BioMed Research International
Volume 2019 (2019), Article ID 4370258, 17 pages
https://doi.org/10.1155/2019/4370258

Research Article

Complete Chloroplast Genome Sequence of Justicia flava: Genome Comparative Analysis and Phylogenetic Relationships among Acanthaceae

Samaila S. Yaradua,1,2 Dhafer A. Alzahrani,¹ Enas J. Albokhary,¹ Abidina Abba,¹ and Abubakar Bello²

¹Department of Biology, King Abdulaziz University, Jeddah, Saudi Arabia
²Centre for Biodiversity and Conservation, Department of Biology, Umaru Musa Yaradua University, Katsina, Nigeria

Correspondence should be addressed to Samaila S. Yaradua; dryaradua@gmail.com

Received 25 March 2019; Accepted 26 June 2019; Published 6 August 2019

Academic Editor: Marcelo A. Soares

Copyright © 2019 Samaila S. Yaradua et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

The complete chloroplast genome of J. flava, an endangered medicinal plant in Saudi Arabia, was sequenced and compared with cp genome of three Acanthaceae species to characterize the cp genome, identify SSRs, and also detect variation among the cp genomes of the sampled Acanthaceae. NOVOPlasty was used to assemble the complete chloroplast genome from the whole genome data. The cp genome of J. flava was 150, 888bp in length with GC content of 38.2%, and has a quadripartite structure; the genome harbors one pair of inverted repeat (IRa and IRb 25, 500bp each) separated by large single copy (LSC, 82, 995 bp) and small single copy (SSC, 16, 893 bp). There are 132 genes in the genome, which includes 80 protein coding genes, 30 tRNA, and 4 rRNA; 113 are unique while the remaining 19 are duplicated in IR regions. The repeat analysis indicates that the genome contained all types of repeats with palindromic occurring more frequently; the analysis also identified total number of 98 simple sequence repeats (SSR) of which majority are mononucleotides A/T and are found in the intergenic spacer. The comparative analysis with other cp genomes sampled indicated that the inverted repeat regions are conserved than the single copy regions and the noncoding regions show high rate of variation than the coding region. All the genomes have ndhF and ycf1 genes in the border junction of IRb and SSC. Sequence divergence analysis of the protein coding genes showed that seven genes (petB, atpF, psaI, rpl32, rpl16, ycf1, and clpP) are under positive selection. The phylogenetic analysis revealed that Justiceae is sister to Ruellieae. This study reported the first cp genome of the largest genus in Acanthaceae and provided resources for studying genetic diversity of J. flava as well as resolving phylogenetic relationships within the core Acanthaceae.

1. Introduction

Justicia L. is one of the largest and the most taxonomically complex genus in Acanthaceae belonging to the tribe Justicieae consisting of ca. 600 species [1–3] typified by J. hyssopifolia (Sp Pl. 15 1753; Gen. Pl. ed. 5, 10 1754; nonsensu Nees 1832, 1847; nec sunsu Kuntze, 1891) distributed in the tropical and subtropical regions of the world, extending into warm temperate zones in Europe, Asia, and North America [4]. The genus is the type specimen of the tribe Justiceae and is characterized by a number of characters which includes corolla having 2 libbeds (bilobed upper lip and trilobed lower lip) and 2 bithecous stamens, one of the two named thecae above the other and the other one contains stur at the base [1, 3]. Several authors [5–7] include small segregate genera in the genus while author [1] in his own view reported a new classification adding more taxa and defined the genus as having 16 sections based on some floral parts and seeds. Recent molecular studies reported that Justicia s. l (Graham 1988) is paraphyletic [8, 9]. Lindau classified Justicia and several closely related species under a tribe that is characterized by androecium having 2 stamens and Knotchenpollen. There is a problem with this system of classification as reported by some student of Acanthaceae because some species in the tribe do not have this character while several taxa with the characters were classified in other tribes. However, there is still a problem in the status of the tribe Justiceae; [6] divided Ruellioideae into Justicieae, Ruellieae, and Lepidagathideae, whereas authors [10] in their study on phylogenetic relationship among Acanthaceae using trnL-trnF reported Ruellia and Justicia as separate lineages. Recently, [11] classified the member of the Justicieae as subtribe under the tribe Ruelloideae.

Justicia flava is among the endangered species in Saudi Arabia; the plant is widely used in traditional medicine in treating various ailments such as cough, paralysis, fever epilepsy, convulsion, and spasm and skin infection disorder. The roots are also reported to be used in treating diarrhea and dysentery [12, 13]. As reported by [14] the plant has antimicrobial and antioxidant activity as well as wound healing activity. Despite the endangered nature and uses in traditional medicine of the species, the complete chloroplast genome of the species was not sequenced until this study.

Comparison of complete chloroplast genome provides very informative information for reconstruction of phylogeny and resolving evolutionary relationships issues at various taxonomic levels [15–19]. This is as a result of the conservative nature of the chloroplast genome [20]; this conservative nature is because the plastome evolves about half the rate of other genomes like the nuclear [21, 22]. However, rearrangements in the sequence of chloroplast genome were reported by various plant chloroplast genome studies [19, 23–25]. These rearrangements occur as a result of contractions, expansions, and inversions in the single copy regions (large single copy and small single copy) and the inverted repeats [26, 27]. The rearrangements of the genes and inversion in the chloroplast genome are reported to be useful in phylogenetic analyses to solve taxonomic problems at various taxonomic levels because they do not occur often and estimation of their homology and inversion event polarity is simple [22, 28–31]. With the importance of complete chloroplast genome in resolving phylogenetic relationship issues and the large number of genera and species in Acanthaceae only complete chloroplast genome of four genera has been so far reported (Andrographis paniculata (Burm.f.) Nees, NC_022451; Ruellia breedlovei T. F. Daniel, KP300014; Strobilanthes cusia (Nees) O. Kuntze, MG874806, and four species Echinocactus MF490441, MH045155, MH045156, and MH045157.)

In this study, we reported the characteristics of the complete chloroplast genome of Justicia flava, the first cp genome in the largest genus of Acanthaceae, and compared the genomes of four Acanthaceae species to understand the variation among the cp genomes, report the simple sequence repeats to provide the tools for genetic diversity and identification of the species, and lastly, resolve the status of Justiceae.

2. Materials and Methods

2.1. Plant Material and DNA Extraction

Plant material (vegetative and floral part) was collected through field survey of J. flava in Taif, Saudi Arabia, and identified based on the herbarium specimens and morphological features seen in relevant literatures, the voucher specimen was deposited in the herbarium of King Abdulaziz University, Jeddah, Saudi Arabia. Leaves were collected from the specimen for genomic DNA extraction. The genomic DNA was extracted using Plant Genomic DNA Kit according to manufacturer’s protocol.

2.2. Library Construction, Sequencing, and Assembly

A total amount of 1.0μg DNA was used as input material for the DNA sample preparations. Sequencing libraries were generated using NEBNext® DNA Library Prep Kit following manufacturer’s recommendations and indices were added to each sample. The genomic DNA is randomly fragmented to a size of 350bp by shearing, then DNA fragments were end polished, A-tailed, and ligated with the NEBNext adapter for Illumina sequencing and further PCR enriched by P5 and indexed P7 oligos. The PCR products were purified (AMPure XP system) and resulting libraries were analyzed for size distribution by Agilent 2100 Bioanalyzer and quantified using real-time PCR. The qualified libraries are fed into Illumina sequencers after pooling according to its effective concentration and expected data volume. The raw reads were filtered to get the clean reads (5 Gb) using PRINSEQ lite v0.20.4 [32] and were subjected to de novo assembly using NOVOPlasty2.7.2 [33] with kmer (K-mer= 31) to assemble the complete chloroplast genome from the whole genome sequence. ndhAx1-ndhAx2 intergenic spacer from Justicia flava (KY632456) was used as seed and the plastome sequence of Ruellia breedlovei (KP300014.1) was used as the reference. Finally, one contig containing the complete chloroplast genome sequence was generated.

2.3. Gene Annotation

The programme DOGMA (Dual Organellar GenoMe Annotator, University of Texas at Austin, Austin, TX, USA) [34] was used to annotate the genes in the assembled chloroplast genome. The positions of start and stop codon were adjusted manually. trnAscan-SE server (http://lowelab.ucsc.edu/tRNAscan-SE/) [35] was used to verified the tRNA genes and finally, the plastome genome circular map was drawn using OGDRAW (Organellar Genome DRAW) [36]. The sequence of the chloroplast genome of J. Flava was deposited in the GenBank database with accession number (MK548577,).

2.4. Sequence Analysis

MEGA 6.0 was used to analyze the relative synonymous codon usage values (RSCU), base composition, and codon usage. Possible RNA editing sites present in the protein coding genes of J. flava cp genome were determined using PREP suite [37] with 0.8 as the cutoff value.

2.5. Repeat Analysis in J. flava Chloroplast Genome

Simple sequence repeats (SSRs) were identified in the J. flava chloroplast genome and genome of other three species of Acanthaceae using the online software MIcroSAtellite (MISA) [38] with the following parameters: eight, five, four, and three repeats units for mononucleotides, dinucleotides, trinucleotides, and tetra-, penta-, hexanucleotides SSR motifs, respectively. For analysis of long repeats (palindromic, forward, reverse, and complement) the program REPuter (https://bibiserv.cebitec.uni-bielefeld.de/reputer) [39] with default parameters was used to identify the size and location of the repeats in J. flava chloroplast genome and genome of other 3 species of Acanthaceae.

2.6. Genome Comparison

The program mVISTA [40] was used to compare the chloroplast genome of J. flava with the cp genomes of E. longzhouensis, R. breedlovei, and S. cusia using the annotation of J. flava as reference in the Shuffle-LAGAN mode [41]. The border region between the large single copy (LSC) and inverted repeat (IR) and small single copy SSC and inverted repeat (IR) junction were compared among the four species of Acanthaceae.

2.7. Characterization of Substitution Rate

DNAsp v5.10.01 [42] was used to analyze synonymous (dS) and nonsynonymous (dN) substitution rate and dN/dS ratio to detect the genes that are under selection pressure; the chloroplast genome of J. flava was compared with the cp genome of E. longzhouensis, R. breedlovei, and S. cusia. Individual protein coding genes were aligned separately in Genious V 8.1.3; the aligned sequences were then translated into protein sequence.

2.8. Phylogenetic Analysis

The complete chloroplast genome of three Acanthaceae and five species from the order Lamiales were downloaded from Genbank. The downloaded sequences were aligned with sequenced cp genome of J. flava using MAFFT v.7 [43]. The data were analyzed using Maximum Parsimony (PAUP version 4.0b10) [44] using heuristic searches with 1000 replicates of random taxon addition, tree bisection-reconnection branch swapping, MulTrees on, saving a maximum of 100 trees each replicate. Missing characters were treated as gaps. Support was assessed using 1000 replicates of nonparametric bootstrap analysis. Bayesian analysis was carried out using MrBayes version 3.2.6 [45]. jModelTest version 3.7 [46] was used to select the suitable model.

3. Results and Discussion

3.1. Characteristics of J. flava Chloroplast Genome

The complete chloroplast genome of J. flava is a circular molecule and has quadripartite structure; the genome was found to have 150, 888bp in length. The genome is divided into four regions, namely, large single copy (LSC), small single copy (SSC), and two inverted repeats (IRa and IRb). The coding region is 98, 671bp in length and constitutes 65.39% of the genome; the rest of 52, 217 bp is the intergenic spacer region including intron (34.60%). The LSC and SSC regions possessed 82, 995bp and 16, 893bp, respectively, the inverted repeats IRa and IRb have 25, 500bp and are separated by SSC region (Figure 1 and Table 1). The organization and structure of the J. flava cp genome are similar to other sequenced Acanthaceae cp genomes [47, 48]. The percentage of occurrence of AT and GC content in the genome showed that the LSC, SSC, IRA, and IRB regions possessed 63.8% and 36.2%, 67.8% and 32.2%, 56.7% and 43.4%, and 56.7% and 3.4, respectively. The whole chloroplast genome has AT content of 61.8% and GC content of 38.2%; this is similar to cp genome of Strobilanthes cusia [49]. The GC content in the inverted repeat is found to be higher than the single copy regions both the LSC and SSC.

Table 1: Base composition in the J. flava chloroplast genome.

Region

T(U) (%)
C (%)
A (%)
G (%)
Total (bp)

cp genome

31
19
31
19
150888

LSC

32
19
31
18
82995

SSC

34
17
34
15
16893

IRA

28
23
28
21
25500

IRB

28
21
28
23
25500

1st Position
31
20
30
19
50296

2nd Position
31
19
31
19
50296

3rd Position
31
20
30
19
50296

Figure 1: Gene map of the J. flava chloroplast genome. Genes outside the circles are transcribed in counterclockwise direction and those inside in clockwise direction. Known functional genes are indicated in the coloured bar. The GC and AT content are denoted by the dark grey and light grey colour in the inner circle, respectively. LSC indicates large single copy; SSC, indicates small single copy, and IR indicates inverted repeat.

The result of the genes annotation in the chloroplast genome of J. flava revealed a total of 132 genes among which 113 are unique; the remaining 19 are duplicated in the inverted region. The genome harbored 80 protein coding genes, 30 tRNA genes, and 4 rRNA genes (Figure 1 and Table 2). The numbers and orientation of the genes in the cp genome are the same as other cp genomes of Acanthaceae [47, 48]. The inverted repeat region contained eight protein coding genes, seven tRNA, and four rRNA while in the single copy region, the LSC contained 61 protein coding genes and 22 tRNA genes; the rest of 12 protein coding genes and 1 tRNA are located within the SSC region. Almost all the protein coding genes start with the ATG codon that code for methionine whereas some of the genes start with codon like ATC, GTG and ACG; this is common in most flowering plant (angiosperms) chloroplast genome [49–51].

Table 2: Genes present in the chloroplast genome of J. flava.

Category
Group of genes
Name of genes

RNA genes
ribosomal RNA genes (rRNA)
rrn5, rrn4.5, rrn16, rrn23

Transfer RNA genes (tRNA)
trnH-GUG, trnK-UUU⁺, trnQ-UUG, trnS-GCU, trnS-CGA⁺, trnR-UCU,trnC-GCA, trnD-GUC, trnY-GUA, trnE-UUC, trnT-GGU, trnS-UGA, trnfM-CAU, trnG-GCC, trnS-GGA, trnL-UAA⁺, trnT-UGU, trnF-GAA, trnV-UAC⁺, trnM-CAU, trnW-CCA, trnP-UGG, trnI-, trnL-, trnV-, trnI-, trnA-, trnR-, trnN-, trnL-UAG,

Ribosomal proteins
Small subunit of ribosome
rps2, rps3, rps4, , rps8, rps11, , rps14, rps15, rps,16⁺, rps18,rps19

Transcription
Large subunit of ribosome
, rpl14, rpl16, rpl20, rpl22, , rpl32, rpl33, rpl36

DNA dependent RNA polymerase
rpoA, rpoB, rpoC1⁺, rpoC2

Protein genes
Photosystem I
psaA, psaB, psaC, psaI,psaJ,ycf3⁺⁺

Photosystem II
psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ

Subunit of cytochrome
petA, petB, petD, petG, petL, petN

Subunit of synthase
atpA, atpB, atpE, atpF⁺, atpH, atpI

Large subunit of rubisco
rbcL

NADH dehydrogenase
ndhA⁺,, ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK

ATP dependent protease subunit P
clpP⁺⁺

Chloroplast envelope membrane protein
cemA

Other genes
Maturase
matK

Subunit acetyl-coA carboxylase
accD

C-type cytochrome synthesis
ccsA

Hypothetical proteins
, ycf4,

Component of TIC complex

with one intron. with two intron. Gene with copies.

The J. flava chloroplast genome is found to contain intron in some of the protein coding and tRNA genes, like other chloroplast genomes of angiosperms [49, 50]. There are 14 genes that contain intron out of the 113 different genes (Table 3); among the 14 genes 8 are protein coding genes while the remaining six are tRNA genes (Table 3). Four genes that have the intron, namely, rpl2, ndhB, trnI-GAU, and trnA-UGC are located in the IR region while the remaining 12 are located in the LSC region. Only two genes ycf3 and clpP have two introns, the other 12 have only one intron, and this is also seen in S. cusia [49]. The tRNA, trnK-UUU has the longest intron of 2460 bp (Table 3); this is as a result of position of the matK gene in the intron.

Table 3: Genes with intron the J. flava chloroplast genome and length of introns and exons.

Gene
Location
Exon I (bp)
Intron I (bp)
Exon II (bp)
Intron II (bp)
Exon III (bp)

atpF
LSC
143
659
467

rpoC1
LSC
443
769
1628

ycf3
LSC
128
682
227
721
152

clpP
LSC
68
763
293
635
227

rpl2
IR
392
664
434

ndhB
IR
776
680
755

ndhA
SSC
551
954
539

trnK-UU
LSC
36
2460
37

trnS-CGA
LSC
31
667
59

trnL-UAA
LSC
36
501
49

trnV-UAC
LSC
37
593
36

trnI-GAU
IR
41
938
34

trnA-UGC
IR
37
818
34

The codon usage bias in the plastome was computed using the protein coding genes and tRNA genes nucleotide sequences 89, 377bp. The relative synonymous codon usage of each codon in the genome is presented in (Table 4); the result revealed that all the genes are encoded by 29, 790 codons. Codons, coding for the amino acids Leucine, are the most frequent codons 3,329 (11.7%) (Figure 2), similar to that of Ailanthus altissima [52], whereas codons coding for Trp are the least 570 (1.91%) in the genome. G- and C-ending are found to be more frequent than their counterpart A and T; this is not the case in other plastomes sequences [53–55]. The result of the analysis (Table 4) showed that codon usage bias is low in the chloroplast genome of J. flava. The RSCU values of 29 codons were >1 and all of them have A/T ending while for 30 codons were <1 and are all of G/C ending. Only two amino acids Tryptophan and Methionine have RSCU value of 1; therefore they are the only amino acids with no codon bias.

Table 4: Codon-anticodon recognition patterns and codon usage of the J. flava chloroplast genome.

Codon
Amino Acid
RSCU
tRNA
Codon
Amino Acid
RSCU
tRNA

UUU
Phe
1.18
trnF-GAA
UAU
Tyr
1.38
trnY-GUA

UUC
Phe
0.82

UAC
Tyr
0.62

UUA
Leu
1.31
trnL-UAA
UAA
Stop
1.01

UUG
Leu
1.31
trnL-CAA
UAG
Stop
1.03

CUU
Leu
1.23
trnL-UAG
CAU
His
1.26
trnH-GUG

CUC
Leu
0.65

CAC
His
0.74

CUA
Leu
0.92

CAA
Gln
1.37
trnQ-UUG

CUG
Leu
0.59

CAG
Gln
0.63

AUU
Ile
1.22
trnI-GAU
AAU
Asn
1.34
trnG-GUU

AUC
Ile
0.82

AAC
Asn
0.66

AUA
Ile
0.95
trnI-CAU
AAA
Lys
1.29
trnK-UUU

AUG
Met
1
trnM-CAU
AAG
Lys
0.71

GUU
Val
1.45
trnV-GAC
GAU
Asp
1.44
trnD-GUC

GUC
Val
0.65

GAC
Asp
0.56

GUG
Val
0.74

GAA
Glu
1.38
trnE-UUC

GUA
Val
1.16
trnV-UAC
GAG
Glu
0.62

UCU
Ser
1.46
trnS-GGA
UGU
Cys
1.14
trnC-GCA

UCC
Ser
0.95

UGC
Cys
0.86

UCG
Ser
0.76

UGA
Stop
0.96

UCA
Ser
1.33
trnS-UGA
UGG
Trp
1
trnW-CCA

CCU
Pro
1.21
trnP-UGG
CGU
Arg
0.84
trnR-ACG

CCC
Pro
0.87

CGC
Arg
0.35
trnR-UCU

CCA
Pro
1.13

CGA
Arg
1.16

CCG
Pro
0.79

CGG
Arg
0.77

ACU
Thr
1.22

AGA
Arg
1.8

ACC
Thr
0.87

AGG
Arg
1.07

ACG
Thr
0.72
trnT-GGU
AGU
Ser
0.92
trnS-GCU

ACA
Thr
1.2
trnT-UGU
AGC
Ser
0.58

GCU
Ala
1.33
trnA-UGC
GGU
Gly
1.18
trnG-GCC

GCC
Ala
0.76

GGC
Gly
0.54

GCA
Ala
1.18

GGA
Gly
1.28

GCG
Ala
0.72

GGG
Gly
0.99
trnG-UCC

Figure 2: Amino acids frequencies in J. flava chloroplast genome protein coding sequences.

The RNA editing site in the J. flava chloroplast genomes was predicted using the program PREP suite; all the analysis was done using the first codon position of the first nucleotide. The result (Table 5) shows that the majority of the conversion in the codon positions is from the amino acid Serine to Leucine (Table 5). In all, the programme revealed 61 editing sites in the genome; the editing sites are distributed among 18 protein coding genes. As reported in previous researches [56–58] the ndhB gene has the highest number of editing sites (9 sites) followed by rpoB (7 site) and ndhG, atpF, rpl2, rpl20, and rps2 have the least 1 site each. Among all the conversion in the RNA editing site, one site changed the amino acid from apolar group to polar group (Proline to Serine). The following genes do not have RNA predicting site in their first codon of the first nucleotides atpB, ccsA, clpP, ndhC, ndhE psaB, petD, petG, rpoA, and petL, among others.

Table 5: Predicted RNA editing site in the J. flava chloroplast genome.

gene
Nucleotide Position
Amino Acid Position
Codon Conversion
Amino Acid Conversion
Score

accD
800
267
TCG => TTG
S => L
0.8

844
282
CCC => TCC
P => S
0.8

atpA
776
259
ACC => ATC
T => I
1

914
305
TCA => TTA
S => L
1

1270
424
CCC => TCC
P => S
1

atpF
92
31
CCA => CTA
P => L
0.86

atpI
404
135
GCT => GTT
A => V
1

620
207
TCA => TTA
S => L
1

matK
640
214
CAT => TAT
H => Y
1

1249
417
CAT => TAT
H => Y
1

ndhA
326
109
ACT => ATT
T => I
1

566
189
TCA => TTA
S => L
1

922
308
CTT => TTT
L => F
1

ndhB
149
50
TCA => TTA
S => L
1

467
156
CCA => CTA
P => L
1

586
196
CAT => TAT
H => Y
1

737
246
CCA => CTA
P => L
1

746
249
TCT => TTT
S => F
1

830
277
TCA => TTA
S => L
1

836
279
TCA => TTA
S => L
1

1292
431
TCC => TTC
S => F
1

1481
494
CCA => CTA
P => L
1

ndhD
2
1
ACG => ATG
T => M
1

32
11
GCA => GTA
A => V
1

878
293
TCA => TTA
S => L
1

1445
482
GCT => GTT
A => V
1

ndhF
124
42
CTT => TTT
L => F
1

671
224
CCA => CTA
P => L
1

713
238
GCT => GTT
A => V
0.8

1505
502
TCT => TTT
S => F
1

1667
556
CCC => CTC
P => L
1

2173
725
CTC => TTC
L => F
1

ndhG
314
105
ACA => ATA
T => I
0.8

petB
617
206
CCA=> CTA
P => L
1

psaI
22
8
CCT => TCT
P => S
1

28
10
CTT => TTT
L => F
0.86

rpl2
596
199
GCG => GTG
A => V
0.86

rpl20
308
103
TCA => TTA
S => L
0.86

rpoB
338
113
TCT => TTT
S => F
1

473
158
TCA => TTA
S => L
0.86

551
184
TCA => TTA
S => L
1

566
189
TCG => TTG
S => L
1

593
198
GCT => GTT
A => V
0.86

2000
667
TCT => TTT
S => F
1

2426
809
TCA => TTA
S => L
0.86

rpoC2
2290
764
CGG => TGG
R => W
1

3202
1068
CTT => TTT
L => F
0.86

3719
1240
TCA => TTA
S => L
0.86

rps2
248
83
TCA => TTA
S => L
1

266
89
ACA => ATA
T => I
0.86

rps14
80
27
TCA => TTA
S => L
1

3.2. Repeat Analysis

3.2.1. Long Repeats

REPuter programme was used to identify the repeat sequence in the chloroplast genome of J. flava using default settings; the result obtained showed that all the four types of repeats (palindromic, forward, reverse, and complement) were present in the genome (Table 6). The analysis showed 16 palindromic repeats, 23 forward repeats, 9 reverse repeats, and only one complement repeat (Table 6). Majority of the repeats size is between 20 and 29bp (46.93%), followed by 10-19bp (40.81%), whereas 30-39bp and 40-49bp are the least with 8.16% and 4.08%, respectively. In total, there are 49 repeats in the chloroplast genome of J. flava. In the first location the intergenic spacer harbored 65.30% of the repeats; this has also been reported in cp genome of Fagopyrum dibotrys [59]. tRNA contained 8 repeats (16.32%); the remaining 9 repeats (18.36%) are located in the protein coding genes specifically atpB, psaB, ndhC, ycf1, and ycf2. Within the protein coding genes ycf2 contained 1 reverse and 2 palindromic and forward repeats.

Table 6: Repeat sequences present in the J. flava chloroplast genome.

S/N
Repeat Size
Repeat Position 1
Repeat Type
Repeat Location 1
Repeat Position 2
Repeat Location 2
E-Value

1
41
97201
F
IGS
117423
IGS
1.32E-15

2
41
117423
P
IGS
136591
IGS
1.32E-15

3
39
42922
F
ycf3 Intron
97203
IGS
2.12E-14

4
39
42922
F
ycf3 Intron
117425
IGS
2.12E-14

5
39
42922
P
ycf3 Intron
136591
IGS
2.12E-14

6
30
7623
P
IGS-trnS-GCU
44420
IGS-trnSGCA
5.55E-09

7
26
86664
P
ycf2
86664
ycf2
1.42E-06

8
26
86664
F
ycf2
147143
ycf2
1.42E-06

9
26
120785
F
IGS
120808
IGS
1.42E-06

10
26
147143
P
ycf2
147143
ycf2
1.42E-06

11
25
118049
F
ndhA Intron
118074
ndhA Intron
5.69E-06

12
24
58638
F
IGS
58661
IGS
2.27E-05

13
23
31057
R
IGS
31057
IGS
9.10E-05

14
22
9120
F
trnG-GCC
35746
trnG-UCC
3.64E-04

15
21
7629
F
trnS-GCU-IGS
34812
trnS-UGA
1.46E-03

16
21
11762
R
IGS
11762
IGS
1.46E-03

17
21
34812
P
trnS-UGA
44423
trnS-GGA
1.46E-03

18
21
45956
R
trnT-UGU
45956
trnT-UGU
1.46E-03

19
21
106777
F
IGS
123747
ycf1
1.46E-03

20
21
123747
P
ycf1
127035
IGS
1.46E-03

21
20
29704
R
IGS
29704
IGS
5.82E-03

22
20
30622
P
IGS
30622
IGS
5.82E-03

23
20
49490
R
ndhC
49490
ndhC
5.82E-03

24
20
51290
P
trnV-UAC
101924
trnA-UGC
5.82E-03

25
20
51290
F
trnV-UAC
131889
trnA-UGC
5.82E-03

26
20
92988
F
IGS
93005
IGS
5.82E-03

27
20
92988
P
IGS
140808
IGS
5.82E-03

28
20
93005
P
IGS
140825
IGS
5.82E-03

29
20
140808
F
IGS
140825
IGS
5.82E-03

30
19
11760
P
IGS
80056
IGS
2.33E-02

31
19
43979
F
IGS
43997
IGS
2.33E-02

32
19
58511
R
IGS
58511
IGS
2.33E-02

33
19
108471
R
ycf2
108471
ycf2
2.33E-02

34
19
120905
R
IGS
120905
IGS
2.33E-02

35
19
123419
F
ycf1
123443
ycf1
2.33E-02

36
18
235
P
IGS
269
IGS
9.32E-02

37
18
5052
F
IGS
5069
IGS
9.32E-02

38
18
7694
F
trnS-GCU
34882
trnS-UGAIGS
9.32E-02

39
18
7758
P
IGS
67000
IGS
9.32E-02

40
18
11761
F
atpF Intron
54061
IGS
9.32E-02

41
18
21857
F
rpoC1 Intron
27884
IGS
9.32E-02

42
18
30254
P
IGS
30254
IGS
9.32E-02

43
18
30895
R
IGS
30895
IGS
9.32E-02

44
18
37376
F
psaB
39591
psaA
9.32E-02

45
18
37925
F
psaB
40149
psaA
9.32E-02

46
18
46008
P
IGS
46008
IGS
9.32E-02

47
18
50038
C
IGS
80055
IGS
9.32E-02

48
18
53019
F
atpB
125148
ycf1
9.32E-02

49
18
54060
F
IGS
106779
IGS
9.32E-02

We compared the frequency of repeats among four Acanthaceae cp genomes and found that all the types of repeats (palindromic, forward, reverse, and complement) are present in all the genomes (Figure 3). S. cusia has the highest frequency of palindromic repeats (23) while J. flava has the lowest with (16). R. breedlovei and S. cusia have the same number of forward repeats 15 each, and number of reverse repeats is the same in the genome of J. flava and S. cusia (Figure 3). Complement repeats are found to be the less type of repeat in the genome with E. longzhouensis and J. flava having 1 and the other two species having 3 each.

Figure 3: Number of different repeats in four chloroplast genome of Acanthaceae. P= palindromic, F = forward, R=reverse, and C= complement.

3.2.2. Simple Sequence Repeats (SSRs)

There are short repeats of nucleotide series (1-6 bp) that are dispensed all through genome called microsatellites (SSRs). This short repeat in plastid genome is passed from a single parent. As a result, they are used as molecular indicators in developmental studies such as genetic heterogeneity and also contribute in recognition of species [60–62]. The sums of 98 microsatellites were found in plastid genome of J. flava in this study (Table 7). Majority of SSRs in the cp genome are mononucleotide (83.67%) of which most are poly T and A (Figure 4). Poly T (polythymine) constituted 58.53% whereas poly A (polyadenine) 40.24%; this is consistent in previous studies [63, 64]. Only a single poly C (polycytosine) 1.21% is present in the genome whereas 2 poly G (polyguanine) is found in the genome. Among the dinucleotide only AT/AT is found in the genome. Reflecting series complementary, three trinucleotide AAG/CTT, ATC/ATG, and AAT/ATT, five tetra AAAC/GTTT, AAAG/CTTT, AAAT/ATTT, AATC/ATTG, and AATT/AATT, and two penta AATAC/ATTGT and AATTC/AATTG were discovered in the genome while no hexanucleotide repeat is present (Figure 4). The intergenic spacer region harbored most of the microsatellite (62.24%) than the coding region (33.67%) (Figure 5). Most but not all the repeats (70.40%) were detected in the LSC region and the SSC region incorporates the least number of repeats in the genome.

Table 7: Simple sequence repeats in the chloroplast genome of J. flava.

Repeat
Length (bp)
Number
Start position

A
8
17
1, 941; 4,077; 7, 866; 11, 688; 13, 037; 17, 946; 41, 684; 43, 752; 51, 259; 54, 200; 68, 846; 66, 730; 95, 631; 110, 063; 111, 010; 113, 860; 118, 261; 150, 742; 150, 809

11
3
7, 592; 14, 787; 15, 599

10
3
14, 665; 21, 867; 28, 269;

9
8
27, 894; 43, 551; 45, 670; 63, 290; 88, 481; 114, 354; 132, 510; 150, 784

14
1
80, 057;

13
1
127, 037;

C
12

4, 487

G
8
2
57, 723; 74, 644

T
8
25
7, 383; 25, 533; 32, 688; 59, 519; 60, 372; 65, 495; 66, 271; 66, 377; 68, 096; 68, 556; 74, 497; 81, 050; 82, 410; 82, 710; 83, 018; 83, 085; 109, 106; 109, 136; 112, 173; 112, 563; 113, 364; 123, 499; 123, 541; 125, 326; 138, 196

10
4
9, 482; 30, 515; 53, 621; 123, 624;

15
1
11, 766;

9
15
12, 488; 15, 906; 17, 805; 70, 662; 74, 580; 75, 746; 81, 096; 83,050; 101, 316; 109, 159; 121, 849; 123, 004; 123, 606; 123, 638; 145, 345

11
3
30, 883; 31, 611; 123, 292;

12
3
35, 533; 77, 108; 124, 143

14
2
50, 040; 54, 066;

13
2
106, 785; 123, 755

AT
6
1
7, 215

5
1
20, 206

TA
5
2
19, 175; 30, 628

TTC
4
1
34, 429

TAT
4
1
84991

TGA
4
1
90, 256

TCT
5
1
124, 548

ATC
4
1
143, 566

ATA
4
1
148, 832

TAA
4
1
62, 980

TTTC
3
1
5, 222

ATTG
3
1
5, 410

ATAA
3
1
58, 759

TAAA
4
1
66, 121

AAAC
3
1
67, 128

AATA
3
1
112, 973

AATC
3
1
118, 083

AATT
3
1
122, 503

CAATA
3
1
30, 293

Figure 4: Frequency of different SSR motifs in different repeat types in J. flava chloroplast genome.

Figure 5: Number of SSR types in complete genome, protein coding regions, and noncoding genes.

The frequency of SSR among the cp genome of the four species was also compared (Figure 6); the comparison showed that mononucleotide occurs more frequently across all the genomes. E. longzhouensis had the highest number of mononucleotides and pentanucleotides with 115 and 9, respectively, but had the lowest number of tetranucleotides with 2. Pentanucleotide is not present in cp genome of R. breedlovei while possessing hexanucleotide which is not present in the remaining 3 species.

Figure 6: Number of different SSR types in the four chloroplast genome of Acanthaceae.

3.3. Comparative Analysis of J. flava Chloroplast to Other Acanthaceae Genomes

The complete chloroplast genome of J. flava was compared with three chloroplast genomes of Acanthaceae available in the Genbank, namely, R. breedlovei, S. cusia, and E. longzhouensis. To examine the degree of DNA sequence divergence among the species of Acanthaceae chloroplast genome, the programme mVISTA was used to align the sequences using annotation of Justicia flava as reference. The result of the alignment showed that the genomes highly conserved with some degree of divergence. The inverted repeat regions are more conserved than the single copy regions, the large single copy and small single copy; on the other hand the protein coding genes were found to be more conserved than the noncoding region, particularly the intergeneric spacer. The nonprotein coding regions that showed high rate of divergence across the genome are trnH-GUG-psbA, rps16-trnQ, trnC-petN, accD-psaI, clpP intron, trnL-trnF, rps15-ycf1, rps12-trnV, and trnL-trnA among others (Figure 7). For the protein coding genes the following genes showed a little sequence variation among the genomes atpE, atpF, rbcL, petA, psbL, petB, and ycf2.

Figure 7: Sequence alignment of four chloroplast genomes in the Acanthaceae family performed with mVISTA using annotation of J. flava as reference. The top arrow shows transcription direction, blue colour indicates protein coding, pink colour shows conserved noncoding sequence CNS, and light green indicates tRNAs and rRNAs. The x-axis represents the coordinates in the cp genome while y-axis represents percentage identity within 50-100%.

The chloroplast genome of angiosperms is reported to be conserved in terms of structure and size [65]; despite the conserved nature there is slightly variation in size and the boundaries of inverted repeats and single copy regions due to evolutionary events such as expansion and contraction in the genome [66, 67]. The comparisons between IR-LCS and IR-SSC boundaries in the four cp genome of Acanthaceae (Justicia flava, Echinocactus longzhouensis, Ruellia breedlovei, and Strobilanthes cusia) are shown in (Figure 8). The result showed that there is slightly variation among the compared cp genomes (Figure 8); four genes, namely, rps19, ndhF, ycf1, and trnH, were located in the junction of inverted repeats and single copy region of J. flava and E. longzhouensis genome with slightly variation in number of base pairs in the borders (Figure 8).Two genes, ndhF and ycf1, are found in the IRb/SSC border among all the four genomes. The IRb/LCS border of R. breedlovei is unique by having ycf2 gene with 390 bp in LSC and 6800 bp in IRb whereas Strobilanthes cusia genome also has unique structural variation by having trnH in IRa and IRb. The ndhF was found to have 70 bp, 59 bp, and 44 bp in the IRb region in J. flava, E. longzhouensis, R. breedlovei, and S. cusia, respectively, whereas the trnH of J. flava and E. longzhouensis starts at exactly IRa/LSC border while the tRNA is 76 bp away the border in R. breedlovei genome.

Figure 8: Comparison of the borders of the IR, SSC, and LSC regions among four chloroplast genome of Acanthaceae.

3.4. Divergence of Protein Coding Gene Sequence

The rates of synonymous (dS) and nonsynonymous (dN) substitution and dN/dS ratio were calculated to detect the selective pressure among the 78 protein coding genes in the cp genome of four Acanthaceae species. The results showed that the dN/dS ration is less than 1 in most of the paired genes except petB, psaI, and ycf1 of J. flava vs. S. cusia having 1.52, 1.24, and 1.12, respectively (Figure 9). Two genes are also found to be greater than 1 in J. flava vs. R. breedlovei atpF, clpP, psaI, and rpl32. petB, rpl16, and psaI genes of J. flava vs. E. longzhouensis are greater than 1 as well. This indicates that most of the genes were under negative selection; only few undergo positive selection. The synonymous (dS) values in all the genes range from 0.02 to 0.44 (Figure 9). The genes, atpH, petG, petN, psaC, psaJ, psbE, psbF, psbI, psbT, and rps12, showed no nonsynonymous change occurs in the cp genome of the four species of Acanthaceae.

Figure 9: The synonymous (dS) and dN/dS ration values of 78 protein coding genes from four Acanthaceae cp genomes (Jf: J. flava; Rb: R. breedlovei; El: E. longzhouensis).

3.5. Phylogenetic Analysis

The phylogenetic relationship within the four species of Acanthaceae was reconstructed using the complete chloroplast genome. The tree from the Bayesian Inference and Maximum Parsimony is congruent with strong support in all the nodes PP, 1.00, and MP, 100. All the species of Acanthaceae sampled clustered in one clade with strong support (Figure 10), as reported by [68]. The result showed that the tribe Justicieae is sister to Ruellieae; this relationship has also been reported by [69] and they should be regarded as independent tribe not as Justiceae under the tribe Ruellieae as proposed by [11]

Figure 10: Phylogenetic tree reconstruction of 9 taxa based on the complete chloroplast genome using Bayesian Inference (BI) and Maximum Parsimony (MP) methods showing relationship within the four species of Acanthaceae. The numbers in the branch nodes represent bootstrap percentage (BP)/posterior probability (PP).

Data Availability

(1) The complete chloroplast genome sequence can be found in Genbank with accession no. MK548577 after publishing the article. (2) The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

References

V. A. Graham, “Delimitation and infra-generic classification of justicia (Acanthaceae),” Kew Bulletin, vol. 43, no. 4, pp. 551–624, 1988.

D. J. Mabberley, Mabberley's Plant-Book, “A Portable Dictionary of Plants, Their Classification and Uses, Cambridge University Press, New York, NY, USA, 3rd edition, 2008.

Z. Y. Wu, P. H. Raven, and D. Y. Hong, Flora of China (Cucurbitaceae–Valerianaceae with Annonaceae and Berberidaceae), vol. 19, Science Press, Beijing, China, 2011.

V. H. Heywood, R. K. Brummit, A. Culham, and O. Seberg, “Flowering plant families of the world,” Royal Botanic Gardens, p. 424, 2007.

C. G. Nees, Acanthaceae, C. F. P. Martius, A. G. Eichler, and I. Urban, Eds., vol. 9, Flora brasiliensis Leipzig, Munchen, Germany, 1847.

C. E. B. Bremekamp, “Notes on the acanthaceae of java,” Verhandelingen Koninklijke Nederlandse Akademie van Wetenschappen Afdeling Natuurkunde, vol. 45, no. 2, pp. 1–78, 1948.

E. C. Leonard, “The acanthaceae of colombia,” Contrubution from the United State National Herbarium, vol. 31, pp. 487–645, 1958.

L. A. McDade, T. F. Daniel, and S. E. Masta, “Phylogenetic relationships within the tribe justicieae (acanthaceae): evidence from molecular sequences, morphology, and cytology,” Annals of the Missouri Botanical Garden, vol. 87, no. 4, pp. 435–458, 2000.

M. Gaom, Phylogenetic Relationship among Acanthaceae from China [Ph.D. Thesis], The Graduate University of the Chinese Academy of Sciences, 2010.

L. A. McDade and M. L. Moody, “Phylogenetic relationships among Acanthaceae: Evidence from noncoding trnL-trnF chloroplast DNA sequences,” American Journal of Botany, vol. 86, no. 1, pp. 70–80, 1999.

R. W. Scotland and K. Vollesen, “Classification of Acanthaceae,” Kew Bulletin, vol. 55, no. 3, pp. 513–589, 2000.

C. Agyare, A. Asase, M. Lechtenberg, M. Niehues, A. Deters, and A. Hensel, “An ethnopharmacological survey and in vitro confirmation of ethnopharmacological use of medicinal plants used for wound healing in Bosomtwi-Atwima-Kwanwoma area, Ghana,” Journal of Ethnopharmacology, vol. 125, no. 3, pp. 393–403, 2009.

H. M. Burkill, The Useful Plants of West Tropical Africa, Royal Botanic Gardens, Kew, 3rd edition, 2000.

C. Agyare, S. B. Bempah, Y. D. Boakye, P. G. Ayande, M. Adarkwa-Yiadom, and K. B. Mensah, “Evaluation of antimicrobial and wound healing potential of Justicia flava and Lannea welwitschii,” Evidence-Based Complementary and Alternative Medicine, vol. 2013, Article ID 632927, 10 pages, 2013.

J. Shaw, E. B. Lickey, E. E. Schilling, and R. L. Small, “Comparison of whole chloroplast genome sequences to choose noncoding regions for phylogenetic studies in angiosperms: The Tortoise and the hare III,” American Journal of Botany, vol. 94, no. 3, pp. 275–288, 2007.

A. V. Mardanov, N. V. Ravin, B. B. Kuznetsov et al., “Complete sequence of the duckweed (Lemna minor) chloroplast genome: structural organization and phylogenetic relationships to other angiosperms,” Journal of Molecular Evolution, vol. 66, no. 6, pp. 555–564, 2008.

M. J. Moore, P. S. Soltis, C. D. Bell, J. G. Burleigh, and D. E. Soltis, “Phylogenetic analysis of 83 plastid genes further resolves the early diversification of eudicots,” Proceedings of the National Acadamy of Sciences of the United States of America, vol. 107, no. 10, pp. 4623–4628, 2010.

I. Park, W. Kim, S. Yang et al., “The complete chloroplast genome sequence of aconitum coreanum and aconitum carmichaelii and comparative analysis with other aconitum species,” PLoS ONE, vol. 12, no. 9, Article ID e0184257, 2017.

M. Sun, J. Li, D. Li, and L. Shi, “Complete chloroplast genome sequence of the medical fern Drynaria roosii and its phylogenetic analysis,” Mitochondrial DNA Part B, vol. 2, no. 1, pp. 7-8, 2017.

K. H. Wolfe, W. H. Li, and P. M. Sharp, “Rates of nucleotide substitution vary greatly among plant mitochondrial, chloroplast, and nuclear DNAs,” Proceedings of the National Acadamy of Sciences of the United States of America, vol. 84, no. 24, pp. 9054–9058, 1987.

R. K. Jansen, L. A. Raubeson, J. L. Boore et al., “Methods for obtaining and analyzing whole chloroplast genome sequences,” in Molecular Evolution: Producing the Biochemical Data, vol. 395 of Methods in Enzymology, pp. 348–384, Elsevier, 2005.

J. F. Walker, M. J. Zanis, and N. C. Emery, “Comparative analysis of complete chloroplast genome sequence and inversion variation in Lasthenia burkei (Madieae, Asteraceae),” American Journal of Botany, vol. 101, no. 4, pp. 722–729, 2014.

J. J. Doyle, J. L. Doyle, J. Ballenger, and J. Palmer, “The distribution and phylogenetic significance of a 50-kb chloroplast DNA inversion in the flowering plant family leguminosae,” Molecular Phylogenetics and Evolution, vol. 5, no. 2, pp. 429–438, 1996.

S. Tangphatsornruang, P. Uthaipaisanwong, D. Sangsrakru et al., “Characterization of the complete chloroplast genome of Hevea brasiliensis reveals genome rearrangement, RNA editing sites and phylogenetic relationships,” Gene, vol. 475, no. 2, pp. 104–112, 2011.

J. F. Walker, R. K. Jansen, M. J. Zanis, and N. C. Emery, “Sources of inversion variation in the small single copy (SSC) region of chloroplast genomes,” American Journal of Botany, vol. 102, no. 11, pp. 1751-1752, 2015.

J. D. Palmer, J. M. Nugent, and L. A. Herbon, “Unusual structure of geranium chloroplast DNA: a triple-sized inverted repeat, extensive gene duplications, multiple inversions, and two repeat families,” Proceedings of the National Acadamy of Sciences of the United States of America, vol. 84, no. 3, pp. 769–773, 1987.

S. Tangphatsornruang, D. Sangsrakru, J. Chanprasert et al., “The chloroplast genome sequence of mungbean (Vigna radiata) determined by high-throughput pyrosequencing: structural organization and phylogenetic relationships,” DNA Research, vol. 17, no. 1, pp. 11–22, 2010.

J. T. Johansson, “There large inversions in the chloroplast genomes and one loss of the chloroplast generps16 suggest an early evolutionary split in the genus Adonis (Ranunculaceae),” Plant Systematics and Evolution, vol. 218, no. 3-4, pp. 133–143, 1999.

H. Lee, R. K. Jansen, T. W. Chumley, and K. Kim, “Gene relocations within chloroplast genomes of jasminum and menodora (Oleaceae) are due to multiple, overlapping inversions,” Molecular Biology and Evolution, vol. 24, no. 5, pp. 1161–1180, 2007.

R. K. Jansen, M. F. Wojciechowski, E. Sanniyasi, S. Lee, and H. Daniell, “Complete plastid genome sequence of the chickpea (Cicer arietinum) and the phylogenetic distribution of rps12 and clpP intron losses among legumes (Leguminosae),” Molecular Phylogenetics and Evolution, vol. 48, no. 3, pp. 1204–1217, 2008.

M. Yan, M. J. Moore, A. Meng, X. Yao, and H. Wang, “The first complete plastome sequence of the basal asterid family Styracaceae (Ericales) reveals a large inversion,” Plant Systematics and Evolution, vol. 303, no. 1, pp. 61–70, 2017.

R. Schmieder and R. Edwards, “Quality control and preprocessing of metagenomic datasets,” Bioinformatics, vol. 27, no. 6, pp. 863-864, 2011.

N. Dierckxsens, P. Mardulyn, and G. Smits, “NOVOPlasty: de novo assembly of organelle genomes from whole genome data,” Nucleic Acids Research, vol. 45, 2016.

S. K. Wyman, R. K. Jansen, and J. L. Boore, “Automatic annotation of organellar genomes with DOGMA,” Bioinformatics, vol. 20, no. 17, pp. 3252–3255, 2004.

P. Schattner, A. N. Brooks, and T. M. Lowe, “The tRNAscan-SE, snoscan and snoGPS web servers for the detection of tRNAs and snoRNAs,” Nucleic Acids Research, vol. 33, no. 2, pp. W686–W689, 2005.

M. Lohse, O. Drechsel, and R. Bock, “OrganellarGenomeDRAW (OGDRAW): a tool for the easy generation of high-quality custom graphical maps of plastid and mitochondrial genomes,” Current Genetics, vol. 52, no. 5-6, pp. 267–274, 2007.

S. Kurtz, J. V. Choudhuri, E. Ohlebusch, C. Schleiermacher, J. Stoye, and R. Giegerich, “REPuter: the manifold applications of repeat analysis on a genomic scale,” Nucleic Acids Research, vol. 29, no. 22, pp. 4633–4642, 2001.

T. Thiel, W. Michalek, R. K. Varshney, and A. Graner, “Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.),” Theoretical and Applied Genetics, vol. 106, no. 3, pp. 411–422, 2003.

S. Kurtz, J. V. Choudhuri, E. Ohlebusch, C. Schleiermacher, J. Stoye, and R. Giegerich, “REPuter: The manifold applications of repeat analysis on a genomic scale,” Nucleic Acids Research, vol. 29, no. 22, pp. 4633–4642, 2001.

C. Mayor, M. Brudno, J. R. Schwartz et al., “VISTA: visualizing global DNA sequence alignments of arbitrary length,” Bioinformatics, vol. 16, no. 11, pp. 1046-1047, 2000.

K. A. Frazer, L. Pachter, A. Poliakov, E. M. Rubin, and I. Dubchak, “VISTA: computational tools for comparative genomics,” Nucleic Acids Research, vol. 32, pp. W273–W279, 2004.

P. Librado and J. Rozas, “DnaSP v5: a software for comprehensive analysis of DNA polymorphism data,” Bioinformatics, vol. 25, no. 11, pp. 1451-1452, 2009.

K. Katoh and D. M. Standley, “MAFFT multiple sequence alignment software version 7: improvements in performance and usability,” Molecular Biology and Evolution, vol. 30, no. 4, pp. 772–780, 2013.

J. Felsenstein, “Cases in which parsimony or compatibility methods will be positively misleading,” Systematic Biology, vol. 27, no. 4, pp. 401–410, 1978.

F. Ronquist, M. Teslenko, P. van der Mark et al., “Mrbayes 3.2: efficient bayesian phylogenetic inference and model choice across a large model space,” Systematic Biology, vol. 61, no. 3, pp. 539–542, 2012.

D. Posada, “jModelTest: phylogenetic model averaging,” Molecular Biology and Evolution, vol. 25, no. 7, pp. 1253–1256, 2008.

C. Gao, Y. Deng, and J. Wang, “The complete chloroplast genomes of echinacanthus species (acanthaceae): phylogenetic relationships, adaptive evolution, and screening of molecular markers,” Frontiers in Plant Science, vol. 1989, no. 9, 2019.

Z. Yongbin and A. T. Erin, “The draft genome of Ruellia speciosa (beautiful wild petunia: acanthaceae),” DNA Research, vol. 24, no. 2, pp. 179–192, 2017.

G. Raman and S. Park, “The complete chloroplast genome sequence of ampelopsis: gene organization, comparative analysis, and phylogenetic relationships to other angiosperms,” Frontiers in Plant Science, vol. 341, no. 7, 2016.

I. Park, W. J. Kim, S.-M. Yeo et al., “The complete chloroplast genome sequences of fritillaria ussuriensis Maxim. and Fritillaria cirrhosa D. Don, and comparative analysis with other fritillaria species,” Molecules, vol. 282, no. 22, 2017.

B. Li, F. Lin, P. Huang, W. Guo, and Y. Zheng, “Complete chloroplast genome sequence of decaisnea insignis: genome organization, genomic resources and comparative analysis,” Scientific Reports, vol. 7, 2017.

J. K. Saina, Z.-Z. Li, A. W. Gichira, and Y.-Y. Liao, “The complete chloroplast genome sequence of tree of heaven (Ailanthus altissima (mill.) (sapindales: Simaroubaceae), an important pantropical tree,” International Journal of Molecular Sciences, vol. 19, no. 4, 2018.

J. Zhou, X. Chen, Y. Cui et al., “Molecular structure and phylogenetic analyses of complete chloroplast genomes of two Aristolochia medicinal species,” International Journal of Molecular Sciences, vol. 1839, no. 18, 2017.

D. Jiang, Z. Zhao, T. Zhang et al., “The chloroplast genome sequence of Scutellaria baicalensis provides insight into intraspecific and interspecific chloroplast genome diversity in Scutellaria,” Gene, vol. 227, no. 8, 2017.

J. Zhou, Y. Cui, X. Chen et al., “Complete chloroplast genomes of papaver rhoeas and papaver orientale: molecular structures, comparative analysis, and phylogenetic analysis,” Molecules, vol. 437, no. 23, 2018.

W. Wang, H. Yu, J. Wang et al., “The complete chloroplast genome sequences of the medicinal plant forsythia suspensa (Oleaceae),” International Journal of Molecular Sciences, vol. 2288, no. 18, 2017.

F. Kumbhar, X. Nie, G. Xing et al., “Identification and characterisation of RNA editing sites in chloroplast transcripts of einkorn wheat ( Triticum monococcum ),” Annals of Applied Biology, vol. 172, no. 2, pp. 197–207, 2018.

M. Park, H. Park, H. Lee, B.-H. Lee, and J. Lee, “The complete plastome sequence of an antarctic bryophyte Sanionia uncinata (Hedw.) loeske,” International Journal of Molecular Sciences, vol. 709, no. 19, 2018.

W. Xumei, Z. Tao, B. Guoqing, and Z. Yuemei, “Complete chloroplast genome sequence of Fagopyrum dibotrys: genome features, comparative analysis and phylogenetic relationships,” Scientific Reports, vol. 8, no. 1, 2018.

G. J. Bryan, J. McNicoll, G. Ramsay, R. C. Meyer, and W. S. De Jong, “Polymorphic simple sequence repeat markers in chloroplast genomes of Solanaceous plants,” Theoretical and Applied Genetics, vol. 99, no. 5, pp. 859–867, 1999.

J. Provan, “Novel chloroplast microsatellites reveal cytoplasmic variation in Arabidopsis thaliana,” Molecular Ecology, vol. 9, no. 12, pp. 2183–2185, 2000.

D. Ebert and R. Peakall, “Chloroplast simple sequence repeats (cpSSRs): technical resources and recommendations for expanding cpSSR discovery and applications to a wide array of plant species,” Molecular Ecology Resources, vol. 9, no. 3, pp. 673–690, 2009.

J. K. Saina, A. W. Gichira, Z.-Z. Li, G.-W. Hu, Q.-F. Wang, and K. Liao, “The complete chloroplast genome sequence of Dodonaea viscosa: comparative and phylogenetic analyses,” Genetica, vol. 146, no. 1, pp. 101–113, 2018.

T. Zhou, C. Chen, Y. Wei et al., “Comparative transcriptome and chloroplast genome analyses of two related dipteronia species,” Frontiers in Plant Science, vol. 1512, no. 7, 2016.

H. Philippe, F. Delsuc, H. Brinkmann, and N. Lartillot, “Phylogenomics,” Annual Review of Ecology, Evolution and Systematics, vol. 36, no. 1, pp. 541–562, 2005.

L. A. Raubeson, R. Peery, T. W. Chumley et al., “Comparative chloroplast genomics: analyses including new sequences from the angiosperms nuphar advena and ranunculus macranthus,” BMC Genomics, vol. 8, pp. 174–201, 2007.

R. J. Wang, C. L. Cheng, C. C. Chang et al., “Dynamics and evolution of the inverted repeat-large single copy junctions in the chloroplast genomes of monocots,” BMC Evolutionary Biology, vol. 8, pp. 36–50, 2008.

A. M. Lucinda, F. D. Thomas, and A. K. Carrie, “Toward a comprehensive understanding of phylogenetic relationships among lineages of Acanthaceae s.l (Lamiales),” American Journal of Botany, vol. 95, no. 9, pp. 1136–1152, 2008.

H. Mikael, W. C. Mark, and G. O. Richard, “Relationships in the acanthaceae and related families as suggested by cladistic analysis of rbcl nucleotide sequences,” Plant Systematics and Evolution, vol. 194, no. 1-2, pp. 93–109, 1995.