Complete chloroplast genomes suggest a single origin of the Indian subcontinent Sonerila (Melastomataceae)

Complete chloroplast genomes suggest a single origin of the Indian subcontinent Sonerila (Melastomataceae) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Complete chloroplast genomes suggest a single origin of the Indian subcontinent Sonerila (Melastomataceae) Jiahong Han, Monique Romeiro-Brito, Resmi Sekarathil, Santhosh Nampy, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7538746/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Sonerila , the second largest genus within the tribe Sonerileae (Melastomaceae), is primarily distributed across tropical and subtropical Asia, from India and Sri Lanka, where it exhibits a remarkably high degree of endemism, to Papua New Guinea. In this study, we assembled and annotated the chloroplast genomes of 15 Sonerila species from India (14 from the subcontinent and one from Sikkim in the Eastern Himalaya) and compared them to 12 previously published genomes representing species from other regions. We performed comparative analyses of gene structure, sequence alignment, nucleotide diversity and phylogenetic reconstructions to evaluate the utility of plastid regions for resolving phylogenetic relationships and understanding plastome evolution within Sonerila and close relatives. Results Our results demonstrate that the complete chloroplast genome exhibit quadripartite structures, with lengths ranging from 154,014 ( S. nervulosa ) to 155,271 bp ( S. annamica ). The genome structure remains relatively conserved, hosting 129 annotated genes, including 84 protein coding genes, 37 tRNA genes, and 8 rRNA genes. The overall GC contents were almost identical (37.1–37.4%), with an average of 37.2%. 51 to 84 SSRs and long repeat sequences were detected. A comparison of nucleotide diversity among all 27 Sonerila chloroplast genomes revealed seven regions ( ycf1 , rps15 , psbE-petL , trnS-GCU-trnR-UCU, rps16-trnQ-UUG, rbcL-accD, rps18-rpl20 ) displayed relatively high nucleotide diversity (Pi > 0.025). In contrast, the chloroplast genomes from Indian Sonerila species showed lower diversity, with only four regions ( ycf1, rps16, rpl32, rpl22 ) exhibiting notable Pi value (> 0.014). Maximum likelihood phylogenetic analysis strongly confirmed the monophyly of Sonerila within the Sonerileae and showed that the structure of the main clades correlated to species geographic distribution, including a single origin for Sonerila in the Indian subcontinent. Conclusion The chloroplast genomes of Sonerila exhibits conserved structural features and gene content and variations were observed in genome size, sequence divergence, and repeat elements. Overall, long repetitive sequences, simple sequence repeats (SSRs), and regions with high variability are phylogenetically informative. Phylogenetic analyses suggest that Sonerila is a monophyletic entity and the Indian subcontinent species originated from a single ancestor. By providing a comprehensive genomic characterization of Sonerila chloroplast, this study delivers essential genetic insights for advancing future research on the evolutionary history and adaptive diversification of Sonerila species. Chloroplast genome Genome structure Indian Sonerila Sonerileae Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Melastomataceae are one of the most species-rich flowering plant families and a major model system for evolutionary studies [ 2 ], with approximately 5857 species representing important ecological components of tropical habitats worldwide [ 1 ]. Sonerileae is the second largest tribe within the family, comprising approximately 1,080 species across 46 genera [ 1 ], with its greatest diversity found in Asia and Oceania [ 3 , 4 , 5 , 6 ]. Members of Sonerileae exhibit remarkable morphological diversity, including herbs, shrubs, small trees, and, in some cases, climbers and epiphytes. Floral characteristics include 3- to 6-merous flowers, isomorphic or dimorphic stamens with various connective appendages including the total loss of these structures, and fruits that are either berries or capsules [ 3 ]. The intergeneric boundaries within Sonerileae have long been problematic, due to extensive morphological convergence [ 3 , 7 , 8 ]. For example, Sonerila and Tashiroea are both small herbs with 3-merous flowers, but molecular studies reveal that they are not closely related [ 9 ]. Similarly, Phyllagathis , Bredia and Scorpiothyrsus were historically grouped together due to similar floral structures, but phylogenetic analyses have shown that these genera belong to distinct evolutionary lineages [ 10 , 11 ]. Moreover, switches in flower merosity (from 3 to 4- to 5-merous), even within species, are not uncommon [ 8 , 12 ]. Recent investigations with expanded sampling across a broader range of Sonerileae species, have improved the genetic delimitation and the identification of several major Asian lineages [ 13 , 14 ]. Advances in high-throughput sequencing technologies have significantly enhanced phylogenetic analyses within Sonerileae, especially by enabling the use of chloroplast genome sequences [ 13 ]. Nonetheless, many challenges persist, particularly regarding species delimitation and infrageneric relationships, reflecting the group’s inherent genetic complexity. Therefore, further research incorporating broader molecular data and advanced phylogenetic approaches is necessary, given the limited phylogenetic resolution currently available for poorly sampled genera. Sonerila is the second largest genus within Sonerileae, having remarkable diversity with approximately 180 species distributed from India and Sri Lanka to Papua New Guinea, across the entire Indomalayan region [ 15 , 16 ]. Regardless of its wide distribution range, this group represents one of the least known entities in paleotropical Melastomataceae. Sonerila typically inhabits moist, shaded environments such as rainforests and montane forest, often occurring along stream banks within these diverse habitats. They are acaulescent and caulescent herbs, often with basal rosettes, with white or brightly colored 3-merous flowers, and a diverse array of capsular fruits. In India, Sonerila exhibit high morphological diversity, with approximately 50 species, over 80% of which are endemics [ 17 , 18 ]. The high level of endemism highlights the group’s significant diversification within the Indian subcontinent. Recent phylogenomic analyses have provided some evidence suggesting that Sonerila may represent a monophyletic entity [ 13 , 14 ]. However, Sonerila species morphological diversity and geographic origin have been largely underrepresented in all phylogenetic studies [ 14 , 48 ]. High-throughput sequencing technology has revolutionized plant systematics, making chloroplast genome sequencing a powerful and increasingly utilized tool. Chloroplast, the photosynthetic organelles in green plants and vital site for molecule synthesis [ 19 ], has a relatively independent and highly conserved structure compared to the nuclear genome [ 20 , 21 , 22 ]. The chloroplast genome of Tigridiopalma magnifica was the first to be reported within Sonerileae, presenting 155,663 bp in length, 130 genes and a GC content of 37.11% [ 23 ]. Similarly, the plastomes of Blastus auriculatus and Blastus cochinchinensis were found to include 155,981bp with 125 genes and GC 37.0% [ 24 ] and 156,005 bp with 129 genes and GC 37.0% [ 25 ], respectively. A tribe-wide phylogenomic study involving 151 complete chloroplast genomes reveled genome size ranging from 153,219 to 158,960 bp, with all plastomes encoding a conserved complement of 129 genes, including 84 protein-coding genes, 37 tRNAs, 8 rRNAs [ 13 , 14 , 48 ]. Despite these advances, plastome sampling across Sonerileae remains sparse and uneven, with most genera represented by only a few species. A comprehensive comparative analysis of chloroplast genome structure within the tribe, especially at the genus level, is still lacking. Focused studies on Sonerila , one of the most taxonomically complex and species-rich genera, are critical for uncovering lineage-specific structural variations, identifying robust molecular markers, and refining phylogenetic relationships across Sonerileae. In an effort to further our insight into the genetic diversity and genomic characteristics of Sonerila , we assembled and annotated the plastome sequences of 15 Sonerila species from India, which represents the westernmost edge of its distribution range. For broader context and comparison, we included 12 publicly available plastomes of Sonerila species from other regions. The objectives of this study are: (1) compare the genome structure and sequence variation of 27 chloroplast genomes in morphologically diverse Sonerila species from different regions across its distribution range; (2) identify simple sequence repeats (SSRs), large repetitive elements, and highly variable regions as potential molecular markers for Sonerila species identification and phylogenetic analysis; and (3) construct a phylogenetic tree using complete chloroplast genome sequences from 72 Sonerileae species to test the utility of the chloroplast and the putative monophyly of Indian Sonerila . Materials and Methods Taxon sampling, DNA extraction, and sequencing We selected 15 species of Sonerila from India and extracted the DNA from fresh leaves stored in silica gel using the modified CTAB protocol [ 26 , 27 , 28 ]. The plant material was identified by Dr. Resmi Sekarathil and Dr. Santhosh Nampy. Voucher specimens of all 15 species were deposited at the University of Calicut Herbarium (CALI and FRC), and the corresponding voucher accession numbers are provided in Table S1 . The quality, integrity, and concentration of DNA were determined by agarose gel electrophoresis and Qubit fluorescence photometer. A complete list of the sampled taxa and their voucher information is provided in supplementary information (Table S1 ). The genome skimming libraries for the newly generated data for Sonerila species were built and sequenced by Rapid Genomics LLC, using the Illumina NovaSeq platform. Chloroplast genome assembly and annotation We filtered the raw sequencing data using fastp [ 29 ] removing low quality base pairs (Phred values < 15) and short sequence length (< 30 bp). Chloroplast genome assembly was performed using GetOrganelle (v 1.7.7.0) [ 30 ]. We used GetOrganelle to first filter plastid-like reads, conduct the de novo assembly, purify the assembly, and finally to generate the complete chloroplast genomes. The complete chloroplast genome sequence of S. annamica (OL813695.1) was subsequently used as a reference and annotated and manually corrected using Geneious (v 11.0.24). For the annotation using Geneious, a minimum of 70% identity cutoff between the genomes was considered. It was used to predict the chloroplast genome for coding proteins, tRNA, and rRNA genes, and then the predicted initial genes were made de-redundant and the first and last genes and exon/intron boundaries were manually corrected to obtain a highly accurate genome. Physical mapping of the chloroplast genomes was performed using OGDRAW ( https://chlorobox.mpimpgolm.mpg.de/OGDraw.html ) software. To reduce potential bias from reference genomes and annotation software, the plastome of twelve Sonerila species obtained from NCBI were reannotated utilizing Geneious, with Sonerila annamica taken as the reference. Codon usage indices To investigate codon usage patterns and nucleotide composition in the twenty-seven Sonerila chloroplast genomes, amino acid frequency, codon usage number, and the relative synonymous codon usage (RSCU) were analyzed and summarized. The RSCU value was used to measure the association between the observed frequency and the anticipated frequency of a particular codon. The RSCU and amino acid frequency in the chloroplast genome was compared using CodonW v1.42 software ( http://codonw.sourceforge.net ). SSRs and repeat sequence analysis SSRs in the chloroplast genomes of 27 Sonerila species were analyzed using the MISA software [ 31 ], with the parameters set as follows: 10 for mono-, 5 for di-, 4 for tri-, and 3 for tetra-, penta-, and hexanucleotides. The Vmatch tool [ 32 ] was used to identify repeats: forward, reverse, palindrome, and complement repeats. The following settings for repeat identification were used: (1) Hamming distance equal to 3; and (2) minimal repeat size set to 30 bp. Tandem repeats were detected using the Tandem Repeats Finder ( http://tandem.bi.edu/trf/ ), an online software. Tandem repeats observed in at least one copy were considered significant. The parameters were set as follows: alignment parameters of matches = 2, mismatches = 3, indels = 5, minimum alignment score = 50, maximum period size = 500, and maximum tandem repeat size = 2. Comparative analysis of chloroplast genomes In this study, Geneious (v11.0.24) software [ 33 ] was used to determine the lengths of the IRa/IRb, LSC, and SSC regions, as well as the boundary genes, in the chloroplast genome of the Sonerila species. The mVISTA program in the shuffle-lagan model [ 34 ] was used to compare the chloroplast genome sequences using S. annamica (OL813695) as a reference. The DnaSP6 software [ 35 ] was used to calculate the Pi values of the LSC, SSC, and IR regions between all the Sonerila species, and to identify divergence hotspot regions within the genome for evolutionary analysis. The step size was set to 200 bp and the window length to 600 bp. The complete chloroplast genome sequences were aligned using MAFFT v 7.49 [ 36 ]. The IRscope software ( https://irscope.shinyapps.io/irapp/ ) [ 37 ] was used to draw an IR boundary map and compare IR boundary characteristics. To visualize and compare the IR boundaries, Adobe illustrator software was employed for creating comparison maps. Phylogenetic analyses Phylogenetic analyses were performed using the 76 complete chloroplast genome sequences form Sonerileae species, the 15 Sonerila species examined in this study and 61 species that were selected from the NCBI database. The complete chloroplast genome sequences including the outgroup were compared into a single file and aligned using MAFFT v 7.49 [ 36 ]. The 76 Sonerileae species in 25 clades were selected, including two species from Dissochaeteae, one species from Pyxidantheae and one species from Melastomateae as outgroup taxa (Table S1 ). Maximum likelihood (ML) analyses were performed using raxmlGUI v 2.0.10 with 1000 bootstrap replicates and the GTR + I + G model [ 38 ], and the best substitution model was determined by the Akaike information criterion (AIC) in Modeltest-ng [ 39 ]. Results Chloroplast genome structure of Sonerila species The chloroplast genomes in Sonerila species ranged from 154,014 bp ( S. nervulosa ) to 155,271 bp ( S. annamica ), displaying a difference of 1,257 bp across different genomes (Table 1 ). All 27 chloroplast genomes displayed the typical quadripartite structure, consisting of a large single copy (LSC) region (84,234–85,272 bp), a small single copy (SSC) region (16,358–16,558 bp), and a pair of inverted repeat (IR) regions (26.638–26,817 bp) (Fig. 1 ). The total GC content was consistent across all plastomes, ranging from 37.1–37.4%, with the average GC content being 37.2%, while the different regions had slightly variable GC content with the LSC, SSC, and IR ranging from 34.9–35.3%, 30.9–31.3%, and 42.6%, respectively (Table 1 ). Our analysis revealed that the chloroplast genomes of Sonerila species appear to be relatively conserved, and all 27 genomes contained 129 unique genes comprising 84 protein coding genes, 37 transfer RNA genes, and 8 ribosomal RNA genes (Table 1 ). A total of 17 genes contained introns; specifically, the genes rpl16 , petD , petB , trnV-UAC , trnL-UAA , rpoC1 , atpF , rps16 , trnK-UUU, ndhA , ndhB , trnA-UGC , trnI-GAU , and rpl2 each contained one intron, and rps12, ycf3 , and clpP1 each contained two introns (Table S2 ). Table 1 Comparison of chloroplast genomes characteristics of Sonerila. Species Length (bp) %GC No. of genes Accession No. Genome size LSC Length SSC Length IR Length Total LSC SSC IR Total CDS tRNAs rRNAs S. cannanorensis 155,026 84,869 16,557 26,800 37.2 35.0 31.0 42.6 129 84 37 8 PX251189 S. ponmudiana 154,940 84,789 16,527 26,812 37.3 35.1 31.1 42.6 129 84 37 8 PX251190 S. speciosa 154,598 84,536 16,486 26,788 37.2 35.0 31.0 42.6 129 84 37 8 PX251191 S. sreenarayaniana 155,100 84,982 16,564 26,777 37.2 35.0 30.9 42.6 129 84 37 8 PX251192 S. janakiana 155,027 84,875 16,552 26,800 37.2 35.0 31.0 42.6 129 84 37 8 PX251193 S. malabarica 155.084 85,045 16.525 26.757 37.2 35.0 31.0 42.6 129 84 37 8 PX251194 S. amabilis 154,811 84,790 16,493 26,764 37.2 35.0 30.9 42.6 129 84 37 8 PX251195 S. lateritica 154,862 84,834 16,536 26,746 37.2 35.1 31.0 42.6 129 84 37 8 PX251196 S. tenella 154,818 84,720 16,528 26,785 37.3 35.1 31.1 42.6 129 84 37 8 PX251197 S. veldkampiana 154,835 84,879 16,488 26,734 37.2 35.0 31.0 42.6 129 84 37 8 PX251198 S. rheedei 154,936 84,912 16.528 26,748 37.2 35.0 31.0 42.6 129 84 37 8 PX251199 S. sadasivanii 155,018 84,900 16.514 26,802 37.3 35.1 31.0 42.6 129 84 37 8 PX251200 S. tinnevelliensis 154,970 84,929 16,527 26,757 37.2 35.1 31.0 42.6 129 84 37 8 PX251201 S. brunonis 155,094 85,040 16,538 26,758 37.2 35.0 31.1 42.6 129 84 37 8 PX251202 S. axillaris 155,019 84,878 16,555 26,793 37.2 35.1 30.9 42.6 129 84 37 8 PX251203 S. cantonensis_1 155,265 85,272 16,475 26,759 37.2 35.0 31.0 42.6 129 84 37 8 MK994791 S. cantonensis_2 155,233 85,229 16,484 26,760 37.2 35.0 31.0 42.6 129 84 37 8 MK994813 S. cantonensis_3 154,858 84,962 16,358 26,769 37.3 35.1 31.3 42.6 129 84 37 8 OL813666 S. plagiocardia 154,428 84,361 16,433 26,817 37.2 35.0 30.9 42.6 129 84 37 8 MK994876 S. pulchella 154,862 84,862 16,492 26,754 37.2 35.1 31.1 42.6 129 84 37 8 MK994884 S. velutina 154,682 84,817 16,391 26,737 37.3 35.1 31.1 42.6 129 84 37 8 MK994892 S. borneensis 154,804 84,872 16,470 26,731 37.3 35.1 31.0 42.6 129 84 37 8 MK994893 S. parviflora 154,752 84,833 16,451 26,734 37.4 35.2 31.2 42.6 129 84 37 8 MK994900 S. obliqua 154,978 85,026 16,518 26,717 37.1 34.9 31.0 42.6 129 84 37 8 OL813672 S. annamica 155,271 85,198 16,545 26,764 37.2 35.0 31.0 42.6 129 84 37 8 OL813695 S. nervulosa_1 154,260 84,306 16,470 26,742 37.4 35.3 31.0 42.6 129 84 37 8 OL813716 S. nervulosa_2 154,014 84,234 16,504 26,638 37.3 35.2 31.1 42.6 129 84 37 8 OL813725 Codon usage analysis The results showed that all protein-coding genes in Sonerila consisted of 64 codons, and encoded 20 amino acids, including three stop codons (UAA (*), UAG (*), UGA (*)) (Table S3 ). The number of encoded codons exhibited minimal variation, the types of codons and amino acids remained consistent. Out of the 64 codons, excluding the three stop codons and the unbiased methionine (Met) and tryptophan (Trp) (RSCU = 1), 32 codons displayed a preference with an RSCU value exceeding 1, indicating a higher priority for these codons. Among them, the AGA codon for Arginine (Arg) had the highest frequency as indicated by an average RSCU value of 1.94. The remaining 30 analyzed codons showed relatively low bias, with RSCU values less than 1 (Fig. 2 ). The codons in the 27 Sonerila chloroplast genomes exhibited a preference for A/T bases and A/T-ending codons, as evidenced by the GC and GC3 content being below 0.5. Moreover, analysis of codon adaptation index values and an effective number of codon values revealed a minor tendency toward biased codon usage. The frequency of optimal codons was relatively low. Furthermore, the hydrophobicity and aromaticity of the protein had a minimal effect on the observed bias in codon usage. Repeat sequence analysis and simple sequence repeats (SSRs) The highest number of SSRs was identified in S. brunonis (84), while the smallest number of SSRs was identified in S. parviflora (51) (Fig. 3 A). The most frequently observed SSR type was mononucleotide repeats, ranging from 32 to 70. All species exhibited mono-, di-, tri-, and tetra-, besides S. janakiana , S. rheedei , and S. axillaris in which no tri-nucleotide repeats were observed. The penta-nucleotide repeats were only observed in 13 species, S. ponmudiana , S. speciosa , S. sreenarayaniana , S. lateritica, S. veldkampiana , S. rheedei , S. tinnevelliensis , S.brunonis , S. cantonensis , S. plagiocardia , S. pulchella , S. parviflora , and S.annamica . The hexa-nucleotide repeats were present in only three species, S. tinnevelliensis , S. pulchella , and S. annamica . Furthermore, the SSRs were mainly distributed in the LSC and SSC regions of the chloroplast genome, while there were few SSRs in the two IR regions (Fig. 3 B). In this study, a total of 1,844 SSRs were detected in the chloroplast genomes of 27 Sonerila species, and the majority of SSRs were found in the LSC region, which might be correlated with the length of the LSC region. Additionally, they were predominantly composed of A/T bases, consistent with the AT richness observed in the whole chloroplast genome. The number of long repeats was 1,617, encompassing 5 types: 422 forward repeats, 132 reverse repeats, 676 palindromic repeats, 130 complement repeats, and 257 random repeats. The forward and palindromic repeats were the most abundant among all 27 species. The number of long repeats in S. obliqua and S. nervulosa was notably higher than in other Sonerila species: S. obliqua showed the highest count for palindromic repeats (52), complement (38) and reverse repeats (27), while S. nervulosa followed closely with 42 palindromic, 24 complement and 29 reverse repeats (Fig. 3 C). Tandem repeats were the common type among repeats. The majority ranged from 30 to 39 bp in length, with a few from 60 to 69 bp within 4 species, S. cantonensis , S. ponmudiana , S. tenella , and S. velutina (Fig. 3 D). Among these, S. obliqua had the highest number of long repeat sequences. The tandem repeats ranged from 6 to 14 in all Sonerila species. Comparative genomic divergence analysis The whole chloroplast genome sequences encoded gene classes, numbers, and alignments that were highly consistent among Sonerila species. These results indicated no significant alterations such as large fragment inversions, duplications, or other structural changes in all 27 chloroplast genomes. The sequence differences were higher in the LSC and SSC regions compared to the IR regions (Fig. 4 ). Sequence divergence and mutation hotspots The Pi values in the IR regions were much lower than those in the LSC and SSC regions. In all 27 Sonerila species, seven regions had a relatively high Pi value (> 0.025). Five regions in LSC ( rps16-trnQ-UUG , trnS-GCU-trnR-UCU , rbcL-accD , psbE-petL , rps18-rpl20 ), one region at the SSC-IRb boundary ( ycf1 ), and one region within the SSC ( rps15 ) were considered as hotspots (Pi > 0.025), and the ycf1 had the highest nucleotide diversity (Pi > 0.03), suggesting a potential useful region for evolutionary studies within Sonerila (Fig. 5 A, Table S4 ). Among the 15 Sonerila species from India, two regions in the LSC ( rps16 and rpl22 ), one region in SSC ( rpl32 ), and one region at SSC-IRb boundary ( ycf1 ) were considered as hotspots (Pi > 0.014) (Fig. 5 B). The ycf1 , rps15 , rps16 , rpl22 , and rpl32 regions not only exhibited high sequence variability but were also coding region sequences. IR contraction and expansion in the Sonerila chloroplast genomes The chloroplast genome is a circular structure consisting of the LSC, SSC, IRa and IRb regions, with four boundaries: LSC-IRb (JLB), IRb-SSC (JSB), SSC-IRa (JSA), and IRa-LSC (JLA). Expansion and contraction of the IR regions are well-known and significant events in plant evolutionary history and directly impact the size of the genome, gene content, order and rearrangement [ 40 , 41 ]. The LSC/IRb/SSC/IRa boundaries of Sonerila species were compared to analyze the expansion and contraction variation in junction regions (Fig. 6 ). The genotypes of the IR/LSC and IR/SSC boundaries were essentially identical, and the lengths of IRs across species were relatively conserved (26,638–26,817 bp) but still with significant expansion or contraction (Fig. 6 ). Six protein-coding genes ( rpl22 , rps19 , rpl2 , ndhF , ycf1 , psbA ) were present near or at the LSC/IR and SSC/IR boundaries. At the LSC/IRb (JLB) boundary, a fragment of rps19 gene was detected with a small change in length. The rps19 in the IRb region varied from 78–115 bp, whereas in the LSC region 201 bp were consistently present across species, except in S. plagiocardia , where it was reduced to164 bp. This indicates a slight IR expansion into the LSC, resulting in the incorporation of a larger portion of the rps19 gene. At the IRb/SSC (JSB) boundary, the pseudogene ycf1 (ψycf1) and ndhF were consistently detected in all the species. The pseudogene ycf1 (ψycf1) spanned the IRb region for 1,827 to 1,848 bp and extended into the SSC region for an additional 56 to 62 bp; the ndhF gene extended across the IRb region for 44 to 50 bp and into the SSC region for 2,185 to 2,200 bp, overlapping with the pseudogene ycf1 (ψycf1) . At the boundary of SSC/IRa (JSA), the full-length ycf1 gene spanned 3,591 to 3,675 bp across the SSC region and 1,827 to 1,848 bp across the IRa region. At the IRa/LSC (JLA) boundary, the trnH gene was located on the right side of the boundary for 0 to 2 bp. The IR/SC boundary differed among the chloroplast genomes of S. lateritica and S. nervulosa (OL813725). In S. lateritica , the apparent absence of rps19 at the boundary was likely due to an annotation gap, as manual inspection confirmed its presence. In contrast, in S. nervulosa (OL813725), rps19 was entirely located within the LSC region, 16 bp from the LSC/IRb (JLB) boundary rather than spanning it. Additionally, the rpl2 was positioned 44 bp closer to the IRa/LSC (JLA), suggesting a contraction of the IR regions. Phylogenetic analyses A phylogenetic tree was constructed using maximum likelihood (ML) methods utilizing the 72 complete chloroplast genomes sequences from tribe Sonerileae, with four additional sequences from the Dissochaeteae, Pyxidantheae, and Melastomateae. Sonerileae species were selected to represent morphological diversity, particularly the variation in floral merosity (ranging from 3- to 6-merous) and stamen morphology (isomorphic and dimorphic types) (Fig. 7 ). Most nodes across the phylogenetic tree showed high support (BS > 70). Sonerila species clustered into a single, strongly supported clade (BS > 99). Sonerila obliqua (Southeast Asia), S. nervulosa (Borneo), and S. plagiocardia (South China, Indochina) were identified as early diverging species, successively sister to the rest of Sonerila . A clade of predominantly Bornean species was recovered sister to a clade that included species from South China, Indochina and India. Fourteen of the 15 species assembled from the Indian subcontinent formed a clade relative to other Sonerila species, with the exception of S. amabilis , which is a Himalayan species from Sikkim (India), and clustered with S. cantonensis (South China) (Fig. 7 ). Overall, the phylogenetic reconstruction of Sonerila revealed strong geographic structuring, with distinct clades representing the Indian subcontinent, South China and Indochina, the Himalayan region, and Southeast Asia (Fig. 7 ). Discussion The aim of this study was to provide an overall assessment of the chloroplast genome structure and sequence variation in Sonerila , one of the least known, yet widespread, paleotropical taxa. Additionally, we attempted to evaluate the utility of the chloroplast genome for generating hypotheses on the origin of Indian species. The origin, diversification, and historical biogeography of Sonerila remains unknown. Only a few phylogenies including minimal taxon sampling [ 7 , 13 , 14 , 64 ] and several independent regional taxonomic studies, mainly new species descriptions [ 15 , 16 , 17 , 18 ], are available. Indian Sonerila represents the westernmost distribution of this group, showcasing remarkable morphological diversity, distinguishing these species from others across the Indomalayan region. Our study represents a first attempt to examine the chloroplast diversity in this group and gather initial evidence in support of either a single or multiple origins of Sonerila in India. Structural characteristics of the chloroplast genome in Sonerila The chloroplast genome is a valuable resource for studying intra- and inter-species evolution and developing molecular markers [ 42 ]. In this study, we sequenced and annotated the chloroplast genomes of Sonerila species from India, and subsequently, compared their genomic features with those of other Sonerila species and close relatives available on public databases. Our results show that Sonerila plastomes are conserved in gene order and length, with quadripartite structure, as previously observed in other representatives of Melatomataceae [ 21 ] and Sonerileae [ 13 , 23 , 24 , 43 ]. The structure included a large single copy region (LSC), a small single copy region (SSC), and two inverted repeat regions (IRs). The major variation in genome size may be attributed to the varying length of LSC and IRs across species of Sonerila . The chloroplast genome of S. nervulosa (OL813725) exhibits contracted IR regions, measuring 26,638 bp in length and resulting in the smallest genome size within the Sonerila species analyzed (154,014bp). Analysis of codon bias and repetitive sequences Relative Synonymous Codon Usage (RSCU) is often used to reflect codon bias [ 44 ]. The GC content of the chloroplast genomes reflects a balance between mutation pressure and adaptation, which is one of the most significant factors contributing to the patterns of codon usage bias [ 45 ]. Although synonymous mutations of the third base of a codon do not alter the resulting amino acid, they are still considered important features due to their influence on factors like codon usage bias, and other molecular processes. Therefore, GC3 is often used as a significant indicator of codon preference [ 46 ]. Herein, the chloroplast genomes of Sonerila species revealed that all optimal synonymous codons (RSCU > 1), except for UUG, CCC, AGG, and GGG, terminated with A or U, indicating a general preference for A/T bases across the genomes. RSCU can affect gene expression by regulating the accuracy and efficiency of translation, with stronger RSCU values associated with increased levels of gene expression. In chloroplast gene expression vector design, optimizing codons according to their bias can boost the expression levels of inserted genes in the chloroplast genome. In addition, known codon usage patterns can help predict the expression and function of unknown genes [ 47 ]. The identified codons with RSCU > 1 can serve as efficient indicators for detecting the expression levels of hypothetical genes or open reading frames. Repetitive sequences affect the transcriptional regulation of genes, protein translation, chromosome formation and metabolism, and may reflect differences between mutation frequencies and evolutionary rates of species [ 48 ]. We observed differences in the number and distribution of repeats types and lengths across Sonerila species. The presence and abundance of repetitive sequences in the chloroplast genome are likely to involve the phylogenetic history and genomic adaptation. Simple Sequence Repeats (SSRs) are abundant and exbibit significant genetic variation, making them effective markers for population genetics and biodiversity studies [ 49 , 50 ]. In S. nervulosa and S. obliqua , the number of reverse and complement repeats is significantly higher than in the other species, which may indicate unique genomic features or evolutionary adaptations. Overall, the SSRs and long repeat sequences identified in this study provide useful information for further research on molecular marker development, population genetics, species delimitation, and conservation studies in Sonerila . Comparative genomics and highly variable regions analysis The contraction and expansion of IR regions during evolution is a relatively common occurrence and has been employed as an evolutionary marker for phylogenetic studies. This dynamic process is a primary factor leading to variations in the length and number of genes in many species [ 51 , 52 , 53 ]. In this study, significant similarities were found in the expansion or contraction of the IR regions in Sonerila species. The LSC/IRb boundary was generally located on the rps19 gene in 26 Sonerila species, except for S. lateritica and S. nervulosa (OL813725), where the contraction of IR regions resulted in the rps19 gene moving from the LSC/IRb boundary to LSC region to decrease the size of the chloroplast genome. Additionally, the expansion of the IR resulted in partially replicated ycf1 genes being in the IRb region, thus generating the pseudogene ycf1 at the IRB/SSC (JSB) boundary. The pseudogenes in the chloroplast genomes at the SSC/IRa boundary are often found in land plants [ 54 , 55 ]. Overall, the conserved IR region in Sonerila may contribute to the length and structural stability of the chloroplast genome. DNA barcoding technology has a wide range of application in the fields of species identification, conservation, phylogeny, and evolution [ 56 , 57 ]. Previous phylogenetic analyses have mostly used the ITS region of nuclear DNA and ycf1 , rps15 , rps16-trnQ , and trnL-trnF in the chloroplast genome [ 58 , 59 ]. Moreover, although the Consortium for the Barcode of Life (CBOL) ( http://www.barcodeoflife.org/ ) has recommended matK and rbcL genes as core plant barcodes [ 60 ], these fail to provide phylogenetic resolution at shallow levels in species-complexes where lineages evolved through recent or rapid radiation or may be the product of hybridization and introgression [ 61 ]. While recent studies recommend the use of complete chloroplast genomes for shallow phylogenetic-level studies [ 13 , 14 , 62 , 64 ], identifying informative regions may still be a challenge, especially for species delimitation. Our results demonstrate that screening for informative chloroplast markers is a difficult task, considering that most phylogenetic informative regions vary even across closely related species [ 63 ]. However, the chloroplast genome represents a valuable resource and provides an important perspective when used in combination with additional genetic data. This study identified one region ( ycf1 ) with consistently high variability value (> 0.03) across all 27 Sonerila species. Additionally, six other regions ( rps15 , psbE - petL , trnS-GCU - trnR-UCU , rps16 - trnQ-UUG , rbcL - accD , rps18 - rpl20 ) showed relatively high variability value (> 0.025). Notably, the ycf1 gene (0.035) located at the SSC-IR boundary, further supports these regions as molecular hotspots. Therefore, all seven regions should be considered most suitable to evaluate phylogenetic relationships among Sonerila species. However, in the specific case of Indian Sonerila , four specific regions ( ycf1 , rps16 , rpl22 , and rpl32 ) presented relatively high Pi values (> 0.014), and therefore, these would be most suitable as DNA barcodes and for resolving phylogenetic relationships. Overall, lower variability in this clade may reflect a recent diversification, which has limited the accumulation of plastome divergence. Alternatively, it may be influenced by historical demographic events, such as a genetic bottleneck. Assessment of phylogenetic resolution We conducted phylogenetic analyses including 72 Sonerileae species and four species from tribes Dissochaeteae, Pyxidantheae, and Melastomateae, utilizing newly generated chloroplast genome data from this study and publicly available sequences. Our findings align well with recent phylogenomic studies of Sonerileae. Recent studies have advanced our understanding of this group's evolutionary relationships. For instance, Zhou et al. [ 14 ] analyzed 151 complete chloroplast genomes, providing strong support for the backbone phylogeny of the Sonerileae/Dissochaeteae clade. Similarly, another study [ 13 ] identified 34 robust lineages within Asian Sonerileae by employing a combination of single-copy orthologs (SCOs), genomic SNPs, and whole plastome data. Independently, Quankenbush et al. [ 64 ] analyzed 385 nuclear and 81 plastid protein-coding loci to explore the relationships among fleshy-fruited genera such as Catanthera , Heteroblemma , Kendrickia , Medinilla , Pachycentria , and Plethiandra revealing their distribution across three distinct evolutionary lineages. Collectively, these studies consistently support the monophyly of Sonerila [ 13 , 14 , 64 ], although it is worth noting that these results might have been influenced by very limited inclusion of Sonerila species. In this study, we selected 15 Sonerila species from India and 12 from published work, representing the wide distribution of the group. We constructed a robust phylogenetic tree based on the whole chloroplast genome sequences of all sampled Sonerileae species. Our results showed that Sonerila is monophyletic, and the species from the Indian subcontinent cluster together forming a well-supported clade. The Indian S. amabilis , endemic to Sikkim, is a Himalayan species, nested with S. cantonensis , which is from nearby southern China and Indochina. It is not surprising that it does not cluster with the rest of the species from the Indian subcontinent. Overall, our phylogenetic reconstruction while confirming the monophyly of Sonerila , reveals some interesting geographic structure. Notably, the placement of Southeast Asian species ( S. obliqua, S. nervulosa ) may suggest that Southeast Asia harbors the potential center of early diversification and ancestral range for the group. This hypothesis is consistent with the high diversity of Sonerila species documented in Malaysia, Sumatra, and Borneo regions recognized as biodiversity hotspots and frequently proposed as centers of origin for tropical plant radiations [ 13 , 14 , 65 , 66 , 67 ]. This also suggests that Sonerila may have diversified and dispersed along an east-west route, with India and Sri Lanka representing the westernmost edge of its distribution, and potentially, the most recently diverging lineages. Further studies integrating nuclear genomic data, biogeographic modeling, and expanded sampling are needed to clarify this evolutionary scenario and test our hypotheses. Overall, these results provide important implications for the assessment of genetic diversity and phylogenetic studies in Sonerila , highlighting the need for continued research. Conclusions In this study, the chloroplast genomes of 15 Sonerila species were assembled and annotated, and a series of comparative analyses were performed using 12 additional published Sonerila genomes. The results revealed that Sonerila chloroplast genomes exhibited conserved structural features and gene content, while variations were observed in genome size, sequence divergence, and repeat elements. The identification of long repetitive sequences, simple sequence repeats (SSRs), and seven regions with high variability are phylogenetically informative. We reconstructed a phylogenetic scenario that suggest Sonerila is a monophyletic entity, with a putative center of origin in the Southeast Asia region. The high level of endemism observed in India highlights the significant evolutionary diversification of this clade in the region. Specifically, we hypothesize a single independent introduction in the Indian subcontinent, with subsequent diversification events, especially in the Western Ghats. This preliminary hypothesis warrants further testing using a much broader taxon sampling that include additional species from the entire distribution range. The results of this study enhance our understanding of chloroplast genome evolution in Sonerila and contribute to future studies focusing on species delimitation and historical evolution of this group. Declarations Competing interests The authors declare that they have no competing interests. Consent for publication Not applicable. Ethics approval and consent to participate Not applicable. Funding This work was supported in part from funding from the National Science Foundation, NSF-DEB 2002270. Author Contribution JH, MRB, NC designed the study; JH and MRB performed the analyses; All authors contributed to the writing of the paper. Acknowledgement RS would like to thank the International Association of plant Taxonomists (IAPT) and the Botanical Survey of India for supporting fieldwork activities. Data Availability Raw reads have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject PRJNA1306189 **,** Study SRP612796). Assembled chloroplast genomes are deposited in GenBank. All accessions are listed in Supplementary Table S5. A reviewer link for accessing the BioProject prior to public release is available here: ( https:/dataview.ncbi.nlm.nih.gov/object/PRJNA1306189?reviewer=2medqtabcjv5gcfglbr8j481cc ). All data will be made publicly available upon publication. References Ulloa Ulloa C, Almeda F, Goldenberg R, Kadereit G, Michelangeli FA, Penneys DS, Stone RD, Veranso-Libalah MC. Melastomataceae: Global Diversity, Distribution, and Endemism. In: Goldenberg R, Michelangeli FA, Almeda F, editors. Systematics, Evolution, and Ecology of Melastomataceae. 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Tropical plant diversity of Borneo: The role of high conservation value area on species conservation in an oil palm plantation. AIP Conference Proceedings . 2018;2019(1):040012. https://doi.org/10.1063/1.5061882 Sun J, Liu B, Rustimai H, Xiao H, Shen X, Ma K. Mapping Asia plants: plant diversity and a checklist of vascular plants in Indonesia. Plants. 2024;13(16):2281. https://doi.org/10.3390/plants13162281 . Additional Declarations No competing interests reported. Supplementary Files TableS1Sonerileaespecieslistcp.xlsx TableS2genesofcpcpsupply.docx TableS3CODONUSAGEBIAS.xlsx TableS4Hotspotcp.xlsx TableS5Data.xlsx CPalignmentv2.fasta Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7538746","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":539426948,"identity":"807caf89-0f5f-44e3-880d-50961a377917","order_by":0,"name":"Jiahong Han","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYBACxmYGNhCdwMDAfIChogDIPEC8FrYEhjMGRGgBApgWHgPitDC3Mz978HFHbR7/7J5vEgcMGOT4biQQchibueHMM8eLJe6c3QbSYixJWAuDmTRv27HEhhu526Q/GDAkbiCshf0bWMv8GznPQLbUE6GFB2RLDdDwHDaQlgQDIrSUSc5sO5C48UaascUBAwmgxx7g12LYf3ybxMe2usR5N5If3jhQYSPPd5yALYYNYOowjC+BXzkIyEOoOsIqR8EoGAWjYOQCAFT2Sm/x/CZrAAAAAElFTkSuQmCC","orcid":"","institution":"University of Florida","correspondingAuthor":true,"prefix":"","firstName":"Jiahong","middleName":"","lastName":"Han","suffix":""},{"id":539426950,"identity":"43f3a12c-d736-4340-b01e-69041df18d55","order_by":1,"name":"Monique Romeiro-Brito","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Monique","middleName":"","lastName":"Romeiro-Brito","suffix":""},{"id":539426953,"identity":"260e51cd-c452-4ce0-94b5-c5854acbab3a","order_by":2,"name":"Resmi Sekarathil","email":"","orcid":"","institution":"Botanical Survey of India, Southern Regional Centre","correspondingAuthor":false,"prefix":"","firstName":"Resmi","middleName":"","lastName":"Sekarathil","suffix":""},{"id":539426954,"identity":"4f0c6991-0a57-461c-a2e0-f46d45ab3ebe","order_by":3,"name":"Santhosh Nampy","email":"","orcid":"","institution":"University of Calicut","correspondingAuthor":false,"prefix":"","firstName":"Santhosh","middleName":"","lastName":"Nampy","suffix":""},{"id":539426956,"identity":"12a95551-539c-4114-b212-7b9aed5991ac","order_by":4,"name":"Lucas C. 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17:25:45","extension":"html","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":227061,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7538746/v1/2241055a91d7e21af2c7c522.html"},{"id":95236024,"identity":"041e2c86-8e53-453d-b6dd-eca0fe0cd205","added_by":"auto","created_at":"2025-11-05 17:25:44","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":329396,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMap of the newly analyzed chloroplast genome of 27 \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSonerila \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003especies.\u003c/strong\u003eGenes inside the circle are transcribed clockwise, and genes outside are transcribed counterclockwise. Genes with different functional groups are distinguished by color coding. The positions of the long single-copy (LSC), short single-copy (SSC), and inverted repeat regions are shown in the inner circles.\u003c/p\u003e","description":"","filename":"11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7538746/v1/c744d838c6556b765638ca29.jpg"},{"id":95312329,"identity":"7047108f-baa0-4683-9a2c-59c98242e956","added_by":"auto","created_at":"2025-11-06 15:48:50","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1253683,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelative synonymous codon usage (RSCU) for the 20 amino acid and stop codons in the chloroplast genomes of 27 \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSonerila\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e species.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7538746/v1/25c977103c3e136b47c2e8ff.jpg"},{"id":95312376,"identity":"d1513a1a-8b67-4d5f-9a77-d843fddf6ad4","added_by":"auto","created_at":"2025-11-06 15:49:03","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1154930,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of the repeated sequences in 27 \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSonerila \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003especies.\u003c/strong\u003eRepeats with different types are visually represented by different colors. A) analysis of the number and type of SSRs (mono, di, tri, tetra, penta, hexa). B) distribution of different types of SSRs in the chloroplast genome. C). types and number of repeats in the 27 chloroplast genomes (Forward, Reverse, Complement, Palindromic). D) Number of tandem repeats in 27 chloroplast genomes.\u003c/p\u003e","description":"","filename":"13.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7538746/v1/c28e66cddd1add9707dd69fd.jpg"},{"id":95236027,"identity":"e936f697-e3ee-4a4a-a513-7d7bfeac0bb6","added_by":"auto","created_at":"2025-11-05 17:25:44","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2203622,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVisualization of the alignment of 27 chloroplast genomes sequences of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSonerila\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003eThe plastome of \u003cem\u003eS. annamica\u003c/em\u003e was used as the reference.\u003cstrong\u003e \u003c/strong\u003eThe vertical scale indicates the percent identity, ranging from 50% to 100%. Coding genes, RNAs, and noncoding regions are marked in purple, sky blue, and red, respectively.\u003c/p\u003e","description":"","filename":"14.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7538746/v1/4d9fd0b066414b8194e5f15b.jpg"},{"id":95312350,"identity":"104e7e34-939b-4864-9847-10ad1f467e40","added_by":"auto","created_at":"2025-11-06 15:48:57","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":255276,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSliding window analysis of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSonerila\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e chloroplast genomes.\u003c/strong\u003eA) Pi values of 27 \u003cem\u003eSonerila\u003c/em\u003e species we studied. B) Pi value of all 15 \u003cem\u003eSonerila\u003c/em\u003especies.\u003c/p\u003e","description":"","filename":"15.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7538746/v1/ccb4c3a1c2adc01001528d60.jpg"},{"id":95236034,"identity":"439ada1c-532d-4445-be54-27ff54dd5d0e","added_by":"auto","created_at":"2025-11-05 17:25:44","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1054860,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of the borders of large singe copy (LSC), small single-copy (SSC), and inverted repeat (IR) regions among \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSonerila\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e chloroplast genomes.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"16.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7538746/v1/fb048655dc1ff0e94c90cfdc.jpg"},{"id":95312438,"identity":"f9dfd31c-af31-4873-935a-38ea36892117","added_by":"auto","created_at":"2025-11-06 15:49:23","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":279821,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eML phylogenetic relationships among 76 \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSonerileae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e species and outgroups based on the whole chloroplast genome. \u003c/strong\u003eIn the figure, the purple circles represent the bootstrap (BP) support values. The legend on the right shows all genera and the distribution in \u003cem\u003eSonerila\u003c/em\u003e. A. \u003cem\u003eSonerila axillaris\u003c/em\u003e, B. \u003cem\u003eSonerila veldkampiana\u003c/em\u003e, C. \u003cem\u003eSonerila brunonis\u003c/em\u003e, D. \u003cem\u003eBlastus cochinchinensis\u003c/em\u003e, E. \u003cem\u003eOxyspora paniculata\u003c/em\u003e, F\u003cem\u003e. Plethiandra robusta\u003c/em\u003e,\u003cem\u003e \u003c/em\u003eG.\u003cem\u003e Phyllagathis stellata\u003c/em\u003e,\u003cem\u003e \u003c/em\u003eH. \u003cem\u003eGravesia subglobosa\u003c/em\u003e. (A) (B) (C) taken by \u003cem\u003eResmi Sekarathil\u003c/em\u003e. (D) adapted from \u003cem\u003eiNaturalist Taiwan, licensed under CC BY-NC 4.0\u003c/em\u003e. (E) adapted from \u003cem\u003eFlickr by Ed Shaw, licensed under CC BY-NC 2.0\u003c/em\u003e. (F) adapted from \u003cem\u003eQuakenbush J.P. et al\u003c/em\u003e. \u003cem\u003e(2024),\u003c/em\u003e \u003cem\u003ethe journal of the International Association of Plant Taxonomy\u003c/em\u003e. (G) adapted from \u003cem\u003eLin C. \u0026amp; Lee C. (2018), journal of Phytotaxa\u003c/em\u003e. (H) adapted from \u003cem\u003eiNaturalist by Bitty A. Roy, licensed under CC BY-NC 4.0.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"17.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7538746/v1/ff9277f573b5f31e4130dd62.jpg"},{"id":108803485,"identity":"f59958f4-4aa7-46e0-90e9-29f5882c4e2e","added_by":"auto","created_at":"2026-05-08 14:56:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7243462,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7538746/v1/267e8317-7f52-4614-8cf0-3904ccb53db5.pdf"},{"id":95312624,"identity":"bda59aa4-4998-4a67-adad-fa0dfbff63d5","added_by":"auto","created_at":"2025-11-06 15:49:50","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14978,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1Sonerileaespecieslistcp.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7538746/v1/ad4275d1766737cc5a0c8a83.xlsx"},{"id":95312304,"identity":"afd06c9d-25d3-4ad8-b444-3b3dc02419f0","added_by":"auto","created_at":"2025-11-06 15:48:46","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":15881,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2genesofcpcpsupply.docx","url":"https://assets-eu.researchsquare.com/files/rs-7538746/v1/40a1f2ae37831a9494ddea84.docx"},{"id":95312473,"identity":"e4fea57a-504e-451c-a58f-2dd2d08a82c4","added_by":"auto","created_at":"2025-11-06 15:49:29","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":19569,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3CODONUSAGEBIAS.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7538746/v1/7d0fa85911216a789add8001.xlsx"},{"id":95236036,"identity":"a7d94aaf-0679-44f0-a707-4a950e814833","added_by":"auto","created_at":"2025-11-05 17:25:44","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":11479,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4Hotspotcp.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7538746/v1/5ca65265c36f52c201b16862.xlsx"},{"id":95236032,"identity":"9515d1d9-f960-40ec-947c-ea12abf1f50e","added_by":"auto","created_at":"2025-11-05 17:25:44","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":10587,"visible":true,"origin":"","legend":"","description":"","filename":"TableS5Data.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7538746/v1/d7b6d5b3c1ce0cb7a495ea86.xlsx"},{"id":95236060,"identity":"54a1b13e-570f-4f44-9c94-5b58fb156c06","added_by":"auto","created_at":"2025-11-05 17:25:45","extension":"fasta","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":15366344,"visible":true,"origin":"","legend":"","description":"","filename":"CPalignmentv2.fasta","url":"https://assets-eu.researchsquare.com/files/rs-7538746/v1/d5323277a788b41c06c39b9c.fasta"}],"financialInterests":"No competing interests reported.","formattedTitle":"Complete chloroplast genomes suggest a single origin of the Indian subcontinent Sonerila (Melastomataceae)","fulltext":[{"header":"Background","content":"\u003cp\u003eMelastomataceae are one of the most species-rich flowering plant families and a major model system for evolutionary studies [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], with approximately 5857 species representing important ecological components of tropical habitats worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Sonerileae is the second largest tribe within the family, comprising approximately 1,080 species across 46 genera [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], with its greatest diversity found in Asia and Oceania [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Members of Sonerileae exhibit remarkable morphological diversity, including herbs, shrubs, small trees, and, in some cases, climbers and epiphytes. Floral characteristics include 3- to 6-merous flowers, isomorphic or dimorphic stamens with various connective appendages including the total loss of these structures, and fruits that are either berries or capsules [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe intergeneric boundaries within Sonerileae have long been problematic, due to extensive morphological convergence [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. For example, \u003cem\u003eSonerila and Tashiroea\u003c/em\u003e are both small herbs with 3-merous flowers, but molecular studies reveal that they are not closely related [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Similarly, \u003cem\u003ePhyllagathis\u003c/em\u003e, \u003cem\u003eBredia\u003c/em\u003e and \u003cem\u003eScorpiothyrsus\u003c/em\u003e were historically grouped together due to similar floral structures, but phylogenetic analyses have shown that these genera belong to distinct evolutionary lineages [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Moreover, switches in flower merosity (from 3 to 4- to 5-merous), even within species, are not uncommon [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Recent investigations with expanded sampling across a broader range of Sonerileae species, have improved the genetic delimitation and the identification of several major Asian lineages [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Advances in high-throughput sequencing technologies have significantly enhanced phylogenetic analyses within Sonerileae, especially by enabling the use of chloroplast genome sequences [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Nonetheless, many challenges persist, particularly regarding species delimitation and infrageneric relationships, reflecting the group\u0026rsquo;s inherent genetic complexity. Therefore, further research incorporating broader molecular data and advanced phylogenetic approaches is necessary, given the limited phylogenetic resolution currently available for poorly sampled genera.\u003c/p\u003e\u003cp\u003e\u003cem\u003eSonerila\u003c/em\u003e is the second largest genus within Sonerileae, having remarkable diversity with approximately 180 species distributed from India and Sri Lanka to Papua New Guinea, across the entire Indomalayan region [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Regardless of its wide distribution range, this group represents one of the least known entities in paleotropical Melastomataceae. \u003cem\u003eSonerila\u003c/em\u003e typically inhabits moist, shaded environments such as rainforests and montane forest, often occurring along stream banks within these diverse habitats. They are acaulescent and caulescent herbs, often with basal rosettes, with white or brightly colored 3-merous flowers, and a diverse array of capsular fruits. In India, \u003cem\u003eSonerila\u003c/em\u003e exhibit high morphological diversity, with approximately 50 species, over 80% of which are endemics [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The high level of endemism highlights the group\u0026rsquo;s significant diversification within the Indian subcontinent. Recent phylogenomic analyses have provided some evidence suggesting that \u003cem\u003eSonerila\u003c/em\u003e may represent a monophyletic entity [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, \u003cem\u003eSonerila\u003c/em\u003e species morphological diversity and geographic origin have been largely underrepresented in all phylogenetic studies [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHigh-throughput sequencing technology has revolutionized plant systematics, making chloroplast genome sequencing a powerful and increasingly utilized tool. Chloroplast, the photosynthetic organelles in green plants and vital site for molecule synthesis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], has a relatively independent and highly conserved structure compared to the nuclear genome [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The chloroplast genome of \u003cem\u003eTigridiopalma magnifica\u003c/em\u003e was the first to be reported within Sonerileae, presenting 155,663 bp in length, 130 genes and a GC content of 37.11% [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Similarly, the plastomes of \u003cem\u003eBlastus auriculatus\u003c/em\u003e and \u003cem\u003eBlastus cochinchinensis\u003c/em\u003e were found to include 155,981bp with 125 genes and GC 37.0% [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and 156,005 bp with 129 genes and GC 37.0% [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], respectively. A tribe-wide phylogenomic study involving 151 complete chloroplast genomes reveled genome size ranging from 153,219 to 158,960 bp, with all plastomes encoding a conserved complement of 129 genes, including 84 protein-coding genes, 37 tRNAs, 8 rRNAs [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Despite these advances, plastome sampling across Sonerileae remains sparse and uneven, with most genera represented by only a few species. A comprehensive comparative analysis of chloroplast genome structure within the tribe, especially at the genus level, is still lacking. Focused studies on \u003cem\u003eSonerila\u003c/em\u003e, one of the most taxonomically complex and species-rich genera, are critical for uncovering lineage-specific structural variations, identifying robust molecular markers, and refining phylogenetic relationships across Sonerileae.\u003c/p\u003e\u003cp\u003eIn an effort to further our insight into the genetic diversity and genomic characteristics of \u003cem\u003eSonerila\u003c/em\u003e, we assembled and annotated the plastome sequences of 15 \u003cem\u003eSonerila\u003c/em\u003e species from India, which represents the westernmost edge of its distribution range. For broader context and comparison, we included 12 publicly available plastomes of \u003cem\u003eSonerila\u003c/em\u003e species from other regions. The objectives of this study are: (1) compare the genome structure and sequence variation of 27 chloroplast genomes in morphologically diverse \u003cem\u003eSonerila\u003c/em\u003e species from different regions across its distribution range; (2) identify simple sequence repeats (SSRs), large repetitive elements, and highly variable regions as potential molecular markers for \u003cem\u003eSonerila\u003c/em\u003e species identification and phylogenetic analysis; and (3) construct a phylogenetic tree using complete chloroplast genome sequences from 72 Sonerileae species to test the utility of the chloroplast and the putative monophyly of Indian \u003cem\u003eSonerila\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eTaxon sampling, DNA extraction, and sequencing\u003c/h2\u003e\u003cp\u003eWe selected 15 species of \u003cem\u003eSonerila\u003c/em\u003e from India and extracted the DNA from fresh leaves stored in silica gel using the modified CTAB protocol [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The plant material was identified by Dr. Resmi Sekarathil and Dr. Santhosh Nampy. Voucher specimens of all 15 species were deposited at the University of Calicut Herbarium (CALI and FRC), and the corresponding voucher accession numbers are provided in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The quality, integrity, and concentration of DNA were determined by agarose gel electrophoresis and Qubit fluorescence photometer. A complete list of the sampled taxa and their voucher information is provided in supplementary information (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The genome skimming libraries for the newly generated data for \u003cem\u003eSonerila\u003c/em\u003e species were built and sequenced by Rapid Genomics LLC, using the Illumina NovaSeq platform.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eChloroplast genome assembly and annotation\u003c/h3\u003e\n\u003cp\u003eWe filtered the raw sequencing data using fastp [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] removing low quality base pairs (Phred values\u0026thinsp;\u0026lt;\u0026thinsp;15) and short sequence length (\u0026lt;\u0026thinsp;30 bp). Chloroplast genome assembly was performed using GetOrganelle (v 1.7.7.0) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. We used GetOrganelle to first filter plastid-like reads, conduct the de novo assembly, purify the assembly, and finally to generate the complete chloroplast genomes. The complete chloroplast genome sequence of \u003cem\u003eS. annamica\u003c/em\u003e (OL813695.1) was subsequently used as a reference and annotated and manually corrected using Geneious (v 11.0.24). For the annotation using Geneious, a minimum of 70% identity cutoff between the genomes was considered. It was used to predict the chloroplast genome for coding proteins, tRNA, and rRNA genes, and then the predicted initial genes were made de-redundant and the first and last genes and exon/intron boundaries were manually corrected to obtain a highly accurate genome. Physical mapping of the chloroplast genomes was performed using OGDRAW (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://chlorobox.mpimpgolm.mpg.de/OGDraw.html\u003c/span\u003e\u003cspan address=\"https://chlorobox.mpimpgolm.mpg.de/OGDraw.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) software. To reduce potential bias from reference genomes and annotation software, the plastome of twelve \u003cem\u003eSonerila\u003c/em\u003e species obtained from NCBI were reannotated utilizing Geneious, with \u003cem\u003eSonerila annamica\u003c/em\u003e taken as the reference.\u003c/p\u003e\n\u003ch3\u003eCodon usage indices\u003c/h3\u003e\n\u003cp\u003eTo investigate codon usage patterns and nucleotide composition in the twenty-seven \u003cem\u003eSonerila\u003c/em\u003e chloroplast genomes, amino acid frequency, codon usage number, and the relative synonymous codon usage (RSCU) were analyzed and summarized. The RSCU value was used to measure the association between the observed frequency and the anticipated frequency of a particular codon. The RSCU and amino acid frequency in the chloroplast genome was compared using CodonW v1.42 software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://codonw.sourceforge.net\u003c/span\u003e\u003cspan address=\"http://codonw.sourceforge.net\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eSSRs and repeat sequence analysis\u003c/h3\u003e\n\u003cp\u003eSSRs in the chloroplast genomes of 27 \u003cem\u003eSonerila\u003c/em\u003e species were analyzed using the MISA software [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], with the parameters set as follows: 10 for mono-, 5 for di-, 4 for tri-, and 3 for tetra-, penta-, and hexanucleotides. The Vmatch tool [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] was used to identify repeats: forward, reverse, palindrome, and complement repeats. The following settings for repeat identification were used: (1) Hamming distance equal to 3; and (2) minimal repeat size set to 30 bp. Tandem repeats were detected using the Tandem Repeats Finder (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://tandem.bi.edu/trf/\u003c/span\u003e\u003cspan address=\"http://tandem.bi.edu/trf/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), an online software. Tandem repeats observed in at least one copy were considered significant. The parameters were set as follows: alignment parameters of matches\u0026thinsp;=\u0026thinsp;2, mismatches\u0026thinsp;=\u0026thinsp;3, indels\u0026thinsp;=\u0026thinsp;5, minimum alignment score\u0026thinsp;=\u0026thinsp;50, maximum period size\u0026thinsp;=\u0026thinsp;500, and maximum tandem repeat size\u0026thinsp;=\u0026thinsp;2.\u003c/p\u003e\n\u003ch3\u003eComparative analysis of chloroplast genomes\u003c/h3\u003e\n\u003cp\u003eIn this study, Geneious (v11.0.24) software [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] was used to determine the lengths of the IRa/IRb, LSC, and SSC regions, as well as the boundary genes, in the chloroplast genome of the \u003cem\u003eSonerila\u003c/em\u003e species. The mVISTA program in the shuffle-lagan model [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] was used to compare the chloroplast genome sequences using \u003cem\u003eS. annamica\u003c/em\u003e (OL813695) as a reference. The DnaSP6 software [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] was used to calculate the Pi values of the LSC, SSC, and IR regions between all the \u003cem\u003eSonerila\u003c/em\u003e species, and to identify divergence hotspot regions within the genome for evolutionary analysis. The step size was set to 200 bp and the window length to 600 bp. The complete chloroplast genome sequences were aligned using MAFFT v 7.49 [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The IRscope software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://irscope.shinyapps.io/irapp/\u003c/span\u003e\u003cspan address=\"https://irscope.shinyapps.io/irapp/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] was used to draw an IR boundary map and compare IR boundary characteristics. To visualize and compare the IR boundaries, Adobe illustrator software was employed for creating comparison maps.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003ePhylogenetic analyses\u003c/h2\u003e\u003cp\u003ePhylogenetic analyses were performed using the 76 complete chloroplast genome sequences form Sonerileae species, the 15 \u003cem\u003eSonerila\u003c/em\u003e species examined in this study and 61 species that were selected from the NCBI database. The complete chloroplast genome sequences including the outgroup were compared into a single file and aligned using MAFFT v 7.49 [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The 76 Sonerileae species in 25 clades were selected, including two species from Dissochaeteae, one species from Pyxidantheae and one species from Melastomateae as outgroup taxa (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Maximum likelihood (ML) analyses were performed using raxmlGUI v 2.0.10 with 1000 bootstrap replicates and the GTR\u0026thinsp;+\u0026thinsp;I\u0026thinsp;+\u0026thinsp;G model [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], and the best substitution model was determined by the Akaike information criterion (AIC) in Modeltest-ng [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eChloroplast genome structure of\u003c/b\u003e \u003cb\u003eSonerila\u003c/b\u003e \u003cb\u003especies\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe chloroplast genomes in \u003cem\u003eSonerila\u003c/em\u003e species ranged from 154,014 bp (\u003cem\u003eS. nervulosa\u003c/em\u003e) to 155,271 bp (\u003cem\u003eS. annamica\u003c/em\u003e), displaying a difference of 1,257 bp across different genomes (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All 27 chloroplast genomes displayed the typical quadripartite structure, consisting of a large single copy (LSC) region (84,234\u0026ndash;85,272 bp), a small single copy (SSC) region (16,358\u0026ndash;16,558 bp), and a pair of inverted repeat (IR) regions (26.638\u0026ndash;26,817 bp) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The total GC content was consistent across all plastomes, ranging from 37.1\u0026ndash;37.4%, with the average GC content being 37.2%, while the different regions had slightly variable GC content with the LSC, SSC, and IR ranging from 34.9\u0026ndash;35.3%, 30.9\u0026ndash;31.3%, and 42.6%, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Our analysis revealed that the chloroplast genomes of \u003cem\u003eSonerila\u003c/em\u003e species appear to be relatively conserved, and all 27 genomes contained 129 unique genes comprising 84 protein coding genes, 37 transfer RNA genes, and 8 ribosomal RNA genes (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A total of 17 genes contained introns; specifically, the genes \u003cem\u003erpl16\u003c/em\u003e, \u003cem\u003epetD\u003c/em\u003e, \u003cem\u003epetB\u003c/em\u003e, \u003cem\u003etrnV-UAC\u003c/em\u003e, \u003cem\u003etrnL-UAA\u003c/em\u003e, \u003cem\u003erpoC1\u003c/em\u003e, \u003cem\u003eatpF\u003c/em\u003e, \u003cem\u003erps16\u003c/em\u003e, \u003cem\u003etrnK-UUU, ndhA\u003c/em\u003e, \u003cem\u003endhB\u003c/em\u003e, \u003cem\u003etrnA-UGC\u003c/em\u003e, \u003cem\u003etrnI-GAU\u003c/em\u003e, and \u003cem\u003erpl2\u003c/em\u003e each contained one intron, and \u003cem\u003erps12, ycf3\u003c/em\u003e, and \u003cem\u003eclpP1\u003c/em\u003e each contained two introns (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComparison of chloroplast genomes characteristics of \u003cem\u003eSonerila.\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"14\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSpecies\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003eLength (bp)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c9\" namest=\"c6\"\u003e\u003cp\u003e%GC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c13\" namest=\"c10\"\u003e\u003cp\u003eNo. of genes\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c14\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eAccession No.\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGenome size\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLSC Length\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSSC Length\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eIR Length\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eLSC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eSSC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eIR\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003eCDS\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c12\"\u003e\u003cp\u003etRNAs\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c13\"\u003e\u003cp\u003erRNAs\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eS. cannanorensis\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e155,026\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e84,869\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16,557\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26,800\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e35.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e31.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e42.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003ePX251189\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eS. ponmudiana\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e154,940\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e84,789\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16,527\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26,812\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e35.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e31.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e42.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003ePX251190\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eS. speciosa\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e154,598\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e84,536\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16,486\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26,788\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e35.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e31.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e42.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003ePX251191\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eS. sreenarayaniana\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e155,100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e84,982\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16,564\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26,777\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e35.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e30.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e42.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003ePX251192\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eS. janakiana\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e155,027\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e84,875\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16,552\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26,800\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e35.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e31.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e42.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003ePX251193\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eS. malabarica\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e155.084\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e85,045\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16.525\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26.757\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e35.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e31.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e42.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003ePX251194\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eS. amabilis\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e154,811\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e84,790\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16,493\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26,764\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e35.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e30.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e42.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003ePX251195\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eS. lateritica\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e154,862\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e84,834\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16,536\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26,746\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e35.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e31.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e42.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003ePX251196\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eS. tenella\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e154,818\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e84,720\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16,528\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26,785\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e35.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e31.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e42.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003ePX251197\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eS. veldkampiana\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e154,835\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e84,879\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16,488\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26,734\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e35.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e31.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e42.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003ePX251198\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eS. rheedei\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e154,936\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e84,912\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16.528\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26,748\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e35.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e31.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e42.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003ePX251199\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eS. sadasivanii\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e155,018\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e84,900\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16.514\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26,802\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e35.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e31.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e42.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003ePX251200\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eS. tinnevelliensis\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e154,970\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e84,929\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16,527\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26,757\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e35.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e31.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e42.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003ePX251201\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eS. brunonis\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e155,094\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e85,040\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16,538\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26,758\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e35.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e31.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e42.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003ePX251202\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eS. axillaris\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e155,019\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e84,878\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16,555\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26,793\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e35.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e30.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e42.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003ePX251203\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eS. cantonensis_1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e155,265\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e85,272\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16,475\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26,759\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e35.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e31.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e42.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003eMK994791\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eS. cantonensis_2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e155,233\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e85,229\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16,484\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26,760\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e35.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e31.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e42.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003eMK994813\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eS. cantonensis_3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e154,858\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e84,962\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16,358\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26,769\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e35.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e31.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e42.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003eOL813666\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eS. plagiocardia\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e154,428\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e84,361\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16,433\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26,817\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e35.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e30.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e42.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003eMK994876\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eS. pulchella\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e154,862\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e84,862\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16,492\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26,754\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e35.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e31.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e42.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003eMK994884\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eS. velutina\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e154,682\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e84,817\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16,391\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26,737\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e35.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e31.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e42.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003eMK994892\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eS. borneensis\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e154,804\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e84,872\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16,470\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26,731\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e35.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e31.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e42.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003eMK994893\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eS. parviflora\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e154,752\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e84,833\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16,451\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26,734\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e35.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e31.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e42.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003eMK994900\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eS. obliqua\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e154,978\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e85,026\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16,518\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26,717\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e34.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e31.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e42.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003eOL813672\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eS. annamica\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e155,271\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e85,198\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16,545\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26,764\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e35.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e31.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e42.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003eOL813695\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eS. nervulosa_1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e154,260\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e84,306\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16,470\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26,742\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e35.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e31.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e42.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003eOL813716\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eS. nervulosa_2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e154,014\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e84,234\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16,504\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26,638\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e35.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e31.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e42.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003eOL813725\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eCodon usage analysis\u003c/h3\u003e\n\u003cp\u003eThe results showed that all protein-coding genes in \u003cem\u003eSonerila\u003c/em\u003e consisted of 64 codons, and encoded 20 amino acids, including three stop codons (UAA (*), UAG (*), UGA (*)) (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). The number of encoded codons exhibited minimal variation, the types of codons and amino acids remained consistent. Out of the 64 codons, excluding the three stop codons and the unbiased methionine (Met) and tryptophan (Trp) (RSCU\u0026thinsp;=\u0026thinsp;1), 32 codons displayed a preference with an RSCU value exceeding 1, indicating a higher priority for these codons. Among them, the AGA codon for Arginine (Arg) had the highest frequency as indicated by an average RSCU value of 1.94. The remaining 30 analyzed codons showed relatively low bias, with RSCU values less than 1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The codons in the 27 \u003cem\u003eSonerila\u003c/em\u003e chloroplast genomes exhibited a preference for A/T bases and A/T-ending codons, as evidenced by the GC and GC3 content being below 0.5. Moreover, analysis of codon adaptation index values and an effective number of codon values revealed a minor tendency toward biased codon usage. The frequency of optimal codons was relatively low. Furthermore, the hydrophobicity and aromaticity of the protein had a minimal effect on the observed bias in codon usage.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eRepeat sequence analysis and simple sequence repeats (SSRs)\u003c/h2\u003e\u003cp\u003eThe highest number of SSRs was identified in \u003cem\u003eS. brunonis\u003c/em\u003e (84), while the smallest number of SSRs was identified in \u003cem\u003eS. parviflora\u003c/em\u003e (51) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The most frequently observed SSR type was mononucleotide repeats, ranging from 32 to 70. All species exhibited mono-, di-, tri-, and tetra-, besides \u003cem\u003eS. janakiana\u003c/em\u003e, \u003cem\u003eS. rheedei\u003c/em\u003e, and \u003cem\u003eS. axillaris\u003c/em\u003e in which no tri-nucleotide repeats were observed. The penta-nucleotide repeats were only observed in 13 species, \u003cem\u003eS. ponmudiana\u003c/em\u003e, \u003cem\u003eS. speciosa\u003c/em\u003e, \u003cem\u003eS. sreenarayaniana\u003c/em\u003e, \u003cem\u003eS. lateritica, S. veldkampiana\u003c/em\u003e, \u003cem\u003eS. rheedei\u003c/em\u003e, \u003cem\u003eS. tinnevelliensis\u003c/em\u003e, \u003cem\u003eS.brunonis\u003c/em\u003e, \u003cem\u003eS. cantonensis\u003c/em\u003e, \u003cem\u003eS. plagiocardia\u003c/em\u003e, \u003cem\u003eS. pulchella\u003c/em\u003e, \u003cem\u003eS. parviflora\u003c/em\u003e, and \u003cem\u003eS.annamica\u003c/em\u003e. The hexa-nucleotide repeats were present in only three species, \u003cem\u003eS. tinnevelliensis\u003c/em\u003e, \u003cem\u003eS. pulchella\u003c/em\u003e, and \u003cem\u003eS. annamica\u003c/em\u003e. Furthermore, the SSRs were mainly distributed in the LSC and SSC regions of the chloroplast genome, while there were few SSRs in the two IR regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn this study, a total of 1,844 SSRs were detected in the chloroplast genomes of 27 \u003cem\u003eSonerila\u003c/em\u003e species, and the majority of SSRs were found in the LSC region, which might be correlated with the length of the LSC region. Additionally, they were predominantly composed of A/T bases, consistent with the AT richness observed in the whole chloroplast genome. The number of long repeats was 1,617, encompassing 5 types: 422 forward repeats, 132 reverse repeats, 676 palindromic repeats, 130 complement repeats, and 257 random repeats. The forward and palindromic repeats were the most abundant among all 27 species. The number of long repeats in \u003cem\u003eS. obliqua\u003c/em\u003e and \u003cem\u003eS. nervulosa\u003c/em\u003e was notably higher than in other \u003cem\u003eSonerila\u003c/em\u003e species: \u003cem\u003eS. obliqua\u003c/em\u003e showed the highest count for palindromic repeats (52), complement (38) and reverse repeats (27), while \u003cem\u003eS. nervulosa\u003c/em\u003e followed closely with 42 palindromic, 24 complement and 29 reverse repeats (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Tandem repeats were the common type among repeats. The majority ranged from 30 to 39 bp in length, with a few from 60 to 69 bp within 4 species, \u003cem\u003eS. cantonensis\u003c/em\u003e, \u003cem\u003eS. ponmudiana\u003c/em\u003e, \u003cem\u003eS. tenella\u003c/em\u003e, and \u003cem\u003eS. velutina\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Among these, \u003cem\u003eS. obliqua\u003c/em\u003e had the highest number of long repeat sequences. The tandem repeats ranged from 6 to 14 in all \u003cem\u003eSonerila\u003c/em\u003e species.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eComparative genomic divergence analysis\u003c/h2\u003e\u003cp\u003eThe whole chloroplast genome sequences encoded gene classes, numbers, and alignments that were highly consistent among \u003cem\u003eSonerila\u003c/em\u003e species. These results indicated no significant alterations such as large fragment inversions, duplications, or other structural changes in all 27 chloroplast genomes. The sequence differences were higher in the LSC and SSC regions compared to the IR regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eSequence divergence and mutation hotspots\u003c/h2\u003e\u003cp\u003eThe Pi values in the IR regions were much lower than those in the LSC and SSC regions. In all 27 \u003cem\u003eSonerila\u003c/em\u003e species, seven regions had a relatively high Pi value (\u0026gt;\u0026thinsp;0.025). Five regions in LSC (\u003cem\u003erps16-trnQ-UUG\u003c/em\u003e, \u003cem\u003etrnS-GCU-trnR-UCU\u003c/em\u003e, \u003cem\u003erbcL-accD\u003c/em\u003e, \u003cem\u003epsbE-petL\u003c/em\u003e, \u003cem\u003erps18-rpl20\u003c/em\u003e), one region at the SSC-IRb boundary (\u003cem\u003eycf1\u003c/em\u003e), and one region within the SSC (\u003cem\u003erps15\u003c/em\u003e) were considered as hotspots (Pi\u0026thinsp;\u0026gt;\u0026thinsp;0.025), and the \u003cem\u003eycf1\u003c/em\u003e had the highest nucleotide diversity (Pi\u0026thinsp;\u0026gt;\u0026thinsp;0.03), suggesting a potential useful region for evolutionary studies within \u003cem\u003eSonerila\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). Among the 15 \u003cem\u003eSonerila\u003c/em\u003e species from India, two regions in the LSC (\u003cem\u003erps16\u003c/em\u003e and \u003cem\u003erpl22\u003c/em\u003e), one region in SSC (\u003cem\u003erpl32\u003c/em\u003e), and one region at SSC-IRb boundary (\u003cem\u003eycf1\u003c/em\u003e) were considered as hotspots (Pi\u0026thinsp;\u0026gt;\u0026thinsp;0.014) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The \u003cem\u003eycf1\u003c/em\u003e, \u003cem\u003erps15\u003c/em\u003e, \u003cem\u003erps16\u003c/em\u003e, \u003cem\u003erpl22\u003c/em\u003e, and \u003cem\u003erpl32\u003c/em\u003e regions not only exhibited high sequence variability but were also coding region sequences.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIR contraction and expansion in the\u003c/b\u003e \u003cb\u003eSonerila\u003c/b\u003e \u003cb\u003echloroplast genomes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe chloroplast genome is a circular structure consisting of the LSC, SSC, IRa and IRb regions, with four boundaries: LSC-IRb (JLB), IRb-SSC (JSB), SSC-IRa (JSA), and IRa-LSC (JLA). Expansion and contraction of the IR regions are well-known and significant events in plant evolutionary history and directly impact the size of the genome, gene content, order and rearrangement [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The LSC/IRb/SSC/IRa boundaries of \u003cem\u003eSonerila\u003c/em\u003e species were compared to analyze the expansion and contraction variation in junction regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The genotypes of the IR/LSC and IR/SSC boundaries were essentially identical, and the lengths of IRs across species were relatively conserved (26,638\u0026ndash;26,817 bp) but still with significant expansion or contraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Six protein-coding genes (\u003cem\u003erpl22\u003c/em\u003e, \u003cem\u003erps19\u003c/em\u003e, \u003cem\u003erpl2\u003c/em\u003e, \u003cem\u003endhF\u003c/em\u003e, \u003cem\u003eycf1\u003c/em\u003e, \u003cem\u003epsbA\u003c/em\u003e) were present near or at the LSC/IR and SSC/IR boundaries. At the LSC/IRb (JLB) boundary, a fragment of \u003cem\u003erps19\u003c/em\u003e gene was detected with a small change in length. The \u003cem\u003erps19\u003c/em\u003e in the IRb region varied from 78\u0026ndash;115 bp, whereas in the LSC region 201 bp were consistently present across species, except in \u003cem\u003eS. plagiocardia\u003c/em\u003e, where it was reduced to164 bp. This indicates a slight IR expansion into the LSC, resulting in the incorporation of a larger portion of the \u003cem\u003erps19\u003c/em\u003e gene. At the IRb/SSC (JSB) boundary, the pseudogene \u003cem\u003eycf1 (ψycf1)\u003c/em\u003e and \u003cem\u003endhF\u003c/em\u003e were consistently detected in all the species. The pseudogene \u003cem\u003eycf1 (ψycf1)\u003c/em\u003e spanned the IRb region for 1,827 to 1,848 bp and extended into the SSC region for an additional 56 to 62 bp; the \u003cem\u003endhF\u003c/em\u003e gene extended across the IRb region for 44 to 50 bp and into the SSC region for 2,185 to 2,200 bp, overlapping with the pseudogene \u003cem\u003eycf1 (ψycf1)\u003c/em\u003e. At the boundary of SSC/IRa (JSA), the full-length \u003cem\u003eycf1\u003c/em\u003e gene spanned 3,591 to 3,675 bp across the SSC region and 1,827 to 1,848 bp across the IRa region. At the IRa/LSC (JLA) boundary, the \u003cem\u003etrnH\u003c/em\u003e gene was located on the right side of the boundary for 0 to 2 bp. The IR/SC boundary differed among the chloroplast genomes of \u003cem\u003eS. lateritica\u003c/em\u003e and \u003cem\u003eS. nervulosa\u003c/em\u003e (OL813725). In \u003cem\u003eS. lateritica\u003c/em\u003e, the apparent absence of \u003cem\u003erps19\u003c/em\u003e at the boundary was likely due to an annotation gap, as manual inspection confirmed its presence. In contrast, in \u003cem\u003eS. nervulosa\u003c/em\u003e (OL813725), \u003cem\u003erps19\u003c/em\u003e was entirely located within the LSC region, 16 bp from the LSC/IRb (JLB) boundary rather than spanning it. Additionally, the \u003cem\u003erpl2\u003c/em\u003e was positioned 44 bp closer to the IRa/LSC (JLA), suggesting a contraction of the IR regions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003ePhylogenetic analyses\u003c/h2\u003e\u003cp\u003eA phylogenetic tree was constructed using maximum likelihood (ML) methods utilizing the 72 complete chloroplast genomes sequences from tribe Sonerileae, with four additional sequences from the Dissochaeteae, Pyxidantheae, and Melastomateae. Sonerileae species were selected to represent morphological diversity, particularly the variation in floral merosity (ranging from 3- to 6-merous) and stamen morphology (isomorphic and dimorphic types) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Most nodes across the phylogenetic tree showed high support (BS\u0026thinsp;\u0026gt;\u0026thinsp;70). \u003cem\u003eSonerila\u003c/em\u003e species clustered into a single, strongly supported clade (BS\u0026thinsp;\u0026gt;\u0026thinsp;99). \u003cem\u003eSonerila obliqua\u003c/em\u003e (Southeast Asia), \u003cem\u003eS. nervulosa\u003c/em\u003e (Borneo), and \u003cem\u003eS. plagiocardia\u003c/em\u003e (South China, Indochina) were identified as early diverging species, successively sister to the rest of \u003cem\u003eSonerila\u003c/em\u003e. A clade of predominantly Bornean species was recovered sister to a clade that included species from South China, Indochina and India. Fourteen of the 15 species assembled from the Indian subcontinent formed a clade relative to other \u003cem\u003eSonerila\u003c/em\u003e species, with the exception of \u003cem\u003eS. amabilis\u003c/em\u003e, which is a Himalayan species from Sikkim (India), and clustered with \u003cem\u003eS. cantonensis\u003c/em\u003e (South China) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Overall, the phylogenetic reconstruction of \u003cem\u003eSonerila\u003c/em\u003e revealed strong geographic structuring, with distinct clades representing the Indian subcontinent, South China and Indochina, the Himalayan region, and Southeast Asia (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe aim of this study was to provide an overall assessment of the chloroplast genome structure and sequence variation in \u003cem\u003eSonerila\u003c/em\u003e, one of the least known, yet widespread, paleotropical taxa. Additionally, we attempted to evaluate the utility of the chloroplast genome for generating hypotheses on the origin of Indian species. The origin, diversification, and historical biogeography of \u003cem\u003eSonerila\u003c/em\u003e remains unknown. Only a few phylogenies including minimal taxon sampling [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e] and several independent regional taxonomic studies, mainly new species descriptions [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], are available. Indian \u003cem\u003eSonerila\u003c/em\u003e represents the westernmost distribution of this group, showcasing remarkable morphological diversity, distinguishing these species from others across the Indomalayan region. Our study represents a first attempt to examine the chloroplast diversity in this group and gather initial evidence in support of either a single or multiple origins of \u003cem\u003eSonerila\u003c/em\u003e in India.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStructural characteristics of the chloroplast genome in\u003c/b\u003e \u003cb\u003eSonerila\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe chloroplast genome is a valuable resource for studying intra- and inter-species evolution and developing molecular markers [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In this study, we sequenced and annotated the chloroplast genomes of \u003cem\u003eSonerila\u003c/em\u003e species from India, and subsequently, compared their genomic features with those of other \u003cem\u003eSonerila\u003c/em\u003e species and close relatives available on public databases. Our results show that \u003cem\u003eSonerila\u003c/em\u003e plastomes are conserved in gene order and length, with quadripartite structure, as previously observed in other representatives of Melatomataceae [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and Sonerileae [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The structure included a large single copy region (LSC), a small single copy region (SSC), and two inverted repeat regions (IRs). The major variation in genome size may be attributed to the varying length of LSC and IRs across species of \u003cem\u003eSonerila\u003c/em\u003e. The chloroplast genome of \u003cem\u003eS. nervulosa\u003c/em\u003e (OL813725) exhibits contracted IR regions, measuring 26,638 bp in length and resulting in the smallest genome size within the \u003cem\u003eSonerila\u003c/em\u003e species analyzed (154,014bp).\u003c/p\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eAnalysis of codon bias and repetitive sequences\u003c/h2\u003e\u003cp\u003eRelative Synonymous Codon Usage (RSCU) is often used to reflect codon bias [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The GC content of the chloroplast genomes reflects a balance between mutation pressure and adaptation, which is one of the most significant factors contributing to the patterns of codon usage bias [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Although synonymous mutations of the third base of a codon do not alter the resulting amino acid, they are still considered important features due to their influence on factors like codon usage bias, and other molecular processes. Therefore, GC3 is often used as a significant indicator of codon preference [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Herein, the chloroplast genomes of \u003cem\u003eSonerila\u003c/em\u003e species revealed that all optimal synonymous codons (RSCU\u0026thinsp;\u0026gt;\u0026thinsp;1), except for UUG, CCC, AGG, and GGG, terminated with A or U, indicating a general preference for A/T bases across the genomes. RSCU can affect gene expression by regulating the accuracy and efficiency of translation, with stronger RSCU values associated with increased levels of gene expression. In chloroplast gene expression vector design, optimizing codons according to their bias can boost the expression levels of inserted genes in the chloroplast genome. In addition, known codon usage patterns can help predict the expression and function of unknown genes [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The identified codons with RSCU\u0026thinsp;\u0026gt;\u0026thinsp;1 can serve as efficient indicators for detecting the expression levels of hypothetical genes or open reading frames.\u003c/p\u003e\u003cp\u003eRepetitive sequences affect the transcriptional regulation of genes, protein translation, chromosome formation and metabolism, and may reflect differences between mutation frequencies and evolutionary rates of species [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. We observed differences in the number and distribution of repeats types and lengths across \u003cem\u003eSonerila\u003c/em\u003e species. The presence and abundance of repetitive sequences in the chloroplast genome are likely to involve the phylogenetic history and genomic adaptation. Simple Sequence Repeats (SSRs) are abundant and exbibit significant genetic variation, making them effective markers for population genetics and biodiversity studies [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. In \u003cem\u003eS. nervulosa\u003c/em\u003e and \u003cem\u003eS. obliqua\u003c/em\u003e, the number of reverse and complement repeats is significantly higher than in the other species, which may indicate unique genomic features or evolutionary adaptations. Overall, the SSRs and long repeat sequences identified in this study provide useful information for further research on molecular marker development, population genetics, species delimitation, and conservation studies in \u003cem\u003eSonerila\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eComparative genomics and highly variable regions analysis\u003c/h2\u003e\u003cp\u003eThe contraction and expansion of IR regions during evolution is a relatively common occurrence and has been employed as an evolutionary marker for phylogenetic studies. This dynamic process is a primary factor leading to variations in the length and number of genes in many species [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. In this study, significant similarities were found in the expansion or contraction of the IR regions in \u003cem\u003eSonerila\u003c/em\u003e species. The LSC/IRb boundary was generally located on the \u003cem\u003erps19\u003c/em\u003e gene in 26 \u003cem\u003eSonerila\u003c/em\u003e species, except for \u003cem\u003eS. lateritica\u003c/em\u003e and \u003cem\u003eS. nervulosa\u003c/em\u003e (OL813725), where the contraction of IR regions resulted in the \u003cem\u003erps19\u003c/em\u003e gene moving from the LSC/IRb boundary to LSC region to decrease the size of the chloroplast genome. Additionally, the expansion of the IR resulted in partially replicated \u003cem\u003eycf1\u003c/em\u003e genes being in the IRb region, thus generating the pseudogene \u003cem\u003eycf1\u003c/em\u003e at the IRB/SSC (JSB) boundary. The pseudogenes in the chloroplast genomes at the SSC/IRa boundary are often found in land plants [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Overall, the conserved IR region in \u003cem\u003eSonerila\u003c/em\u003e may contribute to the length and structural stability of the chloroplast genome.\u003c/p\u003e\u003cp\u003eDNA barcoding technology has a wide range of application in the fields of species identification, conservation, phylogeny, and evolution [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Previous phylogenetic analyses have mostly used the ITS region of nuclear DNA and \u003cem\u003eycf1\u003c/em\u003e, \u003cem\u003erps15\u003c/em\u003e, \u003cem\u003erps16-trnQ\u003c/em\u003e, and \u003cem\u003etrnL-trnF\u003c/em\u003e in the chloroplast genome [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Moreover, although the Consortium for the Barcode of Life (CBOL) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.barcodeoflife.org/\u003c/span\u003e\u003cspan address=\"http://www.barcodeoflife.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) has recommended \u003cem\u003ematK\u003c/em\u003e and \u003cem\u003erbcL\u003c/em\u003e genes as core plant barcodes [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], these fail to provide phylogenetic resolution at shallow levels in species-complexes where lineages evolved through recent or rapid radiation or may be the product of hybridization and introgression [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. While recent studies recommend the use of complete chloroplast genomes for shallow phylogenetic-level studies [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e], identifying informative regions may still be a challenge, especially for species delimitation. Our results demonstrate that screening for informative chloroplast markers is a difficult task, considering that most phylogenetic informative regions vary even across closely related species [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. However, the chloroplast genome represents a valuable resource and provides an important perspective when used in combination with additional genetic data. This study identified one region (\u003cem\u003eycf1\u003c/em\u003e) with consistently high variability value (\u0026gt;\u0026thinsp;0.03) across all 27 \u003cem\u003eSonerila\u003c/em\u003e species. Additionally, six other regions (\u003cem\u003erps15\u003c/em\u003e, \u003cem\u003epsbE\u003c/em\u003e-\u003cem\u003epetL\u003c/em\u003e, \u003cem\u003etrnS-GCU\u003c/em\u003e-\u003cem\u003etrnR-UCU\u003c/em\u003e, \u003cem\u003erps16\u003c/em\u003e-\u003cem\u003etrnQ-UUG\u003c/em\u003e, \u003cem\u003erbcL\u003c/em\u003e-\u003cem\u003eaccD\u003c/em\u003e, \u003cem\u003erps18\u003c/em\u003e-\u003cem\u003erpl20\u003c/em\u003e) showed relatively high variability value (\u0026gt;\u0026thinsp;0.025). Notably, the \u003cem\u003eycf1\u003c/em\u003e gene (0.035) located at the SSC-IR boundary, further supports these regions as molecular hotspots. Therefore, all seven regions should be considered most suitable to evaluate phylogenetic relationships among \u003cem\u003eSonerila\u003c/em\u003e species. However, in the specific case of Indian \u003cem\u003eSonerila\u003c/em\u003e, four specific regions (\u003cem\u003eycf1\u003c/em\u003e, \u003cem\u003erps16\u003c/em\u003e, \u003cem\u003erpl22\u003c/em\u003e, and \u003cem\u003erpl32\u003c/em\u003e) presented relatively high Pi values (\u0026gt;\u0026thinsp;0.014), and therefore, these would be most suitable as DNA barcodes and for resolving phylogenetic relationships. Overall, lower variability in this clade may reflect a recent diversification, which has limited the accumulation of plastome divergence. Alternatively, it may be influenced by historical demographic events, such as a genetic bottleneck.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eAssessment of phylogenetic resolution\u003c/h2\u003e\u003cp\u003eWe conducted phylogenetic analyses including 72 Sonerileae species and four species from tribes Dissochaeteae, Pyxidantheae, and Melastomateae, utilizing newly generated chloroplast genome data from this study and publicly available sequences. Our findings align well with recent phylogenomic studies of Sonerileae. Recent studies have advanced our understanding of this group's evolutionary relationships. For instance, Zhou et al. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] analyzed 151 complete chloroplast genomes, providing strong support for the backbone phylogeny of the Sonerileae/Dissochaeteae clade. Similarly, another study [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] identified 34 robust lineages within Asian Sonerileae by employing a combination of single-copy orthologs (SCOs), genomic SNPs, and whole plastome data. Independently, Quankenbush et al. [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e] analyzed 385 nuclear and 81 plastid protein-coding loci to explore the relationships among fleshy-fruited genera such as \u003cem\u003eCatanthera\u003c/em\u003e, \u003cem\u003eHeteroblemma\u003c/em\u003e, \u003cem\u003eKendrickia\u003c/em\u003e, \u003cem\u003eMedinilla\u003c/em\u003e, \u003cem\u003ePachycentria\u003c/em\u003e, and \u003cem\u003ePlethiandra\u003c/em\u003e revealing their distribution across three distinct evolutionary lineages.\u003c/p\u003e\u003cp\u003eCollectively, these studies consistently support the monophyly of \u003cem\u003eSonerila\u003c/em\u003e [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e], although it is worth noting that these results might have been influenced by very limited inclusion of \u003cem\u003eSonerila\u003c/em\u003e species. In this study, we selected 15 \u003cem\u003eSonerila\u003c/em\u003e species from India and 12 from published work, representing the wide distribution of the group. We constructed a robust phylogenetic tree based on the whole chloroplast genome sequences of all sampled Sonerileae species. Our results showed that \u003cem\u003eSonerila\u003c/em\u003e is monophyletic, and the species from the Indian subcontinent cluster together forming a well-supported clade. The Indian \u003cem\u003eS. amabilis\u003c/em\u003e, endemic to Sikkim, is a Himalayan species, nested with \u003cem\u003eS. cantonensis\u003c/em\u003e, which is from nearby southern China and Indochina. It is not surprising that it does not cluster with the rest of the species from the Indian subcontinent. Overall, our phylogenetic reconstruction while confirming the monophyly of \u003cem\u003eSonerila\u003c/em\u003e, reveals some interesting geographic structure. Notably, the placement of Southeast Asian species (\u003cem\u003eS. obliqua, S. nervulosa\u003c/em\u003e) may suggest that Southeast Asia harbors the potential center of early diversification and ancestral range for the group. This hypothesis is consistent with the high diversity of \u003cem\u003eSonerila\u003c/em\u003e species documented in Malaysia, Sumatra, and Borneo regions recognized as biodiversity hotspots and frequently proposed as centers of origin for tropical plant radiations [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. This also suggests that \u003cem\u003eSonerila\u003c/em\u003e may have diversified and dispersed along an east-west route, with India and Sri Lanka representing the westernmost edge of its distribution, and potentially, the most recently diverging lineages.\u003c/p\u003e\u003cp\u003eFurther studies integrating nuclear genomic data, biogeographic modeling, and expanded sampling are needed to clarify this evolutionary scenario and test our hypotheses. Overall, these results provide important implications for the assessment of genetic diversity and phylogenetic studies in \u003cem\u003eSonerila\u003c/em\u003e, highlighting the need for continued research.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, the chloroplast genomes of 15 \u003cem\u003eSonerila\u003c/em\u003e species were assembled and annotated, and a series of comparative analyses were performed using 12 additional published \u003cem\u003eSonerila\u003c/em\u003e genomes. The results revealed that \u003cem\u003eSonerila\u003c/em\u003e chloroplast genomes exhibited conserved structural features and gene content, while variations were observed in genome size, sequence divergence, and repeat elements. The identification of long repetitive sequences, simple sequence repeats (SSRs), and seven regions with high variability are phylogenetically informative. We reconstructed a phylogenetic scenario that suggest \u003cem\u003eSonerila\u003c/em\u003e is a monophyletic entity, with a putative center of origin in the Southeast Asia region. The high level of endemism observed in India highlights the significant evolutionary diversification of this clade in the region. Specifically, we hypothesize a single independent introduction in the Indian subcontinent, with subsequent diversification events, especially in the Western Ghats. This preliminary hypothesis warrants further testing using a much broader taxon sampling that include additional species from the entire distribution range. The results of this study enhance our understanding of chloroplast genome evolution in \u003cem\u003eSonerila\u003c/em\u003e and contribute to future studies focusing on species delimitation and historical evolution of this group.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported in part from funding from the National Science Foundation, NSF-DEB 2002270.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJH, MRB, NC designed the study; JH and MRB performed the analyses; All authors contributed to the writing of the paper.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eRS would like to thank the International Association of plant Taxonomists (IAPT) and the Botanical Survey of India for supporting fieldwork activities.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eRaw reads have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject PRJNA1306189 **,** Study SRP612796). Assembled chloroplast genomes are deposited in GenBank. All accessions are listed in Supplementary Table S5. A reviewer link for accessing the BioProject prior to public release is available here: ( https:/dataview.ncbi.nlm.nih.gov/object/PRJNA1306189?reviewer=2medqtabcjv5gcfglbr8j481cc ). All data will be made publicly available upon publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eUlloa Ulloa C, Almeda F, Goldenberg R, Kadereit G, Michelangeli FA, Penneys DS, Stone RD, Veranso-Libalah MC. Melastomataceae: Global Diversity, Distribution, and Endemism. In: Goldenberg R, Michelangeli FA, Almeda F, editors. Systematics, Evolution, and Ecology of Melastomataceae. Cham: Springer Nature Switzerland AG; 2022. pp. 3\u0026ndash;28. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-3-030-99742-7_1\u003c/span\u003e\u003cspan address=\"10.1007/978-3-030-99742-7_1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGoldenberg R, Michelangeli FA, Almeda F, editors. Systematics, Evolution, and Ecology of Melastomataceae. Systematics, Evolution, and Ecology of Melastomataceae. 1st ed. 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Plants. 2024;13(16):2281. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/plants13162281\u003c/span\u003e\u003cspan address=\"10.3390/plants13162281\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Chloroplast genome, Genome structure, Indian Sonerila, Sonerileae","lastPublishedDoi":"10.21203/rs.3.rs-7538746/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7538746/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003e\u003cem\u003eSonerila\u003c/em\u003e, the second largest genus within the tribe Sonerileae (Melastomaceae), is primarily distributed across tropical and subtropical Asia, from India and Sri Lanka, where it exhibits a remarkably high degree of endemism, to Papua New Guinea. In this study, we assembled and annotated the chloroplast genomes of 15 \u003cem\u003eSonerila\u003c/em\u003e species from India (14 from the subcontinent and one from Sikkim in the Eastern Himalaya) and compared them to 12 previously published genomes representing species from other regions. We performed comparative analyses of gene structure, sequence alignment, nucleotide diversity and phylogenetic reconstructions to evaluate the utility of plastid regions for resolving phylogenetic relationships and understanding plastome evolution within \u003cem\u003eSonerila\u003c/em\u003e and close relatives.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eOur results demonstrate that the complete chloroplast genome exhibit quadripartite structures, with lengths ranging from 154,014 (\u003cem\u003eS. nervulosa\u003c/em\u003e) to 155,271 bp (\u003cem\u003eS. annamica\u003c/em\u003e). The genome structure remains relatively conserved, hosting 129 annotated genes, including 84 protein coding genes, 37 tRNA genes, and 8 rRNA genes. The overall GC contents were almost identical (37.1\u0026ndash;37.4%), with an average of 37.2%. 51 to 84 SSRs and long repeat sequences were detected. A comparison of nucleotide diversity among all 27 \u003cem\u003eSonerila\u003c/em\u003e chloroplast genomes revealed seven regions (\u003cem\u003eycf1\u003c/em\u003e, \u003cem\u003erps15\u003c/em\u003e, \u003cem\u003epsbE-petL\u003c/em\u003e, \u003cem\u003etrnS-GCU-trnR-UCU, rps16-trnQ-UUG, rbcL-accD, rps18-rpl20\u003c/em\u003e) displayed relatively high nucleotide diversity (Pi\u0026thinsp;\u0026gt;\u0026thinsp;0.025). In contrast, the chloroplast genomes from Indian \u003cem\u003eSonerila\u003c/em\u003e species showed lower diversity, with only four regions (\u003cem\u003eycf1, rps16, rpl32, rpl22\u003c/em\u003e) exhibiting notable Pi value (\u0026gt;\u0026thinsp;0.014). Maximum likelihood phylogenetic analysis strongly confirmed the monophyly of \u003cem\u003eSonerila\u003c/em\u003e within the Sonerileae and showed that the structure of the main clades correlated to species geographic distribution, including a single origin for \u003cem\u003eSonerila\u003c/em\u003e in the Indian subcontinent.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThe chloroplast genomes of \u003cem\u003eSonerila\u003c/em\u003e exhibits conserved structural features and gene content and variations were observed in genome size, sequence divergence, and repeat elements. Overall, long repetitive sequences, simple sequence repeats (SSRs), and regions with high variability are phylogenetically informative. Phylogenetic analyses suggest that \u003cem\u003eSonerila\u003c/em\u003e is a monophyletic entity and the Indian subcontinent species originated from a single ancestor. By providing a comprehensive genomic characterization of \u003cem\u003eSonerila\u003c/em\u003e chloroplast, this study delivers essential genetic insights for advancing future research on the evolutionary history and adaptive diversification of \u003cem\u003eSonerila\u003c/em\u003e species.\u003c/p\u003e","manuscriptTitle":"Complete chloroplast genomes suggest a single origin of the Indian subcontinent Sonerila (Melastomataceae)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-05 17:25:39","doi":"10.21203/rs.3.rs-7538746/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8065e4d7-da08-4b1b-a972-bbb09415a11e","owner":[],"postedDate":"November 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-29T08:26:01+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-05 17:25:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7538746","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7538746","identity":"rs-7538746","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}