Chloroplast genome resources can provide useful information for the evolution of plant species. Tea plant (Camellia sinensis) is among the most economically valuable member of Camellia. Here, we determined the chloroplast genome of the first natural triploid Chinary type tea (‘Wuyi narcissus’ cultivar of Camellia sinensis var. sinensis, CWN) and conducted the genome comparison with the diploid Chinary type tea (Camellia sinensis var. sinensis, CSS) and two types of diploid Assamica type teas (Camellia sinensis var. assamica: Chinese Assamica type tea, CSA and Indian Assamica type tea, CIA). Further, the evolutionary mechanism of the chloroplast genome of Camellia sinensis and the relationships of Camellia species based on chloroplast genome were discussed.
Comparative analysis showed the evolutionary dynamics of chloroplast genome of Camellia sinensis were the repeats and insertion-deletions (indels), and distribution of the repeats, indels and substitutions were significantly correlated. Chinese tea and Indian tea had significant differences in the structural characteristic and the codon usage of the chloroplast genome. Analysis of sequence characterized amplified region (SCAR) using sequences of the intergenic spacers (trnE/trnT) showed none of 292 different Camellia sinensis cultivars had similar sequence characteristic to triploid CWN, but the other four Camellia species did. Estimations of the divergence time showed that CIA diverged from the common ancestor of two Assamica type teas about 6.2 Mya (CI: 4.4–8.1 Mya). CSS and CSA diverged to each other about 0.8 Mya (CI: 0.4–1.5 Mya). Moreover, phylogenetic clustering was not exactly consistent with the current taxonomy of Camellia.
The repeat-induced and indel-induced mutations were two important dynamics contributed to the diversification of the chloroplast genome in Camellia sinensis, which were not mutually exclusive. Chinese tea and Indian tea might have undergone different selection pressures. Chloroplast transfer occurred during the polyploid evolution in Camellia sinensis. In addition, our results supported the three different domestication origins of Chinary type tea, Chinese Assamica type tea and Indian Assamica type tea. And, the current classification of some Camellia species might need to be further discussed.
Because of frequent hybridization and polyploidization, the mechanisms operating in the evolution of Camellia has always been focus of botanical and ecological research [1,2,3]. Tea plant (Camellia sinensis) is a member of the Theaceae family of angiosperms, and is highly regarded as the oldest and most popular nonalcoholic beverage with huge economic values in the world . Cultivated tea plants have been divided into three distinct groups: Camellia sinensis var. sinensis (L.) O. Kuntze (Chinary type), Camellia sinensis var. assamica (Masters) Chang (Assamica type) and C. sinensis var. assamica subssp. Lasiocalyx Planch (Cambodia type). Of which, the most obvious distinction is between C. sinensis var. sinensis and C. sinensis var. assamica. In brief, C. sinensis var. sinensis has small leaves and is major cultivated in China and some Southeast Asian countries, while C. sinensis var. assamica has large leaves and widely grown in India and some hot countries except for southern China [5,6,7]. It has long been suggested that C. sinensis var. sinensis and C. sinensis var. assamica may have distinct origins, but the idea that C. sinensis var. assamica consists of two distinct lineages (Chinese Assamica type and Indian Assamica type) that were domesticated separately is more controversial .
Chloroplast (cp) genomes are highly conserved in sequence and structure due to their non-recombinant, haploid, and uniparentally inherited nature . Nonetheless, the gene losses and/or additions, rearrangements and repeats within cp genomes had been revealed in many angiosperm lineages [10,11,12,13]. Additionally, gene transfer between plastome, chondrome and nucleus had also been found in plants [14, 15]. Therefore, cp genome structural variations are accompanied by speciation over time, which can provide a wealth of evolutionary information . In previous studies, cp genomes had been found to be particularly useful for phylogenetic and phylogeographic studies in the contexts of reticulate evolution (i.e. hybridization) and polyploidization that characterize the history of most plant lineages [17,18,19,20]. Some studies also had found that the cp genome resources could provide useful data for eliciting the evolutionary relationships of tea plants, thus reflecting important evidence for a well-supported hypothesis of classification . Up to now, more than 30 complete cp genomes of Camellia species had been sequenced . These massive data, helped from their conserved evolution, promotes the use of cp sequences as an effective tool for Camellia species phylogenomic analyses.
In addition to interspecific hybridization, polyploidization is another important factor in the diversification of angiosperm plants [23, 24]. cpDNA variation could provide valuable genetic markers for the analysis of polyploids. Non-recombination and uniparental inheritance had made cpDNA marker a good indicator of maternal ancestry which could be easily identified in putative hybrid progeny in the absence of parental information, regardless of how many generations had past [25,26,27,28]. Using cpDNA marker as sequence characterized amplified region (SCAR) to screen for cp differences between species had proven to be utility in analysis of maternal ancestry of polyploid . In a previous study on the evolution of allotetraploid Brassicas, cpDNA data revealed not only the maternal origin of three allotetraploids, but also specific populations of diploids that contributed the cytoplasm to each allotetraploid, and proposed the possibility of introgressive hybridization (chloroplast transfer) . So far, the cp genome of the polyploid tea plant has not been reported, and the possible effects of polyploidization on the cp genome of tea plant need to be further explored.
In this study, we generated the complete cp genome of the first natural triploid tea plant (‘Wuyi narcissus’ cultivar of C. sinensis var. sinensis) which belong to asexual propagation cultivar and was recognized as one of the national quality tea varieties by China National Crop Variety Examination Committee in 1985 (GS13009–1985) . Then, we presented the detailed sequence and structural variations of the cp genome among the four representative tea plants, including ‘Wuyi narcissus’ cultivar of C. sinensis var. sinensis (CWN, a natural triploid Chinary type tea), a diploid C. sinensis var. sinensis (CSS, Chinary type tea) and two diploid C. sinensis var. assamica (CSA, Chinese Assamica type tea and CIA, Indian Assamica type tea). Through comparative analysis, we explored the evolutionary dynamics of cp genome and the effects of polyploidization in C. sinensis. Furthermore, the phylogenetic analysis and the divergence time estimation based on complete cp genomes were conducted to explore the evolutionary relationship between Chinary type tea, Chinese Assamica type tea and Indian Assamica type tea, and to further improve our understanding of the taxonomic classification of Camellia.
Chloroplast genome sequencing and assembly
The cp genome of ‘Wuyi narcissus’ cultivar of C. sinensis var. sinensis was constructed by PacBio long-reads with Illumina paired-ends data support. In total, 46,941,086 Illumina reads (7.04 Gb, Average read length 145 bp) and 364,638 PacBio reads (10,383 reads > 5000 bp, Average read length 1139 bp) were mapped to the complete genome, respectively. The average organelle coverage reached 43,419× and 2650× sequencing depth, respectively. The de novo assembly using error-corrected PacBio reads resulted in a circular genome of 156,762 bp length (Fig. 1). Raw reads, assembled cp genome sequences and accompanying gene annotations had been deposited in the NCBI GenBank (SRA: SRR12002624, Accession numbers: MT612435).
Chloroplast genome structure and characteristics analyses
All four complete cp genomes displayed the typical quadripartite structure of most angiosperms, including the large single copy (LSC), the small single copy (SSC) and a pair of inverted repeats (IRa and IRb). Among these cp genomes, genome size ranged from 156,762 bp to 157,353 bp due to expansion and contraction of cp genomes. The length varied from 86,301 bp to 87,214 bp in the LSC region, from 18,079 bp to 18,285 bp in the SSC region, and from 26,030 bp to 26,090 bp in IR region (Table 1).
Each cp genome contained a total of 137 genes, including 92 protein-coding genes, 37 transfer RNA (tRNA) genes and 8 ribosomal RNA (rRNA) (Supplementary Tab. S1). Of them, 60 protein-coding and 22 tRNA genes were located within LSC, 16 protein-coding genes, 14 tRNA coding genes and eight rRNA coding genes were located within IRs and 11 protein-coding and one tRNA gene were located within SSC. The rps12 gene was a divided gene with the 5′ end exon located in the LSC region while two copies of 3′ end exon and intron were located in the IRs. The ycf1 was located in the boundary regions between IR/SSC, leading to incomplete duplication of the gene within IRs. There were 18 genes containing introns, including 6 tRNA genes and 12 protein-coding genes. Except for two introns in the ycf3 and clpP genes, all other genes contained only one intron. MatK gene was located within the intron of trnK-UUU with the largest intron (2489 bp). Overlaps of adjacent genes were found in the complete genome, rps3-rpl22, atpB-atpE, and psbD-psbC had a 16 bp, 4 bp, and 53 bp overlapping region, respectively. Unusual initiator codons were observed in rps19 with GTG and orf42 with ATC in four cp genomes. The initiation codon of ndhD in CIA was ATG, while that of other three cp genomes was GTG.
Sequence variation analyses
The differences and evolutionary divergences among four cp genomes were compared using nucleotide substitutions and sequence distance. Across all four species, the value of nucleotide differences was 70–185, and the p-distance was 0.00045–0.00118. The value of nucleotide difference (70) and the p-distance (0.00045) between triploid CWN and diploid CSS was smallest (Table 2).
To identify the potential genome rearrangements and inversions, the cp genome sequences of four species were plotted to check their identity using the program mVISTA. No gene rearrangement and inversion events were detected (Fig. 2). Sequence divergence analyses showed four regions (including rp12/trnH-UGU, psaA/ycf3, atpB/rbcL and psbT/psbH) had relatively higher divergence values (Pi > 0.006) (Fig. 3). Mutations of the base replacement or deletion may cause changes in the length of the coding gene sequence, leading to changes in the coding and non-coding regions. Therefore, the variable characters in coding and non-coding regions among four cp genomes were further analyzed. The results showed that the proportion of variability in non-coding regions was with a mean value of 1.82%, while in the coding regions was 1.15%. Five coding genes had over 4% variability proportion, such as rps19, ndhF, ndhD, ndhI and ycf1. Five non-coding regions had over 10% variability proportions, such as rpl2/trnH-GUG, trnE-UUC/trnT-GGU, ndhD/psaC, ndhI/ndhA and rps15/ycf1 (Fig. 4).
To further observe the potential contraction and expansion of IR regions, the gene variation at the IR/SSC and IR/LSC boundary regions of the four plastomes was compared (Fig. 5). The genes rps19, ycf1–5’end/ndhF, ycf1 and rp12/trnH-GUG were located in the junctions of LSC/IR and SSC/IR regions. The rps19 gene in CSS, CSA, and CWN was 279 bp, and crossed the LSC/IR region by 46 bp while the rps19 gene in CIA was just 150 bp, and all located in the LSC region, 1 bp away from the IR region. The ycf1–5’end gene in CSS, CSA, and CWN was 1071 bp, and crossed the IR/SSC region by 2 bp while in CIA was 1065 bp, and crossed the IR/SSC region by 33 bp. The ndhF gene in all four cp genomes was located in the SSC region. The ndhF gene in CSA, CIA, and CWN was 2247 bp while in CSS was 2139. The ndhF gene in CSS was 165 bp away from the IR region, in CSA or CWN was 57 bp away from the IR region while in CIA was 88 bp away from the IR region. The ycf1 gene in CSS or CWN was 5622 bp, in CSA was 5628 bp while in CIA was only 1038 bp. The ycf1 genes in all four cp genomes crossed the IR/SSC region. The ycf1 gene in CSS or CWN was with 4553 bp located in the SSC region and 1069 bp in IR region, in CSA was with 4559 bp located in the SSC region and 1069 bp in IR region while in CIA was with only 6 bp located in the SSC region and 1032 bp in IR region. The rpl2 gene in CSS, CSA or CWN was 107 bp away from the LSC region while in CIA was 82 bp away from the LSC region. The trnH-GUG gene in CSS, CSA or CWN was 2 bp away from the IR region while in CIA was 637 bp away from the IR region.
Repeat and indel sequence analyses
Simple sequence repeats (SSRs) are small repeating units of cpDNA, a total of 671 SSRs were identified in four cp genomes (Fig. 6a), of which 57% were in IGS, 34% were in CDS, and 9% were in Intron (Fig. 6b). 74.0% of these SSRs were monomers, 19.3% of dimers, 0.5% of trimers, 5.3% of tetramers, 0.9% of hexamers and no pentamers found. Comparing the four genomes, except for 167 SSRs of CIA, the other three were all 168. A total of 128 SSRs were identical among four cp genomes (Fig. 6c). There were 47 loci with different SSR types, most of which existed in the LSC region. Among them, CSS had 7 unique types, CSA had 18 unique types, CIA had 9 unique types, and CWN had 14 unique types (Fig. 6c, Supplementary Tab. S2).
A total of 270 long repeats were detected in four plastomes, including three categories of long repeats: tandem, forward and palindromic. The number of the three repeated types was consistent in CSS and CWN, as follows: 23, 20, 23. However, it was 19, 20, 23 in CSA and 21, 23, 32 in CIA. The sizes of repeats ranged from 11 to 82 bp (Fig. 7a, c). The four cp genomes have a total 57 identical long repeat sequences. In addition, CSS had 1 unique long repeat, CIA had 1 unique long repeat, CWN had 2 unique long repeats, while CSA had no unique long repeat (Fig. 7b). These unique repeats were found mainly in the intergenic psaA/ycf3, atpB/rbcL, trnW-CCA/ trnP-UGG, rps19/rpl2, psbT/psbN, rpl2/trnH-GUG and gene rpl2, ycf1, ycf2. Only one repeat was in the intron regions (ndhA) (Supplementary Tab. S3).
A total of 100 indels were found, and indels ranged in size from 1 to 637 bp (Fig. 8a). Most of the indels events occurred in IGS regions (70%), with 23% in CDS regions and only 7% in Intron regions (Fig. 8b). As expected, single-nucleotide indels (1 bp) were the most common, but some long indels also were found. The longest one was an insertion of 637 bp in CIA (intergenic rp12/trnH-GUG), followed by a 335 bp deletion in CWN (intergenic trnE-UUC/trnT-GGU) and a 107 bp deletion in CIA (gene rps19). Paired comparison showed that the CIA had the most indels compared to the other three species (Fig. 8c). In addition, CIA also possessed the most species-specific indels, with 49, followed by CSA with 16, CWN with 11 and CSS with 5 (Fig. 8d, Supplementary Tab. S4).
The regions with relatively high divergence values (rp12/trnH-UGU, psaA/ycf3, atpB/rbcL and psbT/psbH, Pi > 0.006) (Fig. 3) all were associated with the repeat and the indel sequences. For example, the repeat sequences could be found within the region of rp12/trnH-UGU, atpB/rbcL and psbT/psbH. The indel sequences could be found within the region of rp12/trnH-UGU, psaA/ycf3 and psbN/psbH.
Correlation analysis of three types of mutation
Correlations were highly significant in the pairwise comparisons between the three types of mutations: “repeats and substitutions”, “indels and substitutions” and “repeats and indels”. The strength of correlations was greatest for “indels and substitutions” (r: 0.165–0.435) followed by“repeats and indels” (r: 0.090–0.120) and then “repeats and substitutions” (r: 0.028–0.049), and“indels and substitutions” had relatively higher significance value (t: 0.144–0.195) than “repeats and substitutions” (t: 0.103–0.145) (Table 3).
Codon usage analyses
ENc plots analysis showed only a few points lie near the curve, however, most of the genes with lower ENc values than expected values lay below the curve (Fig. 9), suggesting the codon usage bias of the cp genome was slightly affected by the mutation pressure, but selection and other factors play an important role. To further investigate the extent of influence between mutation pressure and natural selection on the codon usage patterns, Neutrality plot (GC12 vs. GC3) was performed. The correlation between GC1 and GC2 was strong (CSS: r = 0.445; CSA: r = 0.453; CIA: r = 0.445; CWN: r = 0.464, p < 0.01). However, no significant correlation was found for GC1 with GC3 (CSS: r = 0.141; CSA: r = 0.139; CIA: r = 0.078; CWN: r = 0.141) or GC2 with GC3 (CSS: r = 0.146; CSA: r = 0.143; CIA: r = 0.078; CWN: r = 0.152), which suggested mutation pressure had a minor effect on the codon usage bias. The slope of Neutrality plot showed that mutation pressure accounts for only 0.52–8.42% on the codon usage patterns in four cp genomes, while natural selection accounts for 91.58–99.48% (Fig. 10).
The distributions of codon usage in four cp genomes showed that RSCU values of the 37 codons (37/64, 57.81%) were identical in the three Chinese teas, but different from those in Indian tea (Table 4).
Analysis of cp sequence characterized amplified region (SCAR)
By comparing with the cp genomes of three representative diploid C. sinensis species, a 335 bp long deletion in the trnE/trnT intergenic spacer was found in triploid CWN (Fig. 11a). We used this marker for SCAR analysis in 292 individuals covering the majority of C. sinensis cultivars in China. No cultivar with similar sequence deletion characteristics to triploid CWN was detected (Fig. 11b, Supplementary Fig. S1, Supplementary Tab. S5). However, we could find similar sequence deletion in C. cuspidate (Accession numbers: NC022459), C. renshanxiangiae (Accession numbers: NC041672), C. elongata (Accession numbers: NC035652) and C. gymnogyna (Accession numbers: NC039626) by comparing the cp genome sequences (Fig. 11a).
Phylogenetic analysis and the divergence time estimation of three tea plants
Phylogenetic trees were generated by ML and BI analysis based on 44 complete cp genomes showed the same topology. Cultivated tea plants were clustered into a single clade, within which Chinary type tea, Chinese Assamica type tea and Indian Assamica type tea were in separate lineages with high support, respectively (Figs. 12 and 13, Supplementary Tab. S6).
Excluding seven non-Camellia species, the sequence variation of the 37 Camellia species associated with the six datasets (Complete cp genome, LSC, SSC, IR, PCGs, and non-PCGs) showed different percentage variation (Supplementary Tab. S7). SSC had the highest percentage variation at 2.32%, followed by non-PCGs at 1.65%. The IR regions were least variable at 0.5%. The cp genome, LSC, and PCGs, were 1.3, 1.54 and 1.21%, respectively. Phylogenetic trees based on six different data sets showed mostly similar topologies. A few individual species were retrieved incongruently among different clades across the six data partitions, but all Camellia species remained grouped separately, except IR regions that were shown to be mixed with Polyspora species of Theaceae. The support values of nodes increased significantly with the increasing of the sequence length in the different data partitions. In terms of interspecific relationships of three tea plants (Chinary type tea, Chinese Assamica type tea and Indian Assamica type tea), the results showed the same topology across all six datasets (Figs. 12 and 13, Supplementary Fig. S2, S3, S4, S5, S6).
Estimated divergence time showed the three types of tea plant were diverged to each other during 0.8–6.2 million years ago (Mya) (CI: 0.3–8.1 Mya). Indian Assamica type tea diverged from the ancestor of Indian Assamica type tea and Chinese Assamica type tea about 6.2 Mya (CI: 4.4–8.1 Mya, Miocene), Chinese Assamica type tea diverged separately about 0.8 Mya (CI: 0.3–1.6 Mya, Quaternary), and Chinary type tea diverged separately from the ancestor of Indian Assamica type tea and Chinary type tea about 0.8 Mya (CI: 0.4–1.5 Mya, Quaternary) (Fig. 12).
Genetic variation and mutational dynamics of the chloroplast genome in tea plant
The four cp genomes of the tea plants showed a high degree of conservation in genome structure, gene content, gene order, intron number, and also GC content. To better understand the sequence variation in tea plant, the three important types of genetic variation in cp genome, inducing nucleotide substitutions, repeats and indels [33,34,35,36], were identified. In addition to nucleotide substitutions, 671 SSRs (simple repeat) were identified (another 32, 31, 31 and 30 SSRs occurred in compound formations for CSS, CSA, CIA and CWN, respectively). The number of SSRs was consistent with a previous study . In addition, a total of 270 long repeats and 100 indels also were identified. The repeats and indels identified here might provide information for markers development to further species identification and population genetic studies [38, 39].
A characteristic feature of eukaryote and prokaryote genomes is the co-occurrence of nucleotide substitution and insertion/deletion (indel) mutations . We also found that the divergent regions of cp genomes were almost all associated with repeat sequences and indel sequences. Therefore, the genome-wide association between repeat, indel and nucleotide substitution was further analyzed. Correlations were significant in the pairwise comparisons between the three types of mutations: substitutions and indels, repeats and indels, and repeats and substitutions, which provided further support for the repeat-and indel-induced mutation hypothesis. The indel-induced mutation hypothesis assumes that the changes are induced by indels that trigger the DNA repair process, in which error-prone DNA polymerases are recruited [41, 42]. Instead, the repeat-induced mutation hypothesis assumes that mutations are produced because the presence of repeat sequences rather than indels per se, which promote replication fork arrest that led to the recruitment of error-prone polymerases and cause indels and nucleotide substitution . Our results showed that while the “repeats and indels” model was significantly correlated, the “indels and substitutions” model exhibited the highest strength and relative statistical significance for correlation (Table 3). Therefore, we considered these two hypotheses as occurring not mutually exclusive in the cp genome evolution of tea plants. It also implied that if the distribution of the repeat or indel sequences had been identified, the mutational hotspot regions and the appropriate sequences for genetic analysis could be predicted based on their location.
The expansion and contraction of cp genome is a common evolutionary phenomenon in plants . Of the four cp genomes, the IR regions of CIA were the smallest, and the more sequence deletions were found in SC/IR regions of CIA, suggesting that deletions might cause the contraction of the IR region. On the other hand, the LSC region of CIA had the longest length, and we found more repeat sequences in LSC/IR region, suggesting the repeats contributed to the length of cp genome. Moreover, a species-specific 335 bp long deletion was found in CWN with a length close to the difference length of cp genome between CWN and two Chinese tea plants (355 and 338 bp for CSS and CSA, respectively). Likewise, the difference length of cp genome between CWN and CIA was 913 bp, which was close to the sum of a species-specific deletion length of CWN (335 bp) plus a species-specific long insertion length of CIA (637 bp). Therefore, the repeats and indels might have critical effects on the structure of cp genome in tea plant, and the indels might play more important role, which was also well supported with the indel-induced mutation having the highest strength of correlation in the pairwise comparisons between the three types of mutations (Table 3).
Difference between Chinese tea and Indian tea
In tea plant, the most obvious division is Chinary type tea (C. sinensis var. sinensis) and Assamica type tea (C. sinensis var. assamica), which have markedly different appearance. Further, we compared the cp genome sequences between them in detail. We found some significant differences between Chinese tea and Indian tea by comparing the four cp genomes (the triploid Chinary type tea, the diploid Chinary type tea, the diploid Chinese Assamica type tea, and the diploid Indian Assamica type tea): (1) The three Chinese teas were more similar in IR/LSC boundary pattern, which was obvious difference from Indian tea (Fig. 5). (2) Chinese teas and Indian tea exhibited the distinct repeat patterns, Indian tea possessed high level of species-specific long repeats, with 18, while three Chinese teas only possessed 0–2, and most of the long repeats in three Chinese teas were shared (Fig. 7b). Similarly, the indel patterns also exhibited obviously different between Chinese teas and Indian tea, Indian tea possessed 49 species-specific indels patterns, accounting for 49% of the total number of indel loci, while only 5–16 indels were specific to each of the three Chinese teas (Fig. 8d). (3) In particular, the analysis showed that selection played an important role in the codon usage of cp genome in all four species (Figs. 9 and 10). However, 37 codons of the three Chinese teas had the identical RSCU value, accounting for 57.8% of the codons, all of which were different from that of Indian tea (Table 4). Codon use bias had long been used to reflect the origin, evolution, and mutation mode of species or genes . Therefore, it suggested that Chinese tea and Indian tea might have undergone different selections, as did Chinese Assamica type tea and Indian Assamica type tea.
Analysis of possible effects of polyploid evolution on cp genome
CpDNA data could be used not only to identify the maternal origin of polyploidy, but also to explore the possible effects of polyploid evolution, such as introgressive hybridization . Our phylogenetic analysis based on complete cp genome showed the triploid CWN was clustered with C. sinensis species together (Fig. 13), and the p-distance between CWN and CSS was only 0.00045 (Table 2), which demonstrated the maternal ancestor of triploid came from C. sinensis species. However, SCAR analysis using a 335 bp deletion of the intergenic spacers (trnE/trnT) in 292 cultivars of C. sinensis showed that none of the cultivars had this sequence deletion like triploid CWN (Fig. 11b, Supplementary Fig. S1). This could be explained by limited sample sizes, possible extinction of its progenitors and/or the occurrence of chloroplast transfer through hybridization with other Camellia species. Chloroplast transfer (or ‘chloroplast capture’), the introgression of a chloroplast from one species into another (introgressive hybridization) , has been reported [47,48,49]. In our study, none of the 292 individuals covering the majority of C. sinensis cultivars in China had a 335 bp long sequence deletion similar to triploid CWN, but it could be found in four another Camellia species, including: C. cuspidate, C. renshanxiangiae, C. elongata and C. gymnogyna (Fig. 11a). Therefore, the triploid CWN should have originated through allopolyploidization involving two parental species. The further field surveys and molecular studies about parental origin remain to be performed. Nevertheless, our study suggested chloroplast transfer occurred during the polyploidization in C. sinensis, that was, the progenitor of the triploid CWN (C. sinensis) itself captured the cp genome of another Camellia species when hybridization occurred where the ranges of the species overlap.
Divergence time estimation and indication for the three different domestication
The controversy over the domestication origin of the tea plant has existed for a long time. Previous studies using nSSRs, SNPs and RADseq data found that there were three different gene pools of tea plant (Chinary type tea, Chinese Assamica type tea and Indian Assamica type tea), considering the breeding history of Assamica type tea was not long enough to produce a new lineage, so suggesting that Chinary type tea, Chinese Assamica type tea and Indian Assamica type tea were likely the result of three independent domestication events [3, 50, 51]. Another view, however, was that the three different gene pools was a far cry from three different domestications. It was also possible that Assamica type tea was domesticated only once and then brought from one region to another, allowing it to evolve separately in both regions . To further understand the domestication origin of these three types of tea plant, in this study, we first used the complete cp genome to estimate the divergence time of tea plant (Fig. 12). The result showed the three types of tea plant (Chinary type tea, Chinese Assamica type tea and Indian Assamica type tea) were in different branches in the phylogenetic tree, and diverged to each other during 0.8–6.2 Mya (CI: 0.3–8.1 Mya). Indian Assamica type tea diverged from the ancestor of Indian Assamica type tea and Chinese Assamica type tea about 6.2 Mya (CI: 4.4–8.1 Mya). Thereafter, Chinary type tea and Chinese Assamica type tea diverged to each other about 0.8 Mya (CI: 0.4–1.5 Mya), which was in good agreement with that previously estimated by Wei et al. using the collinear nuclear genes between Chinary type tea and Chinese Assamica type tea genomes, about 0.38–1.54 Mya . These results suggested that the three types of tea plant diverged from each other much earlier than the known domestication time (2737 BC) , thus supporting the hypothesis of the three different domestication origins [50, 51].
Phylogenetic relationships within Camellia
Camellia is taxonomically and phylogenetically ranked as one of the most challengingly difficult taxa in plants, and the taxonomic classification of Camellia based on morphology has been controversial. In terms of morphological classification, Chang et al. classified the genus Camellia into 4 subgenera with 22 sections , while Ming et al. revised the classification of Chang and classified the genus Camellia into 2 subgenera with a total of 14 sections [54, 55]. Our phylogenetic analysis (Fig. 13) of Camellia did not agree with any of the current traditional classification methods used recently in Camellia taxonomy, such as: C. danzaiensis was clustered with C. pitardii, C. mairei and C. reticulata together (BSML = 100%, PPBI = 1.00), suggesting C. danzaiensis might belong to subgenus Camellia, rather than subgenus Thea. Similarly, C. pubicosta was sister to C. sinensis var. assamica and C. grandibracteata (BSML = 98%, PPBI = 1.00), suggesting this species probably should be classified into sect. Thea by Chang et al. , instead of being classified into sect. Corallina. These results were all consistent with two previous studies of Camellia [21, 56].
In addition, our phylogenetic tree constructed based on complete cp genome in this study showed some incongruence with the previous phylogeny study of Camellia. A previous phylogenetic analysis using RAPDs showed that Camellia species could be divided into 5 ovary groups and 3 ovary groups according to the number of their ovaries, while our phylogenetic relationships of Camellia species did not well follow the number of locule ovary. For example, C. grandibracteata with 5 ovaries and C. sinensis var. assamica with 3 ovaries were well supported as monophyletic (BSML = 100%, PPBI = 1.00). Phylogenetic analysis was not consistent with the taxonomy according to ovarian number, which was also found in Huang et al. ‘s study . However, in Huang et al. ‘s study, C. ptilophylla with 3 ovaries was clustered with some species with five ovaries (C. tachangensis, C. kwangsiensis and C. crassicolumna var. crassicolumna), which did not occur in our results. This might be because the cp gene sequences used in their phylogenetic analysis were incomplete cp genomes (83,585 to 83,835 bp). A recent study had emphasized that the extensive heterogeneity of nucleotide substitution rate among different plastid genes and among different functional groups of genes were likely contributing to phylogenetic ambiguity . In our phylogenetic analysis of the data partitions of cp genome, the sequence variations in the six datasets showed different percentage variation (Supplementary Tab. S7). Although the phylogenetic trees from the six datasets generated mostly similar topological structures, there were still some individual species showing different locations in the six trees, and the support values of nodes increased significantly with the increasing of the sequence length (Fig. 13, Supplementary Fig. S2, S3, S4, S5, S6). It suggested that attention should also be paid to the effects of such heterogeneity when functional genes or plastid fragments were used to study phylogenetic evolution of cp genome of Camellia. In addition, phylogenomic analysis also tend to suffer from the poor sampling , and the number of Camellia species sampled in Huang et al. ‘s study was 18, while ours was more, at 37. Therefore, further taxon sampling and more complete cp genomes of Camellia were needed to resolve the controversial taxonomy of Camellia in future study.
While the analyses of the complete cp genomes provided a feasible way to clarify relationships , it might still be insufficient to fully resolve all phylogenetic relationships [60, 61]. Our results suggested that this type of complete genome phylogenomic analyses would resolve many controversies and guide the way for phylogeny in Camellia. Since plastome was regarded as a linked single locus due to its uniparental inheritance and lack of sexual recombination, in future work researchers should also attach importance to the use of multilocus approaches (including nuclear genes and mitochondrial genes), so as to provide abundant and detailed molecular data for the systematic classification and evolutionary study of Camellia.
In this study, we had found that the repeats and indels were two most important evolutionary dynamics contributed to the diversification of the cp genome, which were not mutually exclusive. Chinese tea and Indian tea exhibited significantly differences in the structural characteristic and the codon usage of the cp genome, suggesting they might have undergone different selection pressures. In addition, our result demonstrated that the chloroplast transfer occurred during the polyploidization. Further, the phylogenomic analysis combined with divergence time estimation implied that Chinary type tea, Chinese Assamica type tea and Indian Assamica type tea might have three different domestication origins, and the current classification of some Camellia species might need to be further discussed. Our data would not only provide insights into the chloroplast genome evolution of C. sinensis but also offer valuable information for taxonomic classification of Camellia.
Plant material and DNA extraction
Fresh leaves of ‘Wuyi narcissus’ cultivar of C. sinensis var. sinensis used in this study were obtained from the tea tree germplasm garden in Wuyi University. The voucher specimen (No. 20200317) was authenticated by Prof. Yongcong Hong (Wuyi University) and deposited in the Wuyi University herbarium.
Genomic DNA was extracted from leaves using CTAB extraction method. Final DNA quality was assessed by a NanoDrop spectrophotometer (Thermo Scientific, Carlsbad, CA, USA), and their integrity was examined by electrophoresis on a 0.8% agarose gel. DNA samples were preserved at − 80 °C at the Key Laboratory of Tea germplasm Genetic Resources of Wuyi University.
Chloroplast genome sequencing, assembly and annotation
The previous studies had shown that cp genomes of closely related species share highly similarity and these sequences could be used as reference genomes to obtain the order of contigs for new cp genome assembly. This efficient strategy did not require organelle DNA isolation and had been well accepted by the scientific community [62,63,64,65,66]. Here, the three published cp genomes of tea plants (Accession number: KJ806281, MH019307 and MH460639) [21, 22, 62] were selected as reference genomes. A combined approach of PacBio sequencing complemented with Illumina sequencing was performed by Biozeron Biotechnology Co., Ltd. (Shanghai, China).
High quality total genomic DNA was applied to 500 bp paired-end library construction using the NEBNext Ultra DNA Library Prep Kit for Illumina sequencing. Sequencing was carried out on the Illumina NovaSeq 6000 platform. For PacBio sequencing, more than 5 μg of sheared and concentrated DNA was applied to size selection by the Blue Pippin (Sage Science, Beverly MA, USA). Approximately 20 kb SMRTbell libraries were prepared according to the manufacturer’s instructions (PacBio, Menlo Park, CA, USA). The samples were sequenced on PacBio Sequel instrument using the P6 polymerase C4 chemistry combination. All raw reads were processed with Trimmomatic v0.39 software  to remove adapter sequences, short reads (length < 75 bp), and to trim low- quality bases (Q-value < 20). The PacBio raw reads (polymerase reads) were filtered by discarding low-quality polymerase reads (Q-value < 0.80), short reads (length < 100 bp), short sub reads (length < 500 bp), and adapters. Both of Illumina reads and PacBio reads were mapped to the cp reference genomes of three closely related species to extract the cp reads. Short reads were aligned to the reference set using NCBI-BLAST-2.2.30 , and long reads were aligned to the reference set using Minimap2 . Prior to the assembly, all filtered PacBio reads were error corrected with Illumina data using Pilon v1.22 . All error-corrected PacBio reads were assembled into a single contig using Canu v2.0 . To estimate the quality and coverage of the assembled genome , we mapped all Illumina and PacBio reads to the assembly using BWA v0.7.12 , respectively.
The cp genes were annotated using CpGAVAS  and verified the sequence coordinates of each of the annotated genes using BLAST search against ref. cp genes. Annotation errors were manually corrected. The four junction regions (SC/IR) and the six randomly selected cp genes were furtherly validated with PCR-based conventional Sanger sequencing. The final annotated cp genome sequence was subjected to OGDRAW software  to generate the circular cp genome map and deposited to NCBI GenBank. The four junction regions (SC/IR) and 6 randomly selected cp genes were validated with PCR-based conventional Sanger sequencing (Supplementary Tab. S8).
Comparative analysis of four cp genomes
Four cp genome sequences of Camellia species were aligned using MAFFT Version 7.017 . Full alignments with annotation were visualized using the mVISTA software . Number of nucleotide substitutions was calculated by MEGA 6.0 . A sliding window analysis was conducted to compare π among the complete cp genomes, using DnaSP v5.0 . The window length was 600 bp with a 200 bp step size. The percentage of variable characters for coding and noncoding regions in the genome was calculated as described previously . The proportion of mutation events = [(NS + ID)/L] × 100, where NS = the number of nucleotide substitutions, ID = the number of indels, L = the aligned sequence length.
Repeat sequences were searched by REPuter  with a minimal size of 30 bp and > 90% identity (Hamming distance equal to 3) between the two repeats. Gap size between the repeats was restricted to a maximal length of 3 kb. Tandem repeats were identified by Tandem Repeats Finder (http://tandem.bu.edu/trf/trf.html)  with default settings. Simple sequence repeats (SSRs) were predicted using MISA  with the parameters: monomer (one nucleotide, n ≥ 8), dimer (two nucleotides, n ≥ 4), trimer (three nucleotides, n ≥ 4), tetramer (four nucleotides, n ≥ 3), pentamer (five nucleotides, n ≥ 3), hexamer (six nucleotides, n ≥ 3). A combination of SSRs separated by the maximum distance of 100 bp was considered as an SSR (i.e. an SSR of the compound formation).
According to Ibrar et al.′s method , the correlations were analyzed in the pairwise comparisons between the three types of mutations: substitutions and indels, repeats and indels, and repeats and substitutions. Setting cp genome of CSS (Accession number: KJ806281) as a reference, indels and substitutions were counted in the comparison of 630 nonoverlapping bins each with a size of 250 bp.
Codon usage analyses
In order to avoid sampling bias, each protein-coding gene (CDS) in cp genome were checked for being full-length and for the presence of proper start and stop codons. CDS < 300 bp were excluded in codon usage calculations .
ENc (Effective number of codons) and GC3s (GC content at the third synonymously variable coding position excluding Met and Trp) were calculated using CodonW v1.4.4 . ENc value is a measure of general non-uniformity of usage within synonymous groups of codons, ranging from 20 (extreme bias where only one codon is used in each amino acid) to 61 (random codon usage) . ENc plot analysis (ENc vs GC3s) was used to examine whether the codon usages were affected only by mutation or other factors. If codon usage is constrained only by mutation pressure, ENc value lie on or slightly below the expected curve, and if codon usage is subject to natural selection, ENc value will lie considerably below the expected curve .
Neutrality plot (GC12 vs. GC3) was used to investigate the effects of mutation pressure and natural selection on codon use patterns. GC12 (the average value of GC contents at the first and second positions of codon) and GC3 (the GC content at the third position) were calculated by Perl script. GC3 was calculated excluding the three termination codons (TAA, TAG and TGA) and the three codons for Ile (ATT, ATC and ATA). Meanwhile, two single codons for Met (ATG) and Trp (TGG) were also excluded in all three patterns . The slope of the plot regression is zero which indicates that there is no effect on directional mutation pressure (complete selection constraint). Slope 1 indicates that the codon usage bias is completely affected by the directional mutation pressure, and represents complete neutrality .
RSCU (Relative synonymous codon usage) for CDS was calculated using CodonW v1.4.4 . RSCU value for a particular codon refers to the ratio of its actual usage frequency to expected frequency when it is used without bias. The preferred codons with RSCU > 1.0 occur when they are used with higher frequencies than random, and the rare codons with RSCU < 1.0 means the opposite .
Analysis of cp sequence characterized amplified region (SCAR)
A sequence part of the chloroplast trnE/trnT intergenic spacer, where a 335 bp long deletion was observed in triploid CWN by comparing with another three tea species, was used to SCAR analysis. Screening of the distribution of the similar deletion in the trnE/trnT intergenic spacer was conducted by PCR amplification of the respective chloroplast region in 292 individuals covering the majority of C. sinensis cultivars in China (Supplementary Tab. S5). All individual samples were collected from the tea germplasm resource garden of Wuyi University and the DNA extraction method was described above. PCR products were observed on 1.5% agarose gels against a D2000 DNA molecular marker. A set of the primers (trnE: 5′-TCCTGAACCACTAGACGATG-3′; trnT: 5′-ATGGCGTTACTCTACCACTG-3′) were designed in conserved regions on either side of the 335 bp long deletion region by comparing cp genome sequences.
Phylogenetic analysis and divergence time estimation
The complete cp genome sequences of 37 Camellia species, 3 Polyspora species, 3 Panax species and the outgroup (Aralia undulata) (Supplementary Tab. S6) were aligned with the program MAFFT version . The ambiguously aligned loci (e.g., ‘N’ or ‘K’) were excluded from the analyses and the poorly aligned regions were removed from the complete plastome dataset using Gblocks v0.91b . Maximum likelihood (ML) analyses were implemented in RAxML version 7.2.6 . Non-parametric bootstrapping test was implemented in the “fast bootstrap” algorithm of RAxML with 1000 replicates. Bayesian inference (BI) analyses were performed using the program MrBayes version 3.1.2 . The best-fitting models were determined by the Akaike Information Criterion  as implemented in the program Modeltest 3.7 . The bootstrap value above 70% (BSML > 70%) for Maximum Likelihood and the posterior probability above 0.95 (PPBI > 0.95) for Bayesian Inference were used to determine a well-supported clade . ML and BI analyses were performed based on the following six datasets: (1) the complete cp DNA sequences, (2) the large single copy region (LSC), (3) the small single copy region (SSC), (4) the inverted repeat region (IR), (5) a set of the common protein coding genes (PCGs) and (6) a set of the common non-coding genes (Non-PCGs). The best-fit models for each data set were showed in Supplementary Tab. S7.
The divergence times of Camellia species were calculated using mcmctree of PAML . The empirical divergence times of P. ginseng/P. quinquefolius (0.8–1.2 Mya), P. ginseng/P. notoginseng (3.5–5.2 Mya) [97,98,99] were assigned to constrain the age of the Panax. Yu et al.  have demonstrated the influence of fossil calibration and divergence time estimation in Theaceae, and tested safety and risk scenarios of 9 scenarios based on a complete cp genome phylogenetic framework. Based on the results of Yu et al., the divergence times of Polyspora species/Camellia species (6.9–21.2 Mya) were assigned to constrain the stem of Camellia family. A Birth-Death prior on branching rates was employed and three independent analyses were run for 10,000 generations.
Availability of data and materials
Raw reads and the complete chloroplast genomes generated (‘Wuyi narcissus’ cultivar of Camellia sinensis var. sinensis) during the current study were deposited in NCBI database (SRA: SRR12002624, Accession number: MT612435). All complete chloroplast genomes adopted in this study, including the three reference chloroplast genomes, were available in NCBI database, and the accession numbers of the complete chloroplast genomes of each species were listed in the Supplementary Tab. S6.
An integrated web server for the annotation, visualization, analysis, and GenBank submission of completely sequenced chloroplast genome sequences
Organellar Genome DRAW soft
Coding DNA sequence
Conserved noncoding sequence
Effective number of codons
GC content at the third synonymously variable coding position
The GC content at the first position
The GC content at the second position
The average value of GC contents at the first and second positions of codon
The GC content at the third position
Relative synonymous codon usage
Large single copy
Small single copy
Simple sequence repeat
Protein coding genes
Non-Protein coding genes
Millions of years ago
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We are very grateful to Bo Zhang (Tea germplasm resource garden of Wuyi University) for his help with experiments and data analyses. We also thank College of Mathematics and Computer Science in Wuyi University for providing the computer resources.
This study was financially supported by Scientific Research Launch Fund of Wuyi University (YJ201902), the Discipline Team Construction Fund of Wuyi University (Wu Yuan Zong  No. 66), Construction Fund of Wuyi Tea Industrial Technology Research Institute (2018 N2004), Open Fund of The Key Laboratory of Cognitive Computing and Intelligent Information Processing of Fujian Education Institutions (No: KLCCIIP2018104) and Fujian province “2011 collaborative innovation center” special fund (Fujian education science  No.75).
Li Li, Yunfei Hu and Min He are first authors.
Li Li, Yunfei Hu and Min He contribute equally to this work.
Authors and Affiliations
College of Tea and Food Science, Wuyi University, 358# Baihua Road, Wuyishan, 354300, China
Li Li, Yunfei Hu, Min He, Bo Zhang, Pumo Cai, Da Huo & Yongcong Hong
College of Mathematics and Computer Science, Wuyi University, 358# Baihua Road, Wuyishan, 354300, China
LL designed the experiments. LL, YFH and MH performed the experiments. LL, YCH, BZ, WW, PMC and DH analyzed data. LL wrote the paper. YHC and LL revised the paper. All authors reviewed and approved the manuscript.
The tea tree of ‘Wuyi narcissus’ cultivars used in this study were planted and grown at Wuyi University in Fujian Province, China. No specific permits were required for plant collection. The study did not require ethical approval or consent, as no endangered or protected plant species were involved.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
SCAR analysis was conducted by PCR amplification of the respective chloroplast region in 292 different cultivars of Camellia sinensis. PCR amplification to screen the distribution of a 335 bp deletion of the intergenic spacers (trnE/trnT) in 292 different cultivars of Camellia sinensis. None of 292 cultivars had this sequence deletion like triploid CWN. M: D2000 DNA molecular marker; C: PCR products of CWN; Lane 1–292: PCR products of 292 different cultivars covering the majority of C. sinensis cultivars in China. CK: Control. The corresponding 292 cultivars were shown in Supplementary Tab. S5.
Phylogeny of Camellia inferred from maximum likelihood (ML) analysis of LSC. Numbers associated with nodes indicated ML bootstrap support (BS)/Bayesian inference (BI) posterior probabilities (PPs) values. Asterisks represented nodes with maximal support values in both analyses. Dash denoted nodes unresolved or with BS/PPs support in the ML/BI trees less than 50%/0.5. C. sinensis var. sinensis (Chinary type tea) and two C. sinensis var. assamica (Chinese Assamica type tea and Indian Assamica type tea) were highlighted with star mark.
Distribution of the different and the shared SSRs in four Camellia chloroplast genomes. CWN: ‘Wuyi narcissus’ cultivar of C. sinensis var. sinensis (natural triploid Chinary type tea); CSS: C. sinensis var. sinensis (diploid Chinary type tea); CSA: C. sinensis var. assamica (diploid Chinese Assamica type tea); CIA: C. sinensis var. assamica (diploid Indian Assamica type tea). Symbol “/” indicated missing.
A list of repeated sequences and their locations identified in four Camellia chloroplast genomes. CWN: ‘Wuyi narcissus’ cultivar of C. sinensis var. sinensis (natural triploid Chinary type tea); CSS: C. sinensis var. sinensis (diploid Chinary type tea); CSA: C. sinensis var. assamica (diploid Chinese Assamica type tea); CIA: C. sinensis var. assamica (diploid Indian Assamica type tea).
Distribution of indel in four chloroplast genomes. CWN: ‘Wuyi narcissus’ cultivar of C. sinensis var. sinensis (natural triploid Chinary type tea); CSS: C. sinensis var. sinensis (diploid Chinary type tea); CSA: C. sinensis var. assamica (diploid Chinese Assamica type tea); CIA: C. sinensis var. assamica (diploid Indian Assamica type tea).
Two hundred ninety two different cultivars of Camellia sinensis in China adopted for SCAR analysis in this study. Two hundred ninety two different cultivars represented the majority of Camellia sinensis varieties from 14 provinces in China.
Primers used for assembly and junction verification of the chloroplast genome.
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Li, L., Hu, Y., He, M. et al. Comparative chloroplast genomes: insights into the evolution of the chloroplast genome of Camellia sinensis and the phylogeny of Camellia.
BMC Genomics22, 138 (2021). https://doi.org/10.1186/s12864-021-07427-2