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

Background 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. Results 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. Conclusion 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. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-023-04670-5.


Introduction
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 hyperaccumulation, 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 subfamilies, 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].
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 Fig. 1 The Sesuvium sesuvioides (Barri Ulwaiti) growing in the Cholistan desert 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.

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 Gen-Bank 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 pairwise 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: https:// www.ncbi.nlm.nih.gov/ nucco re/ MW539 047.1.

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).

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.

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.

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

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   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.

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 Fig. 10 The IRSCOPE analysis of the IRs of the selected five cp genomes Fig. 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 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).

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).

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.

Discussion
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 proteincoding 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.

Conclusion
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.

Fig. 2 Fig. 3
Fig.2The 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

Fig. 4 Fig. 5
Fig. 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

Fig. 6
Fig.6The 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

Fig.
Fig. Multiple alignment petD gene of five species of Aizoaceae family

Fig. 12
Fig. 12 Nucleotide diversity in various areas of Aizoaceae cp genomes

Table 1
The full S. sesuvioides cp genome's comprehensive properties

Table 2
Genes containing introns and their length in S. sesuvioides

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

Table 4
The findings of a cp genome comparison among five Aizoaceae species are shown

Table 5
Transition and Transversion substitutions, their ratio in LSC, IRs, and SSC

Table 6
The detailed InDels analysis for five Aizoaceae cp genomes No. Of inDels, Average InDel Length, InDel Diversity K(i), InDel Diversity per site Pi(i), and alignment length by performing pairwise alignment with four different Aizoaceae species (S. portulacastrum, M. crystallinum, M. cordifolium, and T. tetragonoides)

Table 7
Ten Highly Polymorphic loci of Aizoaceae species