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Article

Interspecific Sharing of Closely Related Chloroplast Genome Haplotypes among Sclerophyllous Oaks in the Hot-Dry Valley of the Jinsha River, Southwestern China

by
Yao Li
1,
Chao Tan
2,
Wenxu Zhang
1,
Lu Wang
2,
Zhi Yang
2,
Yanming Fang
2,
Yong Yang
2 and
Lingfeng Mao
1,*
1
Co-Innovation Center for Sustainable Forestry in Southern China, Laboratory of Biodiversity and Conservation, College of Ecology and Environment, Nanjing Forestry University, 159 Longpan Road, Nanjing 210037, China
2
Key Laboratory of State Forestry and Grassland Administration on Subtropical Forest Biodiversity Conservation, College of Life Sciences, Nanjing Forestry University, 159 Longpan Road, Nanjing 210037, China
*
Author to whom correspondence should be addressed.
Forests 2024, 15(3), 537; https://doi.org/10.3390/f15030537
Submission received: 22 January 2024 / Revised: 7 March 2024 / Accepted: 12 March 2024 / Published: 14 March 2024
(This article belongs to the Section Genetics and Molecular Biology)
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Abstract

:
Evergreen sclerophyllous oak forests (ESOFs) in southwestern China are a special vegetation type developed in response to the expansion of arid habitats after the uplift of the Himalayas. Here, we used chloroplast (cp) DNA and nuclear ribosomal (nr) DNA to investigate the fine-scale genetic variation patterns of six sympatric oaks (Quercus, Fagaceae) in the hot-dry valley ESOFs of the Jinsha River, southwestern China. Three cp genomes were assembled for each species. Nine cp genome haplotypes and 16 nrDNA haplotypes were identified based on single-nucleotide variants and indels. Our results demonstrated that discordance existed between the cpDNA and nrDNA phylogenies of the sclerophyllous oaks in section Ilex. The nrDNA phylogeny was consistent with species boundaries, while the cpDNA phylogeny was decoupled from taxonomy. Interspecific sharing of closely related cp genome haplotypes was detected between Quercus cocciferoides and the other two sclerophyllous oaks, Q. longispica and Q. franchetii. Specifically, Q. cocciferoides and Q. longispica sampled in a mixed stand exhibited two haplotypes that differed by a 9 bp indel. The local distribution of the two highly similar haplotypes suggested that they may have arisen from ancient introgression. Given that the two species have diverged for a long time, it is possible that the ancestral cp genome of one species was captured by another species through asymmetric introgression in early times, and an indel event occurred subsequently. Phylogenetic analyses using more previously published cp genome sequences indicated that Q. cocciferoides and Q. franchetii shared multiple cpDNA lineages of Ilex oaks, which may be caused by shared ancestral polymorphism and/or ancient introgression. Our study showed that at least three highly variable regions (ψycf1, ndhF-rpl32, and trnKUUU-rps16 or rpl32-trnLUAG) can distinguish the nine haplotypes identified by whole-cp genome sequences. These markers are useful for the evolutionary studies of the maternal lineages of oaks in hot-dry valley ESOFs.

1. Introduction

With more than 435 species, oaks (Quercus L., Fagaceae) represent an evolutionarily successful lineage of woody angiosperms in the Northern Hemisphere in terms of species diversity and ecological dominance [1,2,3,4]. These oak trees occupy a wide range of habitats, from swamp forests to Mediterranean maquis communities, and from Neotropical montane forests to subtropical evergreen forests and temperate deciduous forests [4,5,6,7,8]. Several explanations have been proposed to account for the evolutionary success story of oaks [3]. Introgressive hybridization is one of the key mechanisms underlying this story [9,10,11,12,13]. Sympatric oak species within the same section often form a syngameon, a group of ‘good species’ that are genetically coherent across their ranges despite a long history of introgression [14,15,16,17]. Recent studies have shown that introgression among closely related oaks may introduce adaptive alleles to the populations of recipient species. For example, in European temperate white oaks (section Quercus), introgression of Q. robur alleles may have facilitated the expansion of Q. petraea populations to higher altitudes and wetter climates [11]. In East Asian Cerris oaks (section Cerris), Q. acutissima and Q. variabilis populations tend to share the same introgressed genomic regions if they occupy ecologically similar habitats; introgression may contribute to the local adaptation of those Cerris oaks by introducing allelic variation in cis-regulatory elements [13].
Chloroplast (cp) DNA has been widely used to track the historical gene flow among closely related oaks [6,17,18,19,20,21,22,23,24,25,26,27,28,29]. It is expected to be more frequently introgressed than nuclear DNA because maternally inherited cpDNA is dispersed only by seeds and thus experiences a low level of intraspecific gene flow, which would hinder the immediate dilution of introgressed genotypes [30,31]. Consequently, cpDNA variation patterns of oaks, more likely reflecting the imprints of localized interspecific gene flow, are largely influenced by geography rather than by taxonomy [31,32]. Previous studies have shown that interspecific sharing of geographically restricted cpDNA haplotypes occurred within section Quercus [18,19,20,21], section Lobatea [22], section Virentes [23], section Cerris [6,24,25,26], section Ilex [6,27,28,29], and section Cyclobalanopsis [29]. Chloroplast capture events between sections were rare but also reported for sections Quercus and Ponticae, sections Quercus and Virentes, and sections Quercus and Protobalanus [17,32], probably tracking the history of ancient introgression between ancestral populations of oaks. Other processes, like the retention of ancestral polymorphism, may also result in the interspecific sharing of cpDNA haplotypes, but those shared ancestral haplotypes are expected to be randomly and widely distributed across the ranges of the descendant species [26].
Sclerophyllous oaks are among the dominant species of subtropical evergreen sclerophyllous forests and shrublands in Eurasia and North America [33]. These oaks belong to different sections (e.g., Ilex, Cerris, Lobatea, and Quercus) but have morphologically convergent leaves with high tolerance to drought. In southwestern China, evergreen sclerophyllous oak forests (ESOFs) are mainly composed of sclerophyllous oaks in section Ilex (Ilex oaks). These forests were developed in response to the expansion of arid habitats following the rise of the Himalayas [34,35,36,37,38,39]. Two types of ESOFs have been distinguished in this region according to their physiognomic features, species composition, and ecological preferences. The first type is found in cold-dry or sub-humid environments at high altitudes (2600–4300 m a.s.l.), with dominant species such as Q. aquifolioides, Q. pannosa, Q. guyavifolia, Q. longispica, and Q. semecarpifolia [33,34,35,36,37,38]. The second type is similar to Mediterranean maquis and occurs in warm- or hot-dry valleys along the Jinsha River and its tributaries (1500–2600 m a.s.l.), which is composed predominantly of Q. cocciferoides, Q. franchetii, and Q. rehderiana [33,34,35,36,37,38].
Accumulating evidence has revealed that Ilex oaks (mostly sclerophyllous taxa) have multiple interspecifically shared cpDNA lineages that are geographically sorted from the Mediterranean to Japan [17,28,29,40,41]. This pattern may be caused by both incomplete lineage sorting and recurring ancient introgression among ancestral populations of sections Ilex, Cerris, and Cyclobalanopsis [28]. In the ESOFs of southwestern China, subalpine Ilex oaks (e.g., Q. aquifolioides, Q. guyavifolia, Q. longispica) often share the same or closely related cp genome haplotypes within a specific geographical area, reflecting a history of frequent exchange of cp genomes at a local scale [42]. For the Ilex oaks in hot-dry valley ESOFs, previous studies have investigated the genetic structure of some species [43,44], but the patterns of interspecifically shared cpDNA haplotypes are largely unknown. In this study, we focus on the fine-scale cpDNA and nuclear ribosomal (nr) DNA variation patterns of six sympatric oaks in the hot-dry valley of the Jinsha River, southwestern China. Among those, Q. cocciferoides, Q. franchetii, Q. longispica, and Q. dolicholepis are evergreen sclerophyllous oaks in section Ilex, while Q. variabilis and Q. griffithii are deciduous broad-leaved oaks in sections Cerris and Quercus, respectively. All samples were collected in the Panzhihua Cycad National Nature Reserve, where hot-dry valley ESOFs are the main vegetation type. In this reserve, Q. cocciferoides and Q. franchetii are widespread and dominant in the ESOFs, while the other four oak species are narrowly distributed and relatively rare. Our specific aims are to: (1) assess whether discordance exists between cpDNA- and nrDNA-based phylogenies of the six oak species; (2) investigate whether neighboring oak trees interspecifically share the same or closely related cp genome haplotypes; and (3) explore highly variable cp genome regions useful for the evolutionary studies of the maternal lineages of sclerophyllous oaks in hot-dry valley ESOFs.

2. Materials and Methods

2.1. Sampling, DNA Extraction, and Sequencing

Between 20 June 2022 and 4 July 2022, we sampled fresh mature leaves from three trees of each of the six oak species in the Panzhihua Cycad National Nature Reserve, Sichuan Province, China (Figure 1 and Table S1). The two widespread oaks, Q. cocciferoides and Q. franchetii were sampled across the reserve. The four narrowly distributed oaks were collected in Zhulinpo (Q. longispica), Songpingzi (Q. dolicholepis), Fengjialiangzi (Q. griffithii and Q. variabilis), and Baiyanjiao (Q. variabilis) (Figure 1). Spatially explicit information was recorded for each sample using the 2bulu Outdoor Assistant app (Shenzhen, China). All the voucher specimens were deposited in the Herbarium of Nanjing Forestry University (NF). Total genomic DNA was extracted from silica-gel dried leaves using the cetyltrimethylammonium bromide (CTAB) procedure [45]. The DNA integrity was checked on a 1% agarose gel, and the DNA concentration was measured using the OneDrop spectrophotometer (Beijing, China). Library preparation and whole-genome sequencing (WGS) on a DNBSEQ platform were conducted by BGI Genomics (Wuhan, China). Raw reads were filtered using SOAPnuke 2.1.7 with default settings [46].

2.2. Assembly and Annotation of Chloroplast Genomes and Nuclear Ribosomal DNA

We used GetOrganelle v.1.7.6.1 [47] to assemble the cp genomes of the 18 oak trees from high-quality paired-end read data. This toolkit firstly used Bowtie2 v.2.4.5 [48] to obtain seed-mapped reads and then recruited more plastome-associated reads through a modified ‘baiting and iterative mapping’ approach; those extracted reads were employed for the de novo assembly of the cp genomes with SPAdes v.3.13.0 [49] and BLAST v.2.5.0 [50] as dependencies. The k-mer sizes (-k) were set to 21, 45, 65, 85, and 105. The number of maximum extension rounds (-R) was set to 15. The online tool CpGAVAS2 [51] was applied to annotate protein-coding genes, transfer RNA (tRNA) genes, and ribosomal RNA (rRNA) genes with Q. variabilis (GenBank accession number: NC_031356) or Q. cocciferoides (NC_061586) as a reference. The OrganellarGenomeDRAW (OGDRAW) tool v.1.3.1 [52] was used to visualize the circular gene map as well as the positions of the large single-copy (LSC) region, small single-copy (SSC) region, and two inverted repeats (IRs). The 18 newly generated cp genomes were submitted to GenBank under accession numbers OR188388–OR188405.
The nrDNA of the 18 oak trees was also assembled using GetOrganelle v.1.7.6.1 [47], with the k-mer sizes (-k) set to 35, 85, and 115, and the number of maximum extension rounds (-R) set to 10. The generated sequences were manually checked and then aligned with previously published sequences of the 25S-18S rDNA intergenic spacer (IGS) (KC700365; Q. suber) and 18S-5.8S-25S rDNA (FM243875; Q. suber) using BioEdit v.7.2.5 [53,54,55]. After trimming, the final alignment comprised partial 25S-18S rDNA IGS, 18S rDNA, internal transcribed spacer (ITS) 1, 5.8S rDNA, ITS2, and partial 25S rDNA. This alignment is available from Zenodo: https://doi.org/10.5281/zenodo.10656343.

2.3. Sequence Variation and Haplotype Network

We used PhyloSuite v.1.1.152 [56] to extract coding sequences (CDSs), tRNA genes, rRNA genes, introns, and IGSs of the 18 oak cp genomes. These sequences were aligned separately using MAFFT v.7.3.13 [57] and manually adjusted with BioEdit v.7.2.5 [55]. Length variations in mononucleotide repeats were excluded, and inversions were replaced with their reverse complements because of their tendency for homoplasy [58]. Other indels were coded as binary characters according to the simple gap coding method [59] using GapCoder [60]. Separate assignments were concatenated according to their respective positions in the cp genome to obtain the alignments of LSC, SSC, IRb, and the whole-cp genome. Unique cpDNA haplotypes were determined by DnaSP v.6.12.03 [61] based on the alignments with indels considered (dataset 1) or not (dataset 2). To visualize the relationships among cpDNA or nrDNA haplotypes, we used PopART v.1.7 [62] to infer haplotype networks through an integer neighbor-joining algorithm (reticulation tolerance = 0). We also performed principal component analysis (PCA) to explore the cpDNA genetic structure of the four Ilex oaks using the dudi.pca function in R v.4.2.0 [63] package ade4 v.1.7–19 [64]. Dataset 1 was imported as a ‘genind’ object into R using the DNAbin2genind function in package adegenet v.2.1.7 [65].

2.4. Phylogenetic Analyses of Chloroplast Genome Sequences

Phylogenetic analyses were conducted using 53 cp genome sequences belonging to six white oaks (section Quercus) [66,67,68,69], three Cerris oaks (section Cerris) [68,70,71,72,73], six ring-cupped oaks (section Cyclobalanopsis) [74,75,76,77,78,79], 11 Ilex oaks (section Ilex) [67,68,69,71,80,81,82,83], and two outgroups in Fagus [78,84] (Table S2). The HomBlocks v.1.0 pipeline [85] was applied to identify shared locally collinear blocks and generate a phylogenetically informative matrix of 83,130 bp. Both maximum likelihood (ML) and Bayesian Inference (BI) approaches were used to infer the phylogenetic relationships among cp genomes. The BI tree was built using MrBayes 3.2.6 [86]. The GTR+F+I+G4 model was selected as the best-fit substitution model under the Bayesian Information Criterion (BIC) using ModelFinder [87]. Two independent Markov chain Monte Carlo (MCMC) searches were run for 10 million generations with trees sampled every 1000 generations. The initial 25% of the trees were discarded as burn-in, and the remaining trees were used to generate a strict consensus tree. The ML tree was reconstructed using raxmlGUI v.1.5b2 [88,89] under the GTRGAMMA model, with branch support values estimated for each node based on 1000 samples for rapid bootstrap.

2.5. Highly Variable Regions and IR-SC Borders in Chloroplast Genomes

We computed and compared nucleotide diversity (π) in 1-kb sliding windows (step size = 200 bp; gaps not counted) across the cp genome to explore highly variable regions among the six oak species (n = 18) or the four Ilex oaks (n = 12). These analyses were performed with DnaSP v.6.12.03 [61]. The junction sites of LSC, SSC, and IR regions were visualized and compared among species using CPJSdraw v.1.1 [90].

3. Results

3.1. Features of the Newly Sequenced Chloroplast Genomes of Oaks

For each oak sample, a total ranging from 104,594,790 (Q. longispica PL2) to 128,252,558 (Q. dolicholepis PD3) clean reads (150 bp in length) were generated (Table S1), which covered the entire assembled cp genome (100% coverage) with coverage depth ranging from 1084.93× (Q. franchetii PF3) to 8669.27× (Q. longispica PL3) (Table 1). The lengths of the cp genomes varied from 160,873 bp (Q. longispica PL1, PL2, and PL3) to 161,005 bp (Q. griffithii PG1, PG2, and PG3) (Table 1). All the 18 cp genomes possessed a typical quadripartite structure with one LSC region (90,303–90,547 bp), one SSC region (18,894–19,070 bp), and two IRs (25,817–25,871 bp) (Figure 2). In general, the length of the cp genome was positively related to those of LSC (r = 0.970, p < 0.001) and SSC (r = 0.878, p < 0.001), but was insignificantly associated with that of IR (r = −0.151, p = 0.550). The four sclerophyllous oaks in section Ilex had a smaller cp genome size as well as smaller LSC and SSC sizes than the two deciduous broad-leaved oaks in sections Cerris and Quercus (Table 1).
Each of the 18 cp genomes encoded 131 genes (113 were unique), including 86 protein-coding genes (79 were unique), 37 tRNA genes (30 were unique), and eight rRNA genes (four were unique) (Figure 2). Seven protein-coding genes (ndhB, rpl2, rpl23, rps7, rps12, ycf1, and ycf2), seven tRNA genes (trnNGUU, trnRACG, trnAUGC, trnIGAU, trnVGAC, trnLCAA, and trnICAU), and four rRNA genes (rrn5S, rrn4.5S, rrn23S, and rrn16S) were duplicated in IRs. Eighteen genes had intron(s). Among those, nine protein-coding genes (rps16, atpF, rpoC1, petB, petD, rpl16, rpl2, ndhB, and ndhA) and six tRNA genes (trnKUUU, trnGGCC, trnLUAA, trnVUAC, trnIGAU, and trnAUGC) had one intron, while three protein-coding genes (rps12, ycf3, and clpP) had two introns.

3.2. Sequence Variation and Haplotype Network of Chloroplast DNA

The alignment of the 18 cp genome sequences with IRa excluded was 136,423 bp in length (including 2268 sites with gaps). Length variations in mononucleotide repeats were excluded, and 16 inversions (2–35 bp in length) were replaced with their reverse complements (Tables S3 and S4). After this step, a total of 1002 single-nucleotide variants (SNVs) were identified, of which 979 were parsimony informative sites and 23 were singleton variable sites. A total of 743 (74.2%), 226 (22.6%), and 33 (3.3%) were found in LSC, SSC, and IRb, respectively. The SSC (π = 0.00400) and LSC regions (π = 0.00289) had higher nucleotide diversity than IRb (π = 0.00044) (Table 2). A large number of SNVs were detected in IGSs (487, 48.6%), CDSs (380, 37.9%), and introns (129, 12.9%). The concatenated alignments of IGSs (π = 0.00388) and introns (π = 0.00288) had higher nucleotide diversity than that of CDSs (π = 0.00194) (Table 2). More SNVs were detected among sections than within sections (Table S5). A total of 100 SNVs were found among the oak trees in section Ilex, while no SNVs were detected within sections Cerris and Quercus (Table 2 and Table S5). At the species level, SNVs were only detected in Q. cocciferoides (S = 64) and Q. franchetii (S = 8) (Table 2 and Table S5).
When indels were considered and coded as binary characters (dataset 1), 9 cp genome haplotypes (A1–A9) were identified among the 18 cp genomes (Table 1 and Figure 3a). Each of the two widespread oaks (Q. cocciferoides and Q. franchetii) had three haplotypes, while each of the four narrowly distributed oaks (Q. dolicholepis, Q. longispica, Q. variabilis, and Q. griffithii) had only one haplotype. The haplotype A3 was found to be shared by two sclerophyllous oaks in section Ilex (i.e., Q. cocciferoides PC3 and Q. franchetii PF1). When indels were not considered (dataset 2), eight cp genome haplotypes (B1–B8) were identified, of which two were found to be shared by a pair of sclerophyllous oaks (Table 1 and Figure 3b). In addition to B3 (=A3 in Figure 3a), the haplotype B2 was found to be shared by Q. cocciferoides PC2 and Q. longispica PL1–PL3. Their cp genomes only differed by a 9 bp indel (AGAAACCTC; 116,935–116,943 bp) in the ψycf1 region. This indel was found to not involve a tandemly repeated sequence because it was not identical to its flanking sequence on either side.
The integer neighbor-joining networks inferred from datasets 1 and 2 had identical topologies except for the difference between haplotypes A2/A8 and B2 (Figure 3a,b). Both networks grouped the cp genome haplotypes into three major lineages corresponding to the three sections of oaks, i.e., Ilex, Cerris, and Quercus. Within section Ilex, four sublineages separated by not only SNVs but also indels were identified (Figure 3a,b), which were also supported by the PCA results (Figure 3d). Two of those four sublineages were found to be interspecifically shared. The first one (sublineage A) comprised three haplotypes (dataset 1: A3, A5, and A6; dataset 2: B3, B5, and B6) belonging to Q. cocciferoides (PC3) and Q. franchetii (PF1–PF3) (Figure 3a,b). This sublineage was widely distributed across the reserve (Figure 3c). The second one (sublineage B) contained one or two haplotypes (dataset 1: A2 and A8; dataset 2: B2) shared by Q. cocciferoides (PC2) and Q. longispica (PL1–PL3) (Figure 3a,b). This sublineage was narrowly distributed in Zhulinpo (Figure 3c). The remaining two sublineages included only one haplotype belonging to Q. cocciferoides PC1 (sublineage C) or Q. dolicholepis PD1–PD3 (sublineage D) (Figure 3a,b). Notably, Q. cocciferoides was found in three sublineages, while the other three Ilex oaks were only detected in one sublineage. Highly similar integer neighbor-joining networks were also obtained for the concatenated alignment of IGSs and introns, and that of CDSs, tRNA genes, and rRNA genes (Figure S1).

3.3. Sequence Variation and Haplotype Network of Nuclear Ribosomal DNA

The alignment of the nrDNA sequences of the 18 oak trees was 3194 bp in length (including 27 sites with gaps). A much higher level of nucleotide diversity was detected in nrDNA (π = 0.01360) than in cpDNA (π = 0.00259) (Table 2). At the species level, more SNVs were detected in Q. griffithii (S = 28) and Q. cocciferoides (S = 68) than in the other four oak species (Q. variabilis: S = 5; Q. dolicholepis: S = 4; Q. franchetii: S = 3; Q. longispica: S = 2) (Table S5). Sixteen haplotypes (R1–R16) were identified based on 143 SNVs observed among the six oak species (Table 1 and Figure 4). Each species had two or three haplotypes. No haplotypes were found to be shared among species. The integer neighbor-joining network grouped the 16 haplotypes into three clades corresponding to sections Ilex, Cerris, and Quercus (Figure 4). Within section Ilex, four species-specific sublineages were identified.

3.4. Phylogenetic Analyses of Chloroplast Genome Sequences

We reconstructed the ML and BI trees using 53 cp genome sequences representing 26 oak species and two outgroups in Fagus. Both trees showed similar topologies of three main cp genome lineages of oaks (BS = 100, PP = 1.00) (Figure 5). The first lineage consisted of all the taxa in section Quercus (10 accessions), including Q. griffithii PG1–PG3. The second lineage was composed of Ilex oaks (5 accessions) and Cerris oaks (11 accessions) including Q. variabilis PV1–PV3. The third lineage comprised ring-cupped oaks (6 accessions) and Ilex oaks (19 accessions), including all the sequenced trees of Q. cocciferoides, Q. franchetii, Q. longispica, and Q. dolicholepis.
Our cpDNA phylogeny included previously published cp genomes of five of the six sequenced oak species (i.e., Q. griffithii, Q. variabilis, Q. cocciferoides, Q. franchetii, and Q. dolicholepis). We found that none of these five species were resolved as monophyletic (Figure 5). Within the Ilex oak lineage sister to ring-cupped oaks, Q. cocciferoides and Q. franchetii were found in multiple sublineages. Specifically, Q. cocciferoides PC3 and Q. franchetii PF1–PF3 formed the first sublineage (=sublineage A in Figure 3); Q. cocciferoides PC2 and Q. longispica PL1–PL3 formed the second sublineage (=sublineage B in Figure 3); Q. cocciferoides PC1 and MW829652 (Panzhihua) and Q. franchetii MG678018 (Panzhihua) formed the third sublineage (=sublineage C in Figure 3); while Q. cocciferoides MG678016 (Panzhihua) and Q. franchetii MW450869 (Yuanjiang, Yunnan) formed the fourth sublineage. Among the four accessions of Q. dolicholepis, three (PD1–PD3) were found in the Ilex oak lineage closely related to ring-cupped oaks, while another one (KU240010) was nested in the Ilex oak lineage closely related to Cerris oaks. Within the Cerris oak lineage, Q. acutissima MH899015 (Shenyang, Liaoning) and Q. variabilis PV1–PV3 formed a sublineage sister to the cluster containing the remaining accessions of Q. acutissima, Q. variabilis, and Q. chenii. Two accessions of Q. griffithii were observed in two subclades of section Quercus (Figure 5).

3.5. Highly Variable Regions and IR-SC Borders in Chloroplast Genomes

Among the six oak species, the top eight highest peaks of π were located within (1) ycf1 (π = 0.00918); (2) ψycf1 and ndhF (π = 0.00869); (3) ndhF-rpl32, rpl32, and rpl32-trnLUAG (π = 0.00759); (4) trnKUUU and trnKUUU-rps16 (π = 0.00731); (5) trnKUUU-rps16 and rps16 (π = 0.00720); (6) trnGGCC, trnGGCC- trnRUCU, trnRUCU, trnRUCU-atpA, and atpA (π = 0.00700); (7) trnTGGU-psbD and psbD (π = 0.00658); and (8) rpoB and rpoB-trnCGCA (π = 0.00640) (Figure 6 and Table S6). Between four and six haplotypes were determined by each of these regions (Table S6). Among all the CDSs, tRNA genes, rRNA genes, introns, and IGSs, four loci exhibited the most haplotypes, including ndhF-rpl32 (n = 7), trnKUUU-rps16 (n = 6), rpoC2 (n = 6), and clpP intron 2 (n = 6) (Table S3 and Figure S2).
Among the four Ilex oaks, the top eight highest peaks of π were located within (1) ndhF-rpl32, rpl32, and rpl32-trnLUAG (π = 0.00220); (2) trnKUUU intron (π = 0.00145); (3) psbE-petL and petL (π = 0.00145); (4) ycf4-cemA, cemA, and cemA-petA (π = 0.00138); (5) rps11-rpl36, rpl36, rpl36-infA, infA, infA-rps8, and rps8 (π = 0.00130); (6) ψycf1 and ndhF (π = 0.00115); (7) psbK-psbI, psbI, psbI-trnSGCU, trnSGCU, and trnSGCU-trnGGCC (π = 0.00114); and (8) rpoC2 (π = 0.00114) (Figure 6 and Table S7). Between three and four haplotypes were determined by each of these regions (Table S7). Among all the CDSs, tRNA genes, rRNA genes, introns, and IGSs, four loci exhibited the most haplotypes, including ndhF-rpl32 (n = 5), trnKUUU-rps16 (n = 4), rpoC2 (n = 4), and clpP intron 2 (n = 4) (Table S4 and Figure S2).
The IR/LSC junctions were highly conserved among the six oak species. In all the 18 cp genomes, the rps19 and rpl2 genes were separated from the IRb/LSC border by spacers of 10 bp and 62 bp, respectively. The trnH gene was separated from the IRa/LSC border by a spacer of 16 bp (Figure S3). The IR/SSC junctions varied slightly among the six oak species. The ndhF gene was completely located in the SSC region in Q. variabilis and Q. griffithii but extended into the IRb region with 35 bp in Q. dolicholepis and 38 bp in the other three Ilex oak species. The IRa/SSC border was within the ycf1 gene, resulting in a pseudogene fragment ψycf1 with 1041–1073 bp in the IRb region (Figure S3).

4. Discussion

4.1. Incongruence between Chloroplast and Nuclear Phylogenies of Ilex Oaks

Our study demonstrated that discordance existed between the cpDNA- and nrDNA-based phylogenies of the four sympatric Ilex oaks in the hot-dry valley of the Jinsha River, southwestern China. The nrDNA-based phylogeny was consistent with species boundaries (Figure 4). Recent phylogenetic studies using RAD-seq data have shown that the four species belong to different lineages of Ilex oaks [8,91]. Q. franchetii was considered as part of an early diverging lineage of Ilex oaks (=clade I in [91]), which was found to be closest to the common ancestor of Ilex oaks in the nrDNA network (Figure 4). Q. dolicholepis, Q. cocciferoides, and Q. longispica were shown to be the members of the subtropical lineage (=clade II in [91]), the tropical lineage (=clade III in [91]), and the Himalayan subalpine lineage (=clade IV in [91]) of Ilex oaks, respectively. These four species were estimated to have diverged during the Eocene [91]. Such a long evolutionary time suggests that the four species may have developed strong reproductive barriers suppressing recent interspecific gene flow [92]. Although nuclear DNA phylogeny indicates that the four Ilex oaks are not the most closely related species, they were found to share the same or closely related cp genome haplotypes (Figure 3 and Figure 5). The non-monophyly of the cp lineages was also reported for Ilex oaks in other areas [6,17,27,28,29,40,41,42,69,93]. Both the retention of ancestral polymorphism and ancient introgression among ancestral populations of oaks may explain the sharing of closely related haplotypes among Ilex oaks [28,40].
First, our results indicated that Q. cocciferoides and Q. longispica sampled in a mixed stand in Zhulinpo exhibited two haplotypes that only differed by a 9 bp indel (i.e., A2 and A8 in Figure 3a). The local distribution of the two highly similar haplotypes suggested that they may have arisen from ancient introgression [22,26,30,42]. Given that the two species have diverged for more than 35 million years [91], it is possible that the ancestral cp genome of one species was captured by another species through asymmetric introgression in early times, and an indel event occurred subsequently. The sharing of cpDNA lineages between Q. cocciferoides and Q. longispica and its relatives in the Himalayan subalpine lineage of Ilex oaks was also reported by a phylogenetic study using four cpDNA markers [41]. Interestingly, they found that the haplotypes of Q. cocciferoides in Panzhihua were more closely related to those in a neighboring population of Q. longispica (Yuanmou, Yunnan) in the hot-dry valley of the Longchuan River, a tributary of the Jinsha River, while the haplotypes of Q. cocciferoides in the Hengduan Mountains were more closely related to those of Q. longispica in the same area. The geographic sorting of these cpDNA lineages pointed toward ancient introgression among ancestral populations of Q. cocciferoides and Himalayan subalpine Ilex oaks in at least two isolated areas [28,40].
Second, our study showed that Q. cocciferoides and Q. franchetii shared multiple cpDNA lineages of Ilex oaks. Specifically, the haplotype A3 was found to be shared by two Q. cocciferoides and Q. franchetii trees separated by ~2.7 km (Figure 3a,c). Phylogenetic analyses using more previously published cp genome sequences further indicated that Q. cocciferoides shared the other two cpDNA lineages with Q. franchetii in the hot-dry valleys of the Jinsha River or the Yuan River (Figure 5). Given that both species are dominant and common in the hot-dry valley ESOFs [34,36], it is difficult to determine whether the shared cpDNA lineages were locally distributed or widely distributed throughout the species’ ranges using a limited number of samples. If the shared lineages were only observed in a specific geographic area, they were more likely to be a result of ancient introgression [22,26,30,42]; however, if they presented a broad and random distribution, the pattern would be better explained by the retention of ancestral polymorphism [26,94]. Under this scenario, the initial divergence of the cpDNA lineages of the two species was considered to be independent of the speciation process; the two species may share an ancestral plastid gene pool that has split into multiple lineages before the formation of the modern-day species [26,28,95]. A thorough investigation into both nuclear and cp genomes of more samples collected across the species’ range is needed to discriminate between the contributions of shared ancestral polymorphism and ancient introgression [17,32].
Finally, we found that the chloroplast genomes of Q. dolicholepis belonged to two distinct cpDNA lineages of Ilex oaks (Figure 5). Indeed, previous studies have shown that East Asian Ilex oaks had several geographically sorted cpDNA lineages [29,69]. In our case, Q. dolicholepis PD1–PD3 belonged to the southwestern China lineage that was closely related to ring-cupped oaks, while Q. dolicholepis KU240010 (central China) [68] belonged to the Sino-Japan lineage that was closely related to Cerris oaks [29,41]. Our results provide evidence that the phylogeographic split of East Asian Ilex oaks also occurred at the intraspecific level. A similar pattern was also observed in other Ilex oaks such as Q. utilis and Q. spinosa [41,69]. These complex patterns may occur through several steps. A likely scenario was that: (1) Ancient introgression among ancestral lineages of Ilex oaks and the other two sections of oaks (Cerris and Cyclobalanopsis) occurred independently in different areas, e.g., southwestern China and central China [29]. (2) The ancestral oak populations in those areas became geographically isolated [17,29], resulting in the separation of the SW China lineage and the Sino-Japan lineage. (3) Q. dolicholepis may have inherited the highly divergent haplotypes in the two cpDNA lineages or captured the cp genome of its relatives with an overlapping distribution through asymmetric introgression.

4.2. Incongruence between Chloroplast and Nuclear Phylogenies of Cerris Oaks

We found that the cpDNA phylogeny of East Asian Cerris oaks (Q. variabilis, Q. acutissima, and Q. chenii) was decoupled from species boundaries (Figure 5). A recent study has shown that the three species presented three non-species-specific cpDNA lineages with overlapping distributions [25,26]. The ancestral haplotypes of the three lineages were randomly distributed throughout the three species’ ranges, while young haplotypes were locally shared among species, suggesting that both the retention of ancestral polymorphism and introgression have contributed to the sharing of cpDNA haplotypes among East Asian Cerris oaks [26]. In our case, Q. variabilis in southwestern China (PV1–PV3) and Q. acutissima in northeastern China (MH899015) [73] presented haplotypes of the lineage A in [26], while the other Cerris oak trees in southeastern China, northwestern China, and northern China presented haplotypes of the lineage B in [26]. The close relationship between geographically distant samples of different species may reflect the imprint of the retention of ancestral polymorphism [26].

4.3. Highly Variable Chloroplast Genome Regions of Sclerophyllous Oaks in Hot-Dry Valleys

Although cp genome sequences are less efficient in discriminating closely related oaks [41,96], they are useful for the evolutionary studies of maternal lineages of oaks. In our study, we found that at least three highly variable regions can distinguish the nine haplotypes identified by the whole-cp genome sequences of the six oaks, including ψycf1 (separating PC2 from PL1–PL3 through a 9 bp indel), ndhF-rpl32 (separating PF2 from PF3 through a SNV), and trnKUUU-rps16 or rpl32-trnLUAG (separating PF1/PC3 from PF3 through a SNV). These regions also exhibited a high level of nucleotide diversity among other Fagaceae species, e.g., [97,98,99,100]. Together with other highly variable regions, these markers have great potential in tracking the evolutionary history of cp lineages of sclerophyllous oaks in hot-dry valleys.

5. Conclusions

Phylogenetic incongruence between chloroplast and nuclear genome genealogies has been commonly observed among closely related species, including oaks and other Fagaceae species, e.g., [17,30,32,93]. Our study used both cpDNA and nrDNA to explore the genetic variation patterns of six oak species in the hot-dry valley of the Jinsha River, southwestern China. We found that even at a fine scale, discordance existed between the cpDNA- and nrDNA-based phylogenies of sclerophyllous oaks in section Ilex. The nrDNA-based phylogeny was consistent with species boundaries defined by morphology, while the cpDNA-based phylogeny was decoupled from taxonomy. Incongruence between the species boundaries and the cpDNA-based phylogeny was illustrated by the sharing of closely related cp genome haplotypes among Q. cocciferoides, Q. franchetii, and Q. longispica, which may be explained by shared ancestral polymorphism and/or ancient introgression. Future work should use nuclear and chloroplast genome data of more samples collected across the species’ range to discriminate between the contributions of shared ancestral polymorphism and ancient introgression.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f15030537/s1, Figure S1: Integer neighbor-joining network of chloroplast DNA haplotypes identified based on the single-nucleotide variants in (a) the concatenated alignment of intergenic spacers and introns; (b) the concatenated alignment of coding sequences, tRNA genes, and rRNA genes; Figure S2: Integer neighbor-joining network of chloroplast DNA haplotypes identified based on the single-nucleotide variants in the aligned sequences of (a) ndhF-rpl32, (b) trnKUUU-rps16, (c) rpoC2, (d) clpP intron 2; Figure S3: Comparison of the borders of large single-copy, small single-copy, and inverted repeat regions among the six oak species; Table S1: Sampling and sequencing information of the 18 oak trees; Table S2: The 53 chloroplast genome sequences used in the phylogenetic analyses; Table S3: Chloroplast DNA polymorphism information for CDSs, tRNA genes, rRNA genes, introns, and IGSs among the 18 oak trees; Table S4: Chloroplast DNA polymorphism information for CDSs, tRNA genes, rRNA genes, introns, and IGSs among the 12 Ilex oak trees; Table S5: Number of single-nucleotide variants among the chloroplast genome sequences of different groups of oak trees; Table S6: The top eight highest peaks of nucleotide diversity among the 18 chloroplast genomes of the six oak species; Table S7: The top eight highest peaks of nucleotide diversity among the 12 chloroplast genomes of the four Ilex oak species.

Author Contributions

Conceptualization, Y.L., Y.F., Y.Y. and L.M.; formal analysis, Y.L. and L.W.; investigation, Y.L., C.T., W.Z. and Z.Y.; writing—original draft preparation, Y.L.; writing—review and editing, Y.F., Y.Y. and L.M.; supervision, L.M.; funding acquisition, Y.L., Y.Y. and L.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was financially supported by the National Natural Science Foundation of China (32201375), the China Postdoctoral Science Foundation (2020M681629), the Jiangsu Postdoctoral Research Foundation (2021K038A), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Data Availability Statement

Chloroplast genome sequences of the 18 oak trees were submitted to NCBI under GenBank accession numbers OR188388–OR188405. The alignments of cp genomes and nuclear ribosomal DNA are available from Zenodo: https://doi.org/10.5281/zenodo.10656343.

Acknowledgments

We thank Yongqiong Yang, Zhixiang Yu, Chuanfeng Shen, Yibin Yang, Huan Li, Jiangbin Lin, Guangming Bao, and other natural resource managers of the Panzhihua Cycad National Nature Reserve, Sichuan Province, China for their help in the sample collection.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Figure 1. (a) Sampling locations of the 18 oak trees in the Panzhihua Cycad National Nature Reserve, Sichuan Province, China. Taxa are indicated with colors. Squares, diamonds, and circles represent oak species in sections Quercus, Cerris, and Ilex, respectively. (b–g) Leaf photos of Quercus griffithii (b), Q. variabilis (c), Q. cocciferoides (d), Q. dolicholepis (e), Q. franchetii (f), and Q. longispica (g) in the Panzhihua Cycad National Nature Reserve. All photos were taken by Yao Li.
Figure 1. (a) Sampling locations of the 18 oak trees in the Panzhihua Cycad National Nature Reserve, Sichuan Province, China. Taxa are indicated with colors. Squares, diamonds, and circles represent oak species in sections Quercus, Cerris, and Ilex, respectively. (b–g) Leaf photos of Quercus griffithii (b), Q. variabilis (c), Q. cocciferoides (d), Q. dolicholepis (e), Q. franchetii (f), and Q. longispica (g) in the Panzhihua Cycad National Nature Reserve. All photos were taken by Yao Li.
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Figure 2. Gene circle map of the newly sequenced chloroplast genome of Quercus longispica (GenBank accession number: OR188400). Genes of different functions are color-coded. Genes shown inside the circle are transcribed clockwise and those outside are transcribed counterclockwise. The darker gray columns in the inner circle correspond to the GC content.
Figure 2. Gene circle map of the newly sequenced chloroplast genome of Quercus longispica (GenBank accession number: OR188400). Genes of different functions are color-coded. Genes shown inside the circle are transcribed clockwise and those outside are transcribed counterclockwise. The darker gray columns in the inner circle correspond to the GC content.
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Figure 3. (a,b) Integer neighbor-joining network of chloroplast (cp) genome haplotypes identified based on the alignment of cp genomes with IRa excluded and indels considered (dataset 1) (a) or not (dataset 2) (b). When indels were considered, they were coded as binary characters according to the simple gap coding method using GapCoder. Circle size is proportional to the frequency of a haplotype across all samples. Numbers in brackets on branches indicate the number of mutations between haplotypes. Taxa are indicated with colors. Sample IDs are labeled for each haplotype. PC, Quercus cocciferoides; PF, Q. franchetii; PL, Q. longispica; PD, Q. dolicholepis; PV, Q. variabilis; PG, Q. griffithii. (c) Geographic distribution of the four sublineages of Ilex oaks. (d) Principal component analysis (PCA) based on the alignment of cp genomes with IRa excluded and indels coded as binary characters. The four sublineages of Ilex oaks are indicated with colors as in (c).
Figure 3. (a,b) Integer neighbor-joining network of chloroplast (cp) genome haplotypes identified based on the alignment of cp genomes with IRa excluded and indels considered (dataset 1) (a) or not (dataset 2) (b). When indels were considered, they were coded as binary characters according to the simple gap coding method using GapCoder. Circle size is proportional to the frequency of a haplotype across all samples. Numbers in brackets on branches indicate the number of mutations between haplotypes. Taxa are indicated with colors. Sample IDs are labeled for each haplotype. PC, Quercus cocciferoides; PF, Q. franchetii; PL, Q. longispica; PD, Q. dolicholepis; PV, Q. variabilis; PG, Q. griffithii. (c) Geographic distribution of the four sublineages of Ilex oaks. (d) Principal component analysis (PCA) based on the alignment of cp genomes with IRa excluded and indels coded as binary characters. The four sublineages of Ilex oaks are indicated with colors as in (c).
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Figure 4. Integer neighbor-joining network of nuclear ribosomal (nr) DNA haplotypes detected among the six oak species. Circle size is proportional to the frequency of a haplotype across all samples. Numbers in brackets on branches indicate the number of mutations between haplotypes. Taxa are indicated with colors. Sample IDs are labeled for each haplotype. PC, Quercus cocciferoides; PF, Q. franchetii; PL, Q. longispica; PD, Q. dolicholepis; PV, Q. variabilis; PG, Q. griffithii.
Figure 4. Integer neighbor-joining network of nuclear ribosomal (nr) DNA haplotypes detected among the six oak species. Circle size is proportional to the frequency of a haplotype across all samples. Numbers in brackets on branches indicate the number of mutations between haplotypes. Taxa are indicated with colors. Sample IDs are labeled for each haplotype. PC, Quercus cocciferoides; PF, Q. franchetii; PL, Q. longispica; PD, Q. dolicholepis; PV, Q. variabilis; PG, Q. griffithii.
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Figure 5. Cladogram of the cpDNA phylogeny of 53 accessions representing 26 Quercus species and two outgroups from Fagus. The phylogeny was inferred using maximum-likelihood (ML) and Bayesian inference (BI) methods based on trimmed locally collinear blocks (LCBs) shared by chloroplast genomes. Numbers associated with nodes indicate ML bootstrap support (BS) values (left) and BI posterior probability (PP) values (right). Colored blocks indicate the four sections of oaks including Ilex, Cerris, Cyclobalanopsis (Cyclo.), and Quercus. Dark blue bars indicate the four sublineages of Ilex oaks (A–D) identified in the integer neighbor-joining networks of cp genome haplotypes in Figure 3. Accessions with identical sequences of LCBs have been collapsed. Newly generated sequences are marked by asterisks.
Figure 5. Cladogram of the cpDNA phylogeny of 53 accessions representing 26 Quercus species and two outgroups from Fagus. The phylogeny was inferred using maximum-likelihood (ML) and Bayesian inference (BI) methods based on trimmed locally collinear blocks (LCBs) shared by chloroplast genomes. Numbers associated with nodes indicate ML bootstrap support (BS) values (left) and BI posterior probability (PP) values (right). Colored blocks indicate the four sections of oaks including Ilex, Cerris, Cyclobalanopsis (Cyclo.), and Quercus. Dark blue bars indicate the four sublineages of Ilex oaks (A–D) identified in the integer neighbor-joining networks of cp genome haplotypes in Figure 3. Accessions with identical sequences of LCBs have been collapsed. Newly generated sequences are marked by asterisks.
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Figure 6. Sliding-window analyses showing the nucleotide diversity (π) values among the 18 oak trees (a) and among the 12 Ilex oak trees (b). (a) The top eight highest peaks of π were located within (1) ycf1; (2) ψycf1 and ndhF; (3) ndhF-rpl32, rpl32, and rpl32-trnLUAG; (4) trnKUUU and trnKUUU-rps16; (5) trnKUUU-rps16 and rps16; (6) trnGGCC, trnGGCC-trnRUCU, trnRUCU, trnRUCU-atpA, and atpA; (7) trnTGGU-psbD and psbD; and (8) rpoB and rpoB-trnCGCA. (b) The top eight highest peaks of π were located within (1) ndhF-rpl32, rpl32, and rpl32-trnLUAG; (2) trnKUUU intron; (3) psbE-petL and petL; (4) ycf4-cemA, cemA, and cemA-petA; (5) rps11-rpl36, rpl36, rpl36-infA, infA, infA-rps8, and rps8; (6) ψycf1 and ndhF; (7) psbK-psbI, psbI, psbI-trnSGCU, trnSGCU, and trnSGCU-trnGGCC; and (8) rpoC2.
Figure 6. Sliding-window analyses showing the nucleotide diversity (π) values among the 18 oak trees (a) and among the 12 Ilex oak trees (b). (a) The top eight highest peaks of π were located within (1) ycf1; (2) ψycf1 and ndhF; (3) ndhF-rpl32, rpl32, and rpl32-trnLUAG; (4) trnKUUU and trnKUUU-rps16; (5) trnKUUU-rps16 and rps16; (6) trnGGCC, trnGGCC-trnRUCU, trnRUCU, trnRUCU-atpA, and atpA; (7) trnTGGU-psbD and psbD; and (8) rpoB and rpoB-trnCGCA. (b) The top eight highest peaks of π were located within (1) ndhF-rpl32, rpl32, and rpl32-trnLUAG; (2) trnKUUU intron; (3) psbE-petL and petL; (4) ycf4-cemA, cemA, and cemA-petA; (5) rps11-rpl36, rpl36, rpl36-infA, infA, infA-rps8, and rps8; (6) ψycf1 and ndhF; (7) psbK-psbI, psbI, psbI-trnSGCU, trnSGCU, and trnSGCU-trnGGCC; and (8) rpoC2.
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Table 1. Summary of the chloroplast (cp) genomes and nuclear ribosomal (nr) DNA sequences for the six oak species found in the hot-dry valley of the Jinsha River, southwestern China. Unique cpDNA haplotypes were identified based on the alignment of cp genomes with IRa excluded and indels considered (dataset 1) or not (dataset 2). LSC, large single-copy; SSC, small single-copy; IR, inverted repeat.
Table 1. Summary of the chloroplast (cp) genomes and nuclear ribosomal (nr) DNA sequences for the six oak species found in the hot-dry valley of the Jinsha River, southwestern China. Unique cpDNA haplotypes were identified based on the alignment of cp genomes with IRa excluded and indels considered (dataset 1) or not (dataset 2). LSC, large single-copy; SSC, small single-copy; IR, inverted repeat.
SpeciesSectionVoucherChloroplast GenomesnrDNA Haplotype
GenBank
Acc. No.		Mean
Coverage		Length (bp)GC (%)cpDNA Haplotype
WholeLSCIRa/IRbSSCDataset 1Dataset 2
Q. cocciferoidesIlexPC1OR1883882270.98×160,90990,35725,82618,90036.90A1B1R1
Q. cocciferoidesIlexPC2OR1883893963.72×160,89090,30325,84618,89536.91A2B2R2
Q. cocciferoidesIlexPC3OR1883903229.87×160,96590,37825,84618,89536.90A3B3R3
Q. franchetiiIlexPF1OR1883942037.66×160,96790,38025,84618,89536.90A3B3R7
Q. franchetiiIlexPF2OR1883953679.56×160,97290,38625,84618,89436.90A5B5R8
Q. franchetiiIlexPF3OR1883961084.93×160,97290,38625,84618,89436.90A6B6R9
Q. longispicaIlexPL1OR1884006627.02×160,87390,30425,83718,89536.91A8B2R13
Q. longispicaIlexPL2OR1884015043.55×160,87390,30425,83718,89536.91A8B2R13
Q. longispicaIlexPL3OR1884028669.27×160,87390,30425,83718,89536.91A8B2R14
Q. dolicholepisIlexPD1OR1883916479.03×161,00490,35625,87118,90636.89A4B4R4
Q. dolicholepisIlexPD2OR1883922321.80×161,00590,35725,87118,90636.89A4B4R5
Q. dolicholepisIlexPD3OR1883934866.05×161,00590,35725,87118,90636.89A4B4R6
Q. variabilisCerrisPV1OR1884031826.09×161,13990,43525,81719,07036.78A9B8R15
Q. variabilisCerrisPV2OR1884041864.74×161,13990,43525,81719,07036.78A9B8R16
Q. variabilisCerrisPV3OR1884051659.92×161,13990,43525,81719,07036.78A9B8R15
Q. griffithiiQuercusPG1OR1883972196.73×161,27790,54725,84419,04236.82A7B7R10
Q. griffithiiQuercusPG3OR1883982581.35×161,27790,54725,84419,04236.82A7B7R11
Q. griffithiiQuercusPG5OR1883992070.87×161,27790,54725,84419,04236.82A7B7R12
Table 2. Number of single-nucleotide variants (SNVs) and nucleotide diversity (π) in different regions of chloroplast (cp) genomes and nuclear ribosomal (nr) DNA sequences. The alignment of whole-cp genome sequences with IRa excluded was used. Inversions were replaced with their reverse complements.
Table 2. Number of single-nucleotide variants (SNVs) and nucleotide diversity (π) in different regions of chloroplast (cp) genomes and nuclear ribosomal (nr) DNA sequences. The alignment of whole-cp genome sequences with IRa excluded was used. Inversions were replaced with their reverse complements.
RegionSix Oak Species
(n = 18)		Four Ilex Oaks
(n = 12)		Q. cocciferoides
(n = 3)		Q. franchetii
(n = 3)
SπSπSπSπ
Cp genome10020.002591000.00027640.0003280.00004
LSC7430.00289730.00029510.0003840.00003
SSC226 *0.00400230.0004690.0003230.00011
IRb330.0004440.0000640.0001010.00003
CDS3800.00194300.00017210.0002020.00002
tRNA30.0004700.0000000.0000000.00000
rRNA30.0001910.0000410.0001500.00000
IGS4870.00388580.00048330.0005060.00009
Introns1290.00288110.0002590.0003900.00000
nrDNA1430.01360860.01092160.0033630.00063
n, number of sequences; S, number of SNVs; LSC, large single-copy; SSC, small single-copy; IR, inverted repeat; CDS, coding sequence; IGS, intergenic spacer. * This number included two SNVs flanking the IRb/SSC junction. The numbers of SNVs in each CDS, tRNA gene, rRNA gene, IGS, and intron are shown in Tables S3 and S4.
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Li, Y.; Tan, C.; Zhang, W.; Wang, L.; Yang, Z.; Fang, Y.; Yang, Y.; Mao, L. Interspecific Sharing of Closely Related Chloroplast Genome Haplotypes among Sclerophyllous Oaks in the Hot-Dry Valley of the Jinsha River, Southwestern China. Forests 2024, 15, 537. https://doi.org/10.3390/f15030537

AMA Style

Li Y, Tan C, Zhang W, Wang L, Yang Z, Fang Y, Yang Y, Mao L. Interspecific Sharing of Closely Related Chloroplast Genome Haplotypes among Sclerophyllous Oaks in the Hot-Dry Valley of the Jinsha River, Southwestern China. Forests. 2024; 15(3):537. https://doi.org/10.3390/f15030537

Chicago/Turabian Style

Li, Yao, Chao Tan, Wenxu Zhang, Lu Wang, Zhi Yang, Yanming Fang, Yong Yang, and Lingfeng Mao. 2024. "Interspecific Sharing of Closely Related Chloroplast Genome Haplotypes among Sclerophyllous Oaks in the Hot-Dry Valley of the Jinsha River, Southwestern China" Forests 15, no. 3: 537. https://doi.org/10.3390/f15030537

APA Style

Li, Y., Tan, C., Zhang, W., Wang, L., Yang, Z., Fang, Y., Yang, Y., & Mao, L. (2024). Interspecific Sharing of Closely Related Chloroplast Genome Haplotypes among Sclerophyllous Oaks in the Hot-Dry Valley of the Jinsha River, Southwestern China. Forests, 15(3), 537. https://doi.org/10.3390/f15030537

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