Comparative analysis of the complete chloroplast genomes of thirteen Bougainvillea cultivars from South China with implications for their genome structures and phylogenetic relationships

Xiao-Ye Wu; He-Fa Wang; Shui-Ping Zou; Lan Wang; Gen-Fa Zhu; Dong-Mei Li

doi:10.1371/journal.pone.0310091

. 2024 Sep 11;19(9):e0310091. doi: 10.1371/journal.pone.0310091

Comparative analysis of the complete chloroplast genomes of thirteen Bougainvillea cultivars from South China with implications for their genome structures and phylogenetic relationships

Xiao-Ye Wu ¹, He-Fa Wang ², Shui-Ping Zou ¹, Lan Wang ¹, Gen-Fa Zhu ^3,^*, Dong-Mei Li ^3,^*

Editor: Pankaj Bhardwaj⁴

PMCID: PMC11389920 PMID: 39259741

Abstract

Bougainvillea spp., belonging to the Nyctaginaceae family, have high economic and horticultural value in South China. Despite the high similarity in terms of leaf appearance and hybridization among Bougainvillea species, especially Bougainvillea × buttiana, their phylogenetic relationships are very complicated and controversial. In this study, we sequenced, assembled and analyzed thirteen complete chloroplast genomes of Bougainvillea cultivars from South China, including ten B. × buttiana cultivars and three other Bougainvillea cultivars, and identified their phylogenetic relationships within the Bougainvillea genus and other species of the Nyctaginaceae family for the first time. These 13 chloroplast genomes had typical quadripartite structures, comprising a large single-copy (LSC) region (85,169–85,695 bp), a small single-copy (SSC) region (18,050–21,789 bp), and a pair of inverted-repeat (IR) regions (25,377–25,426 bp). These genomes each contained 112 different genes, including 79 protein-coding genes, 29 tRNAs and 4 rRNAs. The gene content, codon usage, simple sequence repeats (SSRs), and long repeats were essentially conserved among these 13 genomes. Single-nucleotide polymorphisms (SNPs) and insertions/deletions (indels) were detected among these 13 genomes. Four divergent regions, namely, trnH-GUG_psbA, trnS-GCU_trnG-UCC-exon1, trnS-GGA_rps4, and ccsA_ndhD, were identified from the comparative analysis of 16 Bougainvillea cultivar genomes. Among the 46 chloroplast genomes of the Nyctaginaceae family, nine genes, namely, rps12, rbcL, ndhF, rpoB, rpoC2, ndhI, psbT, ycf2, and ycf3, were found to be under positive selection at the amino acid site level. Phylogenetic relationships within the Bougainvillea genus and other species of the Nyctaginaceae family based on complete chloroplast genomes and protein-coding genes revealed that the Bougainvillea genus was a sister to the Belemia genus with strong support and that 35 Bougainvillea individuals were divided into 4 strongly supported clades, namely, Clades Ⅰ, Ⅱ, Ⅲ and Ⅳ. Clade Ⅰ included 6 individuals, which contained 2 cultivars, namely, B. × buttiana ‘Gautama’s Red’ and B. spectabilis ‘Flame’. Clades Ⅱ only contained Bougainvillea spinosa. Clade Ⅲ comprised 7 individuals of wild species. Clade Ⅳ included 21 individuals and contained 11 cultivars, namely, B. × buttiana ‘Mahara’, B. × buttiana ‘California Gold’, B. × buttiana ‘Double Salmon’, B. × buttiana ‘Double Yellow’, B. × buttiana ‘Los Banos Beauty’, B. × buttiana ‘Big Chitra’, B. × buttiana ‘San Diego Red’, B. × buttiana ‘Barbara Karst’, B. glabra ‘White Stripe’, B. spectabilis ‘Splendens’ and B. × buttiana ‘Miss Manila’ sp. 1. In conclusion, this study not only provided valuable genome resources but also helped to identify Bougainvillea cultivars and understand the chloroplast genome evolution of the Nyctaginaceae family.

Introduction

The Nyctaginaceae family, also called the four o’clock family, contains approximately 31 genera [1,2]. San Jiao Mei and Le Du Juan, which are well known in China, belong to the Bougainvillea genus of the Nyctaginaceae family. Bougainvillea plants are tropical and subtropical shrubs or small trees armed with simple or forked thorns, commonly with colorful bracts [2,3]. The colorful bracts surrounding small tubular flowers are often mistakenly treated as flowers. Due to the demand of the commercial market, garden growers have obtained new cultivars with bright bracts through hybridization or grafting [4,5].

To date, more than 200 Bougainvillea cultivars have been produced and introduced to China. Bougainvillea cultivars with large bracts of various colors have been seen in many cities of South China, such as Zhangzhou of Fujian, Guangzhou of Guangdong, Haikou of Hainan, and Nanning of Guangxi. Bougainvillea × buttiana cultivars have also been used in many cities in South China. B. × buttiana is named a new species based on a plant cultivated in the Singapore Botanical Garden [4]. It was originally from a garden in Cartagena, Colombia, and was introduced to Trinidad in 1910 as the cultivar ‘Mrs. Butt’. It is presumed by Gillis [5] to be a hybrid between Bougainvillea peruviana and B. glabra. These Bougainvillea cultivars have been widely used for horticultural landscaping in cities of South China. However, identification of these Bougainvillea cultivars based mainly on leaf morphology has been challenging because of the high similarity of their leaf appearances [2,3].

In previous studies, although the phylogenetic relationships of the Nyctaginaceae family, including the Bougainvillea genus, were identified using several chloroplast genes (ndhF, rps16, and rpl16) and one nuclear region (ITS), low-resolution branches among different genera existed [1,6]. With recent advancements in sequencing, complete chloroplast genome sequencing has become convenient. Complete chloroplast genomes have been extensively used for phylogenetic analyses of ornamental plants, such as Caryophyllales [7], Aglaonema [8] and Hyacinthus [9]. More recently, the phylogenetic relationships of wild Bougainvillea species have been explored using complete chloroplast genomes [2,3,10–13] and even up to one hybrid cultivar [3]. However, the phylogenetic relationships of B. × buttiana cultivars and the molecular evolution of chloroplast genomes from the Nyctaginaceae family remain to be elucidated [2–5,9–13]. Therefore, it is worthwhile to investigate the phylogenetic relationships of B. × buttiana cultivars and the molecular evolution of chloroplast genomes in the Nyctaginaceae family.

In this study, the complete chloroplast genomes of thirteen Bougainvillea cultivars were newly sequenced, assembled and annotated. These thirteen cultivars from South China [14,15], included ten B. × buttiana cultivars, namely, B. × buttiana ‘Mahara’, B. × buttiana ‘Gautama’s Red’, B. × buttiana ‘California Gold’, B. × buttiana ‘Double Salmon’, B. × buttiana ‘Double Yellow’, B. × buttiana ‘Big Chitra’, B. × buttiana ‘Los Banos Beauty’, B. × buttiana ‘Barbara Karst’, B. × buttiana ‘San Diego Red’, and B. × buttiana ‘Miss Manila’ sp. 1, which was one bud mutation armed with simple or no thorns and derived from B. × buttiana ‘Miss Manila’; and three commonly used cultivars, namely, B. glabra ‘White Stripe’, B. spectabilis ‘Flame’ and B. spectabilis ‘Splendens’ (Fig 1). Then, we performed comparative genomics and phylogenomic analyses by integrating three published complete chloroplast genomes of Bougainvillea cultivars from the NCBI. In this study, five objectives were targeted: (1) to characterize and investigate the 13 newly sequenced complete chloroplast genome structures; (2) to detect variations in simple sequence repeats (SSRs), long repeats, and codon usage among these 13 chloroplast genomes; (3) to identify highly variable regions for potential DNA marker development among Bougainvillea cultivars; (4) to understand the molecular evolution of chloroplast genomes in the Nyctaginaceae family; and (5) to infer the phylogenetic relationships among Bougainvillea species and cultivars and other species of the Nyctaginaceae family.

Fig 1 — A, *Bougainvillea*×*buttiana* ‘Mahara’; B, B.×*buttiana* ‘Gautama’s Red’; C, B. ×*buttiana* ‘California Gold’; D, B.×*buttiana* ‘Double Salmon’; E, B.×*buttiana* ‘Double Yellow’; F, B. ×*buttiana* ‘Big Chitra’; G, B.×*buttiana* ‘Los Banos Beauty’; H, *Bougainvillea glabra ‘*White Stripe’; I, *Bougainvillea spectabilis* ‘Flame’; J, B. *spectabilis* ‘Splendens’; K, B. × *buttiana* ‘Barbara Karst’; L, B. × *buttiana* ‘San Diego Red’; M and N, B. × *buttiana* ‘Miss Manila’ sp. 1. armed with simple or no thorns, derived from B. × *buttiana* ‘Miss Manila’.

Materials and methods

Plant materials, DNA extraction, and sequencing

Fresh leaves of twelve Bougainvillea cultivars, namely, B.× buttiana ‘Mahara’, B. × buttiana ‘Gautama’s Red’, B. × buttiana ‘California Gold’, B. × buttiana ‘Double Salmon’, B. × buttiana ‘Double Yellow’, B. × buttiana ‘Big Chitra’, B. × buttiana ‘Los Banos Beauty’, B. glabra ‘White Stripe’, B. spectabilis ‘Flame’, B. spectabilis ‘Splendens’, B. × buttiana ‘Barbara Karst’, and B. × buttiana ‘San Diego Red’ (Fig 1, S1 Table), were collected from the Provincial Flower Germplasm Resources Bank of San Jiao Mei in Zhangzhou (117°37′47″E, 24°28′35″N), Fujian Province, China. One bud mutation armed with simple or no thorns and derived from B. × buttiana ‘Miss Manila’, given name, B. × buttiana ‘Miss Manila’ sp. 1 (Fig 1, S1 Table), was collected from the cultivation factoty of Zhangzhou (117°49′9″E, 24°31′33″N) in Xiamen Qianrihong Horticulture Co., Ltd, Fujian Province, China. Fresh leaves were quickly frozen on dry ice, sent to the laboratory of the Environmental Horticulture Research Institute (113°20′39″E, 23°8′51″N) at the Guangdong Academy of Agricultural Sciences, Guangzhou, China, and stored at −80° until use. Genomic chloroplast DNA was extracted from each sample using the modified sucrose gradient centrifugation method [16]. Then, the DNA quality and quantity were checked through agarose gel electrophoresis and the NanoDrop microspectrometer method, respectively. Each qualified DNA sample was sheared to fragments of approximately 350 bp. Short-insert (350 bp) paired-end libraries were constructed, and sequencing was performed on an Illumina NovaSeq 6000 platform with a paired read length of 150 bp (Biozeron, Shanghai, China). The raw data from each sample were checked using FastQC v. 0.11.9 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and adaptors and low-quality reads were subsequently deleted by Trimmomatic v. 0.39 [17] with default parameters. The remaining materials, including the leaves and DNA, were deposited in the laboratory of the Environmental Horticulture Research Institute (store sheet code: B2023, 113°20′39″E, 23°8′51″N), Guangdong Academy of Agricultural Sciences, Guangzhou, China, as vouchers (S1 Table).

Chloroplast genome assembly and annotation

At least 5.6 Gb of clean data were obtained from each sample (S1 Table). Chloroplast genome assembly and annotation were conducted using previously reported methods [18]. In brief, the clean paired-end reads were assembled using GetOrganelle v. 1.7.6.1 [19] with default parameters. The published complete chloroplast genomes of Bougainvillea peruviana (GenBank MT407463) and B. glabra (GenBank MN888961) were used as references for sequence correction by Geneious Prime 2022.10 [20]. Gene annotation was carried out using GeSeq [21] and the online Dual Organellar Genome Annotator (DOGMA) [22] with default parameters. The transfer RNA (tRNA) and ribosomal RNA (rRNA) sequences were confirmed by tRNAscanSE v. 2.0.5 [23] and BLAST v. 2.13.0 [24]. The annotated complete chloroplast genome sequences were first validated using online GB2sequin [25], then verified and formatted using Sequin v. 15.50 from NCBI and deposited in GenBank (accession numbers are shown in Table 1). Chloroplast genome maps were drawn using Organellar Genome Draw (OGDRAW) v. 1.3.1 [26].

Table 1. Characteristics of the 13 newly sequenced complete chloroplast genomes of Bougainvillea cultivars.

Cultivars	GenBank accession	Size (bp)	LSC (bp)	SSC (bp)	IR (bp)	GC content (%)					Number of genes (different)	Number of CDSs (different)	Number of tRNAs (different)	Number of rRNAs (different)
Cultivars	GenBank accession	Size (bp)	LSC (bp)	SSC (bp)	IR (bp)	Total	LSC	SSC	IR	CDS	Number of genes (different)	Number of CDSs (different)	Number of tRNAs (different)	Number of rRNAs (different)
Bougainvillea × buttiana ‘Mahara’	OR344376	154,541	85,694	18,077	25,385	36.46	34.17	29.47	42.81	37.21	131 (112)	86 (79)	37 (29)	8 (4)
*B. × buttiana* ‘Gautama’s Red’**	OR344371	154,465	85,563	18,050	25,426	36.49	34.19	29.53	42.85	37.85	131 (112)	86 (79)	37 (29)	8 (4)
*B. × buttiana* ‘California Gold’**	OR344368	154,542	85,695	18,077	25,385	36.46	34.17	29.47	42.81	37.19	131 (112)	86 (79)	37 (29)	8 (4)
*B. × buttiana* ‘Double Salmon’**	OR344375	154,542	85,695	18,077	25,385	36.46	34.17	29.47	42.81	37.18	131 (112)	86 (79)	37 (29)	8 (4)
*B. × buttiana* ‘Double Yellow’**	OR344373	154,542	85,695	18,077	25,385	36.46	34.17	29.47	42.81	37.18	131 (112)	86 (79)	37 (29)	8 (4)
*B. × buttiana* ‘Big Chitra’**	OR344367	154,542	85,695	18,077	25,385	36.46	34.17	29.47	42.81	37.18	131 (112)	86 (79)	37 (29)	8 (4)
*B. × buttiana* ‘Los Banos Beauty’**	OR344374	154,542	85,695	18,077	25,385	36.46	34.17	29.47	42.81	37.18	131 (112)	86 (79)	37 (29)	8 (4)
*B. glabra ‘White Stripe’*	OR344370	154,520	85,688	18,078	25,377	36.46	34.18	29.47	42.81	37.18	131 (112)	86 (79)	37 (29)	8 (4)
*B. spectabilis* ‘Flame’**	OR344366	153,994	85,169	18,043	25,391	36.55	34.30	29.53	42.83	37.16	131 (112)	86 (79)	37 (29)	8 (4)
*B. spectabilis* ‘Splendens’**	OR344372	154,520	85,688	18,078	25,377	36.46	34.18	29.47	42.81	37.18	131 (112)	86 (79)	37 (29)	8 (4)
*× buttiana* ‘Barbara Karst’**	OR344369	158,231	85,688	21,789	25,377	36.34	34.18	29.81	42.81	37.18	131 (112)	86 (79)	37 (29)	8 (4)
*B. × buttiana* ‘San Diego Red’**	OR344377	154,542	85,695	18,077	25,385	36.46	34.17	29.47	42.81	37.18	131 (112)	86 (79)	37 (29)	8 (4)
*B. × buttiana* ‘Miss Manila’ sp. 1**	OR344378	154,520	85,688	18,078	25,377	36.46	34.18	29.47	42.81	37.18	131 (112)	86 (79)	37 (29)	8 (4)

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Note: CDS, protein-coding gene; GC, guanine-cytosine; LSC, large single-copy region; SSC, small single-copy region; IR, inverted repeat.

Analyses of SSRs and long repeats

MIcroSAtellite (MISA) was used to identify simple sequence repeats (SSRs) in the thirteen newly sequenced Bougainvillea chloroplast genomes [27]. The parameters for di-, tri-, tetra-, penta-, and hexa-nucleotide SSRs and the minimum number of repeats were set to 10, 5, 4, 3, 3, and 3, respectively.

REPuter software [28] was used to identify and analyze the sizes and positions of long repeats, including forward, palindrome, reverse and complement repeat units, within the thirteen newly sequenced Bougainvillea chloroplast genomes. Long repeats were detected with a minimum repeat size of 30 bp, a Hamming distance of 3, and a repeat identity of more than 90%.

Analysis of codon usage

The relative synonymous codon usage (RSCU) and amino acid frequencies of the thirteen newly sequenced Bougainvillea chloroplast genomes were analyzed using MEGA v. 7.0 [29] with default parameters. A clustered heatmap of the RSCU values of the thirteen newly sequenced Bougainvillea chloroplast genomes was constructed with R v. 4.0.2 (https://www.R-project.org) (accessed on 10 August 2023).

Comparative genomics and sequence divergence analyses

For comparison, 13 newly sequenced Bougainvillea chloroplast genomes were obtained using the CGView server [30]. GC contents were detected based on GC skew using the equation: GC skew = (G − C)/(G + C). To further evaluate the variations among these 13 complete genomes of Bougainvillea, first, single-nucleotide polymorphisms (SNPs) and insertions/deletions (indels) were also identified and located using MUMmmer 4 [31] and Geneious Prime 2022.10 [32], using the annotated B. glabra ‘White Stripe’ as the reference; second, except B. glabra ‘White Stripe’, the rest 12 Bougainvillea cultivar chloroplast genomes were compared and analyzed to identify SNPs and indels using the annotated chloroplast genome of B. × buttiana ‘Mahara’ as the reference; third, to identify SNPs and indels between two complete chloroplast genomes of B. spectabilis ‘Splendens’, of which one was sequenced in this study and the other one was reported in a previous study [13], the reported one (OR253994) was used as the reference.

The mVISTA program in the Shuffle-LAGAN mode [33] and sliding window analysis using DnaSP v. 6.12.03 [34] were also employed to compare the complete chloroplast genome divergence among Bougainvillea cultivars. In total, 16 complete chloroplast genomes of Bougainvillea cultivars were analyzed, including 13 newly sequenced chloroplast genomes and 3 from the GenBank database (GenBank numbers MW557548, MW557549, and MW557550). The chloroplast genome of B. × buttiana ‘Mahara’ was used as the reference. Among these 16 chloroplast genomes of Bougainvillea, the LSC/IR and SSC/IR boundaries and their adjacent genes were also analyzed using IRscope [35].

Selection pressure analysis in the Nyctaginaceae family

Selection pressure was applied following a previously described method [18]. In short, to detect positively selected amino acid sites among 46 complete chloroplast genomes of the Nyctaginaceae family (S2 Table), the nonsynonymous (dN) and synonymous (dS) substitution rates of consensus protein-coding genes were calculated by using the CodeML program implemented in EasyCodeML [36]. Gene selective pressure analysis was based on 79 consensus protein-coding gene sequences after removing all stop codons. The positive selection model of M8 (β & ω > 1) was used to detect positively selected sites based on both the dN and dS ratios (ω) and likelihood ratio test (LRT) values [37]. The Bayesian empirical Bayes (BEB) method was used to identify the codons most likely under positive selection, with posterior probabilities higher than 0.95 and 0.99 indicating sites under positive selection and strong positive selection, respectively [38].

Phylogenetic relationships in the Bougainvillea genus and the Nyctaginaceae family

To reconstruct the phylogenetic relationships of the Nyctaginaceae family, 46 chloroplast genomes, including the 13 genomes generated in this study and 33 genomes downloaded from the GenBank database, were analyzed (S2 Table). Seguieria aculeata (NC_041418), Rivina humilis (NC_041300), Petiveria alliacea (NC_041417), and Monococcus echinophorus (NC_041414) were used as outgroups. Chloroplast genome sequences and protein-coding sequences were aligned using MAFFT v. 7.458 [39] with default parameters. Phylogenetic trees were constructed using the maximum likelihood (ML) and Bayesian inference (BI) methods. The best nucleotide substitution model (GTR + G + I) was determined using the Akaike information criterion (AIC) in jModelTest v. 2.1.10 [40]. ML analysis was conducted in PhyML v. 3.0 [41] with 1000 bootstrap replicates. BI analysis was performed in MrBayes v. 3.2.6 [42]. Two Markov chain Monte Carlo (MCMC) algorithm runs were conducted simultaneously with 200,000 generations and four Markov chains, starting from random trees, sampling trees every 100 generations, and discarding the first 10% of samples as burn-in. The phylogenetic trees were edited and visualized using iTOL v. 3.4.3 (http://itol.embl.de/itol.cgi) (accessed on 15 September 2023).

Results

General characteristics of the thirteen complete chloroplast genomes

In this study, the 13 newly sequenced chloroplast genomes of Bougainvillea cultivars exhibited a typical quadripartite structure containing one large single-copy (LSC), one small single-copy (SSC) and two inverted-repeat regions (IRa and IRb) according to the OGDRAW and CGView tools (Fig 2, Table 1). The sizes of these 13 Bougainvillea chloroplast genomes ranged from 153,994 bp (B. spectabilis ‘Flame’) to 158,231 bp (B. × buttiana ‘Barbara Karst’) (Table 1). Among these 13 chloroplast genomes, four junction regions were identified, namely, one LSC region of 85,169–85,695 bp, one SSC region of 18,050–21,789 bp, and a pair of IR regions (IRa and IRb) of 25,377–25,426 bp each (Fig 2, Table 1). The GC contents of these 13 chloroplast genomes varied from 36.34% to 36.55% (Table 1). The IR region had the highest GC content (42.81–42.85%), followed by the LSC region (34.17–34.30%), while the SSC region had the lowest GC content (29.47–29.81%) (Table 1). The GC content of the protein-coding regions varied from 37.16% to 37.85%. All 13 Bougainvillea chloroplast genomes were submitted to the GenBank database (accession numbers OR344366–OR344378) (Table 1).

Fig 2 — Genes shown on the outside of the outermost first ring are transcribed counter-clockwise, and those on the inside are transcribed clockwise. The second ring with the darker gray color corresponds to the GC content, whereas the third ring with the lighter gray color corresponds to the AT content of the B. *× buttiana* ‘Mahara’ chloroplast genome generated by OGDRAW. The gray arrowheads indicate the directions of the genes. LSC, large single -copy region; IR, inverted repeat; SSC, small single-copy region. The innermost first black ring indicates the chloroplast genome size of B. *× buttiana* ‘Mahara’. The innermost second and third rings indicate deviations in the GC content and GC skew, respectively, in the chloroplast genome of C. *barbatus*: GC skew + indicates G > C, and GC skew − indicates G < C. CGView comparison of the 13 complete chloroplast genomes of *Bougainvillea* cultivars displayed from the innermost 4th colored ring to the outer 16th ring: B. *× buttiana* ‘Mahara’, B. *× buttiana* ‘Gautama’s Red’, B. *× buttiana* ‘California Gold’, B. *× buttiana* ‘Double Salmon’, B. *× buttiana* ‘Double Yellow’, B. *× buttiana* ‘Big Chitra’, B. *× buttiana* ‘Los Banos Beauty’, B. *glabra ‘*White Stripe’, B. *spectabilis* ‘Flame’, B. *spectabilis* ‘Splendens’, B.× buttiana ‘Barbara Karst’, B.× buttiana ‘San Diego Red’, and B. × *buttiana* ‘Miss Manila’ sp. 1, respectively. Chloroplast genome similar and highly divergent locations are represented by continuous and interrupted track lines, respectively.

Among these 13 Bougainvillea chloroplast genomes, each contained 131 annotated functional genes, which consisted of 86 protein-coding genes, 37 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes (Tables 1, 2 and S3). Among these genes, a total of 112 different genes were found in these 13 genomes, including 79 protein-coding genes, 29 tRNA genes, and 4 rRNA genes (Tables 1, 2 and S3). Overall, 17 genes contained introns in each of these 13 genomes. Fifteen genes (atpF, ndhA, ndhB, petB, petD, rpl16, rpoC1, rps12, rps16, trnA-UGC, trnG-UCC, trnI-GAU, trnK-UUU, trnL-UAA, and trnV-UAC) contained one intron, while clpP and ycf3 each contained two introns (Tables 2 and S3). Among the 17 intron-containing genes in these 13 genomes, three genes (ndhB, trnA-UGC and trnI-GAU) occurred in both IRs; 12 genes (atpF, clpP, petB, petD, rpl16, rpoC1, rps16, trnG-UCC, trnK-UUU, trnL-UAA, trnV-UAC and ycf3) were distributed in the LSC; one gene (ndhA) was in the SSC; and one gene (rps12) in the first exon was located in the LSC, with the other two exons in both IRs (S3 Table).

Table 2. Genes present in the 13 newly sequenced chloroplast genomes of Bougainvillea cultivars.

Gene category	Gene group	Gene names
Self-replication	DNA-dependent RNA polymerase	rpoA, rpoB, rpoC1, rpoC2*
	Large subunit of ribosomal proteins	rpl2 (×2), rpl14, rpl16, rpl20, rpl22, rpl23* (×2), rpl32, rpl33, rpl36
	Small subunit of ribosomal proteins	rps2, rps3, rps4, rps7 (×2), rps8, rps11, rps12 (×2), rps14, rps15, rps16*, rps18, rps19*
RNA genes	Ribosomal RNA	rrn4.5 (×2), rrn5 (×2), rrn16 (×2), rrn23 (×2)
RNA genes	Transfer RNA	trnA-UGC (×2), trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnfM-CAU, trnG-GCC, trnG-UCC*, trnH-GUG, trnI-GAU* (×2), trnK-UUU*, trnL-CAA* (×2), trnL-UAA, trnL-UAG, trnM-CAU* (×3), trnN-GUU (×2), trnP-UGG, trnQ-UUG, trnR-ACG (×2), trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU, trnT-UGU, trnV-GAC (×2), trnV-UAC, trnW-CCA, trnY-GUA*
Photosynthesis related genes	Subunits of photosystem Ⅰ	psaA, psaB, psaC, psaI, psaJ
	Subunits of photosystem Ⅱ	psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ, infA
	Subunits of cytochrome b/f complex	petA, petB, petD*, petG, petL, petN*
	Subunits of ATP synthase	atpA, atpB, atpE, atpF, atpH, atpI*
	Subunits of NADH dehydrogenase	ndhA, ndhB* (×2), ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK*
	Subunit of rubisco	rbcL
Other genes	Subunit of acetyl-CoA-carboxylase	accD
	c-type cytochrome synthesis gene	ccsA
	Envelope membrane protein	cemA
	Protease	clpP**
	Maturase	matK
Genes of unknown function	Conserved open reading frames	ycf1 (×2), ycf2 (×2), ycf3*, ycf4*

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Note: ×2: Gene with two copies; ×3: Gene with three copies; *: Gene containing only one intron; **: Gene containing two introns.

SSRs and long repeats analyses

In the present study, the number of detected SSRs ranged from 86 (B. spectabilis ‘Flame’) to 98 (B. × buttiana ‘Barbara Karst’) among these 13 genomes (Fig 3A). Five types of SSRs were identified, including mononucleotide, dinucleotide, trinucleotide, tetranucleotide, and pentanucleotide (Fig 3A, S4 Table). There were no hexanucleotides in any of the 13 sequenced genomes (Fig 3A). Among these 13 genomes, most SSRs were located in the LSC regions (67–76 loci) rather than in the SSC regions (13–14 loci) and IR regions (3 loci) (Fig 3B, S4 Table). Among each sequenced chloroplast genome, mononucleotide repeats were the most frequent, with numbers ranging from 61 to 72, followed by tetranucleotides ranging from 10 to 11, dinucleotides ranging from 6 to 9, trinucleotides ranging from 3 to 7, and pentanucleotides ranging from 1 to 2 (Fig 3C, S4 Table). Most of the mononucleotide SSRs were A/T repeats, which accounted for 68.18–75.58% of all SSRs among these 13 chloroplast genomes (Fig 3C, S4 Table). Among dinucleotide repeats, AT/AT repeats were observed most frequently, accounting for 6.98–10.22% of all SSRs (Fig 3C, S4 Table). In the trinucleotide category, AAT/ATT repeats were the most abundant type, accounting for 6.82–7.37% of all SSRs (Fig 3C, S4 Table).

Additionally, four different types of long repeats, including forward, complement, reverse, and palindromic repeats, were detected among these 13 chloroplast genomes. The total number of long repeats ranged from 48 to 65 (Fig 4A, S5 Table). The number of forward repeats varied from 19 to 34, the number of palindromic repeats varied from 27 to 29, and the number of reverse repeats varied from 2 to 4 (Fig 4A, S5 Table). There were no complement repeats in these 13 chloroplast genomes. The length of long repeats varied among these 13 chloroplast genomes (Fig 4B, S5 Table). Long repeats of 30–34 bp were found to be the most common, and those with lengths of 35–39 bp and 40–44 bp were the second and third most common, respectively (Fig 4B, S5 Table). These results indicated that the number, length and distribution of long repeats varied among these 13 chloroplast genomes in this study.

Fig 4 — (A) Total numbers of four long repeat types. (B) Length distribution of long repeats in each sequenced chloroplast genome.

Codon usage analysis

In this study, the codon usage, amino acid frequency, and relative synonymous codon usage (RSCU) of the 13 chloroplast genomes of Bougainvillea were analyzed (Fig 5). Methionine (Met) and tryptophan (Trp) are each encoded by one codon, showing no codon bias, with RSCU values of 1.00, while the others are encoded by multiple synonymous codons, ranging from two to six (Fig 5A). The codons with the four lowest RSCU values (AGC, CGC, CTG and GAC) and four with the highest RSCU values (TTA, TCT, GCT and AGA) were identified in these 13 chloroplast genomes of Bougainvillea (Fig 5B). With the exception of Met and Trp, codon usage bias was detected for 29 codons with RSCU > 1.00 in the genes of these 13 chloroplast genomes (S6 Table). Interestingly, of the 29 codons, 28 were A/T-ending codons. Therefore, our RSCU results indicated that all 13 chloroplast genomes of Bougainvillea had a greater frequency of A/T-ending than G/C-ending codons.

Fig 5 — (A) Codon content and codon usage of the 20 amino acids and stop codons of all protein-coding genes. Each histogram from left to right is shown for B. *× buttiana* ‘Mahara’, B. *× buttiana* ‘Gautama’s Red’, B. *× buttiana* ‘California Gold’, B. *× buttiana* ‘Double Salmon’, B. *× buttiana* ‘Double Yellow’, B. *× buttiana* ‘Big Chitra’, B. *× buttiana* ‘Los Banos Beauty’, B. *glabra ‘*White Stripe’, B. *spectabilis* ‘Flame’, B. *spectabilis* ‘Splendens’, B.× buttiana ‘Barbara Karst’, B.× buttiana ‘San Diego Red’, and B. × *buttiana* ‘Miss Manila’ sp. 1, respectively. (B) Heatmap analysis of the codon distribution of all protein-coding genes in the 13 newly sequenced chloroplast genomes.

SNPs and indels analyses among the thirteen complete chloroplast genomes

Using the chloroplast genome of B. glabra ‘White Stripe’ as the reference, SNP/indel loci of the other 12 chloroplast genomes of Bougainvillea were detected (S1 Fig, Tables 3 and S7). Three comparisons, B. glabra ‘White Stripe’ vs. B. spectabilis ‘Splendens’, B. glabra ‘White Stripe’ vs. B. × buttiana ‘Barbara Karst’, and B. glabra ‘White Stripe’ vs. B. × buttiana ‘Miss Manila’ sp. 1, had no SNPs/indels. Five comparisons, B. glabra ‘White Stripe’ vs. B. × buttiana ‘California Gold’, B. glabra ‘White Stripe’ vs. B. × buttiana ‘Double Salmon’, B. glabra ‘White Stripe’ vs. B. × buttiana ‘Double Yellow’, B. glabra ‘White Stripe’ vs. B. × buttiana ‘Los Banos Beauty’, and B. glabra ‘White Stripe’ vs. B. × buttiana ‘San Diego Red’, identified the same numbers of SNPs and indels, with 1 protein-coding gene SNP, 4 intergenic SNPs, and 3 indels (S1 Fig, Tables 3 and S7). Two comparisons revealed slightly more SNPs and indels than did the other five comparisons. Regarding B. glabra ‘White Stripe’ vs. B. × buttiana ‘Big Chitra’, 1 protein-coding gene SNP, 6 intergenic SNPs, and 3 indels were identified; for B. glabra ‘White Stripe’ vs. B. × buttiana ‘Mahara’, 1 protein-coding gene SNP, 8 intergenic SNPs, and 4 indels were identified (S1 Fig, Tables 3 and S7). Between B. glabra ‘White Stripe’ and B. × buttiana ‘Gautama’s Red’, 287 protein-coding gene SNPs, 516 intergenic SNPs, and 125 indels were detected (S1 Fig, Tables 3 and S7). With respect to B. glabra ‘White Stripe’ vs. B. spectabilis ‘Flame’, 279 protein-coding gene SNPs, 509 intergenic SNPs, and 130 indels were found (S1 Fig, Tables 3 and S7).

Table 3. Distribution of SNPs and indels among the 13 newly sequenced complete chloroplast genomes of Bougainvillea cultivars.

Comparison pairs	Insertions	Deletions	Indels	Protein- coding genes SNPs	Intergenic regions SNPs	Total SNPs
‘White Stripe’ vs. ‘Big Chitra’	1	2	3	1	6	7
‘White Stripe’ vs. ‘California Gold’	1	2	3	1	4	5
‘White Stripe’ vs. ‘Double Salmon’	1	2	3	1	4	5
‘White Stripe’ vs. ‘Double Yellow’	1	2	3	1	4	5
‘White Stripe’ vs. ‘Gautama’s Red’	49	76	125	287	516	803
‘White Stripe’ vs. ‘Los Banos Beauty’	1	2	3	1	4	5
‘White Stripe’ vs. ‘Mahara’	1	3	4	1	8	9
‘White Stripe’ vs. ‘San Diego Red’	1	2	3	1	4	5
‘White Stripe’ vs. ‘Flame’	54	76	130	279	509	788
‘Mahara’ vs. ‘Gautama’s Red’	50	74	124	284	492	776
‘Mahara’ vs. ‘Big Chitra’	0	0	0	0	2	2
‘Mahara’ vs. ‘California Gold’	0	0	0	0	2	2
‘Mahara’ vs. ‘Double Salmon’	0	0	0	0	2	2
‘Mahara’ vs. ‘Double Yellow’	0	0	0	0	2	2
‘Mahara’ vs. ‘Los Banos Beauty’	0	0	0	0	2	2
‘Mahara’ vs. ‘Barbara Karst’	4	1	5	1	6	7
‘Mahara’ vs. ‘San Diego Red’	0	0	0	0	2	2
‘Mahara’ vs. ‘Miss Manila’ sp. 1	3	1	4	1	6	7
‘Mahara’ vs. ‘Flame’	53	74	127	278	496	774
‘Mahara’ vs. ‘Splendens’	3	1	4	1	6	7

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Note: ‘White Stripe’, ‘Big Chitra’, ‘California Gold’, ‘Double Salmon’, ‘Double Yellow’, ‘Gautama’s Red’, ‘Los Banos Beauty’, ‘Mahara’, ‘Barbara Karst’,‘San Diego Red’, ‘Flame’, ‘Splendens’ and ‘Miss Manila’ sp. 1 represent B. glabra ‘White Stripe’, B.× buttiana ‘Big Chitra’, B.× buttiana ‘California Gold’, B.× buttiana ‘Double Salmon’, B.× buttiana ‘Double Yellow’, B.× buttiana ‘Gautama’s Red’, B. × buttiana ‘Los Banos Beauty’, B.× buttiana ‘Mahara’, B.× buttiana ‘Barbara Karst’, B.× buttiana ‘San Diego Red’, B. spectabilis ‘Flame’, B. spectabilis ‘Splendens’ and B.× buttiana ‘Miss Manila’ sp. 1, respectively.

Except B. glabra ‘White Stripe’, the rest 12 Bougainvillea cultivar chloroplast genomes were also compared using the chloroplast genome of B. × buttiana ‘Mahara’ as the reference. Concerning B. × buttiana ‘Mahara’ vs. B. × buttiana ‘Gautama’s Red’, 284 protein-coding gene SNPs, 492 intergenic SNPs, and 124 indels were found (S1 Fig, Tables 3 and S7). Six comparisons, B. × buttiana ‘Mahara’ vs. B. × buttiana ‘Big Chitra’, B. × buttiana ‘Mahara’ vs. B. × buttiana ‘California Gold’, B. × buttiana ‘Mahara’ vs. B. × buttiana ‘Double Salmon’, B. × buttiana ‘Mahara’ vs. B. × buttiana ‘Double Yellow’, B. × buttiana ‘Mahara’ vs. B. × buttiana ‘Los Banos Beauty’, and B. × buttiana ‘Mahara’ vs. B.× buttiana ‘San Diego Red’ had no indels (S1 Fig, Tables 3 and S7). However, these six comparisons had the same SNPs, with 2 intergenic SNPs each (S1 Fig, Tables 3 and S7). Interestingly, these 2 SNPs were both in trnS-GCU_trnG-UCC-exon1 (S7 Table), suggesting that trnS-GCU_trnG-UCC-exon1 can be used to identify these 7 B. × buttiana cultivars. Three comparisons, B. × buttiana ‘Mahara’ vs. B. × buttiana ‘Barbara Karst’, B. × buttiana ‘Mahara’ vs. B. × buttiana ‘Miss Manila’ sp. 1, and B. × buttiana ‘Mahara’ vs. B. spectabilis ‘Splendens’, had the same numbers of SNPs, with 1 protein-coding gene SNP and 6 intergenic SNPs. But these three comparisons identified different numbers of indels, with 5, 4, and 4 indels, respectively (S1 Fig, Tables 3 and S7). With respect to B. × buttiana ‘Mahara’ vs. B. spectabilis ‘Flame’, 278 protein-coding gene SNPs, 496 intergenic SNPs, and 127 indels were identified (S1 Fig, Tables 3 and S7).

Intraspecific analyses of two chloroplast genomes of B. spectabilis ‘Splendens’

The two chloroplast genomes from B. spectabilis ‘Splendens’ were found to show a 349 bp difference in length (OR344372 in Table 1 and OR253994 in [13]). With the total length difference, SNPs and indels were identified between the two complete chloroplast genomes of B. spectabilis ‘Splendens’. Through intraspecific comparison, a total of 119 indels were identified between the two B. spectabilis ‘Splendens’ accessions (S8 Table). There were 55 insertions and 64 deletions between them (S8 Table). Among them, cemA, rpl23, ycf1 and ycf2 exhibited the same number indels, each of which showed 2 indels. There were 504 SNPs identified in the two complete chloroplast genomes of B. spectabilis ‘Splendens’ (S8 Table). The most frequently occurring mutations were G/T substitutions (72 times), followed by T/G (60 times), C/A (59 times), and T/C (57 times), respectively. Among these SNPs, ycf1 contained the highest number of SNPs (36), followed by rpoC2 and ndhF, which showed 11 and 9 SNPs, respectively (S8 Table).

IR expansion and contraction

Comparisons of the LSC/IR and SSC/IR boundaries among these 13 chloroplast genomes and 3 published chloroplast genomes of Bougainvillea cultivars were performed (Fig 6). Regarding the LSC/IRb borders, the rps19 gene was located at the boundaries of the LSC/IRb borders in all 16 Bougainvillea chloroplast genomes. The rps19 gene expanded into the IRb region with a distance of 114 bp in all 16 Bougainvillea chloroplast genomes (Fig 6). Regarding the IRa/LSC borders, the rpl2 and trnH-GUG genes were located at the boundaries of the IRa/LSC borders in all 16 Bougainvillea chloroplast genomes. The distances between the ends of the rpl2 and IRa/LSC borders ranged from 176 bp to 177 bp (Fig 6). The distances between the ends of the trnH-GUG and IRa/LSC borders ranged from 18 bp to 23 bp (Fig 6).

Fig 6 — The 13 newly sequenced *Bougainvillea* chloroplast genomes identified in this study are shown in blue.

The SSC/IRa border was located in the ycf1 region, which crossed into the IRa region in all 16 Bougainvillea chloroplast genomes, with distances ranging from 1332 bp to 1371 bp (Fig 6). For the IRb/SSC borders, ycf1 expanded into the SSC regions in all 16 Bougainvillea chloroplast genomes and overlapped with the ndhF gene. A total distance of 2 or 3 bp was detected between the end of ycf1 and the IRb/SSC border, and a 21 bp distance was detected between the start of ndhF and the IRb/SSC border (Fig 6). Overall, the LSC/IR boundary regions of the 16 Bougainvillea chloroplast genomes were highly conserved, but the SSC/IR boundary regions exhibited slight variations.

Sequence divergence analysis

Multiple alignments of these 13 sequenced Bougainvillea chloroplast genomes were first compared by using CGView with the annotated genome sequence of B. × buttiana ‘Mahara’ as the reference (Fig 2). The CGView results indicated that no significant rearrangements were observed among these 13 chloroplast genomes, but several regions showed more divergence than others (the innermost 4th color ring to the outer 14th ring in Fig 2). Specifically, trnT-GGU_psbD and trnT-GGU_trnE-UUC in the LSC region were highly divergent.

To further detect sequence divergence in the chloroplast genomes of Bougainvillea cultivars, highly divergent regions in the 13 sequenced genomes in this study and 3 from the GenBank database were analyzed using mVISTA and DnaSP, with the annotated genome sequence of B. × buttiana ‘Mahara’ used as the reference (Fig 7). The mVISTA results showed that the LSC and SSC regions were more divergent than the two IR regions and that a greater divergence was found in the non-coding regions than in the coding regions (Fig 7). The main divergences for the coding regions were psbJ, psaI, and ycf1. For the non-coding regions, the strongly divergent regions were trnH-GUG_psbA, psbI_trnS-GCU, trnS-GCU_trnG-UCC, and ccsA_ndhD (Fig 7). For nucleotide diversity (Pi) values, the Pi values for the protein-coding regions ranged from 0 to 0.00990, with an average value of 0.00078 (S9 Table). Of these protein-coding regions, 7 regions (psaI, psbJ, petG, clpP-exon1, rps19, ndhF, and ycf1) exhibited remarkably high values (Pi > 0.0038; Fig 8A). For the intron and intergenic regions, the Pi values ranged from 0 to 0.02047, with an average of 0.00298 (S9 Table). Among these intron and intergenic regions, the 8 most divergent regions, trnH-GUG_psbA, psbI_trnS-GCU, trnS-GCU_trnG-UCC-exon1, trnR-UCU_atpA, trnS-GGA_rps4, petD-exon2_rpoA, ccsA_ndhD, and ndhI_ndhA-exon2, with Pi values ranging from 0.01130 to 0.02047, were identified (Fig 8B). Additionally, using a region length ≥ 200 bp and a Pi value ≥ 01130 for the selection of potential molecular markers, 4 regions were obtained: trnH-GUG_psbA, trnS-GCU_trnG-UCC-exon1, trnS-GGA_rps4, and ccsA_ndhD (S9 Table).

Fig 7 — The gray arrows and thick black lines above the alignment indicate gene orientation. Purple bars represent exons, sky-blue bars represent untranslated regions (UTRs), red bars represent non-coding sequences (CNS), gray bars represent mRNAs, and white regions represent sequence differences among the analyzed chloroplast genomes. The y-axis represents the identity percentage ranging from 50% to 100%. The 13 sequenced *Bougainvillea* chloroplast genomes in this study are shown in bold.

Fig 8 — (A) Protein-coding genes. Protein-coding genes with Pi values > 0.0038 are labeled with gene names. (B) Intergenic regions. Intergenic regions with Pi values > 0.0113 are labeled with intergenic region names.

Selection pressure analysis of the Nyctaginaceae family

The ratios (ω) of non-synonymousnonsynonymous (dN) to synonymous (dS) substitutions (dN/dS) for all 79 shared protein-coding genes were analyzed across 46 complete chloroplast genomes in the Nyctaginaceae family. These 46 genomes belonged to 9 genera of Nyctaginaceae, namely, Bougainvillea, Belemia, Mirabilis, Nyctaginia, Boerhavia, Acleisanthes, Pisonia, Guapira and Salpianthus. According to the M8 (β & ω > 1) model, a total of 9 protein-coding genes were under positive selection with a posterior probability greater than 0.95 according to the Bayes empirical Bayes (BEB) method (Table 4). Among these genes, rps12 harbored the greatest number of positive amino acid sites (33), followed by rbc L (7), ndhF (6), ycf2 (3), rpoB (2), rpoC2 (2), ndhI (1), psbT (1), and ycf3 (1) (Table 4).

Table 4. Positively selected sites detected in 46 complete chloroplast genomes of the Nyctaginaceae family.

Gene	Positively selected sites (* p > 95%; ** p > 99%)
*ndhF*	462 L 0.969, 502 A 0.960, 508 T 0.955, 518 F 0.958, 573 L 0.980, 576 Y 0.996*
*ndhI*	166 E 0.999**
*psbT*	34 M 0.982*
*rbcL*	23 T 0.994, 28 N 1.000, 32 Q 0.959, 225 L 0.998, 359 N 0.994, 439 R 0.995, 477 K 1.000*
*rpoB*	88 Q 0.990, 363 W 0.951
*rpoC2*	556 L 0.992, 706 Q 0.991
*rps12*	1 M 1.000, 2 P 1.000, 3 T 1.000, 4 N 1.000, 5 T 1.000, 6 R 1.000, 7 Q 1.000, 8 P 1.000, 9 I 1.000, 10 K 1.000, 11 N 1.000, 12 V 1.000, 13 T 1.000, 14 K 1.000, 15 S 1.000, 16 P 1.000, 17 A 1.000, 18 L 1.000, 19 R 1.000, 20 G 1.000, 21 C 1.000, 22 P 1.000, 23 Q 1.000, 24 R 1.000, 25 R 1.000, 26 G 1.000, 27 T 1.000, 28 C 1.000, 29 T 1.000, 30 R 1.000, 31 V 1.000, 32 Y 1.000, 110 K 0.997**
*ycf2*	531 E 1.000, 534 Y 0.999, 1548 Q 0.999**
*ycf3*	116 Q 0.997**

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Note: Each gene was assumed to have 95 degrees of freedom.

Phylogenetic relationships

Four phylogenetic trees were constructed using chloroplast genome sequences and protein-coding genes via the ML and BI methods, respectively (Figs 9 and S2). Four species of Petiveriaceae were used as outgroups. The ML and BI trees from complete chloroplast genomes and protein-coding genes showed similar topological structures within 9 genera of Nyctaginaceae and differed in support values and positions among several Bougainvillea cultivars (Figs 9 and S2). The 9 genera within Nyctaginaceae, Bougainvillea, Belemia, Mirabilis, Nyctaginia, Boerhavia, Acleisanthes, Pisonia, Guapira and Salpianthus, were strongly supported based on complete chloroplast genomes and protein-coding genes (bootstrap values, BS = 98–100% for the ML trees and posterior probabilities, PP = 1 for the BI trees) (Figs 9 and S2).

Fig 9 — (A) ML tree. (B) BI tree. The 13 newly sequenced *Bougainvillea* chloroplast genomes identified in this study are shown in bold.

Within Nyctaginaceae, Bougainvillea was strongly supported as a sister to Belemia (BS = 98–99% for the ML trees and PP = 1 for the BI trees) (Figs 9 and S2). The 35 Bougainvillea individuals analyzed were divided into four clades, namely, Clades Ⅰ, Ⅱ, Ⅲ, and Ⅳ, with strongly supported values (BS = 85–100% for the ML trees and PP = 0.99–1 for the BI trees) (Figs 9 and S2). Two cultivars, B. × buttiana ‘Gautama’s Red’ and B. spectabilis ‘Flame’, were clustered into clade Ⅰ, and the other 11 cultivars, including B. × buttiana ‘Mahara’, B. × buttiana ‘California Gold’, B. × buttiana ‘Double Salmon’, B. × buttiana ‘Double Yellow’, B. × buttiana ‘Los Banos Beauty’, B. × buttiana ‘Big Chitra’, B. × buttiana ‘Barbara Karst’, B. glabra ‘White Stripe’, B. spectabilis ‘Splendens’, B. × buttiana ‘San Diego Red’, and B. × buttiana ‘Miss Manila’ sp. 1, were clustered into Clade Ⅳ (Figs 9 and S2). In Clade Ⅰ, B. spectabilis ‘Flame’ was sister to B. peruviana MW123901 and then formed a strong sister cluster to B. pachyphylla, both based on chloroplast genome sequences and protein-coding genes (Figs 9 and S2). However, the position of B. × buttiana ‘Gautama’s Red’ in the ML tree constructed from chloroplast genome sequences differed from those in the other three phylogenetic trees in this study. For the former, B. × buttiana ‘Gautama’s Red’ was sister to B. spectabilis ‘Pixie Pink’ and then clustered with B. peruviana MT407463 with strong support (BS = 90%) (Fig 9A). For the latter, B. spectabilis ‘Pixie Pink’ was sister to B. peruviana MT407463 and then clustered with B. × buttiana ‘Gautama’s Red’ with strong support (BS = 93–100%, and PP = 0.93–1) (Figs 9 and S2). In Clade Ⅱ, it only contained B. spinosa (Figs 9 and S2). In Clade Ⅲ, there were 7 individuals of wild species, including B. campanulata, B. berberidifolia, B. infesta, B. modesta OM44398, B. modesta OM044396, B. stipitata, and B. stipitata var. grisebachiana (Figs 9 and S2). In Clade Ⅳ, in the ML tree based on the chloroplast genome sequences, B. × buttiana ‘Mahara’, B. × buttiana ‘California Gold’, B. × buttiana ‘Double Salmon’, B. × buttiana ‘Double Yellow’, B. × buttiana ‘Los Banos Beauty’, B. × buttiana ‘Big Chitra’, B. × buttiana ‘San Diego Red’, B. spectabilis ‘Ratana Red’, B. glabra MN888961, and B. peruviana ‘Mona Lisa Red’ were clustered together in one cluster with strong support (BS = 88–92%). B. × buttiana ‘Barbara Karst’, B. glabra ‘White Stripe’, B. spectabilis ‘Splendens’, B. × buttiana ‘Miss Manila’ sp. 1, B. spectabilis MN315508, B. spectabilis China MW167297, and B. hybrid cultivar MW123903 were clustered together in another cluster with strong support (BS = 88–95%) (Fig 9A). However, in the ML tree based on protein-coding genes, B. × buttiana ‘Mahara’ was sister to the other cultivars in Clade Ⅳ with strong support (BS = 100%) (S2A Fig). In both BI trees, B. × buttiana ‘Mahara’, B. × buttiana ‘California Gold’, B. × buttiana ‘Double Salmon’, B. × buttiana ‘Double Yellow’, B. × buttiana ‘Los Banos Beauty’, B. × buttiana ‘Big Chitra’, B. × buttiana ‘San Diego Red’, B. spectabilis ‘Ratana Red’, B. glabra, B. peruviana ‘Mona Lisa Red’, B. × buttiana ‘Barbara Karst’, B. glabra ‘White Stripe’, B. spectabilis ‘Splendens’, B. × buttiana ‘Miss Manila’ sp. 1, B. spectabilis MN315508, B. spectabilis MW167297, and B. hybrid cultivar MW123903 were clustered together in one cluster in Clade Ⅳ with moderate to strong support (PP = 0.84–1) (Figs 9B and S2B Fig). In the four phylogenetic trees, Clades Ⅲ and Ⅳ were clustered together, forming a cluster with strong support (BS = 85–99%, and PP = 0.99–1); and then the cluster, Clade Ⅱ, and Clade Ⅰ were clustered step by step in the Bougainvillea genus with strong support (BS = 99–100%, and PP = 0.99–1) (Figs 9 and S2).

Discussion

This study first analyzed the complete chloroplast genome sequences of ten B. × buttiana cultivars. Herein, all 13 sequenced chloroplast genomes possessed quadripartite structures, including one LSC, one SSC and two IR regions, and showed the same numbers of total genes, protein-coding genes, tRNA and rRNA genes, and introns, consistent with other reported chloroplast genomes of Bougainvillea [2,3,12,13]. There were some variations in the chloroplast genome lengths of these 13 cultivars, with B. × buttiana ‘Barbara Karst’ having the longest genome of 158,231 bp, and that of B. spectabilis ‘Flame’ having the shortest genome of 153,994 bp (Table 1). The genome sizes of the seven B. × buttiana cultivars in this study, B. × buttiana ‘Mahara’, B. × buttiana ‘California Gold’, B. × buttiana ‘Double Salmon’, B. × buttiana ‘Double Yellow’, B. × buttiana ‘Los Banos Beauty’, B. × buttiana ‘Big Chitra’, and B. × buttiana ‘San Diego Red’ were almost identical to the reported chloroplast genome of B. glabra (MN888961), which is 154,542 bp in length [11]. For B. × buttiana ‘Gautama’s Red’, the genome size was the same as that of the chloroplast genome of B. peruviana (MT407463), which was 154,465 bp. The genome sizes of three cultivars, B. spectabilis ‘Splendens’, B. glabra ‘White Stripe’, and B. × buttiana ‘Miss Manila’ sp. 1, were the same as those of the reported chloroplast genome of B. spectabilis (MW167297), which is 154,520 bp [10]. Similar findings were also reported for the Hyacinthus and Aglaonema cultivars [8,9]. Among the three cultivars of Hyacinthus, ‘Woodstock’, ‘Delft Blue’ and ‘Aiolos’ had the same chloroplast genome size of 154,640 bp [9]. Among the two Aglaonema cultivars, ‘Hong Jian’ and ‘Red Valentine’, also displayed the same genome size of 165,797 bp [8]. In the present study, seven B. × buttiana cultivars had the same chloroplast genome sizes, possibly because these chloroplast genomes did not undergo recombination through hybridization or grafting.

Because B. × buttiana cultivars are difficult to differentiate by their leaf appearance, developing molecular markers to identify them is important. In previous studies, highly divergent regions, SSRs, long repeats, SNPs, and indels were investigated among 20 wild species of Bougainvillea and one cultivar [2,3]. However, no studies on SNPs or indels among B. × buttiana cultivars have been previously reported. In the present study, 776 SNPs and 124 indels were found between B. × buttiana ‘Mahara’ and B. × buttiana ‘Gautama’s Red’ (Tables 3 and S7). These SNPs and indels could be useful in the identification of these two B. × buttiana cultivars. Additionally, using B. × buttiana ‘Mahara’ as the reference, the other 6 B. × buttiana cultivars each had 2 intergenic SNPs (S1 Fig, Tables 3 and S7). These 2 SNPs were both in trnS-GCU_trnG-UCC-exon1 (S7 Table). Therefore, trnS-GCU_trnG-UCC-exon1 could be used to differentiate these 7 B. × buttiana cultivars. The other 9 comparisons, B. glabra ‘White Stripe’ vs. B. × buttiana ‘California Gold’, B. glabra ‘White Stripe’ vs. B. × buttiana ‘Double Salmon’, B. glabra ‘White Stripe’ vs. B. × buttiana ‘Double Yellow’, B. glabra ‘White Stripe’ vs. B. × buttiana ‘Los Banos Beauty’, B. glabra ‘White Stripe’ vs. B. × buttiana ‘San Diego Red’, B. glabra ‘White Stripe’ vs. B. × buttiana ‘Big Chitra’, B. glabra ‘White Stripe’ vs. B. × buttiana ‘Mahara’, B. glabra ‘White Stripe’ vs. B. × buttiana ‘Gautama’s Red’, and B. glabra ‘White Stripe’ vs. B. spectabilis ‘Flame’, also contained SNPs and indels (Tables 3 and S7). These SNPs and indels could be used to identify these 10 cultivars. However, 3 comparisons, B. glabra ‘White Stripe’ vs. B. spectabilis ‘Splendens’, B. glabra ‘White Stripe’ vs. B. × buttiana ‘Barbara Karst’, and B. glabra ‘White Stripe’ vs. B. × buttiana ‘Miss Manila’ sp. 1 had no SNPs/indels. These 3 comparisons indicated that the chloroplast genomes of these 4 Bougainvillea cultivars did not undergo recombination during hybridization or grafting. The leaf color of B. glabra ‘White Stripe’ was yellow‒green with white spots, while the leaf colors of B. spectabilis ‘Splendens’, B. × buttiana ‘Barbara Karst’ and B. × buttiana ‘Miss Manila’ sp. 1 were dark green (Fig 1). The bract color of B. glabra ‘White Stripe’ was white, while the bract colors of the three cultivars were red (Fig 1). The molecular regulatory mechanisms of leaf and bract color variations in these 4 cultivars need further study.

Highly divergent regions can be used as potential DNA markers for studies on phylogenetic relationships and species identification [43,44]. However, for some Nyctaginaceae species, phylogenetic relationships determined using universal DNA markers include multiple poor-resolution branches [1,6]. For example, three chloroplast DNA markers, namely, ndhF, rps16, and rpl16, and one nuclear ITS could not be used to identify Acleisanthes lanceolatus and A. longiflora [1]. Additionally, based on the Pi values studied here, it was also obvious that frequently used chloroplast markers, including ndhF, rps16, and rpl16, exhibited low polymorphism (0.0038, 0, and 0.0003, respectively) at the genus level in Bougainvillea. Therefore, it will be necessary to explore more highly divergent regions that represent potential markers for future studies. Currently, based on the results of mVISTA and nucleotide diversity analyses, 4 divergent regions among the 16 chloroplast genomes of the Bougainvillea cultivars in this study have been identified, including trnH-GUG_psbA, trnS-GCU_trnG-UCC-exon1, trnS-GGA_rps4, and ccsA_ndhD (Figs 2, 7 and 8). In comparison, trnH-GUG_psbA was also reported in wild Bougainvillea species and cultivars [2,13]; ccsA-ndhD was reported as a potential molecular marker in Amomum [45] and Alpinia [46]; and trnS_trnG was reported in Kaempferia [47]. Hence, based on these results, we suggest that these four divergent regions can be used as potential marker resources for Bougainvillea cultivar identification and phylogenetic analysis.

In this study, the ω ratio (ω = dN/dS) was used for measuring selective pressure in the Nyctaginaceae family. For most of the protein-coding genes, the value of ω was less than one, revealing that they were under purifying selection. Additionally, 9 genes, namely, rps12, rbcL, ndhF, rpoB, rpoC2, ndhI, psbT, ycf2, and ycf3, were identified as having positive selection sites in the Nyctaginaceae family (Table 3). Recent studies have indicated that these 9 genes are commonly undergoing positive selection in higher plants [43,48–54]. For example, rpoC2, rps12, rbcL, and ycf2 have also been identified as being under positive selection in orchid species [43]; rbcL, rpoC2, rps12, and ycf2 have been reported as being under positive selection in some Zingiberaceae species [48]; ndhF, rbcL, rpoC2, rps12 and ycf2 have also been identified as being under positive selection in Papilionoideae species [49]; rpoC2, rps12, rbcL, and ycf3 have also been identified as being under positive selection in Zingiber [50,51]; rpoB and rps12 have also been identified as being under positive selection in Begonia [52]; rbcL and ycf2 have also been identified as being under positive selection in Monsteroideae [53]; and ndhI has also been identified as being under positive selection in Saxifraga [54]. The analyzed species of the Nyctaginaceae family possess diverse morphological and ecological characteristics; for instance, some species are distributed in the tropics, while other species are distributed in the subtropics; some species are high-elevation trees, while other species are low-elevation shrubs and herbs [1,2]. In other words, Nyctaginaceae species live in diverse habitats and have high levels of plant diversity. Therefore, Nyctaginaceae species may face different types of stresses in their ecological habitats, and these 9 positively selected genes may play important roles in the evolution and adaptation of Nyctaginaceae species to their respective ecological habitats.

In the present study, our four phylogenetic trees obtained from chloroplast genome sequences and protein-coding genes revealed that Bougainvillea was a sister to Belemia with strong support (BS = 98–99%, and PP = 1) (Figs 9 and S2). This result was broadly consistent with those of previous studies [1,6]. We also found that 35 Bougainvillea individuals within the Bougainvillea genus were divided into four clades with strong support (Figs 9 and S2). This finding was in agreement with a previous study [3], but it had difference with recently reported study [13]. For the former, B. spinosa was sister to clade Ⅱ (the ‘cultivated’ Bougainvillea clade) or clade Ⅲ (the ‘wild’ Bougainvillea clade) based on protein-coding genes of chloroplast genomes [3], whereas for the latter, the 19 Bougainvillea plants were clustered into 3 clades based on complete chloroplast genomes, and B. spinosa was classified into the third clade [13]. This might because the latter study did not use plenty of Bougainvillea samples for phylogenetic analysis. However, the four phylogenetic trees in this study displayed some inconsistencies in the Bougainvillea genus, such as the shifting position of B. × buttiana ‘Gautama’s Red’ in Clade Ⅰ (Figs 9 and S2). Therefore, more Bougainvillea cultivar chloroplast genomes may need to be sequenced to resolve their positions. Nonetheless, based on our phylogenetic results, we propose that B. × buttiana ‘Gautama’s Red’ may be from B. peruviana and B. spectabilis. Additionally, the other eleven cultivars, including the remaining nine B. × buttiana cultivars, were clustered in Clade Ⅳ in the Bougainvillea genus (Figs 9 and S2). From the SNPs/indels analyses, seven of these nine B. × buttiana cultivars had no indels and only 2 SNPs (Table 3). Surprisingly, between B. × buttiana ‘Mahara’ and B. spectabilis ‘Splendens’, there existed relatively low numbers of SNPs/indels, with only 1 protein-coding gene SNP, 6 intergenic SNPs and 4 indels (S1 Fig, Tables 3 and S7). These results indicated that these analyzed B. × buttiana cultivars and B. spectabilis ‘Splendens’ showed close relationships. Considering that grafting techniques are often used in the cultivation processes of B. × buttiana cultivars, and based on the results of our four phylogenetic trees and SNPs/indels, we speculated that these nine B. × buttiana cultivars may come from hybrids involving B. peruviana, B. spectabilis and B. glabra. These results were, to some extent, in agreement with a previous hypothesis, which presumed that the B. × buttiana cultivars may be hybrids of B. peruviana and B. glabra [5].

Conclusions

In this study, 13 complete chloroplast genomes of 13 Bougainvillea cultivars from South China were sequenced, assembled and compared for genome structural characteristics. Furthermore, the molecular evolution of chloroplast genomes in the Nyctaginaceae family was studied, and the phylogenetic relationships of the Nyctaginaceae family, including Bougainvillea cultivars, were reconstructed with high-resolution branches. The 13 newly sequenced chloroplast genomes had a typical quadripartite structure, and each contained 112 different genes, including 79 protein-coding genes, 29 tRNA genes and 4 rRNA genes, with a chloroplast genome length of 153,994–158,231 bp. Comparative analyses of Bougainvillea cultivar chloroplast genomes revealed 4 highly divergent regions that can be used as potential markers for phylogenetic analyses and cultivar identification. Among the 46 chloroplast genomes of the Nyctaginaceae family, 9 protein-coding genes, namely, rps12, rbcL, ndhF, rpoB, rpoC2, ndhI, psbT, ycf2, and ycf3, were found to be undergoing positive selection at the amino acid site level. Based on complete chloroplast genomes and protein-coding genes, phylogenetic trees divided the Bougainvillea species and cultivars into 4 clades with strong support. These assembled chloroplast genomes enrich genomic resources and will help with the identification and utilization of Bougainvillea germplasm resources.

Supporting information

S1 Fig. Indels statistics of 13 newly sequenced complete chloroplast genomes of the Bougainvillea cultivars.

First, the B. glabra ‘White Stripe’ chloroplast genome was used as the reference sequence for indels analyses for the other twelve Bougainvillea chloroplast genomes. Second, except B. glabra ‘White Stripe’, the rest 12 Bougainvillea cultivar chloroplast genomes were compared using the chloroplast genome of B. × buttiana ‘Mahara’ as the reference. (A) Total indels statistics. (B) Insertion statistics. (C) Deletion statistics.

(DOCX)

pone.0310091.s001.docx^{(1.6MB, docx)}

S2 Fig. Phylogenetic relationships of Nyctaginaceae species based on protein-coding genes reconstructed using ML and BI methods.

(A) ML tree. (B) BI tree. The 13 newly sequenced Bougainvillea chloroplast genomes identified in this study are shown in bold.

(DOCX)

pone.0310091.s002.docx^{(2.4MB, docx)}

S1 Table. Information on the 13 Bougainvillea cultivars.

(DOCX)

pone.0310091.s003.docx^{(18.5KB, docx)}

S2 Table. The 46 complete chloroplast genomes in the Nyctaginaceae family used for determining the selective pressure and phylogenetic relationships.

(DOCX)

pone.0310091.s004.docx^{(19KB, docx)}

S3 Table. Genes distribution in the 13 chloroplast genomes of the Bougainvillea cultivars.

(XLSX)

pone.0310091.s005.xlsx^{(239.8KB, xlsx)}

S4 Table. SSRs detected in the 13 chloroplast genomes of the Bougainvillea cultivars.

(XLSX)

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S5 Table. Long repeats detected in the 13 chloroplast genomes of the Bougainvillea cultivars.

(XLSX)

pone.0310091.s007.xlsx^{(151KB, xlsx)}

S6 Table. Codon usage in the 13 chloroplast genomes of the Bougainvillea cultivars.

(XLSX)

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S7 Table. SNPs and indels detection among the 13 chloroplast genomes of the Bougainvillea cultivars.

(XLSX)

pone.0310091.s009.xlsx^{(339.3KB, xlsx)}

S8 Table. SNPs and indels detection between the 2 chloroplast genomes of B. spectabilis ‘Splendens’.

(XLSX)

pone.0310091.s010.xlsx^{(69.4KB, xlsx)}

S9 Table. Nuclear diversity of 16 chloroplast genomes from the 16 Bougainvillea cultivars.

(XLSX)

pone.0310091.s011.xlsx^{(34.5KB, xlsx)}

Data Availability

Data Availability Statement: The data presented in this study were submitted to the NCBI repository (https://www.ncbi.nlm.nih.gov) under accession numbers OR344366–OR344378.

Funding Statement

This research was financially supported by the Collection, Identification and Utilization of New and Superior Flower Germplasm Resources (2023-2025), Science and Technology Program from Forestry Administration of Guangdong Province (2024KJQT0014) and the Guangdong Province Modern Agriculture Industry Technical System–Flower Innovation Team Construction Project (2023KJ121). The collection, identification and utilization of new and superior flower germplasm resources (2023-2025) and Science and Technology Program from Forestry Administration of Guangdong Province (2024KJQT0014) were funded by Guangdong Bailin Ecology and Technology Co., Ltd. The Guangdong Province Modern Agriculture Industry Technical System–Flower Innovation Team Construction Project (2023KJ121) was funded by the Environmental Horticulture Research Institute, Guangdong Academy of Agricultural Sciences. There was no additional external funding received for this study.

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Pankaj Bhardwaj

4 Mar 2024

PONE-D-23-43492Comparative analysis of the complete chloroplast genomes of thirteen Bougainvillea cultivars from South China with implications for their genome structures and phylogenetic relationshipsPLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The study of Wu et al. domenstrates the application of chloroplast genomes in explore the germplasm of Bougainvillea cultivars. Apart from the phylogenetic relationships between the cultivars, potential molecular markers including SSRs, LSRs and hotspot regions have been investigated from the chloroplast genomes. The study shows merits on the research of Bougainvillea, that could serve as a reference in studying other horticultural crops with diversified morphologies. However, prior to a make a further decision, the following major and minor issues should be resolved.

Major issues:

1) The cultivars of Bougainvillea are regulated by the International Code for the Nomenclature for Cultivated Plants (ICNCP). The registration of Bougainvillea cultivars is designated to the Indian Agricultural Research Institute (IARI). The authors are responsible to carefully check if the cultivar epithets are well established and registered. Illegitimate epithets (e.g. misspelling and homonyms) and unestablished epithets are common in horticultural germplasms. The authors should refer to the Articles 25 to 27 of ICNCP (9th Edition) which is available online (https://www.ishs.org/sites/default/files/static/ScriptaHorticulturae_18.pdf). The publications of The Bougainvillea Society of India (BSI) of IARI (http://www.bsi-iari.com/publication.htm) will help the authors in checking. The authors should indicate those unestablished and unregistered cultivars in the manuscript as precaution for the readers.

2) I wonder if the mutant (Bougainvillea sp.1) is discovered by the authors themselves? If so, what is the original cultivar of this mutant? The authors should elaborate why this mutant is included in this study. Also, Figure S1 (the images of 13 cultivars) should be included in the main text, prior to the chloroplast genome map. Vouchers of the studied cultivars, either live and dried specimens, should be listed out with their collection location, GPS, date of collection, collector numbers, and deposited herbarium or institution.

3) I noticed that Bougainvillea spinosa was included in Clade II by the authors. However, it should not be regarded as a member of Clade II, as it is sister to all other members from both clade II and III (in both Figure 8 A&B and S3 A&B). The authors should discuss the potential reasons in the Discussion.

4) The definition of SNPs and InDels in this manuscript should be well defined. Could the nucleotide differences between one and another cultivar be considered as SNPs and InDels? Molecular Diagnostic Characters (MDCs) of a cultivar to differentiate itself from the other 12 cultivars could be more meaningful. Also, haplotype analysis could aid in visualizing the figures in this tables by grouping the cultivars. The authors could refer to the following article:

Wong, K. H., Wu, H. Y., Kong, B. L. H., But, G. W. C., Siu, T. Y., Hui, J. H. L., Shaw, P. C., & Lau, D. T. W. (2022). Characterisation of the complete chloroplast genomes of seven Hyacinthus orientalis L. cultivars: Insights into cultivar phylogeny. Horticulturae, 8(5), 1-28.

Minor issues:

The writing style and language of the manuscript, particularly the abstracts, introduction and the discussion, should be meticulously improved. I have identified a number of mistakes in grammar and use of wordings which deteriorate the comprehensibility of the manuscript. In addition, I highly recommend the authors to employ a native English speaker or professional editing agency to proofread the manuscript. The style in presenting figures and tables should also be improved. Please kindly refer to the following:

L16-18: Having high similarity in leaf appearance and hybridization among Bougainvillea species, the phylogenetic relationships of the genus are complicated and controversial.

L20-21: Their phylogenetic relationships within the genus Bougainvillea and other species of the family Nyctaginaceae are identified for the first time.

L27-28: Four divergent regions, including ......, were identified from sliding window analysis of 16 Bougainvillea cultivar genomes.

L36: replace "which contained" by "including"

L39: ..., but also helped to identify Bougainvillea

L51: "Le Du Juan", which is well-known in China, belongs to the genus Bougainvillea of the family Nyctaginaceae.

L53-54: with colored bracts [2,3]. The colorful bracts surrounding the small tubular flowers are often mistakenly treated as flowers.

L59: remove "value"

L60: replace "only" by "mainly"

L60-61: ... challenging because of high similarity

L68: replace "identified by" by "explored using"

L69: Why is this sentence concerning the usage of Bougainvillea is placed here? Recommend placing it in the previous paragraphs.

L70: delete "that has been"

L71: replace "taken" by "introduce"

L75: ... B. x buttiana cultivars and the molecular evolution ...

L77-78: In this study, complete chloroplast genomes of thirteen Bougaivillea cultivars were newly sequence, assembled and annotated. These thirteen cultivars from South China include seven ....

L84: ... integrating three published ...

L85: Bougainvillea cultivars on the NCBI. In this study, five objectives were targeted: (1) ... [Aim =/= objectives!]

L97: please state the full name of the "resource garden" here.

L99: remove "finally"

L101-102: ... the DNA quality and quantity was checked through ... method, respectively. ...

L104: is the read in 150 bp paired or not???

L109: assembly and annotation were conducted using the methods previously reported [15].

L125: ... The long repeats were detected with a minimum ....

L143: remove "ones"

L163: Phylogenetic trees were constructed using ...

L186 and elsewhere throughout the manuscript: "different genes" should be better replaced as "unique genes".

L253 and the parts reporting IR: please keep consistency with the border titles with Fig. 5. For example, in L253 "IRb/LSC" should be replaced as "LSC/IRb".

L303 and elsewhere throughout the manuscript: the authority of genera and species should be shown for the first appearance in the manuscript. For example, Bougainvillea Comm. ex Juss., Belemia Pires, Mirabilis L. Latin should be italic.

L310-311: the authors could discuss more concerning the different phylogentic positions of the cultivars of B x buttiana 'Gautama's Red' and 'Mahara'. Would it suggest a different origin of them?

L325: Why B. glabra 'White Stripe' is not clustered with other cultivars of B. glabra?

L374: replace "with" by "in"

Fig 1. It might be meaningless to include the GCView Comparison results since they are too thin which are hardly can see. The layout of chloroplast genome map would be too complicated. The authors could consider removing the GC comparison from this figure and submit it as a supplementary figure.

Table 4: The authors should define what the numbers and alphabets represent in the table. It is recommended to rearrange the table in a more easy-understanding manner e.g. by dividing the items of a positively selected sites in columns:

Positively selected sites

Gene xx |x| xxx

ndhF 462 |L|0.969*

502 |A|0.960*

...

Reviewer #2: Dear authors

I have reviewed this study from the beginning to the end and I did not see any deficiency in terms of the main headings (introduction, material-method, conclusion and discussion). The methods were selected in accordance with the purpose of the study and applied correctly. The language of the article is simple and understandable. There are no stylistic errors. The main objectives of the study are: (1) to characterise and study the structures of 13 newly sequenced complete chloroplast genomes; (2) to detect variation in simple sequence repeats 87 (SSR), long repeats and codon usage among these 13 chloroplast genomes; (3) to identify highly variable 88 regions for potential development of DNA markers among Bougainvillea cultivars; (4) to understand the molecular 89 evolution of chloroplast genomes in the Nyctaginaceae family; and (5) to reveal the phylogenetic relationships 90 between Bougainvillea species and varieties and other species in the Nyctaginaceae family. Therefore, I would like to say that the study is acceptable in its present form. Sincerely.

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Reviewer #1: Yes: Kwan-Ho WONG

Reviewer #2: No

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PLoS One. 2024 Sep 11;19(9):e0310091. doi: 10.1371/journal.pone.0310091.r003

Author response to Decision Letter 0

11 Apr 2024

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Yes

Response: We revised the whole text and conclusion carefully using red markers.

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

3. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

4. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: No

Reviewer #2: Yes

Response: We revised the English of the text carefully using red markers by American Journal Experts (AJE). We believe that the revised manuscript is more readable.

5. Review Comments to the Author

Major issues:

Response: Thank you for this question. We can’t open the BSI of IARI. So the cultivars in the manuscript were unregistered in this web. We wrote in Data Availability Statement as following: The 13 Bougainvillea cultivars studied in this study are unregistered in the Bougainvillea Society of India (BSI) of IARI (http://www.bsi-iari.com/publication.htm).

2) I wonder if the mutant (Bougainvillea sp.1) is discovered by the authors themselves?

Response: Thank you for this question. Yes, the mutant (Bougainvillea sp.1) was discovered by the author He-Fa Wang.

If so, what is the original cultivar of this mutant?

Response: The mutant (Bougainvillea sp.1) was from the other cultivar (mother line), of which the bract colour looked like the bract colour of the cultivar Bougainvillea ‘Barbara Karst’. This mother line has thorns and was sold out in commercial activities. However, the bud mutant was kept in live and grown by grafting. Because the author He-Fa Wang hasn’t yet got certificate of registered commercial name for the mutant. In the manuscript, we gave the mutant with the name of Bougainvillea sp.1.

The authors should elaborate why this mutant is included in this study.

Response: In this study, we want to know the phylogenetic relationships among the mutant (Bougainvillea sp.1) and other individuals of the Bougainvillea genus by using complete chloroplast genomes.

Also, Figure S1 (the images of 13 cultivars) should be included in the main text, prior to the chloroplast genome map. Vouchers of the studied cultivars, either live and dried specimens, should be listed out with their collection location, GPS, date of collection, collector numbers, and deposited herbarium or institution.

Response: Thank you for this question. We revised Figure 1 instead Figure S1 in the manuscript. In the Plant materials, DNA extraction, and sequencing section, we wrote detail information of the studied cultivars, either live and dried specimens, with their collection location, GPS, date of collection, collector numbers, and deposited herbarium or institution.

Fresh leaves of twelve Bougainvillea cultivars and one bud mutation, namely, B.× buttiana ‘Mahara’ , B. × buttiana ‘Gautama's Red’, B. × buttiana ‘California Gold’, B. × buttiana ‘Double Salmon’, B. × buttiana ‘Double Yellow’, B. × buttiana ‘Big Chitra’, B. × buttiana ‘Los Banos Beauty’, B. glabra ‘White Stripe’, B. spectabilis ‘Flame’, B. spectabilis ‘Splendens’, B. ‘Barbara Karst’, B. ‘San Diego Red’, and B. sp. 1 (Fig 1, S1 Table), were collected from the Provincial Flower Germplasm Resources Bank of San Jiao Mei (117°37′47″E, 24°28′35″N) in Zhangzhou, Fujian Province, China. Fresh leaves were quickly frozen on dry ice, sent to the laboratory of the Environmental Horticulture Research Institute at the Guangdong Academy of Agricultural Sciences (113°21′8″E, 23°9′2″N), Guangzhou, China, and stored at −80 ℃ until use. Genomic chloroplast DNA was extracted from each sample using the modified sucrose gradient centrifugation method [13]. Then, the DNA quality and quantity were checked through agarose gel electrophoresis and the NanoDrop microspectrometer method, respectively. Each qualified DNA sample was sheared to fragments of approximately 350 bp. Short-insert (350 bp) paired-end libraries were constructed, and sequencing was performed on an Illumina NovaSeq 6000 platform with a paired read length of 150 bp (Biozeron, Shanghai, China). The raw data from each sample were checked using FastQC v. 0.11.9 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and adaptors and low-quality reads were subsequently deleted by Trimmomatic v. 0.39 [14] with default parameters. The remaining materials, including the leaves and DNA, were deposited in the laboratory of the Environmental Horticulture Research Institute (store sheet code: B2023), Guangdong Academy of Agricultural Sciences, Guangzhou, China, as vouchers (S1 Table).

Fig 1. Morphologies among 13 cultivars of the Bougainvillea genus. A, Bougainvillea glabra ‘White Stripe’; B, Bougainvillea×buttiana ‘Mahara’; C, Bougainvillea×buttiana ‘Double Yellow’; D, Bougainvillea×buttiana ‘Double Salmon’; E, Bougainvillea×buttiana ‘Los Banos Beauty’; F, Bougainvillea ×buttiana ‘California Gold’; G, Bougainvillea ×buttiana ‘Big Chitra’; H, Bougainvillea×buttiana ‘Gautama's Red’; I, Bougainvillea spectabilis ‘Flame’; J, Bougainvillea spectabilis ‘Splendens’; K, Bougainvillea ‘Barbara Karst’; L, Bougainvillea ‘San Diego Red’; M and N, Bougainvillea sp1. armed with simple or no thorns.

Response: Thank you for this question. Bougainvillea spinosa should be clustered in Clade Ⅰ. We revised the results of phylogenetic relationships as following.

In Clade Ⅰ, B. spectabilis ‘Flame’ was sister to B. peruviana MW123901, then forming a cluster strongly sister to B. pachyphylla, both based on chloroplast genome sequences and protein-coding genes (Fig 9, S2 Fig). However, the position of B. × buttiana ‘Gautama's Red’ in ML tree constructed by chloroplast genomes sequences was different from the other three phylogenetic trees in this study. For the former, B. × buttiana ‘Gautama's Red’ was sister to B. spectabilis ‘Pixie Pink’, then clustered with B. peruviana MT407463 with strong support (BS = 90%) (Fig 9A). For the latter, B. spectabilis ‘Pixie Pink’ was sister to B. peruviana MT407463, then clustered with B. × buttiana ‘Gautama's Red’ with strong support (BS = 93-100%, and PP = 0.93-1) (Fig 9B, S2 Fig). Then, B. spinosa and these 6 individuals were clustered together with strong support in Clade Ⅰ (Fig 9, S2 Fig). In Clade Ⅱ, there were 7 individuals of wild species, including B. campanulata, B. berberidifolia, B. infesta, B. modesta OM44398, B. modesta OM044396, B. stipitata, and B. stipitata var. grisebachiana.

4)The definition of SNPs and InDels in this manuscript should be well defined. Could the nucleotide differences between one and another cultivar be considered as SNPs and InDels?

Molecular Diagnostic Characters (MDCs) of a cultivar to differentiate itself from the other 12 cultivars could be more meaningful. Also, haplotype analysis could aid in visualizing the figures in this tables by grouping the cultivars. The authors could refer to the following article:

Response: Thank you for this question. We didn’t use the presentation of SNP and indels as described in cultivars of Hyacinthus orientalis. For one reason, in my opinion, the background of Hyacinthus orientalis was less complicated than the background of the cultivars of Bougainvillea. The breeding of Hyacinthus orientalis didn’t use grafting; however, grafting is very common in cultivation of Bougainvillea cultivars. For other reason, too many SNPs and indels (more than 100), such as B. glabra ‘White Stripe’ vs. B. × buttiana ‘Gautama's Red’, B. glabra ‘White Stripe’ vs. B. spectabilis ‘Flame’ and B. × buttiana ‘Mahara’ vs. B. × buttiana ‘Gautama's Red’, were not suitable in the text. We also showed the SNPs and indels of different comparisons in the supplementary file (S7 Table).

Minor issues:

L16-18: Having high similarity in leaf appearance and hybridization among Bougainvillea species, the phylogenetic relationships of the genus are complicated and controversial.

Response: Thank you for this idea. We revised as following:

Having high similarity in leaf appearance and hybridization among Bougainvillea species, especially from Bougainvillea × buttiana, their phylogenetic relationships were very complicated and controversial.

L20-21: Their phylogenetic relationships within the genus Bougainvillea and other species of the family Nyctaginaceae are identified for the first time.

Response: Thank you for this idea. We revised the sentence as following:

In this study, we sequenced, assembled and analyzed thirteen complete chloroplast genomes of Bougainvillea cultivars from South China, including seven B. × buttiana cultivars and six other Bougainvillea cultivars, and identified their phylogenetic relationships within the Bougainvillea genus and other species of the Nyctaginaceae family for the first time.

L27-28: Four divergent regions, including ......, were identified from sliding window analysis of 16 Bougainvillea cultivar genomes.

Response: Thank you for this idea. Comparative analysis not only contained sliding window analysis (mVISTA), but also contained CGView analysis and nuclear diversity analysis (Pi). Therefore, we revised the sentence as following:

Four divergent regions, including trnH-GUG_psbA, trnS-GCU_trnG-UCC-exon1, trnS-GGA_rps4, and ccsA_ndhD, were identified from comparative analysis of 16 Bougainvillea cultivars genomes.

L36: replace "which contained" by "including"

Response: Thank you for this idea. We revised ‘including’ instead of ‘which contained’.

L39: ..., but also helped to identify Bougainvillea

Response: Thank you for this idea. We revised ‘helped to identify Bougainvillea’

L51: "Le Du Juan", which is well-known in China, belongs to the genus Bougainvillea of the family Nyctaginaceae.

Response: Thank you for this idea. We revised as following:

“Le Du Juan”, which is well-known in China, belongs to the Bougainvillea genus of the Nyctaginaceae family.

L53-54: with colored bracts [2,3]. The colorful bracts surrounding the small tubular flowers are often mistakenly treated as flowers.

Response: Thank you for this idea. We revised as following:

The colorful bracts surrounding the small tubular flowers are often mistakenly treated as flowers.

L59: remove "value"

Response: Thank you for this idea. We removed ‘value’.

L60: replace "only" by "mainly"

Response: Thank you for this idea. We revised ‘mainly’ instead of ‘only’.

L60-61: ... challenging because of high similarity

Response: Thank you for this idea. We revised ‘challenging because of high similarity’.

L68: replace "identified by" by "explored using"

Response: Thank you for this idea. We revised ‘explored using’ instead of ‘identified by’.

L69: Why is this sentence concerning the usage of Bougainvillea is placed here? Recommend placing it in the previous paragraphs.

Response: Thank you for this idea. We placed this sentence in the previous paragraph.

L70: delete "that has been"

Response: Thank you for this idea. We deleted ‘that has been’.

L71: replace "taken" by "introduce"

Response: Thank you for this idea. We revised ‘introduce’ instead of ‘taken’.

L75: ... B. x buttiana cultivars and the molecular evolution ...

Response: Thank you for this idea. We revised as following:

However, phylogenetic relationships of B. × buttiana cultivars and molecular evolution of chloroplast genomes from the Nyctaginaceae family still remains to be unveiled [2-5,9-12].

L77-78: In this study, complete chloroplast genomes of thirteen Bougaivillea cultivars were newly sequence, assembled and annotated. These thirteen cultivars from South China include seven ....

Response: Thank you for this idea. We revised as following:

In this study, complete chloroplast genomes of thirteen Bougainvillea cultivars were newly sequenced, assembled and annotated. These thirteen cultivars from South China included seven B. × buttiana cultivars,

L84: ... integrating three published ...

Response: Thank you for this idea. We revised as following:

by integrating three published complete chloroplast genomes

Attachment

Submitted filename: response to reviewers.docx

pone.0310091.s013.docx^{(6MB, docx)}

PLoS One. doi: 10.1371/journal.pone.0310091.r004

Decision Letter 1

Pankaj Bhardwaj

7 May 2024

PONE-D-23-43492R1Comparative analysis of the complete chloroplast genomes of thirteen Bougainvillea cultivars from South China with implications for their genome structures and phylogenetic relationshipsPLOS ONE

Dear Dr. Li,

Thank you for submitting your manuscript. Following a detailed review, our editorial team and reviewers have identified several critical areas that require revision before we can reconsider your manuscript for publication. We believe that your work has potential, but significant changes are needed to ensure the quality and accuracy of your research.

Please review the comments from our reviewers below, and provide a detailed response letter outlining how you have addressed each point. The revised manuscript should reflect these changes and adhere to our journal's guidelines.

Key Issues for Revision:

One of the reviewers noted inconsistencies in the cultivar nomenclature. You must confirm the legitimacy of the names used and correct any discrepancies. Additionally, the clustering of Bougainvillea spinosa into clades appeared inconsistent, suggesting a need for further consultation with experts in phylogeny or re-analysis.

Another reviewer highlighted the absence of references to existing studies on the chloroplast genome of Bougainvillea. Please ensure you incorporate relevant literature, including a comparative analysis with studies like Lin et al. (2023). Discuss any differences in findings and explain their significance.

The reviewers requested more context regarding the significance of the thornless mutant. Additionally, they suggested examining intraspecific genetic distances among cultivars derived from the same species, particularly B. x buttiana and B. spectabilis.

It was noted that you constructed multiple phylogenetic trees, but the differences between Maximum Likelihood and Bayesian Inference were not explained. Please clarify why these methods were used and which types of sequences are most suitable for constructing phylogenetic trees.

Reviewers identified additional minor issues, such as the correct presentation of GPS coordinates, language and format consistency, and a more reader-friendly presentation of SNPs and Indels. Address these issues to improve the clarity and readability of the manuscript.

Given these concerns, we request that you revise your manuscript and submit a detailed response letter that explains how you have addressed each point. Please submit your revised manuscript by [Deadline Date]. If you need more time, do let us know, and we will be happy to discuss an extension.

We look forward to receiving your revised manuscript. If you have any questions or need further guidance, please do not hesitate to contact us.

Thank you for your understanding and cooperation.

Sincerely,

Please submit your revised manuscript by Jun 21 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Pankaj Bhardwaj, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #3: (No Response)

Reviewer #4: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #3: Yes

Reviewer #4: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

6. Review Comments to the Author

Reviewer #1: It is highly appreciated that the authors have made substantial efforts in addressing most of the minor issues and improving comprehensibility of the manuscript. However, the major issues were not settled, particularly the cultivar nomenclature and the clustering of clades. It is extremely irresponsible that the authors calimed all the cultivars unregistered only because of their inaccessiblity to the website of Bougainvillea Society of India. Some of the studied cultivars were registered while some are not. This act is seriously misleading to the readers since they claimed some legitimate cultivar epithets as illegitimate. The authors should try their best to reach to the pieces of information regardless of any format (e.g. books, printed literatures and any electronic means), but not just giving up and leave an incorrect and irrespinsible statement. For the clustering of Bougainvillea spinosa, the authors obviously showed their wrong concept on monophyly. The claims of the authors (Bougainvillea spinosa was in either clade I or II) is very misleading to the readers outside the field of phylogeny.

The authors clearly failed to address all four major issues. For major #2, the significancy in including the thornless mutant was not well explained and elaborated. For major #4, the breeding techniques do not affect the presentation of SNPs and Indels which are significant in cultivar authentication. It is understandable their are plenty of SNPs discovered from the analysis. Yet, the authors failed the listed out those valuable in cultivar authentication in an reader-friendly manner. For major #1 and #3, the authors should obtain more suggestions froms experienced taxonomists and phylogenists, respectively. A numbers of minor issues were also not well addressed. The authors should read more articles to find out which format "the xxx genus/family" or "the genus/family xxx" should be correct in English. The authors also failed to add authorities after genus name and species epithets, which are commonly adopted in the scientific research articles. The presentation of GPS coordinates was in wrong order. Suggestions on figure and table presentation were sadly ignored.

This interesting sutdy in exploring cultivar phylogeny of Bougainvillea would potentially contribute to the science community. Although the authors have made plenties of efforts in improving the manuscript, it is sorrow that I have to suggest a rejection from publishing this articles delivering incorrect information.

Reviewer #3: The overall write up is convincing. As this work is on the characterization of Bougainvillea cultivars, it should not be related to anything about taxonomy. Eventually, I have two concerns for this manuscripts:

1. There are a number of published work on the complete chloroplast genome of Bougainvillea. Considering that all information are important, I would be looking at a discussion on the difference of findings between this work and other published works, and the reasons for the difference identified in these analyses. For example, Lin et al. 2023, International Journal of Molecular Science 24:15138 should be the latest work before this work. I neither see it cited, nor being discussed. Lin et al. had similar analyses when compared to the current one. Please make sure all published work on the complete chloroplast genome of Bougainvillea are included in this work and compared for their findings.

2. Since some of the cultivars are named along with their parent (perhaps they are mutant clones), it would be reasonable to at least investigate the intraspecific genetic distance of these cultivar derived from the (presumably) same species. At least the analysis is conducted on the species assembled in this study, i.e., B. x buttiana and B. spectabilis.

Reviewer #4: The manuscript titled 'Comparative analysis of the complete chloroplast genomes of thirteen Bougainvillea cultivars from South China with implications for their genome structures and phylogenetic relationships' presents the assembly and annotation of 13 cp genomes of Bougainvillea. The study is well-organized and offers valuable insights for analyzing genetic diversity and phylogenetic relationships within the Nyctaginaceae family. I highly recommend this work for publication in the PLoS One journal.

I think the following minor suggestions and comments will help the authors improve their work.

1. The authors should provide additional information of the origin of the bud mutation, such as which plant accession did this mutation derive from?

2. I wondered why these authors constructed so many types of phylogenetic trees, including two ML and BI trees based on whole cp genome sequences and coding sequences. These authors should point out what’s the difference between ML and BI trees, and which sequence is more appropriate to construct phylogenetic tree.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #3: No

Reviewer #4: No

**********

PLoS One. 2024 Sep 11;19(9):e0310091. doi: 10.1371/journal.pone.0310091.r005

Author response to Decision Letter 1

11 Jun 2024

Reviewer #1: It is highly appreciated that the authors have made substantial efforts in addressing most of the minor issues and improving comprehensibility of the manuscript. However, the major issues were not settled, particularly the cultivar nomenclature and the clustering of clades. It is extremely irresponsible that the authors claimed all the cultivars unregistered only because of their inaccessiblity to the website of Bougainvillea Society of India. Some of the studied cultivars were registered while some are not. This act is seriously misleading to the readers since they claimed some legitimate cultivar epithets as illegitimate. The authors should try their best to reach to the pieces of information regardless of any format (e.g. books, printed literatures and any electronic means), but not just giving up and leave an incorrect and irrespinsible statement.

Response: We removed the statement. The 12 Bougainvillea cultivars included B. × buttiana ‘Mahara’ , B. × buttiana ‘California Gold’, B. × buttiana ‘Double Yellow’, B. × buttiana ‘Los Banos Beauty’, B. × buttiana ‘San Diego Red’, B. ×buttiana ‘Barbara Karst’, B. spectabilis ‘Flame’, B. spectabilis ‘Splendens’, B. × buttiana ‘Gautama's Red’, B. × buttiana ‘Double Salmon’, B. × buttiana ‘Big Chitra’, and B. glabra ‘White Stripe’. Their names came from following information:

Liu Y, Ruan L, Zhou H, Yu M. Cultivar classification of Bougainvillea. China Forestry Press, Beijing, China, 2020

Sun L. Molecular identification of cultivars and transcriptome analysis of bracts in Bougainvillea. Ph. D. Chinese Academy of Forestry, Beijing, China, 2019, pp38-39

From the description of Sun in 2019, B.× buttiana ‘San Diego Red’ and B. ×buttiana ‘Barbara Karst’ were the correct names for Bougainvillea ‘San Diego Red’ and Bougainvillea ‘Barbara Karst’, respectively. We used B. × buttiana ‘San Diego Red’ and B. ×buttiana ‘Barbara Karst’ in the whole text instead of B. ‘San Diego Red’ and B. ‘Barbara Karst’, respectively.

Because the thornless mutant was a variety from Bougainvillea × buttiana ‘Miss Manila’ and the author He-Fa Wang hasn’t yet got certificate of registered commercial name for the mutant, we gave the mutant with the name of Bougainvillea × buttiana ‘Miss Manila’ sp.1 in the text.

For the clustering of Bougainvillea spinosa, the authors obviously showed their wrong concept on monophyly. The claims of the authors (Bougainvillea spinosa was in either clade I or II) is very misleading to the readers outside the field of phylogeny.

Response: Thanks for your opinion on the clustering of Bougainvillea spinosa.

In Bautista et al. (2022), B. spinosa was sister to clade II or wild clade Bougainvillea in that study. However, Lin et al. (2023) clustered the B. spinosa into clade 3, which was the same clade in our study, clade I.

Bautista MAC, Zheng Y, Boufford DE, Hu Z, Deng Y, Chen T. Phylogeny and taxonomic synopsis of the genus Bougainvillea (Nyctaginaceae). Plants (Basel). 2022; 11, 1700. https://doi.org/10.3390/plants11131700 PMID: 35807654

Lin X, Lee SY, Ni J, Zhang X, Hu X, Zou P, Wang W, Liu G. Comparative analyses of chloroplast genome provide effective molecular markers for species and cultivar identification in Bougainvillea. Int J Mol Sci. 2023; 24(20),15138. https://doi.org/10.3390/ijms242015138. PMID: 37894819

We revised the section Phylogenetic relationships in the Bougainvillea genus as following:

The 35 Bougainvillea individuals analyzed were divided into four clades, namely, Clades Ⅰ, Ⅱ, Ⅲ, and Ⅳ, with strongly supported values (BS = 85–100% for the ML trees and PP = 0.99–1 for the BI trees) (Fig 9, S2 Fig). Two cultivars, B. × buttiana ‘Gautama's Red’ and B. spectabilis ‘Flame’, were clustered into clade Ⅰ, and the other 11 cultivars, including B. × buttiana ‘Mahara’, B. × buttiana ‘California Gold’, B. × buttiana ‘Double Salmon’, B. × buttiana ‘Double Yellow’, B. × buttiana ‘Los Banos Beauty’, B. × buttiana ‘Big Chitra’, B. × buttiana ‘Barbara Karst’, B. glabra ‘White Stripe’, B. spectabilis ‘Splendens’, B. × buttiana ‘San Diego Red’, and B. × buttiana ‘Miss Manila’ sp. 1, were clustered into clade Ⅳ (Fig 9, S2 Fig). In Clade Ⅰ, B. spectabilis ‘Flame’ was sister to B. peruviana MW123901 and then formed a strong sister cluster to B. pachyphylla, both based on chloroplast genome sequences and protein-coding genes (Fig 9, S2 Fig). However, the position of B. × buttiana ‘Gautama's Red’ in the ML tree constructed from chloroplast genome sequences differed from those in the other three phylogenetic trees in this study. For the former, B. × buttiana ‘Gautama's Red’ was sister to B. spectabilis ‘Pixie Pink’ and then clustered with B. peruviana MT407463 with strong support (BS = 90%) (Fig 9A). For the latter, B. spectabilis ‘Pixie Pink’ was sister to B. peruviana MT407463 and then clustered with B. × buttiana ‘Gautama's Red’ with strong support (BS = 93–100%, and PP = 0.93–1) (Fig 9B, S2 Fig). In Clade Ⅱ, it only contained B. spinosa (Fig 9, S2 Fig). In Clade Ⅲ, there were 7 individuals of wild species, including B. campanulata, B. berberidifolia, B. infesta, B. modesta OM44398, B. modesta OM044396, B. stipitata, and B. stipitata var. grisebachiana. In Clade Ⅳ, in the ML tree based on the chloroplast genome sequences, B. × buttiana ‘Mahara’, B. × buttiana ‘California Gold’, B. × buttiana ‘Double Salmon’, B. × buttiana ‘Double Yellow’, B. × buttiana ‘Los Banos Beauty’, B. × buttiana ‘Big Chitra’, B. × buttiana ‘San Diego Red’, B. spectabilis ‘Ratana Red’, B. glabra MN888961, and B. peruviana ‘Mona Lisa Red’ were clustered together in one cluster with strong support (BS = 88–92%). B. × buttiana ‘Barbara Karst’, B. glabra ‘White Stripe’, B. spectabilis ‘Splendens’, B. × buttiana ‘Miss Manila’ sp. 1, B. spectabilis MN315508, B. spectabilis China MW167297, and B. hybrid cultivar MW123903 were clustered together in another cluster with strong support (BS =88–95%) (Fig 9A). However, in the ML tree based on protein-coding genes, B. × buttiana ‘Mahara’ was sister to the other cultivars in Clade Ⅳ with strong support (BS = 100%) (S2A Fig). In both BI trees, B. × buttiana ‘Mahara’, B. × buttiana ‘California Gold’, B. × buttiana ‘Double Salmon’, B. × buttiana ‘Double Yellow’, B. × buttiana ‘Los Banos Beauty’, B. × buttiana ‘Big Chitra’, B. × buttiana ‘San Diego Red’, B. spectabilis ‘Ratana Red’, B. glabra, B. peruviana ‘Mona Lisa Red’, B. × buttiana ‘Barbara Karst’, B. glabra ‘White Stripe’, B. spectabilis ‘Splendens’, B. × buttiana ‘Miss Manila’ sp. 1, B. spectabilis MN315508, B. spectabilis MW167297, and B. hybrid cultivar MW123903 were clustered together in one cluster in Clade Ⅳ with moderate to strong support (PP = 0.84–1) (Fig 9B, S2B Fig). In the four phylogenetic trees, Clades Ⅲ and Ⅳ were clustered together, forming a cluster with strong support (BS =85–99%, and PP = 0.99–1); then the cluster, Clade Ⅱ, and Clade Ⅰ were clustered step by step in the Bougainvillea genus with strong support (BS = 99-100%, and PP = 0.99-1) (Fig 9, S2 Fig).

The authors clearly failed to address all four major issues. For major #2, the significancy in including the thornless mutant was not well explained and elaborated.

Response: Thanks for your idea. The thornless mutant was a variety from Bougainvillea × buttiana ‘Miss Manila’. The mother line of Bougainvillea × buttiana ‘Miss Manila’ has been sold out in commercial activities so far. However, the bud mutant has been kept in live and cultivated by grafting. Because the author He-Fa Wang hasn’t yet got certificate of registered commercial name for the mutant. In the manuscript, we gave the mutant with the name of Bougainvillea × buttiana ‘Miss Manila’ sp.1.

For major #4, the breeding techniques do not affect the presentation of SNPs and Indels which are significant in cultivar authentication. It is understandable their are plenty of SNPs discovered from the analysis. Yet, the authors failed the listed out those valuable in cultivar authentication in an reader-friendly manner.

Response: Thanks for your idea. We didn’t use the presentation of SNP and indels as described in cultivars of Hyacinthus orientalis. For three comparisons, there were more than 700 SNPs and 100 indels. This situation is not fit in the text. However, we listed out these SNPs and indels in detail information in the supplementary file: S7 Table, including base information of SNPs, mutant type, gene name, gene start, gene end, indel sequence, indel start, indel end, alignment strand and so on.

For major #1 and #3, the authors should obtain more suggestions froms experienced taxonomists and phylogenists, respectively. A numbers of minor issues were also not well addressed. The authors should read more articles to find out which format "the xxx genus/family" or "the genus/family xxx" should be correct in English.

Response: Thanks for your idea. These two forms have appeared in many articles. We selected the form of “the XXX genus/family” in the text.

The authors also failed to add authorities after genus name and species epithets, which are commonly adopted in the scientific research articles.

Response: The name of 12 Bougainvillea cultivar were adopted in the scientific book as following.

Liu Y, Ruan L, Zhou H, Yu M. Cultivar classification of Bougainvillea. China Forestry Press, Beijing, China, 2020

Sun L. Molecular identification of cultivars and transcriptome analysis of bracts in Bougainvillea. Ph. D. Chinese Academy of Forestry, Beijing, China, 2019, pp38-39

The presentation of GPS coordinates was in wrong order.

Response: We checked and revised the correct GPS in correct order. We reported details about the GPS for sample collection, DNA sample store and the rest leaf materials store.

Fresh leaves of twelve Bougainvillea cultivars, namely, B.× buttiana ‘Mahara’ , B. × buttiana ‘Gautama's Red’, B. × buttiana ‘California Gold’, B. × buttiana ‘Double Salmon’, B. × buttiana ‘Double Yellow’, B. × buttiana ‘Big Chitra’, B. × buttiana ‘Los Banos Beauty’, B. glabra ‘White Stripe’, B. spectabilis ‘Flame’, B. spectabilis ‘Splendens’, B. × buttiana ‘Barbara Karst’, and B. × buttiana ‘San Diego Red’ (Fig 1, S1 Table), were collected from the Provincial Flower Germplasm Resources Bank of San Jiao Mei in Zhangzhou (117°37′47″E, 24°28′35″N), Fujian Province, China. One bud mutation armed with simple or no thorns and derived from B. × buttiana ‘Miss Manila’, given namely, B. × buttiana ‘Miss Manila’ sp. 1 (Fig 1, S1 Table), was collected from the cultivation factoty of Zhangzhou (117°49′9″E, 24°31′33″N) in Xiamen Qianrihong Horticulture Co., Ltd, Fujian Province, China. Fresh leaves were quickly frozen on dry ice, sent to the laboratory of the Environmental Horticulture Research Institute (113°20′39″E, 23°8′51″N) at the Guangdong Academy of Agricultural Sciences, Guangzhou, China, and stored at −80 ℃ until use. Genomic chloroplast DNA was extracted from each sample using the modified sucrose gradient centrifugation method [13]. Then, the DNA quality and quantity were checked through agarose gel electrophoresis and the NanoDrop microspectrometer method, respectively. Each qualified DNA sample was sheared to fragments of approximately 350 bp. Short-insert (350 bp) paired-end libraries were constructed, and sequencing was performed on an Illumina NovaSeq 6000 platform with a paired read length of 150 bp (Biozeron, Shanghai, China). The raw data from each sample were checked using FastQC v. 0.11.9 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and adaptors and low-quality reads were subsequently deleted by Trimmomatic v. 0.39 [14] with default parameters. The remaining materials, including the leaves and DNA, were deposited in the laboratory of the Environmental Horticulture Research Institute (store sheet code: B2023, 113°20′39″E, 23°8′51″N), Guangdong Academy of Agricultural Sciences, Guangzhou, China, as vouchers (S1 Table).

Suggestions on figure and table presentation were sadly ignored.

Response: We revised Figure 1 to Figure 9 as following.

We did not ignore the presentation of Table 4. For table 4, ‘462 L 0.969’ and ‘462 |L|0.969’ were two different forms for presentation. We use the form of ‘462 L 0.969’, not used the form of ‘462 |L|0.969’. Because the analyzed result from the CodeML program used the form of ‘462 L 0.969’, not used the form of ‘462 |L|0.969’.

Response: Thank you for this idea. We cited the article (Lin et al. 2023), compared and discussed the chloroplast genome, phylogenetic tree and related results.

2.Since some of the cultivars are named along with their parent (perhaps they are mutant clones), it would be reasonable to at least investigate the intraspecific genetic distance of these cultivar derived from the (presumably) same species. At least the analysis is conducted on the species assembled in this study, i.e., B. x buttiana and B. spectabilis.

Response: This is a good question. There were two main reasons that we did not sequence and assemble the wild species of B.×buttiana and B. spectabilis. First, wild species of B. spectabilis had been sequenced and reported in previous studies before, such as in Wang et al. 2019 and Bautista et al. 2020, 2022. Second, in the Provincial Flower Germplasm Resources Bank of San Jiao Mei in Zhangzhou (117°37′47″E, 24°28′35″N), Fujian Province, China, there are so many cultivars of B.×buttiana. However, the wild species of B.×buttiana was not easy to be obtained and identified. Therefore, we used the published chloroplast genomes of B. spectabilis in NCBI for phylogenetic analysis.

Based on this suggestion, we added the SNPs/indels analyses among groups of B.×buttiana ‘Mahara’ vs. B. spectabilis ‘Flame’, B.×buttiana ‘Mahara’ vs. B. spectabilis ‘Splendens’, and B. spectabilis ‘Splendens’-OR253994 (Lin et al. 2023) vs. B. spectabilis ‘Splendens’-OR344372 (this study). The details results can be found in two sections of SNPs and indels analyses among the thirteen complete chloroplast genomes, and Intraspecific analyses of two chloroplast genomes of B. spectabilis ‘Splendens’.

Bautista MAC, Zheng Y, Hu Z, Deng Y, Chen T. Comparative analysis of complete chloroplast genome sequences of wild and cultivated Bougainvillea (Nyctaginaceae). Plants (Basel). 2020; 9, 1671. https://doi.org/10.3390/plants9121671 PMID: 33260641

Wang N, Qiu MY, Yang Y, Li JW, Zou XX. Complete chloroplast genome sequence of Bougainvillea spectabilis (Nyctaginaceae). Mitochondrial DNA B Resour. 2019; 4, 4010-4011. https://doi.org/ 10.1080/23802359.2019.1688716 PMID: 33366293

Reviewer #4: The manuscript titled 'Comparative analysis

Attachment

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PLoS One. doi: 10.1371/journal.pone.0310091.r006

Decision Letter 2

Pankaj Bhardwaj

26 Aug 2024

Comparative analysis of the complete chloroplast genomes of thirteen Bougainvillea cultivars from South China with implications for their genome structures and phylogenetic relationships

PONE-D-23-43492R2

Dear Dr. Li,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Pankaj Bhardwaj, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #4: All comments have been addressed

Reviewer #5: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #4: Yes

Reviewer #5: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #4: Yes

Reviewer #5: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #4: Yes

Reviewer #5: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #4: Yes

Reviewer #5: Yes

**********

6. Review Comments to the Author

Reviewer #4: (No Response)

Reviewer #5: After reading the revised MS, they have answered all raised questions. the plastid genome and all kinds of methods to build the phylogeny is solid for this paper.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #4: No

Reviewer #5: No

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PLoS One. doi: 10.1371/journal.pone.0310091.r007

Acceptance letter

Pankaj Bhardwaj

1 Sep 2024

PONE-D-23-43492R2

PLOS ONE

Dear Dr. Li,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team.

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Indels statistics of 13 newly sequenced complete chloroplast genomes of the Bougainvillea cultivars.

(DOCX)

pone.0310091.s001.docx^{(1.6MB, docx)}

S2 Fig. Phylogenetic relationships of Nyctaginaceae species based on protein-coding genes reconstructed using ML and BI methods.

(A) ML tree. (B) BI tree. The 13 newly sequenced Bougainvillea chloroplast genomes identified in this study are shown in bold.

(DOCX)

pone.0310091.s002.docx^{(2.4MB, docx)}

S1 Table. Information on the 13 Bougainvillea cultivars.

(DOCX)

pone.0310091.s003.docx^{(18.5KB, docx)}

S2 Table. The 46 complete chloroplast genomes in the Nyctaginaceae family used for determining the selective pressure and phylogenetic relationships.

(DOCX)

pone.0310091.s004.docx^{(19KB, docx)}

S3 Table. Genes distribution in the 13 chloroplast genomes of the Bougainvillea cultivars.

(XLSX)

pone.0310091.s005.xlsx^{(239.8KB, xlsx)}

S4 Table. SSRs detected in the 13 chloroplast genomes of the Bougainvillea cultivars.

(XLSX)

pone.0310091.s006.xlsx^{(167.8KB, xlsx)}

S5 Table. Long repeats detected in the 13 chloroplast genomes of the Bougainvillea cultivars.

(XLSX)

pone.0310091.s007.xlsx^{(151KB, xlsx)}

S6 Table. Codon usage in the 13 chloroplast genomes of the Bougainvillea cultivars.

(XLSX)

pone.0310091.s008.xlsx^{(48.6KB, xlsx)}

S7 Table. SNPs and indels detection among the 13 chloroplast genomes of the Bougainvillea cultivars.

(XLSX)

pone.0310091.s009.xlsx^{(339.3KB, xlsx)}

S8 Table. SNPs and indels detection between the 2 chloroplast genomes of B. spectabilis ‘Splendens’.

(XLSX)

pone.0310091.s010.xlsx^{(69.4KB, xlsx)}

S9 Table. Nuclear diversity of 16 chloroplast genomes from the 16 Bougainvillea cultivars.

(XLSX)

pone.0310091.s011.xlsx^{(34.5KB, xlsx)}

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Submitted filename: PONE-D-23-37508-point-by-point response.docx

pone.0310091.s012.docx^{(11.3KB, docx)}

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pone.0310091.s013.docx^{(6MB, docx)}

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pone.0310091.s014.pdf^{(4.6MB, pdf)}

Data Availability Statement

Data Availability Statement: The data presented in this study were submitted to the NCBI repository (https://www.ncbi.nlm.nih.gov) under accession numbers OR344366–OR344378.

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PERMALINK

Comparative analysis of the complete chloroplast genomes of thirteen Bougainvillea cultivars from South China with implications for their genome structures and phylogenetic relationships

Xiao-Ye Wu

He-Fa Wang

Shui-Ping Zou

Lan Wang

Gen-Fa Zhu

Dong-Mei Li

Roles

Abstract

Introduction

Fig 1. Morphologies among 13 cultivars of the Bougainvillea genus.

Materials and methods

Plant materials, DNA extraction, and sequencing

Chloroplast genome assembly and annotation

Table 1. Characteristics of the 13 newly sequenced complete chloroplast genomes of Bougainvillea cultivars.

Analyses of SSRs and long repeats

Analysis of codon usage

Comparative genomics and sequence divergence analyses

Selection pressure analysis in the Nyctaginaceae family

Phylogenetic relationships in the Bougainvillea genus and the Nyctaginaceae family

Results

General characteristics of the thirteen complete chloroplast genomes

Fig 2. Chloroplast genome map of B. × buttiana ‘Mahara’ (the outermost three rings) and CGView comparison of 13 complete chloroplast genomes of Bougainvillea cultivars (the inner rings with different colors).

Table 2. Genes present in the 13 newly sequenced chloroplast genomes of Bougainvillea cultivars.

SSRs and long repeats analyses

Fig 3. Distribution of SSRs in the 13 newly sequenced Bougainvillea chloroplast genomes.

Fig 4. Analysis of long repeat sequences in the 13 newly sequenced Bougainvillea chloroplast genomes.

Codon usage analysis

Fig 5. Codon contents of all protein-coding genes of 13 newly sequenced complete chloroplast genomes of the Bougainvillea cultivars.

SNPs and indels analyses among the thirteen complete chloroplast genomes

Table 3. Distribution of SNPs and indels among the 13 newly sequenced complete chloroplast genomes of Bougainvillea cultivars.

Intraspecific analyses of two chloroplast genomes of B. spectabilis ‘Splendens’

IR expansion and contraction

Fig 6. Comparison of the borders of the LSC, SSC, and IR regions among the 16 Bougainvillea chloroplast genomes.

Sequence divergence analysis

Fig 7. Complete chloroplast genome comparison of the 16 Bougainvillea chloroplast genomes using B. × buttiana ‘Mahara’ as a reference.

Fig 8. Comparisons of nucleotide diversity (Pi) values among 16 complete chloroplast genomes of Bougainvillea cultivars.

Selection pressure analysis of the Nyctaginaceae family

Table 4. Positively selected sites detected in 46 complete chloroplast genomes of the Nyctaginaceae family.

Phylogenetic relationships

Fig 9. Phylogenetic relationships of Nyctaginaceae species based on chloroplast genomes sequences reconstructed using maximum likelihood (ML) and Bayesian inference (BI) methods.

Discussion

Conclusions

Supporting information

Data Availability

Funding Statement

References

Author response to previous submission

Decision Letter 0

Pankaj Bhardwaj

Roles

Author response to Decision Letter 0

Decision Letter 1

Pankaj Bhardwaj

Roles

Author response to Decision Letter 1

Decision Letter 2

Pankaj Bhardwaj

Roles

Acceptance letter

Pankaj Bhardwaj

Roles

Associated Data

Supplementary Materials

Data Availability Statement

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