two groups exhibit extensive overlap and transitional forms in their distribution ranges and morphological features other than cupule morphology, suggesting shared ancestry and co-evolutionary history [63]. Finally, it is essential to recognize methodological limitations: the high bootstrap support values obtained in this study (e.g, BS = 100%) do not necessarily equate to genuine phylogenetic signal. The complete chloroplast genome is approximately 160 kb in length, and the large alignment size, combined with non-independence among sites, may lead to systematic overestimation of bootstrap values. Therefore, high support alone cannot substitute for independent validation of phylogenetic topology stability. In summary, the molecular evidence presented in this study adds chloroplast genomic data to clarify the phylogenetic position of Cyclobalanopsis. However, due to the highly conserved nature of the chloroplast genome (with the highest Pi value of approximately 0.004), its resolving power at the species level is limited, and the uniparentally inherited plastid tree may be influenced by evolutionary events such as chloroplast capture. Consequently, ultimately resolving this controversy will require integration of nuclear genomic data, broader population sampling, and morphological and biogeographical evidence to better elucidate the evolutionary relationships between Cyclobalanopsis and other Quercus lineages.
In this study, we compared the chloroplast genomes of eight Quercus species. Among these, the complete chloroplast genome of Quercus pachyloma is reported for the first time, and for the remaining seven species, we provide independent genomic resources from new geographical populations (Hunan and Jiangxi). Quercus species are widely distributed globally and play important ecological and economic roles; however, their taxonomic system has long remained unresolved. Our results revealed that the chloroplast genomes of these eight Quercus species are highly conserved in structure, with highly consistent composition and gene content. The eight species in this study consistently contained 131 chloroplast genes, which may reflect annotation consistency rather than biological variation across studies. Three highly variable regions: psaI, infA, and rpl22 were identified through comparative analysis. The phylogenetic tree indicates that this study reconstructed a phylogenetic relationship with high node support among the eight species based on complete chloroplast genome data. However, due to the highly conserved nature of the chloroplast genome (with the highest Pi value of approximately 0.004), its resolving power at the species level is limited. Therefore, this result should be regarded as preliminary molecular evidence. The distinctive clustering of Quercus shennongii and Cyclobalanopsis edithae provides referable molecular evidence for further exploring the taxonomic status of Cyclobalanopsis. This result is compatible with, but does not uniquely support, treating Cyclobalanopsis as a subgenus of Quercus. Given the genetic characteristics of chloroplast genomes and their complexity in Quercus evolution, final taxonomic decisions require careful evaluation integrating multiple lines of evidence, including nuclear genomic, morphological, and biogeographical data.
Acknowledgement:
Funding Statement: This research was funded by the Changsha Natural Science Foundation, grant number kq2202356 and the Hunan Forestry Science and Technology Innovation Plan Project, grant number XLK202106-2 and XLK202616.
Author Contributions: The authors confirm contribution to the paper as follows: Conceptualization, Xin Li, Xiaoming Tian and Hao Lv; methodology, Xiaoming Tian and Gaofei Li; software, Lu Zhu and Chao Liu; validation, Xin Li, Guangfeng Xiang and Lan Zhou; investigation, Xin Li, Li Liu and Tianyi Jiang; resources: Hao Lv and Cun Mou; data curation, Xiangpeng Li and Li Liu; writing—original draft preparation, Xin Li and Xiaoming Tian; writing—review and editing, Xin Li, Hao Lv and Lan Zhou. All authors reviewed and approved the final version of the manuscript.
Availability of Data and Materials: The data that support the findings of this study are available from the corresponding author upon reasonable request.
Ethics Approval: Not applicable.
Conflicts of Interest: The authors declare no conflicts of interest.
Supplementary Materials: The supplementary material is available online at https://www.techscience.com/doi/10.32604/phyton.2026.079853/s1.
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