Complete chloroplast genome sequence of Pachystachys lutea Nees: genome structure, adaptive evolution, and phylogenetic relationships

Complete chloroplast genome sequence of Pachystachys lutea Nees: genome structure, adaptive evolution, and phylogenetic relationships | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Complete chloroplast genome sequence of Pachystachys lutea Nees: genome structure, adaptive evolution, and phylogenetic relationships Changmei Du, Yan Dong, Haishuo Gao, Tingting Fang, Jianhua Yue, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5848411/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Nov, 2025 Read the published version in BMC Genomic Data → Version 1 posted 10 You are reading this latest preprint version Abstract Background Pachystachys lutea Nees is a typical species of the family Acanthaceae, native to tropical South America. As an evergreen shrub, it has found extensive application in landscape greening due to its unique ornamental value. However, there are few available phylogenetic and genetic studies about the chloroplast (cp) genome of P. lutea . Results This study characterized the cp genome of P. lutea by using Illumina sequencing technology and inferred the phylogenetic position of the species. The results indicated that the cp genome had a high degree of conservation in gene structure and gene content, with a typical quadripartite structure. Its total length is 151,574 bp and the total GC content is 38.18%. A total of 132 genes were annotated, including 87 protein-coding genes (PCGs), 37 tRNAs and eight rRNA genes. Through the comparative analysis, the diversity and variation of large single-copy (LSC) and small single-copy (SSC) regions were significantly higher than those of inverted repeat (IR) regions. Genes with high nucleotide polymorphism, such as rps19 , ycf1 , and ndhF provided potential reference loci for molecular identification within the P. lutea . The phylogenetic analysis showed that the P. lutea and Clinacanthus nutans forms a sister group with 100% bootstrap value, which proves that P. lutea develops conservatively in the course of evolution. Conclusion This paper for the first time reports the phylogenetic study of the complete cp genome within the genus Pachystachys . The study provides a theoretical basis for the research on genetic diversity, molecular markers, and species identification of plants in the Acanthaceae family. It enriches the genetic information and supports the evolutionary relationships among plants in this family. Pachystachys lutea Acanthaceae chloroplast genome comparative analysis phylogenetic analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction The Acanthaceae family contains approximately 250 genera and more than 4000 species [ 1 , 2 ], mainly distributed in tropical and subtropical areas [ 3 , 4 ]. Approximately 50 genera and more than 400 species are distributed in China [ 5 ]. Many species of the Acanthaceae family are remarkable flowering ornamental plants, characterized by diverse forms, vivid colors, and extended ornamental periods. Pachystachys lutea Nees is one of them, is a perennial evergreen flowering shrub that prefers high temperatures, high humidity and sunny environments and is mainly distributed in the southern areas of the Yangtze River in China. The flower has a unique shape and blooms continuously throughout the year in warm environments (the greenhouse of Shanghai Chenshan Botanical Garden), so it is often planted as an ornamental plant. P. lutea has very large spikelike inflorescences at the top of each new branch, and the golden bracts are stacked and spread out with small white flowers like shrimp bodies. Furthermore, its roots, leaves, and flowers are also used for medicinal purposes, treating pneumonia [ 6 ], diarrhea, worms [ 7 ], etc., and are the important medicinal plant resources to be developed. In addition, it has economic value. The leaves serve as the source of endophytic fungi [ 8 , 9 ] and blue dye [ 10 ], and nectar has the potential to serve as a substitute for sugar production in the future [ 11 ]. Currently, there are relatively few species within the genus Pachystachys that have been named, yet its outstanding ornamental characteristics have made P. lutea highly recommended in China, where it has become an extremely valuable ornamental plant resource, widely loved by the public, and effectively enhancing the diversity of landscape species applications. The chloroplast (cp), an important organelle in green plants [ 12 ], houses the cp genome which is of great significance in plant evolutionary biology research. It presents remarkable benefits due to its genetic stability, well-preserved genome structure, and faster rate of evolutionary change compared to mitochondria. Consequently, the cp genome is widely utilized in the study of genetic relationships and species evolution between different species [ 13 , 14 ]. Despite its significant ornamental and medicinal value, the complete cp genome of P. lutea has not yet been sequenced, limiting our understanding of its evolutionary history and genetic traits. P. lutea is a species within the genus Pachystachys of the family Acanthaceae. Research on P. lutea has mainly focused on its horticultural applications, cultivation techniques, and medicinal properties, leaving its genetic and phylogenetic aspects largely unexplored. Nevertheless, scant knowledge exists regarding its phylogenetic relationship and evolutionary history, primarily due to the dearth of research on the modern taxonomic system, especially in the scarcely explored realm of molecular evolution. Scotland and Vollesen (2000) [ 15 ], initially applied molecular systematics in conjunction with floral organ development to establish a classification system for the family Acanthaceae. Through a comprehensive study of trnL-trnF and internal transcribed spacers (ITS), it was demonstrated that Pachystachys shares a common lineage with Henrya , Carlowrightia , Anisacanthus , and Tetramurium , and this branch is further grouped with the one encompassing Dicliptera and Justicia . However, the position of P. lutea within the Acanthaceae requires further validation. This study aims to: (1) analyze the structural characteristics of the cp genome of P. lutea , (2) investigate its adaptive evolutionary traits, and (3) clarify its phylogenetic relationships with closely related species. To achieve these aims, we employed molecular biology and high-throughput sequencing techniques to characterize the cp genome of P. lutea . This involved using the Illumina NovaSeq 6000 platform for sequencing and a suite of bioinformatics tools for genome assembly, annotation, and analysis. We also compared the results with those obtained from traditional morphological taxonomic methods. Traditional morphological taxonomic methods have limitations, often leading to ambiguous or inconsistent classifications. Studies at molecular level can provide more precise genetic information. By filling this research gap, we aim to lay a solid foundation for more accurately determining its taxonomic position and facilitating future research endeavors. Results Genome structure of the P. lutea cp genome In this study, we assembled and annotated the complete cp genome, analyzed the sequencing depth, which reached an average of 3,012× sequencing depth (Fig. S1 ). Compared with that of other species [ 16 , 17 ], this higher sequencing depth has enhanced the reliability of gene annotation and structural analysis. The accuracy of the assembly and annotation of P. lutea cp genome was evaluated, respectively (Fig. S2 ; Fig. S3 ). The complete cp genome of P. lutea was 151,574 bp in length (Fig. 1 ), and it had a typical quadripartite structure with junction regions: a LSC region of 83,348 bp, an SSC region of 17,226 bp, and a pair of IR regions (IRa and IRb) of 25,500 bp each. The overall GC content of the complete P. lutea cp genome was 38.18%, and the corresponding values in the LSC, SSC, and IR regions were 36.21%, 32.36%, and 43.36%, respectively. The complete cp genome was found to contain 132 genes, which comprised 87 PCGs, 37 tRNA genes, and eight rRNA genes (Table 1 ). Nine PCGs, six tRNA genes, and four rRNA genes were duplicated in the IR regions. Nineteen genes contained two exons, and four genes ( clpP , ycf3 , and two rps12 ) contained three exons. Among 87 PCGs, 45 photosynthesis genes, 29 genes related to self-replication, six other genes and seven genes with unknown function were identified. Table 1 Gene function statistics table of chloroplast genome in P. lutea . Gene category Gene function Gene name Photosynthesis gene Subunits of photosystem I psaA, psaB, psaC, psaI, psaJ Subunits of photosystem II psbL, psbZ, psbM, psbN, psbA, psbB, psbC, psbD, psbE, psbF, psbT, psbH, psbI, psbJ, psbK Subunits of NADH-dehydrogenase ndhG, ndhH, ndhI, ndhJ, ndhK, ndhA*, ndhB*(2), ndhC, ndhD, ndhE, ndhF Subunits of cytochrome b/f complex petL, petN, petA, petB*, petD*, petG Subunit for ATP synthase atpI, atpA, atpB, atpE, atpF*, atpH Large subunit of rubisco rbcL Self-replication Large subunit of ribosome rpl20, rpl22, rpl32, rpl23(2), rpl14, rpl33, rpl16*, rpl36, rpl2*(2) Small subunit of ribosome rps11, rps14, rps15, rps16*, rps2, rps3, rps18, rps4, rps19, rps7(2), rps8, rps12**(2) DNA dependent RNA polymerase rpoA, rpoB, rpoC1*, rpoC2 rRNA gene rrn5(2), rrn4.5(2), rrn16(2), rrn23(2) tRNA gene trnR-UCU, trnE-UUC, trnT-GGU, trnS-GGA, trnV-GAC(2), trnR-ACG(2), trnL-UAA*, trnG-GCC, trnD-GUC, trnY-GUA, trnP-UGG, trnM-CAU, trnL-CAA(2), trnS-GCU, trnW-CCA, trnF-GAA, trnT-UGU, trnS-UGA, trnV-UAC*, trnG-UCC*, trnL-UAG, trnI-GAU*(2), trnH-GUG, trnH-CAU(2), trnQ-UUG, trnN-GUU(2), trnK-UUU*, trnA-UGC*(2), trnC-GCA Other genes Translational initiation factor infA Maturase matK Protease clpP** Envelope membrane protein cemA Subunit of Acetyl-carboxylase accD C-type cytochrome synthesis gene ccsA Unknown gene Open reading frames (ORF,ycf) ycf1, ycf2(2), ycf3**, ycf4, ycf15(2) Notes: Gene *: Gene with one intron and two exons; Gene **: Gene with two introns and three exons; Gene (2): Number of copies of multi-copy genes; Simple sequence repeats analysis SSRs analysis identified 47 SSRs in the P. lutea cp genome. They were mainly of three types: mononucleotides, dinucleotides, trinucleotides, tetranucleotide, pentanucleotide, and hexanucleotide types were not found. Among them, the mononucleotides repeat sequence (A/C/T/G) type was the most abundant, with 40, which were mainly dominated by A and T bases; there were six dinucleotide repeat sequences (AT/TA), and one trinucleotide repeat sequence (TCT) (Fig. 2 ). Out of 47 SSR loci, 37 were concentrated in the LSC region, eight were located in the SSC region, and only two were located in the IR regions (Table S1 ). Codon preference analysis A total of 24,537 codons in 87 PCGs of the cp genome of P. lutea were participated in translation protein expression. Codons with a RSCU over 1 are thought to be favored by amino acids. Overall, 31 codons had an RSCU > 1.0, with 29 codons ending in A or T and two codons ending in G or C. In addition, among the amino acid codes, leucine (Leu) had the highest encoding rate, with six synonymous codon codes (TTA, TTG, CTT, CTC, CTA, CTG), with a total of 2,629. The encoding rate of cysteine (Cys) was low, and there were two synonymous codons (TGT, TGC), totaling 279. methionine (Met) and tryptophan (Trp) had only one codon, while the rest of the amino acids had two to six codons (Fig. 3 ; Table S2 ). In addition, the ENC value of the PCGs was 45.83, and the GC1, GC2 and GC3 contents were 47.20%, 40.07% and 28.62%, respectively. Neutral-plot analysis showed that all genes were located above the diagonal line, indicating a weak correlation between GC12 (average of GC1 and GC2) and GC3; ENC-plot analysis showed that most of the genes were located below the standard curve and far away from the curve; and PR2-plot analysis showed that the genes were unevenly distributed in the four regions, and most of the genes were located in the lower right of the plot (Fig. 4 ). These results indicated that the codon preference of P. lutea was influenced more by natural selection [ 18 – 20 ]. IR/SC boundary comparison analysis The sequence of the IR boundary regions may extend outward and expand inward, resulting in changes in the copy number of related genes or the generation of pseudogenes in the boundary regions, which is a common phenomenon in the evolution of the cp genome. In this study, we compared the boundary expansion and contraction of IR and SSC regions in eight species from six genus of the family Acanthaceae (Fig. 5 ). The results revealed little variation in the size of the cp genomes across the eight species, with the exception of S. cusia , which exhibited a reduction of 5,094 to 7,536 bp compared to the other species. Similarly, the size of the IR regions of S. cusia showed a distinct shorter length. In six species (except C. nutans and S. cusia ), rps19 straddled the IRb/LSC (JLB) boundary, ndhF straddled the IRb/SSC (JSB), and ycf1 straddled the IRa/SSC (JSA). rpl2 was located within the IR boundary, and trnH was located near the LSC-IRa boundary junction. The changes in genes at the four boundaries of P. lutea were consistent with two species of Dicliptera genus, two species of Justicia genus and one species of Peristrophe genus. Compared with C. nutans , rps19 at the JLB boundary expanded to the LSC region but contracted in the IRb direction, and ndhF showed obvious contraction in the IRb direction. The IR/SC borders were validated using Sanger sequencing [ 21 ], which was completely consistent with the sequencing results from the Illumina sequencing platform (Fig. S4 ). Analysis of cp genome sequence variation Visual analysis of the similarity among the eight cp genome sequences showed that there was little difference in the gene interval composition of the cp genomes of Acanthaceae plants, which was relatively consistent (Fig. 6 ). Among the four regions, the LSC region had the highest variability in changes, followed by SSC region, and the IR regions had the lowest variability and was the most conserved, which was consistent with the results of boundary analysis. From the perspective of the non-gene coding regions and gene coding regions, the degree of variation in the non-gene coding regions was relatively high, while the gene coding regions were relatively conserved. However, there was a significant difference in the degree of variation in the gene coding regions, such as those of rps19 , ycf1 , ndhF , ndhA , clpP , and ycf3 . This indicated the existence of single nucleotide polymorphisms (SNPs) in the coding regions of genes such as rps19 , ndhF and ycf1 , which was further confirmed by gene sequence comparison (Fig. S5 ). Substitution rates of PCGs The Ka/Ks ratio in genetics is used to evaluate the existence of selection pressure on a certain PCG during evolution. A Ka/Ks value greater than 1 implies positive selection. A Ka/Ks value of 1 suggests neutral selection. When the Ka/Ks ratio is below 1, it signifies negative selection [ 22 ]. Selected 61 PCGs from P. lutea cp genome were compared with seven species from Acanthaceae for Ka/Ks calculation (Fig. 7 ; Table S3 ). To enhance the presentation of the data, only Ka/Ks values greater than 0.5 were shown. Overall, most genes of P. lutea cp genome experienced negative selection throughout evolution, as shown by the Ka/Ks values of 58 PCGs, being less than 1 when compared to other seven species. The high Ka/Ks values of petD from P. lutea compared to two Dicliptera species suggested that positive selection occurred during evolution. Furthermore, petA exhibited positive selection when comparing P. lutea with P. japonica , whereas rpl20 showed positive selection when comparing P. lutea with C. nutans . Phylogenetic analysis To elucidate the evolutionary dynamics within the family Acanthaceae, particularly focusing on P. lutea , a comprehensive phylogenetic analysis was conducted, encompassing P. lutea and 34 other samples from 16 distinct genus, with two species assigned as outgroups for comparative context. This investigation was founded on the analysis of conserved PCGs (Fig. 8 ) and cp genomes (Fig. S6 ), facilitating a dual perspective on phylogenetic relationships. Overall, both phylogenetic trees exhibited the same evolutionary pattern. Namely, the species from the Pseuderanthemum , Clinacanthus , Pachystachys , Justicia , Hypoestes , Peristrophe , and Dicliptera exhibited well-defined clustering, indicating a clear evolutionary relationship among them. P. lutea and C. nutans were found to be especially closely related, as evidenced by a bootstrap support (BS) value of 100, which strongly supports the robustness of this phylogenetic relationship. Discussion The study of cp genome sequences gives extensive information for phylogenetic research of plants, and DNA barcoding utilizing cp markers allows for accurate identification of plant species [ 12 ]. In this study, the cp genome of P. lutea was sequenced, assembled and annotated by high-throughput sequencing technology, and its genome structure, SSR sites, codon preference and phylogeny were analyzed. The findings will provide valuable insights into the evolutionary relationships and genetic variability of P. lutea , which is essential for its preservation. Genomic data may provide references for the development of plans for habitat restoration and the preservation of genetic reservoirs. The complete cp genome obtained had a total length of 151,574 bp, a total of 132 annotated genes, and a GC content of 38.18%. Compared with the reported homologous plants such as C. nutans (151,669 bp, 38.40%) [ 23 ], D. peruviana (150,811 bp, 38.00%) [ 24 ], D. mucronata (150,720 bp, 38.00%) [ 24 ], J. flava (150,888 bp, 38.20%) [ 25 ] and P. japonica (151,374 bp, 38.07%) [ 26 ], the Acanthaceae plants have highly similar genome sizes, structures, compositions, and GC contents, indicating that the plants of the Acanthaceae family exhibit good conservatism during evolution. SSRs are widely distributed in most plants, mainly in the external and noncoding regions of genes, and are often used in species identification, genetic diversity analysis and molecular marker-assisted breeding [ 27 ]. In this study, a total of 47 SSR sites were found in the cp genome of P. lutea , of which mononucleotides were the most abundant (85.11%), followed by dinucleotides (12.76%). The repetitive units of SSRs were mainly composed of A and T base combinations, with fewer G/C repeats. This further indicated that SSRs in cp genomes exhibit significant AT preference, which may be related to the difficulty of AT and GC chain uncoupling. Combined with the results of cp genome sequence variation analysis, the degree of variation in the IR regions with higher GC content was significantly lower than that in the LSC and SSC regions with lower GC content. Therefore, it can be inferred that base preference may be positively correlated with the degree of sequence variation, and it also indicated that the structure of the cp genome of P. lutea was highly conserved. Consequently, the identified SSR loci hold great potential as they can offer a solid theoretical basis for the identification of P. lutea , phylogenetic analysis, and the development of molecular markers related to this particular plant species. Codons serve as a crucial link among nucleic acids, proteins, and genetic material, thereby playing a significant role in the transmission of genetic information within organisms. Their preferred usage patterns offer dependable information for investigations into gene function, species evolution, and other related aspects [ 28 , 29 ]. In the present study, Leu was identified as the most abundant amino acid, accounting for 10.71% within the cp genome of P. lutea . The cp genome of P. lutea demonstrated a preference for using codons ending with A/T. The occurrence of codon usage bias during the evolution of the cp genome is attributed to natural selection and mutations [ 30 ]. It is well-documented that an ENc value greater than 40 implies a weak codon preference [ 31 , 32 ]. Given that the GC3 contents were less than 50%, it could be inferred that the codons in this genome tended to utilize A and T bases. This finding was in alignment with the results of the codon preference analysis conducted on J. flava , suggesting that the closer the phylogenetic relationship between species, the more similar their codon usage preferences are. This further corroborated the conclusions put forward by Parvathy et al [ 33 ]. Moreover, through Neutral-plot, ENC-plot, and PR2-plot analyses, it was determined that natural selection constitutes the primary factor influencing the codon usage bias observed in P. lutea . The IR region of the cp genome is considered to be the most conserved region and plays an important role in maintaining the stability of the cp genome [ 34 ]. The contraction, expansion and deletion of IR boundaries can cause differences in cp genomes [ 35 ]. Comparisons between LSC-IR and SSC-IR boundaries, and the genome sequence variation in the eight species’ complete cp genomes showed that the LSC/SSC regions had higher variability, while the IR regions had lower variability, but the whole genome was still relatively conserved. In addition, there was variation among genomes, variation was manifested as the location and number of base pairs in the borders of four genes, rps19 , ndhF , ycf1 , and trnH . This may be caused by the instability of LSC-IR and SSC-IR boundaries and the different degrees of expansion and contraction during the historical evolution of species. The rps19 gene is a key component of the ribosome biogenesis process, which is of vital importance for plant protein synthesis. Any variation in rps19 may have an impact on the overall growth rate of the plant. The ycf1 is located on the plastid membrane and promotes the transmembrane transport of various molecules. Mutations in ycf1 will alter the plant’s response to environmental stress. The ndhF gene is associated with the NADH dehydrogenase-like complex, which is involved in the electron transport during photosynthesis. Changes in ndhF may affect the photosynthetic efficiency of the plant. These genetic variations may have enabled P. lutea to adapt to its native tropical habitats in South America. They may also have contributed to the successful cultivation of P. lutea in the southern regions of the Yangtze River Basin in China. Additionally, these regions with partial differences can provide molecular bases for the identification and phylogenetic analysis of different species in the family Acanthaceae. Adaptive evolution has a profound implication on the study of structural and functional variation of genes, and Ka/Ks is an effective method to evaluate whether the adaptive evolution of PCGs has occurred [ 36 ]. Ks occurs more frequently than Ka in most genes of organisms, so Ka/Ks values are usually less than 1 [ 37 ]. In this study, we detected that the majority of genes in Acanthaceae species had Ka/Ks < 1, indicating that the cp genes of Acanthaceae species had been subjected to strong purifying selection during the long evolutionary process. The Ka/Ks of photosynthetic related genes such as atpE , ndhE , psbH and psbJ were less than 1, indicating that they were subjected to strong purifying selection during the evolutionary process. While petD , petA and rpl20 showed positive selection in other species, indicating that the aforementioned genes had a strong influence on the evolution trend of different species. Furthermore, PCGs of the cp genome encodes many critical proteins involved in photosynthesis and other metabolic processes, playing a role in the development of plant defense against pathogen ingress, stress tolerance, and ornamental traits [ 38 , 39 ]. For example, the photosynthesis gene psbA encodes a critical and highly conserved component of the photosystem II reaction center, polypeptide D1, which is participated in the photosynthetic electron transport chain. Under conditions of high-light stress, the increased production of the psbA protein helps to protect the photosynthetic machinery from damage caused by excess light energy, thereby maintaining photosynthetic efficiency and ensuring the plant's survival and growth. Thus, the transcription and translation of psbA play an important role in high-light stress responses [ 40 ]. Ribosomal proteins are essential for cell survival, among which rps15 and rpl33 are important components of the ribosome, responsible for protein synthesis in the cell, and their absence is also causally responsible for the high chilling sensitivity in plants [ 41 ]. For example, tobacco plants lacking the ribosomal proteins rps15 or rpl33 exhibited heightened sensitivity to cold stress [ 42 ]. In this study, psbA , rps15 and rpl33 genes were found in cp genome of P. lutea , which may play a role in photosynthesis and resistance. The psbA gene can maintain highly efficient photosynthesis under the intense light conditions typical of tropical regions, ensuring a stable energy supply for the plants. Adequate energy serves as the cornerstone for the growth of P. lutea and also contributes to its continuous blooming. With stable energy input, P. lutea can sustain the metabolic activities necessary for flower production throughout the year. Tropical regions are characterized by frequent fluctuations in temperature, humidity, and light intensity. The rps15 and rpl33 genes facilitate rapid environmental adaptation of P. lutea by promoting efficient protein synthesis. When these two genes function properly, P. lutea can synthesize proteins in a timely manner to cope with environmental changes. This significantly enhances the plant’s tolerance to various environmental stresses, thereby providing support for its year-round growth and blooming. Overall, the genetic variations in the cp genome of P. lutea , especially the presence and functionality of the psbA , rps15 , and rpl33 genes, may represent crucial factors underlying the unique growth and blooming characteristics of P. lutea . The family Acanthaceae has many species and genera, wide distribution, outstanding morphological diversity, including shrubs, herbs, and even vines, and outstanding habitat diversity. For a long time, the family was considered difficult to study. Since 1789, when French botanist Antoine-Laurent de Jussieu (1789) [ 43 ] proposed the natural classification of plants according to the relative positions of stamens and ovaries, the family Acanthaceae was published. Many scholars, such as Nees (1832) [ 44 ], Bentham (1876) [ 45 ], Lindau (1895) [ 46 ], and Bremekamp (1965) [ 47 ] have proposed different classification systems according to different classification characteristics. In recent years, the study of Acanthaceae evolutionary biology has benefited greatly from the utilization of cp genomes. Gao et al. (2019) [ 1 ] determined the cp genomes of four Echinacanthus species and resolved the phylogenetic relationship within Acanthaceae, which exhibited that Echinacanthus was sister to Strobilanthes . Similarly, Huang et al. (2020) [ 24 ] demonstrated that Justicia , Clinacanthus , and Dicliptera belong to one branch, whereas Echinacanthus and Strobilanthes belong to another branch. Our findings were consistent with the evolutionary connection among the five aforementioned taxa. Furthermore, our study identified the position of P. lutea in Acanthaceae for the first time. That is, P. lutea was closely related to members of Justiciinae, but it was most closely related to C. nutans of Diclipterinae, and they clustered into a small branch to form a sister relationship with a support value of 100%. McDade et al. (2000) [ 48 ] classified Pachystachys and Clinacanthus into the same subtribe. In this study, it was found that there was a close relationship between P. lutea , Justiciinae and Diclipterinae. As a monospecific genus, Pachystachys was suggested to be included in the Justicieae, but whether it can be divided into Justiciinae or Diclipterinae can be further discussed in future studies. Conclusions As a newly emerged species resulting from natural selection during the evolutionary process of the Acanthaceae family, P. lutea boasts a genome structure that is highly conserved and bears a remarkable similarity to those of other related species. Phylogenetic analysis has revealed that P. lutea shares a close kinship with C. nutans . Moreover, it can be grouped together with species from Justicia , Hypoestes , Peristrophe , and Dicliptera within the Justicieae tribe. The cp genome data obtained in this research are of significant importance for the conservation as well as the rational development of the germplasm resources of Pachystachys , thus playing a crucial role in safeguarding the biodiversity and facilitating the sustainable utilization of these valuable genetic materials within the Acanthaceae family. Subsequent research will focus on validating the functions of the adaptive genes identified in the cp genome of P. lutea . These genomic data can directly serve conservation and breeding efforts. By leveraging the identified genetic variations, it is promising to cultivate new varieties with outstanding ornamental traits, strong stress resistance, and excellent ecological adaptability, thus promoting the diverse applications of plants in the Acanthaceae family. Materials and Methods Genomic DNA extraction and sequencing Fresh leaves of P. lutea were collected from Xinyang, Henan Province, China (the experimental base of Xinyang Agriculture and Forestry University: 114° 12' E, 32° 16' N, altitude: 102 m) (Fig. 9 ). The leaves were frozen in liquid nitrogen before DNA extraction. Total genomic DNA was extracted from leaves using the CTAB method [ 49 ] and was sent to Shanghai Origingene Biotechnology Co., Ltd. for DNA library construction. The DNA library was constructed according to the instructions of the TruSeq RNA Sample Prep Kit, with an average length of 500 bp. Then, the samples were sequenced by using the Illumina NovaSeq 6000 sequencing platform (Illumina, San Diego, CA). A total of 8.9 GB raw data were produced and deposited in the SRA database (Sequence Read Archive, http://www.ncbi.nlm.nih.gov/Traces/sra ). Genome assembly and annotation The quality of the raw paired-end reads was assessed using FastQC v0.11.7 [ 50 ] software. To ensure the accuracy of subsequent biological information analysis, the sequencing data for adapter sequences, low-quality reads, sequences with a high N rate, and sequences with insufficient lengths included in the raw data reads were filtered to obtain high-quality clean reads. After the quality evaluation, the reads were assembled by using both Fast-plast v.1.2.8 ( https://github.com/mrmckain/Fast-Plast ) [ 51 ] and GetOrganelle v.1.7.0+ [ 52 ]. Single contigs containing the complete cp genome were generated. PGA and Geseq were used for gene prediction and annotation of the P. lutea cp genome [ 53 , 54 ], with default parameters and percent identity cut-off for protein-coding genes (PCGs) and RNAs set at ≥ 60 and ≤ 85, respectively. All sample annotation results were manually corrected. BWA (v.0.7.17-r1188) was used to process comparison data, SAMtools (v.1.9) was used to calculate coverage depth, and then visualized using ggplot2 in R [ 55 ]. After the annotation was completed, the sequence of the cp genome of P. lutea was deposited in the GenBank database with accession number OP546128. CPGview ( http://www.1kmpg.cn/cpgview/ ) was used to generate a circular cp genome map [ 56 ]. Simple sequence repeats analysis MISA v1.0 software [ 57 ] was used for simple sequence repeats (SSRs) analysis in the cp genome of P. lutea , and the number of repetitions was set to 10, 5, 5, 5, 5, and 5 from mononucleotide to hexanucleotide, respectively. The maximum cardinality between any two SSRs was set to 100 bp. MISA was also used to determine the specific location of SSRs in the cp genome of P. lutea . Codon preference analysis The PCGs sequence of cp genome in P. lutea were manually screened to remove duplicates and coding sequences less than 300 bp in length, and the eligible sequences were used for subsequent analysis. Using CodonW V1.4.4 software [ 58 ] and the online software CUSP ( http://emboss.toulouse.inra.fr/cgi-bin/emboss/cusp ) [ 59 ] calculated the effective number of codons (ENC), the GC content of codon 1, 2 and 3 bases (GC1, GC2, GC3, respectively) and relative synonymous codon usage (RSCU), and Neutral-plot, ENC-plot, and PR2-plot analysis were performed. Sequence variation map and variations of inverted repeat regions (IRs) sequences The comparison between the genome of P. lutea and seven Acanthaceae species’ cp genomic sequences was performed using the mVISTA program ( http://genome.lbl.gov/vista/mvista/submit.shtml ) to find interspecific variation [ 60 ]; the annotation of P. lutea was used as a reference in the Shuffle-LAGAN mode. Furthermore, comparisons between the borders of the IR (IRa and IRb), the small single-copy (SSC), and the large single-copy (LSC) regions were generated using the IRscope online program ( https://irscope.shinyapps.io/irapp/ ) [ 61 ]. The IR/SC borders of the P. lutea were validated using Sanger sequencing [ 62 ]. Non-synonymous substitutions (Ka)/Synonymous substitutions (Ks) analysis The Ka/Ks substitution rates of the PCGs in P. lutea cp genome were compared with seven related species from Acanthaceae. Sequence alignment was conducted using Mega 7.0 [ 63 ], while Ka/Ks calculations were performed using DnaSP v.6.12 [ 64 ]. Phylogenetic analysis In this study, to determine the phylogenetic position of P. lutea , the complete cp genomes of 33 species were downloaded from NCBI. Among these, 31 from four subfamilies within the Acanthaceae family, and two ( Catalpa bungei and Catalpa fargesii ) from outside this family, serving as outgroup species. The Multiple sequence alignment of the 34 species including P. lutea was performed by using MAFFT v7.158b [ 65 ] software and Gblock software [ 66 ] to find the conserved sequences between different species. The best substitution model, GTR + G, was selected in the jModelTest v2.1.7 program [ 67 ]. The phylogenetic tree was constructed with the maximum-likelihood (ML) method using RAxML v8.2.12 software [ 68 ]. Bootstrap analysis was used to evaluate the support for individual clades with 1000 replicates. Abbreviations cp chloroplast LSC large single-copy SSC small single-copy IRs inverted repeat regions PCGs protein-coding genes SSRs simple sequence repeats ML maximum-likelihood method ITS internal transcribed spacers Enc effective number of codons GC1, GC2, GC3 GC content of codon 1, 2 and 3 bases RSCU relative synonymous codon usage Ka Non-synonymous substitutions Ks Synonymous substitutions Leu leucine Cys cysteine Met methionine Trp tryptophan JLB IRb/LSC JSB IRb/SSC JSA IRa/SSC JLA IRa/LSC SNPs single nucleotide polymorphisms BS bootstrap support Declarations Statement Our experimental research and field studies on plants comply with relevant institutional, national, and international guidelines and legislation. Author Contributions: C.D. designed and executed experiments, completed data analysis, and wrote the first draft of the paper. Y.D. and H.G. contributed to the experimental design and analysis. T.F. assisted in sample collection and species identification. J.Y. and Y.Z. were the project developer and leader, guiding the experimental design, data analysis, and paper writing and revision. The final text has been read and approved by all authors. Funding: This work was supported by the Program for Innovative Research Team of Horticultural Plant Resources and Utilization in Xinyang Agriculture and Forestry University (XNKJTD-012), the Young Key Teachers Training Program in Xinyang Agriculture and Forestry University (2021), the Academic Core Teachers Program in Xinyang Agriculture and Forestry University (2022), and the Scientific Research Foundation for Young Teachers of Xinyang Agriculture and Forestry University (QN2023023). Availability of data and materials The original sequencing data have been submitted to the NCBI database and received GenBank accession number OP546128. The data used in this study are available in the public domain (https://www.ncbi.nlm.nih.gov). And the associated Bio-project, SRA, Bio-sample numbers are PRJNA884896, SRX17730640, and SAMN31059961, respectively. Ethics approval and consent to participate Pachystachys lutea were collected in July 2022 from Xinyang, Henan Province, China (the experimental base of Xinyang Agriculture and Forestry University: 114° 12' E, 32° 16' N, altitude: 102 m). The plant specimen was deposited at the Herbarium of the Horticultural Plant Biotechnology Laboratory, Xinyang Agriculture and Forestry University under voucher code PL20220716. Consent for publication Not applicable. Competing interests The authors declare no competing interests. References Gao CM, Deng YF, Wang, J. The complete chloroplast genomes of Echinacanthus species (Acanthaceae): phylogenetic relationships, adaptive evolution, and screening of molecular markers. Front. Plant Sci. 2019; 9:1989. https://doi.org/10.3389/fpls.2018.01989 Manzitto-Tripp EA, Darbyshire I, Daniel TF, Kiel CA, McDade LA. Revised classification of Acanthaceae and worldwide dichotomous keys. TAXON. 2022; 71(1):103–153. https://doi.org/10.1002/tax.12600 Kiel CA, Daniel TF, McDade LA. Phylogenetics of new world ‘justicioids’ (Justicieae: Acanthaceae): major lineages, morphological patterns, and widespread lncongruence with classification. Syst Bot. 2018; 43(2):459–484. https://doi.org/10.1600/036364418X697201 McDade LA, Daniel TF, Kiel CA. Toward a comprehensive understanding of phylogenetic relationships among lineages of Acanthaceae s.l. (Lamiales). Am J Bot. 2008; 95(9):1136–1152. https://doi.org/10.3732/ajb.0800096 Grant WF. A cytogenetic study in the Acanthaceae. Brittonia. 1955; 8(2):121–149. https://doi.org/10.2307/2804856 Das RJ, Pathak K. Use of indigenous plants in traditional health care systems by Mishing tribe of Dikhowmukh, Sivasagar district, Assam. Int J Herb Med. 2013; 1(3):50–57. Kartika T. Pemanfaatan tanaman hias pekarangan berkhasiat obat di Kecamatan Tanjung Batu. Sainmatika. 2018; 15(1):48–55. da Silva Ribeiro A, Polonio JC, Costa AT, dos Santos CM, Rhoden SA, Azevedo JL, Pamphile JA. Bioprospection of culturable endophytic fungi associated with the ornamental plant Pachystachys lutea . Curr Microbiol. 2018; 75:588–596. https://doi.org/10.1007/s00284-017-1421-9 da Silva Ribeiro A, Domingos Polli A, Oliveira A, dos Santos de Oliveira JA, Emmer A, Hamamura Alves L, Neves Pereira OC, Pamphile JA. Ornamental plant Pachystachys lutea as a source of promising endophytes for plant growth and phytoprotective activity. Acta Scientiarum. Biological Sciences. 2021; 43(1):e51737. https://doi.org/10.4025/actascibiolsci.v43i1.51737 Rosdewi M, Sada M, Fitriah F. Inventory and identification of natural dyes of ikat woven fabrics at Sanggar Bliran Sina Watublapi. Jurnal Riset Ilmu Pendidikan. 2023; 3:6–19. Wicaksono A, Raihandhany R. Bioprospeksi bunga lolipop ( Pachystachys lutea Nees) sebagai sumber alternatif untuk produksi gula: Bioprospecting of the lollipop plant ( Pachystachys lutea Nees) as alternative source for sugar production. Jurnal Sumberdaya Hayati. 2023; 9(2):63–69. https://doi.org/10.29244/jsdh.9.2.63-69 Daniell H, Lin CS, Yu M, Chang WJ. Chloroplast genomes: diversity, evolution, and applications in genetic engineering. Genome Biol. 2016; 17:134. https://doi.org/10.1186/s13059-016-1004-2 Li YT, Zhang J, Li LF, Gao LJ, Xu JT, Yang MS. Structural and comparative analysis of the complete chloroplast genome of Pyrus hopeiensis - “wild plants with a tiny population”-and three other Pyrus species. Int J Mol Sci. 2018; 19(10):3262. https://doi.org/10.3390/ijms19103262 Raman G, Park S. Analysis of the complete chloroplast genome of a medicinal plant, Dianthus superbus var. longicalyncinus , from a comparative genomics perspective. PLoS ONE. 2015; 10(10):e0141329. https://doi.org/10.1371/journal.pone.0141329 Scotland RW, Vollesen K. Classification of Acanthaceae. Kew Bul1. 2000; 55(3):513–589. https://doi.org/10.2307/4118776 Wu WW, Li JL, Liu Y, Jiang M, Lan MS, Liu C. Peculiarities of the inverted repeats in the complete chloroplast genome of Strobilanthes bantonensis Lindau. Mitochondrial DNA B. 2021; 6(4):1440–1447. https://doi.org/10.1080/23802359.2021.1911699 Kirankumar SI, Balaji R, Tanuja, Parani M. The complete chloroplast genome of Ocimum basilicum L. var. basilicum (Lamiaceae) and its phylogenetic analysis. Mitochondrial DNA B, 2023; 8(11):1169–1173. https://doi.org/10.1080/23802359.2023.2275835 Sueoka N. Near homogeneity of PR2-bias fingerprints in the human genome and their implications in phylogenetic analyses. J Mol Evol. 2001; 53:469–476. https://doi.org/10.1007/s002390010237 Gao Y, Lu Y, Song Y, Jing L. Analysis of codon usage bias of WRKY transcription factors in Helianthus annuus . BMC Genom Data. 2022; 23:46. https://doi.org/10.1186/s12863-022-01064-8 He B, Dong H, Jiang C, Gao FL, Tao ST, Xu LA. Analysis of codon usage patterns in Ginkgo biloba reveals codon usage tendency from A/U-ending to G/C-ending. Sci Rep. 2016; 6:35927. https://doi.org/10.1038/srep35927 Chen HM, Shao JJ, Zhang H, Jiang M, Huang LF, Zhang Z, Yang D, He M, Ronaghi M, Luo X, Sun BT, Wu WW, Liu C. Sequencing and analysis of Strobilanthes cusia (Nees) Kuntze chloroplast genome revealed the rare simultaneous contraction and expansion of the inverted repeat region in angiosperm. Front. Plant Sci. 2018; 9:324. https://doi.org/10.3389/fpls.2018.00324 Zhang Z, Li J, Zhao XQ, Wang J, Wong GKS, Yu J. KaKs_Calculator: calculating Ka and Ks through model selection and model averaging. Genom Proteom Bioinf. 2006; 4(4):259–263. https://doi.org/10.1016/S1672-0229(07)60007-2 Li MN, Pei HQ, Wang JH, Zhao KK, Zhu ZX, Wang HF. Complete plastome sequence of Clinacanthus nutans (Acanthaceae): a medicinal species in Southern China. Mitochondrial DNA B. 2019; 4(1):118–119. https://doi.org/10.1080/23802359.2018.1532349 Huang SN, Ge XJ, Cano A, Salazar BGM, Deng YF. Comparative analysis of chloroplast genomes for five Dicliptera species (Acanthaceae): molecular structure, phylogenetic relationships, and adaptive evolution. PeerJ. 2020; 8:e8450. https://doi.org/10.7717/peerj.8450 Yaradua SS, Alzahrani DA, Albokhary EJ, Abba A, Bello A. Corrigendum to “Complete chloroplast genome sequence of Justicia flava : genome comparative analysis and phylogenetic relationships among Acanthaceae”. BioMed Res Int. 2020; 2020:2049759. https://doi.org/10.1155/2020/2049759 Chen JX, Wang L, Zhao YC, Qin MJ. The complete chloroplast genome of a Chinese medicinal plant, Peristrophe japonica (Thunb.) Bremek. (Lamiales: Acanthaceae) from Nanjing, China. Mitochondrial DNA B. 2021; 6(7):1888–1889. https://doi.org/10.1080/23802359.2021.1934157 Fernandez-Pozo N, Menda N, Edwards JD, Saha S, Tecle IY, Strickler SR, Bombarely A, Fisher-York T, Pujar A, Foerster H, Yan A, Mueller LA. The Sol Genomics Network (SGN)--from genotype to phenotype to breeding. Nucleic Acids Res. 2015; 43:D1036–D1041. https://doi.org/10.1093/nar/gku1195 Powell W, Morgante M, McDevitt R, Vendramin GG, Rafalski JA. Polymorphic simple sequence repeat regions in chloroplast genomes: applications to the population genetics of pines. P Natl Acad Sci U S A. 1995; 92(17):7759–7763. https://doi.org/10.1073/pnas.92.17.7759 Pugh T, Fouet O, Risterucci AM, Brottier P, Abouladze M, Deletrez C, Courtois B, Clement D, Larmande P, N'Goran JAK, Lanaud C. A new cacao linkage map based on codominant markers: development and integration of 201 new microsatellite markers. Theor Appl Genet. 2004; 108:1151–1161. https://doi.org/10.1007/s00122-003-1533-4 Li B, Lin FR, Huang P, Guo WY, Zheng YQ. Complete chloroplast genome sequence of Decaisnea insignis : genome organization, genomic resources and comparative analysis. Sci Rep. 2017; 7:10073. https://doi.org/10.1038/s41598-017-10409-8 Ikemura T. Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes: a proposal for a synonymous codon choice that is optimal for the E. coli translational system. J Mol Biol. 1981; 151(3): 389–409. https://doi.org/10.1016/0022-2836(81)90003-6 Chen ZY, Liu Q, Xiao Y, Zhou GH, Yu PH, Bai J, Huang H, Gong YH. Correction to: Complete chloroplast genome sequence of Camellia sinensis : genome structure, adaptive evolution, and phylogenetic relationships. J Appl Genet. 2023; 64:601. https://doi.org/10.1007/s13353-023-00769-5 Parvathy ST, Udayasuriyan V, Bhadana V. Codon usage bias. Mol Biol Rep. 2022; 49:539–565. https://doi.org/10.1007/s11033-021-06749-4 Fu JM, Liu HM, Hu JJ, Liang YQ, Liang JJ, Wuyun TN, Tan XF. Five complete chloroplast genome sequences from Diospyros : genome organization and comparative analysis. PLoS One. 2016; 11(7):e0159566. https://doi.org/10.1371/journal.pone.0159566 Wang WC, Chen SY, Zhang XZ. Whole-genome comparison reveals divergent IR borders and mutation hotspots in chloroplast genomes of herbaceous bamboos (Bambusoideae: Olyreae). Molecules. 2018; 23(7):1537. https://doi.org/10.3390/molecules23071537 Fay JC, Wu CI. Sequence divergence, functional constraint, and selection in protein evolution. Annu Rev Genomics Hum Genet. 2003; 4:213–235. https://doi.org/10.1146/annurev.genom.4.020303.162528 Makałowski W, Boguski MS. Evolutionary parameters of the transcribed mammalian genome: an analysis of 2,820 orthologous rodent and human sequences. P Natl Acad Sci U S A. 1998; 95(16):9407–9412. https://doi.org/10.1073/pnas.95.16.9407 Bobik K, Burch-Smith TM. Chloroplast signaling within, between and beyond cells. Front. Plant Sci. 2015; 6:781. https://doi.org/10.3389/fpls.2015.00781 Dobrogojski J, Adamiec M, Luciński R. The chloroplast genome: a review. Acta Physiol Plant. 2020; 42:98. https://doi.org/10.1007/s11738-020-03089-x Pfannschmidt T, Nilsson A, Tullberg A, Link G, Allen JF. Direct transcriptional control of the chloroplast genes psbA and psaAB adjusts photosynthesis to light energy distribution in plants. IUBMB Life. 1999; 48:271–276. https://doi.org/10.1080/713803507 Rogalski M, Schöttler MA, Thiele W, Schulze WX, Bock R. Rpl33, a nonessential plastid-encoded ribosomal protein in tobacco, is required under cold stress conditions. Plant Cell. 2008; 20(8): 2221–2237. https://doi.org/10.1105/tpc.108.060392 Fleischmann TT, Scharff LB, Alkatib S, Hasdorf S, Schöttler MA, Bock R. Nonessential plastid-encoded ribosomal proteins in tobacco: a developmental role for plastid translation and implications for reductive genome evolution. Plant Cell. 2011; 23(9):3137–3155. https://doi.org/10.1105/tpc.111.088906 Jussieu AL de. Genera plantarum secundum ordines naturales disposita. 2nd ed, Apud viduam Herissant et Theophilum Barrois: Paris, France, 1789; 307. Nees Von Esenbeck CG. Acanthaceae. In Plantae Asiaticae Rariores; Wallich, N. Eds, Treuttel, Würtz, Richter, London. 1832; 3:77–l12. Bentham G. Acanthaceae. In Genera Plantarum; Bentham, G., Hooker, J.D. Eds, Reeve & Co.: London, 1876; v.2: pt.2:1066–1122. Lindau Q. Acanthaceae. In: Die Natürlichen Pflanzenfamilien; Engler, A., Prantl, K, Verlag von Wilhelm Englemann: Leipzig, 1895; IV(3b):274–354. Bremekamp CEB. Delimitation and subdivision of the Acanthaceae. Bulletin of the Botanical Survey of India. 1965; 7:21–30. McDade LA, Masta SE, Moody ML, Waters E. Phylogenetic relationships among Acanthaceae: evidence from two genomes. Syst Bot. 2000; 25(1):106–121. https://doi.org/10.2307/2666677 Doyle J. DNA protocols for plants. In Molecular Techniques in Taxonomy, Nato ASI Subseries H: Cell Biology; 1st ed, Hewitt, G.M., Johnston, A.W.B., Young, J.P.W. Eds, Springer: Berlin, Heidelberg, Germany, 1991; 57:283–293. https://doi.org/10.1007/978-3-642-83962-7_18 Wingett SW, Andrews S. FastQ Screen: a tool for multi-genome mapping and quality control [version 2; peer review: 4 approved]. F1000Research. 2018; 7:1338. https://doi.org/10.12688/f1000research.15931.2 McKain MR, Wilson M. Fast-Plast: rapid de novo assembly and finishing for whole chloroplast genomes. https://github.com/mrmckain/Fast-Plast (2017) Jin JJ, Yu WB, Yang JB, Song Y, dePamphilis CW, Yi TS, Li DZ. GetOrganelle: a fast and versatile toolkit for accurate de novo assembly of organelle genomes. Genome Biol. 2020; 21:241. https://doi.org/10.1186/s13059-020-02154-5 Qu XJ, Moore MJ, Li DZ, Yi TS. PGA: a software package for rapid, accurate, and flexible batch annotation of plastomes. Plant Methods. 2019; 15:50. https://doi.org/10.1186/s13007-019-0435-7 Tillich M, Lehwark P, Pellizzer T, Ulbricht-Jones ES, Fischer A, Bock R, Greiner S. GeSeq - versatile and accurate annotation of organelle genomes. Nucleic Acids Res. 2017; 45(W1):W6–W11. https://doi.org/10.1093/nar/gkx391 Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009; 25(14):1754–1760. https://doi.org/10.1093/bioinformatics/btp324 Liu SY, Ni Y, Li JL, Zhang XY, Yang HY, Chen HM, Liu C. CPGView: a package for visualizing detailed chloroplast genome structures. Mol Ecol Resour. 2023; 23(3):694–704. https://doi.org/10.1111/1755-0998.13729 Thiel T, Michalek W, Varshney RK, Graner A. Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley ( Hordeum vulgare L.). Theor Appl Genet. 2003; 106:411–422. https://doi.org/10.1007/s00122-002-1031-0 Sun XY, Yang Q, Xia XH. An improved implementation of Effective Number of Codons ( Nc ). Mol Biol Evol. 2013; 30(1):191–196. https://doi.org/10.1093/molbev/mss201 Wang YH, Wei QY, Xue TY, He SX, Fang J, Zeng CL. Comparative and phylogenetic analysis of the complete chloroplast genomes of 10 Artemisia selengensis resources based on high-throughput sequencing. BMC Genomics. 2024; 25:561. https://doi.org/10.1186/s12864-024-10455-3 Mayor C, Brudno M, Schwartz JR, Poliakov A, Rubin EM, Frazer KA, Pachter LS, Dubchak I. VISTA : visualizing global DNA sequence alignments of arbitrary length. Bioinformatics. 2000; 16(11):1046–1047. https://doi.org/10.1093/bioinformatics/16.11.1046 Amiryousefi A, Hyvönen J, Poczai P. IRscope: an online program to visualize the junction sites of chloroplast genomes. Bioinformatics. 2018; 34(17):3030–3031. https://doi.org/10.1093/bioinformatics/bty220 Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. P Natl Acad Sci U S A. 1977; 74(12):5463–5467. https://doi.org/10.1073/pnas.74.12.5463 Kumar S, Stecher G, Tamura K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016; 33(7):1870–1874. https://doi.org/10.1093/molbev/msw054 Rozas J, Ferrer-Mata A, Sánchez-DelBarrio JC, Guirao-Rico S, Librado P, Ramos-Onsins SE, Sánchez-Gracia A. DnaSP 6: DNA sequence polymorphism analysis of large data sets. Mol Biol Evol. 2017; 34(12):3299–3302. https://doi.org/10.1093/molbev/msx248 Katoh K, Rozewicki J, Yamada KD. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinform. 2019; 20(4):1160–1166. https://doi.org/10.1093/bib/bbx108 Castresana J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol biol Evol. 2000; 17(4):540–552. https://doi.org/10.1093/oxfordjournals.molbev.a026334 Darriba D, Taboada GL, Doallo R, Posada D. jModelTest 2: more models, new heuristics and parallel computing. Nat Methods. 2012; 9:772. https://doi.org/10.1038/nmeth.2109 Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014; 30(9):1312–1313. https://doi.org/10.1093/bioinformatics/btu033 Additional Declarations No competing interests reported. Supplementary Files FigureS1.tif FigureS2.tif FigureS3.tif FigureS4.tif FigureS5.tif FigureS6.tif TableS1.xlsx TableS2.xlsx TableS3.xlsx SupplementaryInformationLegends.docx Cite Share Download PDF Status: Published Journal Publication published 19 Nov, 2025 Read the published version in BMC Genomic Data → Version 1 posted Editorial decision: Revision requested 15 Jul, 2025 Reviews received at journal 15 Jul, 2025 Reviews received at journal 07 Jul, 2025 Reviewers agreed at journal 04 Jul, 2025 Reviewers agreed at journal 02 Jun, 2025 Editor assigned by journal 30 May, 2025 Reviewers agreed at journal 20 Apr, 2025 Reviewers invited by journal 16 Apr, 2025 Submission checks completed at journal 16 Apr, 2025 First submitted to journal 09 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5848411","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":443900783,"identity":"f8705c87-9589-438b-bb24-153a13c79a9f","order_by":0,"name":"Changmei Du","email":"","orcid":"","institution":"Xinyang Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Changmei","middleName":"","lastName":"Du","suffix":""},{"id":443900784,"identity":"6075b233-5f75-4b88-803f-fe2b6bd75340","order_by":1,"name":"Yan Dong","email":"","orcid":"","institution":"Xinyang Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Dong","suffix":""},{"id":443900785,"identity":"b6e62597-52ee-4318-836c-f2edcd3f1efd","order_by":2,"name":"Haishuo Gao","email":"","orcid":"","institution":"Xinyang Camellia oleifera Industry Development Center","correspondingAuthor":false,"prefix":"","firstName":"Haishuo","middleName":"","lastName":"Gao","suffix":""},{"id":443900786,"identity":"aaf3ecd2-9a83-4dbf-aead-4875dd433270","order_by":3,"name":"Tingting Fang","email":"","orcid":"","institution":"Xinyang Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Tingting","middleName":"","lastName":"Fang","suffix":""},{"id":443900789,"identity":"fc930c19-380d-453f-9006-fc115b293427","order_by":4,"name":"Jianhua Yue","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAq0lEQVRIiWNgGAWjYBACxhlgyoaHn7+BNC1pMpIzDhBrjQSYPGxj0JBApA7m2T1mEh/+nOcxYDjA+OFjDjEOm3PGTHJm220ec+YGZsmZ24jRMiPHTJq34TaPZcMBNmZeorX8+XOOx+BAAilaGNgOkKQlrdiyty2ZR3LGwWbi/GI4I3njjR9/7Oz5+ZsPfvhIlJYGDgOYhQ1EqAcCeQb2B8SpHAWjYBSMgpELANbbNUPDv/V9AAAAAElFTkSuQmCC","orcid":"","institution":"Xinyang Agriculture and Forestry University","correspondingAuthor":true,"prefix":"","firstName":"Jianhua","middleName":"","lastName":"Yue","suffix":""},{"id":443900791,"identity":"72dad464-d2ed-454f-9f50-e1456c0157d1","order_by":5,"name":"Yan Zhang","email":"","orcid":"","institution":"Shanghai Vocational College of Agriculture and Forestry","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-01-17 10:38:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5848411/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5848411/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12863-025-01380-9","type":"published","date":"2025-11-19T15:58:27+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80810134,"identity":"6cdcaed0-cf14-4439-9d4c-0af312385526","added_by":"auto","created_at":"2025-04-17 10:13:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1247382,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe chloroplast genome map of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. lutea\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003eGenes outside the circle are transcribed clockwise, while genes inside the circle are transcribed counterclockwise. Different functional groups are represented by different colors. The darker and lighter gray in the inner indicated the GC and AT content, respectively.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5848411/v1/2c59b9704fd1abc1f9aeec5b.png"},{"id":80810125,"identity":"d2fa92cc-f8f8-43f5-b962-00d7a4d7d445","added_by":"auto","created_at":"2025-04-17 10:13:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":79356,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSSRs analysis of chloroplast genome in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. lutea\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003eThe x-axis represents the types of repetitive sequences in base pairs, while the y-axis indicates the number of SSRs found within each category.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5848411/v1/b297a244586db29e83828556.png"},{"id":80810140,"identity":"f4fd3583-5777-40e5-bdec-46011eb8dfe3","added_by":"auto","created_at":"2025-04-17 10:13:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":183160,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCodon usage mode of chloroplast genes in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. lutea\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003eBoxes below the graphs represent all codons encoding each amino acid, with the colors of the histograms corresponding to the colors of the codons.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5848411/v1/f8c84b863d47703581b382fc.png"},{"id":80810164,"identity":"48ebc891-b946-43b7-b558-ce44ba4286fb","added_by":"auto","created_at":"2025-04-17 10:13:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":71560,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe analysis of evolutionary forces of chloroplast genes in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. lutea\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e (A) Neutral-plot analysis (GC3 vs GC12). (B) ENC-plot analysis (GC3 vs ENC). (C) PR2-plot analysis (G3/ (G3 + C3) vs A3/ (A3 + T3)). G3/ (G3 + C3): the probability of G when the third base of codon is G or C. A3/ (A3 + T3): the probability of A when the third base of the codon is A or T.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5848411/v1/50effbd33180c32f76825e98.png"},{"id":80810502,"identity":"6987bd5c-f4da-4bb8-b86c-9f94e6eae451","added_by":"auto","created_at":"2025-04-17 10:21:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":997480,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of the boundaries of LSC, IR and SSC among chloroplast genome of eight Acanthaceae species. \u003c/strong\u003eThe genes around the borders are shown above or below the main line. The JLB, JSB, JSA, and JLA represent junction sites of LSC/IRb, IRb/SSC, SSC/IRa, and IRa/LSC, respectively.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5848411/v1/58ce9b0cdc0345726c0032a5.png"},{"id":80810506,"identity":"23b848d7-a080-498a-b24d-390fdeec6a78","added_by":"auto","created_at":"2025-04-17 10:21:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1191679,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGlobal alignment analysis of chloroplast genome in eight Acanthaceae species.\u003c/strong\u003eAnnotated genes are displayed along the top. The horizontal axis indicates the coordinates in the chloroplast genome and the vertical axis indicates the percentage identity (between 50 and 100%). Genome regions are color-coded as exon, intron, and conserved non-coding sequences (CDS).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-5848411/v1/54111c1c78e237c91fb98fb3.png"},{"id":80810141,"identity":"24035af4-49a0-4d67-8bf6-58711e60bc81","added_by":"auto","created_at":"2025-04-17 10:13:27","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":260454,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKa/Ks values of selected PCGs of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. lutea\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e with close species. \u003c/strong\u003eThe x-axis represents genes, and\u003cstrong\u003e \u003c/strong\u003ethe order is alphabetical, while the y-axis represents the Ka/Ks value of each gene.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-5848411/v1/717bc55c7d6c06da183d051e.png"},{"id":80810162,"identity":"58ef0e75-f351-4bf2-8334-ccb310cd8e9a","added_by":"auto","created_at":"2025-04-17 10:13:28","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":386171,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe phylogenetic tree of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. lutea\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e was constructed by ML method based on the analysis of conserved PCGs of 34 species.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-5848411/v1/9ec14da8be4d22d5c16c90f1.png"},{"id":80810135,"identity":"53139fbc-5d8f-4ced-a673-56698457a8af","added_by":"auto","created_at":"2025-04-17 10:13:27","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":2063560,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlant morphological characteristics (A) and the floral organ morphology (B) of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. lutea\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-5848411/v1/98a3e62a147ff6f922ece0e2.png"},{"id":96650967,"identity":"a2e0a99c-4880-4da8-8b8e-94384e517f34","added_by":"auto","created_at":"2025-11-24 16:13:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7399766,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5848411/v1/10950abd-4220-4391-b8eb-c8dadfc3d6b8.pdf"},{"id":80811790,"identity":"808beead-5cac-49bf-8a2a-8044d1bb3633","added_by":"auto","created_at":"2025-04-17 10:29:27","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2955224,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-5848411/v1/2e62532659599b1c42575996.tif"},{"id":80810503,"identity":"28bfcac1-1256-4205-b651-e8a71e32e388","added_by":"auto","created_at":"2025-04-17 10:21:27","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1725336,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-5848411/v1/286b9cd83ca73c26f3be3d74.tif"},{"id":80810507,"identity":"96e43d8e-34ef-4c83-9283-df81ac488aca","added_by":"auto","created_at":"2025-04-17 10:21:27","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":3600864,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-5848411/v1/096038d5dc7bf7e96f086716.tif"},{"id":80810182,"identity":"8694cea6-326d-4432-a9c6-b162a85069fa","added_by":"auto","created_at":"2025-04-17 10:13:29","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":4143844,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS4.tif","url":"https://assets-eu.researchsquare.com/files/rs-5848411/v1/cbc4fd8dd19b4587e5829d69.tif"},{"id":80810132,"identity":"3f86dddc-159d-4767-9c93-f33c0c2ec6f9","added_by":"auto","created_at":"2025-04-17 10:13:27","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1954592,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS5.tif","url":"https://assets-eu.researchsquare.com/files/rs-5848411/v1/09bf14214684f935c33c24b6.tif"},{"id":80810149,"identity":"d88c716a-d818-4141-a4af-2566d0f0e719","added_by":"auto","created_at":"2025-04-17 10:13:28","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":3018424,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS6.tif","url":"https://assets-eu.researchsquare.com/files/rs-5848411/v1/37f5a2be468bc75bb88fe9b2.tif"},{"id":80811791,"identity":"5d2c2b4f-9540-4ce2-9c43-0f7d880ab687","added_by":"auto","created_at":"2025-04-17 10:29:28","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":11569,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5848411/v1/37b0b19b895777ff15f95b1d.xlsx"},{"id":80810126,"identity":"75f46fd5-3c08-4946-9c28-2560da37a3d1","added_by":"auto","created_at":"2025-04-17 10:13:27","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":12429,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5848411/v1/3232b5b3cf2d4f5a2aaec6a3.xlsx"},{"id":80810133,"identity":"319b4778-691e-4197-a4c0-d6d8222454e7","added_by":"auto","created_at":"2025-04-17 10:13:27","extension":"xlsx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":12478,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5848411/v1/3abbebe8622674e12656a68c.xlsx"},{"id":80812360,"identity":"9843e8dc-97f0-41ed-ae79-e83eda854dea","added_by":"auto","created_at":"2025-04-17 10:37:28","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":14291,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationLegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-5848411/v1/06604672fe73c81941680088.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Complete chloroplast genome sequence of Pachystachys lutea Nees: genome structure, adaptive evolution, and phylogenetic relationships","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe Acanthaceae family contains approximately 250 genera and more than 4000 species [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], mainly distributed in tropical and subtropical areas [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Approximately 50 genera and more than 400 species are distributed in China [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Many species of the Acanthaceae family are remarkable flowering ornamental plants, characterized by diverse forms, vivid colors, and extended ornamental periods. \u003cem\u003ePachystachys lutea\u003c/em\u003e Nees is one of them, is a perennial evergreen flowering shrub that prefers high temperatures, high humidity and sunny environments and is mainly distributed in the southern areas of the Yangtze River in China. The flower has a unique shape and blooms continuously throughout the year in warm environments (the greenhouse of Shanghai Chenshan Botanical Garden), so it is often planted as an ornamental plant. \u003cem\u003eP. lutea\u003c/em\u003e has very large spikelike inflorescences at the top of each new branch, and the golden bracts are stacked and spread out with small white flowers like shrimp bodies. Furthermore, its roots, leaves, and flowers are also used for medicinal purposes, treating pneumonia [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], diarrhea, worms [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], etc., and are the important medicinal plant resources to be developed. In addition, it has economic value. The leaves serve as the source of endophytic fungi [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and blue dye [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and nectar has the potential to serve as a substitute for sugar production in the future [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Currently, there are relatively few species within the genus \u003cem\u003ePachystachys\u003c/em\u003e that have been named, yet its outstanding ornamental characteristics have made \u003cem\u003eP. lutea\u003c/em\u003e highly recommended in China, where it has become an extremely valuable ornamental plant resource, widely loved by the public, and effectively enhancing the diversity of landscape species applications.\u003c/p\u003e \u003cp\u003eThe chloroplast (cp), an important organelle in green plants [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], houses the cp genome which is of great significance in plant evolutionary biology research. It presents remarkable benefits due to its genetic stability, well-preserved genome structure, and faster rate of evolutionary change compared to mitochondria. Consequently, the cp genome is widely utilized in the study of genetic relationships and species evolution between different species [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Despite its significant ornamental and medicinal value, the complete cp genome of \u003cem\u003eP. lutea\u003c/em\u003e has not yet been sequenced, limiting our understanding of its evolutionary history and genetic traits.\u003c/p\u003e \u003cp\u003e \u003cem\u003eP. lutea\u003c/em\u003e is a species within the genus \u003cem\u003ePachystachys\u003c/em\u003e of the family Acanthaceae. Research on \u003cem\u003eP. lutea\u003c/em\u003e has mainly focused on its horticultural applications, cultivation techniques, and medicinal properties, leaving its genetic and phylogenetic aspects largely unexplored. Nevertheless, scant knowledge exists regarding its phylogenetic relationship and evolutionary history, primarily due to the dearth of research on the modern taxonomic system, especially in the scarcely explored realm of molecular evolution. Scotland and Vollesen (2000) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], initially applied molecular systematics in conjunction with floral organ development to establish a classification system for the family Acanthaceae. Through a comprehensive study of \u003cem\u003etrnL-trnF\u003c/em\u003e and internal transcribed spacers (ITS), it was demonstrated that \u003cem\u003ePachystachys\u003c/em\u003e shares a common lineage with \u003cem\u003eHenrya\u003c/em\u003e, \u003cem\u003eCarlowrightia\u003c/em\u003e, \u003cem\u003eAnisacanthus\u003c/em\u003e, and \u003cem\u003eTetramurium\u003c/em\u003e, and this branch is further grouped with the one encompassing \u003cem\u003eDicliptera\u003c/em\u003e and \u003cem\u003eJusticia\u003c/em\u003e. However, the position of \u003cem\u003eP. lutea\u003c/em\u003e within the Acanthaceae requires further validation.\u003c/p\u003e \u003cp\u003eThis study aims to: (1) analyze the structural characteristics of the cp genome of \u003cem\u003eP. lutea\u003c/em\u003e, (2) investigate its adaptive evolutionary traits, and (3) clarify its phylogenetic relationships with closely related species.\u003c/p\u003e \u003cp\u003eTo achieve these aims, we employed molecular biology and high-throughput sequencing techniques to characterize the cp genome of \u003cem\u003eP. lutea\u003c/em\u003e. This involved using the Illumina NovaSeq 6000 platform for sequencing and a suite of bioinformatics tools for genome assembly, annotation, and analysis. We also compared the results with those obtained from traditional morphological taxonomic methods. Traditional morphological taxonomic methods have limitations, often leading to ambiguous or inconsistent classifications. Studies at molecular level can provide more precise genetic information. By filling this research gap, we aim to lay a solid foundation for more accurately determining its taxonomic position and facilitating future research endeavors.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eGenome structure of the\u003c/b\u003e \u003cb\u003eP. lutea\u003c/b\u003e \u003cb\u003ecp genome\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn this study, we assembled and annotated the complete cp genome, analyzed the sequencing depth, which reached an average of 3,012\u0026times; sequencing depth (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Compared with that of other species [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], this higher sequencing depth has enhanced the reliability of gene annotation and structural analysis. The accuracy of the assembly and annotation of \u003cem\u003eP. lutea\u003c/em\u003e cp genome was evaluated, respectively (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e; Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). The complete cp genome of \u003cem\u003eP. lutea\u003c/em\u003e was 151,574 bp in length (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and it had a typical quadripartite structure with junction regions: a LSC region of 83,348 bp, an SSC region of 17,226 bp, and a pair of IR regions (IRa and IRb) of 25,500 bp each. The overall GC content of the complete \u003cem\u003eP. lutea\u003c/em\u003e cp genome was 38.18%, and the corresponding values in the LSC, SSC, and IR regions were 36.21%, 32.36%, and 43.36%, respectively. The complete cp genome was found to contain 132 genes, which comprised 87 PCGs, 37 tRNA genes, and eight rRNA genes (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Nine PCGs, six tRNA genes, and four rRNA genes were duplicated in the IR regions. Nineteen genes contained two exons, and four genes (\u003cem\u003eclpP\u003c/em\u003e, \u003cem\u003eycf3\u003c/em\u003e, and two \u003cem\u003erps12\u003c/em\u003e) contained three exons. Among 87 PCGs, 45 photosynthesis genes, 29 genes related to self-replication, six other genes and seven genes with unknown function were identified.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eGene function statistics table of chloroplast genome in\u003c/b\u003e \u003cb\u003eP. lutea\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene category\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGene function\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGene name\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003ePhotosynthesis gene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSubunits of photosystem I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003epsaA, psaB, psaC, psaI, psaJ\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSubunits of photosystem II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003epsbL, psbZ, psbM, psbN, psbA, psbB, psbC, psbD, psbE, psbF, psbT, psbH, psbI, psbJ, psbK\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSubunits of NADH-dehydrogenase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003endhG, ndhH, ndhI, ndhJ, ndhK, ndhA*, ndhB*(2), ndhC, ndhD, ndhE, ndhF\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSubunits of cytochrome b/f complex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003epetL, petN, petA, petB*, petD*, petG\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSubunit for ATP synthase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eatpI, atpA, atpB, atpE, atpF*, atpH\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLarge subunit of rubisco\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003erbcL\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eSelf-replication\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLarge subunit of ribosome\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003erpl20, rpl22, rpl32, rpl23(2), rpl14, rpl33, rpl16*, rpl36, rpl2*(2)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSmall subunit of ribosome\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003erps11, rps14, rps15, rps16*, rps2, rps3, rps18, rps4, rps19, rps7(2), rps8, rps12**(2)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDNA dependent RNA polymerase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003erpoA, rpoB, rpoC1*, rpoC2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003erRNA gene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003errn5(2), rrn4.5(2), rrn16(2), rrn23(2)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etRNA gene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003etrnR-UCU, trnE-UUC, trnT-GGU, trnS-GGA, trnV-GAC(2), trnR-ACG(2), trnL-UAA*, trnG-GCC, trnD-GUC, trnY-GUA, trnP-UGG, trnM-CAU, trnL-CAA(2), trnS-GCU, trnW-CCA, trnF-GAA, trnT-UGU, trnS-UGA, trnV-UAC*, trnG-UCC*, trnL-UAG, trnI-GAU*(2), trnH-GUG, trnH-CAU(2), trnQ-UUG, trnN-GUU(2), trnK-UUU*, trnA-UGC*(2), trnC-GCA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eOther genes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTranslational initiation factor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003einfA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaturase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ematK\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProtease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eclpP**\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEnvelope membrane protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ecemA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSubunit of Acetyl-carboxylase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eaccD\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC-type cytochrome synthesis gene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eccsA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUnknown gene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOpen reading frames (ORF,ycf)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eycf1, ycf2(2), ycf3**, ycf4, ycf15(2)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eNotes: Gene *: Gene with one intron and two exons; Gene **: Gene with two introns and three exons; Gene (2): Number of copies of multi-copy genes;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSimple sequence repeats analysis\u003c/h2\u003e \u003cp\u003eSSRs analysis identified 47 SSRs in the \u003cem\u003eP. lutea\u003c/em\u003e cp genome. They were mainly of three types: mononucleotides, dinucleotides, trinucleotides, tetranucleotide, pentanucleotide, and hexanucleotide types were not found. Among them, the mononucleotides repeat sequence (A/C/T/G) type was the most abundant, with 40, which were mainly dominated by A and T bases; there were six dinucleotide repeat sequences (AT/TA), and one trinucleotide repeat sequence (TCT) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Out of 47 SSR loci, 37 were concentrated in the LSC region, eight were located in the SSC region, and only two were located in the IR regions (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCodon preference analysis\u003c/h3\u003e\n\u003cp\u003eA total of 24,537 codons in 87 PCGs of the cp genome of \u003cem\u003eP. lutea\u003c/em\u003e were participated in translation protein expression. Codons with a RSCU over 1 are thought to be favored by amino acids. Overall, 31 codons had an RSCU\u0026thinsp;\u0026gt;\u0026thinsp;1.0, with 29 codons ending in A or T and two codons ending in G or C. In addition, among the amino acid codes, leucine (Leu) had the highest encoding rate, with six synonymous codon codes (TTA, TTG, CTT, CTC, CTA, CTG), with a total of 2,629. The encoding rate of cysteine (Cys) was low, and there were two synonymous codons (TGT, TGC), totaling 279. methionine (Met) and tryptophan (Trp) had only one codon, while the rest of the amino acids had two to six codons (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). In addition, the ENC value of the PCGs was 45.83, and the GC1, GC2 and GC3 contents were 47.20%, 40.07% and 28.62%, respectively. Neutral-plot analysis showed that all genes were located above the diagonal line, indicating a weak correlation between GC12 (average of GC1 and GC2) and GC3; ENC-plot analysis showed that most of the genes were located below the standard curve and far away from the curve; and PR2-plot analysis showed that the genes were unevenly distributed in the four regions, and most of the genes were located in the lower right of the plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These results indicated that the codon preference of \u003cem\u003eP. lutea\u003c/em\u003e was influenced more by natural selection [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eIR/SC boundary comparison analysis\u003c/h3\u003e\n\u003cp\u003eThe sequence of the IR boundary regions may extend outward and expand inward, resulting in changes in the copy number of related genes or the generation of pseudogenes in the boundary regions, which is a common phenomenon in the evolution of the cp genome. In this study, we compared the boundary expansion and contraction of IR and SSC regions in eight species from six genus of the family Acanthaceae (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The results revealed little variation in the size of the cp genomes across the eight species, with the exception of \u003cem\u003eS. cusia\u003c/em\u003e, which exhibited a reduction of 5,094 to 7,536 bp compared to the other species. Similarly, the size of the IR regions of \u003cem\u003eS. cusia\u003c/em\u003e showed a distinct shorter length. In six species (except \u003cem\u003eC. nutans\u003c/em\u003e and \u003cem\u003eS. cusia\u003c/em\u003e), \u003cem\u003erps19\u003c/em\u003e straddled the IRb/LSC (JLB) boundary, \u003cem\u003endhF\u003c/em\u003e straddled the IRb/SSC (JSB), and \u003cem\u003eycf1\u003c/em\u003e straddled the IRa/SSC (JSA). \u003cem\u003erpl2\u003c/em\u003e was located within the IR boundary, and \u003cem\u003etrnH\u003c/em\u003e was located near the LSC-IRa boundary junction. The changes in genes at the four boundaries of \u003cem\u003eP. lutea\u003c/em\u003e were consistent with two species of \u003cem\u003eDicliptera\u003c/em\u003e genus, two species of \u003cem\u003eJusticia\u003c/em\u003e genus and one species of \u003cem\u003ePeristrophe\u003c/em\u003e genus. Compared with \u003cem\u003eC. nutans\u003c/em\u003e, rps19 at the JLB boundary expanded to the LSC region but contracted in the IRb direction, and \u003cem\u003endhF\u003c/em\u003e showed obvious contraction in the IRb direction. The IR/SC borders were validated using Sanger sequencing [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], which was completely consistent with the sequencing results from the Illumina sequencing platform (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eAnalysis of cp genome sequence variation\u003c/h3\u003e\n\u003cp\u003eVisual analysis of the similarity among the eight cp genome sequences showed that there was little difference in the gene interval composition of the cp genomes of Acanthaceae plants, which was relatively consistent (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Among the four regions, the LSC region had the highest variability in changes, followed by SSC region, and the IR regions had the lowest variability and was the most conserved, which was consistent with the results of boundary analysis. From the perspective of the non-gene coding regions and gene coding regions, the degree of variation in the non-gene coding regions was relatively high, while the gene coding regions were relatively conserved. However, there was a significant difference in the degree of variation in the gene coding regions, such as those of \u003cem\u003erps19\u003c/em\u003e, \u003cem\u003eycf1\u003c/em\u003e, \u003cem\u003endhF\u003c/em\u003e, \u003cem\u003endhA\u003c/em\u003e, \u003cem\u003eclpP\u003c/em\u003e, and \u003cem\u003eycf3\u003c/em\u003e. This indicated the existence of single nucleotide polymorphisms (SNPs) in the coding regions of genes such as \u003cem\u003erps19\u003c/em\u003e, \u003cem\u003endhF\u003c/em\u003e and \u003cem\u003eycf1\u003c/em\u003e, which was further confirmed by gene sequence comparison (Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eSubstitution rates of PCGs\u003c/h3\u003e\n\u003cp\u003eThe Ka/Ks ratio in genetics is used to evaluate the existence of selection pressure on a certain PCG during evolution. A Ka/Ks value greater than 1 implies positive selection. A Ka/Ks value of 1 suggests neutral selection. When the Ka/Ks ratio is below 1, it signifies negative selection [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Selected 61 PCGs from \u003cem\u003eP. lutea\u003c/em\u003e cp genome were compared with seven species from Acanthaceae for Ka/Ks calculation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e; Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). To enhance the presentation of the data, only Ka/Ks values greater than 0.5 were shown. Overall, most genes of \u003cem\u003eP. lutea\u003c/em\u003e cp genome experienced negative selection throughout evolution, as shown by the Ka/Ks values of 58 PCGs, being less than 1 when compared to other seven species. The high Ka/Ks values of \u003cem\u003epetD\u003c/em\u003e from \u003cem\u003eP. lutea\u003c/em\u003e compared to two \u003cem\u003eDicliptera\u003c/em\u003e species suggested that positive selection occurred during evolution. Furthermore, \u003cem\u003epetA\u003c/em\u003e exhibited positive selection when comparing \u003cem\u003eP. lutea\u003c/em\u003e with \u003cem\u003eP. japonica\u003c/em\u003e, whereas \u003cem\u003erpl20\u003c/em\u003e showed positive selection when comparing \u003cem\u003eP. lutea\u003c/em\u003e with \u003cem\u003eC. nutans\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis\u003c/h2\u003e \u003cp\u003eTo elucidate the evolutionary dynamics within the family Acanthaceae, particularly focusing on \u003cem\u003eP. lutea\u003c/em\u003e, a comprehensive phylogenetic analysis was conducted, encompassing \u003cem\u003eP. lutea\u003c/em\u003e and 34 other samples from 16 distinct genus, with two species assigned as outgroups for comparative context. This investigation was founded on the analysis of conserved PCGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e) and cp genomes (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e), facilitating a dual perspective on phylogenetic relationships. Overall, both phylogenetic trees exhibited the same evolutionary pattern. Namely, the species from the \u003cem\u003ePseuderanthemum\u003c/em\u003e, \u003cem\u003eClinacanthus\u003c/em\u003e, \u003cem\u003ePachystachys\u003c/em\u003e, \u003cem\u003eJusticia\u003c/em\u003e, \u003cem\u003eHypoestes\u003c/em\u003e, \u003cem\u003ePeristrophe\u003c/em\u003e, and \u003cem\u003eDicliptera\u003c/em\u003e exhibited well-defined clustering, indicating a clear evolutionary relationship among them. \u003cem\u003eP. lutea\u003c/em\u003e and \u003cem\u003eC. nutans\u003c/em\u003e were found to be especially closely related, as evidenced by a bootstrap support (BS) value of 100, which strongly supports the robustness of this phylogenetic relationship.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe study of cp genome sequences gives extensive information for phylogenetic research of plants, and DNA barcoding utilizing cp markers allows for accurate identification of plant species [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In this study, the cp genome of \u003cem\u003eP. lutea\u003c/em\u003e was sequenced, assembled and annotated by high-throughput sequencing technology, and its genome structure, SSR sites, codon preference and phylogeny were analyzed. The findings will provide valuable insights into the evolutionary relationships and genetic variability of \u003cem\u003eP. lutea\u003c/em\u003e, which is essential for its preservation. Genomic data may provide references for the development of plans for habitat restoration and the preservation of genetic reservoirs.\u003c/p\u003e \u003cp\u003eThe complete cp genome obtained had a total length of 151,574 bp, a total of 132 annotated genes, and a GC content of 38.18%. Compared with the reported homologous plants such as \u003cem\u003eC. nutans\u003c/em\u003e (151,669 bp, 38.40%) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], \u003cem\u003eD. peruviana\u003c/em\u003e (150,811 bp, 38.00%) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], \u003cem\u003eD. mucronata\u003c/em\u003e (150,720 bp, 38.00%) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], \u003cem\u003eJ. flava\u003c/em\u003e (150,888 bp, 38.20%) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and \u003cem\u003eP. japonica\u003c/em\u003e (151,374 bp, 38.07%) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], the Acanthaceae plants have highly similar genome sizes, structures, compositions, and GC contents, indicating that the plants of the Acanthaceae family exhibit good conservatism during evolution.\u003c/p\u003e \u003cp\u003eSSRs are widely distributed in most plants, mainly in the external and noncoding regions of genes, and are often used in species identification, genetic diversity analysis and molecular marker-assisted breeding [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In this study, a total of 47 SSR sites were found in the cp genome of \u003cem\u003eP. lutea\u003c/em\u003e, of which mononucleotides were the most abundant (85.11%), followed by dinucleotides (12.76%). The repetitive units of SSRs were mainly composed of A and T base combinations, with fewer G/C repeats. This further indicated that SSRs in cp genomes exhibit significant AT preference, which may be related to the difficulty of AT and GC chain uncoupling. Combined with the results of cp genome sequence variation analysis, the degree of variation in the IR regions with higher GC content was significantly lower than that in the LSC and SSC regions with lower GC content. Therefore, it can be inferred that base preference may be positively correlated with the degree of sequence variation, and it also indicated that the structure of the cp genome of \u003cem\u003eP. lutea\u003c/em\u003e was highly conserved. Consequently, the identified SSR loci hold great potential as they can offer a solid theoretical basis for the identification of \u003cem\u003eP. lutea\u003c/em\u003e, phylogenetic analysis, and the development of molecular markers related to this particular plant species.\u003c/p\u003e \u003cp\u003eCodons serve as a crucial link among nucleic acids, proteins, and genetic material, thereby playing a significant role in the transmission of genetic information within organisms. Their preferred usage patterns offer dependable information for investigations into gene function, species evolution, and other related aspects [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In the present study, Leu was identified as the most abundant amino acid, accounting for 10.71% within the cp genome of \u003cem\u003eP. lutea\u003c/em\u003e. The cp genome of \u003cem\u003eP. lutea\u003c/em\u003e demonstrated a preference for using codons ending with A/T. The occurrence of codon usage bias during the evolution of the cp genome is attributed to natural selection and mutations [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. It is well-documented that an ENc value greater than 40 implies a weak codon preference [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Given that the GC3 contents were less than 50%, it could be inferred that the codons in this genome tended to utilize A and T bases. This finding was in alignment with the results of the codon preference analysis conducted on \u003cem\u003eJ. flava\u003c/em\u003e, suggesting that the closer the phylogenetic relationship between species, the more similar their codon usage preferences are. This further corroborated the conclusions put forward by Parvathy et al [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Moreover, through Neutral-plot, ENC-plot, and PR2-plot analyses, it was determined that natural selection constitutes the primary factor influencing the codon usage bias observed in \u003cem\u003eP. lutea\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe IR region of the cp genome is considered to be the most conserved region and plays an important role in maintaining the stability of the cp genome [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The contraction, expansion and deletion of IR boundaries can cause differences in cp genomes [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Comparisons between LSC-IR and SSC-IR boundaries, and the genome sequence variation in the eight species\u0026rsquo; complete cp genomes showed that the LSC/SSC regions had higher variability, while the IR regions had lower variability, but the whole genome was still relatively conserved. In addition, there was variation among genomes, variation was manifested as the location and number of base pairs in the borders of four genes, \u003cem\u003erps19\u003c/em\u003e, \u003cem\u003endhF\u003c/em\u003e, \u003cem\u003eycf1\u003c/em\u003e, and \u003cem\u003etrnH\u003c/em\u003e. This may be caused by the instability of LSC-IR and SSC-IR boundaries and the different degrees of expansion and contraction during the historical evolution of species. The \u003cem\u003erps19\u003c/em\u003e gene is a key component of the ribosome biogenesis process, which is of vital importance for plant protein synthesis. Any variation in \u003cem\u003erps19\u003c/em\u003e may have an impact on the overall growth rate of the plant. The \u003cem\u003eycf1\u003c/em\u003e is located on the plastid membrane and promotes the transmembrane transport of various molecules. Mutations in \u003cem\u003eycf1\u003c/em\u003e will alter the plant\u0026rsquo;s response to environmental stress. The \u003cem\u003endhF\u003c/em\u003e gene is associated with the NADH dehydrogenase-like complex, which is involved in the electron transport during photosynthesis. Changes in \u003cem\u003endhF\u003c/em\u003e may affect the photosynthetic efficiency of the plant. These genetic variations may have enabled \u003cem\u003eP. lutea\u003c/em\u003e to adapt to its native tropical habitats in South America. They may also have contributed to the successful cultivation of \u003cem\u003eP. lutea\u003c/em\u003e in the southern regions of the Yangtze River Basin in China. Additionally, these regions with partial differences can provide molecular bases for the identification and phylogenetic analysis of different species in the family Acanthaceae.\u003c/p\u003e \u003cp\u003eAdaptive evolution has a profound implication on the study of structural and functional variation of genes, and Ka/Ks is an effective method to evaluate whether the adaptive evolution of PCGs has occurred [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Ks occurs more frequently than Ka in most genes of organisms, so Ka/Ks values are usually less than 1 [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In this study, we detected that the majority of genes in Acanthaceae species had Ka/Ks\u0026thinsp;\u0026lt;\u0026thinsp;1, indicating that the cp genes of Acanthaceae species had been subjected to strong purifying selection during the long evolutionary process. The Ka/Ks of photosynthetic related genes such as \u003cem\u003eatpE\u003c/em\u003e, \u003cem\u003endhE\u003c/em\u003e, \u003cem\u003epsbH\u003c/em\u003e and \u003cem\u003epsbJ\u003c/em\u003e were less than 1, indicating that they were subjected to strong purifying selection during the evolutionary process. While \u003cem\u003epetD\u003c/em\u003e, \u003cem\u003epetA\u003c/em\u003e and \u003cem\u003erpl20\u003c/em\u003e showed positive selection in other species, indicating that the aforementioned genes had a strong influence on the evolution trend of different species. Furthermore, PCGs of the cp genome encodes many critical proteins involved in photosynthesis and other metabolic processes, playing a role in the development of plant defense against pathogen ingress, stress tolerance, and ornamental traits [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. For example, the photosynthesis gene \u003cem\u003epsbA\u003c/em\u003e encodes a critical and highly conserved component of the photosystem II reaction center, polypeptide D1, which is participated in the photosynthetic electron transport chain. Under conditions of high-light stress, the increased production of the \u003cem\u003epsbA\u003c/em\u003e protein helps to protect the photosynthetic machinery from damage caused by excess light energy, thereby maintaining photosynthetic efficiency and ensuring the plant's survival and growth. Thus, the transcription and translation of \u003cem\u003epsbA\u003c/em\u003e play an important role in high-light stress responses [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Ribosomal proteins are essential for cell survival, among which \u003cem\u003erps15\u003c/em\u003e and \u003cem\u003erpl33\u003c/em\u003e are important components of the ribosome, responsible for protein synthesis in the cell, and their absence is also causally responsible for the high chilling sensitivity in plants [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. For example, tobacco plants lacking the ribosomal proteins \u003cem\u003erps15\u003c/em\u003e or \u003cem\u003erpl33\u003c/em\u003e exhibited heightened sensitivity to cold stress [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In this study, \u003cem\u003epsbA\u003c/em\u003e, \u003cem\u003erps15\u003c/em\u003e and \u003cem\u003erpl33\u003c/em\u003e genes were found in cp genome of \u003cem\u003eP. lutea\u003c/em\u003e, which may play a role in photosynthesis and resistance. The \u003cem\u003epsbA\u003c/em\u003e gene can maintain highly efficient photosynthesis under the intense light conditions typical of tropical regions, ensuring a stable energy supply for the plants. Adequate energy serves as the cornerstone for the growth of \u003cem\u003eP. lutea\u003c/em\u003e and also contributes to its continuous blooming. With stable energy input, \u003cem\u003eP. lutea\u003c/em\u003e can sustain the metabolic activities necessary for flower production throughout the year. Tropical regions are characterized by frequent fluctuations in temperature, humidity, and light intensity. The \u003cem\u003erps15\u003c/em\u003e and \u003cem\u003erpl33\u003c/em\u003e genes facilitate rapid environmental adaptation of \u003cem\u003eP. lutea\u003c/em\u003e by promoting efficient protein synthesis. When these two genes function properly, \u003cem\u003eP. lutea\u003c/em\u003e can synthesize proteins in a timely manner to cope with environmental changes. This significantly enhances the plant\u0026rsquo;s tolerance to various environmental stresses, thereby providing support for its year-round growth and blooming. Overall, the genetic variations in the cp genome of \u003cem\u003eP. lutea\u003c/em\u003e, especially the presence and functionality of the \u003cem\u003epsbA\u003c/em\u003e, \u003cem\u003erps15\u003c/em\u003e, and \u003cem\u003erpl33\u003c/em\u003e genes, may represent crucial factors underlying the unique growth and blooming characteristics of \u003cem\u003eP. lutea\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe family Acanthaceae has many species and genera, wide distribution, outstanding morphological diversity, including shrubs, herbs, and even vines, and outstanding habitat diversity. For a long time, the family was considered difficult to study. Since 1789, when French botanist Antoine-Laurent de Jussieu (1789) [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] proposed the natural classification of plants according to the relative positions of stamens and ovaries, the family Acanthaceae was published. Many scholars, such as Nees (1832) [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], Bentham (1876) [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], Lindau (1895) [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], and Bremekamp (1965) [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] have proposed different classification systems according to different classification characteristics. In recent years, the study of Acanthaceae evolutionary biology has benefited greatly from the utilization of cp genomes. Gao et al. (2019) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] determined the cp genomes of four \u003cem\u003eEchinacanthus\u003c/em\u003e species and resolved the phylogenetic relationship within Acanthaceae, which exhibited that \u003cem\u003eEchinacanthus\u003c/em\u003e was sister to \u003cem\u003eStrobilanthes\u003c/em\u003e. Similarly, Huang et al. (2020) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] demonstrated that \u003cem\u003eJusticia\u003c/em\u003e, \u003cem\u003eClinacanthus\u003c/em\u003e, and \u003cem\u003eDicliptera\u003c/em\u003e belong to one branch, whereas \u003cem\u003eEchinacanthus\u003c/em\u003e and \u003cem\u003eStrobilanthes\u003c/em\u003e belong to another branch. Our findings were consistent with the evolutionary connection among the five aforementioned taxa. Furthermore, our study identified the position of \u003cem\u003eP. lutea\u003c/em\u003e in Acanthaceae for the first time. That is, \u003cem\u003eP. lutea\u003c/em\u003e was closely related to members of Justiciinae, but it was most closely related to \u003cem\u003eC. nutans\u003c/em\u003e of Diclipterinae, and they clustered into a small branch to form a sister relationship with a support value of 100%. McDade et al. (2000) [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] classified \u003cem\u003ePachystachys\u003c/em\u003e and \u003cem\u003eClinacanthus\u003c/em\u003e into the same subtribe. In this study, it was found that there was a close relationship between \u003cem\u003eP. lutea\u003c/em\u003e, Justiciinae and Diclipterinae. As a monospecific genus, \u003cem\u003ePachystachys\u003c/em\u003e was suggested to be included in the Justicieae, but whether it can be divided into Justiciinae or Diclipterinae can be further discussed in future studies.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eAs a newly emerged species resulting from natural selection during the evolutionary process of the Acanthaceae family, \u003cem\u003eP. lutea\u003c/em\u003e boasts a genome structure that is highly conserved and bears a remarkable similarity to those of other related species. Phylogenetic analysis has revealed that \u003cem\u003eP. lutea\u003c/em\u003e shares a close kinship with \u003cem\u003eC. nutans\u003c/em\u003e. Moreover, it can be grouped together with species from \u003cem\u003eJusticia\u003c/em\u003e, \u003cem\u003eHypoestes\u003c/em\u003e, \u003cem\u003ePeristrophe\u003c/em\u003e, and \u003cem\u003eDicliptera\u003c/em\u003e within the Justicieae tribe. The cp genome data obtained in this research are of significant importance for the conservation as well as the rational development of the germplasm resources of \u003cem\u003ePachystachys\u003c/em\u003e, thus playing a crucial role in safeguarding the biodiversity and facilitating the sustainable utilization of these valuable genetic materials within the Acanthaceae family. Subsequent research will focus on validating the functions of the adaptive genes identified in the cp genome of \u003cem\u003eP. lutea\u003c/em\u003e. These genomic data can directly serve conservation and breeding efforts. By leveraging the identified genetic variations, it is promising to cultivate new varieties with outstanding ornamental traits, strong stress resistance, and excellent ecological adaptability, thus promoting the diverse applications of plants in the Acanthaceae family.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eGenomic DNA extraction and sequencing\u003c/h2\u003e \u003cp\u003eFresh leaves of \u003cem\u003eP. lutea\u003c/em\u003e were collected from Xinyang, Henan Province, China (the experimental base of Xinyang Agriculture and Forestry University: 114\u0026deg; 12' E, 32\u0026deg; 16' N, altitude: 102 m) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). The leaves were frozen in liquid nitrogen before DNA extraction. Total genomic DNA was extracted from leaves using the CTAB method [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] and was sent to Shanghai Origingene Biotechnology Co., Ltd. for DNA library construction. The DNA library was constructed according to the instructions of the TruSeq RNA Sample Prep Kit, with an average length of 500 bp. Then, the samples were sequenced by using the Illumina NovaSeq 6000 sequencing platform (Illumina, San Diego, CA). A total of 8.9 GB raw data were produced and deposited in the SRA database (Sequence Read Archive, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/Traces/sra\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/Traces/sra\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eGenome assembly and annotation\u003c/h2\u003e \u003cp\u003eThe quality of the raw paired-end reads was assessed using FastQC v0.11.7 [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] software. To ensure the accuracy of subsequent biological information analysis, the sequencing data for adapter sequences, low-quality reads, sequences with a high N rate, and sequences with insufficient lengths included in the raw data reads were filtered to obtain high-quality clean reads. After the quality evaluation, the reads were assembled by using both Fast-plast v.1.2.8 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/mrmckain/Fast-Plast\u003c/span\u003e\u003cspan address=\"https://github.com/mrmckain/Fast-Plast\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] and GetOrganelle v.1.7.0+ [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Single contigs containing the complete cp genome were generated. PGA and Geseq were used for gene prediction and annotation of the \u003cem\u003eP. lutea\u003c/em\u003e cp genome [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], with default parameters and percent identity cut-off for protein-coding genes (PCGs) and RNAs set at \u0026ge;\u0026thinsp;60 and \u0026le;\u0026thinsp;85, respectively. All sample annotation results were manually corrected. BWA (v.0.7.17-r1188) was used to process comparison data, SAMtools (v.1.9) was used to calculate coverage depth, and then visualized using ggplot2 in R [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. After the annotation was completed, the sequence of the cp genome of \u003cem\u003eP. lutea\u003c/em\u003e was deposited in the GenBank database with accession number OP546128. CPGview (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.1kmpg.cn/cpgview/\u003c/span\u003e\u003cspan address=\"http://www.1kmpg.cn/cpgview/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to generate a circular cp genome map [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSimple sequence repeats analysis\u003c/h2\u003e \u003cp\u003eMISA v1.0 software [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] was used for simple sequence repeats (SSRs) analysis in the cp genome of \u003cem\u003eP. lutea\u003c/em\u003e, and the number of repetitions was set to 10, 5, 5, 5, 5, and 5 from mononucleotide to hexanucleotide, respectively. The maximum cardinality between any two SSRs was set to 100 bp. MISA was also used to determine the specific location of SSRs in the cp genome of \u003cem\u003eP. lutea\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCodon preference analysis\u003c/h2\u003e \u003cp\u003eThe PCGs sequence of cp genome in \u003cem\u003eP. lutea\u003c/em\u003e were manually screened to remove duplicates and coding sequences less than 300 bp in length, and the eligible sequences were used for subsequent analysis. Using CodonW V1.4.4 software [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] and the online software CUSP (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://emboss.toulouse.inra.fr/cgi-bin/emboss/cusp\u003c/span\u003e\u003cspan address=\"http://emboss.toulouse.inra.fr/cgi-bin/emboss/cusp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] calculated the effective number of codons (ENC), the GC content of codon 1, 2 and 3 bases (GC1, GC2, GC3, respectively) and relative synonymous codon usage (RSCU), and Neutral-plot, ENC-plot, and PR2-plot analysis were performed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSequence variation map and variations of inverted repeat regions (IRs) sequences\u003c/h2\u003e \u003cp\u003eThe comparison between the genome of \u003cem\u003eP. lutea\u003c/em\u003e and seven Acanthaceae species\u0026rsquo; cp genomic sequences was performed using the mVISTA program (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://genome.lbl.gov/vista/mvista/submit.shtml\u003c/span\u003e\u003cspan address=\"http://genome.lbl.gov/vista/mvista/submit.shtml\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to find interspecific variation [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]; the annotation of \u003cem\u003eP. lutea\u003c/em\u003e was used as a reference in the Shuffle-LAGAN mode. Furthermore, comparisons between the borders of the IR (IRa and IRb), the small single-copy (SSC), and the large single-copy (LSC) regions were generated using the IRscope online program (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://irscope.shinyapps.io/irapp/\u003c/span\u003e\u003cspan address=\"https://irscope.shinyapps.io/irapp/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. The IR/SC borders of the \u003cem\u003eP. lutea\u003c/em\u003e were validated using Sanger sequencing [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eNon-synonymous substitutions (Ka)/Synonymous substitutions (Ks) analysis\u003c/h2\u003e \u003cp\u003eThe Ka/Ks substitution rates of the PCGs in \u003cem\u003eP. lutea\u003c/em\u003e cp genome were compared with seven related species from Acanthaceae. Sequence alignment was conducted using Mega 7.0 [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], while Ka/Ks calculations were performed using DnaSP v.6.12 [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis\u003c/h2\u003e \u003cp\u003eIn this study, to determine the phylogenetic position of \u003cem\u003eP. lutea\u003c/em\u003e, the complete cp genomes of 33 species were downloaded from NCBI. Among these, 31 from four subfamilies within the Acanthaceae family, and two (\u003cem\u003eCatalpa bungei\u003c/em\u003e and \u003cem\u003eCatalpa fargesii\u003c/em\u003e) from outside this family, serving as outgroup species. The Multiple sequence alignment of the 34 species including \u003cem\u003eP. lutea\u003c/em\u003e was performed by using MAFFT v7.158b [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e] software and Gblock software [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e] to find the conserved sequences between different species. The best substitution model, GTR\u0026thinsp;+\u0026thinsp;G, was selected in the jModelTest v2.1.7 program [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. The phylogenetic tree was constructed with the maximum-likelihood (ML) method using RAxML v8.2.12 software [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Bootstrap analysis was used to evaluate the support for individual clades with 1000 replicates.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ecp chloroplast\u003c/p\u003e \u003cp\u003eLSC large single-copy\u003c/p\u003e \u003cp\u003eSSC small single-copy\u003c/p\u003e \u003cp\u003eIRs inverted repeat regions\u003c/p\u003e \u003cp\u003ePCGs protein-coding genes\u003c/p\u003e \u003cp\u003eSSRs simple sequence repeats\u003c/p\u003e \u003cp\u003eML maximum-likelihood method\u003c/p\u003e \u003cp\u003eITS internal transcribed spacers\u003c/p\u003e \u003cp\u003eEnc effective number of codons\u003c/p\u003e \u003cp\u003eGC1, GC2, GC3 GC content of codon 1, 2 and 3 bases\u003c/p\u003e \u003cp\u003eRSCU relative synonymous codon usage\u003c/p\u003e \u003cp\u003eKa Non-synonymous substitutions\u003c/p\u003e \u003cp\u003eKs Synonymous substitutions\u003c/p\u003e \u003cp\u003eLeu leucine\u003c/p\u003e \u003cp\u003eCys cysteine\u003c/p\u003e \u003cp\u003eMet methionine\u003c/p\u003e \u003cp\u003eTrp tryptophan\u003c/p\u003e \u003cp\u003eJLB IRb/LSC\u003c/p\u003e \u003cp\u003eJSB IRb/SSC\u003c/p\u003e \u003cp\u003eJSA IRa/SSC\u003c/p\u003e \u003cp\u003eJLA IRa/LSC\u003c/p\u003e \u003cp\u003eSNPs single nucleotide polymorphisms\u003c/p\u003e \u003cp\u003eBS bootstrap support\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eStatement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur experimental research and field studies on plants comply with relevant institutional, national, and international guidelines and legislation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e C.D. designed and executed experiments, completed data analysis, and wrote the first draft of the paper. Y.D. and H.G. contributed to the experimental design and analysis. T.F. assisted in sample collection and species identification. J.Y. and Y.Z. were the project developer and leader, guiding the experimental design, data analysis, and paper writing and revision. The final text has been read and approved by all authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was supported by the Program for Innovative Research Team of Horticultural Plant Resources and Utilization in Xinyang Agriculture and Forestry University (XNKJTD-012), the Young Key Teachers Training Program in Xinyang Agriculture and Forestry University (2021), the Academic Core Teachers Program in Xinyang Agriculture and Forestry University (2022), and the Scientific Research Foundation for Young Teachers of Xinyang Agriculture and Forestry University (QN2023023).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original sequencing data have been submitted to the NCBI database and received GenBank accession number OP546128. The data used in this study are available in the public domain (https://www.ncbi.nlm.nih.gov). And the associated Bio-project, SRA, Bio-sample numbers are PRJNA884896, SRX17730640, and SAMN31059961, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePachystachys lutea\u003c/em\u003e were collected in July 2022 from Xinyang, Henan Province, China (the experimental base of Xinyang Agriculture and Forestry University: 114\u0026deg; 12\u0026apos; E, 32\u0026deg; 16\u0026apos; N, altitude: 102 m). The plant specimen was deposited at the Herbarium of the Horticultural Plant Biotechnology Laboratory, Xinyang Agriculture and Forestry University under voucher code PL20220716.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGao CM, Deng YF, Wang, J. The complete chloroplast genomes of \u003cem\u003eEchinacanthus\u003c/em\u003e species (Acanthaceae): phylogenetic relationships, adaptive evolution, and screening of molecular markers. Front. Plant Sci. 2019; 9:1989. https://doi.org/10.3389/fpls.2018.01989\u003c/li\u003e\n\u003cli\u003eManzitto-Tripp EA, Darbyshire I, Daniel TF, Kiel CA, McDade LA. Revised classification of Acanthaceae and worldwide dichotomous keys. TAXON. 2022; 71(1):103\u0026ndash;153. https://doi.org/10.1002/tax.12600\u003c/li\u003e\n\u003cli\u003eKiel CA, Daniel TF, McDade LA. Phylogenetics of new world \u0026lsquo;justicioids\u0026rsquo; (Justicieae: Acanthaceae): major lineages, morphological patterns, and widespread lncongruence with classification. Syst Bot. 2018; 43(2):459\u0026ndash;484. https://doi.org/10.1600/036364418X697201\u003c/li\u003e\n\u003cli\u003eMcDade LA, Daniel TF, Kiel CA. Toward a comprehensive understanding of phylogenetic relationships among lineages of Acanthaceae s.l. (Lamiales). Am J Bot. 2008; 95(9):1136\u0026ndash;1152. https://doi.org/10.3732/ajb.0800096\u003c/li\u003e\n\u003cli\u003eGrant WF. A cytogenetic study in the Acanthaceae. Brittonia. 1955; 8(2):121\u0026ndash;149. https://doi.org/10.2307/2804856\u003c/li\u003e\n\u003cli\u003eDas RJ, Pathak K. Use of indigenous plants in traditional health care systems by Mishing tribe of Dikhowmukh, Sivasagar district, Assam. Int J Herb Med. 2013; 1(3):50\u0026ndash;57.\u003c/li\u003e\n\u003cli\u003eKartika T. Pemanfaatan tanaman hias pekarangan berkhasiat obat di Kecamatan Tanjung Batu. Sainmatika. 2018; 15(1):48\u0026ndash;55.\u003c/li\u003e\n\u003cli\u003eda Silva Ribeiro A, Polonio JC, Costa AT, dos Santos CM, Rhoden SA, Azevedo JL, Pamphile JA. Bioprospection of culturable endophytic fungi associated with the ornamental plant \u003cem\u003ePachystachys lutea\u003c/em\u003e. Curr Microbiol. 2018; 75:588\u0026ndash;596. https://doi.org/10.1007/s00284-017-1421-9\u003c/li\u003e\n\u003cli\u003eda Silva Ribeiro A, Domingos Polli A, Oliveira A, dos Santos de Oliveira JA, Emmer A, Hamamura Alves L, Neves Pereira OC, Pamphile JA. Ornamental plant \u003cem\u003ePachystachys lutea\u003c/em\u003e as a source of promising endophytes for plant growth and phytoprotective activity. Acta Scientiarum. Biological Sciences. 2021; 43(1):e51737. https://doi.org/10.4025/actascibiolsci.v43i1.51737\u003c/li\u003e\n\u003cli\u003eRosdewi M, Sada M, Fitriah F. Inventory and identification of natural dyes of ikat woven fabrics at Sanggar Bliran Sina Watublapi. Jurnal Riset Ilmu Pendidikan. 2023; 3:6\u0026ndash;19.\u003c/li\u003e\n\u003cli\u003eWicaksono A, Raihandhany R. Bioprospeksi bunga lolipop (\u003cem\u003ePachystachys lutea\u003c/em\u003e Nees) sebagai sumber alternatif untuk produksi gula: Bioprospecting of the lollipop plant (\u003cem\u003ePachystachys lutea\u003c/em\u003e Nees) as alternative source for sugar production. Jurnal Sumberdaya Hayati. 2023; 9(2):63\u0026ndash;69. https://doi.org/10.29244/jsdh.9.2.63-69\u003c/li\u003e\n\u003cli\u003eDaniell H, Lin CS, Yu M, Chang WJ. Chloroplast genomes: diversity, evolution, and applications in genetic engineering. Genome Biol. 2016; 17:134. https://doi.org/10.1186/s13059-016-1004-2\u003c/li\u003e\n\u003cli\u003eLi YT, Zhang J, Li LF, Gao LJ, Xu JT, Yang MS. Structural and comparative analysis of the complete chloroplast genome of \u003cem\u003ePyrus hopeiensis\u003c/em\u003e- \u0026ldquo;wild plants with a tiny population\u0026rdquo;-and three other \u003cem\u003ePyrus\u003c/em\u003e species. Int J Mol Sci. 2018; 19(10):3262. https://doi.org/10.3390/ijms19103262\u003c/li\u003e\n\u003cli\u003eRaman G, Park S. Analysis of the complete chloroplast genome of a medicinal plant, \u003cem\u003eDianthus\u003c/em\u003e \u003cem\u003esuperbus\u003c/em\u003e var. \u003cem\u003elongicalyncinus\u003c/em\u003e, from a comparative genomics perspective. PLoS ONE. 2015; 10(10):e0141329. https://doi.org/10.1371/journal.pone.0141329\u003c/li\u003e\n\u003cli\u003eScotland RW, Vollesen K. Classification of Acanthaceae. Kew Bul1. 2000; 55(3):513\u0026ndash;589. https://doi.org/10.2307/4118776\u003c/li\u003e\n\u003cli\u003eWu WW, Li JL, Liu Y, Jiang M, Lan MS, Liu C. Peculiarities of the inverted repeats in the complete chloroplast genome of \u003cem\u003eStrobilanthes bantonensis\u003c/em\u003e Lindau. Mitochondrial DNA B. 2021; 6(4):1440\u0026ndash;1447. https://doi.org/10.1080/23802359.2021.1911699\u003c/li\u003e\n\u003cli\u003eKirankumar SI, Balaji R, Tanuja, Parani M. The complete chloroplast genome of \u003cem\u003eOcimum basilicum\u003c/em\u003e L. var. \u003cem\u003ebasilicum\u003c/em\u003e (Lamiaceae) and its phylogenetic analysis. Mitochondrial DNA B, 2023; 8(11):1169\u0026ndash;1173. https://doi.org/10.1080/23802359.2023.2275835\u003c/li\u003e\n\u003cli\u003eSueoka N. Near homogeneity of PR2-bias fingerprints in the human genome and their implications in phylogenetic analyses. J Mol Evol. 2001; 53:469\u0026ndash;476. https://doi.org/10.1007/s002390010237\u003c/li\u003e\n\u003cli\u003eGao Y, Lu Y, Song Y, Jing L. Analysis of codon usage bias of WRKY transcription factors in \u003cem\u003eHelianthus annuus\u003c/em\u003e. BMC Genom Data. 2022; 23:46. https://doi.org/10.1186/s12863-022-01064-8\u003c/li\u003e\n\u003cli\u003eHe B, Dong H, Jiang C, Gao FL, Tao ST, Xu LA. Analysis of codon usage patterns in \u003cem\u003eGinkgo biloba\u003c/em\u003e reveals codon usage tendency from A/U-ending to G/C-ending. Sci Rep. 2016; 6:35927. https://doi.org/10.1038/srep35927\u003c/li\u003e\n\u003cli\u003eChen HM, Shao JJ, Zhang H, Jiang M, Huang LF, Zhang Z, Yang D, He M, Ronaghi M, Luo X, Sun BT, Wu WW, Liu C. Sequencing and analysis of \u003cem\u003eStrobilanthes cusia\u003c/em\u003e (Nees) Kuntze chloroplast genome revealed the rare simultaneous contraction and expansion of the inverted repeat region in angiosperm. Front. Plant Sci. 2018; 9:324. https://doi.org/10.3389/fpls.2018.00324\u003c/li\u003e\n\u003cli\u003eZhang Z, Li J, Zhao XQ, Wang J, Wong GKS, Yu J. KaKs_Calculator: calculating Ka and Ks through model selection and model averaging. Genom Proteom Bioinf. 2006; 4(4):259\u0026ndash;263. https://doi.org/10.1016/S1672-0229(07)60007-2\u003c/li\u003e\n\u003cli\u003eLi MN, Pei HQ, Wang JH, Zhao KK, Zhu ZX, Wang HF. Complete plastome sequence of \u003cem\u003eClinacanthus nutans\u003c/em\u003e (Acanthaceae): a medicinal species in Southern China. Mitochondrial DNA B. 2019; 4(1):118\u0026ndash;119. https://doi.org/10.1080/23802359.2018.1532349\u003c/li\u003e\n\u003cli\u003eHuang SN, Ge XJ, Cano A, Salazar BGM, Deng YF. Comparative analysis of chloroplast genomes for five \u003cem\u003eDicliptera\u003c/em\u003e species (Acanthaceae): molecular structure, phylogenetic relationships, and adaptive evolution. PeerJ. 2020; 8:e8450. https://doi.org/10.7717/peerj.8450\u003c/li\u003e\n\u003cli\u003eYaradua SS, Alzahrani DA, Albokhary EJ, Abba A, Bello A. Corrigendum to \u0026ldquo;Complete chloroplast genome sequence of \u003cem\u003eJusticia flava\u003c/em\u003e: genome comparative analysis and phylogenetic relationships among Acanthaceae\u0026rdquo;. BioMed Res Int. 2020; 2020:2049759. https://doi.org/10.1155/2020/2049759\u003c/li\u003e\n\u003cli\u003eChen JX, Wang L, Zhao YC, Qin MJ. The complete chloroplast genome of a Chinese medicinal plant, \u003cem\u003ePeristrophe japonica\u003c/em\u003e (Thunb.) Bremek. (Lamiales: Acanthaceae) from Nanjing, China. Mitochondrial DNA B. 2021; 6(7):1888\u0026ndash;1889. https://doi.org/10.1080/23802359.2021.1934157\u003c/li\u003e\n\u003cli\u003eFernandez-Pozo N, Menda N, Edwards JD, Saha S, Tecle IY, Strickler SR, Bombarely A, Fisher-York T, Pujar A, Foerster H, Yan A, Mueller LA. The Sol Genomics Network (SGN)--from genotype to phenotype to breeding. Nucleic Acids Res. 2015; 43:D1036\u0026ndash;D1041. https://doi.org/10.1093/nar/gku1195\u003c/li\u003e\n\u003cli\u003ePowell W, Morgante M, McDevitt R, Vendramin GG, Rafalski JA. Polymorphic simple sequence repeat regions in chloroplast genomes: applications to the population genetics of pines. P Natl Acad Sci U S A. 1995; 92(17):7759\u0026ndash;7763. https://doi.org/10.1073/pnas.92.17.7759\u003c/li\u003e\n\u003cli\u003ePugh T, Fouet O, Risterucci AM, Brottier P, Abouladze M, Deletrez C, Courtois B, Clement D, Larmande P, N\u0026apos;Goran JAK, Lanaud C. A new cacao linkage map based on codominant markers: development and integration of 201 new microsatellite markers. Theor Appl Genet. 2004; 108:1151\u0026ndash;1161. https://doi.org/10.1007/s00122-003-1533-4\u003c/li\u003e\n\u003cli\u003eLi B, Lin FR, Huang P, Guo WY, Zheng YQ. Complete chloroplast genome sequence of \u003cem\u003eDecaisnea insignis\u003c/em\u003e: genome organization, genomic resources and comparative analysis. Sci Rep. 2017; 7:10073. https://doi.org/10.1038/s41598-017-10409-8\u003c/li\u003e\n\u003cli\u003eIkemura T. Correlation between the abundance of \u003cem\u003eEscherichia coli\u003c/em\u003e transfer RNAs and the occurrence of the respective codons in its protein genes: a proposal for a synonymous codon choice that is optimal for the \u003cem\u003eE. coli\u003c/em\u003e translational system. J Mol Biol. 1981; 151(3): 389\u0026ndash;409. https://doi.org/10.1016/0022-2836(81)90003-6\u003c/li\u003e\n\u003cli\u003eChen ZY, Liu Q, Xiao Y, Zhou GH, Yu PH, Bai J, Huang H, Gong YH. Correction to: Complete chloroplast genome sequence of \u003cem\u003eCamellia sinensis\u003c/em\u003e: genome structure, adaptive evolution, and phylogenetic relationships. J Appl Genet. 2023; 64:601. https://doi.org/10.1007/s13353-023-00769-5\u003c/li\u003e\n\u003cli\u003eParvathy ST, Udayasuriyan V, Bhadana V. Codon usage bias. Mol Biol Rep. 2022; 49:539\u0026ndash;565. https://doi.org/10.1007/s11033-021-06749-4\u003c/li\u003e\n\u003cli\u003eFu JM, Liu HM, Hu JJ, Liang YQ, Liang JJ, Wuyun TN, Tan XF. Five complete chloroplast genome sequences from \u003cem\u003eDiospyros\u003c/em\u003e: genome organization and comparative analysis. PLoS One. 2016; 11(7):e0159566. https://doi.org/10.1371/journal.pone.0159566\u003c/li\u003e\n\u003cli\u003eWang WC, Chen SY, Zhang XZ. Whole-genome comparison reveals divergent IR borders and mutation hotspots in chloroplast genomes of herbaceous bamboos (Bambusoideae: Olyreae). Molecules. 2018; 23(7):1537. https://doi.org/10.3390/molecules23071537\u003c/li\u003e\n\u003cli\u003eFay JC, Wu CI. Sequence divergence, functional constraint, and selection in protein evolution. Annu Rev Genomics Hum Genet. 2003; 4:213\u0026ndash;235. https://doi.org/10.1146/annurev.genom.4.020303.162528\u003c/li\u003e\n\u003cli\u003eMakałowski W, Boguski MS. Evolutionary parameters of the transcribed mammalian genome: an analysis of 2,820 orthologous rodent and human sequences. P Natl Acad Sci U S A. 1998; 95(16):9407\u0026ndash;9412. https://doi.org/10.1073/pnas.95.16.9407\u003c/li\u003e\n\u003cli\u003eBobik K, Burch-Smith TM. Chloroplast signaling within, between and beyond cells. Front. Plant Sci. 2015; 6:781. https://doi.org/10.3389/fpls.2015.00781\u003c/li\u003e\n\u003cli\u003eDobrogojski J, Adamiec M, Luciński R. The chloroplast genome: a review. Acta Physiol Plant. 2020; 42:98. https://doi.org/10.1007/s11738-020-03089-x\u003c/li\u003e\n\u003cli\u003ePfannschmidt T, Nilsson A, Tullberg A, Link G, Allen JF. Direct transcriptional control of the chloroplast genes \u003cem\u003epsbA\u003c/em\u003e and \u003cem\u003epsaAB\u003c/em\u003e adjusts photosynthesis to light energy distribution in plants. IUBMB Life. 1999; 48:271\u0026ndash;276. https://doi.org/10.1080/713803507\u003c/li\u003e\n\u003cli\u003eRogalski M, Sch\u0026ouml;ttler MA, Thiele W, Schulze WX, Bock R. Rpl33, a nonessential plastid-encoded ribosomal protein in tobacco, is required under cold stress conditions. Plant Cell. 2008; 20(8): 2221\u0026ndash;2237. https://doi.org/10.1105/tpc.108.060392\u003c/li\u003e\n\u003cli\u003eFleischmann TT, Scharff LB, Alkatib S, Hasdorf S, Sch\u0026ouml;ttler MA, Bock R. Nonessential plastid-encoded ribosomal proteins in tobacco: a developmental role for plastid translation and implications for reductive genome evolution. Plant Cell. 2011; 23(9):3137\u0026ndash;3155. https://doi.org/10.1105/tpc.111.088906\u003c/li\u003e\n\u003cli\u003eJussieu AL de. Genera plantarum secundum ordines naturales disposita. 2nd ed, Apud viduam Herissant et Theophilum Barrois: Paris, France, 1789; 307.\u003c/li\u003e\n\u003cli\u003eNees Von Esenbeck CG. Acanthaceae. In Plantae Asiaticae Rariores; Wallich, N. Eds, Treuttel, W\u0026uuml;rtz, Richter, London. 1832; 3:77\u0026ndash;l12.\u003c/li\u003e\n\u003cli\u003eBentham G. Acanthaceae. In Genera Plantarum; Bentham, G., Hooker, J.D. Eds, Reeve \u0026amp; Co.: London, 1876; v.2: pt.2:1066\u0026ndash;1122.\u003c/li\u003e\n\u003cli\u003eLindau Q. Acanthaceae. In: Die Nat\u0026uuml;rlichen Pflanzenfamilien; Engler, A., Prantl, K, Verlag von Wilhelm Englemann: Leipzig, 1895; IV(3b):274\u0026ndash;354.\u003c/li\u003e\n\u003cli\u003eBremekamp CEB. Delimitation and subdivision of the Acanthaceae. Bulletin of the Botanical Survey of India. 1965; 7:21\u0026ndash;30.\u003c/li\u003e\n\u003cli\u003eMcDade LA, Masta SE, Moody ML, Waters E. Phylogenetic relationships among Acanthaceae: evidence from two genomes. Syst Bot. 2000; 25(1):106\u0026ndash;121. https://doi.org/10.2307/2666677\u003c/li\u003e\n\u003cli\u003eDoyle J. DNA protocols for plants. In Molecular Techniques in Taxonomy, Nato ASI Subseries H: Cell Biology; 1st ed, Hewitt, G.M., Johnston, A.W.B., Young, J.P.W. Eds, Springer: Berlin, Heidelberg, Germany, 1991; 57:283\u0026ndash;293. https://doi.org/10.1007/978-3-642-83962-7_18\u003c/li\u003e\n\u003cli\u003eWingett SW, Andrews S. FastQ Screen: a tool for multi-genome mapping and quality control [version 2; peer review: 4 approved]. F1000Research. 2018; 7:1338. https://doi.org/10.12688/f1000research.15931.2\u003c/li\u003e\n\u003cli\u003eMcKain MR, Wilson M. Fast-Plast: rapid de novo assembly and finishing for whole chloroplast genomes. https://github.com/mrmckain/Fast-Plast (2017)\u003c/li\u003e\n\u003cli\u003eJin JJ, Yu WB, Yang JB, Song Y, dePamphilis CW, Yi TS, Li DZ. GetOrganelle: a fast and versatile toolkit for accurate de novo assembly of organelle genomes. Genome Biol. 2020; 21:241. https://doi.org/10.1186/s13059-020-02154-5\u003c/li\u003e\n\u003cli\u003eQu XJ, Moore MJ, Li DZ, Yi TS. PGA: a software package for rapid, accurate, and flexible batch annotation of plastomes. Plant Methods. 2019; 15:50. https://doi.org/10.1186/s13007-019-0435-7\u003c/li\u003e\n\u003cli\u003eTillich M, Lehwark P, Pellizzer T, Ulbricht-Jones ES, Fischer A, Bock R, Greiner S. GeSeq - versatile and accurate annotation of organelle genomes. Nucleic Acids Res. 2017; 45(W1):W6\u0026ndash;W11. https://doi.org/10.1093/nar/gkx391\u003c/li\u003e\n\u003cli\u003eLi H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009; 25(14):1754\u0026ndash;1760. https://doi.org/10.1093/bioinformatics/btp324\u003c/li\u003e\n\u003cli\u003eLiu SY, Ni Y, Li JL, Zhang XY, Yang HY, Chen HM, Liu C. CPGView: a package for visualizing detailed chloroplast genome structures. Mol Ecol Resour. 2023; 23(3):694\u0026ndash;704. https://doi.org/10.1111/1755-0998.13729\u003c/li\u003e\n\u003cli\u003eThiel T, Michalek W, Varshney RK, Graner A. Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (\u003cem\u003eHordeum vulgare\u003c/em\u003e L.). Theor Appl Genet. 2003; 106:411\u0026ndash;422. https://doi.org/10.1007/s00122-002-1031-0\u003c/li\u003e\n\u003cli\u003eSun XY, Yang Q, Xia XH. An improved implementation of Effective Number of Codons (\u003cem\u003eNc\u003c/em\u003e). Mol Biol Evol. 2013; 30(1):191\u0026ndash;196. https://doi.org/10.1093/molbev/mss201\u003c/li\u003e\n\u003cli\u003eWang YH, Wei QY, Xue TY, He SX, Fang J, Zeng CL. Comparative and phylogenetic analysis of the complete chloroplast genomes of 10 \u003cem\u003eArtemisia selengensis\u003c/em\u003e resources based on high-throughput sequencing. BMC Genomics. 2024; 25:561. https://doi.org/10.1186/s12864-024-10455-3\u003c/li\u003e\n\u003cli\u003eMayor C, Brudno M, Schwartz JR, Poliakov A, Rubin EM, Frazer KA, Pachter LS, Dubchak I. \u003cem\u003eVISTA\u003c/em\u003e: visualizing global DNA sequence alignments of arbitrary length. Bioinformatics. 2000; 16(11):1046\u0026ndash;1047. https://doi.org/10.1093/bioinformatics/16.11.1046\u003c/li\u003e\n\u003cli\u003eAmiryousefi A, Hyv\u0026ouml;nen J, Poczai P. IRscope: an online program to visualize the junction sites of chloroplast genomes. Bioinformatics. 2018; 34(17):3030\u0026ndash;3031. https://doi.org/10.1093/bioinformatics/bty220\u003c/li\u003e\n\u003cli\u003eSanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. P Natl Acad Sci U S A. 1977; 74(12):5463\u0026ndash;5467. https://doi.org/10.1073/pnas.74.12.5463\u003c/li\u003e\n\u003cli\u003eKumar S, Stecher G, Tamura K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016; 33(7):1870\u0026ndash;1874. https://doi.org/10.1093/molbev/msw054\u003c/li\u003e\n\u003cli\u003eRozas J, Ferrer-Mata A, S\u0026aacute;nchez-DelBarrio JC, Guirao-Rico S, Librado P, Ramos-Onsins SE, S\u0026aacute;nchez-Gracia A. DnaSP 6: DNA sequence polymorphism analysis of large data sets. Mol Biol Evol. 2017; 34(12):3299\u0026ndash;3302. https://doi.org/10.1093/molbev/msx248\u003c/li\u003e\n\u003cli\u003eKatoh K, Rozewicki J, Yamada KD. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinform. 2019; 20(4):1160\u0026ndash;1166. https://doi.org/10.1093/bib/bbx108\u003c/li\u003e\n\u003cli\u003eCastresana J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol biol Evol. 2000; 17(4):540\u0026ndash;552. https://doi.org/10.1093/oxfordjournals.molbev.a026334\u003c/li\u003e\n\u003cli\u003eDarriba D, Taboada GL, Doallo R, Posada D. jModelTest 2: more models, new heuristics and parallel computing. Nat Methods. 2012; 9:772. https://doi.org/10.1038/nmeth.2109\u003c/li\u003e\n\u003cli\u003eStamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014; 30(9):1312\u0026ndash;1313. https://doi.org/10.1093/bioinformatics/btu033\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomic-data","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gtic","sideBox":"Learn more about [BMC Genomic Data](http://bmcgenet.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gtic/default.aspx","title":"BMC Genomic Data","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Pachystachys lutea, Acanthaceae, chloroplast genome, comparative analysis, phylogenetic analysis","lastPublishedDoi":"10.21203/rs.3.rs-5848411/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5848411/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e \u003cem\u003ePachystachys lutea\u003c/em\u003e Nees is a typical species of the family Acanthaceae, native to tropical South America. As an evergreen shrub, it has found extensive application in landscape greening due to its unique ornamental value. However, there are few available phylogenetic and genetic studies about the chloroplast (cp) genome of \u003cem\u003eP. lutea\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults \u003c/strong\u003eThis study characterized the cp genome of \u003cem\u003eP. lutea\u003c/em\u003e by using Illumina sequencing technology and inferred the phylogenetic position of the species. The results indicated that the cp genome had a high degree of conservation in gene structure and gene content, with a typical quadripartite structure. Its total length is 151,574 bp and the total GC content is 38.18%. A total of 132 genes were annotated, including 87 protein-coding genes (PCGs), 37 tRNAs and eight rRNA genes. Through the comparative analysis, the diversity and variation of large single-copy (LSC) and small single-copy (SSC) regions were significantly higher than those of inverted repeat (IR) regions. Genes with high nucleotide polymorphism, such as \u003cem\u003erps19\u003c/em\u003e, \u003cem\u003eycf1\u003c/em\u003e, and \u003cem\u003endhF\u003c/em\u003e provided potential reference loci for molecular identification within the \u003cem\u003eP. lutea\u003c/em\u003e. The phylogenetic analysis showed that the \u003cem\u003eP. lutea\u003c/em\u003e and \u003cem\u003eClinacanthus nutans\u003c/em\u003e forms a sister group with 100% bootstrap value, which proves that \u003cem\u003eP. lutea\u003c/em\u003e develops conservatively in the course of evolution.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion \u003c/strong\u003eThis paper for the first time reports the phylogenetic study of the complete cp genome within the genus \u003cem\u003ePachystachys\u003c/em\u003e. The study provides a theoretical basis for the research on genetic diversity, molecular markers, and species identification of plants in the Acanthaceae family. It enriches the genetic information and supports the evolutionary relationships among plants in this family.\u003c/p\u003e","manuscriptTitle":"Complete chloroplast genome sequence of Pachystachys lutea Nees: genome structure, adaptive evolution, and phylogenetic relationships","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-17 10:13:21","doi":"10.21203/rs.3.rs-5848411/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-15T10:33:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-15T05:40:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-08T02:07:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"52868065308767456210818425810667456826","date":"2025-07-04T04:33:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"62121851927273600146937132558112174286","date":"2025-06-03T02:57:55+00:00","index":"hide","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-30T16:02:25+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"245467891809263480493069810401804397118","date":"2025-04-20T15:10:26+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-16T13:57:09+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-16T10:26:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomic Data","date":"2025-04-09T09:16:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomic-data","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gtic","sideBox":"Learn more about [BMC Genomic Data](http://bmcgenet.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gtic/default.aspx","title":"BMC Genomic Data","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3ad4c47e-890a-43bd-928a-17609256cb8c","owner":[],"postedDate":"April 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-24T16:09:19+00:00","versionOfRecord":{"articleIdentity":"rs-5848411","link":"https://doi.org/10.1186/s12863-025-01380-9","journal":{"identity":"bmc-genomic-data","isVorOnly":false,"title":"BMC Genomic Data"},"publishedOn":"2025-11-19 15:58:27","publishedOnDateReadable":"November 19th, 2025"},"versionCreatedAt":"2025-04-17 10:13:21","video":"","vorDoi":"10.1186/s12863-025-01380-9","vorDoiUrl":"https://doi.org/10.1186/s12863-025-01380-9","workflowStages":[]},"version":"v1","identity":"rs-5848411","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5848411","identity":"rs-5848411","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}