Comparative chloroplast genomes of eleven Schima (Theaceae) species: Insights into DNA barcoding and phylogeny

Abstract

Schima is an ecologically and economically important woody genus in tea family (Theaceae). Unresolved species delimitations and phylogenetic relationships within Schima limit our understanding of the genus and hinder utilization of the genus for economic purposes. In the present study, we conducted comparative analysis among the complete chloroplast (cp) genomes of 11 Schima species. Our results indicate that Schima cp genomes possess a typical quadripartite structure, with conserved genomic structure and gene order. The size of the Schima cp genome is about 157 kilo base pairs (kb). They consistently encode 114 unique genes, including 80 protein-coding genes, 30 tRNAs, and 4 rRNAs, with 17 duplicated in the inverted repeat (IR). These cp genomes are highly conserved and do not show obvious expansion or contraction of the IR region. The percent variability of the 68 coding and 93 noncoding (>150 bp) fragments is consistently less than 3%. The seven most widely touted DNA barcode regions as well as one promising barcode candidate showed low sequence divergence. Eight mutational hotspots were identified from the 11 cp genomes. These hotspots may potentially be useful as specific DNA barcodes for species identification of Schima. The 58 cpSSR loci reported here are complementary to the microsatellite markers identified from the nuclear genome, and will be leveraged for further population-level studies. Phylogenetic relationships among the 11 Schima species were resolved with strong support based on the cp genome data set, which corresponds well with the species distribution pattern. The data presented here will serve as a foundation to facilitate species identification, DNA barcoding and phylogenetic reconstructions for future exploration of Schima.

Introduction

The chloroplast (cp) is a type of plastid that is critical to the growth of most plants, playing a major role in photosynthesis and fixation of CO₂ [1]. The cp genomes in angiosperms are circular DNA molecules with a highly conserved gene order and gene content, and range from 120 to 160 kb in length [2]. These genomes typically include two copies of an inverted repeat (IR) region that is separated by a large-single-copy (LSC) region and a small-single-copy (SSC) region [3]. Due to the rapid accumulation of genomic data gleaned from next-generation sequencing (NGS) technologies [4–6], more than 800 complete cp genomes of land plants have been sequenced (up to December 2016 from NCBI). The cp genome can provide valuable information for species identification, phylogeny and population genetic analyses [7–9]. It has also been postulated to be a potential ultra- or organelle-scale barcode for efficient plant species identification, especially for the taxonomically complex groups [10, 11].

Schima, with ca. 20 species, is an economically and ecologically important genus of the tea family (Theaceae). The genus is distributed in subtropical and tropical areas of East Asia, with 13 species (6 endemic) present in China [12]. Species of Schima are large trees and dominant elements of the subtropical evergreen broadleaved forests (SEBLFs) in East Asia [13, 14]. Some species are used as biological fire-resistant trees, and the wood is used for building and furniture [15, 16]. Schima is distinct from other genera within Theaceae, characterized by globose to oblate fruits and small reniform seeds with a marginal membranous wing. However, the infrageneric classification of Schima is complex and controversial due to a dearth of taxonomically diagnostic characters and high morphological similarity among species. This taxonomic uncertainty may hinder our exploitation and utilization of the genus.

Since its establishment as a genus, there has been much debate regarding the number of species within Schima [17]. Eighteen species were proposed in the second edition of the “Die Natürlichen Pflanzenfamilien” [18]. Bloembergen [19] regarded the genus as monotypic and subdivided Schima wallichii into nine geographically separated subspecies and three varieties. Airy-Shaw [20] recognized 15 species in Schima. Keng [21] accepted most of Bloembergen’s subspecies and raised them to the species level, and proposed that there were 10–15 species within the genus. The most recent treatment recognized ca. 20 species in Schima [12]. Schima is placed in tribe Gordonieae based on the results of molecular phylogenetic studies [22–24]. However, phylogenetic relationships within Schima are still unclear due to limited species sampling in previous studies, thus both species delimitations and phylogenetic reconstruction within Schima require further exploration.

Complete cp genomes have been shown to be effective in resolving interspecies phylogenetic relationships within Camellia, a genus in the sister tribe (Theeae) to Gordonieae [25, 26]. Here, we sequenced 11 cp genomes of the 13 Chinese Schima species. This study aims to: (1) investigate structural patterns of Schima cp genomes, (2) screen sequence divergence hotspots in the 11 Schima cp genomes, (3) explore simple sequence repeats (SSRs) among the 11 Schima cp genomes, (4) and reconstruct phylogenetic relationships among the 11 Schima species using the cp genome sequences. The results will provide abundant information for further studies regarding taxonomy, phylogeny, and population genetics of Schima, and will also assist in the exploration and utilization of the resources within the genus.

Materials and methods

Taxon sampling

In this study, we follow the classification of Schima from Min and Bartholomew [12]. Healthy and fresh leaves from 11 species of Schima were sampled from various localities across southern China (Table 1). Voucher specimens of each species were collected and deposited in the Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences (KUN). Gordonia lasianthus and Franklinia alatamaha were used as outgroups in the phylogenetic analyses, and the cp genomes of these two species were obtained from our previous work (Yu et al., unpublished work).

Table 1. List of taxa sampled in this study, with the voucher, chloroplast genome size, Illumina reads and coverage depth information.

Taxon	Voucher specimen	Sources	Genome size	LSC length (bp)	SSC length (bp)	IR length (bp)	GC content (%)	No. reads (trimmed)	Mean coverage	GenBank No.
Schima argentea	YXQ041	Yunnan, China	157,245	87,222	18,091	25,966	37.43	7,448,533	620.1	KY406780
Schima brevipedicellata	YXQ069	Yunnan, China	157,227	87,202	18,089	25,968	37.44	687,249	1538.6	KY406758
Schima crenata	YXQ103	Hainan, China	157,288	87,232	18,104	25,976	37.44	701,233	1679.4	KY406755
Schima khasiana	YXQ070	Yunnan, China	157,252	87,208	18,112	25,966	37.43	459,809	1106.5	KY406794
Schima multibracteata	YXQ146	Guangxi, China	157,278	87,233	18,103	25,971	37.44	737,292	1882.5	KY406763
Schima noronhae	YXQ034	Yunnan, China	157,278	87,217	18,091	25,985	37.43	527,901	1284.3	KY406787
Schima remotiserrata	YXQ186	Hunan, China	157,284	87,229	18,103	25,976	37.43	562,801	1389.5	KY406749
Schima sericans	YXQ053	Yunnan, China	157,302	87,272	18,122	25,954	37.45	748,129	1735.2	KY406779
Schima sinensis	YXQ2902	Sichuan, China	157,297	87,243	18,102	25,976	37.45	10,152,425	716.8	KY406762
Schima superba	YXQ142	Guangxi, China	157,254	87,202	18,100	25,976	37.44	457,215	1221.9	KY406788
Schima wallichii	YXQ001	Yunnan, China	157,240	87,204	18,104	25,966	37.44	28,866	71.7	KY406795

Voucher specimens were deposited in the Herbarium of Kunming Institute of Botany (KUN), Chinese Academy of Sciences.

DNA extraction, sequencing, chloroplast genome assembly

Total genomic DNA was isolated from fresh leaves (~100 mg) using the modified CTAB method (Doyle and Doyle 1987). Subsequently, the cp genomes were amplified using long-range PCR with fifteen primers [27]. The PCR products were fragmented for constructing short-insert (500 bp) libraries following the Illumina Nextera XT DNA library preparation instructions. Paired-end sequencing (250 bp) was performed on the Illumina MiSeq 2000 at the Laboratory of Molecular Biology of Germplasm Bank of Wild Species in Southwest China. Quality control of the raw sequence reads was performed using the NGS QC Tool Kit [28], with a cut-off value for percentage of read length and PHRED quality score as 80 and 30 following Yang et al. [5]. High-quality reads were assembled into contigs using the de novo assembler in CLC Genomics Workbench v6.5 (CLC Bio), using a k-mer of 64 and a minimum contig length of 500 base pairs (bp). The de novo contigs were assembled into complete chloroplast genomes followed the procedure of Yang et al. [5].

Chloroplast genome annotation and comparisons

The complete cp genomes were annotated with the identification of introns and exons using DOGMA [29]. The positions of start and stop codons and boundaries between introns and exons were investigated according to the published cp genome of Camellia taliensis (NC022264). The annotated GenBank files were used to draw the circular chloroplast genome maps using OrganellarGenomeDRAW [30]. The mVISTA program [31] was employed in the LAGAN mode to detect the variation of the chloroplast genomes. The cp genome of Schima sinensis was used as a reference. Microsatellites (mono-, di-, tri-, tetra-, penta- and hexanucleotide repeats) were detected using Phobos v3.311 [32], with the parameters set to ten repeat units (≥ 10) for mononucleotide SSRs, six repeat units (≥ 6) for dinucleotide, four repeat units (≥ 4) for trinucleotide, four repeat units (≥ 4) for tetranucleotide, and three repeat units (≥ 3) for pentanucleotide and hexanucleotide SSRs. The percent variability for all protein-coding and noncoding (intergenic spacers and introns) regions of the cp genomes with an aligned length larger than 150 bp among the 11 Schima species was estimated in Geneious [33].

Phylogenetic inference

The cp genomes were aligned using MAFFT v7.221 [34] under default settings (FFT-NS-2 strategy). One of the IRs was removed from the data set for the phylogenetic analysis. Poorly aligned regions (mainly introns and spacers) of the data set were realigned using the G-INS-i (accurate strategy) to improve the quality of the alignment. We used jModelTest v0.11 [35] to select the best-fitting nucleotide substitution models for maximum-likelihood (ML) according to the Akaike information criterion (AIC; Akaike, 1974). ML analysis was implemented in RAxML v8.20 [36]. We conducted a rapid bootstrap analysis (1000 replicates) and searched for the best-scoring ML tree simultaneously (the “-f a” option). Numbers of variable and informative sites were calculated in DnaSP v5.10 [37].

Results

Chloroplast genome features

Illumina paired-end sequencing of long-range PCR amplified cp DNA generated 28,866–10,152,425 clean reads for the 11 sampled Schima species, with mean coverage from 71.7 to 1882.5. The genome size ranged from 157,227 bp in Schima brevipedicellata to 157,302 bp in Schima sericans (Table 1). All of the 11 cp genomes showed typical quadripartite structure consisting of a pair of IR (25,954–25,985 bp) separated by the LSC (87,202–87,272 bp) and SSC (18,089–18,122 bp) regions (Table 1). The cp genome map of Schima superba is presented as a representative (Fig 1). Excluding the duplicated IR region, the 11 Schima cp genomes identically encoded 114 different genes that were arranged in the same order, including 80 protein-coding genes, 30 tRNAs and 4 rRNAs. Seventeen genes were duplicated in the IRs, with six protein-coding genes, four rRNA and seven tRNA genes. Twelve of the protein-coding genes and six of the tRNA genes contained introns. Fifteen out of those eighteen genes contained a single intron, while the other three (clpP, rps12 and ycf3) had two introns. The 11 Schima cp genomes exhibited high similarity at the LSC/IR/SSC boundaries (Fig 2). The rps19 gene crossed the LSC/IR_B (J_LB) region with no variation of sequence length within the two parts. The SSC/IR_B (J_SB) junction occurred between the ycf1_like (incompletely duplicated in IR_B) and the 3’ end of ndhF gene, with the sequence length of ycf1_like gene within IR_B as 1388 or 1394. The ycf1 gene crossed the SSC/IR_A (J_SA) region, with 1388 or 1394 bp of ycf1 within IR_A. The ycf1 related length changes were the only variation detected in these junctions. The LSC/IR_A (J_LA) junction was located at the 3’ end of the rps19_like (6 bp; incompletely duplicated in IR_A), with a 14 bp noncoding sequence between J_LA and trnH gene. In addition, we identified unusual start codons for four genes, ACG for ndhD, ATC for psbI, ATT for psbT and GTG for rps19.

Fig 1. Gene map of Schima superba chloroplast genome.

Fig 2. Comparisons of the border regions among the chloroplast genomes of 11 Schima species.

ycf1^* (ycf1_like) and rps19^* (rps19_like) represent the incomplete duplication of the gene within the IR region.

Chloroplast genome comparisons and divergence hotspots

Sequence identity plots of the 11 Schima cp genomes, generated using mVISTA, are shown in Fig 3. The plots illustrate the high sequence similarity across the Schima cp genomes, with a sequence identity of 99.1%. Two (ccsA and rps15) of the 49 variable protein-coding (>150 bp) genes had a percentage of variation above 1.00% (Table 2), while 19 (>150 bp) had no variation. Both of the two core DNA barcodes (rbcL and matK) [38] showed extremely low sequence divergence (0.21% and 0.33%, respectively). Furthermore, the variation of ycf1, the proposed “most promising chloroplast DNA barcode” of land plants [39], was only 0.67%. Among the 79 noncoding (>150 bp) regions, the percentage of variation ranged from 0.11% to 2.85% (Fig 4 and Table 3). Fourteen fragments (atpI-rps2, trnS (UGA)-psbZ, rps4-trnT (UGU), trnL (UAA)-trnF (GAA), petB-petD, rpl2 intron, rpl23-trnI (CAU), ycf15-trnL (CAA), trnL (CAA)-ndhB, ndhB intron, trnV (GAC)-rrn16, rrn16-trnI (GAU), trnA (UGC) intron, trnN (GUU)-ndhF) did not show any sequence variation. Eight potential mutational hotspots (trnW (CCA)-trnP (UGG), trnT (UGU)-trnL (UAA), trnG (UCC)-trnfM (CAU), petD-rpoA, psbB-psbT, ndhE-ndhG, ndhC-trnV (UAC), rpl32-trnL (UAG)) were identified, with the variation percentage exceeding 2.0% among the 11 sampled species (Fig 4 and Table 3). These eight highly variable hotspots may have the potential to be used as special DNA barcodes for identifying Schima species.

Fig 3. mVISTA percent identity plot comparison among the chloroplast genomes with S. sinensis as a reference.

Table 2. Sequence divergence of 49 variable coding regions (>150 bp) from 11 chloroplast genomes of Schima, with one of the Inverted Repeat regions removed.

Fragments	Length (bp)	Aligned length (bp)	Variable positions	Nucleotide substitutions	Number of indels	Total length of indels	Percent variability (%)
matK	1527	1527	5	5	0	0	0.33
psbK	186	186	1	1	0	0	0.54
psbI	153–156	156	3	0	1	3	0.64
atpA	1524	1524	3	3	0	0	0.20
atpF	567	567	2	2	0	0	0.35
atpI	744	744	1	1	0	0	0.13
rps2	711	711	1	1	0	0	0.14
rpoC2	4137	4137	11	11	0	0	0.27
rpoC1	2061	2061	3	3	0	0	0.15
rpoB	3213	3213	11	11	0	0	0.34
psbC	1422	1422	3	3	0	0	0.21
psaB	2205	2205	2	2	0	0	0.09
psaA	2253	2253	6	6	0	0	0.27
rps4	606	606	2	2	0	0	0.33
ndhK	678	678	1	1	0	0	0.15
ndhC	363	363	2	2	0	0	0.55
atpE	402	402	1	1	0	0	0.25
atpB	1497	1497	1	1	0	0	0.07
rbcL	1428	1428	3	3	0	0	0.21
accD	1542	1542	4	4	0	0	0.26
ycf4	555	555	3	3	0	0	0.54
cemA	690	690	2	2	0	0	0.29
petA	963	963	2	2	0	0	0.21
rpl20	354	354	1	1	0	0	0.28
rps12	372	372	1	1	0	0	0.27
clpP	645	645	3	3	0	0	0.47
psbB	1527	1527	2	2	0	0	0.13
petB	663	663	4	4	0	0	0.60
rpoA	1014	1014	2	2	0	0	0.20
rps11	417	417	3	3	0	0	0.72
infA	234	234	1	1	0	0	0.43
rps8	408	408	1	1	0	0	0.25
rpl14	369	369	3	3	0	0	0.81
rpl16	411	411	2	2	0	0	0.49
rps3	657	657	2	2	0	0	0.30
rpl22	474	474	1	1	0	0	0.21
ycf2	6867–6873	6873	8	2	1	6	0.04
rps7	468	468	1	1	0	0	0.21
ndhF	2247–2253	2253	26	14	2	12	0.71
rpl32	162	162	1	1	0	0	0.62
ccsA	963	963	10	10	0	0	1.04
ndhD	1530	1530	3	3	0	0	0.20
psaC	246	246	1	1	0	0	0.41
ndhE	306	306	1	1	0	0	0.33
ndhG	531	531	3	3	0	0	0.56
ndhA	1092	1092	2	2	0	0	0.18
ndhH	1182	1182	2	2	0	0	0.17
rps15	273	273	3	3	0	0	1.10
ycf1	5652–5658	5658	43	37	1	6	0.67

Fig 4. Percentage of variation in 79 variable noncoding regions of the 11 Schima chloroplast genomes.

These regions are oriented according to their locations in the genome.

Table 3. Sequence divergence of 79 variable noncoding loci (>150 bp) from 11 chloroplast genomes of Schima, with one of the invert repeat regions removed.

Fragments	Length (bp)	Aligned length (bp)	Variable positions	Nucleotide substitutions	Number of indels	Total length of indels	Percent variability (%)
trnH (GUG)-psbA	395–426	428	37	4	1	33	1.17
psbA-trnK (UUU)	220	220	1	1	0	0	0.45
trnK (UUU)-matK	270	270	2	2	0	0	0.74
matK-trnK (UUU)	712–713	713	2	1	1	1	0.28
trnK (UUU)-rps16	809–818	819	22	10	4	12	1.71
rps16 intron	838–844	845	10	3	2	7	0.59
rps16-trnQ (UUG)	1689–1698	1700	36	18	4	18	1.29
trnQ (UUG)-psbK	333	333	2	2	0	0	0.60
psbK-psbI	345	345	3	3	0	0	0.87
trnS (GCU)-trnG (UCC)	681–682	682	3	3	0	0	0.44
trnG (UCC) intron	690–696	696	8	2	1	6	0.43
trnG (UCC)-trnR (UCU)	276–311	311	37	2	1	35	0.96
atpF intron	701	701	2	2	0	0	0.29
atpF-atpH	387–389	389	7	5	1	2	1.54
atpH-atpI	1143–1153	1153	15	4	4	11	0.69
rps2-rpoC2	254–255	255	1	0	1	1	0.39
rpoC1 intron	732–734	734	2	0	1	2	0.14
rpoB-trnC (GCA)	1211–1222	1222	26	9	4	17	1.06
trnC (GCA)-petN	722–727	727	8	3	1	5	0.55
petN-psbM	1123–1125	1125	15	13	1	2	1.24
psbM-trnD (GUC)	1136–1153	1153	27	8	5	19	1.13
trnE (UUC)-trnT (GGU)	473	473	5	5	0	0	1.06
trnT (GGU)-psbD	1513–1517	1519	19	11	3	8	0.92
psbC-trnS (UGA)	234–239	239	6	1	1	5	0.84
psbZ-trnG (UCC)	283	283	1	1	0	0	0.35
trnG (UCC)-trnfM (CAU)	157–159	160	5	2	2	3	2.50
trnfM (CAU)-rps14	154	154	2	2	0	0	1.30
psaA-ycf3	747–755	755	17	3	3	14	0.79
ycf3 intron1	721	721	2	2	0	0	0.28
ycf3 intron2	726	726	2	2	0	0	0.28
ycf3-trnS (GGA)	839–842	842	8	4	3	4	0.83
trnS (GGA)-rps4	293	293	1	1	0	0	0.34
trnT (UGU)-trnL (UAA)	982–999	999	46	19	6	27	2.50
trnL (UAA) intron	522–529	529	7	0	1	7	0.19
trnF (GAA)-ndhJ	704–714	715	21	3	4	18	0.98
ndhC-trnV (UAC)	400–414	417	36	3	6	33	2.16
trnV (UAC) intron	585	585	2	2	0	0	0.34
trnV (UAC)-trnM (CAU)	166	166	1	1	0	0	0.60
trnM (CAU)-atpE	229–238	240	13	0	2	13	0.83
atpB-rbcL	765–768	768	5	2	1	3	0.39
rbcL-accD	525–526	526	4	3	1	1	0.76
accD-psaI	681–683	683	4	2	1	2	0.44
psaI-ycf4	423–425	425	3	1	1	2	0.47
ycf4-cemA	909–915	915	13	7	1	6	0.87
cemA-petA	220–228	228	8	0	2	8	0.88
petA-psbJ	1035–1042	1043	12	4	3	8	0.67
psbE-petL	1277–1287	1287	22	12	2	10	1.09
petL-petG	185–186	186	1	0	1	1	0.54
trnW (CCA)-trnP (UGG)	170–175	175	9	4	1	5	2.86
trnP (UGG)-psaJ	391–393	393	2	0	1	2	0.25
psaJ-rpl33	455–457	457	5	3	1	2	0.88
rpl33-rps18	175–176	176	2	1	1	1	1.14
rps18-rpl20	284	284	1	1	0	0	0.35
rpl20-rps12	786	786	4	4	0	0	0.51
clpP intron1	598–605	607	13	3	5	10	1.32
clpP intron2	797–798	798	3	2	1	1	0.38
clpP-psbB	473–479	479	8	2	1	6	0.63
psbB-psbT	172–174	174	5	3	1	2	2.30
petB intron	787	787	3	3	0	0	0.38
petD intron	711	711	7	7	0	0	0.98
petD-rpoA	200–213	215	15	0	5	15	2.33
rps8-rpl14	196	196	1	1	0	0	0.51
rpl16 intron	996–998	998	13	11	1	2	1.20
rpl16-rps3	150–200	200	52	2	1	50	1.50
rps12 intron	536	536	2	2	0	0	0.37
rps12-trnV (GAC)	1602–1619	1619	29	2	5	27	0.43
trnI (GAU) intron	938	938	1	1	0	0	0.11
trnA (UGC)-rrn23	152	152	1	1	0	0	0.66
rrn4.5-rrn5	256–275	275	19	0	1	19	0.36
rrn5-trnR (ACG)	248–249	249	2	1	1	1	0.80
trnR (ACG)-trnN (GUU)	595	595	7	7	0	0	1.18
ndhF-rpl32	825–847	851	32	6	4	26	1.18
rpl32-trnL (UAG)	908–926	928	36	13	6	23	2.05
ccsA-ndhD	240–244	245	6	1	2	5	1.22
psaC-ndhE	251–254	254	4	1	1	3	0.79
ndhE-ndhG	230	230	5	5	0	0	2.17
ndhG-ndhI	359–360	360	6	5	1	1	1.67
ndhA intron	1106–1111	1112	16	8	3	8	0.99
rps15-ycf1	379	379	5	5	0	0	1.32

The aligned length of the complete cp genome (with one of the IR removed) among the 11 Schima species was 130,508 bp, with the total number of variable and parsimony informative (PI) sites being 1,121 bp and 261 bp, respectively. This data set contained 131 indels with a total length of 586 bp, and the percent variability was 0.51%. These results indicate that the global variation of the cp genome within Schima is extremely low. A similar pattern was reported in other long-lived plants [40–42]. The ability to identify species within the genus using cp genome data needs to be assessed by sampling multiple individuals per species, even though the phylogenetic analyses have most of the species separated from each other (see below).

SSR polymorphisms

In total, 58 cpSSRs, including 55 mononucleotide (A, T), 1 dinucleotide (AT) and 2 trinucleotide (ATT, TTA) repeats were detected within the 11 Schima cp genomes. No tetranucleotide, pentanucleotide or hexanucleotide repeats were observed. The mononucleotide repeat (A, T) was found to be the most abundant, with repeat numbers of 10, 11 and 12 (Table 4). The proportion of A and T repeats in mononucleotide repeat unit was 43.64% and 56.36%, respectively. Only one SSR locus with a different repeat unit (C) was detected in the trnG (UCC)-trnfM (CAU) intergenic spacer region. Within the 11 Schima cp genomes, SSR loci were primarily located in the LSC region (89.09%), followed by the SSC portion (14.55%), with only one present in the IR region (rrn5-trnR (ACG)) (Table 4). One SSR locus was detected in the protein-coding gene psbI, with all others located in gene spacers and introns. No SSRs were found in the tRNAs and rRNAs. The mononucleotide repeat (A) in trnH-psbA was the most variable SSR, with the size ranging from 12 to 42 bp. The cpSSRs of the 11 Schima species represented here showed abundant variation, and could be useful for research at the population level. They will provide complementary data to the SSR markers of Schima identified from the nuclear genome [43].

Table 4. Location of SSR loci within the 11 Schima genomes.

No.	Motif	Location	Region	Repeat length
1	A	trnH-psbA	LSC	12–43
2	A	trnK (UUU) intron	LSC	10–11
3	A	trnK (UUU)-rps16	LSC	9–10
4	A	trnK (UUU)-rps16	LSC	11–14
5	A	trnK (UUU)-rps16	LSC	8–10
6	T	rps16-trnQ (UUG)	LSC	9–14
7	A	rps16-trnQ (UUG)	LSC	8–10
8	T	rps16-trnQ (UUG)	LSC	8–11
9	T	psbI	LSC	10, 13
10	A	atpA-atpF	LSC	14,15
11	T	atpF-atpH	LSC	9–12
12	AT	atpF-atpH	LSC	12,14
13	A	atpH-atpI	LSC	13–20
14	T	atpH-atpI	LSC	12,13
15	A	rps2-rpoC2	LSC	10,11
16	A	rpoC2-trnC (GCA)	LSC	9,10
17	T	psbM-trnD (GUC)	LSC	10,11
18	T	trnT (GGU)-psbD	LSC	9,10
19	A	trnT (GGU)-psbD	LSC	9–14
20	T	psbC-trnS (UGA)	LSC	11–16
21	C	trnG (UCC)-trnfM (CAU)	LSC	8, 10
22	A	trnG (UCC)-trnfM (CAU)	LSC	9,10
23	A	psaA-ycf3	LSC	10–14
24	A	ycf3-trnS (GGA)	LSC	11,12
25	A	ycf3-trnS (GGA)	LSC	11,12
26	A	trnT (UGU)-trnL (UAA)	LSC	13,14
27	T	trnF (GAA)-ndhJ	LSC	9, 10
28	T	ndhC-trnV (UAC)	LSC	8–16
29	T	ndhC-trnV (UAC)	LSC	9–12
30	TTA	ndhC-trnV (UAC)	LSC	3,12
31	T	ndhC-trnV (UAC)	LSC	9,10
32	T	trnM (CAU)-atpE	LSC	9–12
33	T	atpB-rbcL	LSC	12–15
34	T	rbcL-accD	LSC	11,12
35	T	accD-psaI	LSC	13–15
36	T	psaI-ycf4	LSC	10–12
37	T	petA-psbJ	LSC	10–13
38	T	petA-psbJ	LSC	10,11
39	T	petL-petG	LSC	10,11
40	T	trnP (UGG)-psaJ	LSC	10–12
41	T	rpl20-rps12	LSC	6,10
42	T	clpP intron	LSC	10,11
43	A	clpP intron	LSC	9–11
44	A	clpP intron	LSC	9,10
45	ATT	clpP-psbB	LSC	6,12
46	A	psbB-psbT	LSC	8–10
47	T	petD-rpoA	LSC	9,10
48	A	petD-rpoA	LSC	9–11
49	A	petD-rpoA	LSC	9,10
50	A	rrn5-trnR (ACG)	IR	9,10
51	T	ndhF-rpl32	SSC	9–12
52	T	ndhF-rpl32	SSC	10,11
53	A	rpl32-trnL (UAG)	SSC	10,11
54	T	rpl32-trnL (UAG)	SSC	10–13
55	T	ccsA-ndhD	SSC	10–13
56	T	psaC-ndhE	SSC	9–12
57	T	ndhG-ndhI	SSC	10,11
58	A	ndhA intron	SSC	9,10

Phylogenetic analyses

The data matrix we used for phylogenetic estimation consisted of an alignment containing entire cp genomes with one of the IRs removed. This data set was comprised of 131,113 nucleotide positions, with 2,508 variable sites (1.91%) and 427 PI sites (0.33%). ML analysis resulted in a well-resolved tree, with eight of the 10 nodes supported by 100% bootstrap values (BS). All Schima species grouped into a strongly supported clade (BS = 100%, Fig 5), indicating Schima is monophyletic. Two main clades were recovered, with Schima sericans being sister to those two lineages. Five species (S. argentea, S. brevipedicellata, S. khasiana, S. noronhae, S. wallichii) formed clade I (BS = 100%, Fig 5). The remaining five species (S. sinensis, S. superba, S. remotiserrata, S. multibracteata and S. crenata) grouped in clade II (BS = 100%, Fig 5). The branch leading to S. superba and three closely related species is extremely short, and bootstrap support values for two internal nodes within this clade are less than 80%.

Fig 5. Phylogenetic relationships among the 11 Schima species.

The phylogenetic tree was reconstructed using the whole chloroplast genome data set minus a copy of the IR region. Numbers above the branches show bootstrap support values that are above 80%.

Discussion

Chloroplast genome features and comparison within Theaceae

Prior to this study, Camellia was the only genus within Theaceae to have its cp genome sequenced [25, 26]. In the present study, we sequenced cp genomes of 11 species from Schima. The cp genomes all displayed typical quadripartite structure (Fig 1), which is consistent amongst most lineages of angiosperms [2]. The expansion and contraction of the IR region is considered to be the primary mechanism affecting length variation of angiosperm cp genomes, as demonstrated in Trochodendraceae [44] and Apiales [45]. However, only minor variation was detected at the SSC/IR_A boundary of all of the 11 Schima cp genomes (Fig 2). Although the genes located at the IR junctions are identical in cp genomes of Schima and Camellia, the overall cp genome sequences of Schima are more homogenous as compared to Camellia, which was suggested to show more differences at the junction regions [26]. The cp genomes of Schima encode the same set of protein-coding genes as previously reported Camellia species, with the exception of Orf 42 and Orf 188 which were reported in Camellia [25], but not in other Ericales members such as Actinidia (Actinidiaceae) and Ardisia (Primulaceae) [46, 47]. For the whole cp genomes of Schima, 37 tRNA genes were annotated, which is consistent with Huang et al. [26]. However, 38 tRNA genes were found in Yang et al. [25], due to a redundant annotation of trnP (UGG) in their study. As compared with sequences of Camellia, no significant structural rearrangements such as inversions or changes of gene locations were found in the 11 Schima cp genomes. The high sequence similarity across the Schima cp genomes (Fig 3) may be associated with long generation time and recent radiation.

Potentially specific DNA barcodes for Schima

Since the concept of DNA barcoding was proposed over a decade ago [48], substantial efforts have been made to develop DNA barcodes possessing both high universality and efficiency. Kress et al. [49] suggested that the internal transcribed spacer (ITS) and trnH-psbA spacer region had potential as useful DNA barcode regions for flowering plants. Hollingsworth et al. [38] later advocated matK and rbcL as a two-locus core barcode for land plants after comparing seven leading candidate loci, subsequently the nrDNA ITS was recommended to be incorporated into core barcode based on a large-scale sapmpling of seed plants [50]. Dong et al. [39] proposed that ycf1 was the most variable loci of the cp genome, which might be a promising DNA barcode performing better than existing plastid candidate barcodes of land plants. However, all of the five candidate protein-coding DNA barcodes (matK, rbcL, rpoB, rpoC1 and ycf1) showed extremely low sequence variation (<1.00%), and the other three fragments are also not among the most variable spacers. The eight potential mutational hotspots (trnW (CCA)-trnP (UGG), trnT (UGU)-trnL (UAA), trnG (UCC)-trnfM (CAU), petD-rpoA, psbB-psbT, ndhE-ndhG, ndhC-trnV (UAC), rpl32-trnL (UAG)) (Fig 4 and Table 3) identified in this study could be suitable barcodes for Schima. Recently, using the cp genome as a possible ultra- or organelle-scale barcode for efficient plant species identification was discussed [10, 11]. The high phylogenetic resolution among closely related species of Schima (Fig 5) suggests that the cp genome may indeed be useful as an organelle-scale barcode for species identification of Schima. Further studies based on sampling at the population scale are needed to evaluate the efficiency of the barcodes mentioned above and also the cp genome as an organelle-scale barcode.

Phylogenetic relationships among species of Schima

The cp genome has been suggested to be useful for phylogenetic reconstructions at low taxonomic levels [7, 8, 10, 51]. Interspecies phylogenetic relationships within Camellia (Theaceae) were well-resolved using cp genome data [25, 26]. In the present study, based on a recent classification of the genus [12], 11 out of 13 Schima species occurring in China were represented. The phylogenetic relationships within Schima were well resolved with strong support based on cp genome sequences (Fig 5). Therefore, our study indicates that the complete cp genome has significant potential to resolve the low level phylogenetic relationships. Schima sericans, the first diverging lineage among sampled species, is distributed in southeastern Xizang and northwestern Yunnan in China. Schima sericans was sister to the remaining taxa, which formed two clades. Clade I includes five species (S. argentea, S. brevipedicellata, S. khasiana, S. noronhae and S. wallichii) that are primarily distributed in southwestern China and Indochina. Clade II comprises five species (S. sinensis, S. superba, S. remotiserrata, S. multibracteata and S. crenata) that mainly occur within central and eastern China (Fig 5). The phylogenetic relationships within Schima found here correspond well with the geographic distribution pattern, but do not match well with morphology. Schima was classified into two groups based on the shape of the leaf margin (entire or serrate) [12]. However, all of the species within clade I possess a serrate leaf margin except S. khasiana. Likewise, S. multibracteata is the only species with an entire leaf margin in clade II (Fig 5). Our results suggest that the taxonomic value of leaf margin shape should be reassessed for classification of Schima. Additionally, the branch length of the clade including S. superba and three closely related species is extremely short, indicating that these four species have recently diversified, or perhaps illustrating past hybridization within the group. These results indicate that the current treatment of the genus needs to be reevaluated by integrating more types of evidence.

Data Availability

The GenBank accession numbers are listed in Table 1 of the paper.

Funding Statement

This work was supported by grants from the Applied Fundamental Research Foundation of Yunnan Province (2014FB167; 2014GA003), the National Key Basic Research Program of China (2014CB954100) and the China Postdoctoral Science Foundation (2014M562352).