Introduction

Camellia oleifera L. (Abel) is a woody oil tree of the genus Camellia in the family Theaceae. C. oleifera seeds are harvested for the extraction of an edible tea oil that has high nutritional value and healthful properties including blood cholesterol reduction and the prevention of hypertension and arteriosclerosis (Feás et al. 2013; Zeng et al. 2015; Qu et al. 2019). For the reasons that its chemical composition and unsaturated fatty acid contents are similar to those of olive oil, C. oleifera oil is known as “eastern olive oil” (Gao et al. 2015; Li et al. 2016). C. oleifera seed meal can be used to extract saponin for feed production, and the shells can be used to produce potassium carbonate or to cultivate edible and medicinal fungi (Hu et al. 2012; Zhu et al. 2018). C. oleifera is the most valued oil-producing plant in China (Tan et al. 2018). In recent years, C. oleifera has been planted over large areas in hilly regions of southern China with red soil (Wang et al. 2019).

C. Oleifera cultivars with desirable traits have been planted on a large scale by farmers. High-yield cultivars C. oleifera ‘Huashuo’, ‘Huajin’ and ‘Huaxin’ were bred from C. oleifera in 2009. C. oleifera ‘Huashuo’ has large fruit, high yield, strong resistance, and late maturity (Tan et al. 2011) (Fig. 1A); C. oleifera ‘Huajin’ has rapid growth, large green leaves, high yield, precocity, and pear-shaped fruit (Yuan 2012) (Fig. 1B); and C. oleifera ‘Huaxin’ has high and stable yield, strong resistance, precocity, and red fruit (Tan et al. 2012) (Fig. 1C). These three C. oleifera cultivars have been cultivated widely in the hilly red soil region of Hunan Province in recent years (Wu et al. 2020). However, the genetic backgrounds of these cultivars are poorly known, and genetic resources are scarce.

Chloroplasts are key organelles that act as the plant metabolic center; they contain the complete enzymatic machinery for plant growth and development, with carbon fixation and oxygen release. The chloroplast genome, one of three DNA genomes in the plant body, has a highly conserved circular DNA arrangement that encodes many key proteins related to photosynthesis (Bobik and Burch-Smith 2015; Zhang et al. 2017; Liu et al. 2018). Since publication of the first chloroplast genome from Marchantia polymorpha (Kazuhiko et al. 1984; Wang et al. 2016), over 2,500 chloroplast genomes have been sequenced (http://www.ncbi.nlm.nih.gov/genomes/), providing insights into plant diversity, and evolution, and have been applied in DNA barcoding and genetic engineering of biomedical products (Kang et al. 2017; Song et al. 2017). Most chloroplast genomes range from 115 to 165 kb and have a quadripartite organization, including a large single-copy (LSC) region, a small single-copy (SSC) region and a pair of inverted repeats (IRs) (Li et al. 2017; Xu et al. 2017). Chloroplast genomes do not undergo recombination; they exhibit maternal inheritance and greater conservation than observed in nuclear and mitochondrial genomes (Palmer et al. 1988; Wu et al. 2010). C. oleifera is a widely distributed self-incompatible plant with extremely complex cross-pollination characteristics and intraspecific variation. The C. oleifera genome has not been sequenced, and most C. oleifera cultivars are polyploid, with complex genetic backgrounds and evolutionary processes. Moreover, the chloroplast genome sequences of the three C. oleifera cultivars have not yet been elucidated; therefore, it is important to clarify the phylogenetic and evolutionary relationships among different Camellia species to improve and expand the range of existing cultivars.

In this study, we assembled the complete chloroplast genome sequences of three important C. oleifera cultivars (‘Huashuo’, ‘Huaxin’ and ‘Huajin’) and characterized their genomes using Illumina high-throughput sequencing (HiSeq) technology. Genome maps of the obtained sequencing data were mapped using bioinformatics analysis to reveal the photosynthesis mechanisms and phylogenetic relationships of these cultivars relative to other C. oleifera cultivars. We also performed comparative analyses using known chloroplast genomes to improve our understanding of the C. oleifera chloroplast genome. The objective of this study was to investigate plant molecular markers, species relationships, and the structure and origin of chloroplast DNA, and to further explore the evolution of Camellia species using molecular methods.

Materials and Methods

Plant materials and DNA sequencing

Fresh leaves of the three C. oleifera cultivars were collected from 8-year-old trees growing at the Central South University of Forestry Science and Technology (112°40'E, 28°29'N, Wang Cheng, Hunan, China). Approximately 5 g of fresh leaves were harvested for chloroplast DNA isolation using an improved extraction method (McPherson et al. 2013). After DNA isolation, 1 μg purified DNA was fragmented to construct short-insert libraries (insert size, 430 bp) according to the manufacturer’s instructions (Illumina) and then sequenced using the Illumina HiSeq4000 system (Borgstrom et al. 2011; Shanghai Biozeron Biotechnology). High-molecular-weight DNA was purified and used to prepare the PacBio library and for BluePippin size selection and then sequenced using a Sequel sequencer. Additional sequencing was performed by Nextomics (Wuhan, China) using the PacBio RS II platform.

Genome assembly

Before assembly, the Illumina raw reads were filtered to remove reads with adaptors, low-quality reads (Q<20), reads containing ≥10% N characters, and duplicate sequences. We assembled the genome framework using both Illumina and PacBio data with SPAdes v. 3.10.1 (Antipovet al. 2016). Next, we verified the assembly and circular character of the chloroplast genomes, filling any gaps that occurred.

Genome annotation

We annotated the chloroplast genes using the DOGMA online tool (Wyman et al. 2004). A whole-chloroplast genome blast search was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) (Minoru et al. 2014), Clusters of Orthologous Groups (COG) (Tatusov et al. 2003), Non-Redundant (NR) Protein, Swiss-Prot (Magrane and Consortium 2011) and Gene Ontology (GO) (Ashburner et al. 2000) databases. A circular chloroplast genome map was drawn using Organellar Genome DRAW v. 1.2 (Lohse et al. 2007).

Chloroplast genome sequence analysis

The programs Mauve (Ravi et al. 2006) and mVISTA programs were used to identify similarities among different chloroplast genomes (Mayor et al. 2000). The REPuter program was used to identify and locate forward (direct) repeats, reverse sequences, complementary sequences, and palindromic sequences with lengths of at least 22 bp and sequence identity ≥90% (Kurtz et al. 2001). SSR distributions were predicted using the MISA microsatellite search tool (Beier et al. 2017). IR expansion/contraction regions were compared among C. oleifera ‘Huashuo’, C. oleifera ‘Huajin’, C. oleifera ‘Huaxin’, N. tabacum, C. sinensis, C. petelotii, C. pitardii and C. oleifera.

Phylogenetic analysis

The maximum likelihood (ML) analysis was performed using RAxML v. 7.2.6 with the default parameters (Stamatakis 2006). The maximum parsimony (MP) analyses were performed using PAUP 4.0.

Results

General features of C. oleifera chloroplast DNA

The C. oleifera ‘Huashuo’, ‘Huajin’ and ‘Huaxin’ chloroplast genomes were 156,965, 156,975 and 156,975 bp in length, respectively. These genomes were similar to those of most angiosperms, with an LSC region of 86,650 bp in ‘Huashuo’ and 86,660 bp in both ‘Huajin’ and ‘Huaxin’ and an SSC region of 18,409 bp in ‘Huashuo’ and 18,406 bp in both ‘Huajin’ and ‘Huaxin’ separated by a pair of IRs of 51,906 bp in ‘Huashuo’ and 51,908 bp in both ‘Huajin’ and ‘Huaxin’ (Fig. 2 and Table 1). The genomes had guanine–cytosine (GC) contents of 37.29%; however, the GC content was higher in the rRNA region (55.41% in all three cultivars) than in the overall genome (Table 1).

Gene content, orientation, and order were similar among the three C. oleifera cultivars. A total of 133 genes consisting of 88 protein-coding genes, 37 tRNA genes and 8 rRNA genes were identified from each genome (Table 1 and 2). A total of 20 genes (8 tRNA, 4 rRNA, and 8 protein-coding genes) were duplicated in the IR regions of each genome (Fig. 2). ‘Huashuo’ had 16 genes with introns, whereas both ‘Huajin’ and ‘Huaxin’ had only 15 genes with introns. GC protein-coding genes were 77,866 bp in length in ‘Huashuo’ and 76,648 bp in ‘Huajin’ and ‘Huaxin’. Therefore, the final chloroplast genome sequences of the three C. oleifera were obtained (Submitted to the NCBI database).

Comparison with other Camellia species

Fig. 1: The three Camellia oleifera cultivars, fruiting in September. A: ‘Huashuo’; B: ‘Huajin’; C: ‘Huaxin’

Fig. 2: Gene map of the three C. oleifera cultivar chloroplast genomes determined using the PacBio RS II platform. Thick lines indicate inverted repeats (IRa and IRb), which separate the genome into large single-copy (LSC) and small single-copy (SSC) regions. Genes on the outside of the map are transcribed in a clock wise direction; those inside the map are transcribed in a counter clock wise direction

Table 1: Characteristics of the plastome genomes of three Camellia oleifera cultivars. cp, chloroplast. LSC, large single copy; SSC, small single copy; IR, inverted repeat; IGS, intergenic spacer. GC, guanine–cytosine content

Sequence region	Length (bp)/Percent (%)
	C. oleifera ‘Huashuo’	C. oleifera ‘Huaxin’	C. oleifera ‘Huajin’
Total cp genome	156,965	156,975	156,975
LSC region	86,650	86,661	86,661
SSC region	18,409	18,406	18,406
IR region	51,906	51,908	51,908
Coding regions	79,500	79,504	79,504
Introns	110,882	110,879	110,879
rRNA	9,046	9,046	9,046
tRNA	2,802	2,802	2,802
IGS	77,357	77,364	77,364
GC content	Length (bp)/Percent (%)
Overall GC size	58,532/37.29	58,535/37.29	58,537/37.29
Overall A size	48,745/31.05	48,749/31.06	48,752/31.06
Overall T size	49,688/31.66	49,691/31.66	49,686/31.65
Overall G size	28,677/18.27	28,678/18.27	28,681/18.27
Overall C size	29,855/19.02	29,857/19.02	29,856/19.02
GC content in protein-coding regions	77,866 (40.90)	76,648 (40.26)	76,648 (40.26)
GC content in introns	38.97	38.97	38.97
GC content in rRNA	5,012/55.41	5,012/55.41	5,012/55.41
GC content in tRNA	1,482/52.89	1,482/52.89	1,482/52.89
GC content in IGS	28,638/37.02	28,640/37.02	28,640/37.02
Gene classification	Number
Total genes	133	133	133
Protein-coding genes	88	88	88
tRNA genes	37	37	37
rRNA genes	8	8	8
Genes with introns	16	15	15

We compared the lengths of 10 Camellia chloroplast genomes, which ranged from 156,585 to 157,121 bp. The GC contents of C. oleifera ‘Huashuo’, ‘Huajin’ and ‘Huaxin’ were similar to those of C. oleifera samples collected in Hainan. C. oleifera from Hainan had the longest chloroplast genome among these four Camellia samples, at 156,995 bp, with GC content of 37.31%. The average size of the 10 Camellia chloroplast genomes was 156,983 bp. Of the 10 Camellia samples, C. petelotii had the longest chloroplast genome, and C. pitardii the shortest (Table 3). The C. oleifera ‘Huashuo’ chloroplast genome had the smallest IR region (51,906 bp), while C. sinensis had the longest (52,180 bp). The C. oleifera ‘Huashuo’ chloroplast genome had the longest SSC region (18,409 bp) and C. pitardii had the shortest (18,260 bp) (Table 3). C. pitardii had the highest GC content (37.34%). The chloroplast genomes of all 10 samples encoded 37 of tRNAs, except for that of C. pitardii, which encoded 40 (Table 3).

Repeat sequence analysis

Table 2: Genes identified from the chloroplast genomes of the three Camellia oleifera cultivars

Gene categories	Gene groups	Gene names
Genes for photosynthesis	Photosystem I subunits	psaA, psaB, psaC, psaI, psaJ
	Photosystem II subunits	psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbK, psbL, psbM, psbN, psbT, psbZ
	ATP synthase subunits	atpA, atpB, atpE, atpF,atpH, atpI
	Cytochrome b6/f complex subunits	petA, petB, petD, petG, petL, petN
	NADH dehydrogenase subunits	ndhA, ndhB, ndhB-D2, ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK
	Large rubisco subunit	rbcL
	Small ribosomal subunit proteins	rps11, rps12, rps12-D2, rps14, rps15, rps16, rps18, rps19, rps2, rps3, rps4, rps7, rps7-D2, rps8
	Large ribosomal subunit proteins	rpl14, rpl16, rpl2, rpl2-D2, rpl20, rpl22, rpl23, rpl23-D2, rpl32, rpl33, rpl36
	RNA polymerase subunits	rpoA, rpoB, rpoC1, rpoC2
Other genes	Acetyl-CoA carboxylase	accD
	Cytochrome c biogenesis	ccsA
	Envelope membrane protein	cemA
	Maturase	matK
	Protease	clpP
	Translation initiation factor	infA
Unknown genes	Conserved hypothetical chloroplast reading frame	orf42, orf42-D2, ycf1, ycf15, ycf15-D2, ycf2, ycf2-D2, ycf3, ycf4

Table 3: Comparison of the Camellia chloroplast genome characteristics

Genome feature	C. oleifera‘Huashuo’	C. oleifera‘Huajin’	C. oleifera‘Huaxin’	C. sinensis	C. petelotii	C. azalea	C. pitardii	C. oleifera	C. oleifera in Hainan	C. sinensiscv. Longjing 43
Total length (bp)	156,965	156,975	156,975	157,103	157,121	157,039	156,585	156,971	156,995	157,103
LSC length (bp)	86,650	86,661	86,661	86,646	86,660	86,675	86,213	86,472	86,649	86,646
SSC length (bp)	18,409	18,406	18,406	18,277	18,283	18,282	18,260	18,280	18,298	18,277
IR length (bp)	51,906	51,908	51,908	52,180	52,178	52,082	52,112	52,056	52,048	52,180
GC content (%)	37.29	37.29	37.29	37.31	37.29	37.30	37.34	37.31	37.29	37.31
Total genes	133	133	133	132	132	132	137	132	132	132
Protein genes	88	88	88	87	87	87	89	87	87	87
tRNA genes	37	37	37	37	37	37	40	37	37	37
rRNA genes	8	8	8	8	8	8	8	8	8	8

Repeat sequence analysis showed 37 repeats with at least 18 bp per repeat unit in the three Camellia chloroplast genomes (Tables S1–S3). These repeats included 19 direct (forward) repeats in C. oleifera ‘Huashuo’ and ‘Huaxin’ and 18 direct (forward) repeats in Camellia ‘Huajin’. Fifteen palindrome repeats were detected in C. oleifera ‘Huashuo’ and ‘Huajin’, and 14 in C. oleifera ‘Huaxin’. Forward and palindrome repeats were more abundant than reverse and complement repeats in all three C. oleifera cultivars. C. oleifera ‘Huajin’ and ‘Huaxin’ each had two reverse repeats and two complement repeats, whereas C. oleifera ‘Huashuo’ had one reverse repeat (Fig. 4); most were 19–20 bp in length, although C. oleifera ‘Huashuo’ had one 18 bp repeat. In each of the three Camellia chloroplast genomes, we also found one repeat each of 23, 24, 26 and 30 bp, four repeats of 38 bp, and two repeats of 42 bp.

Fig. 3: Comparison of 10 chloroplast genomes using mVISTA. Gray arrows and thick black lines above the alignment indicate gene orientation and inverted repeat (IR) positions, respectively. The vertical scale indicates the percentage identity (50–100%)

Fig. 4: Repeat sequences in three C. oleifera chloroplast genomes. A: Repeated sequences in the three C. oleifera chloroplast genomes; B: Frequencies of four repeat types according to length in the three C. oleifera chloroplast genomes

SSR analysis

Fifty SSR loci were identified in the chloroplast genomes of C. oleifera ‘Huashuo’ and ‘Huaxin’, and 51 SSR loci in that of C. oleifera ‘Huajin’ (Tables S4–S6). The maximum length of mononucleotide SSRs among the three C. oleifera chloroplast genomes was 17 bp (Fig. 5). These SSR loci were all identified as mononucleotide SSR loci, except for one complicated SSR locus in ‘Huashuo’. These mononucleotides repeat units were all type A or T; no G type repeat units were found. These SSR loci contributed to the A/T richness of the three C. oleifera chloroplast genomes. These results are similar to those from previous studies of tung tree chloroplast genomes (Li et al. 2017). Mononucleotide motif repeat numbers generally range from 10 to 14 bp. In the ‘Huashuo’, ‘Huajin’ and ‘Huaxin’ C. oleifera cultivars, the repeat numbers of mononucleotide motifs ranged from 10 to 17, except that ‘Huashuo’ and ‘Huaxin’ had no 16-mononucleotide repeats (Fig. 5).

IR expansion/contraction

The IR-SSC and IR-LSC borders of the three C. oleifera cultivar chloroplast genomes were compared to those of four other Camellia species (C. petelotii, C. sinensis, C. oleifera, and C. pitardii) and N. tabacum. The ycf1 pseudogenes were 962 bp long in the three C.oleifera cultivars, 1,068 bp in C. petelotii, C. sinensis, and C. oleifera, 1,042 bp in C. pitardii, and 1,027 bp in N. tabacum. The IRb/SSC borders of eight Camellia chloroplast genomes were nested in the ycf1 gene (962–1068 bp), extending into the IRb region.

Phylogenetic analysis

The three C. oleifera cultivars (‘Huashuo’, ‘Huajin’ and ‘Huaxin’) and Camellia oleifera formed a strongly supported monophyly (100%). A sister relationship was revealed among Camellia oleifera and the three C. oleifera cultivars (‘Huashuo’, ‘Huajin’ and ‘Huaxin’) (100%) (Fig. 7). ML indicated that C. oleifera ‘Huashuo’ was highly supported as a sister to a clade consisting of C. oleifera ‘Huaxin’, C. oleifera, and C. oleifera ‘Huajin’. C. oleifera from Hainan was identified as sister to C. azalea using bootstrapping (91%) (Fig. S1). C. oleifera was suggested to be more closely related to C. oleifera ‘Huajin’ than to C. oleifera ‘Huaxin’ or C. oleifera ‘Huashuo’ (Fig. 7 and S1).

Discussion

Fig. 5: Distribution of A/T simple-sequence repeats (SSRs) in three C. oleifera chloroplast genomes

Fig. 6: Comparison of the LSC, IR, and SSC border regions among eight Camellia chloroplast genomes

Fig. 7: Phylogenetic tree of 65 taxa based on 50 protein-coding chloroplast genes using the maximum parsimony (MP) default parameters. Bootstrap values (1,000 replications) are shown at the nodes

The entire chloroplast genomes of three C. oleifera cultivars were determined using Illumina HiSeq 4000 Sequencing and third-generation sequencing (PacBio RS II System). Illumina PE (300~500 bp) and PacBio (8~10 kb) libraries were constructed. The obtained sequencing data were mapped using bioinformatics analysis. With Illumina HiSeq sequencing platform to sequence samples results in some low-quality raw data. To ensure that the subsequent analysis was more accurate and reliable, were moved the adapter sequence from the reads, reads with N contents of up to 10%, and those with non-AGCT bases at the 5' end to ensure the accuracy of chloroplast genome assembly. We found that the chloroplast genome sizes were similar among the three C. oleifera cultivars (Table 1), ranging from about 156 to 160 kb, which is typical of Camellia species (Wang et al. 2017). Whole-chloroplast genome alignment revealed conserved organization and linear gene order among the three C. oleifera cultivars and seven other representative Camellia chloroplast genomes (Fig. 2 and Table 3). These results are consistent with those reported for herbaceous bamboo (Wang et al. 2018) and paleotropical plants (Vieira et al. 2015).

The differences in whole-chloroplast genome length are mainly due to differences in the IR region length (Guo et al. 2018). The cp genome sequences of C. oleifera ‘Huashuo’, ‘Huajin’ and ‘Huaxin’ were then compared with those of seven Camellia species using mVISTA. The alignment showed that the 10 cp genomes were conserved, with high gene order. Sequence comparison shows greater divergence is in the LSC and SSC regions than in the IR region, and lower in the coding region than then on-coding region. The 10 Camellia chloroplast genomes contained highly differentiated regions in the intergenic spacers. These results are consistent with those for other species (Ni et al. 2016; Guo et al. 2018; Jian et al. 2018). We detected slight variation in the coding regions of some genes including psbN and ycf1 (Fig. 3); variation in the ycf1 gene has been reported (Jian et al. 2018).

Repeat sequences such as SSRs play an important role in the rearrangement and stabilization of cp genome sequences and the copy number variation in different species, even in the same species, characteristics that make them suitable molecular markers for studying genetic diversity (Vieira et al. 2014; Su et al. 2017). In each of the three C. oleifera chloroplast genomes, we found one repeat each of 23, 24, 26, and 30 bp, four repeats of 38 bp, and two repeats of 42 bp. In the three C. oleifera chloroplast genomes, most repeats were located in IGS (Tables S1–S3), consistent with results reported by Li et al. (2018). In the three Camellia oleifera cp genomes, there were 50, 50, and 51 SSR loci at least 10 bp long in C. oleifera ‘Huashuo’, ‘Huaxin’ and ‘Huajin’, respectively (Tables S4–S6). Most SSR loci were located in the noncoding regions in the three C. oleifera cp genomes. These results are consistent with findings that SSR loci in the cp genome are usually located in IGS regions (Sithichoke et al. 2011; Li et al. 2017).

Although, the chloroplast genome has a nearly collinear gene order in most land plants, changes in the genome occur in the course of evolution, such as gene loss, sequence inversion, and expansion at the borders of the SSC, LSC, and IR regions (Choi et al. 2016; Su et al. 2017). The IRs/LSC boundary is a highly informative region for population and phylogenetic studies; for example, the distance between the end edge of ycf1 and IRb was 257 bp in Oenothera argillicola (Gu et al. 2018). In all chloroplast genomes examined in this study, the ndhF gene was located in the SSC region, 6–69 bp from the IRb/SSC border; it was farthest from the IRb/SSC border in the three C. oleifera cultivar chloroplast genomes, and it was nearest to the IRb/SSC border in C. pitardii. The rps19 gene overlapped at IRs in all Camellia chloroplast genomes by 45 bp, whereas the rps19 gene of N. tabacum was located in the LSC region, 4 bp from the IRb/LSC border (Fig. 6). Some rps19 genes are located in the LSC region, some in IR region, especially in monocotyledons, and some at the IRb/LSC border (Wang et al. 2016; Li et al. 2017). We found that the rps19 gene positions were exactly the same in seven Camellia species, indicating that the rps19 gene is very stable in Camellia. In chloroplast DNA, IRa and IRb are the relatively conserved regions, while expansion and contraction at the borders of IR regions are the main reasons for size variation in chloroplast genomes (Raubeson et al. 2007).

Several studies have analyzed phylogenetic relationships within the family Theaceae based on chloroplast coding or non-coding sequences (Yang et al. 2013; Huang et al. 2014). The chloroplast genome sequence is a useful resource for studying taxonomic status and evolutionary relationships within families (Prince and Parks 2001; Liu et al. 2018). The three C. oleifera cultivar chloroplast genomes used in this study provide sequence information that can be used in future studies of Camellia molecular evolution and phylogeny. To identify the phylogenetic position of Camellia within the as terid lineage, we performed multiple sequence alignments using 50 protein-coding genes present in 65 complete chloroplast genome sequences representing 31 orders. Additional chloroplast genomes from Ginkgo biloba, Wollemianobilis and Pinus sp were included as outgroups.

Conclusion

This study analyzed the complete chloroplast genomes of three C. oleifera cultivars (‘Huashuo’, ‘Huajin’, and ‘Huaxin’) that are cultivated widely in China. The genome structure, gene content, and gene number were similar in the three chloroplast genomes and those of other Camellia species. Phylogenetic analysis indicated a sister relationship among the three C. oleifera cultivars and C. oleifera, and C. oleifera from Hainan was identified as a sister to Camellia azalea. The results provide valuable whole-chloroplast genome information for Camellia species that may helpful further phylogenetic analyses of Camellia evolutionary relationships and facilitate the genetics and breeding of modern Camellia.

Acknowledgments

This work was supported by the Major Projects of Science and Technology Project of Hunan Province (2018NK1030).

Author Contributions

Lingli Wu and Jian’an Li analyzed the results. Ze Li and Xiaofeng Tan prepared plant materials and collected the samples. Fanhang Zhang prepared Fig. 1–4. Yiyang Gu prepared Fig. 5–7. Lingli Wu, Ze Li, and Xiaofeng Tan wrote the main manuscript text. All authors reviewed the manuscript.

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