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Genomic exploration of Sesuvium sesuvioides: comparative study and phylogenetic analysis within the order Caryophyllales from Cholistan desert, Pakistan



The Aizoaceae family’s Sesuvium sesuvioides (Fenzl) Verdc is a medicinal species of the Cholistan desert, Pakistan. The purpose of this study was to determine the genomic features and phylogenetic position of the Sesuvium genus in the Aizoaceae family. We used the Illumina HiSeq2500 and paired-end sequencing to publish the complete chloroplast sequence of S. sesuvioides.


The 155,849 bp length cp genome sequence of S. sesuvioides has a 36.8% GC content. The Leucine codon has the greatest codon use (10.6%), 81 simple sequence repetitions of 19 kinds, and 79 oligonucleotide repeats. We investigated the phylogeny of the order Caryophyllales’ 27 species from 23 families and 25 distinct genera. The maximum likelihood tree indicated Sesuvium as a monophyletic genus, and sister to Tetragonia. A comparison of S. sesuvioides, with Sesuvium portulacastrum, Mesembryanthemum crystallinum, Mesembryanthemum cordifolium, and Tetragonia tetragonoides was performed using the NCBI platform. In the comparative investigation of genomes, all five genera revealed comparable cp genome structure, gene number and composition. All five species lacked the rps15 gene and the rpl2 intron. In most comparisons with S. sesuvioides, transition substitutions (Ts) were more frequent than transversion substitutions (Tv), producing Ts/Tv ratios larger than one, and the Ka/Ks ratio was lower than one. We determined ten highly polymorphic regions, comprising rpl22, rpl32-trnL-UAG, trnD-GUC-trnY-GUA, trnE-UUC-trnT-GGU, trnK-UUU-rps16, trnM-CAU-atpE, trnH-GUG-psbA, psaJ-rpl33, rps4-trnT-UGU, and trnF-GAA-ndhJ.


The whole S. sesuvioides chloroplast will be examined as a resource for in-depth taxonomic research of the genus when more Sesuvium and Aizoaceae species are sequenced in the future. The chloroplast genomes of the Aizoaceae family are well preserved, with little alterations, indicating the family’s monophyletic origin. This study’s highly polymorphic regions could be utilized to build realistic and low-cost molecular markers for resolving taxonomic discrepancies, new species identification, and finding evolutionary links among Aizoaceae species. To properly comprehend the evolution of the Aizoaceae family, further species need to be sequenced.

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The Caryophyllales order is made up of 37 families, 749 genera, and around 11,600 species [1, 2]. The clade is famous not only for its rich diversity and wide ecological and geographic distribution but also for a variety of distinct morphological and eco-physiological adaptations [3]. Many Caryophyllales species are well-known for their drought resistance, but the clade also includes species with high adaptations to cold, heavy metal hyper-accumulation, salt tolerance, carnivory, CAM and C4 metabolism, and succulence [3,4,5,6]. The majority of these characteristics are known to have evolved at different times throughout the group, making Caryophyllales an important natural laboratory for studying evolutionary traits in angiosperms [3]. Aizoaceae is the largest succulent family of the Caryophyllales order with five sub-families, 143 genera, and more than 2300 species that are typically located in tropical and subtropical climates, especially along the shore or in arid areas [7]. The five Aizoaceae sub-families are Tetragonioideae, Mesembryanthemoideae, Sesuvioideae, Ruschioideae and Aizooideae [4, 8]. The subfamily Sesuvioideae is reported to have two tribes (Sesuvieae and Anisostigmateae) and five genera (Zaleya, Sesuvium, Trianthema, Cypselea, and Tribulocarpus) [4, 6]. Molecular and morphological phylogenetic studies demonstrate that Sesuvioideae is closely associated with the other four Aizoaceae subfamilies [6]. Sesuvioideae and its sibling group separated at the beginning of the Miocene period [9] [10];. During the late Miocene, the sister clades, especially the Ruschioideae, produced about 1770 species, whereas the Sesuvioideae retained a low number of species [4, 9, 10]. Molecular phylogenetic studies also demonstrated that Mesembryanthemoideae and Ruschioideae species are monophyletic [8, 9].

One of the most widespread genera in the Sesuvioideae subfamily, Sesuvium L. is found all over the world in diverse subtropical and tropical climates [11, 12]. According to previous morphological and molecular phylogenetic investigations, there were fourteen to seventeen accepted species of the Sesuvium genus [13]. Due to the absence of well-defined species boundaries, Sesuvium is one of Aizoaceae’s more challenging genera to categorize [5]. Barri Ulwaiti is the local name for Sesuvium sesuvioides (Fenzl) Verdc. (Aizoaceae). S. sesuvioides is a short-lived perennial herb and one of the succulent halophytes found mostly in salty areas and inter-dunal clayey plains of Pakistan’s Cholistan desert [14] shown in Fig. 1. It has 2n = 16 chromosomes [15]. S. sesuvioides is a herbal medicine utilized by indigenous practitioners in the Cholistan desert for folkloric cures such as arthritis, gout, epistaxis, bleeding, smallpox, chickenpox, cold, flu, haemorrhage, thyroid malfunction, inflammation, fever, ulcer, measles, and nasal bleeding [16,17,18]. Aqueous extracts of this plant contained phenolic compounds, flavonoids, glycosides, coumarin, terpenes, saponins, triglycerides, and carbohydrates [19]. Recent research has demonstrated its anti-inflammatory, analgesic, and antipyretic effects [19].

Fig. 1
figure 1

The Sesuvium sesuvioides (Barri Ulwaiti) growing in the Cholistan desert

Chloroplasts in higher plants serve as metabolic hubs for photosynthesis, which merely keeps life on Earth alive [20]. Chloroplast (cp.) genomes are significant and relevant data sources for evolutionary biology, and they have been utilized extensively in plant phylogenetic investigations [21]. The cp genome has significantly conserved gene content and genome order [22]. Due to its modest size, the chloroplast genome was the inaugural plant genome to be thoroughly sequenced [23]. Furthermore, the cp genome has fewer nucleotide alterations and genome sequence reorganizations than the nuclear genome [24, 25] making it a great tool for figuring out how genomes evolve and how phylogenetics relations work in complicated angiosperm families [21, 26, 27]. The phylogenetic investigations of various plant families have benefited tremendously from the use of chloroplast genomes, which have also made it easier to understand the evolutionary connections between various phylogenetic clades [21]. Our perception of plant science and heterogeneity has been expanded by the whole chloroplast genome sequencing [28].

Even though Aizoaceae species have received significant research attention in terms of evolutionary studies [6, 9, 29, 30], there is very little data for chloroplast genomes on NCBI, and information for phylogenetic connections within major subfamilies is currently not sufficient [9]. Only four cp genomes of Aizoaceae species (Sesuvium portulacastrum, Mesembryanthemum crystallinum, Mesembryanthemum cordifolium, and Tetragonia tetragonoides) have been completely sequenced available on NCBI [1]. Hassan et al., (2005) previously explored the phylogeny of Sesuvioideae (Aizoaceae) based on nuclear DNA, but the location of the genus Sesuvium remained unknown [29]. To resolve phylogenetic difficulties, more chloroplast genomes from the genus Sesuvium (Aizoaceae family) must be sequenced and analyzed. Understanding the evolutionary link between S. sesuvioides and other Aizoaceae plants can help in the sequencing and identification of other members of the family. This study aimed to fill some knowledge gaps by (1) gaining insights into the structure of the S. sesuvioides plastid genome and performing comparative analysis to explore mutations within the Aizoaceae family, (2) reconstructing the phylogenetic tree of the Caryophyllales order to resolve phylogenetic issues in both the genus and the family, and (3) determining highly polymorphic loci for the creation of reliable, significant, and affordable species detection markers. This study will aid in adding substantial molecular and phylogenetic data to the Aizoaceae family to accommodate species detection in the future.

Materials and methods

DNA extraction and chloroplast genome sequencing

Plants of S. sesuvioides were collected from Pakistan’s Lesser Cholistan desert (28.7719699, 71.3346211) and the regulatory verification procedure was conducted at the Cholistan Institute of Desert Studies (CIDS) of Islamia University Bahawalpur. The DNA was extracted from fresh and young leaves using the SDS-based DNA extraction method [31]. The quantity and quality of isolated DNA were assessed using nanodrop and 1% agarose gel electrophoresis. A whole genome shotgun was created at the Beijing Institute of Genomics (BIG) in Beijing, China, using an Illumina Hiseq2500 with a Paired-end database containing 150 bp.

Annotation of genes and assembly of the chloroplast genome

FastQC analysis was used to assess the quality of the raw readings data [32]. We used NOVOPlasty to extract the chloroplast genome from whole genome sequence (WGS) data and reconstructed the full chloroplast genome [33]. By evaluating the sequence scaffolding, the borders of the LSC, SSC, and IR regions were observed. GeSeq [34] and CpGAVAS with standard settings were used to annotate the cp genome sequence [35]. For further verification of annotations, the S. sesuvioides genome was pairwise aligned with other genomes of the Aizoaceae family, S. portulacastrum (MK330004), M. crystallinum (KM016695), M. cordifolium (MK397873), and T. tetragonoides (MF975369) by MAFFT alignment (Multiple Alignment with Fast Fourier Transform) [36] in Geneious Prime 2021.1.1 [37]. The tRNAscan-SE 1.23 programme was employed to validate the tRNA genes [38]. By mapping sequencing short sequences to their corresponding de novo assembled cp genomes using BWA software [39], the average sequencing coverage depth for the assembled S. sesuvioides genome was obtained and visualized in Tablet [40]. OGDraw v1.2 [40] was used to build the circular map of the cp genome. The cp genome of S. sesuvioides was submitted to GenBank and assigned the accession number MW539047. The raw data acquired in this investigation was uploaded to Sequence Read Archive (SRA) under project number PRJNA660981.

Determination of amino acid frequency, and codon usage

MEGA-X [41] was used to examine Relative Synonymous Codon Usage (RSCU) in S. sesuvioides protein-coding sequences, whereas Geneious Prime 2021.1.1 was used to examine amino acid frequency [37].

Detection of simple sequence repeats (SSRs) and oligonucleotide repeats

The Perl script MIcroSAtellite Identification Tools (MISA) [42] was used to detect SSRs, with minimum repetition counts of ten for mono-, five for di-, four for tri-, three tetra-, three Penta-, and three for hexanucleotides. The REPuter programme [43] was used to find forward (F), reverse (R), complementary (C), and palindromic (P) oligonucleotide repeats with an edit distance of two, a minimum repeat size of 10 bp, and a maximum computed repeat of 100.

Phylogenetic analysis of Caryophyllales

The cp genomes of 26 species of order Caryophyllales from 23 families were used to construct phylogenetic connections (Table S5). Asclepias nivea and Asclepias syriaca from the Apocynaceae family were chosen as an outgroup. The phylogenetic tree contains 29 species in total (1 S. sesuvioides plus 28 NCBI species). The selected species were downloaded from NCBI (National Center for Biotechnology Information), protein-coding sequences from each species were extracted, and the sequences were concatenated in Geneious Prime 2021.1.1. To align these protein-coding sequences, MAFFT was employed. The best-fit model GTR + F + R6 according to AIC (Akaike information criterion) was used to build the phylogenetic tree [44]. The maximum likelihood tree was generated online in Galaxy using IQ-TREE [45], and Ultrafast bootstrap settings with 1000 bootstrap replications [46]. To complete the tree display, we utilized the iTOL (interactive tree of life) software [47].

Species selected for basic comparison with S. Sesuvioides

Based on the results of phylogenetic studies, the complete cp genomes of four Aizoaceae species including S. portulacastrum, M. crystallinum, M. cordifolium, and T. tetragonoides were compared to that of S. sesuvioides. The Geneious Prime 2021.1.1 was used to analyze the cp genomes for the basic comparison. The Mafft alignment was used to make the multiple alignments of the selected species, to detect the arrangement and show the comparison between these cp genomes.

IR contraction and expansion

The junctions of cp genomes of five Aizoaceae species, including S. sesuvioides, S. portulacastrum, M. crystallinum, M. cordifolium, and T. tetragonoides, were compared. With the use of IRScope, the expansion and contraction of IRs areas at the intersections of the four major cp genome components (LSC/IRb/SSC/IRa) were studied [48].

Estimation of synonymous (Ks) and non-synonymous (Ka) substitution rates

We analyzed synonymous (Ks), non-synonymous substitutions (Ka), and Ka/Ks values by making pair-wise alignments of protein-coding sequences of S. sesuvioides cp genome with the other four species of Aizoaceae. To do this, we extracted the protein-coding sequences for each genome and performed the pairwise alignment using Geneious Prime 2021.1.1. Pairwise alignments for each analysis were carried out using S. sesuvioides as the reference member. DnaSP was utilized to assess the pairwise alignment and to identify Ka, and Ks substitutions [49].

Single nucleotide polymorphisms (SNPs) and InDels mutations

IR, SSC, and LSC areas of the S. sesuvioides chloroplast genome were pair-wise aligned with corresponding regions of the other four Aizoaceae species cp genomes chosen for comparative study using MAFFT alignment implemented in Geneious Prime 2021.1.1. The reference genome for this investigation was the S. sesuvioides cp genome. The Geneious Prime 2021.1.1 was used to calculate the number, spatial positions, and kind of SNPs (transition and transversion). The pairwise aligned cp genomes were used to find InDels mutations using DnaSP [49]. For each site, it also calculated the alignment length, no. of inDels, k(i) inDel diversity, inDel average length, and Pi(i) inDel diversity per site.

Determination of nucleotide diversity and high polymorphism loci

Nucleotide diversity (π) was calculated in 130 common regions of S. sesuvioides, S. portulacastrum, M. crystallinum, M. cordifolium, and T. tetragonoides. We made 130 multiple alignments in MAFFT from 650 regions extracted from the five Aizoaceae species (60 CDS regions, 14 intronic locations and 50 IGS sequences). Nucleotide diversity (π) was calculated using DnaSP [49]. To observe highly polymorphic regions between the species chosen for comparative study, ten loci with greater nucleotide diversity were selected.


Chloroplast genome structure of Sesuvium sesuvioides

The raw data for S. sesuvioides from the Illumina HiSeq2500 paired-end sequencing with 150 bp reads was 10.9 GB. The de novo assembled S. sesuvioides cp genome had an average coverage depth of 870. The S. sesuvioides cp genome (155,849 bp) is composed of the SSC region (18,736 bp), the LSC region (89,619 bp), and two inverted repeats (25,947 bp). It has 36.8% GC overall, with IRs having 42.8% greater GC than the LSC (34.6%) and SSC (30.2%). The 132 genes found in the chloroplast genome of S. sesuvioides comprise 37 tRNA, eight rRNA, and 87 CDS genes. In the inverted repeat regions, 19 genes are duplicated (Table 1). The genome of S. sesuvioides cp comprises 21 genes with introns, including 13 CDS genes and 8 tRNA genes. There are 18 genes with one intron and three with two introns (Table 2; Fig. 2), including ycf3, rps12, and clpP. The rps12 gene showed evidence of trans-splicing. The S. sesuvioides cp genome lacked the rpl2 intron and the rps15 genes. The ycf1 gene began in the inverted repeats and ended in the SSC domain, with a pseudo copy found in the IRB region. Similarly, at the IRB/LSC junction, the gene rps19 has a functional copy of 279 bp and a pseudo copy of 150 bp. The S. sesuvioides cp genome is available on this NCBI link:

Table 1 The full S. sesuvioides cp genome’s comprehensive properties
Table 2 Genes containing introns and their length in S. sesuvioides
Fig. 2
figure 2

The cp genomic map of S. sesuvioides. Genes are translated in two directions: clockwise for those on the circle’s rim and anticlockwise for those within. Protein-coding genes are distinguished by their shade based on their function. In the inner circle, the AT and GC components of the genome are depicted as light grey and dark grey, respectively. Inverted Repeats are denoted by the letters IRb and IRa, whereas Single-copy regions are denoted by the letters SSC and LSC

Amino acid frequencies and RSCU values

The S. sesuvioides contain a coding sequence of 80,376 bp and 51,949 codons. Leucine (11%) was the most prevalent amino acid in the S. sesuvioides cp genome, followed by isoleucine (9%), while cysteine (1%) was the least abundant amino acid (see Fig. 3). We discovered RSCU values for 64 codons, 31 of which were with RSCU values less than one. The RSCU values ranged from 0.44 to 1.81. The AGA codon, which encodes Arginine, had the highest usage bias (1.81), whereas the CGC codon, which also encodes Arginine, had the lowest (0.44). In the S. sesuvioides plastid genome, 12 codons exhibited the lowest preference (1.0 < RSCU< 1.2), two with no preference (RSCU = 1) 6 demonstrated modest preference (1.2 < RSCU< 1.3), and 13 exhibited intense preference (RSCU> 1.3). The highest frequencies and RSCU values were observed for the codons ending at U and A. Apart from methionine and tryptophan which had RSCU = 1, most amino acids with numerous codons were strongly biased for one or two A/U ending codons. Stop codons in S. sesuvioides are UAA, UGA, and UAG (Table 3).

Fig. 3
figure 3

Comparison of amino acid frequency for S. sesuvioides

Table 3 Frequency and RSCU values of 64 codons in S. sesuvioides cp genome. Stop codons are donated by "*"

Determination of SSRs

We discovered 81 SSRs of 19 distinct kinds in S. sesuvioides using the Perl script MISA (Table S1). The number of SSRs present in compound form was 7. The S. sesuvioides included 55 (68%) mononucleotides, 11 (14%) di-nucleotide, 4 (5%) tri-nucleotide, 10 (12%) tetra-nucleotide, and 1 (1%) pentanucleotide (Fig. 4a & Table S3). No hexanucleotides were observed in the cp genome of S. sesuvioides. All mononucleotide SSRs included A/T motifs, but no C/G motifs (Table S2). Di-nucleotides of four different forms have been identified i.e. AT, AT, AC, GT. In the cp genome of S. sesuvioides, four tri-nucleotide variants (AAT, ATT, AAC, GTT) were identified, as well as eleven tetra-nucleotide forms (AAAT, ATTT, AAAG, CTTT, AATT, AATT, AGAT, ATCT, ACCT, AGGT), and two pentanucleotide types (AAATT, AATTT). The LSC has the most SSRs (64), followed by the SSC (9), and then the inverted repeats (IRs) (8 SSRs) (Fig. 4c). The proportion of SSRs in designated areas in this scenario was as follows: 72% SSRs (58) in intergenic areas > 12% SSRs (10) in intronic regions > 11% SSRs (9) in the rrn region (Fig. 4b). We found two CDS/IGS mutual SSRs that were mononucleotides at ycf4-cemA and petD-rpoA.

Fig. 4
figure 4

SSRs detail of cp genome of S. sesuvioides. a Types of SSRs. b Represent SSRs in functional cp genome regions. c Location of the SSRs

Oligonucleotide repeats analysis

We identified 79 unique oligonucleotide repeat sequences in the S. sesuvioides cp genome utilizing the REPuter program. We detected four types of oligonucleotide repetitions, as shown in Fig. 5(a): 31 Forward, 10 Reverse, 36 Palindromic, and two Complementary repeats. The length of the repeats varied from 19 to 50 bp (Fig. 5b). The LSC region had the most oligonucleotide repeats at 63% (50) and then IRs at 8% (6), whereas the SSC region had the lowest at 6% (5). LSC and SSC shared eight oligonucleotide repeat sequences, SSC and IR four, and LSC and IR seven (Fig. 5c). The intergenic spacer region contains the greatest oligonucleotide repeats (48%), following the trn region (11%), the CDS (9%), and the intronic region (5%). Mutual repetitions were also found in the domains CDS/IGS (9%), Intron/IGS (14%), and trn/IGS (4%), as shown in Fig. 5(d). Table S4 shows the position, arrangement, and area of the repetitions.

Fig. 5
figure 5

The detail of ORs of S. sesuvioides cp genome. a The amount of oligonucleotide repeats found in S. sesuvioides is classified into four types: reverse, complementary, palindromic, and forward repetitions. b Described the S. sesuvioides oligonucleotide repeats size range. c Estimation of the no. of oligonucleotide repeats in regions of the cp genome. d Demonstrated the functional locations of oligonucleotide repeats

Phylogenetic study of the Caryophyllales

There were 161,453 bp of consensus sequence nucleotide locations in the alignment of 29 species (Table S5) with a pairwise similarity of 55.7%. There were 86,947 consistent sites, 25, 280 instructive parsimony sites, and 31,809 unique site patterns discovered. The phylogenetic tree resulted in 26 branches with bootstrap values ranging from 59 to 100 (See Fig. 6). The bootstrap value for 22 among these branches was 100. The phylogenetic analysis showed the Sesuvium and the other two genera of the Aizoaceae family, Tetragonia, and Mesembryanthemum, were closely linked and monophyletic. A reasonable bootstrap support value for the five species of Aizoaceae was 100. The phylogenetic tree also showed that members of the Aizoaceae family were closely related to the genus Portulaca of the Portulacaceae family and the genus Nyctaginia of the Nyctaginaceae family. The Aizoaceae genera chosen were all closely connected to the Sesuvium genus (Fig. 6). This phylogenetic tree also highlighted the monophyly of all the represented sub-families of Aizoaceae i.e. Tetragonioideae (Tetragonia), Mesembryanthemoideae (Mesembryanthemum), Sesuvioideae (Sesuvium). This phylogenetic tree further demonstrated the tight connections of all Caryophyllales families, confirming the order’s monophyletic nature.

Fig. 6
figure 6

The order Caryophyllales’ maximum likelihood (ML) tree. The out-group is represented by Asclepias nivea and Asclepias syriaca (Family Apocynaceae). The genus Sesuvium and the genus Tetragonia have a close connection

Comparative analysis of S. Sesuvioides with other species

We picked four Aizoaceae species to perform a comparison with S. sesuvioides, including Sesuvium portulacastrum, Mesembryanthemum crystallinum, Mesembryanthemum cordifolium, and Tetragonia tetragonoides, shown in Table 4. Overall, the length of the chloroplast sequences spans from 155,934 bp (S. portulacastrum) to 149,506 bp (T. tetragonoides), and every segment of the quadripartite cycle was comparable among these cp genomes. M. crystallinum has the greatest number of genes (134), while T. tetragonoides has the fewest (127). These cp genomes’ total GC content varied between 36.6 to 37.3%. These cp genomes have similar gene compositions, with only a small number of genes being added or deleted. The rps15 and rpl2 introns were entirely missing in all five Aizoaceae species. The rps19 gene has one functional copy in all genomes, but S. sesuvioides has an additional pseudo copy. The infA gene was missing in T. tetragonoides and was extremely small in S. sesuvioides. The ycf3 gene is found only in M. crystallinum. The introns of the petB, petD, and rpl16 genes are missing in M. crystallinum but found in the other four Aizoaceae species (Figs. 7, 8, 9). MAFFT alignment of five species was employed in Geneious Prime 2021.1.1, 162,218 bp consensus sequence had 134,189 (82.7%) identical sites and 90.5% pairwise identity.

Table 4 The findings of a cp genome comparison among five Aizoaceae species are shown
Fig. 7
figure 7

Multiple alignment petD gene of five species of Aizoaceae family

Fig. 8
figure 8

Multiple alignment petB gene of five species of Aizoaceae family

Fig. 9
figure 9

Multiple alignment rpl16 gene of five species of Aizoaceae family

IR contraction and expansion

Chloroplast genome evolution is influenced by variations in IR region length throughout time. In S. sesuvioides and four other cp genomes (S. portulacastrum, M. crystallinum, M. cordifolium, and T. tetragonoides), the contraction and expansion of IRs regions at the junctions of four main regions were examined (LSC, IRB, SSC, IRB). At the SSC/IRa border, all species contain a functional copy of the ycf1 gene, as well as a false copy of ycf1Ψ at the IRb/SSC interface. In two taxa, S. portulacastrum and M. cordifolium, the ycf1Ψ gene was not annotated. The size of the functional ycf1 copy spanned from 1689 to 5730 bp. The ycf1Ψ copy had a length that varied from 1365 to 1395 bp. Near the IRb/SSC junction, the ndhF gene, with a length ranging from 2139 to 2244 kb, was present in four species. The SSC region of M. cordifolium contains the entire ndhF gene. The rps19 was located at the LSC/IRb junction in all species. The rpl2 gene was located in the IRs. The rpl22 was present in the LSC region of all cp genomes. At the IRa/LSC junction, the trnH and psbA genes were completely visible. These IRScope findings indicated that all Aizoaceae cp genomes are quite similar, with just minor differences in gene sizes and positions. The identification of similar genes at each chloroplast genome junction also indicated genome size correlations in these species. A thorough analysis of the contractions and expansions of IRs is shown in Fig. 10.

Fig. 10
figure 10

The IRSCOPE analysis of the IRs of the selected five cp genomes

The rate of Ka, and Ks substitutions

The Ka/Ks ratios for the S. sesuvioides and four Aizoaceaecp genomes were analyzed including S. portulacastrum, M. crystallinum, M. cordifolium, and T. tetragonoides (Fig. 11). S. sesuvioides was utilized as the reference member in each pairwise alignment. With the help of MAFFT alignment 308 pair-wise alignments on 77 related protein-coding genes were performed to examine the Ka/Ks for the selected cp genomes. Genes with irrelevant (N/A) Ka/Ks values were changed with 0 (See also Table S6). The Ka/Ks ratio varied from 0.01-1.70. The average Ka/Ks ratio was 0.212, indicating the genes underwent considerable purifying selection processes in cp genomes of the Aizoaceae family. 11 genes out of 77 (clpP, petN, psaC, psbA, psbI, psbJ, psbM, psbN, psbZ, rpl23, and rpl36) had Ka/Ks ratios equal to zero, confirming that these genes were preserved in all five Aizoaceae cp genomes. There were 63 genes with more synonymous substitutions (Ks) than non-synonymous substitutions (Ka). In all comparisons, all genes had lower than one Ka/Ks ratio; however, only three genes, rpl22, rpl32, and ycf2, showed unusual behaviour. The ycf2 has a Ka/Ks ratio of one in S. portulacastrum and less than one in the other three comparisons. Similarly, the rpl22 gene has more than one Ka/Ks value in M. crystallinum (1.35), and M. cordifolium (1.30). The rpl32 gene has more than one Ka/Ks ratio observed in S. portulacastrum (1.47) and T. tetragonoides (1.70) while it was below one in the other two assessments (Table S6).

Fig. 11
figure 11

The Ka/Ks value of five Aizoaceae cp genomes. Ka/Ks values for 66 genes are mentioned; genes with 0 Ka/Ks are not presented

Analyzing SNPs and InDel mutations in S. Sesuvioides

Pairwise alignments of the pertinent areas were used to compare S. sesuvioides to four additional Aizoaceae species to find SNPs and InDels mutations in the IRs, SSC and LSC. When S. sesuvioides compared to T. tetragonoides, had the most SNPs (7146), while S. portulacastrum had the fewest (1441 SNPs). A transition-to-transversion ratio greater than one resulted from the observation that the average transition rate is higher than the rate of a transversion. The Ts/Tv values in just two comparisons were less than one (Table 5). The LSC part had the greatest substitution rate, followed by the SSC and IR sections (Table S7). The LSC area had the most inDels, followed by the SSC region, and the least in the IRs (Table 6). The pairwise alignment of S. sesuvioides and T. tetragonoides produced the most inDels (11,978). The M. cordifolium has the second most inDels (10,148), whereas S. portulacastrum has the fewest (2184).

Table 5 Transition and Transversion substitutions, their ratio in LSC, IRs, and SSC
Table 6 The detailed InDels analysis for five Aizoaceae cp genomes

The estimation of nucleotide diversity and highly polymorphic loci

Nucleotide diversity (π) was calculated in 130 common regions of S. sesuvioides, S. portulacastrum, M. crystallinum, M. cordifolium, and T. tetragonoides (Fig. 12). It ranged from 0.0024 to 0.4789. The rpl22 gene has the greatest rate (0.4789) of nucleotide diversity (Table S8). Intergenic spacer areas had the maximum average nucleotide diversity (0.1913), then intronic regions (0.0865) and the lowest values for CDS regions (0.0598). Ten highly polymorphic areas were found (Table 7), nine of which were IGS polymorphic sites, and one was a protein-coding site.

Fig. 12
figure 12

Nucleotide diversity in various areas of Aizoaceae cp genomes

Table 7 Ten Highly Polymorphic loci of Aizoaceae species


The organizational structure and evolution of chloroplast genomes are useful for offering a deeper understanding of the plant genome and phylogenetic studies. This study reported on the complete cp genome of S. sesuvioides and compared it to the genomes of four other Aizoaceae species: S. portulacastrum, M. crystallinum, M. cordifolium, and T. tetragonoides. The plastid genome of S. sesuvioides was generated using advanced sequencing technology and has a typical quadripartite composition, with notable similarities to the cp genomes of S. portulacastrum and other Aizoaceae species [1, 50].

Leucine was shown to be the most common amino acid and cysteine is an exceptionally rare amino acid. In plants, leucine is essential for ATP production, protein synthesis, chlorophyll fluorescence modulation, tissue regeneration, net photosynthesis rate and photochemical efficiency [51, 52]. The fact that cysteine is scarce in the cp genome does not imply that it is unimportant; in fact, cysteine appears to be crucial for the redox control of the chloroplast under particular illumination circumstances [53]. The codon use bias in the cp genomes has been identified as a critical evolutionary feature for mRNA translation, new gene recognition, and molecular research [54]. In addition, certain genes’ codon usage bias in plastoms is probably a reaction to external factors [55]. All codons do not contribute equally to amino acid coding; for example, AGA coding Arginine had the largest usage bias, whereas CGC coding Arginine had the lowest. Unequal codon distribution among particular amino acids in the genome demonstrates that nucleotide mutation is not at random and that there are mutations preferred and selection pressure, leading to synonymous codon use bias [56]. Previous studies on the nucleic acid composition of many angiosperm plants [56,57,58,59,60] found that codons ending in amino acids U(T) and A were the most common and had the greatest relative synonymous codon usage (RSCU); our findings confirmed this. The results presented here are compatible with prior cp genomic data, suggesting that the usage bias of certain codons was caused by adaptive evolution or the configuration bias of the high A/T intensity [21, 61,62,63].

SSRs and oligonucleotide repeats may be useful in phylogenetics and functional genomics [21, 57, 64]. SSRs were engaged in a variety of cp genome alterations, deletions, insertions, and substantial variants [65, 66]. The most prevalent SSR type was mononucleotides, which had two repeat patterns, A and T. Several higher plants showed comparable outcomes, revealing the prevalence of adenine and thymine repeats in cp genomes [21, 57, 61, 64,65,66]. Hexanucleotide SSRs, which are lacking in some other cp genomes [57, 61, 65, 67] were not found in the S. sesuvioides genome. Sequence repeats were more abundant in single-copy sections than in inverted repeats, supporting the notion that IRs are preserved. The IGS has the most repetitions of any section of the cp genome [21, 68,69,70]. As a result, we concluded that IGS areas are more vulnerable to alterations and recombination of genes than protein-coding regions [71]. The abundance of palindromic sequence repeats in the cp genome suggests the occurrence of several types of similar or equivalent sequences that are either continuous or separated by a spacer region [72, 73]. Several angiosperms have a corresponding figure of SSRs and oligonucleotide repeat transmission [21, 71, 74]. These findings show that sequence repeats change chloroplast genomes and are critical for recognizing species-specific genomic diversity [75].

The cp genome sequences are an important source of data for phylogenetic studies [21, 55, 57, 76]. The emergence of scale-up sequencing methods has increased access to cp genomes with massive amounts of genetic influence [55, 63, 77]. Previously, researchers used plastomes and nuclear genome data to conduct molecular phylogenetic studies for the order Caryophyllales [2, 78,79,80,81]. According to the results, the genera Sesuvium and Tetragonia are closely related. All of the other Aizoaceae family genera are thought to be quite comparable to Sesuvium, confirming the family’s monophyletic nature of the family [1, 9, 82]. This phylogenetic tree also revealed that all the sub-families of the Aizoaceae family are monophyletic. Several molecular investigations, however, have established the evolutionary position and monophyly of the Aizoaceae subfamilies, namely Tetragonioideae, Aizooideae, and Sesuvioideae [29, 30, 82]. The evolutionary tree also revealed close links between all Caryophyllales families, confirming the previously stated fact that Caryophyllales is a monophyletic group based on genomic evidence [2, 78, 82]. Additionally, more comprehensive plastome data are required for a deeper comprehension of the evolutionary relationships and phylogeny of Aizoaceae.

This study found that S. sesuvioides and the other four Aizoaceae cp genomes had equal gene content, gene organization, GC content, and a uniform trend of intron/intron existence in the genes. Several angiosperm lineages have been demonstrated to have comparable gene organization and content for the consistent structure of the cp genome [21, 48, 71, 83]. In the Aizoaceae species, the infA gene was determined to be functional, non-functional, or missing from the chloroplast genome. The chloroplast genomes of many other angiosperm species have undergone gene loss and pseudogenization during evolution [81, 84,85,86]. The rps15 gene and the rpl2 intron were absent in all Aizoaceae species. However, intron loss has been seen in several angiosperm species [21, 87, 88]. The rpoC2, atpF, rpl2, rps12, rps16, and clpP are examples of previously known protein-coding genes with intron deletion [82, 87,88,89,90]. Introns play an important function in gene expression regulation and can increase exogenous gene expression in plant areas to attain specific agronomic characteristics [72]. The absence of particular introns may result in alterations in gene function [72]. The genes rps12, ycf3, and clpP exhibited multiple introns in all Aizoaceae species. More than one intron for ycf3, rps12, and clp was discovered in earlier chloroplast genome investigations [58,59,60, 91]. Additional introns have been suggested to be helpful for investigations of photosynthetic evolution [58]. The GC content is an essential measure for establishing familial connections [92, 93]. The AT content in all Aizoaceae species cp genomes was higher than the GC percentage. Our data revealed that the proportion of GC in cpDNA was not spread evenly among chloroplast genomic regions. The IR area had a higher GC content than the other areas, owing to the high GC concentration identified in the four rRNAs in the inverted repeats [94].

The IRs junctions of S. sesuvioides were compared to those of four other Aizoaceae species. The presence of comparable genes at similar positions in all Aizoaceae species corroborated their tight association and this study’s phylogenetic findings. Our findings are also supported by the fact that the IRs are more conserved and the majority of modifications occurred in the SSC and LSC parts [21, 68,69,70]. Almost all of the genes in S. sesuvioides and chosen Aizoaceae species were subjected to purifying selection pressure, indicating that the protein-coding genes in this family are conserved in nature. This study’s findings complement previous research findings in a variety of different angiosperms that the rate of occurrence of synonymous substitutions in protein-coding genes in cp genomes is higher than that of nonsynonymous changes [55, 75, 95, 96]. In general, synonymous substitutions are allowed in cp genomes, while nonsynonymous substitutions are eliminated by purifying selection forces, resulting in protein-coding genes being more conserved than non-coding regions [97, 98].

Throughout the majority of species comparisons with S. sesuvioides, the number of inDels and SNPs was greatest in the LSC and lowest in the IRs. These findings support previous findings that inDels and substitutions are much more prevalent in single-copy regions than in inverted repeats [21, 99]. We calculated a Ts/Tv ratio greater than one, which is consistent with previous research findings that the highest ratio of transitions exists at the mutational level and requires much-reduced distortions of the DNA double-helix structure than transversions, implying that transitions happen more commonly in the replication of DNA [21, 100]. The transitions tend to be less disruptive than transversions because they do not produce substantial modifications in amino acid fundamental properties [100].

To determine nucleotide diversity, we investigated IGS regions, CDS regions, and intronic regions in S. sesuvioides and four other Aizoaceae species. The average nucleotide diversity was smallest in coding regions and highest in intergenic spacer regions. The fact that protein-coding genes have limited nucleotide variation adds validity to the hypothesis that Aizoaceae cp genomes are relatively conservative. Nucleotide diversity varies from 0.0024 to 0.4789; lower values indicate plastome architectural retention in Aizoaceae species, and a decreased rate of nucleotide diversity has also been seen in multiple other cp genomes [21, 101,102,103]. Ten highly polymorphic sites with the greatest nucleotide diversity were identified as suitable molecular markers. These highly polymorphic loci might be used to create valid and reliable DNA barcodes for the Sesuvium and Aizoaceae family. More research into the Sesuvium genus and Aizoaceae family is required to corroborate these markers.


The cp genome of S. sesuvioides is sequenced and analysed for the first time. The outcomes of this investigation will give detailed taxonomic information regarding the structure, genetic content, and evolutionary history of the Aizoaceae cp genomes. The comparison of S. sesuvioides to other Aizoaceae species adds to our understanding of genetic variation, substitutions, and the evolution of the family. The phylogenetic analyses of this work show significant support for the Caryophyllales order’s monophyly and the sister group connection amongst Aizoaceae subfamilies. The phylogenetic analysis will help to resolve and enhance the scant data on these family members. This study’s findings will also assist in identifying new species and creating novel molecular markers for Aizoaceae chloroplasts in plant genetic manipulation. More species must be sequenced to properly understand the Aizoaceae family’s evolutionary history.

Availability of data and materials

The datasets generated and/or analyzed during the current study are available in the NCBI repository, ACCESSION MW539047. Raw data submitted Sequence Read Archive (SRA) under the project number PRJNA660981. Further data is present in the manuscript. For more in-depth details, there is a supplementary file.


  1. Choi KS, Kwak M, Lee B, Park SJ. Complete chloroplast genome of Tetragonia tetragonioides: molecular phylogenetic relationships and evolution in Caryophyllales. PLoS One. 2018;13(6):1–11.

    Article  CAS  Google Scholar 

  2. Brockington SF, et al. Phylogeny of the Caryophyllales sensu lato: revisiting hypotheses on pollination biology and perianth differentiation in the core Caryophyllales. Int J Plant Sci. 2009;170(5):627–43.

    Article  Google Scholar 

  3. Walker JF, et al. From cacti to carnivores: improved phylotranscriptomic sampling and hierarchical homology inference provide further insight into the evolution of Caryophyllales. Am J Bot. 2018;105(3):446–62.

    Article  PubMed  Google Scholar 

  4. Klak C, Hanáček P, Bruyns PV. Disentangling the Aizooideae: new generic concepts and a new subfamily in Aizoaceae. Taxon. 2017;66(5):1147–70.

    Article  Google Scholar 

  5. Hernández-Ledesma P, et al. A taxonomic backbone for the global synthesis of species diversity in the angiosperm order Caryophyllales. Willdenowia. 2015;45(3):281–383.

    Article  Google Scholar 

  6. Bohley K, Schröder T, Kesselmeier J, Ludwig M, Kadereit G. C 4-like photosynthesis and the effects of leaf senescence on C 4-like physiology in Sesuvium sesuvioides (Aizoaceae). J Exp Bot. 2019;70(5):1567–80.

    Article  CAS  Google Scholar 

  7. El-Raouf HSA. Taxonomic significance of leaves in family Aizoaceae. Saudi J Biol Sci. 2021;28(1):512–22.

    Article  CAS  PubMed  Google Scholar 

  8. Klak C, Hanáček P, Bruyns PV. A recircumscription of Jacobsenia (Aizoaceae): re-instating Drosanthemopsis, with two new quartz-endemics from Namaqualand, South Africa and sinking Knersia. South African J Bot. 2018;116:67–81.

    Article  Google Scholar 

  9. Liede-Schumann S, Grimm GW, Nürk NM, Potts AJ, Meve U, Hartmann HEK. Phylogenetic relationships in the southern African genus Drosanthemum (Ruschioideae, Aizoaceae). PeerJ. 2020;3:1–29.

    Article  Google Scholar 

  10. Valente LM, Britton AW, Powell MP, Papadopulos AST, Burgoyne PM, Savolainen V. Correlates of hyperdiversity in southern African ice plants (Aizoaceae). Bot J Linn Soc. 2014;174(1):110–29.

    Article  PubMed  Google Scholar 

  11. G Bohley K, Winter PJD, Kadereit G. A Revision of Sesuvium (Aizoaceae , Sesuvioideae). Syst Bot. 2017;42(1):124–47.

    Article  Google Scholar 

  12. Sukhorukov AP, et al. Chorological and taxonomic notes on African plants, 2. Bot Lett. 2017;164(2):135–53.

    Article  Google Scholar 

  13. Sukhorukov AP, et al. Diagnostics, taxonomy, nomenclature and distribution of perennial Sesuvium (Aizoaceae) in Africa. PhytoKeys. 2018;92:45–88.

    Article  Google Scholar 

  14. Khan AA, Chaudhry MS, Aziz S. Natural resource diversity in Cholistan Desert (Pakistan) and possible conservational measures. J Pure Appl Sci. 2004;23(1):25–47.

    Google Scholar 

  15. Filfilan TASA, Mehmood SF. A cytological study of flowering plants from Saudi Arabia. Willdenowia. 2000;30:339–58.

    Article  Google Scholar 

  16. Ahmad M, Wariss S, Alam HM, Anjum K, Mukhtar S. Ethnobotanical studies of plant resources of Cholistan desert, Pakistan. Int J Sci Res. 2014;3(6):1782–8.

    Google Scholar 

  17. Rehman F, et al. Ethnobotanical survey; common medicinal plants used by people of Cholistan Desert. Prof Med J. 2015;20(10):1367–72.

    Article  Google Scholar 

  18. Javed F, Jabeen Q, Aslam N, Mehmood A. Pharmacological evaluation of analgesic, anti-inflammatory and antipyretic activities of ethanolic extract of Indigofera argentea Burm. f. J Ethnopharmacol. 2020;259:1–8.

    Article  CAS  Google Scholar 

  19. Sajid-ur-Rehman M, et al. Phytochemical profiling, in vitro and in vivo anti-inflammatory, analgesic and antipyretic potential of Sesuvium sesuvioides (Fenzl) Verdc. (Aizoaceae). Inflammopharmacology. 2021;29(3):789–800.

    Article  CAS  PubMed  Google Scholar 

  20. Freudenthal JA, Pfaff S, Terhoeven N, Korte A, Ankenbrand MJ, Förster F. A systematic comparison of chloroplast genome assembly tools. Genome Biol. 2020;21(1):1–21.

    Article  CAS  Google Scholar 

  21. Javaid N, Ramzan M, Khan IA, Alahmadi TA, Datta R. The chloroplast genome of Farsetia hamiltonii Royle, phylogenetic analysis, and comparative study with other members of clade C of Brassicaceae. BMC Plant Biol. 2022;22(384):1–19.

    Article  CAS  Google Scholar 

  22. Wicke S, Schneeweiss GM, dePamphilis CW, Müller KF, Quandt D. The evolution of the plastid chromosome in land plants: gene content, gene order, gene function. Plant Mol Biol. 2011;76(3–5):273–97.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Shinozaki K, et al. The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression. EMBO J. 1986;5(9):2043–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Duchene D, Bromham L. Rates of molecular evolution and diversification in plants: chloroplast substitution rates correlate with species-richness in the Proteaceae. BMC Evol Biol. 2013;13(1)

  25. Smith DR. Mutation rates in plastid genomes: they are lower than you might think. Genome Biol Evol. 2015;7(5):1227–34.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Walker JF, Zanis MJ, Emery NC. Comparative analysis of complete chloroplast genome sequence and inversion variation in Lasthenia burkei (Madieae, Asteraceae). Am J Bot. 2014;101(4):722–9.

    Article  PubMed  Google Scholar 

  27. Androsiuk P, et al. Evolutionary dynamics of the chloroplast genome sequences of six Colobanthus species. Sci Rep. 2020;10(1):1–14.

    Article  CAS  Google Scholar 

  28. Wambugu PW, Brozynska M, Furtado A, Waters DL, Henry RJ. Relationships of wild and domesticated Rices (Oryza AA genome species) based upon whole chloroplast genome sequences. Sci Rep. 2015;5:1–9.

    Article  Google Scholar 

  29. Hassan NS, Thiede J, Liede-Schumann S. Phylogenetic analysis of Sesuvioideae (Aizoaceae) inferred from nrDNA internal transcribed spacer (ITS) sequences and morphological data. Plant Syst Evol. 2005;255:121–43.

    Article  CAS  Google Scholar 

  30. Powell RF, Magee AR, Forest F, Cowan RS, Boatwright JS. A phylogeographic study of the stoneplant Conophytum (Aizoaceae; Ruschioideae; Ruschieae) in the Bushmanland inselberg region (South Africa) suggests anemochory. Syst Biodivers. 2019;17(2):110–23.

    Article  Google Scholar 

  31. Xia Y, et al. A modified SDS-based DNA extraction method from raw soybean. Biosci Rep. 2019;39(2):1–10.

    Article  Google Scholar 

  32. S. Andrews, “FastQC: A Quality Control Tool for High Throughput Sequence Data.” 2010. [Online]. Available:

  33. Dierckxsens N, Mardulyn P, Smits G. NOVOPlasty: De novo assembly of organelle genomes from whole genome data. Nucleic Acids Res. 2017;45(4):1–9.

    Article  CAS  Google Scholar 

  34. Tillich M, et al. GeSeq - versatile and accurate annotation of organelle genomes. Nucleic Acids Res. 2017;45(W1):W6–W11.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Shi L, et al. CPGAVAS2, an integrated plastome sequence annotator and analyzer. Nucleic Acids Res. 2019;47(W1):W65–73.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Katoh K, Kuma KI, Toh H, Miyata T. MAFFT version 5: improvement in accuracy of multiple sequence alignment. Nucleic Acids Res. 2005;33(2):511–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Kearse M, et al. Geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012;28(12):1647–9.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Schattner P, Brooks AN, Lowe TM. The tRNAscan-SE, snoscan and snoGPS web servers for the detection of tRNAs and snoRNAs. Nucleic Acids Res. 2005;33(2):686–9.

    Article  CAS  Google Scholar 

  39. Li H, Durbin R. Fast and accurate long-read alignment with burrows-wheeler transform. Bioinformatics. 2010;26(5):589–95.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Milne I, et al. Tablet-next generation sequence assembly visualization. Bioinformatics. 2009;26(3):401–2.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013;30(12):2725–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. 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(3):411–22.

    Article  CAS  PubMed  Google Scholar 

  43. Kurtz S, Choudhuri JV, Ohlebusch E, Schleiermacher C, Stoye J, Giegerich R. REPuter: the manifold applications of repeat analysis on a genomic scale. Nucleic Acids Res. 2001;29(22):4633–42.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Kalyaanamoorthy S, Minh BQ, Wong TKF, Von Haeseler A, Jermiin LS. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat Methods. 2017;14(6):587–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Minh BQ, et al. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Mol Biol Evol. 2020;37(5):1530–4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Hoang DT, Chernomor O, Von Haeseler A, Minh BQ, Vinh LS. UFBoot2: improving the ultrafast bootstrap approximation. Mol Biol Evol. 2018;35(2):518–22.

    Article  CAS  PubMed  Google Scholar 

  47. Letunic I, Bork P. Interactive tree of life (iTOL) v4: recent updates and new developments. Nucleic Acids Res. 2019;47(256–259)

  48. Amiryousefi A, Hyvönen J, Poczai P. The chloroplast genome sequence of bittersweet (Solanum dulcamara): plastid genome structure evolution in Solanaceae. PLoS One. 2018;13(4):1–23.

    Article  CAS  Google Scholar 

  49. Rozas J, et al. DnaSP 6: DNA sequence polymorphism analysis of large data sets. Mol Biol Evol. 2017;34(12):3299–302.

    Article  CAS  PubMed  Google Scholar 

  50. Xu H. The complete chloroplast genome of newly alien medicinal and toxic species, Zaleya pentandra (L.) C. Jeffrey (Aizoaceae), in China. Mitochondrial DNA Part B. 2019;4(2):3412–3.

    Article  PubMed  PubMed Central  Google Scholar 

  51. Pedroso JAB, Zampieri TT, Donato J. Reviewing the effects of l-leucine supplementation in the regulation of food intake, energy balance, and glucose homeostasis. Nutrients. 2015;7(5):3914–37.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Sun M, Li S, Gong Q, Xiao Y, Peng F. Leucine contributes to copper stress tolerance in peach (Prunus persica) seedlings by enhancing photosynthesis and the antioxidant defense system. Antioxidants. 2022;11(12)

  53. Gotor C, Álvarez C, Bermúdez MÁ, Moreno I, García I, Romero LC. Low abundance does not mean less importance in cysteine metabolism. Plant Signal Behav. 2010;5(8):1028–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Song W, et al. Comparative chloroplast genome analysis of wax gourd (Benincasa hispida) with three Benincaseae species, revealing evolutionary dynamic patterns and phylogenetic implications. Genes (Basel). 2022;13(3)

  55. B. Zhu, F. Qian, Y. Hou, W. Yang, M. Cai, and X. Wu, “Complete chloroplast genome features and phylogenetic analysis of Eruca sativa (Brassicaceae),” PLoS One, vol. 16, no. 3 March, pp. 1–19, 2021, doi:

  56. Zuo LH, et al. The first complete chloroplast genome sequences of Ulmus species by de novo sequencing: genome comparative and taxonomic position analysis. PLoS One. 2017;12(2):1–19.

    Article  CAS  Google Scholar 

  57. Zarei A, Ebrahimi A, Mathur S, Lawson S. The first complete chloroplast genome sequence and phylogenetic analysis of pistachio (Pistacia vera). Diversity. 2022;14(7):1–16.

    Article  CAS  Google Scholar 

  58. Guo S, et al. Complete chloroplast genome sequence and phylogenetic analysis of Paeonia ostii. Molecules. 2018;23(2):1–14.

    Article  CAS  Google Scholar 

  59. Li DM, Zhao CY, Liu XF. Complete chloroplast genome sequences of Kaempferia galanga and Kaempferia elegans: molecular structures and comparative analysis. Molecules. 2019;24(3)

  60. Zhou T, et al. The complete chloroplast genome of Euphrasia regelii, Pseudogenization of ndh genes and the phylogenetic relationships within Orobanchaceae. Front Genet. 2019;10:1–15.

    Article  CAS  Google Scholar 

  61. Yan C, Du J, Gao L, Li Y, Hou X. The complete chloroplast genome sequence of watercress (Nasturtium officinale R. Br.): genome organization, adaptive evolution and phylogenetic relationships in Cardamineae. Gene. 2019;699:24–36.

    Article  CAS  PubMed  Google Scholar 

  62. Wang Z, et al. Comparative analysis of codon usage patterns in chloroplast genomes of six Euphorbiaceae species. PeerJ. 2020;2020(1):1–17.

    Article  Google Scholar 

  63. Saina JK, Gichira AW, Li ZZ, Hu GW, Wang QF, Liao K. The complete chloroplast genome sequence of Dodonaea viscosa: comparative and phylogenetic analyses. Genetica. 2018;146(1):101–13.

    Article  CAS  PubMed  Google Scholar 

  64. Shen X, et al. Complete chloroplast genome sequence and phylogenetic analysis of Aster tataricus. Molecules. 2018;23(10):1–14.

    Article  CAS  Google Scholar 

  65. Bi Y, Zhang MF, Xue J, Dong R, Du YP, Zhang XH. Chloroplast genomic resources for phylogeny and DNA barcoding: a case study on Fritillaria. Sci Rep. 2018;8(1):1–12.

    Article  CAS  Google Scholar 

  66. Keller J, et al. The evolutionary fate of the chloroplast and nuclear rps16 genes as revealed through the sequencing and comparative analyses of four novel legume chloroplast genomes from Lupinus. DNA Res. 2017;24(4):343–58.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Hu ZY, Hua W, Huang SM, Wang HZ. Complete chloroplast genome sequence of rapeseed (Brassica napus L.) and its evolutionary implications. Genet Resour Crop Evol. 2011;58(6):875–87.

    Article  CAS  Google Scholar 

  68. Q. jie Li et al., “Chloroplast genomes elucidate diversity, phylogeny, and taxonomy of Pulsatilla (Ranunculaceae),” Sci Rep, vol. 10, no. 1, pp. 1–12, 2020, doi:

  69. Cao J, et al. Development of chloroplast genomic resources in Chinese yam (Dioscorea polystachya). Biomed Res Int. 2018:1–11.

  70. Liu L, et al. Chloroplast genome analyses and genomic resource development for epilithic sister genera Oresitrophe and Mukdenia (Saxifragaceae), using genome skimming data. BMC Genomics. 2018;19(1):1–17.

    Article  CAS  Google Scholar 

  71. Menezes APA, et al. Chloroplast genomes of Byrsonima species (Malpighiaceae): comparative analysis and screening of high divergence sequences. Sci Rep. 2018;8(1):1–12.

    Article  CAS  Google Scholar 

  72. Liang C, et al. A comparative analysis of the chloroplast genomes of four Salvia medicinal plants. Engineering. 2019;5(5):907–15.

    Article  CAS  Google Scholar 

  73. Alzahrani D, Albokhari E, Yaradua S, Abba A. Complete chloroplast genome sequences of Dipterygium glaucum and Cleome chrysantha and other Cleomaceae species, comparative analysis and phylogenetic relationships. Saudi J Biol Sci. 2021;28(4):2476–90.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Saina JK, Li ZZ, Gichira AW, Liao YY. The complete chloroplast genome sequence of tree of heaven (Ailanthus altissima (mill.)) (sapindales: Simaroubaceae), an important pantropical tree. Int J Mol Sci. 2018;19(4)

  75. Du X, et al. The complete chloroplast genome sequence of Yellow Mustard (Sinapis alba L.) and its phylogenetic relationship to other Brassicaceae species. Gene. 2020;731(2019):144340.

    Article  CAS  PubMed  Google Scholar 

  76. Yu X, Tan W, Zhang H, Gao H, Wang W, Tian X. Complete chloroplast genomes of Ampelopsis humulifolia and Ampelopsis japonica: molecular structure, comparative analysis, and phylogenetic analysis. Plants. 2019;8(10):1–15.

    Article  CAS  Google Scholar 

  77. Saarela JM, et al. A 250 plastome phylogeny of the grass family (Poaceae): topological support under different data partitions. PeerJ. 2018;2018(2):1–71.

    Article  CAS  Google Scholar 

  78. Cuénoud P, Savolainen V, Chatrou LW, Powell M, Grayer RJ, Chase MW. Molecular phylogenetics of Caryophyllales based on nuclear 18S rDNA and plastid rbcL, atpB, and matK DNA sequences. Am J Bot. 2002;89(1):132–44.

    Article  PubMed  Google Scholar 

  79. Ruhlman T, et al. Complete plastid genome sequence of Daucus carota: implications for biotechnology and phylogeny of angiosperms. BMC Genomics. 2006;7:222.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Wang X, Zhou T, Bai G, Zhao Y. Complete chloroplast genome sequence of Fagopyrum dibotrys: genome features, comparative analysis and phylogenetic relationships. Sci Rep. 2018;8(1):1–12.

    Article  CAS  Google Scholar 

  81. Scobeyeva VA, et al. Gene loss, pseudogenization in plastomes of genus Allium (Amaryllidaceae), and putative selection for adaptation to environmental conditions. Front Genet. 2021;12(July)

  82. Downie SR, Palmer JD. A chloroplast DNA phylogeny of the Caryophyllales based on structural and inverted repeat restriction site variation. Syst Bot. 1994;19(2):236–52.

    Article  Google Scholar 

  83. Li W, et al. Interspecific chloroplast genome sequence diversity and genomic resources in Diospyros. BMC Plant Biol. 2018;210(18):1–11.

    Article  CAS  Google Scholar 

  84. Li Y, et al. Gene losses and partial deletion of small single-copy regions of the chloroplast genomes of two hemiparasitic Taxillus species. Sci Rep. 2017;7(1):1–12.

    Article  CAS  Google Scholar 

  85. Frailey DC, Chaluvadi SR, Vaughn JN, Coatney CG, Bennetzen JL. Gene loss and genome rearrangement in the plastids of five Hemiparasites in the family Orobanchaceae. BMC Plant Biol. 2018;18(1):1–12.

    Article  CAS  Google Scholar 

  86. Millen RS, et al. Many parallel losses of infA from chloroplast DNA during angiosperm evolution with multiple independent transfers to the nucleus. Plant Cell. 2001;13(3):645–58.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Sloan DB, Triant DA, Forrester NJ, Bergner LM, Wu M, Taylor DR. A recurring syndrome of accelerated plastid genome evolution in the angiosperm tribe Sileneae (Caryophyllaceae). Mol Phylogenet Evol. 2014;72(1):82–9.

    Article  CAS  PubMed  Google Scholar 

  88. Jansen RK, Wojciechowski MF, Sanniyasi E, Lee SB, Daniell H. Complete plastid genome sequence of the chickpea (Cicer arietinum) and the phylogenetic distribution of rps12 and clpP intron losses among legumes (Leguminosae). Mol Phylogenet Evol. 2008;48(3):1204–17.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. He P, Huang S, Xiao G, Zhang Y, Yu J. Abundant RNA editing sites of chloroplast protein-coding genes in Ginkgo biloba and an evolutionary pattern analysis. BMC Plant Biol. 2016;16(1):1–12.

    Article  CAS  Google Scholar 

  90. Jansen RK, et al. Analysis of 81 genes from 64 plastid genomes resolves relationships in angiosperms and identifies genome-scale evolutionary patterns. Proc Natl Acad Sci U S A. 2007;104(49):19369–74.

    Article  PubMed  PubMed Central  Google Scholar 

  91. Song Y, Chen Y, Lv J, Xu J, Zhu S, Li M. Comparative chloroplast genomes of Sorghum species: sequence divergence and phylogenetic relationships. Biomed Res Int. 2019;2019

  92. Asaf S, et al. Chloroplast genomes of Arabidopsis halleri ssp. gemmifera and Arabidopsis lyrata ssp. petraea: structures and comparative analysis. Sci Rep. 2017;7(1):1–15.

    Article  CAS  Google Scholar 

  93. Xu C, et al. Comparative analysis of six Lagerstroemia complete chloroplast genomes. Front Plant Sci. 2017;8:1–12.

    Article  Google Scholar 

  94. Zhang Z, et al. Characterization of the complete chloroplast genome of Brassica oleracea var. italica and phylogenetic relationships in Brassicaceae. PLoS One. 2022;17:1–18.

    Article  CAS  Google Scholar 

  95. Odago WO, et al. Analysis of the complete Plastomes of 31 species of Hoya group: insights into their comparative genomics and phylogenetic relationships. Front Plant Sci. 2022;12:1–12.

    Article  Google Scholar 

  96. Huang CH, et al. Resolution of Brassicaceae phylogeny using nuclear genes uncovers nested radiations and supports convergent morphological evolution. Mol Biol Evol. 2016;33(2):394–412.

    Article  CAS  PubMed  Google Scholar 

  97. Lawrie DS, Messer PW, Hershberg R, Petrov DA. Strong purifying selection at synonymous sites in D. Melanogaster. PLoS Genet. 2013;9(5):33–40.

    Article  CAS  Google Scholar 

  98. Firetti F, Zuntini AR, Gaiarsa JW, Oliveira RS, Lohmann LG. Complete chloroplast genome sequences contribute to plant species delimitation : A case study of the Anemopaegma species complex 1. 2017;104(10):1493–509.

  99. Kim HT, Kim KJ. Chloroplast genome differences between Asian and American Equisetum arvense (Equisetaceae) and the origin of the hypervariable trnY-trnE intergenic spacer. PLoS One. 2014;9(8):e103898.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Zou Z, Zhang J. Are nonsynonymous transversions generally more deleterious than nonsynonymous transitions? Mol Biol Evol. 2021;38(1):181–91.

    Article  CAS  PubMed  Google Scholar 

  101. Odintsova MS, Yurina NP. Plastid genomes of higher plants and algae: structure and functions. Mol Biol. 2003;37(5):649–62.

    Article  CAS  Google Scholar 

  102. Cai J, Ma PF, Li HT, Li DZ. Complete plastid genome sequencing of four Tilia species (Malvaceae): a comparative analysis and phylogenetic implications. PLoS One. 2015;10(11):1–13.

    Article  CAS  Google Scholar 

  103. Smith DR, Keeling PJ. Mitochondrial and plastid genome architecture: reoccurring themes, but significant differences at the extremes. Proc Natl Acad Sci U S A. 2015;112(33):10177–84.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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The authors acknowledge the contribution of the Beijing Institute of Genomics (BIG), China in sequencing the chloroplast genome of Sesuvium sesuvioides.

Research and Development (R&D) Institute, Alpha Genomics Private Limited, Islamabad 45720, Pakistan is acknowledged for providing training for Bio-informatics analysis.

The authors extend their appreciation to the Researchers Supporting Project number (RSPD2023R677), King Saud University, Riyadh, Saudi Arabia for financial support.


The authors extend their appreciation to the Researchers Supporting Project number (RSPD2023R677), King Saud University, Riyadh, Saudi Arabia for financial support.

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Conceptualization = Nida Javaid, Musarrat Ramzan, Subhan Danish. Methodology = Nida Javaid, Musarrat Ramzan, Shagufta Jabeen, Subhan Danish. Writing original draft preparation = Nida Javaid, Musarrat Ramzan, Shagufta Jabeen. Data collection and analysis, Nida Javaid, Subhan Danish. Supervision = Musarrat Ramzan. Revise draft preparation = M.N.S, A.H.H,Data validation = M.N.S, A.H.H, All authors have read and agreed to the published version of the manuscript.

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Correspondence to Musarrat Ramzan or Subhan Danish.

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We all declare that manuscript reporting studies do not involve any human participants, human data, or human tissue. So, it is not applicable.

This study complies with relevant institutional, national, and international guidelines. Plant voucher number AizoaceaeSesuvium sesuvioides (Fenzl) VerdcBari Ulwati is CIDS/ IUB-0601/84.

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Not applicable (NA).

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The authors declare no competing interests.

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Tables S1-S8.

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Javaid, N., Ramzan, M., Jabeen, S. et al. Genomic exploration of Sesuvium sesuvioides: comparative study and phylogenetic analysis within the order Caryophyllales from Cholistan desert, Pakistan. BMC Plant Biol 23, 658 (2023).

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