The Complete Chloroplast Genome of Hypoestes forskaolii (Vahl) R.Br: Insights into Comparative and Phylogenetic Analyses within the Tribe Justiceae

Hypoestes forskaolii is one of the most important species of the family Acanthaceae, known for its high economic and medicinal importance. It is well distributed in the Arab region as well as on the African continent. Previous studies on ethnomedicine have reported that H. forskaolii has an anti-parasitic effect as well as antimalarial and anthelmintic activities. Previous studies mainly focused on the ethnomedicinal properties, hence, there is no information on the genomic architecture and phylogenetic positions of the species within the tribe Justiceae. The tribe Justicieae is the most taxonomically difficult taxon in Acanthoideae due to its unresolved infratribal classification. Therefore, by sequencing the complete chloroplast genome (cp genome) of H. forskaolii, we explored the evolutionary patterns of the cp genome and reconstructed the phylogeny of Justiceae. The cp genome is quadripartite and circular in structure and has a length of 151,142 bp. There are 130 genes (86 coding for protein, 36 coding for tRNA and 8 coding for rRNA) present in the plastome. Analyses of long repeats showed only three types of repeats: forward, palindromic and reverse were present in the genome. Microsatellites analysis revealed 134 microsatellites in the cp genome with mononucleotides having the highest frequency. Comparative analyses within Justiceae showed that genomes structure and gene contents were highly conserved but there is a slight distinction in the location of the genes in the inverted repeat and single copy junctions. Additionally, it was discovered that the cp genome includes variable hotspots that can be utilized as DNA barcodes and tools for determining evolutionary relationships in the Justiceae. These regions include: atpH-atpI, trnK-rps16, atpB-rbcL, trnT-trnL, psbI-trnS, matK, trnH-psbA, and ndhD. The Bayesian inference phylogenetic tree showed that H. forskaolii is a sister to the Dicliptra clade and belongs to Diclipterinae. The result also confirms the polyphyly of Justicia and inclusion of Diclipterinae within justicioid. This research has revealed the phylogenetic position of H. forskaolii and also reported the resources that can be used for evolutionary and phylogenetic studies of the species and the Justicieae.


Introduction
Hypoestes forskaolii (Vahl) R.Br is a herbaceous plant and one of the most important Acanthaceaous species mainly distributed in some part of Africa and the Arab region [1]. The plant has been used in folk medicine in the treatment of various diseases like cancer, gonorrhea, heart and chest diseases [2,3]. The species is also reported to have several biological properties which includes antifungal, atrypanosomal, antileishmanial, antiplasmodial and cytotoxic properties [2,4]. A leaf decoction from the plant is used by Herdsmen to kill insect and parasite on sheep [5,6]. Fresh leaves of H. forskaolii are used to heal wounds and are also reported to have insecticidal activity [7]. Despite the traditional medicinal value of the plant, its complete chloroplast genome (cp genome) has not been sequenced, thus the genome evolution remains unexplored.
Genes 2022, 13, 2259 3 of 17 bag containing silica gel and the specimen was taken to Herbarium Center for Biodiversity and Conservation, Umaru Musa Yaradua University for identication. The specimen was authenticated by the Curator and was given accession number UMH0145. The dried leaf material was used to extract the DNA using a Qiagen genomic DNA extraction kit, following the manufacturer's instructions.

Library Construction, Sequencing and Assembly
For the DNA sample preparations, 1.0 µg of DNA was used as input material. Following the manufacturer's instructions, the NEBNext DNA Library Prep Kit was used to create the sequencing libraries, followed by addition of indices to the sample. The genomic DNA was sheared at random into segments of 350 bp, which were then prepared for further PCR enrichment and sequencing. The resulting PCR products were subject to purification (using the AMPure XP system), then the resultant libraries were measured using real-time PCR and evaluated for size distribution using an Agilent 2100 Bio analyzer. After pooling, the qualified libraries based on projected data volume and effective concentration, were fed onto Illumina sequencers. Using PRINSEQlite v0.20.4 [24], clean reads sequences (5.2 GB) were obtained from the raw data through filtering, which were then subjected to assembly using NOVOPlasty 4.2. [25] using kmer (K-mer = 39) and the cp genome of Justicia flava (NC 044862.1) was used as the seed and reference.

Gene Annotation
The online tool GeSeq [26] was used to annotate all the genes present in the cp genome of H. forskaolii using the plastome sequence of Justicia flava as reference. The sequin program was used to correct the errors in the genes that were not successfully annotated in the Geseq by adjusting the position of the codons. The cp genome architecture was drawn in OGDRAW (Organellar Genome DRAW) [26].

Codon Usage Analysis
The software MEGA version 6 [27] was used to calculate the relative synonymous codon usage values (RSCU), codon usage and base composition.

Repeat Analysis
MicroSAtellite (MISA) (http://webblast.ipk-gatersleben.de/misa/index.php accessed on 24 November 2022) was used to identify Simple Sequence Repeats (SSRs) in the H. forskaolii cp genome [28]. For mononucleotides, dinucleotides, trinucleotides, and tetra, penta, and hexa nucleotides SSR motifs eight, five, four, and three repetitions units were used, respectively. The online software REPuter [29] was used to identify the types of long repeats present in the cp genome.

Genome Comparison
Using the annotation of H. forskaolii as a reference in the Shuffle-LAGAN mode [30], the cp genomes of seven species of Justiceae were compared using the tool mVISTA [31]. The online software IR scope (https://irscope.shinyapps.io/irapp/ accessed on 1 November 2022) was used to compare the border regions of the cp genome.

Characterization of Substitution Rate
To identify the genes that are under selection pressure, DNAsp version 6 [32] was used to examine synonymous (dS) and nonsynonymous (dN) substitution rate and dN/dS ratio.

Sequence Divergence
To determine sequence divergence and identify the variable regions in the cp genome, sliding window analysis was used to evaluate the diversity of nucleotide in the cp genome using DnaSP version 6 with the following parameters: 600 bp for window length and 200 bp for step size.

Phylogenetic Analysis
The cp genome of seventeen Acanthoideae species as well as four species, Erythranthe lutea (Phrymaceae), Scrophularia dentata (Scrophulariaceae), Lysionotus pauciflorus (Gesneriaceae) and Tanaecium tetragonolobum (Bignoniaceae) were downloaded from Genbank. The Acanthoideae species are the in group while the other four were used as the out group. Using MAFFT version 7 [33], the newly sequenced cp genome of H. forskaolii was aligned with the downloaded cp genomes. Using MrBayes version 3.2.6, a Bayesian inference strategy was used to reconstruct the phylogenetic tree [34]. The appropriate model was chosen using jModelTest version 3.7 [35].

Characteristics of H. forskaolii Chloroplast Genome
Previous findings revealed that the cp genome of flowering plants is extremely wellpreserved in both the gene content and structural organization; however, expansion and contraction in the inverted repeat and single copy junctions are among the evolutionary events leading to variations in the cp genomes [36,37]. The cp genome of H. forskaolii is quadripartite and circular in structure and has a total length of 151,142 bp. The cp genome consists of a pair of inverted repeats (IRb and IRa) 25,477 bp, Small Single Copy (SSC) 17,012 and Large Single Copy 83,176 bp ( Figure 1). A total of 72,852 bp make up the genome's non-coding region which is comprised of introns and intergenic spacers, and the 78,290 bp code for protein genes. The GC percentage of the LSC and SSC regions was 36.0% and 32.3%, respectively, whereas the GC content of the inverted repeats IRa and IRb was 43.4% and 43.3%, respectively (Table 1). It is discovered that the IRs have a larger proportion of GC than SSC and LSC regions. The annotation of H. forskaolii cp genome revealed a total of 130 genes (86 proteincoding genes, 36 tRNAs genes and 8 rRNAs genes), 96 genes are present in the LSC (82 protein coding genes and 21 tRNA genes) and SSC (14 protein coding genes and 1 tRNA) while the remaining 17 genes (7 tRNAs, 4 rRNAs and 7 protein-coding genes) are repeated in the IRa and IRb regions (Table 2 and Figure 1). Almost all the protein-coding genes in the cp genome have ATG as their start codon and few of them have alternative start codon, GTG, ACG and ATC; this has been reported to occur in the cp genome of angiosperms [38][39][40].
Some of the coding genes present in the cp genome of H. forskaolii have introns. Introns are reported to occur in some of the protein-coding and tRNAs genes of flowering plants cp genomes [38,39]. Out of the 130 coding genes, 16 are characterized with one or two introns (Table 3). Among the 16 genes, six are tRNAs and 11 are protein coding genes. Ten of the intron-containing genes are located in the LSC, one gene in the SSC while the remaining five are in the inverted repeat region. ATP dependent protease subunit p gene (clpP) and one of the Photosysem I gene (ycf3) possessed two introns while the remaining 14 genes have only one intron. The tRNA, trnK-UUU is the gene with longest intron which is due to the inclusion of matK in the gene.
1 Figure 1. The structure of the H. forskaolii cp genome. Genes inside the circles are transcribed clockwise, while those outside the circles are transcribed counterclockwise. The colorful bar displays genes that are known to be functioning. The inner circle's dark grey and light grey colors, respectively, designate the GC and AT contents.
The frequency of a codon that encodes for a specific amino acid was compared using codon usage analysis [41]. A codon is a sequence of trinucleotides that encodes for specific amino acids that are used in protein synthesis [42]. Because of bias in mutation, codon use is a factor influencing the development of the chloroplast genome [43] and it differs between species [44]. The nucleotide sequence of the protein-coding genes (78,290 bp) was used to calculate the frequency of the codons present in the cp genome. The relatively synonymous codon usage (RSCU) of the genes in the cp genome is shown in (Table 4, Figure 2). The codon usage analysis revealed that 26,095 codons encode for the genes in the cp genome. All the 20 known amino acids are encoded by 61 codons (Figure 2). Codons coding for the amino acid leucine are more prevalent in the cp genome while codons that code for Cysteine are the less common ( Table 4). The Cytosine (C) and Guanine (G) endings are more prevalent than the Thymine (T) and Adenine (A) endings; the cp genomes of other angiosperms have Thymine and Adenine endings occurring more frequently [45][46][47]. The result of the analysis (

Long Repeats
Long repeats sequences present in the cp genome of H. forskaolii were identified using the program REPuter; from the results it was discovered that tandem repeats and three  Table 3. Genes with introns in the H. forskaolii chloroplast genome and length of introns and exons.  Long repeats sequences present in the cp genome of H. forskaolii were identified using the program REPuter; from the results it was discovered that tandem repeats and three types of long repeats (forward, reverse and palindromic) were present in the plastome H. forskaolii (Figure 3). In total, there are 88 repeats in the cp genome of H. forskaolii (19 palindromic repeats, 27 forward repeats, 4 reverse repeats and 38 tandem repeats). Most of the palindromic and forward repeats sizes are between 20-29 bp, followed 10-19 bp. The length of repeated sequences in H. forskaolii cp genome ranges from 10 to 44 bp, are analogous to the lengths in other angiosperm cp genomes [48][49][50].  We compared the frequency of repeats among seven Justiceae cp genomes, result indicated that only three species, namely, D. acuminata, P. japonica and R. p contained complement, forward, reverse and palindromic repeats ( Figure 4D). C and P. haikangense have the highest frequency of forward repeats (37) while R. p has the lowest (12). D. acuminata and J. procumbes have the same number of repeats, five each. R. pectinata has the highest number of reverse repeats (9) w forskaolii has the lowest (4). Complement repeats are found to be the less numero of repeat across the genome with D. acuminata, P. japonica and R. pectinata having o and five, respectively.

Simple Sequence Repeats (SSRs)
Simple sequence repeats (SSRs) are short repeats of sequences usually 1-6 bp very useful at evaluating genetic variation among species. These SSRs are presen genomes of angionsperms and are uniparently inherited. They are therefore empl molecular markers in developmental research such as genetic hetero investigations, and they also aid in the identification of species [51][52][53]. This discovered 136 microsatellites in the cp genome of H. forskaolii (Table 5). Mononucl are the most frequent SSRs in the cp genome, constituting about 78.67%, of which m are polythymine (41.91%) and polyadenine (30.14%); this is consistent with p studies [53]. Among the dinucleotide only AT/AT is found in the genome. Reflectin complementary, only two trinucleotide AAT/ATT and ATC/ATG, five tetra AAAC AAAG/CTTT, AAAT/ATTT, AATC/ATTG, AATT/AATT were present in the cp g Penta nucleotide and hexa nucleotide were not discovered in the cp genome (Figu The LSC region harbored most of the microsatellites, followed by SSC ( Figure 4B)  We compared the frequency of repeats among seven Justiceae cp genomes, and the result indicated that only three species, namely, D. acuminata, P. japonica and R. pectinata contained complement, forward, reverse and palindromic repeats ( Figure 4D). C. nutans and P. haikangense have the highest frequency of forward repeats (37) while R. pectinata has the lowest (12). D. acuminata and J. procumbes have the same number of reverse repeats, five each. R. pectinata has the highest number of reverse repeats (9) while H. forskaolii has the lowest (4). Complement repeats are found to be the less numerous type of repeat across the genome with D. acuminata, P. japonica and R. pectinata having one, two and five, respectively.

Simple Sequence Repeats (SSRs)
Simple sequence repeats (SSRs) are short repeats of sequences usually 1-6 bp that are very useful at evaluating genetic variation among species. These SSRs are present in cp genomes of angionsperms and are uniparently inherited. They are therefore employed as molecular markers in developmental research such as genetic heterogeneity investigations, and they also aid in the identification of species [51][52][53]. This study discovered 136 microsatellites in the cp genome of H. forskaolii (Table 5). Mononucleotides are the most frequent SSRs in the cp genome, constituting about 78.67%, of which majority are polythymine (41.91%) and polyadenine (30.14%); this is consistent with previous studies [53]. Among the dinucleotide only AT/AT is found in the genome. Reflecting series complementary, only two trinucleotide AAT/ATT and ATC/ATG, five tetra AAAC/GTTT, AAAG/CTTT, AAAT/ATTT, AATC/ATTG, AATT/AATT were present in the cp genome.
Penta nucleotide and hexa nucleotide were not discovered in the cp genome ( Figure 4A). The LSC region harbored most of the microsatellites, followed by SSC ( Figure 4B). The frequency of SSRs among the cp genomes of seven species of Justiceae was analyzed ( Figure 4C); the results revealed that mononucleotides repeats are the most frequent across all the cp genomes. D. acuminata and H. forskaolii are the species with the highest frequency of mononucleotide; 108 and 107, respectively. Pentanucleotides were not present in the cp genomes of H. forskaolii, D. nutans and D. acuminata while hexanucleotide was only present in P. japonica.  The frequency of SSRs among the cp genomes of seven species of Justiceae was analyzed ( Figure 4C); the results revealed that mononucleotides repeats are the most frequent across all the cp genomes. D. acuminata and H. forskaolii are the species with the highest frequency of mononucleotide; 108 and 107, respectively. Pentanucleotides were not present in the cp genomes of H. forskaolii, D. nutans and D. acuminata while hexanucleotide was only present in P. japonica.

Comparative Analysis of Justiceae Species Cp Genome
To evaluate the level of genome divergence in Justiceae, the newly sequenced cp genome H. forskaolii was compared with six Justiceae species cp genomes downloaded from the GenBank. The cp genomes were aligned using mVISTA with the annotation of H. forskaolii as a reference. The result of the anaysis revealed that the compared cp genomes are well-preserved in terms of genome structure and gene content; however, there was some level of variation. The protein-coding regions were found to be more conserved than the introns and intergenic spacers. In terms of the four regions, the IRa and IRb were more conserved than the SSC and LSC ( Figure 5). This has been reported to occur in certain taxa cp genomes in earlier studies [54,55]. The most divergent noncoding regions among the seven cp genomes are trnH-psbA, trnK-rps16, rps16-trnQ-UUG, trnE-UUC-psbD, atpH-atpI, trnT-trnL, ndhC-trnV, accD-psaI, petA-psbJ, atpB-rbcL, rps12 and trnL-rpl32. A slight sequence variation was observed in the following genes psbM, matK, ycf1, trnA-UGC, ndhH, and rrn16. These regions can be used as a source of potential barcode for identification/authentication of Justiceae species as well as resources for inferring phylogenetic relationships of the Acanthoideae. UUC-psbD, atpH-atpI, trnT-trnL, ndhC-trnV, accD-psaI, petA-psbJ, atpB-rbcL, rps12 and trnL-rpl32. A slight sequence variation was observed in the following genes psbM, matK, ycf1, trnA-UGC, ndhH, and rrn16. These regions can be used as a source of potential barcode for identification/authentication of Justiceae species as well as resources for inferring phylogenetic relationships of the Acanthoideae. The structure and size of the chloroplast genome is often retained by angiosperms [53]; however, due to evolutionary processes, including genome contraction and expansion, there can be subtle variations in the size and location of the boundaries of inverted repeats and single copy regions [56,57]. We compared the JLB, JSB, JSA and JLA boundaries of the seven cp genomes of Justiceae and the results ( Figure 6) showed some degree of similarity and variation among the compared cp genomes. The length of the seven cp genomes ranged from 149,627 bp (R. pectinata) to 152,849 bp (P. haikangense). Three species J. flava, P. japonica and D. acuminata have their JLB within the gene rps19 with 102 bp overlapping into the IR. The JLB was bordered by rps19 (LSC) and rpl2 in C. nutans due to the contraction of the IR. In contrast, due to the IR expansion, the JLB of the R. pectinata was located within the rpl22 while P. haikangense was between the rpl22 (LSC) and rps19 (IR). The JSB was found within ndhF with 2170-2184 bp overlapping into the IR except for the C. nutans. The trnH is located at the junction of the LSC/IRa border of all the Figure 5. Variable regions in the cp genome of seven Justiceae species. The top arrow represents the direction of transcription; the colors blue and pink denote protein coding and conserved non-coding sequence, respectively; light green denotes tRNAs and rRNAs. The cp genome coordinates are shown by the x-axis, and the percentage identity ranges from 50% to 100% on the y-axis.
The structure and size of the chloroplast genome is often retained by angiosperms [53]; however, due to evolutionary processes, including genome contraction and expansion, there can be subtle variations in the size and location of the boundaries of inverted repeats and single copy regions [56,57]. We compared the JLB, JSB, JSA and JLA boundaries of the seven cp genomes of Justiceae and the results ( Figure 6) showed some degree of similarity and variation among the compared cp genomes. The length of the seven cp genomes ranged from 149,627 bp (R. pectinata) to 152,849 bp (P. haikangense). Three species J. flava, P. japonica and D. acuminata have their JLB within the gene rps19 with 102 bp overlapping into the IR. The JLB was bordered by rps19 (LSC) and rpl2 in C. nutans due to the contraction of the IR. In contrast, due to the IR expansion, the JLB of the R. pectinata was located within the rpl22 while P. haikangense was between the rpl22 (LSC) and rps19 (IR). The JSB was found within ndhF with 2170-2184 bp overlapping into the IR except for the C. nutans. The trnH is located at the junction of the LSC/IRa border of all the compared cp genomes with the exception of H. forskaolii, which might be due to an expansion which leads to the loss of rps19. The cp genome of C. nutans varied with the other cp genomes by having the ndhF gene located in the SSC region. The ycf1 pseudogene extended through the SSC and IRa with about 4300 bp in the SSC and 812 bp in the IRa in all the compared genomes except for C. nutans. The JSA and JSB had the most conserved borders among the compared cp genomes. The cp genome of R. pectinata was unique by having rps3 in LSC/IRb border. The cp genome of R. pectinata had the smallest LSC region 81,979 bp while P. haikangese had the longest 83,878 bp. H. forskaolii was the only species that lost the rps19, which is located LSC/IR border. compared cp genomes with the exception of H. forskaolii, which might be due to an expansion which leads to the loss of rps19. The cp genome of C. nutans varied with the other cp genomes by having the ndhF gene located in the SSC region. The ycf1 pseudogene extended through the SSC and IRa with about 4300 bp in the SSC and 812 bp in the IRa in all the compared genomes except for C. nutans. The JSA and JSB had the most conserved borders among the compared cp genomes. The cp genome of R. pectinata was unique by having rps3 in LSC/IRb border. The cp genome of R. pectinata had the smallest LSC region 81,979 bp while P. haikangese had the longest 83,878 bp. H. forskaolii was the only species that lost the rps19, which is located LSC/IR border.

Divergence of Protein Coding Genes Sequence
To determine the genes that were undergoing selective pressure in the cp genome of H. forskaolii, DNAsp was used to calculate the dN/dS ratio, nonsynonymous (dN) and synonymous (dS) rates. The results showed that the dN/dS ratio was less than one in almost all of the paired genes except atpF and clpP (Figure 7), indicating that most of the genes were under negative selection with the exception of clpP and atpF. The synonymous (dS) values rangee from 0.0053 to 0.1628 in all the protein-coding genes (Figure 7). Twentynine genes, including ycf15, ycf3, rps18, rps14, rpl36, rpl32, psbM, psbN, psbJ, psaI, petG, ndhJ, ndhC, infA and atpH showed no nonsynonymous changes occurring in the plastome of H. forskaolii; comparable findings were reported for other cp genomes [58][59][60]

Divergence of Protein Coding Genes Sequence
To determine the genes that were undergoing selective pressure in the cp genome of H. forskaolii, DNAsp was used to calculate the dN/dS ratio, nonsynonymous (dN) and synonymous (dS) rates. The results showed that the dN/dS ratio was less than one in almost all of the paired genes except atpF and clpP (Figure 7), indicating that most of the genes were under negative selection with the exception of clpP and atpF. The synonymous (dS) values rangee from 0.0053 to 0.1628 in all the protein-coding genes (Figure 7). Twentynine genes, including ycf15, ycf3, rps18, rps14, rpl36, rpl32, psbM, psbN, psbJ, psaI, petG, ndhJ, ndhC, infA and atpH showed no nonsynonymous changes occurring in the plastome of H. forskaolii; comparable findings were reported for other cp genomes [58][59][60].

Identification of Sequence Divergence
Variable regions of the chloroplast genome are very useful in infering phylogenetic relationships and identification of species at the lowest taxonomic rank. These regions also play a vital role in providing information that helps in detecting differences between species and revealing the changes in the population structure [61,62]. The plastome sequence of H. forskaolii was found to be similar with that of related Justiceae species. The calculated pi values ranged from 0 to 0.79 (Figure 8), which shows slightly variation among the chloroplast genome and are relatively conserved. This pattern of variation was reported to occur in the plastomes of angiosperm [40]. Comparing the sequence divergence in the single copy regions and the inverted repeat region, the single copy region showed a higher variability than the invterted repeat. Highly variable protein-coding genes in the plastomes include psbM, ndhH and ycf1. Similarly, two intergenic spacers, trnE-UUC-psbD and rps16-trnQ-UUG were found to be highly variable. These results agree with the mVISTA divergence analysis and show that the regions could be used in the identification and authentication of Justiceae species.

Identification of Sequence Divergence
Variable regions of the chloroplast genome are very useful in infering phylogenetic relationships and identification of species at the lowest taxonomic rank. These regions also play a vital role in providing information that helps in detecting differences between species and revealing the changes in the population structure [61,62]. The plastome sequence of H. forskaolii was found to be similar with that of related Justiceae species. The calculated pi values ranged from 0 to 0.79 (Figure 8), which shows slightly variation among the chloroplast genome and are relatively conserved. This pattern of variation was reported to occur in the plastomes of angiosperm [40]. Comparing the sequence divergence in the single copy regions and the inverted repeat region, the single copy region showed a higher variability than the invterted repeat. Highly variable protein-coding genes in the plastomes include psbM, ndhH and ycf1. Similarly, two intergenic spacers, trnE-UUC-psbD and rps16-trnQ-UUG were found to be highly variable. These results agree with the mVISTA divergence analysis and show that the regions could be used in the identification and authentication of Justiceae species.

Phylogenetic Analysis
The cp genome has been well utilized in inferring phylogenetic relationship d its conserved nature and the presence of informative sites [63][64][65]. Various taxon complexes at different taxonomic levels have been resolved using phylogenetic reconstructed from cp genomes [66,67]. To reconstruct the phylogeny of Justiceae infer the phylogenetic position of H. forskaolii, the cp genome sequences of 21 taxa downloaded from Genbank. MAFFT version 7 was used to align the cp genome gen of H. forskaolii and all the downloaded cp genomes. Bayesian inference was use reconstructing the phylogenetic tree. The results in (Figure 9), show the lineage of just

Phylogenetic Analysis
The cp genome has been well utilized in inferring phylogenetic relationship due to its conserved nature and the presence of informative sites [63][64][65]. Various taxonomic complexes at different taxonomic levels have been resolved using phylogenetic trees reconstructed from cp genomes [66,67]. To reconstruct the phylogeny of Justiceae and infer the phylogenetic position of H. forskaolii, the cp genome sequences of 21 taxa were downloaded from Genbank. MAFFT version 7 was used to align the cp genome genome of H. forskaolii and all the downloaded cp genomes. Bayesian inference was used in reconstructing the phylogenetic tree. The results in (Figure 9), show the lineage of justiciod is strongly supported (1.0 PP) with all the twelve species belonging to the tribe Justiceae clustering in one clade (monophyletic). This is congruent with previous studies using molecular, nrITS and some of the chloroplast genes [0,17]. Withing the clade, there are six major sub clades, all with strongly supported posterior probability [PP] = 1.00. H. forskaolii forms a sister relationship with a sub clade containing Peristrophe and Dicleptera (Diclipterinae) with a strong support. A similar tree was reported by [17] and this confirmed that the species is a member of the Diclipterinae subtribe as reported by Bremekamp in his ealier classification. The core Diclipterianae (Dicliptera, Hypoestes and Peristrophe) is strongly supported in this study and members of this genera are reported to be united by some of the inflorescence part [68]. The sister relationship between Peristrophe and Dicleptera needs to be revisited by looking at their phylogenetic positions, though [69,70] suggested that the two taxa should be treated as different genera due to their differences in capsule dehiscence. The New World Justicia species forms a sister relationship with Diclipterinae with strong support. The phylogenetic tree clearly showed that Justicia is paraphyletic, as reported previously by [0,17], and there is a need to combine the phylogenetic approach with a morphological study to resolve this complexity.

Conclusions
In this study, we have sequenced and reported on the cp genome of H. forskaolii to provide a valuable plastome genomic resources for the species. The plastomes have a

Conclusions
In this study, we have sequenced and reported on the cp genome of H. forskaolii to provide a valuable plastome genomic resources for the species. The plastomes have a typical gymonosperm cp genome structure they are comparable to another p genome of Acanthaceae. Simple sequence repeats used for evolutionary studies within the species were identified. The genome comparative analyses of seven Justiceae species revealed variable hotspot that could be used to develop a DNA barcode for the identification of the species. These hotspots will also be useful in phylogenetic relationship studies of the family Acanthaceae. The study has also revealed that only a few genes are under positive selection. The findings of the study have reported and confirmed the tribal position of major genera within Justiceae and has called for a further phylogenetic approach and morphological study to resolve the taxonomic complexities of the polyphyletic Justicia and the Justiceae.