CP genome and phylogenetic relationships of Bougainvillea spectabilis and B. glabra

Citation: Zhang, H.; Huang, T.; Zhou,
Q.; Sheng, Q.; Zhu, Z. Complete
Chloroplast Genomes and
Phylogenetic Relationships of
Bougainvillea spectabilis and
Bougainvillea glabra (Nyctaginaceae).
Int. J. Mol. Sci. 2023, 24, 13044.
https://doi.org/10.3390/
ijms241713044
Academic Editor: Pedro
Martínez-Gómez
Received: 21 July 2023
Revised: 15 August 2023
Accepted: 17 August 2023
Published: 22 August 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
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Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
International Journal of
Molecular Sciences
Article
Complete Chloroplast Genomes and Phylogenetic Relationships of
Bougainvillea spectabilis and Bougainvillea glabra (Nyctaginaceae)
Huihui Zhang 1,2, Tao Huang 1,2, Qi Zhou 1, Qianqian Sheng 1,2 and Zunling Zhu 1,2,3,*
1 College of Landscape Architecture, Nanjing Forestry University, Nanjing 210037, China;
zhanghuihui@njfu.edu.cn (H.Z.); bougainvillea97@163.com (T.H.); zhouqi514@njfu.edu.cn (Q.Z.);
qqs@njfu.edu.cn (Q.S.)
2 Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University,
Nanjing 210037, China
3 Jinpu Research Institute, Nanjing Forestry University, Nanjing 210037, China
* Correspondence: zhuzunling@njfu.edu.cn; Tel.: +86-25-68224603
Abstract: Bougainvillea L. (Nyctaginaceae) is a South American native woody flowering shrub of high
ornamental, economic, and medicinal value which is susceptible to cold damage. We sequenced
the complete chloroplast (cp) genome of B. glabra and B. spectabilis, two morphologically similar
Bougainvillea species differing in cold resistance. Both genomes showed a typical quadripartite
structure consisting of one large single-copy region, one small single-copy region, and two inverted
repeat regions. The cp genome size of B. glabra and B. spectabilis was 154,520 and 154,542 bp, re-
spectively, with 131 genes, including 86 protein-coding, 37 transfer RNA, and 8 ribosomal RNA
genes. In addition, the genomes contained 270 and 271 simple sequence repeats, respectively, with
mononucleotide repeats being the most abundant. Eight highly variable sites (psbN, psbJ, rpoA, rpl22,
psaI, trnG-UCC, ndhF, and ycf1) with high nucleotide diversity were identified as potential molecular
markers. Phylogenetic analysis revealed a close relationship between B. glabra and B. spectabilis. These
findings not only contribute to understanding the mechanism by which the cp genome responds to
low-temperature stress in Bougainvillea and elucidating the evolutionary characteristics and phyloge-
netic relationships among Bougainvillea species, but also provide important evidence for the accurate
identification and breeding of superior cold-tolerant Bougainvillea cultivars.
Keywords: Bougainvillea; Nyctaginaceae; chloroplast genome; genome comparative analysis; phylogeny
1. Introduction
Species of the genus Bougainvillea L., belonging to the family Nyctaginaceae, are
of high horticultural ornamental value. They are tropical and subtropical woody vines
characterized by vibrant bracts, a long flowering period, and high stress tolerance, mak-
ing them ideal ornamental horticultural plants [1]. Recent studies have discovered that
Bougainvillea potentially has anti-inflammatory, anticancer, antioxidant, antimicrobial, and
antihyperglycemic properties [2–6]. This plant group has attracted widespread attention in
horticulture, the pharmaceutical industry, and environmental research [7]. Bougainvillea is
native to Peru, southern Argentina, and Brazil in South America, but is widely cultivated as
landscape plants in other warm climate regions such as the Pacific Islands, Southeast Asia,
the Mediterranean, Australia, and the Caribbean Islands [8]. The genus comprises approx-
imately 18 species, among which Bougainvillea spectabilis Willdenow, Bougainvillea glabra
Choisy, and Bougainvillea peruviana Humboldt and Bonpland are native species and serve as
breeding materials for major horticultural cultivars [9]. By hybridizing and mutating these
three native species and one hybrid species, Bougainvillea x buttiana Holttum & Standley,
many modern horticultural cultivars with different colors, shapes, and bract sizes have been
developed. Currently, there are more than 400 Bougainvillea cultivars worldwide. However,
the frequent hybridization of Bougainvillea species because of commercial demands has led
Int. J. Mol. Sci. 2023, 24, 13044. https://doi.org/10.3390/ijms241713044 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2023, 24, 13044 2 of 15
to intricate genetic relationships among many hybrid varieties, resulting in unclear phy-
logenetic and evolutionary relationships [1,10]. Additionally, due to their origin in South
America, Bougainvillea plants exhibit a relatively low tolerance to cold temperatures. They
are susceptible to cold-related damage throughout their growth, significantly constraining
their potential for promotion, application, and the overall development of the Bougainvillea
industry chain.
Chloroplasts (cps), which are the organelles responsible for photosynthesis in most
green plants, participate in developmental processes and secondary metabolic activities,
and coordinate gene expression between organelles and the nuclear genome [11,12]. More
and more studies have shown that cps play a very important role in plants’ resistance to
various environmental stresses [13]. They act as sensors of environmental stresses, con-
necting diverse stress responses and cellular signaling pathways [14]. The cp genome of
angiosperms typically exhibits a characteristic quadripartite structure, consisting of a large
single-copy region (LSC), a small single-copy region (SSC), and a pair of inverted repeat
regions (IRa and IRb) [15]. It is characterized by structural stability, high conservation,
slow molecular evolution, low molecular weight, and maternal inheritance, making it
widely applicable in research areas such as molecular marker development and phylogenet-
ics [13,16,17]. Remarkably, research reports regarding the genome of Bougainvillea remain
conspicuously absent, which seriously hinders the process of improving its cold resistance.
However, the cp genome of Bougainvillea is relatively small and simple. Therefore, studying
the cp genome of Bougainvillea can help promote the improvement of its cold resistance,
especially in terms of molecular marker selection. Although there have been studies on
the cp genomes of species within this genus [7,18–20], current research has predominantly
focused on Bougainvillea species, emphasizing the study of the phylogenetic and classifica-
tion of Bougainvillea from the perspective of plastids. Research reports on the cp genome
structure, composition, and variation characteristics among key cultivated varieties are
very limited. Furthermore, the response of cps to low-temperature stress in Bougainvillea
remains to be unveiled.
Bougainvillea glabra and B. spectabilis are well-known cultivated Bougainvillea vari-
eties [21]. Strong evidence indicates that the morphological and horticultural diversity of
Bougainvillea is primarily due to the variability within B. glabra and B. spectabilis, which
have undergone indistinguishable hybridization with each other and with B. peruviana.
Bougainvillea glabra and B. spectabilis are highly similar in terms of their basic morphological
characteristics, which has consequently led to confusion in the identification of their culti-
vated varieties. The former has elliptical, hairless leaves and the flower bracts are covered
with fine pubescence or are smooth, whereas the latter has larger, ovate leaves with hairs
and the flower bracts are covered with long stiff or soft hairs [22,23]. It was not until the
mid-1980s that botanists classified them as distinct species [24]. Studies have shown that
B. glabra exhibits the strongest cold resistance and has the widest range of applications,
whereas B. spectabilis has the poorest cold resistance [25,26]. ‘Brasiliensis’ and ‘Auratus’ are
excellent cultivars of B. glabra and B. spectabilis, respectively.
In this study, we sequenced the complete cp genomes of B. glabra ‘Brasiliensis’ and
B. spectabilis ‘Auratus’, two morphologically similar Bougainvillea cultivars differing in cold
resistance. We comparatively analyzed the cp genomes of B. glabra, B. spectabilis, and other
Bougainvillea species aiming to explore the differences in cp genome structure, composition,
and variation characteristics. We also studied the phylogenetic relationships among these
two and other Bougainvillea species. The results of this study not only establish a foundation
for understanding the mechanism by which the cp genome responds to low-temperature
stress in Bougainvillea, but also provide important evidence for the accurate identification
and breeding of superior cold-tolerant Bougainvillea cultivars.

Int. J. Mol. Sci. 2023, 24, 13044 3 of 15
2. Results
2.1. Basic Characteristics and Comparative Analysis of the cp Genomes
The cp genomes of B. glabra and B. spectabilis were 154,520 and 154,542 base pairs (bp)
in length, respectively (Figure 1). These cp genomes, similar to those of other Bougainvillea
species, were covalently closed double-stranded circular molecules with a typical quadri-
partite structure comprising (i) an LSC with a length of 85,688 and 85,695 bp, respectively,
accounting for 55.5% of the total genome length in both species; (ii) an SSC with a length of
18,078 and 18,077 bp, respectively, accounting for 11.7% of the total genome length in both
species; and (iii) a pair of IRs separating the SSC and LSC regions, with a size of 25,377 and
25,385 bp, respectively, covering 16.4% of the total genome in both species.
The cp genomes of B. glabra and B. spectabilis were 154,520 and 154,542 base pairs (bp)
in length, respectively (Figure 1). These cp genomes, similar to those of other Bougainvillea
species, were covalently closed double-stranded circular molecules with a typical quadri-
partite structure comprising (i) an LSC with a length of 85,688 and 85,695 bp, respectively,
accounting for 55.5% of the total genome length in both species; (ii) an SSC with a length
of 18,078 and 18,077 bp, respectively, accounting for 11.7% of the total genome length in
both species; and (iii) a pair of IRs separating the SSC and LSC regions, with a size of
25,377 and 25,385 bp, respectively, covering 16.4% of the total genome in both species.
A comparative analysis of the cp genomes of B. glabra and B. spectabilis and four re-
lated Bougainvillea species revealed that the cp genome size ranged from 153,966 (B. peru-
viana) to 154,872 bp (B. spinosa) (Table 1). Their gene structure, GC content, gene num-
ber, mRNA, tRNA, and rRNA were similar, indicating a slow evolution of species
within Bougainvillea. The GC content of the cp genomes of B. glabra and B. spectabilis
was identical (36.46%). Co-linearity analysis using Mauve software (http://dar-
linglab.org/mauve, accessed on 24 March 2023) revealed that the structure and gene
arrangement sequences of the cp genomes among the six species of Bougainvillea were
largely similar, with no evident gene rearrangements or inversions. This indicated a
high conservation of cp genome sequences in Bougainvillea species (Figure 2).
Figure 1. Genome maps of the (A) B. glabra and (B) B. spectabilis cp genomes. Genes placed outside
the circle are transcribed clockwise, whereas genes inside the circle are transcribed counterclock-
wise. Gene colors diﬀerentiate protein-coding genes based on their respective functions. LSC, large
single-copy region; SSC, small single-copy region; IRA and IRB, two inverted repeats; GC content,
dark grey area in inner circle; AT content, light grey area in inner circle.
Figure 1. Genome maps of the (A) B. glabra and (B) B. spectabilis cp genomes. Genes placed outside
the circle are transcribed clockwise, whereas genes inside the circle are transcribed counterclockwise.
Gene colors differentiate protein-coding genes based on their respective functions. LSC, large single-
copy region; SSC, small single-copy region; IRA and IRB, two inverted repeats; GC content, dark grey
area in inner circle; AT content, light grey area in inner circle.
A comparative analysis of the cp genomes of B. glabra and B. spectabilis and four related
Bougainvillea species revealed that the cp genome size ranged from 153,966 (B. peruviana) to
154,872 bp (B. spinosa) (Table 1). Their gene structure, GC content, gene number, mRNA,
tRNA, and rRNA were similar, indicating a slow evolution of species within Bougainvillea.
The GC content of the cp genomes of B. glabra and B. spectabilis was identical (36.46%).
Co-linearity analysis using Mauve software (http://darlinglab.org/mauve, accessed on
24 March 2023) revealed that the structure and gene arrangement sequences of the cp
genomes among the six species of Bougainvillea were largely similar, with no evident gene
rearrangements or inversions. This indicated a high conservation of cp genome sequences
in Bougainvillea species (Figure 2).

Int. J. Mol. Sci. 2023, 24, 13044 4 of 15
Table 1. Complete cp genome features of B. glabra, B. spectabilis, B. peruviana, B. pachyphylla, B. praecox,
and B. spinosa.
Genome Feature Bougainvillea
glabra
Bougainvillea
spectabilis
Bougainvillea
peruviana
Bougainvillea
pachyphylla
Bougainvillea
praecox
Bougainvillea
spinosa
Genome size (bp) 154,520 154,542 153,966 154,062 154,306 154,872
LSC length (bp) 85,688 85,695 85,159 85,181 85,474 85,846
SSC length (bp) 18,078 18,077 18,025 18,027 18,014 18,020
IR length (bp) 25,377 25,385 25,391 25,427 25,409 25,503
Number of genes 131 131 131 131 131 131
Number of protein-coding genes 86 86 86 86 86 86
Number of rRNA genes 8 8 8 8 8 8
Number of tRNA genes 37 37 37 37 37 37
GC content (%) 36.5 36.5 36.6 36.5 36.5 36.4
GC content in LSC (%) 34.2 34.2 34.3 34.3 34.3 34.1
GC content in SSC (%) 29.5 29.5 29.6 29.6 29.5 29.4
GC content in IR (%) 42.8 42.8 42.8 42.8 42.8 42.9
LSC, large single copy; SSC, small single copy; IR, inverted repeat.
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 4 of 18
Table 1. Complete cp genome features of B. glabra, B. spectabilis, B. peruviana, B. pachyphylla, B. prae-
cox, and B. spinosa.
Genome Feature
Bougainvillea
glabra
Bougainvillea
spectabilis
Bougainvillea
peruviana
Bougainvillea
pachyphylla
Bougainvillea
praecox
Bougainvillea
spinosa
Genome size (bp) 154,520 154,542 153,966 154,062 154,306 154,872
LSC length (bp) 85,688 85,695 85,159 85,181 85,474 85,846
SSC length (bp) 18,078 18,077 18,025 18,027 18,014 18,020
IR length (bp) 25,377 25,385 25,391 25,427 25,409 25,503
Number of genes 131 131 131 131 131 131
Number of protein-coding
genes
86 86 86 86 86 86
Number of rRNA genes 8 8 8 8 8 8
Number of tRNA genes 37 37 37 37 37 37
GC content (%) 36.5 36.5 36.6 36.5 36.5 36.4
GC content in LSC (%) 34.2 34.2 34.3 34.3 34.3 34.1
GC content in SSC (%) 29.5 29.5 29.6 29.6 29.5 29.4
GC content in IR (%) 42.8 42.8 42.8 42.8 42.8 42.9
LSC, large single copy; SSC, small single copy; IR, inverted repeat.
Figure 2. Co-linearity analysis of cp genomes among six Bougainvillea species. Within the align-
ments, local collinear blocks are represented by blocks of the same color connected by lines.
2.2. Gene Composition of the cp Genomes
Gene annotation revealed that both the B. glabra and B. spectabilis cp genomes con-
tained 131 genes, including 86 protein-coding, 37 transfer (t)RNA, and 8 ribosomal
(r)RNA genes (Tables 1 and 2). These genes could be categorized into four groups: photo-
synthesis-related genes, self-expression-related genes, other genes, and unknown genes.
The types and number of genes in these four categories were identical between the B. gla-
bra and B. spectabilis cp genomes. There were 45 photosynthesis-related genes, including
five (ndhA, ndhB, petB, petD, and atpF) with introns. ndhB was present in two copies in the
IR regions. There were 74 self-expression-related genes, with one intron each in rpl16,
rps16, rpoC1, trnA-UGC, trnI-GAU, trnK-UUU, trnL-UAA, and trnV-UAC and two introns
Figure 2. Co-linearity analysis of cp genomes among six Bougainvillea species. Within the alignments,
local collinear blocks are represented by blocks of the same color connected by lines.
2.2. Gene Composition of the cp Genomes
Gene annotation revealed that both the B. glabra and B. spectabilis cp genomes contained
131 genes, including 86 protein-coding, 37 transfer (t)RNA, and 8 ribosomal (r)RNA genes
(Tables 1 and 2). These genes could be categorized into four groups: photosynthesis-
related genes, self-expression-related genes, other genes, and unknown genes. The types
and number of genes in these four categories were identical between the B. glabra and
B. spectabilis cp genomes. There were 45 photosynthesis-related genes, including five (ndhA,
ndhB, petB, petD, and atpF) with introns. ndhB was present in two copies in the IR regions.
There were 74 self-expression-related genes, with one intron each in rpl16, rps16, rpoC1,
trnA-UGC, trnI-GAU, trnK-UUU, trnL-UAA, and trnV-UAC and two introns in rps12. Fifteen
genes (rpl2, rpl23, rps7, rps12, rrn4.5, rrn5, rrn16, rrn23, trnA-UGC, trnI-CAU, trnI-GAU,
trnL-CAA, trnN-GUU, trnR-ACG, and trnV-GAC) were present in two copies in the IR
regions. clpP in the ‘other genes’ category contained two introns. The unknown genes ycf1
and ycf2 were located in the IR regions and existed in two copies, whereas ycf3 contained
two introns (Figure 1, Tables 2, S1 and S2). Except for three introns with different lengths,

Int. J. Mol. Sci. 2023, 24, 13044 5 of 15
the remaining introns had the same length in both species. ndhB had two introns of the
same length in each species: 660 bp in B. glabra and 668 bp in B. spectabilis. Additionally,
the introns in rps16 and petB of B. glabra were 887 and 777 bp in size, respectively, which
were one base pair longer than those in B. spectabilis (Tables S1 and S2).
Table 2. Annotated genes and their classification in the cp genomes of B. glabra and B. spectabilis.
Category Group Genes
Photosynthesis 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
Subunits of NADH dehydrogenase ndhA *, ndhB * (2), ndhC, ndhD, ndhE, ndhF, ndhG,
ndhH, ndhI, ndhJ, ndhK
Subunits of cytochrome b/f complex petA, petB *, petD *, petG, petL, petN
Subunits of ATP synthase atpA, atpB, atpE, atpF *, atpH, atpI
Large subunit of rubisco rbcL
Subunits protochlorophyllide reductase -
Self-replication Proteins of large ribosomal subunit rpl14, rpl16 *, rpl2 * (2), rpl20, rpl22, rpl23 (2), rpl32,
rpl33, rpl36
Proteins of small ribosomal subunit rps11, rps12 ** (2), rps14, rps15, rps16 *, rps18, rps19,
rps2, rps3, rps4, rps7 (2), rps8
Subunits of RNA polymerase rpoA, rpoB, rpoC1 *, rpoC2
Ribosomal RNAs rrn16 (2), rrn23 (2), rrn4.5 (2), rrn5 (2)
Transfer RNAs
trnA-UGC * (2), trnC-GCA, trnD-GUC,
trnE-UUC, trnF-GAA, trnG-GCC, trnG-UCC *,
trnH-GUG, trnI-CAU (2), trnI-GAU * (2), trnK-UUU *,
trnL-CAA (2), trnL-UAA *, trnL-UAG,
trnM-CAU, 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, trnfM-CAU
Other genes Maturase matK
Protease clpP **
Envelope membrane protein cemA
Acetyl-CoA carboxylase accD
c-type cytochrome synthesis gene ccsA
Translation initiation factor infA
Other -
Genes of unknown function
Conserved hypothetical chloroplast open
reading frame
ycf1 (2), ycf2 (2), ycf3 **, ycf4
Gene *, gene with one intron; Gene **, gene with two introns; Gene (2), number of copies of multi-copy genes.
2.3. Codon Usage
Relative synonymous codon usage (RSCU) was used to assess the usage of synony-
mous codons in the coding sequences, with a higher RSCU value indicating stronger
preference [27]. Statistical analysis of the codon numbers and RSCU in the cp DNA of
B. glabra and B. spectabilis revealed that they shared the same number of codons (26,599)
and different amino acid types encoded by these codons (21). With respect to the codon
numbers encoding other amino acid types, the codon numbers were the same except for
lysine (Lys) and asparagine (Asn), which had different codon numbers (1477 and 1296 in
B. glabra, 1476 and 1297 in B. spectabilis, respectively). Leucine (Leu) was the most abundant
amino acid, with 2800 codons (accounting for 10.53% of the total codons), followed by
isoleucine (Ile) with 2317 codons (8.71% of the total). Cysteine (Cys) was the least abundant,
with 297 codons (1.12% of the total) (Table S3). As shown in Figure 3, except for tryptophan
(Trp), all amino acids were encoded by two or more synonymous codons, and methionine
(Met) was encoded by seven synonymous codons. The preferred synonymous codons
(RSCU > 1) mainly ended with A or U, i.e., A/T bases.
2.4. Simple Sequence Repeat Analysis
Three types of repetitive sequences were detected in the cp genomes of B. glabra
and B. spectabilis: forward repeats, reverse repeats, and palindromic repeats. There were
19 forward repeats, 27 palindromic repeats, and 2 reverse repeats in both species (Figure 4,
Tables S4 and S5).

Int. J. Mol. Sci. 2023, 24, 13044 6 of 15
Figure 3. Relative synonymous codon usage (RSCU) values of 20 amino acids and stop codons in
the protein-coding genes of the (A) B. glabra and (B) B. spectabilis cp genomes. Boxes below the
graphs represent all codons encoding each amino acid. The colors of the histograms correspond to
the colors of the codons.
2.4. Simple Sequence Repeat Analysis
Three types of repetitive sequences were detected in the cp genomes of B. glabra and
B. spectabilis: forward repeats, reverse repeats, and palindromic repeats. There were 19
forward repeats, 27 palindromic repeats, and 2 reverse repeats in both species (Figure 4,
Tables S4 and S5).
Figure 3. Relative synonymous codon usage (RSCU) values of 20 amino acids and stop codons in the
protein-coding genes of the (A) B. glabra and (B) B. spectabilis cp genomes. Boxes below the graphs
represent all codons encoding each amino acid. The colors of the histograms correspond to the colors
of the codons.
In total, 270 and 271 simple sequence repeat (SSR) loci were identified in the cp
genomes of B. glabra and B. spectabilis, respectively. Among them, there were 183 mononu-
cleotide repeats (67.78% and 67.53% of the total, respectively), 8 dinucleotide repeats (2.96%
and 2.95%), 68 trinucleotide repeats (25.19% and 25.09%), 10 tetranucleotide repeats (3.70%
and 3.69%), and 1 and 2 pentanucleotide repeats (0.37% and 0.74%) (Figure 5A, Tables
S6 and S7). In the LSC region, there were 177 and 178 SSR loci (65.60% and 65.70%) in
B. glabra and B. spectabilis, respectively, whereas the SSC region had 49 SSR loci (18.10%)
and the IR region had 44 SSR loci (16.30%) in both species (Figure 5B). The majority of

Int. J. Mol. Sci. 2023, 24, 13044 7 of 15
single-base repeats were A/T, and the dinucleotide repeats were mostly AT/TA (Figure 5C).
A total of 128 (47.41%) and 129 (47.60%) SSRs were located in intergenic regions in B.
glabra and B. spectabilis, respectively, whereas 99 SSRs (36.67% and 36.53%) were located in
protein-coding genes and 43 SSRs (15.92% and 15.87%) were located in introns (Figure 5D).
Figure 4. Numbers of diﬀerent repeats in the cp genomes of (A) B. glabra and (B) B. spectabilis. F,
forward repeat; P, palindromic repeat; R, reverse repeat; C, complement repeat.
In total, 270 and 271 simple sequence repeat (SSR) loci were identified in the cp ge-
nomes of B. glabra and B. spectabilis, respectively. Among them, there were 183 mononu-
cleotide repeats (67.78% and 67.53% of the total, respectively), 8 dinucleotide repeats
(2.96% and 2.95%), 68 trinucleotide repeats (25.19% and 25.09%), 10 tetranucleotide re-
peats (3.70% and 3.69%), and 1 and 2 pentanucleotide repeats (0.37% and 0.74%) (Figure
5A, Tables S6 and S7). In the LSC region, there were 177 and 178 SSR loci (65.60% and
65.70%) in B. glabra and B. spectabilis, respectively, whereas the SSC region had 49 SSR loci
(18.10%) and the IR region had 44 SSR loci (16.30%) in both species (Figure 5B). The ma-
jority of single-base repeats were A/T, and the dinucleotide repeats were mostly AT/TA
(Figure 5C). A total of 128 (47.41%) and 129 (47.60%) SSRs were located in intergenic re-
gions in B. glabra and B. spectabilis, respectively, whereas 99 SSRs (36.67% and 36.53%)
were located in protein-coding genes and 43 SSRs (15.92% and 15.87%) were located in
introns (Figure 5D).
Figure 4. Numbers of different repeats in the cp genomes of (A) B. glabra and (B) B. spectabilis. F,
forward repeat; P, palindromic repeat; R, reverse repeat; C, complement repeat.
2.5. Nucleotide Diversity of Genes
Nucleotide diversity (Pi) values for B. glabra and B. spectabilis were calculated using
DnaSP software v5.10.1. The results showed that the Pi values in the two cp genomes
ranged from 0 to 0.20282, with an average of 0.00401. Eight highly variable regions with
Pi > 0.007 were detected. Among them, six were located in the LSC region (psbN, psbJ,
rpoA, rpl22, psaI, and trnG-UCC) and two were located in the SSC region (ndhF and ycf1)
(Figure 6).
2.6. Analysis of IR Boundary Changes
As shown in Figure 7, the IR boundaries exhibited a high degree of conservation
between B. glabra and B. spectabilis. The gene content and expansion extent of the boundary
regions were identical. When the cp genomes were compared with those of four other
Bougainvillea species, all six species were found to have the same genes at the boundaries,
but with slightly different lengths.
The LSC/IRb (JLB) boundaries of all six species were located in the ycf1-coding region.
Except for B. spinosa, whose ycf1 gene crossed the JLB boundary by 152 bp, the ycf1 genes
of the other five species crossed the JLB boundary by 114 bp. The expansion range of the
SSC/IRb (JSB) boundary showed that in all six Bougainvillea species, the JSB boundary was
located between ycf1 and ndhF, with slight differences in the extent of expansion. The ycf1
genes of all six species extended 3 bp beyond the boundary into the SSC region. The IRb
region was 1371 bp in length in B. glabra and B. spectabilis; 1374 bp in B. praecox, B. pachyphylla,
and B. spinosa; and 1335 bp in B. peruviana. ndhF was located near the boundary on the SSC
side, and the ndhF genes of all species extended 21 bp beyond the boundary into the IRb
region. The expansion range of the SSC/IRa (JSA) boundary showed that the JSB boundary in
all six Bougainvillea species was located within ycf1, with trnN on the right side, but with slight
differences in the extent of expansion. The expansion range of the LSC/IRa (JLA) boundary
showed that the JLA boundary had the same genes, with rpl2 on the left side and trnH on the
right side, but with slight differences in the extent of expansion. rpl2 of B. glabra, B. spectabilis,
and B. praecox was located 176 bp from the boundary, whereas trnH was located 22 bp from
the boundary. rpl2 of B. peruviana and B. pachyphylla was located 177 bp from the boundary,
and trnH was located 17 bp from the boundary. rpl2 of B. spinosa was located 219 bp from the
boundary, and trnH was located 10 bp from the boundary.

Int. J. Mol. Sci. 2023, 24, 13044 8 of 15
Figure 5. Analysis of simple sequence repeats (SSRs) in the cp genomes of B. glabra and B. spectabilis.
(A) Number of different types of SSRs identified in the cp genomes. (B) SSR distributions in the LSC,
SSC, and IR regions of the cp genomes. (C) Frequencies of various SSR types identified in the cp
genomes. (D) Positional distribution of SSRs in B. glabra and B. spectabilis.

Int. J. Mol. Sci. 2023, 24, 13044 9 of 15
Nucleotide diversity (Pi) values for B. glabra and B. spectabilis were calculated using
DnaSP software v5.10.1. The results showed that the Pi values in the two cp genomes
ranged from 0 to 0.20282, with an average of 0.00401. Eight highly variable regions with
Pi > 0.007 were detected. Among them, six were located in the LSC region (psbN, psbJ, rpoA,
rpl22, psaI, and trnG-UCC) and two were located in the SSC region (ndhF and ycf1) (Figure
6).
Figure 6. Nucleotide polymorphism analysis of the cp genomes of B. glabra and B. spectabilis. Names
of protein-coding genes and genes of the intergenic region are along the X-axis, and the nucleotide
diversity (Pi) value in each window is along the Y-axis.
2.6. Analysis of IR Boundary Changes
As shown in Figure 7, the IR boundaries exhibited a high degree of conservation be-
tween B. glabra and B. spectabilis. The gene content and expansion extent of the boundary
regions were identical. When the cp genomes were compared with those of four other
Bougainvillea species, all six species were found to have the same genes at the boundaries,
but with slightly different lengths.
The LSC/IRb (JLB) boundaries of all six species were located in the ycf1-coding re-
gion. Except for B. spinosa, whose ycf1 gene crossed the JLB boundary by 152 bp, the ycf1
genes of the other five species crossed the JLB boundary by 114 bp. The expansion range
of the SSC/IRb (JSB) boundary showed that in all six Bougainvillea species, the JSB bound-
ary was located between ycf1 and ndhF, with slight differences in the extent of expansion.
The ycf1 genes of all six species extended 3 bp beyond the boundary into the SSC region.
The IRb region was 1371 bp in length in B. glabra and B. spectabilis; 1374 bp in B. praecox,
B. pachyphylla, and B. spinosa; and 1335 bp in B. peruviana. ndhF was located near the bound-
ary on the SSC side, and the ndhF genes of all species extended 21 bp beyond the boundary
into the IRb region. The expansion range of the SSC/IRa (JSA) boundary showed that the
JSB boundary in all six Bougainvillea species was located within ycf1, with trnN on the right
side, but with slight differences in the extent of expansion. The expansion range of the
Figure 6. Nucleotide polymorphism analysis of the cp genomes of B. glabra and B. spectabilis. Names
of protein-coding genes and genes of the intergenic region are along the X-axis, and the nucleotide
diversity (Pi) value in each window is along the Y-axis.
LSC/IRa (JLA) boundary showed that the JLA boundary had the same genes, with rpl2 on
the left side and trnH on the right side, but with slight differences in the extent of expan-
sion. rpl2 of B. glabra, B. spectabilis, and B. praecox was located 176 bp from the boundary,
whereas trnH was located 22 bp from the boundary. rpl2 of B. peruviana and B. pachyphylla
was located 177 bp from the boundary, and trnH was located 17 bp from the boundary.
rpl2 of B. spinosa was located 219 bp from the boundary, and trnH was located 10 bp from
the boundary.
Figure 7. Comparison of IR boundaries in the cp genomes of six Bougainvillea species. JLA, junction
between LSC and IRa; JLB, junction between LSC and IRb; JSA, junction between SSC and IRa; JSB,
junction between SSC and IRb.
2.7. Phylogenetic Relationships
To determine the phylogenetic positions and relationships of the cp genomes of B.
glabra and B. spectabilis, the two reassembled Bougainvillea cp genomes were compared
with the published cp genomes of 15 Caryophyllales species, and a phylogenetic tree was
constructed (Figure 8). The results showed high support (>90%) for all branch nodes ex-
Figure 7. Comparison of IR boundaries in the cp genomes of six Bougainvillea species. JLA, junction
between LSC and IRa; JLB, junction between LSC and IRb; JSA, junction between SSC and IRa; JSB,
junction between SSC and IRb.

Int. J. Mol. Sci. 2023, 24, 13044 10 of 15
2.7. Phylogenetic Relationships
To determine the phylogenetic positions and relationships of the cp genomes of
B. glabra and B. spectabilis, the two reassembled Bougainvillea cp genomes were compared
with the published cp genomes of 15 Caryophyllales species, and a phylogenetic tree was
constructed (Figure 8). The results showed high support (>90%) for all branch nodes except
one. The outgroup species Buxus microphylla and Pachysandra terminalis formed one branch,
whereas the 15 Caryophyllales (including Caryophyllaceae, Amaranthaceae, and Nyctag-
inaceae) species formed a larger branch, clearly distinct from the outgroup. The branch
formed by the two Caryophyllaceae species Silene wilfordii and Silene latifolia (branch A) was
sister to the branch formed by the Amaranthaceae species Amaranthus hypochondriacus and
Amaranthus caudatus (branch B). The branch consisting of branch A and branch B formed a
sister group with the larger branch C formed by 11 other Nyctaginaceae species. Within
branch C, Nyctaginia capitata, Mirabilis jalapa, and Acleisanthes obtusa formed a sister group
with 100% support. Guapira discolor and Pisonia aculeata were separated on another branch,
and the remaining six Bougainvillea species formed a sister group. Bougainvillea pachyphylla
and B. peruviana were basal to the Nyctaginaceae clade. Bougainvillea glabra and B. spectabilis
formed a sister group, which was sister to B. praecox with 100% support. The branch formed
by B. glabra, B. spectabilis, and B. praecox was sister to B. spinosa with 89% support.
Bougainvillea pachyphylla and B. peruviana were basal to the Nyctaginaceae clade. Bougain-
villea glabra and B. spectabilis formed a sister group, which was sister to B. praecox with
100% support. The branch formed by B. glabra, B. spectabilis, and B. praecox was sister to B.
spinosa with 89% support.
Figure 8. Maximum likelihood phylogenetic tree reconstructed based on the complete cp genome
sequences of 17 species. Bougainvillea microphylla and Pachysandra terminalis were used as the out-
group. Numbers represent bootstrap values (%). A, B and C represent three branches formed by
Caryophyllales species. The black stars represent the positions of B. glabra and B. spectabilis.
3. Discussion
For the first time, this study sequenced, assembled, and analyzed the cp genomes of
B. glabra ‘Brasiliensis’ and B. spectabilis ‘Auratus’, two morphologically similar Bougainvil-
lea cultivars diﬀering in cold resistance. The results revealed that the cp genomes of these
two species possess a typical quadripartite structure, with one LSC, one SSC, and two IR
regions, consistent with the cp genome structures of other Bougainvillea species and the
most common structure in plant cp genomes [7,18,19]. The cp genome size of B. glabra and
B. spectabilis was 154,520 and 154,542 bp, respectively, which was relatively large for Bou-
gainvillea. The size of the sequenced cp genomes was found to be similar to the size of the
earlier reported cp genomes of B. glabra (154,536 bp; 154,763 bp) and B. spectabilis (154,541
bp) [18,20]. Bougainvillea glabra and B. spectabilis exhibit the same total GC content in their
cp genomes, and the GC content is significantly higher in the IR region than in the LSC
and SSC regions. GC content plays an important role in genome variation, and its uneven
distribution may contribute to the conservation of the LSC, SSC, and IR regions. Addition-
ally, B. glabra and B. spectabilis show consistency in the number of total, protein-coding,
rRNA and tRNA genes and introns, indicating a high similarity in their cp genome se-
quences, which partially explains the similarity in their morphological characteristics.
During biological evolution, codon usage bias is commonly observed among species
Figure 8. Maximum likelihood phylogenetic tree reconstructed based on the complete cp genome
sequences of 17 species. Bougainvillea microphylla and Pachysandra terminalis were used as the out-
group. Numbers represent bootstrap values (%). A, B and C represent three branches formed by
Caryophyllales species. The black stars represent the positions of B. glabra and B. spectabilis.
3. Discussion
For the first time, this study sequenced, assembled, and analyzed the cp genomes of
B. glabra ‘Brasiliensis’ and B. spectabilis ‘Auratus’, two morphologically similar Bougainvillea
cultivars differing in cold resistance. The results revealed that the cp genomes of these
two species possess a typical quadripartite structure, with one LSC, one SSC, and two IR
regions, consistent with the cp genome structures of other Bougainvillea species and the
most common structure in plant cp genomes [7,18,19]. The cp genome size of B. glabra
and B. spectabilis was 154,520 and 154,542 bp, respectively, which was relatively large for
Bougainvillea. The size of the sequenced cp genomes was found to be similar to the size
of the earlier reported cp genomes of B. glabra (154,536 bp; 154,763 bp) and B. spectabilis
(154,541 bp) [18,20]. Bougainvillea glabra and B. spectabilis exhibit the same total GC content
in their cp genomes, and the GC content is significantly higher in the IR region than in the

Int. J. Mol. Sci. 2023, 24, 13044 11 of 15
LSC and SSC regions. GC content plays an important role in genome variation, and its
uneven distribution may contribute to the conservation of the LSC, SSC, and IR regions.
Additionally, B. glabra and B. spectabilis show consistency in the number of total, protein-
coding, rRNA and tRNA genes and introns, indicating a high similarity in their cp genome
sequences, which partially explains the similarity in their morphological characteristics.
During biological evolution, codon usage bias is commonly observed among species
and can be used to infer phylogenetic relationships among different species or within the
same genus [28]. The cp genomes of B. glabra and B. spectabilis consist of 26,599 codons,
with Leu being the most frequently encoded amino acid. The RSCU values showed that
the majority of optimal synonymous codons end with A or U, leading to an increased AT
content in the genes, supporting the widespread occurrence of A/T codon bias in the cp
genomes of higher plants [29]. These results are consistent with those from previous studies
on codon usage bias in the cp genomes of Bougainvillea species [7,18,19] and suggest that it
may be a result of natural selection and gene mutation [30].
SSRs in plant cp genomes are characterized by their abundance, high conservation,
and rich genetic information, and variations in their copy numbers can serve as important
molecular markers for studying plant polymorphisms, population structure, and popula-
tion genetic evolution [31]. SSR analysis of the cp genomes of B. glabra and B. spectabilis
revealed 270 and 271 SSR loci, respectively, with mononucleotide repeats, mainly composed
of poly-A and poly-T sequences, being the most abundant. This may explain the differences
in the base composition of the cp genomes in these two species. Previous studies have also
revealed that plastid SSRs are generally composed of poly-A and poly-T repeats and rarely
contain guanine (G) and cytosine (C) repeats [32,33]. No hexanucleotide or longer repeats
were detected in either species, which is consistent with the findings of Bautista et al. [7],
but differs from the results of Yang et al. [19], who detected no tetranucleotide or longer
repeats. This difference may be due to different parameter settings in SSR analysis, as Yang
et al. [19] set the minimum repeat number for tetranucleotide to hexanucleotide repeats
to five. In addition to SSRs, we identified eight highly variable regions in B. glabra and B.
spectabilis using a sliding window approach, six of which were located in the LSC region and
the remaining in the SSC region. This indicates that the IR regions of the two genomes are
relatively conserved, which is consistent with the research results of Bautista et al. [7]. This
is possibly due to the corrective effect of repeated genes in the IR regions on variations [34].
Among these highly variable sites, protein-coding regions ycf1 and ndhF are also highly vari-
able in Bougainvillea plants and other plant species, making them recommended candidate
regions for DNA barcoding [35,36]. Other fragments, particularly trnG-UCC, also exhibit
high variability, suggesting their potential as DNA barcoding regions in Bougainvillea and
suggesting directions for future research.
The contraction and expansion of the IR regions are major factors causing variation in
the size of angiosperm cp genomes, as well as gene variation and loss, and pseudogene
formation. Therefore, the cp genome size varies among species [37–39]. The IR region in
angiosperm cp genomes is typically between 20,000 and 30,000 bp, and longer IR regions
result in less impact from structural rearrangements on the cp genome [40]. The IR regions
of B. glabra and B. spectabilis were 25,377 and 25,385 bp, respectively, falling within the longer
range, indicating higher conservation in this region. Species with minor differences in cp
genome junctions are generally closely related [41]. The JLB, JSB, JSA, and JLA boundaries
of B. glabra and B. spectabilis share identical flanking genes, and the expansion lengths of
each boundary gene sequence are also consistent. This suggests a high conservation of
the IR boundaries between B. glabra and B. spectabilis and indicates a close phylogenetic
relationship. Bougainvillea glabra, B. spectabilis, and four other Bougainvillea species share
the same genes at the boundaries, but there are slight differences in the contraction and
expansion lengths of the genes, indicating that the contraction and expansion of the IR
boundaries in Bougainvillea cp genomes are relatively conserved.
To determine the phylogenetic relationship between B. glabra and B. spectabilis and
their systematic positions within Bougainvillea, a phylogenetic tree was constructed based

Int. J. Mol. Sci. 2023, 24, 13044 12 of 15
on the complete cp genomes of B. glabra, B. spectabilis, and 15 Caryophyllales species. The
results showed that all Bougainvillea species formed a major clade, with B. pachyphylla
and B. peruviana as the basal groups of the genus, which is consistent with the findings
of Bautista et al. [7] and Bautista et al. [18]. Bougainvillea glabra and B. spectabilis formed a
sister group, indicating a close relationship between them, and this branch was sister to
B. praecox with 100% support, suggesting a relatively close phylogenetic relationship.
4. Materials and Methods
4.1. Sampling, DNA Extraction, and Sequencing
Bougainvillea glabra ‘Brasiliensis’ (voucher specimen: NJFU220918) and B. spectabilis
‘Auratus’ (voucher specimen: NJFU220919) plants were obtained from Zhangzhou Shengx-
iang Landscape and Greening Co., Ltd. (Zhang’zhou, China) and planted at Nanjing
Forestry University (118◦81 E, 32◦07 N) (Nanjing, China). Voucher specimens were de-
posited in the VR Laboratory, College of Landscape Architecture, Nanjing Forestry Uni-
versity. Healthy mature leaves were collected from a single plant of both B. glabra and B.
spectabilis, rapidly frozen in liquid nitrogen, and stored at −80 ◦C until use. Total DNA was
extracted from the leaves using a modified cetyltrimethylammonium bromide method [42].
The volume and concentration of B. glabra ‘Brasiliensis’ were 40 µL and 54.59 ng/µL, re-
spectively, and those of B. spectabilis ‘Auratus’ were 40 µL and 43.86 ng/µL, respectively.
After a successful quality assessment of the DNA, it was mechanically disrupted using
an ultrasonic homogenizer. Fragment purification, end repair, A-tailing of the 30 ends,
and adapter ligation were performed to generate sequencing libraries. The libraries were
sequenced using the Illumina NovaSeq PE150 platform (Genepioneer Biotechnologies,
Nanjing, China).
4.2. Chloroplast Genome Assembly and Annotation
We used fastp v0.20.0 (https://github.com/OpenGene/fastp (accessed on 24 March
2023)) software [43] to filter the raw data, with the following filtering criteria: (1) sequencing
connectors and primer sequences from reads; (2) reads with an average quality value of
less than Q5; and (3) reads with a quantity of N greater than 5. After removing adapters
and low-quality data from the raw reads, the cp genomes were assembled using SPAdes
software v3.10.1 (http://cab.spbu.ru/software/spades/ (accessed on 24 March 2023)) [44],
with k-mer sizes of 55, 87, and 121. Contigs were scaffolded using SSPACE v2.0 (https:
//www.baseclear.com/services/bioinformatics/basetools/sspace-standard/ (accessed on
24 March 2023)) [45]. The software Gapfiller v2.1.1 (https://jaist.dl.sourceforge.net/project/
gapfiller/v2.1.1/gapfiller-2.1.1.tar.gz (accessed on 24 March 2023)) was used to fill gaps [46].
Two methods were used for cp genome annotation. First, the cp coding sequences
were annotated using Prodigal v2.6.3 (https://www.github.com/hyattpd/Prodigal (ac-
cessed on 24 March 2023)), rRNAs were predicted using HMMER software v3.1b2 (http:
//www.hmmer.org/ (accessed on 24 March 2023)) [47], and tRNAs were predicted using
ARAGORN v1.2.38 (http://130.235.244.92/ARAGORN/ (accessed on 24 March 2023)) [48].
Second, gene sequences from closely related species available in the National Center for
Biotechnology Information (NCBI) database were extracted and compared with the as-
sembled sequences using BLAST v2.6 (https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed
on 24 March 2023)) to obtain an alternative annotation [49]. The two annotation results
were manually inspected to identify discordant gene annotations, remove erroneous and
redundant annotations, and determine the boundaries of multi-exonic genes, resulting
in the final annotation. The cp genome maps were generated using OGDraw (https:
//chlorobox.mpimp-golm.mpg.de/OGDraw.html (accessed on 24 March 2023)) [50]. The
cp genome sequences have been deposited in GenBank under accession numbers OR233065
(B. glabra) and OR233066 (B. spectabilis). Mauve software (http://darlinglab.org/mauve,
accessed on 24 March 2023) [51] was used for a global comparison of six Bougainvillea
species and gene rearrangement in the genomes was detected using collinearity analysis.

Int. J. Mol. Sci. 2023, 24, 13044 13 of 15
Codon usage was analyzed using CodonW (http://codonw.sourceforge.net/, accessed on
24 March 2023) [52].
4.3. Analysis of SSRs, Nucleotide Polymorphisms, and IR Boundary Changes
Vmatch software v2.3.0 (http://www.vmatch.de/ (accessed on 24 March 2023)), in combi-
nation with Perl scripts, was used to identify types of repetitive sequences (forward, palindromic,
reverse, complement). The parameter settings were as follows: minimum length = 30 bp and
Hamming distance = 3 [53]. MISA v1.0 (MIcroSAtellite identification tool, http://pgrc.ipk-
gatersleben.de/misa/misa.html (accessed on 24 March 2023)) was used to identify the types
and numbers of SSR loci in the cp genomes [54]. The parameter settings were as follows:
mononucleotides ≥ 8; dinucleotides ≥ 5; and trinucleotides, tetranucleotides, pentanucleotides,
and hexanucleotides ≥ 3. Nucleic acid variations among the cp genomes were determined
using DnaSP v5.10 [55]. Differences in boundary sequences were visualized using IRscope
(https://ir-scope.shinyapps.io/irapp/ (accessed on 24 March 2023)) [50].
4.4. Phylogenetic Analysis
Fifteen published cp genome sequences of Caryophyllales species were downloaded from
the NCBI GenBank. Bougainvillea microphylla and P. terminalis were chosen as outgroups. Multiple
sequence alignment was performed using MAFFT v7.427 (auto mode) [56]. Trimmed align-
ments were obtained using trimAl v1.4.rev15. The maximum likelihood phylogenetic tree was
constructed using RAxML v8.2.10 (https://cme.h-its.org/exelixis/software.html (accessed on
24 March 2023)), with the GTRGAMMA model and rapid bootstrap analysis (bootstrap = 1000).
5. Conclusions
In this study, the complete cp genomes of ‘Brasiliensis’ and ‘Auratus’, cultivars of B. glabra
and B. spectabilis, respectively, which are important horticultural species, were sequenced and
analyzed. The results indicated that the cp genomes of these two species were highly conserved
in terms of structure and gene content. A total of 270 and 271 SSR loci were identified in the cp
genomes of B. glabra and B. spectabilis, respectively, alongside eight highly variable regions (psbN,
psbJ, rpoA, rpl22, psaI, trnG-UCC, ndhF, and ycf1), which can serve as potential molecular markers.
Phylogenetic analysis showed a close relationship between B. glabra and B. spectabilis. The
findings of this study not only provide important evidence for the further genetic improvement
and breeding of cold tolerance in Bougainvillea plants and the selection of superior varieties, but
also contribute to elucidating the evolutionary and systematic relationships among species in
Bougainvillea.
Supplementary Materials: The supporting information can be downloaded at: https://www.mdpi.
com/article/10.3390/ijms241713044/s1.
Author Contributions: Z.Z. and H.Z. designed the research. H.Z. and T.H. performed the research.
H.Z. collected and analyzed the data. H.Z. wrote the manuscript. Z.Z., Q.S. and Q.Z. revised the
manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This study was supported by the National Natural Science Foundation of China (No. 31770752),
the National Natural Science Foundation for Young Scientists of China (No. 32101582), the Art
Program of National Social Science Foundation of China (No. 22BG110), and the Research Innovation
Plan Project for Graduates of Jiangsu Province (No. KYCX23_1259).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The assembled chloroplast genome sequences of Bougainvillea glabra
and Bougainvillea spectabilis have been uploaded to and deposited in GenBank under accession number
OR233065 and OR233066, respectively.
Conflicts of Interest: The authors declare no conflict of interest.

Int. J. Mol. Sci. 2023, 24, 13044 14 of 15
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