Plastome Evolution at the Edge: Structural Rearrangements, IR Expansion, and Gene Flux in Hypericaceae (Malpighiales)

Plastome Evolution at the Edge: Structural Rearrangements, IR Expansion, and Gene Flux in Hypericaceae (Malpighiales) | 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 Plastome Evolution at the Edge: Structural Rearrangements, IR Expansion, and Gene Flux in Hypericaceae (Malpighiales) Sivagami Jean Claude, Kyutae Park, SeonJoo Park This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7295767/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Dec, 2025 Read the published version in BMC Plant Biology → Version 1 posted 13 You are reading this latest preprint version Abstract Background The evolutionary history of the Hypericaceae Juss. family remains poorly understood despite previous phylogenomic efforts. A prior study on Hypericum ascyron revealed exceptional plastome rearrangements and gene loss events, prompting questions about whether such genomic patterns are unique to Hypericum or reflect broader evolutionary trends within the family. Results To explore plastome evolution across Hypericaceae , we sequenced 14 complete chloroplast genomes representing seven genera from the three major tribes: Hypericeae , Vismieae , and Cratoxyleae , alongside two genera from the related family Clusiaceae . Comparative analyses revealed extensive variation in plastome structure and gene content, including lineage-specific rearrangements, inversions, and expansions of inverted repeat (IR) regions. Notably, species within Vismieae exhibited significantly expanded IR regions. In Hypericum , unique lineage-specific open reading frames (ORFs) were identified, with genes such as accD and matK relocated into or near the IR regions, likely driven by repeat-mediated recombination. Multiple independent losses of genes ( rpl23 , rpl32 , rps16 , infA , ycf1 , ycf2 ) and introns were observed across the family, particularly in Hypericeae , often accompanying structural rearrangements. Additionally, matK was translocated from its typical position within the trnK-UUU intron into the IR region, a rare event in angiosperm plastomes. The protein-coding genes accD and clpP also showed domain-disrupting expansions, potentially impacting their functional roles. Conclusions Our results demonstrate that plastome evolution in Hypericaceae is highly dynamic, characterized by substantial structural plasticity, gene loss, and lineage-specific innovation. These findings provide new insights into plastome diversification across the family and lay the groundwork for further phylogenomic and evolutionary studies within Malpighiales. Plastome evolution Hypericaceae Hypericum Genome rearrangements matK relocation IR expansion Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The order Malpighiales Juss ex Bercht & J. Presl, one of the most taxonomically and ecologically diverse clades of flowering plants, was first resolved through molecular phylogenetic studies by Chase et al. ( 1993 ). This reclassification reshaped the understanding of angiosperm relationships and highlighted the potential of molecular tools in resolving deep evolutionary lineages (Chase et al., 1993 , Savolainen et al., 2000 , Soltis et al., 2000 ). Among its major subclades, the clusioid clade comprising five families: Bonnetiaceae L. Beauvis Ex Nakai, Calophyllaceae J.Agardh, Clusiaceae Lindl., Hypericaceae Juss, and Podostemaceae Rich., represents a morphologically and biogeographically complex lineage with over 1,900 species spread across 94 genera (Ruhfel et al., 2011 , Stevens, 2007a , Wurdack and Davis, 2009 ). Members of this clade are predominantly tropical, though certain genera extend into temperate and high-altitude regions, often adapting to extreme or specialized ecological niches such as montane forests and fast-flowing aquatic systems (Davis et al., 2005, Ruhfel et al., 2016 ). Within the clusioid clade, Hypericaceae stands out due to its taxonomic complexity and unevenly studied genera. Historically treated as a subfamily within Clusiaceae, Hypericaceae was elevated to family rank based on distinct morphological and molecular features (Kubitzki et al., 2007 ). It currently comprises approximately 518–700 species across nine genera and is taxonomically divided into three tribes: Hypericeae Choisy, Vismieae Vandelli, and Cratoxyleae Engl. APG III, 2009 (Crockett and Robson, 2011 ). The genus Hypericum , the most species-rich lineage with a cosmopolitan distribution, accounts for nearly 80% of the family’s diversity. In contrast, the other genera, many of which are confined to tropical regions, have remained poorly sampled in molecular studies, leaving tribal and intergeneric relationships largely unresolved (Nürk et al., 2013 , Stevens, 2007b ). Previous phylogenetic efforts in Hypericaceae have focused heavily on Hypericum , employing morphological data (Robson, 1977 , Robson, 1981a , Robson, 1981b , Robson, 1985 , Robson, 1990 , Robson, 2006 , Robson, 2010a , Robson, 2010b , Robson, 2012 ), ITS markers (Nurk and Blattner, 2010 , Park and Kim, 2004 ), and plastid regions (e.g., trn L- trn F, psb A- trn H). These studies provided key insights into biogeographic patterns, such as the disjunction between New World and Old World clades, and proposed evolutionary scenarios involving ancient Gondwanan dispersal and multiple niche shifts (Meseguer et al., 2015 , Nürk et al., 2015 , Meseguer et al., 2014 ). However, these single- or low-copy gene datasets offered limited resolution at deeper nodes and often failed to resolve intertribal relationships, especially for Vismieae and Cratoxyleae. Plastid genomes (plastomes) have emerged as powerful tools for resolving angiosperm relationships at both deep and shallow phylogenetic levels (Jansen et al., 2007 ). Their quadripartite structure, gene content, and substitution dynamics offer evolutionary signals that are often inaccessible through nuclear markers alone. Furthermore, increasing evidence suggests that plastome structural variation, such as inversions, IR boundary shifts, and gene loss, is not just phylogenetically informative but may also reflect lineage-specific ecological adaptations and genome evolution under selection (Guisinger et al., 2011a, Wicke et al., 2011 ). In this context, H . ascyron . was recently shown to possess one of the most highly rearranged plastomes among angiosperms, with large inversions, pseudogenization, and accelerated substitution rates (Claude et al., 2022 ). Whether this pattern is unique to H. ascyron or indicative of a broader trend across Hypericaceae remains unclear. To address this question and improve resolution of phylogenetic relationships within Hypericaceae, we sequenced and analyzed complete chloroplast genomes from 14 species across all three tribes: Hypericeae (including Hypericum , Triadenum Raf., and Thornea Breedlove & E.M.McClint), Vismieae ( Harungana Lam., Vismia Vand.), and Cratoxyleae ( Cratoxylum Blume., Eliea (Lam.) Cambess.). Two species from Clusiaceae were included as outgroups. By combining comparative plastome analysis with phylogenomic reconstruction based on 67 plastid protein-coding genes, we aimed to: Assess the extent and pattern of structural plastome variation across the family, Investigate gene loss, pseudogenization, and possible functional relocation events and Resolve tribal and infrageneric relationships in light of previous nuclear and morphological findings. Our results provide new insight into plastome evolution in Hypericaceae and reveal lineage-specific trends in genome reorganization that align with ecological transitions and biogeographic structuring. These findings not only refine the phylogenetic backbone of the family but also shed light on how structural plastome dynamics correlate with environmental adaptation and diversification. Results Variation in Plastome Structure and Size across Hypericaceae Tribes Plastome sizes in the tribe Hypericeae ranged from 138,163 bp in Hypericum patulum to 167,482 bp in Triadenum fauriei (Table 1 ). The large single-copy (LSC) region exhibited substantial variation, spanning from 103,150 bp in H. patulum to 85,948 bp in H. calcicola . The inverted repeat (IR) region reached its maximum length in H. laxum (32,823 bp) and its minimum in H. patulum (12,032 bp). The small single-copy (SSC) region showed a narrower range, from 10,423 bp in H. laxum to 12,421 bp in T. calcicola . In the Vismieae tribe, plastome sizes were generally larger, ranging from 166,590 bp in Vismia baccifera to 176,170 bp in Harungana madagascariensis (Fig. 1 ). The LSC region measured 67,585 bp in V. baccifera and 57,619 bp in H. madagascariensis , while the IR region was largest in H. madagascariensis (52,124 bp) and smallest in V. baccifera (42,583 bp). The SSC region in both species remained within the size range observed in Hypericeae. For the Cratoxyleae tribe, plastome sizes were more conserved, with a median length of approximately 156–157 kb. The LSC region measured 85,588 bp in Eliea articulata and 86,024 bp in Cratoxylum maingayi . The SSC regions ranged from 17 to 18 kb, and the IR regions ranged from 25 to 26 kb. These values are comparable to those found in the outgroup plastomes of Mesua riparia and Rheedia edulis . Across all three tribes, plastome sizes varied from 138,163 bp ( H. patulum ) to 176,170 bp ( H. madagascariensis ), reflecting both lineage-specific expansions and reductions in chloroplast genome architecture (Table 1 ). Table 1 able 1. Summary of plastome features for 15 Hypericaceae species used in this study for phylogenetic analysis. The table includes total plastome size, lengths of the large single- copy (LSC), inverted repeat (IR), and small single-copy (SSC) regions, GC content, and the number of protein-coding sequences (CDS) and tRNA genes. Species Total LSC IR SSC GC % CDS tRNA H . ascyron 162,286 97,542 26,846 11,052 37.40% 74 34 H. erectum 143,167 99,272 16,370 11,155 37.10% 72 34 H. chejuense 142,029 98,131 16,375 11,148 37.20% 73 34 H. patulum 138,163 103,150 12,032 10,949 38.10% 72 34 H . laxum 165,288 89,219 32,823 10,423 38.10% 73 34 H. japonicum 165,190 89,123 32,805 10,457 32.00% 75 34 T. fauriei 167,482 92,077 31,916 11,191 37.40% 75 34 T . calcicola 150,935 85,948 26,283 12,421 37.90% 75 34 H . madagascariensis 176,170 57,619 52,124 14,303 36.90% 75 35 H . rubescens 174,661 57,746 51,809 13,297 37.10% 75 35 V . baccifera 166,590 67,585 42,583 13,839 36.90% 75 35 E . articulata 156,123 85,588 25,640 19,254 36.40% 77 35 C . maingayi 157,279 86,024 26,155 18,945 36.30% 77 35 M . riparia 156,826 84,914 27,057 17,798 36.00% 77 35 R . edulis 158,160 86,384 27,010 17,756 35.80% 78 35 Plastome Size Variation in Hypericaceae and Related Malpighiales Lineages To evaluate patterns of plastome size evolution within Hypericaceae and across the broader order Malpighiales, we compared plastome sizes among 381 species, including representatives from both focal and outgroup families (Fig. 2 ). The distribution of plastome sizes revealed substantial inter- and intra-lineage variability. Species within Hypericum , including H. ascyron , H. laxum , and H. patulum , clustered within a relatively narrow and conserved size range. The genera Triadenum and Eliea , also members of Hypericaceae, showed a similar pattern. In contrast, taxa from the genera Harungana and Vismia exhibited significantly larger plastome sizes, appearing as outliers relative to the general distribution of Hypericaceae and most Malpighiales families. These enlarged plastomes may reflect lineage-specific structural expansions or accumulation of non-coding sequences. At the lower end of the spectrum, H. chejuense and H. erectum displayed comparatively reduced plastome sizes within Hypericaceae, suggesting possible genome contraction events. Overall, the plastome size comparison highlights both the evolutionary conservation and structural divergence across Malpighiales, with H. ascyron and its close relatives occupying a distinct position within this landscape. Distribution of Tandem Repeats Across Hypericaceae Plastomes The comparative analysis of tandem repeat content revealed notable variability across Hypericaceae plastomes and outgroup species from Podostemaceae, Calophyllaceae, and Clusiaceae (Fig. 3 ). Species within Hypericaceae, particularly T . japonicum , H . ascyron , and H . hookerianum , exhibited a markedly higher number of tandem repeats, with over 90 repeats detected in some cases. These were predominantly within the 100–199 bp and 200–499 bp length classes. In contrast, outgroup taxa such as M . riparia and R . edulis displayed relatively low repeat abundance, primarily in the 50–199 bp range. Notably, several Hypericaceae species harbored long repeats (≥ 500 bp), which were either rare or absent in outgroup plastomes. The presence of longer and more numerous repeats in Hypericaceae suggests lineage-specific repeat proliferation, potentially contributing to the structural rearrangements and plastome plasticity observed in this family. Structural Evolution of the Hypericaceae Plastome Hypericeae Tribe Comparison of plastomes from the Hypericeae tribe with Mesua ferrea used as a reference for the ancestral plastome structure revealed dynamic structural rearrangements, including multiple inversions, segmental rearrangements, and inverted repeat (IR) boundary shifts ( Supplementary Fig. 1 ). Mauve alignment analysis identified 25 locally collinear blocks (LCBs), suggesting at least seven inversion events involving 21 breakpoints, including: trnH–psbA , psbA–trnK , rps16–psbL , atpA–rps2 , rpoC2–rpoC1 , rpoC1–rpoB , petN–psbM , rps7–psbD , psbD–ndhC , ndhC–ndhJ , atpE–rbcL , accD , psaI–petA , clpP–psbJ , psbB–rps19 , rpl2 , petG , ndhG , ndhF , rpl32–rps15 , and ycf1 . In total, we identified ten separate inversion events across the Hypericeae plastomes within LCB regions 2–11 and 17. Specifically: H. ascyron exhibited eight inversions, H. chejuense and H. erectum showed eleven inversions, T. calcicola displayed ten inversions, H. patulum showed seven inversions, H. laxum and T. fauriei had six inversions each. The largest inversion identified across Hypericeae was LCB 8 (~ 14 kb), present in all species, and LCB 18 (~ 15 kb) in H. ascyron , H. chejuense , and H. patulum . Notably, one inversion located in the IR region was associated with IR expansion. A prominent structural rearrangement involved the relocation of the matK gene from the LSC region into the IR region, consistently observed across all species in the tribe. This rearrangement was accompanied by the apparent loss of the trnK-UUU gene, leaving only a 655 bp remnant of the 5′ intron with 73.1% sequence identity ( Supplementary Fig. 5 ). Vismieae Tribe The plastomes of the Vismieae tribe also demonstrated dynamic structural variation compared to Mesua ferrea , including six inversion events (LCBs 3, 4, 5, 7, 9, and 12), IR boundary shifts, and a notably large inversion spanning 54 kb (LCB 4). Mauve alignment revealed 12 LCBs corresponding to seven inversions and ten breakpoints, including: trnH–psbA , trnK–rps16 , trnG–trnQ , rbcL–trnR , petA–accD , psbJ–rps18 , clpP–rpl20 , psbB–trnN , rps15–ndhF , and ycf1 ( Supplementary Fig. 2 ). Despite these rearrangements, a high degree of synteny was maintained among Vismieae plastomes. The IR regions were markedly expanded (~ 54 kb), distinct from other Hypericaceae tribes. As a consequence of this expansion, a partial duplication of the ycf1 gene was detected only in Vismieae plastomes. Cratoxyleae Tribe Plastomes from the Cratoxyleae tribe exhibited a single large inversion spanning ~ 65 kb compared to Mesua ferrea . Mauve alignment identified four LCBs, suggesting two inversion events with three major breakpoints: trnH–psbA , rbcL–trnK , accD–trnN , and ndhF–ycf1 ( Supplementary Fig. 3 ). The two representative species— Cratoxylum maingayi and Eliea articulata , displayed identical syntenic block structures, indicating structural conservation within the tribe. Ancestral Plastome Structural Evolution in Hypericaceae Phylogenomic analysis suggests that the Cratoxyleae tribe, comprising the genera Eliea and Cratoxylum , represents the most ancestral lineage within the Hypericaceae family. A defining event in this lineage is a single large inversion spanning approximately 65 kb, representing the earliest major plastome structural rearrangement (Fig. 4 ). This inversion distinctly positions the matK gene within the large single-copy (LSC) region and the accD gene within the inverted repeat (IR) region, a configuration not found in the Hypericeae tribe, where a unique rearrangement consistently relocates matK into the IR region and positions accD within the LSC. This pattern is characterized by a matK relocation to the IR—is specific to the Hypericeae tribe and is not observed in the ancestral Cratoxyleae or in the structurally intermediate Vismieae tribe. In Cratoxyleae plastomes, this arrangement contrasts with the canonical organization found in the core Hypericum group. Notably, 22 plastomes across Hypericaceae show shifts in gene order near the LSC–IR boundaries, particularly involving the repositioning of trnH and rbcL , often replacing the ancestral location of matK . This rearranged configuration appears to be a defining structural signature of the Hypericeae lineage. Another ancestral structural event occurred in the Vismieae tribe, which exhibits a ~ 54 kb inversion encompassing two large- locally collinear blocks (LCBs) (Fig. 4 ). This inversion appears to represent a secondary rearrangement relative to the Cratoxyleae event, with breakpoints near the accD and trnK-UUU genes. The Vismieae plastomes also display a pronounced IR expansion, extending into the ycf1 region, resulting in a partial duplication of the ycf1 gene in the opposing IR copy, a unique feature restricted to this lineage. In contrast, plastomes from the Hypericeae tribe reveal extensive structural complexity, characterized by a greater number of LCBs and at least 15 breakpoints localized primarily within the LSC region ( Supplementary Figs. 1–3 ). These rearrangements reflect multiple independent inversion events and IR boundary shifts, marking Hypericeae as the most structurally dynamic lineage among the three tribes. This pattern suggests a progressive accumulation of plastome complexity throughout the diversification of Hypericaceae. IR Expansion and Contraction in the Hypericaceae Family Structural variation in the IR boundaries of Hypericaceae plastomes, relative to the ancestral plastome of Mesua ferrea , reveals dynamic patterns of expansion and contraction, resulting in gene duplication and relocation events across lineages (Fig. 3 ; Supplementary Fig. 4 ). Within the Hypericeae tribe, significant IR contraction was observed at the LSC/IRA junction in Hypericum ascyron and H. chejuense , reducing the IR by approximately 11–13 kb and excluding a block of eight genes (from rpl2 to rps12 ). An even more pronounced contraction (~ 17 kb) was detected in H. erectum , affecting the rps11–rps7 region at the LSC/IRA/SSC boundaries. In contrast, species such as H. laxum and T. japonicum within Hypericeae, as well as all examined members of the Vismieae tribe, showed no evidence of IR contraction. Across the family, IR expansions at both the IRB/SSC and IRA/SSC boundaries were more consistently observed. These expansions led to the duplication of ndhF in most species across Hypericeae, Vismieae, and Cratoxyleae tribes. The only exception was Thornea calcicola , which retained a full copy and a partial copy of ndhF near the IRB/SSC boundary. In addition, a complete duplication of ycf1 was detected only in H. ascyron and T. japonicum , suggesting lineage-specific expansions in these taxa (Fig. 4 ). A particularly notable case of large-scale IR expansion occurred in the Vismieae tribe, where the IR region extended from IRB/LSC to the rpl2–psaJ region, expanding to approximately 56 kb—nearly double the IR size compared to other tribes. This boundary shift resulted in the duplication of up to 30 genes, with no contraction events observed in this tribe. Similarly, the Cratoxyleae tribe showed no evidence of contraction but exhibited an expansion at the IRB/SSC junction, leading to the duplication of the entire ycf1 gene on one side. A partial copy (~ 981 bp) of ycf1 was found at the opposite IR boundary near the LSC/IRA junction (Fig. 4 ). These IR boundary dynamics of expansions, contractions, and gene duplications that demonstrate the extensive plastome structural evolution across the Hypericaceae family and highlight the lineage-specific mechanisms shaping IR architecture. Comparative analysis of the accD gene across Hypericaceae plastomes revealed the presence of internal insertions comprising tandem repeat sequences, with notable variation in both length and position. Two species, H . erectum and H. chejuense , exhibited exceptionally long insertions of 1,468 and 1,484 bp , respectively, disrupting the conserved coding region of acc D. In contrast, most other species contained shorter insertions ranging from 79 bp to 459 bp . These repeat-derived expansions fractured the gene's conserved domains and contributed to significant structural divergence among lineages. The pattern suggests that repeat proliferation has played a key role in acc D evolution, potentially impacting gene function and driving plastome plasticity within Hypericaceae. Gene Relocation and ORF Emergence in Hypericum Plastomes Comparative analysis of plastid genomes among five Hypericum species ( H . ascyron , H . chejuense, H . erectum , H . laxum , and H . patulum ) revealed lineage-specific structural rearrangements associated with the relocation of acc D and mat K genes and the emergence of novel open reading frames (ORFs) ( Supplementary Fig. 5 ). In H . ascyron , both mat K and acc D are translocated into or near the inverted repeat (IR) regions. This rearrangement is accompanied by the presence of lineage-specific ORFs, including ORF13 (between acc D and mat K) and ORF15 (downstream of mat K). These novel ORFs are absent in other species, suggesting they may have originated from IR-mediated recombination or structural rearrangement at the IR junctions. In H . chejuense and H. erectum , similar patterns are observed. While mat K remains conserved, ORF66/67 ( H . chejuense ) and ORF8/9 ( H . erectum ) are located downstream, suggesting independent ORF formation following structural rearrangement events. In H . laxum , acc D is placed upstream of ORF1, again implying that the disruption of plastome structure near acc D may promote ORF emergence. In contrast, H . patulum shows a markedly different organization. Both acc D and mat K are located adjacently within the large single-copy (LSC) region, and no novel ORFs are detected in their vicinity. This more conserved structure likely reflects the absence of recombination-prone IR junctions near these genes, supporting the hypothesis that relocation of coding genes into the IR and disruption of neighboring intergenic regions play a key role in the generation of novel ORFs in specific Hypericum lineages. Parallel Structural Placement of matK and accD Genes An unusual ~ 65 kb inversion in the basal clade of the Cratoxyleae tribe positions the trnH–rbcL region at the beginning of the large single-copy (LSC) region, placing trnH and matK in a canonical configuration typically observed in chloroplast genomes (Fig. 4 ). Structural comparisons across Cratoxyleae, Vismieae, and Hypericeae tribes revealed a pattern of parallel rearrangements affecting the relative positions of matK and accD genes, which may suggest signatures of biparental structural inheritance or convergent rearrangement events. In Cratoxyleae, the matK and accD genes are positioned adjacently in the LSC region ( matK:accD ), a configuration inferred to represent the ancestral state. In contrast, a second large inversion (~ 54 kb) in the Vismieae tribe results in the reversal of this order to accD:matK , positioned adjacent to the inverted repeat (IR) region, along with a substantial IR expansion. This modified accD:matK orientation is also retained in the basal genus of the Hypericeae tribe ( Triadenum ), accompanied by the incorporation of ycf1 into the IR, further supporting a shared structural signature between Vismieae and early diverging Hypericeae plastomes. Interestingly, Thornea and H. patulum retain the ancestral matK:accD configuration, suggesting structural conservation with the Cratoxyleae lineage. However, pronounced divergence is evident in the Hypericum core clade, particularly the Trigynobrathys section ( H. laxum , H. japonicum ), where multiple inversion events appear to have disrupted the original gene order. In this group, the repositioning of accD into the LSC and matK into the IR region is observed, except in H. patulum , which uniquely retains the ancestral Cratoxyleae -like arrangement. These findings indicate that the matK–accD gene pair serves as a phylogenetically informative marker reflecting structural divergence within Hypericaceae. Selective Retention of matK–trnK-UUU In plastomes of the Hypericeae tribe, substantial restructuring occurred around the trnK-UUU region. The trnK-UUU gene was lost in all examined species, and matK was retained as a free-standing gene, uncoupled from its usual intronic context. Despite the loss of intron, matK was preserved across all species, indicating strong functional conservation ( Supplementary Figs. 5 and 6 ). The Hypericeae plastomes retained only five introns overall, with both the cis-spliced rps12 and trnK-UUU introns being absent. Selection analysis showed dN/dS ratios > 1 for matK along the branches leading to Hypericaceae and related Clusioid lineages. Likelihood ratio tests (LRTs) supported the hypothesis that matK underwent positive selection, likely driven by structural reorganization and the functional compensation for the loss of trnK-UUU (Supplementary Figure ). Furthermore, although most plastid genes showed conservation, some rpo genes (e.g., rpoC1 ) displayed intron loss and signs of positive selection. These genes encode the RNA polymerase type I enzyme, which is essential for plastid tRNA and mRNA synthesis. The adaptive evolution of this gene group may reflect compensatory mechanisms associated with plastome structural reconfiguration in Hypericaceae. Collectively, these findings suggest that the relocation and selection of matK , coupled with functional shifts in rpo genes, are key evolutionary responses to plastome rearrangement in Hypericaceae. A more comprehensive sampling across this family will be essential to fully elucidate the evolutionary mechanisms underlying elevated substitution rates and gene reorganization. Gene and Intron Loss Patterns in Hypericaceae and Related Lineages The plastomes of Hypericeae species contained 69–75 protein-coding genes, 29 tRNA genes, and four rRNA genes (Figs. 2 –8; Table 1 ). Notably, the translation initiation factor ( inf A), ribosomal protein S16 ( rps 16), and tRNA-Lys ( trn K-UUU) genes were completely absent across all examined Hypericeae plastomes ( Supplementary Fig. 7 ). Additionally, rps 7 was found to be pesduogenized only in H . ascyron , H . erectum , and H . patulum , while rpl 23 was pesduogenized in all Hypericeae species. The rpl 32 gene also appeared to be pesduogenized in all Hypericeae members, likely due to frameshift mutations and internal stop codons. Complete or near- loss of ycf 1 and ycf 2 was detected in H . erectum , H . chejuens e, H . patulum , and T . calcicola . Intron loss was also widespread in Hypericeae plastomes. Specifically, all species lacked both introns of the clp P gene, the second (cis-spliced) intron of rps 12, the rpo C1 intron, and the second intron of ycf 3. In the Cratoxyleae tribe, gene loss was limited to inf A and rps16, while intron losses mirrored those in Hypericeae, with absence of introns in rps 12, ycf 3, and the second intron of clp P. The Vismieae tribe also exhibited complete loss of inf A and rps 16, and pseudogenization of rpl 23, rpl 32, and rps 7, as well as ycf 1 and ycf 2 in all sampled species of Harungana and Vismia . Intron losses in Vismieae included rps 12, ycf 3, clp P, rpo C1, and atp F, suggesting extensive structural reduction across the plastomes of this lineage. In the related Clusiaceae family, plastome gene losses were more limited: inf A was absent in both Mammea riparia and Rheedia edulis , while rps 16 was lost only in M . riparia . Both species exhibited intron loss in ycf 3 but retained other intron-containing genes intact. Phylogenetic Reconstruction of Hypericaceae and Related Malpighiales To reconstruct evolutionary relationships within the Hypericaceae family and among related lineages in the order Malpighiales, a maximum likelihood (ML) approach was applied using a concatenated alignment of 59 plastid protein-coding genes (supermatrix length: 46,929 bp) across 29 genera, including 25 Hypericaceae species and 4 outgroup taxa (Fig. 4 ). The outgroup comprised representatives from the clusioid clade, including Hypericum monogynum and Calophyllum cochinchinense (Hypericaceae), Tristicha trifaria (Podostemaceae), Mesua ferrea (Calophyllaceae), and Moronobea and Rheedia species (Clusiaceae) (Fig. 5 ). The ML analysis yielded an optimal tree topology with a log-likelihood of − 226352.109, while both ML and BI trees displayed highly congruent topologies. All major branches were strongly supported with bootstrap support (BS) = 100 and Bayesian posterior probability (PP) = 1.00 (Fig. 5 ). Phylogenetic analyses recovered the Cratoxyleae tribe as a monophyletic group and sister to a clade composed of Vismieae and Hypericeae, confirming tribe-level relationships within Hypericaceae. Within this framework, Vismieae was recovered as sister to Hypericeae, indicating a closer evolutionary affinity between these two tribes. Among the Hypericum species: H. laxum and H. japonicum grouped closely with Triadenum fauriei and T. japonicum , forming a well-supported clade corresponding to the Trigynobrathys section (BS/PP = 100/1.00). Thornea species also clustered within this clade, confirming their close phylogenetic relationship to Triadenum and the Trigynobrathys section. H. patulum and H. monogynam formed a clade within the Ascyreia s.l. + campylosporous section, which was resolved as sister to the Roscyna section represented by H. ascyron (BS/PP = 100/1.00). In the core Hypericum , H. erectum and H. chejuense formed a distinct clade (BS/PP = 100/1.00), which was recovered as sister to the combined Ascyreia s.l., Roscyna, and campylosporous lineages. Within Vismieae, Harungana and Vismia species were recovered as a well-supported monophyletic group. In Cratoxyleae, Cratoxylum and Eliea formed a strongly supported sister clade to the Hypericeae–Vismieae clade (BS/PP = 100/1.00), further confirming tribal boundaries. Additional ML phylogenies constructed using individual plastid gene sets (e.g., atp , ndh , pet , psa , psb , rpl , rps , rpo , ycf ) yielded consistent topologies (Supplementary Fig. 1), though variation was observed in certain marker-based trees (e.g., ccsA , clpP , pet , psb , ycf ), which showed slight topological shifts in relationships among tribes. Analysis of nuclear ITS sequences provided complementary evidence, particularly supporting the separation of H. laxum and H. japonicum from core Hypericum , instead of grouping them with Triadenum , thereby corroborating plastome-based results ( Supplementary Fig. 8 ). Notably, the relationships among Thornea , Triadenum , and the Trigynobrathys section appeared controversial in certain gene trees, particularly those constructed using rps , pet , and ycf genes. Collectively, both coalescent-based and concatenated tree methods support a scenario in which: The New World Hypericum clades (e.g., Thornea , Triadenum , Trigynobrathys ) diverge early, While both New and Old-World clades (e.g., Androsaemum , Ascyria , Roscyna , and the Hypericum core) form a monophyletic group sister to Vismieae. These results provide robust phylogenomic evidence for major taxonomic realignments within Hypericaceae and reinforce plastome structure and gene order as useful phylogenetic markers for resolving complex tribal and sectional relationships. Elevated Substitution Rates in Hypericaceae Analysis of substitution rates showed that plastid genes in Hypericeae experienced significantly elevated rates of molecular evolution in specific bordered disturbed genes among the tribe. Nonsynonymous (dN) and synonymous (dS) substitution rates were observed higher, respectively, than those observed in Cratoxylum . H. ascyron exhibited especially high substitution rates across most plastid genes, with dN and dS values are greater than in Cratoxylum , respectively. The genes accD , clpP , and matK , which also underwent structural modifications, showed markedly accelerated substitution rates compared to those in Tristicha ( Supplementary Fig. 10–15 ). Branch-site models and RELAX analyses further revealed that several genes in Hypericaceae underwent episodic positive selection ( Supplementary Table 2–3 ). The genes accD , clpP , matK , cemA , psbN , rpl33 , rps3 , rps12 , rpoA , and rpoC2 showed elevated dN/dS ratios along branches leading to Hypericum , Cratoxylum , and Marathrum . Likelihood ratio tests (LRTs) indicated significant positive selection (p < 0.05, Bonferroni-corrected) in branches leading to ( Hypericum + Cratoxylum + Marathrum ) for clpP , ( Hypericum + Cratoxylum ) for accD , clpP , and matK , and at the Hypericum terminal branch for accD , clpP , and matK ( Supplementary Fig. 10–16 ). Discussion In a previous study, we reported that the plastid genome of H. ascyron exhibited extensive structural disruption, including multiple large inversions, gene and intron loss, and significantly elevated substitution rates (Claude et al., 2022 ). These features positioned H. ascyron as one of the most highly rearranged plastomes among angiosperms and raised questions about whether such plastomes instability was species-specific or indicative of a broader evolutionary trend within Hypericaceae. The present study addresses this by expanding the taxonomic scope to include representatives from all three tribes of Hypericaceae (Hypericeae, Vismieae, and Cratoxyleae) and conducting comparative analyses with outgroups from Clusiaceae. Our results clearly demonstrate that the patterns observed in H. ascyron are reflective of recurring evolutionary mechanisms operating across the family. Plastome Structural Dynamics and Gene Evolution in Hypericaceae Structural variation was widespread. Plastome sizes ranged from 138,163 bp ( H. patulum ) to 176,170 bp ( H. madagascariensis ), with much of this variation attributable to IR expansions and contractions. Members of the Vismieae tribe exhibited dramatic IR expansions exceeding 50 kb, leading to the duplication of over 20 genes. Conversely, significant IR contraction in Hypericeae resulted in the loss of multiple boundary-associated genes. These findings are paralleled in other lineages of Malpighiales, most notably in Passifloraceae, which exhibit IR loss and extensive structural reorganization (Cauz-Santos et al., 2020 ), and Podostemaceae, which display compact, gene-reduced plastomes with high levels of rearrangement (Bedoya et al., 2019 ). Such recurring patterns suggest that plastome instability may be an emergent trait in several clades of Malpighiales, especially those undergoing ecological specialization. Gene loss and pseudogenization were most pronounced in the Hypericeae tribe, where inf A, rpl 23, rpl 32, and rps 16 were absent in all species, while rps 7, ycf 1, and ycf 2 were pesduogenized in several lineages. These losses likely reflect functional transfers to the nuclear genome, a process previously documented in other angiosperm families such as Ranunculaceae and Passifloraceae (Park et al., 2015, Shrestha et al., 2020). For example, rpl 32 was functionally replaced by a nuclear-encoded SOD fusion in Populus (Ueda et al., 2007 ), and rps 16 loss occurred repeatedly with the emergence of dual-targeting mechanisms (Ueda et al., 2008 ). In Geraniaceae, multiple plastid genes, including clp P, underwent loss or pseudogenization, often associated with nuclear transfer or coevolutionary constraints (Weng et al., 2016). These examples collectively highlight that gene loss in plastid genomes is not stochastic but frequently accompanied by functional compensation and structural rearrangement (Wicke et al., 2011 , Jansen et al., 2007 ). The consistent relocation of matK into the IR region and its decoupling from the trn K-UUU intron further suggest a functional repurposing under lineage-specific selective pressures. This inference is supported by our dN/dS analysis, which revealed elevated substitution rates in mat K across multiple branches in the Hypericeae tribe. Notably, in several species where matK has relocated, we observed the presence of adjacent partial or large open reading frames (ORFs), a feature absents in closely related species where mat K remains embedded within the trn K-UUU intron. These ORFs may represent truncated pseudogenes, novel gene fusions, or potentially co-evolving loci that have emerged in tandem with mat K displacement. Their consistent association with relocated matK implies a possible structural or regulatory role, although further functional characterization is needed. The relocation of matK or accD into IR regions, as seen in Hypericum species, may lead to structural disruption of neighboring intergenic regions and generation of lineage-specific ORFs, a phenomenon documented in other taxa (Guisinger et al., 2011b, Wicke et al., 2011 , Zhu et al., 2016 ). Underlying this process is the presence of repeat elements that including palindromic and inverted repeats at IR junctions, which are known hotspots for homologous or illegitimate recombination, facilitating genomic rearrangements and the creation of novel ORFs (Kolodner and Tewari, 1979 , Day and Madesis, 2007 , Odahara et al., 2015 ). Evidence from previous studies reinforces the idea that matK is subject to adaptive evolution. Hao et al. ( 2009 ) found that mat K evolves under positive selection across multiple angiosperm lineages, particularly in domains critical to its maturase function(Hao et al., 2009 ). Furthermore, cases such as Epifagus virginiana , Cuscuta , and leptosporangiate ferns demonstrate that matK can function as a freestanding gene, independent of trn K-UUU, and still retain its splicing role (Kuo et al., 2008 , Wicke et al., 2011 ). These findings align with our observations in Hypericaceae, suggesting that matK relocation is not a random genomic event but rather a potentially adaptive response accompanied by secondary structural or functional changes. In addition, structural alterations in coding genes like acc D, clp P, and rpoC 1 were associated with sequence divergence and loss of introns, a pattern consistent with accelerated substitution rates. These genes, essential for plastid function and organelle-nuclear communication, appear to be hotspots for adaptive evolution. Their modification, often via insertion or fragmentation, may be linked to selection acting on plastid performance in stress-prone habitats. Phylogenetic Relationships and Tribal Evolution in Hypericaceae Phylogenomic reconstruction based on 67 plastid protein-coding genes yielded a well-resolved tree that supports the monophyly of the three recognized tribes of Hypericaceae: Hypericeae, Vismieae, and Cratoxyleae. Our analyses resolved Cratoxyleae as the earliest diverging lineage and sister to a clade comprising Vismieae and Hypericeae. This branching pattern provides strong support for tribal distinctions and clarifies deep evolutionary splits within the family, which were previously ambiguous in studies based on nuclear ITS and plastid intergenic spacers (Nurk and Blattner, 2010 , Nürk and Crockett, 2011 , Park and Kim, 2004 ). Cratoxyleae, with its conserved plastome structure and geographically restricted range in tropical Southeast Asia, likely represents an ancestral lineage within Hypericaceae. In contrast, Hypericeae and Vismieae, both show greater plastome rearrangements including IR expansion and gene loss that share a more recent common ancestor and reflect derived evolutionary trends. These patterns correspond with ecological transitions, as Hypericeae includes both tropical and temperate species, while Vismieae is largely tropical(Nurk and Blattner, 2010 ). Within Hypericeae, our plastome phylogeny revealed unexpected relationships. H. laxum and H. japonicum form a strongly supported clade with Triadenum and Thornea , contradicting traditional genus-level assignments and indicating that these taxa may be better classified within a redefined Hypericum . This finding aligns with earlier ITS and low-copy nuclear marker studies suggesting the paraphyly of core Hypericum and potential instances of incomplete lineage sorting or ancient hybridization (Meseguer et al., 2014 , Norman, 2016 ). Morphological similarities among these species may therefore reflect retained ancestral traits or convergent evolution rather than deep taxonomic splits(Nürk et al., 2013 ). Moreover, our results mirror geographic lineage structuring previously described in the genus. The H. laxum–Triadenum clade corresponds to the New World lineage (e.g., section Trigynobrathys ), while species such as H. erectum and H. patulum group with Old World clades like Ascyreia and Roscyna . This East–West divergence has been attributed to climatic shifts during the Oligocene–Miocene, which drove altitudinal migrations, ecological niche shifts, and speciation events in Hypericum (Nürk et al., 2015 , Meseguer et al., 2015 ). Our genome-scale phylogeny supports this biogeographic scenario and suggests that these divergent events were accompanied by plastome evolution and structural remodeling. Our study provides the most robust plastome-based framework to date for Hypericaceae. It not only affirms tribal relationships but also highlights hidden paraphyly within Hypericeae , signals the need for genus-level taxonomic revision, and corroborates ecological and geographic hypotheses about the group’s diversification. The concordance between plastome evolution, previous nuclear-based reconstructions, and biogeographic history reinforces a model of adaptive radiation in Hypericum driven by historical climate change and niche expansion across continents. Environmental shifts may be a major driver of the plastome dynamism observed. Nürk et al. ( 2015 ) demonstrated that cold-tolerant Hypericum species radiated rapidly in montane habitats such as the Andes and East Asia, likely in response to Oligocene–Miocene climate changes. Structural genome evolution, including gene relocations, intron losses, and inversions may reflect adaptive responses to new ecological pressures, as similarly seen in Geraniaceae and Silene (Erixon and Oxelman, 2008 , Park et al., 2017 ). Such plastome remodeling could facilitate transcriptional or regulatory flexibility under fluctuating environmental regimes. In summary, this study provides the first family-wide plastome-based phylogenomic assessment of Hypericaceae, revealing extensive structural dynamism, recurrent gene loss, and adaptive rearrangements across its three major tribes. By expanding the scope beyond the previously analyzed H. ascyron , our data demonstrate that genome instability—manifested as IR expansion/contraction, gene relocation, pseudogenization, and intron loss—is not species-specific but a recurrent feature across the family, particularly within the Hypericeae and Vismieae lineages. The consistent relocation of mat K and its association with novel open reading frames, as well as elevated dN/dS ratios in mat K, acc D, and clp P, suggest that plastome remodeling is not a random consequence of genome decay, but rather reflects selective responses to ecological pressures. These structural changes are paralleled in other lineages of Malpighiales, such as Passifloraceae and Podostemaceae, where genome reconfiguration has also been linked to adaptation in specialized habitats. Phylogenetically, The placement of Triadenum and Thornea within the Hypericum clade calls for taxonomic revision and reflects historical processes such as hybridization and incomplete lineage sorting. These findings are consistent with previous nuclear and morphological studies but now gain stronger support through genome-scale data. Biogeographically, our phylogeny reinforces the pattern of East–West lineage divergence within Hypericum , supporting a scenario of rapid radiation in response to Miocene climate shifts and montane expansion. The convergence of genomic rearrangements with ecological transitions suggests that plastome evolution played a significant role in enabling the widespread distribution and diversification of Hypericum . Altogether, this study not only provides a resolved phylogenetic framework for Hypericaceae but also advances our understanding of how plastid genome structure, gene content, and selection intersect with environmental history. These findings lay the groundwork for future investigations into plastome–nuclear interactions, functional consequences of gene loss, and the genomic basis of ecological adaptation in angiosperms. Materials and Methods Plant material and Next generation sequencing: This study analyzed the plastid genomes of 14 species across the families Hypericaceae and Clusiaceae, including eight species from the tribe Hypericeae, three from Vismieae, and two from Cratoxyleae . Additionally, two species from Clusiaceae served as outgroups. Plant material used in this study was obtained either as leaf tissue or DNA from the Kew DNA Bank. Wild-collected samples from Korea were collected from common, non-protected species and therefore did not require special permits under Korean biodiversity regulations. Formal identification of the species was performed by SeonJoo Park, and voucher information is provided in Supplementary Table 1 . Additional specimens were obtained from established DNA and herbaria, including the Missouri Botanical Garden, the Kew DNA Bank, and the University of Texas Herbarium, with voucher details available in Supplementary Table 1 .This study did not involve the collection of any species listed in the Convention on the International Trade in Endangered Species of Wild Fauna and Flora (CITES). Fresh leaves were used for several species, including H. erectum Thunb., H. patulum Thunb., H. chejuense S.J.Park & K.J. Kim, H. laxum Blume., H. japonicum Thunp., and Triadenum fauriei R.Keller, with genomic DNA extracted using the GeneAll Plant SV Mini Kit (GeneAll Biotechnology, Seoul, Korea). Herbarium specimens leaves such as Harungana madagascariensis Lam. Ex Poir., Vismia baccifera (L.) Triana & Planch., Eliea articulata (Lam.), Thornea calcicola Standl. & Steyerm., Rheedia edulis (Seem.) Planch. & Triana., and Moronobea riparia Planch. & Triana. were processed using a modified 3× CTAB protocol with a 3-hour incubation, as described by Doyle and Doyle DNA extraction method (Allen et al., 2006 ). For Cratoxylum maingayi and H. rubescens (Oliv.) Byng & Christenh., DNA was sourced from the Kew DNA Bank. High-throughput sequencing was carried out on the Illumina HiSeq 2500 platform (Illumina Inc., San Diego, CA), generating approximately 6 Gb of 150 bp paired-end reads per sample from 550 bp insert libraries. Assembly and genome size comparison Chloroplast genome assembly was conducted using GetOrganelle v1.7.5.3 and Velvet v1.2.10, with k-mer sizes ranging from 97 to 147 optimized for genome coverage and accuracy (Jin et al., 2020 , Zerbino and Birney, 2008 ). Assembly quality was evaluated by mapping paired-end reads to the assembled plastomes using Bowtie2 v2.2.6(Langmead and Salzberg, 2012 ). The results were visually inspected and verified in Geneious R11.0.5 ( https://www.geneious.com ). To analyze genome size variation, 381 complete chloroplast genomes from the order Malpighiales were downloaded from the NCBI nucleotide database as of February 28, 2022. Genome size statistics and visualizations were generated in R v4.0.4 using the ggplot2 package(Wickham, 2016 ). All newly assembled plastome sequences have been deposited in the NCBI GenBank database under accession numbers [PQ010624–PQ010637]. Plastid gene annotation and Genome rearrangement Gene annotation was initially performed in Geneious R11.0.5 using Nicotiana tabacum as the reference genome(Shinozaki et al., 1986 ). Gene boundaries were verified by BLASTN searches using NCBI-BLAST + v2.7.1(Camacho et al., 2009 ). Transfer RNA genes were annotated with tRNAscan-SE v2.0.3 (Chan and Lowe, 2019 ) and ARAGORN v1.2.38 (Laslett and Canback, 2004 ), and circular genome maps were constructed using OGDraw v1.3.1.(Greiner et al., 2019 ). Structural rearrangements were investigated by comparing species from each tribe of Hypericaceae with an outgroup species ( Mesua ferrea L., Clusiaceae) using the progressive Mauve algorithm implemented in Geneious(Darling et al., 2010 , Geneious). Phylogenetic and substitution rate estimation Phylogenetic reconstruction was based on 67 plastid protein-coding genes extracted from 29 plastomes. Each gene was aligned using MAFFT v7.450 with the G-INS-i strategy and concatenated into a supermatrix(Katoh and Standley, 2013 ). Phylogenetic inference was carried out using maximum likelihood in IQ-TREE v1.6.2 under the GTR + GAMMA + I model with 1,000 ultrafast bootstrap replicates(Minh et al., 2020 ), and Bayesian inference using MrBayes v3.3.7a(Ronquist et al., 2020 ). Coalescent-based species trees were generated using ASTRAL v5.7.8 with 67 gene trees derived from IQ-TREE, incorporating models selected by ModelFinder (Mirarab et al., 2014 ). Additionally, nuclear ITS regions were retrieved using GetOrganelle and aligned with MUSCLE(Edgar, 2004 ). ITS based phylogenies were reconstructed using IQ-TREE with the GTR + GAMMA + I model. Selection analysis To detect selection, nonsynonymous (dN) and synonymous (dS) substitution rates were estimated for each plastid gene using the CODEML program within the PAML v4.8 package under the F3×4 codon frequency model(Yang, 1997 ). Branch-site positive selection was tested using the adaptive Branch-Site Random Effects Likelihood (absREL) model in HyPhy v2.5 via the Datamonkey server, with Holm-Bonferroni correction for multiple testing(Pond et al., 2020 ). A total of 70 protein-coding genes were aligned using the Translation Align option in MAFFT, and likelihood ratio tests (LRTs) were conducted to evaluate variation in dN/dS ratios across branches. Visualization of substitution rates was performed in R v4.0.4 using ggplot2(Wickham, 2016 ). To further explore divergence in the inverted repeat (IR) regions, the ycf1 and ycf2 genes from 11 genera of Malpighiales, including a known pseudogene in Passiflora edulis , were aligned using MAFFT. Phylogenetic trees were constructed using PhyML with 100 bootstrap replicates to assess evolutionary patterns associated with IR-specific gene dynamics(Guindon et al., 2010 ). Declarations Contributions JCS performed the experiments, generated datasets and figures, and wrote the first draft of the manuscript. KTP contributed to the data assembled, performed analyses, and read/edited the manuscript. SJP contributed to project design and read/edited the manuscript. All authors read and approved the final draft of the manuscript. Ethics declarations Ethics approval and consent to participate Experimental study on the plant, including collection of the material, comply with institutional, national, and international guidelines. Clinical trial number Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Funding: This work was supported by the 2024 Yeungnam University Grant (221A061009), South Korea. Author Contribution JCS performed the experiments, generated datasets and figures, and wrote the first draft of the manuscript. KTP contributed to the data assembled, performed analyses, and read/edited the manuscript. SJP contributed to project design and read/edited the manuscript. All authors read and approved the final draft of the manuscript.All authors read and approved the final manuscript. Acknowledgements Not applicable. 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SHINOZAKI K, OHME M, TANAKA M, WAKASUGI T, HAYASHIDA N, MATSUBAYASHI–SHINOZAKI K, ZAITA N, YAMAGUCHI–SHINOZAKI CHUNWONGSEJ, K., SUGIURA M. The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression. EMBO J. 1986;5:2043–9. SHRESTHA B, GILBERT, L. E., RUHLMAN, T. A., JANSEN RK. Rampant Nuclear Transfer and Substitutions of Plastid Genes in Passiflora. Genome Biol Evol. 2020;12:1313–29. SOLTIS DE, SOLTIS PS, MORT CHASEMW, ALBACH ME, ZANIS DC, FAY MSAVOLAINENVHAHNWHHOOTSB, AXTELL MF, NIXON MSWENSENSMPRINCELMKRESSWJ, K. C., FARRIS JS. Angiosperm phylogeny inferred from 18S rDNA, rbcL, and atpB sequences. Bot J Linn Soc. 2000;133:381–461. STEVENS PF. Clusiaceae-Guttiferae. In: KUBITZKI K, editor. 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Substitution of the gene for chloroplast RPS16 was assisted by generation of a dual targeting signal. Mol Biol Evol. 2008;25:1566–75. WENG ML, RUHLMAN, T. A., JANSEN RK. Plastid–Nuclear Interaction and Accelerated Coevolution in Plastid Ribosomal Genes in Geraniaceae. Genome Biol Evol. 2016;8:1824–38. WICKE S, SCHNEEWEISS GM, DEPAMPHILIS, C. W., MÜLLER, K. F., QUANDT D. The evolution of the plastid chromosome in land plants: gene content, gene order, gene function. Plant Mol Biol. 2011;76:273–97. WICKHAM H. ggplot2: Elegant Graphics for Data Analysis. New York: Springer- New York; 2016. WURDACK KJ, DAVIS CC. Malpighiales phylogenetics: Gaining ground on one of the most recalcitrant clades in the angiosperm tree of life. Am J Bot. 2009;96:1551–70. YANG Z. PAML: a program package for phylogenetic analysis by maximum likelihood. Comput Appl Biosci (CABIOS). 1997;13:555–6. ZERBINO DR, BIRNEY E. Velvet: Algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 2008;18:821–9. ZHU A, GUO W, GUPTA, S., FAN, W., MOWER JP. Evolutionary dynamics of the plastid inverted repeat: the effects of expansion, contraction, and loss on substitution rates. New Phytol. 2016;209:1747–56. Additional Declarations No competing interests reported. Supplementary Files SupplementaryTableFigure.pdf Cite Share Download PDF Status: Published Journal Publication published 23 Dec, 2025 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 30 Oct, 2025 Reviews received at journal 23 Oct, 2025 Reviews received at journal 19 Oct, 2025 Reviews received at journal 12 Oct, 2025 Reviewers agreed at journal 08 Oct, 2025 Reviewers agreed at journal 08 Oct, 2025 Reviewers agreed at journal 07 Oct, 2025 Reviewers agreed at journal 26 Sep, 2025 Reviewers invited by journal 25 Sep, 2025 Editor invited by journal 01 Sep, 2025 Editor assigned by journal 28 Aug, 2025 Submission checks completed at journal 27 Aug, 2025 First submitted to journal 27 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-7295767","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":526033864,"identity":"738ddbe7-f479-453d-b7e7-d2f48b143132","order_by":0,"name":"Sivagami Jean Claude","email":"","orcid":"","institution":"Gachon University","correspondingAuthor":false,"prefix":"","firstName":"Sivagami","middleName":"Jean","lastName":"Claude","suffix":""},{"id":526033866,"identity":"05bd6a53-79ed-4897-9aac-f6f4be088284","order_by":1,"name":"Kyutae Park","email":"","orcid":"","institution":"Kyungpook National University","correspondingAuthor":false,"prefix":"","firstName":"Kyutae","middleName":"","lastName":"Park","suffix":""},{"id":526033867,"identity":"c2e8549f-3641-4fec-9682-c66c6d4c9d18","order_by":2,"name":"SeonJoo Park","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIie3PsQrCMBCA4XNxCtbxQqW+QqRQFEp9lUohU3V2cIiLLuJc8GUihU6pc3wHh4IguIitoKPpKJifZAjkgzsAm+0nk81F4rzf2JKEAyrakxfjIZNtyXBdHq9LlRNfzwuEVQT0IL8TJk8JVTongV5whCIBtxcbCChGRdWQNEDoSvCIaTCh/HtD/KwhjxYEpAqo0JwwrElnI8E1kXoXPhEqJKgu/ni2TwjdmQbLyvwsCpw623Skq1vkoTIN9vnRjwHqY9wEwHnP4UjzZ5vNZvvPnu5VQqtUgUmOAAAAAElFTkSuQmCC","orcid":"","institution":"Yeungnam University","correspondingAuthor":true,"prefix":"","firstName":"SeonJoo","middleName":"","lastName":"Park","suffix":""}],"badges":[],"createdAt":"2025-08-05 03:23:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7295767/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7295767/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-025-07888-7","type":"published","date":"2025-12-23T15:57:48+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":93080366,"identity":"4a7162ef-35ab-4830-b35c-7418f682d1de","added_by":"auto","created_at":"2025-10-08 23:21:52","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4589035,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscriptedited2025.docx","url":"https://assets-eu.researchsquare.com/files/rs-7295767/v1/2ea972d7a22b6667dc3d273c.docx"},{"id":93080406,"identity":"952c7d78-b467-4a10-aaaa-16cc74bf1741","added_by":"auto","created_at":"2025-10-08 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23:21:30","extension":"xml","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":185626,"visible":true,"origin":"","legend":"","description":"","filename":"6429eb4c46894a248ea4ac9ab12a3ac21structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7295767/v1/213cd6ce5d78cbfba7a12d13.xml"},{"id":93080288,"identity":"773fa237-cec8-4af9-b013-4648364ba9d8","added_by":"auto","created_at":"2025-10-08 23:21:44","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":207557,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7295767/v1/3f141de14fcff25f9a3407ac.html"},{"id":93080265,"identity":"b6871af3-af13-460c-8957-e87da2af57ae","added_by":"auto","created_at":"2025-10-08 23:21:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1006439,"visible":true,"origin":"","legend":"\u003cp\u003eChloroplast genome map of \u003cem\u003eVismia and Harungana\u003c/em\u003e (Vismieae) showing an expanded inverted repeat (IR). The circular plastome structure of \u003cem\u003eVismia baccifera\u003c/em\u003e displays a notable expansion of the IR region to ~50kb, leading to extensive duplication of genes beyond the typical IR boundary. Genes are color-coded by functional category, with arrows indicating transcriptional direction. LSC, SSC, and IR regions are labeled, and the inner grey circle indicates GC content.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7295767/v1/1f0390157564bbed91717869.png"},{"id":93080409,"identity":"2b468be0-2d24-44d8-8c60-ca3453a66a82","added_by":"auto","created_at":"2025-10-08 23:22:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1287526,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of plastome sizes across 381 species from Hypericaceae (red) and other families within the order Malpighiales (blue). Each panel represents a different comparative subset or phylogenetic grouping. Red dots and error bars highlight species and genera within Hypericaceae, while blue points represent plastome sizes from other Malpighiales lineages. Notable species and genera are labeled for reference.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7295767/v1/6e3db04ef71b581d9a98b3a9.png"},{"id":93080319,"identity":"26fa3e3c-6410-4907-9947-a0d6bc22fb06","added_by":"auto","created_at":"2025-10-08 23:21:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":220625,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of tandem repeats in plastomes of Hypericaceae species compared to outgroup species from Podostemaceae, Calophyllaceae, and Clusiaceae. The bar plot shows the number of tandem repeats grouped by repeat length categories. Colors indicate different repeat size ranges, with Hypericaceae species generally exhibiting a higher abundance and broader size range of repeats than outgroups.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7295767/v1/864b7725dd8d18e638212199.png"},{"id":93080254,"identity":"6309e36e-1b37-4456-ab98-5693fb33233c","added_by":"auto","created_at":"2025-10-08 23:21:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":508736,"visible":true,"origin":"","legend":"\u003cp\u003eAncestral reconstruction of plastome structural variation across Hypericaceae tribes compared to \u003cem\u003eMesua\u003c/em\u003e (Calophyllaceae) as the outgroup. The diagram illustrates major evolutionary changes, including inverted repeat (IR) boundary shifts, gene relocations, and multiple inversions. Key events are annotated by colored arrows and boxes, with major inversions labeled (e.g., 65 kb and 54 kb) and lineage-specific rearrangements highlighted. Notable changes in the \u003cem\u003eaccD\u003c/em\u003e, \u003cem\u003ematK\u003c/em\u003e, \u003cem\u003eclpP\u003c/em\u003e, and \u003cem\u003eycf\u003c/em\u003e gene regions are shown across Cratoxyleae, Vismieae, Hypericeae, and \u003cem\u003eHypericum\u003c/em\u003e core lineages.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7295767/v1/471fb223ef8c5e3fff2bfa14.png"},{"id":93080396,"identity":"a77479d1-69af-4fbe-aa1e-dda9291bff86","added_by":"auto","created_at":"2025-10-08 23:21:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":633916,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum likelihood phylogeny of Hypericaceae based on 79 plastid protein-coding genes. The tree was reconstructed using concatenated nucleotide sequences of 79 plastid protein-coding genes from 29 Hypericaceae species and one outgroup (Calophyllaceae, Clusiaceae and Podostemaceae). The tree resolves three major tribes of Hypericaceae: Hypericeae, Vismieae, and Cratoxyleae, each indicated with colored blocks and corresponding floral images. Bootstrap support values from 1,000 replicates are shown at the nodes. Species of \u003cem\u003eHypericum\u003c/em\u003e are further classified into “old” and “new” world lineages. The genus \u003cem\u003eHypericum\u003c/em\u003e exhibits multiple distinct clades, including \u003cem\u003eH\u003c/em\u003e. \u003cem\u003eascyron\u003c/em\u003e, which forms a separate lineage within the new world clade. Representative floral images are shown for each tribal group. The tree is rooted with Clusiaceae and other family members, and branch lengths are proportional to nucleotide substitutions per site.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-7295767/v1/5855ff860fd9428cd5e9dcd8.png"},{"id":99172535,"identity":"2bed506b-491d-4991-9252-f35b48a382db","added_by":"auto","created_at":"2025-12-29 16:10:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4297041,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7295767/v1/2dc7a46f-7649-43dd-a3e8-8b2adb747765.pdf"},{"id":93080201,"identity":"bcf5d82e-cf10-42cb-82a1-5bc7dec040e9","added_by":"auto","created_at":"2025-10-08 23:21:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":5117131,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableFigure.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7295767/v1/58586f5e03bfda492705c6eb.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Plastome Evolution at the Edge: Structural Rearrangements, IR Expansion, and Gene Flux in Hypericaceae (Malpighiales)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe order Malpighiales Juss ex Bercht \u0026amp; J. Presl, one of the most taxonomically and ecologically diverse clades of flowering plants, was first resolved through molecular phylogenetic studies by Chase et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). This reclassification reshaped the understanding of angiosperm relationships and highlighted the potential of molecular tools in resolving deep evolutionary lineages (Chase et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1993\u003c/span\u003e, Savolainen et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2000\u003c/span\u003e, Soltis et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Among its major subclades, the clusioid clade comprising five families: Bonnetiaceae L. Beauvis Ex Nakai, Calophyllaceae J.Agardh, Clusiaceae Lindl., Hypericaceae Juss, and Podostemaceae Rich., represents a morphologically and biogeographically complex lineage with over 1,900 species spread across 94 genera (Ruhfel et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Stevens, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2007a\u003c/span\u003e, Wurdack and Davis, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Members of this clade are predominantly tropical, though certain genera extend into temperate and high-altitude regions, often adapting to extreme or specialized ecological niches such as montane forests and fast-flowing aquatic systems (Davis et al., 2005, Ruhfel et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Within the clusioid clade, Hypericaceae stands out due to its taxonomic complexity and unevenly studied genera. Historically treated as a subfamily within Clusiaceae, Hypericaceae was elevated to family rank based on distinct morphological and molecular features (Kubitzki et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). It currently comprises approximately 518\u0026ndash;700 species across nine genera and is taxonomically divided into three tribes: Hypericeae Choisy, Vismieae Vandelli, and Cratoxyleae Engl. APG III, 2009 (Crockett and Robson, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The genus \u003cem\u003eHypericum\u003c/em\u003e, the most species-rich lineage with a cosmopolitan distribution, accounts for nearly 80% of the family\u0026rsquo;s diversity. In contrast, the other genera, many of which are confined to tropical regions, have remained poorly sampled in molecular studies, leaving tribal and intergeneric relationships largely unresolved (N\u0026uuml;rk et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Stevens, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2007b\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePrevious phylogenetic efforts in Hypericaceae have focused heavily on \u003cem\u003eHypericum\u003c/em\u003e, employing morphological data (Robson, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1977\u003c/span\u003e, Robson, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1981a\u003c/span\u003e, Robson, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1981b\u003c/span\u003e, Robson, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1985\u003c/span\u003e, Robson, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1990\u003c/span\u003e, Robson, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2006\u003c/span\u003e, Robson, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2010a\u003c/span\u003e, Robson, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2010b\u003c/span\u003e, Robson, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), ITS markers (Nurk and Blattner, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, Park and Kim, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), and plastid regions (e.g., \u003cem\u003etrn\u003c/em\u003eL-\u003cem\u003etrn\u003c/em\u003eF, \u003cem\u003epsb\u003c/em\u003eA-\u003cem\u003etrn\u003c/em\u003eH). These studies provided key insights into biogeographic patterns, such as the disjunction between New World and Old World clades, and proposed evolutionary scenarios involving ancient Gondwanan dispersal and multiple niche shifts (Meseguer et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, N\u0026uuml;rk et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Meseguer et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). However, these single- or low-copy gene datasets offered limited resolution at deeper nodes and often failed to resolve intertribal relationships, especially for Vismieae and Cratoxyleae.\u003c/p\u003e\u003cp\u003ePlastid genomes (plastomes) have emerged as powerful tools for resolving angiosperm relationships at both deep and shallow phylogenetic levels (Jansen et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Their quadripartite structure, gene content, and substitution dynamics offer evolutionary signals that are often inaccessible through nuclear markers alone. Furthermore, increasing evidence suggests that plastome structural variation, such as inversions, IR boundary shifts, and gene loss, is not just phylogenetically informative but may also reflect lineage-specific ecological adaptations and genome evolution under selection (Guisinger et al., 2011a, Wicke et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In this context, \u003cem\u003eH\u003c/em\u003e. \u003cem\u003eascyron\u003c/em\u003e. was recently shown to possess one of the most highly rearranged plastomes among angiosperms, with large inversions, pseudogenization, and accelerated substitution rates (Claude et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Whether this pattern is unique to \u003cem\u003eH. ascyron\u003c/em\u003e or indicative of a broader trend across Hypericaceae remains unclear.\u003c/p\u003e\u003cp\u003eTo address this question and improve resolution of phylogenetic relationships within Hypericaceae, we sequenced and analyzed complete chloroplast genomes from 14 species across all three tribes: \u003cem\u003eHypericeae\u003c/em\u003e (including \u003cem\u003eHypericum\u003c/em\u003e, \u003cem\u003eTriadenum\u003c/em\u003e Raf., and \u003cem\u003eThornea\u003c/em\u003e Breedlove \u0026amp; E.M.McClint), \u003cem\u003eVismieae\u003c/em\u003e (\u003cem\u003eHarungana\u003c/em\u003e Lam., \u003cem\u003eVismia\u003c/em\u003e Vand.), and Cratoxyleae (\u003cem\u003eCratoxylum\u003c/em\u003e Blume., \u003cem\u003eEliea\u003c/em\u003e (Lam.) Cambess.). Two species from Clusiaceae were included as outgroups. By combining comparative plastome analysis with phylogenomic reconstruction based on 67 plastid protein-coding genes, we aimed to:\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eAssess the extent and pattern of structural plastome variation across the family,\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eInvestigate gene loss, pseudogenization, and possible functional relocation events and\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eResolve tribal and infrageneric relationships in light of previous nuclear and morphological findings.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003eOur results provide new insight into plastome evolution in Hypericaceae and reveal lineage-specific trends in genome reorganization that align with ecological transitions and biogeographic structuring. These findings not only refine the phylogenetic backbone of the family but also shed light on how structural plastome dynamics correlate with environmental adaptation and diversification.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eVariation in Plastome Structure and Size across Hypericaceae Tribes\u003c/h2\u003e\u003cp\u003ePlastome sizes in the tribe Hypericeae ranged from 138,163 bp in \u003cem\u003eHypericum patulum\u003c/em\u003e to 167,482 bp in \u003cem\u003eTriadenum fauriei\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The large single-copy (LSC) region exhibited substantial variation, spanning from 103,150 bp in \u003cem\u003eH. patulum\u003c/em\u003e to 85,948 bp in \u003cem\u003eH. calcicola\u003c/em\u003e. The inverted repeat (IR) region reached its maximum length in \u003cem\u003eH. laxum\u003c/em\u003e (32,823 bp) and its minimum in \u003cem\u003eH. patulum\u003c/em\u003e (12,032 bp). The small single-copy (SSC) region showed a narrower range, from 10,423 bp in \u003cem\u003eH. laxum\u003c/em\u003e to 12,421 bp in \u003cem\u003eT. calcicola\u003c/em\u003e. In the Vismieae tribe, plastome sizes were generally larger, ranging from 166,590 bp in \u003cem\u003eVismia baccifera\u003c/em\u003e to 176,170 bp in \u003cem\u003eHarungana madagascariensis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The LSC region measured 67,585 bp in \u003cem\u003eV. baccifera\u003c/em\u003e and 57,619 bp in \u003cem\u003eH. madagascariensis\u003c/em\u003e, while the IR region was largest in \u003cem\u003eH. madagascariensis\u003c/em\u003e (52,124 bp) and smallest in \u003cem\u003eV. baccifera\u003c/em\u003e (42,583 bp). The SSC region in both species remained within the size range observed in Hypericeae. For the Cratoxyleae tribe, plastome sizes were more conserved, with a median length of approximately 156\u0026ndash;157 kb. The LSC region measured 85,588 bp in \u003cem\u003eEliea articulata\u003c/em\u003e and 86,024 bp in \u003cem\u003eCratoxylum maingayi\u003c/em\u003e. The SSC regions ranged from 17 to 18 kb, and the IR regions ranged from 25 to 26 kb. These values are comparable to those found in the outgroup plastomes of \u003cem\u003eMesua riparia\u003c/em\u003e and \u003cem\u003eRheedia edulis\u003c/em\u003e. Across all three tribes, plastome sizes varied from 138,163 bp (\u003cem\u003eH. patulum\u003c/em\u003e) to 176,170 bp (\u003cem\u003eH. madagascariensis\u003c/em\u003e), reflecting both lineage-specific expansions and reductions in chloroplast genome architecture (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\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\u003eable 1. Summary of plastome features for 15 Hypericaceae species used in this study for phylogenetic analysis. The table includes total plastome size, lengths of the large single- copy (LSC), inverted repeat (IR), and small single-copy (SSC) regions, GC content, and the number of protein-coding sequences (CDS) and tRNA genes.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" 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=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" 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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSpecies\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLSC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eIR\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSSC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eGC %\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eCDS\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003etRNA\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eH\u003c/em\u003e. \u003cem\u003eascyron\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e162,286\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e97,542\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e26,846\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e11,052\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.40%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eH. erectum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e143,167\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e99,272\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16,370\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e11,155\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.10%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eH. chejuense\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e142,029\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e98,131\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16,375\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e11,148\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.20%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eH. patulum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e138,163\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e103,150\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e12,032\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e10,949\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e38.10%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eH\u003c/em\u003e. \u003cem\u003elaxum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e165,288\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e89,219\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e32,823\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e10,423\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e38.10%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eH. japonicum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e165,190\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e89,123\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e32,805\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e10,457\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e32.00%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eT. fauriei\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e167,482\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e92,077\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e31,916\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e11,191\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.40%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eT\u003c/em\u003e. \u003cem\u003ecalcicola\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e150,935\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e85,948\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e26,283\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e12,421\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.90%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eH\u003c/em\u003e. \u003cem\u003emadagascariensis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e176,170\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e57,619\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e52,124\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e14,303\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e36.90%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eH\u003c/em\u003e. \u003cem\u003erubescens\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e174,661\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e57,746\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e51,809\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e13,297\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e37.10%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eV\u003c/em\u003e. \u003cem\u003ebaccifera\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e166,590\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e67,585\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e42,583\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e13,839\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e36.90%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eE\u003c/em\u003e. \u003cem\u003earticulata\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e156,123\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e85,588\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e25,640\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e19,254\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e36.40%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eC\u003c/em\u003e. \u003cem\u003emaingayi\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e157,279\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e86,024\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e26,155\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e18,945\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e36.30%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eM\u003c/em\u003e. \u003cem\u003eriparia\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e156,826\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e84,914\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e27,057\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e17,798\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e36.00%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eR\u003c/em\u003e. \u003cem\u003eedulis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e158,160\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e86,384\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e27,010\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e17,756\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e35.80%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e35\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\u003c/div\u003e\n\u003ch3\u003ePlastome Size Variation in Hypericaceae and Related Malpighiales Lineages\u003c/h3\u003e\n\u003cp\u003eTo evaluate patterns of plastome size evolution within \u003cem\u003eHypericaceae\u003c/em\u003e and across the broader order Malpighiales, we compared plastome sizes among 381 species, including representatives from both focal and outgroup families (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The distribution of plastome sizes revealed substantial inter- and intra-lineage variability. Species within\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eHypericum\u003c/em\u003e, including \u003cem\u003eH. ascyron\u003c/em\u003e, \u003cem\u003eH. laxum\u003c/em\u003e, and \u003cem\u003eH. patulum\u003c/em\u003e, clustered within a relatively narrow and conserved size range. The genera \u003cem\u003eTriadenum\u003c/em\u003e and \u003cem\u003eEliea\u003c/em\u003e, also members of Hypericaceae, showed a similar pattern. In contrast, taxa from the genera \u003cem\u003eHarungana\u003c/em\u003e and \u003cem\u003eVismia\u003c/em\u003e exhibited significantly larger plastome sizes, appearing as outliers relative to the general distribution of Hypericaceae and most Malpighiales families. These enlarged plastomes may reflect lineage-specific structural expansions or accumulation of non-coding sequences. At the lower end of the spectrum, \u003cem\u003eH. chejuense\u003c/em\u003e and \u003cem\u003eH. erectum\u003c/em\u003e displayed comparatively reduced plastome sizes within Hypericaceae, suggesting possible genome contraction events. Overall, the plastome size comparison highlights both the evolutionary conservation and structural divergence across Malpighiales, with \u003cem\u003eH. ascyron\u003c/em\u003e and its close relatives occupying a distinct position within this landscape.\u003c/p\u003e\n\u003ch3\u003eDistribution of Tandem Repeats Across Hypericaceae Plastomes\u003c/h3\u003e\n\u003cp\u003eThe comparative analysis of tandem repeat content revealed notable variability across Hypericaceae plastomes and outgroup species from Podostemaceae, Calophyllaceae, and Clusiaceae (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Species within Hypericaceae, particularly \u003cem\u003eT\u003c/em\u003e. \u003cem\u003ejaponicum\u003c/em\u003e, \u003cem\u003eH\u003c/em\u003e. \u003cem\u003eascyron\u003c/em\u003e, and \u003cem\u003eH\u003c/em\u003e. \u003cem\u003ehookerianum\u003c/em\u003e, exhibited a markedly higher number of tandem repeats, with over 90 repeats detected in some cases. These were predominantly within the 100\u0026ndash;199 bp and 200\u0026ndash;499 bp length classes. In contrast, outgroup taxa such as \u003cem\u003eM\u003c/em\u003e. \u003cem\u003eriparia\u003c/em\u003e and \u003cem\u003eR\u003c/em\u003e. \u003cem\u003eedulis\u003c/em\u003e displayed relatively low repeat abundance, primarily in the 50\u0026ndash;199 bp range. Notably, several Hypericaceae species harbored long repeats (\u0026ge;\u0026thinsp;500 bp), which were either rare or absent in outgroup plastomes. The presence of longer and more numerous repeats in Hypericaceae suggests lineage-specific repeat proliferation, potentially contributing to the structural rearrangements and plastome plasticity observed in this family.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eStructural Evolution of the Hypericaceae Plastome\u003c/h3\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eHypericeae Tribe\u003c/h2\u003e\u003cp\u003eComparison of plastomes from the Hypericeae tribe with \u003cem\u003eMesua ferrea\u003c/em\u003e used as a reference for the ancestral plastome structure revealed dynamic structural rearrangements, including multiple inversions, segmental rearrangements, and inverted repeat (IR) boundary shifts \u003cb\u003e(\u003c/b\u003e\u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e). Mauve alignment analysis identified 25 locally collinear blocks (LCBs), suggesting at least seven inversion events involving 21 breakpoints, including: \u003cem\u003etrnH\u0026ndash;psbA\u003c/em\u003e, \u003cem\u003epsbA\u0026ndash;trnK\u003c/em\u003e, \u003cem\u003erps16\u0026ndash;psbL\u003c/em\u003e, \u003cem\u003eatpA\u0026ndash;rps2\u003c/em\u003e, \u003cem\u003erpoC2\u0026ndash;rpoC1\u003c/em\u003e, \u003cem\u003erpoC1\u0026ndash;rpoB\u003c/em\u003e, \u003cem\u003epetN\u0026ndash;psbM\u003c/em\u003e, \u003cem\u003erps7\u0026ndash;psbD\u003c/em\u003e, \u003cem\u003epsbD\u0026ndash;ndhC\u003c/em\u003e, \u003cem\u003endhC\u0026ndash;ndhJ\u003c/em\u003e, \u003cem\u003eatpE\u0026ndash;rbcL\u003c/em\u003e, \u003cem\u003eaccD\u003c/em\u003e, \u003cem\u003epsaI\u0026ndash;petA\u003c/em\u003e, \u003cem\u003eclpP\u0026ndash;psbJ\u003c/em\u003e, \u003cem\u003epsbB\u0026ndash;rps19\u003c/em\u003e, \u003cem\u003erpl2\u003c/em\u003e, \u003cem\u003epetG\u003c/em\u003e, \u003cem\u003endhG\u003c/em\u003e, \u003cem\u003endhF\u003c/em\u003e, \u003cem\u003erpl32\u0026ndash;rps15\u003c/em\u003e, and \u003cem\u003eycf1\u003c/em\u003e. In total, we identified ten separate inversion events across the Hypericeae plastomes within LCB regions 2\u0026ndash;11 and 17. Specifically:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eH. ascyron\u003c/em\u003e exhibited eight inversions,\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eH. chejuense\u003c/em\u003e and \u003cem\u003eH. erectum\u003c/em\u003e showed eleven inversions,\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eT. calcicola\u003c/em\u003e displayed ten inversions,\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eH. patulum\u003c/em\u003e showed seven inversions,\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eH. laxum\u003c/em\u003e and \u003cem\u003eT. fauriei\u003c/em\u003e had six inversions each.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eThe largest inversion identified across Hypericeae was LCB 8 (~\u0026thinsp;14 kb), present in all species, and LCB 18 (~\u0026thinsp;15 kb) in \u003cem\u003eH. ascyron\u003c/em\u003e, \u003cem\u003eH. chejuense\u003c/em\u003e, and \u003cem\u003eH. patulum\u003c/em\u003e. Notably, one inversion located in the IR region was associated with IR expansion. A prominent structural rearrangement involved the relocation of the \u003cem\u003ematK\u003c/em\u003e gene from the LSC region into the IR region, consistently observed across all species in the tribe. This rearrangement was accompanied by the apparent loss of the \u003cem\u003etrnK-UUU\u003c/em\u003e gene, leaving only a 655 bp remnant of the 5\u0026prime; intron with 73.1% sequence identity (\u003cb\u003eSupplementary Fig.\u0026nbsp;5\u003c/b\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eVismieae Tribe\u003c/h2\u003e\u003cp\u003eThe plastomes of the Vismieae tribe also demonstrated dynamic structural variation compared to \u003cem\u003eMesua ferrea\u003c/em\u003e, including six inversion events (LCBs 3, 4, 5, 7, 9, and 12), IR boundary shifts, and a notably large inversion spanning 54 kb (LCB 4). Mauve alignment revealed 12 LCBs corresponding to seven inversions and ten breakpoints, including: \u003cem\u003etrnH\u0026ndash;psbA\u003c/em\u003e, \u003cem\u003etrnK\u0026ndash;rps16\u003c/em\u003e, \u003cem\u003etrnG\u0026ndash;trnQ\u003c/em\u003e, \u003cem\u003erbcL\u0026ndash;trnR\u003c/em\u003e, \u003cem\u003epetA\u0026ndash;accD\u003c/em\u003e, \u003cem\u003epsbJ\u0026ndash;rps18\u003c/em\u003e, \u003cem\u003eclpP\u0026ndash;rpl20\u003c/em\u003e, \u003cem\u003epsbB\u0026ndash;trnN\u003c/em\u003e, \u003cem\u003erps15\u0026ndash;ndhF\u003c/em\u003e, and \u003cem\u003eycf1\u003c/em\u003e (\u003cb\u003eSupplementary Fig.\u0026nbsp;2 ).\u003c/b\u003e Despite these rearrangements, a high degree of synteny was maintained among Vismieae plastomes. The IR regions were markedly expanded (~\u0026thinsp;54 kb), distinct from other Hypericaceae tribes. As a consequence of this expansion, a partial duplication of the \u003cem\u003eycf1\u003c/em\u003e gene was detected only in Vismieae plastomes.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCratoxyleae Tribe\u003c/h3\u003e\n\u003cp\u003ePlastomes from the Cratoxyleae tribe exhibited a single large inversion spanning\u0026thinsp;~\u0026thinsp;65 kb compared to \u003cem\u003eMesua ferrea\u003c/em\u003e. Mauve alignment identified four LCBs, suggesting two inversion events with three major breakpoints: \u003cem\u003etrnH\u0026ndash;psbA\u003c/em\u003e, \u003cem\u003erbcL\u0026ndash;trnK\u003c/em\u003e, \u003cem\u003eaccD\u0026ndash;trnN\u003c/em\u003e, and \u003cem\u003endhF\u0026ndash;ycf1\u003c/em\u003e (\u003cb\u003eSupplementary Fig.\u0026nbsp;3\u003c/b\u003e). The two representative species\u0026mdash;\u003cem\u003eCratoxylum maingayi\u003c/em\u003e and \u003cem\u003eEliea articulata\u003c/em\u003e, displayed identical syntenic block structures, indicating structural conservation within the tribe.\u003c/p\u003e\n\u003ch3\u003eAncestral Plastome Structural Evolution in Hypericaceae\u003c/h3\u003e\n\u003cp\u003ePhylogenomic analysis suggests that the Cratoxyleae tribe, comprising the genera \u003cem\u003eEliea\u003c/em\u003e and \u003cem\u003eCratoxylum\u003c/em\u003e, represents the most ancestral lineage within the Hypericaceae family. A defining event in this lineage is a single large inversion spanning approximately 65 kb, representing the earliest major plastome structural rearrangement (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This inversion distinctly positions the \u003cem\u003ematK\u003c/em\u003e gene within the large single-copy (LSC) region and the \u003cem\u003eaccD\u003c/em\u003e gene within the inverted repeat (IR) region, a configuration not found in the Hypericeae tribe, where a unique rearrangement consistently relocates \u003cem\u003ematK\u003c/em\u003e into the IR region and positions \u003cem\u003eaccD\u003c/em\u003e within the LSC. This pattern is characterized by a \u003cem\u003ematK\u003c/em\u003e relocation to the IR\u0026mdash;is specific to the Hypericeae tribe and is not observed in the ancestral Cratoxyleae or in the structurally intermediate Vismieae tribe.\u003c/p\u003e\u003cp\u003eIn Cratoxyleae plastomes, this arrangement contrasts with the canonical organization found in the core \u003cem\u003eHypericum\u003c/em\u003e group. Notably, 22 plastomes across Hypericaceae show shifts in gene order near the LSC\u0026ndash;IR boundaries, particularly involving the repositioning of \u003cem\u003etrnH\u003c/em\u003e and \u003cem\u003erbcL\u003c/em\u003e, often replacing the ancestral location of \u003cem\u003ematK\u003c/em\u003e. This rearranged configuration appears to be a defining structural signature of the Hypericeae lineage.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAnother ancestral structural event occurred in the Vismieae tribe, which exhibits a\u0026thinsp;~\u0026thinsp;54 kb inversion encompassing two large- locally collinear blocks (LCBs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This inversion appears to represent a secondary rearrangement relative to the Cratoxyleae event, with breakpoints near the \u003cem\u003eaccD\u003c/em\u003e and \u003cem\u003etrnK-UUU\u003c/em\u003e genes. The Vismieae plastomes also display a pronounced IR expansion, extending into the \u003cem\u003eycf1\u003c/em\u003e region, resulting in a partial duplication of the \u003cem\u003eycf1\u003c/em\u003e gene in the opposing IR copy, a unique feature restricted to this lineage.\u003c/p\u003e\u003cp\u003eIn contrast, plastomes from the Hypericeae tribe reveal extensive structural complexity, characterized by a greater number of LCBs and at least 15 breakpoints localized primarily within the LSC region ( \u003cb\u003eSupplementary Figs.\u0026nbsp;1\u0026ndash;3\u003c/b\u003e). These rearrangements reflect multiple independent inversion events and IR boundary shifts, marking Hypericeae as the most structurally dynamic lineage among the three tribes. This pattern suggests a progressive accumulation of plastome complexity throughout the diversification of Hypericaceae.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eIR Expansion and Contraction in the Hypericaceae Family\u003c/h2\u003e\u003cp\u003eStructural variation in the IR boundaries of Hypericaceae plastomes, relative to the ancestral plastome of \u003cem\u003eMesua ferrea\u003c/em\u003e, reveals dynamic patterns of expansion and contraction, resulting in gene duplication and relocation events across lineages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; \u003cb\u003eSupplementary Fig.\u0026nbsp;4\u003c/b\u003e). Within the Hypericeae tribe, significant IR contraction was observed at the LSC/IRA junction in \u003cem\u003eHypericum ascyron\u003c/em\u003e and \u003cem\u003eH. chejuense\u003c/em\u003e, reducing the IR by approximately 11\u0026ndash;13 kb and excluding a block of eight genes (from \u003cem\u003erpl2\u003c/em\u003e to \u003cem\u003erps12\u003c/em\u003e). An even more pronounced contraction (~\u0026thinsp;17 kb) was detected in \u003cem\u003eH. erectum\u003c/em\u003e, affecting the \u003cem\u003erps11\u0026ndash;rps7\u003c/em\u003e region at the LSC/IRA/SSC boundaries. In contrast, species such as \u003cem\u003eH. laxum\u003c/em\u003e and \u003cem\u003eT. japonicum\u003c/em\u003e within Hypericeae, as well as all examined members of the Vismieae tribe, showed no evidence of IR contraction. Across the family, IR expansions at both the IRB/SSC and IRA/SSC boundaries were more consistently observed. These expansions led to the duplication of \u003cem\u003endhF\u003c/em\u003e in most species across Hypericeae, Vismieae, and Cratoxyleae tribes. The only exception was \u003cem\u003eThornea calcicola\u003c/em\u003e, which retained a full copy and a partial copy of \u003cem\u003endhF\u003c/em\u003e near the IRB/SSC boundary. In addition, a complete duplication of \u003cem\u003eycf1\u003c/em\u003e was detected only in \u003cem\u003eH. ascyron\u003c/em\u003e and \u003cem\u003eT. japonicum\u003c/em\u003e, suggesting lineage-specific expansions in these taxa (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). A particularly notable case of large-scale IR expansion occurred in the Vismieae tribe, where the IR region extended from IRB/LSC to the \u003cem\u003erpl2\u0026ndash;psaJ\u003c/em\u003e region, expanding to approximately 56 kb\u0026mdash;nearly double the IR size compared to other tribes. This boundary shift resulted in the duplication of up to 30 genes, with no contraction events observed in this tribe. Similarly, the Cratoxyleae tribe showed no evidence of contraction but exhibited an expansion at the IRB/SSC junction, leading to the duplication of the entire \u003cem\u003eycf1\u003c/em\u003e gene on one side. A partial copy (~\u0026thinsp;981 bp) of \u003cem\u003eycf1\u003c/em\u003e was found at the opposite IR boundary near the LSC/IRA junction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These IR boundary dynamics of expansions, contractions, and gene duplications that demonstrate the extensive plastome structural evolution across the Hypericaceae family and highlight the lineage-specific mechanisms shaping IR architecture. Comparative analysis of the \u003cem\u003eaccD\u003c/em\u003e gene across Hypericaceae plastomes revealed the presence of internal insertions comprising tandem repeat sequences, with notable variation in both length and position. Two species, \u003cem\u003eH\u003c/em\u003e. \u003cem\u003eerectum\u003c/em\u003e and \u003cem\u003eH. chejuense\u003c/em\u003e, exhibited exceptionally long insertions of 1,468 and 1,484 \u003cem\u003ebp\u003c/em\u003e, respectively, disrupting the conserved coding region of \u003cem\u003eacc\u003c/em\u003eD. In contrast, most other species contained shorter insertions ranging from 79 bp to 459 \u003cem\u003ebp\u003c/em\u003e. These repeat-derived expansions fractured the gene's conserved domains and contributed to significant structural divergence among lineages. The pattern suggests that repeat proliferation has played a key role in \u003cem\u003eacc\u003c/em\u003eD evolution, potentially impacting gene function and driving plastome plasticity within Hypericaceae.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGene Relocation and ORF Emergence in\u003c/b\u003e \u003cb\u003eHypericum\u003c/b\u003e \u003cb\u003ePlastomes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eComparative analysis of plastid genomes among five \u003cem\u003eHypericum\u003c/em\u003e species (\u003cem\u003eH\u003c/em\u003e. \u003cem\u003eascyron\u003c/em\u003e, \u003cem\u003eH\u003c/em\u003e. chejuense, \u003cem\u003eH\u003c/em\u003e. \u003cem\u003eerectum\u003c/em\u003e, \u003cem\u003eH\u003c/em\u003e. \u003cem\u003elaxum\u003c/em\u003e, and \u003cem\u003eH\u003c/em\u003e. \u003cem\u003epatulum\u003c/em\u003e) revealed lineage-specific structural rearrangements associated with the relocation of \u003cem\u003eacc\u003c/em\u003eD and \u003cem\u003emat\u003c/em\u003eK genes and the emergence of novel open reading frames (ORFs) (\u003cb\u003eSupplementary Fig.\u0026nbsp;5\u003c/b\u003e). In \u003cem\u003eH\u003c/em\u003e. \u003cem\u003eascyron\u003c/em\u003e, both \u003cem\u003emat\u003c/em\u003eK and \u003cem\u003eacc\u003c/em\u003eD are translocated into or near the inverted repeat (IR) regions. This rearrangement is accompanied by the presence of lineage-specific ORFs, including ORF13 (between \u003cem\u003eacc\u003c/em\u003eD and \u003cem\u003emat\u003c/em\u003eK) and ORF15 (downstream of \u003cem\u003emat\u003c/em\u003eK). These novel ORFs are absent in other species, suggesting they may have originated from IR-mediated recombination or structural rearrangement at the IR junctions. In \u003cem\u003eH\u003c/em\u003e. \u003cem\u003echejuense\u003c/em\u003e and H. \u003cem\u003eerectum\u003c/em\u003e, similar patterns are observed. While \u003cem\u003emat\u003c/em\u003eK remains conserved, ORF66/67 (\u003cem\u003eH\u003c/em\u003e. \u003cem\u003echejuense\u003c/em\u003e) and ORF8/9 (\u003cem\u003eH\u003c/em\u003e. \u003cem\u003eerectum\u003c/em\u003e) are located downstream, suggesting independent ORF formation following structural rearrangement events. In \u003cem\u003eH\u003c/em\u003e. \u003cem\u003elaxum\u003c/em\u003e, \u003cem\u003eacc\u003c/em\u003eD is placed upstream of ORF1, again implying that the disruption of plastome structure near \u003cem\u003eacc\u003c/em\u003eD may promote ORF emergence. In contrast, \u003cem\u003eH\u003c/em\u003e. \u003cem\u003epatulum\u003c/em\u003e shows a markedly different organization. Both \u003cem\u003eacc\u003c/em\u003eD and \u003cem\u003emat\u003c/em\u003eK are located adjacently within the large single-copy (LSC) region, and no novel ORFs are detected in their vicinity. This more conserved structure likely reflects the absence of recombination-prone IR junctions near these genes, supporting the hypothesis that relocation of coding genes into the IR and disruption of neighboring intergenic regions play a key role in the generation of novel ORFs in specific \u003cem\u003eHypericum\u003c/em\u003e lineages.\u003c/p\u003e\u003cp\u003e\u003cb\u003eParallel Structural Placement of\u003c/b\u003e \u003cb\u003ematK\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eaccD\u003c/b\u003e \u003cb\u003eGenes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAn unusual\u0026thinsp;~\u0026thinsp;65 kb inversion in the basal clade of the Cratoxyleae tribe positions the \u003cem\u003etrnH\u0026ndash;rbcL\u003c/em\u003e region at the beginning of the large single-copy (LSC) region, placing \u003cem\u003etrnH\u003c/em\u003e and \u003cem\u003ematK\u003c/em\u003e in a canonical configuration typically observed in chloroplast genomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Structural comparisons across Cratoxyleae, Vismieae, and Hypericeae tribes revealed a pattern of parallel rearrangements affecting the relative positions of \u003cem\u003ematK\u003c/em\u003e and \u003cem\u003eaccD\u003c/em\u003e genes, which may suggest signatures of biparental structural inheritance or convergent rearrangement events. In Cratoxyleae, the \u003cem\u003ematK\u003c/em\u003e and \u003cem\u003eaccD\u003c/em\u003e genes are positioned adjacently in the LSC region (\u003cem\u003ematK:accD\u003c/em\u003e), a configuration inferred to represent the ancestral state. In contrast, a second large inversion (~\u0026thinsp;54 kb) in the Vismieae tribe results in the reversal of this order to \u003cem\u003eaccD:matK\u003c/em\u003e, positioned adjacent to the inverted repeat (IR) region, along with a substantial IR expansion. This modified \u003cem\u003eaccD:matK\u003c/em\u003e orientation is also retained in the basal genus of the Hypericeae tribe (\u003cem\u003eTriadenum\u003c/em\u003e), accompanied by the incorporation of \u003cem\u003eycf1\u003c/em\u003e into the IR, further supporting a shared structural signature between Vismieae and early diverging Hypericeae plastomes. Interestingly, \u003cem\u003eThornea\u003c/em\u003e and \u003cem\u003eH. patulum\u003c/em\u003e retain the ancestral \u003cem\u003ematK:accD\u003c/em\u003e configuration, suggesting structural conservation with the Cratoxyleae lineage. However, pronounced divergence is evident in the \u003cem\u003eHypericum\u003c/em\u003e core clade, particularly the Trigynobrathys section (\u003cem\u003eH. laxum\u003c/em\u003e, \u003cem\u003eH. japonicum\u003c/em\u003e), where multiple inversion events appear to have disrupted the original gene order. In this group, the repositioning of \u003cem\u003eaccD\u003c/em\u003e into the LSC and \u003cem\u003ematK\u003c/em\u003e into the IR region is observed, except in \u003cem\u003eH. patulum\u003c/em\u003e, which uniquely retains the ancestral \u003cem\u003eCratoxyleae\u003c/em\u003e-like arrangement. These findings indicate that the \u003cem\u003ematK\u0026ndash;accD\u003c/em\u003e gene pair serves as a phylogenetically informative marker reflecting structural divergence within Hypericaceae.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSelective Retention of\u003c/b\u003e \u003cb\u003ematK\u0026ndash;trnK-UUU\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn plastomes of the Hypericeae tribe, substantial restructuring occurred around the \u003cem\u003etrnK-UUU\u003c/em\u003e region. The \u003cem\u003etrnK-UUU\u003c/em\u003e gene was lost in all examined species, and \u003cem\u003ematK\u003c/em\u003e was retained as a free-standing gene, uncoupled from its usual intronic context. Despite the loss of intron, \u003cem\u003ematK\u003c/em\u003e was preserved across all species, indicating strong functional conservation (\u003cb\u003eSupplementary Figs.\u0026nbsp;5 and 6\u003c/b\u003e). The Hypericeae plastomes retained only five introns overall, with both the cis-spliced \u003cem\u003erps12\u003c/em\u003e and \u003cem\u003etrnK-UUU\u003c/em\u003e introns being absent. Selection analysis showed dN/dS ratios\u0026thinsp;\u0026gt;\u0026thinsp;1 for \u003cem\u003ematK\u003c/em\u003e along the branches leading to Hypericaceae and related Clusioid lineages. Likelihood ratio tests (LRTs) supported the hypothesis that \u003cem\u003ematK\u003c/em\u003e underwent positive selection, likely driven by structural reorganization and the functional compensation for the loss of \u003cem\u003etrnK-UUU\u003c/em\u003e (Supplementary Figure ). Furthermore, although most plastid genes showed conservation, some \u003cem\u003erpo\u003c/em\u003e genes (e.g., \u003cem\u003erpoC1\u003c/em\u003e) displayed intron loss and signs of positive selection. These genes encode the RNA polymerase type I enzyme, which is essential for plastid tRNA and mRNA synthesis. The adaptive evolution of this gene group may reflect compensatory mechanisms associated with plastome structural reconfiguration in Hypericaceae. Collectively, these findings suggest that the relocation and selection of \u003cem\u003ematK\u003c/em\u003e, coupled with functional shifts in \u003cem\u003erpo\u003c/em\u003e genes, are key evolutionary responses to plastome rearrangement in Hypericaceae. A more comprehensive sampling across this family will be essential to fully elucidate the evolutionary mechanisms underlying elevated substitution rates and gene reorganization.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eGene and Intron Loss Patterns in Hypericaceae and Related Lineages\u003c/h2\u003e\u003cp\u003eThe plastomes of Hypericeae species contained 69\u0026ndash;75 protein-coding genes, 29 tRNA genes, and four rRNA genes (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;8; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Notably, the translation initiation factor (\u003cem\u003einf\u003c/em\u003eA), ribosomal protein S16 (\u003cem\u003erps\u003c/em\u003e16), and tRNA-Lys (\u003cem\u003etrn\u003c/em\u003eK-UUU) genes were completely absent across all examined Hypericeae plastomes (\u003cb\u003eSupplementary Fig.\u0026nbsp;7\u003c/b\u003e). Additionally, \u003cem\u003erps\u003c/em\u003e7 was found to be pesduogenized only in \u003cem\u003eH\u003c/em\u003e. \u003cem\u003eascyron\u003c/em\u003e, \u003cem\u003eH\u003c/em\u003e. \u003cem\u003eerectum\u003c/em\u003e, and \u003cem\u003eH\u003c/em\u003e. \u003cem\u003epatulum\u003c/em\u003e, while \u003cem\u003erpl\u003c/em\u003e23 was pesduogenized in all Hypericeae species. The \u003cem\u003erpl\u003c/em\u003e32 gene also appeared to be pesduogenized in all Hypericeae members, likely due to frameshift mutations and internal stop codons. Complete or near- loss of \u003cem\u003eycf\u003c/em\u003e1 and \u003cem\u003eycf\u003c/em\u003e2 was detected in \u003cem\u003eH\u003c/em\u003e. \u003cem\u003eerectum\u003c/em\u003e, \u003cem\u003eH\u003c/em\u003e. \u003cem\u003echejuens\u003c/em\u003ee, \u003cem\u003eH\u003c/em\u003e. \u003cem\u003epatulum\u003c/em\u003e, and \u003cem\u003eT\u003c/em\u003e. \u003cem\u003ecalcicola\u003c/em\u003e. Intron loss was also widespread in Hypericeae plastomes. Specifically, all species lacked both introns of the \u003cem\u003eclp\u003c/em\u003eP gene, the second (cis-spliced) intron of \u003cem\u003erps\u003c/em\u003e12, the \u003cem\u003erpo\u003c/em\u003eC1 intron, and the second intron of \u003cem\u003eycf\u003c/em\u003e3. In the Cratoxyleae tribe, gene loss was limited to \u003cem\u003einf\u003c/em\u003eA and rps16, while intron losses mirrored those in Hypericeae, with absence of introns in \u003cem\u003erps\u003c/em\u003e12, \u003cem\u003eycf\u003c/em\u003e3, and the second intron of \u003cem\u003eclp\u003c/em\u003eP. The Vismieae tribe also exhibited complete loss of \u003cem\u003einf\u003c/em\u003eA and \u003cem\u003erps\u003c/em\u003e16, and pseudogenization of \u003cem\u003erpl\u003c/em\u003e23, \u003cem\u003erpl\u003c/em\u003e32, and \u003cem\u003erps\u003c/em\u003e7, as well as \u003cem\u003eycf\u003c/em\u003e1 and \u003cem\u003eycf\u003c/em\u003e2 in all sampled species of \u003cem\u003eHarungana\u003c/em\u003e and \u003cem\u003eVismia\u003c/em\u003e. Intron losses in Vismieae included \u003cem\u003erps\u003c/em\u003e12, \u003cem\u003eycf\u003c/em\u003e3, \u003cem\u003eclp\u003c/em\u003eP, \u003cem\u003erpo\u003c/em\u003eC1, and \u003cem\u003eatp\u003c/em\u003eF, suggesting extensive structural reduction across the plastomes of this lineage. In the related Clusiaceae family, plastome gene losses were more limited: \u003cem\u003einf\u003c/em\u003eA was absent in both \u003cem\u003eMammea riparia\u003c/em\u003e and \u003cem\u003eRheedia edulis\u003c/em\u003e, while \u003cem\u003erps\u003c/em\u003e16 was lost only in \u003cem\u003eM\u003c/em\u003e. \u003cem\u003eriparia\u003c/em\u003e. Both species exhibited intron loss in \u003cem\u003eycf\u003c/em\u003e3 but retained other intron-containing genes intact.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003ePhylogenetic Reconstruction of Hypericaceae and Related Malpighiales\u003c/h2\u003e\u003cp\u003eTo reconstruct evolutionary relationships within the Hypericaceae family and among related lineages in the order Malpighiales, a maximum likelihood (ML) approach was applied using a concatenated alignment of 59 plastid protein-coding genes (supermatrix length: 46,929 bp) across 29 genera, including 25 Hypericaceae species and 4 outgroup taxa (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The outgroup comprised representatives from the clusioid clade, including \u003cem\u003eHypericum monogynum\u003c/em\u003e and \u003cem\u003eCalophyllum cochinchinense\u003c/em\u003e (Hypericaceae), \u003cem\u003eTristicha trifaria\u003c/em\u003e (Podostemaceae), \u003cem\u003eMesua ferrea\u003c/em\u003e (Calophyllaceae), and \u003cem\u003eMoronobea\u003c/em\u003e and \u003cem\u003eRheedia\u003c/em\u003e species (Clusiaceae) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe ML analysis yielded an optimal tree topology with a log-likelihood of \u0026minus;\u0026thinsp;226352.109, while both ML and BI trees displayed highly congruent topologies. All major branches were strongly supported with bootstrap support (BS)\u0026thinsp;=\u0026thinsp;100 and Bayesian posterior probability (PP)\u0026thinsp;=\u0026thinsp;1.00 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Phylogenetic analyses recovered the Cratoxyleae tribe as a monophyletic group and sister to a clade composed of Vismieae and Hypericeae, confirming tribe-level relationships within Hypericaceae. Within this framework, Vismieae was recovered as sister to Hypericeae, indicating a closer evolutionary affinity between these two tribes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAmong the \u003cem\u003eHypericum\u003c/em\u003e species:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eH. laxum\u003c/em\u003e and \u003cem\u003eH. japonicum\u003c/em\u003e grouped closely with \u003cem\u003eTriadenum fauriei\u003c/em\u003e and \u003cem\u003eT. japonicum\u003c/em\u003e, forming a well-supported clade corresponding to the Trigynobrathys section (BS/PP\u0026thinsp;=\u0026thinsp;100/1.00).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eThornea\u003c/em\u003e species also clustered within this clade, confirming their close phylogenetic relationship to \u003cem\u003eTriadenum\u003c/em\u003e and the Trigynobrathys section.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eH. patulum\u003c/em\u003e and \u003cem\u003eH. monogynam\u003c/em\u003e formed a clade within the Ascyreia s.l. + campylosporous section, which was resolved as sister to the Roscyna section represented by \u003cem\u003eH. ascyron\u003c/em\u003e (BS/PP\u0026thinsp;=\u0026thinsp;100/1.00).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eIn the core \u003cem\u003eHypericum\u003c/em\u003e, \u003cem\u003eH. erectum\u003c/em\u003e and \u003cem\u003eH. chejuense\u003c/em\u003e formed a distinct clade (BS/PP\u0026thinsp;=\u0026thinsp;100/1.00), which was recovered as sister to the combined Ascyreia s.l., Roscyna, and campylosporous lineages.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eWithin Vismieae, \u003cem\u003eHarungana\u003c/em\u003e and \u003cem\u003eVismia\u003c/em\u003e species were recovered as a well-supported monophyletic group. In Cratoxyleae, \u003cem\u003eCratoxylum\u003c/em\u003e and \u003cem\u003eEliea\u003c/em\u003e formed a strongly supported sister clade to the Hypericeae\u0026ndash;Vismieae clade (BS/PP\u0026thinsp;=\u0026thinsp;100/1.00), further confirming tribal boundaries. Additional ML phylogenies constructed using individual plastid gene sets (e.g., \u003cem\u003eatp\u003c/em\u003e, \u003cem\u003endh\u003c/em\u003e, \u003cem\u003epet\u003c/em\u003e, \u003cem\u003epsa\u003c/em\u003e, \u003cem\u003epsb\u003c/em\u003e, \u003cem\u003erpl\u003c/em\u003e, \u003cem\u003erps\u003c/em\u003e, \u003cem\u003erpo\u003c/em\u003e, \u003cem\u003eycf\u003c/em\u003e) yielded consistent topologies (Supplementary Fig.\u0026nbsp;1), though variation was observed in certain marker-based trees (e.g., \u003cem\u003eccsA\u003c/em\u003e, \u003cem\u003eclpP\u003c/em\u003e, \u003cem\u003epet\u003c/em\u003e, \u003cem\u003epsb\u003c/em\u003e, \u003cem\u003eycf\u003c/em\u003e), which showed slight topological shifts in relationships among tribes. Analysis of nuclear ITS sequences provided complementary evidence, particularly supporting the separation of \u003cem\u003eH. laxum\u003c/em\u003e and \u003cem\u003eH. japonicum\u003c/em\u003e from core \u003cem\u003eHypericum\u003c/em\u003e, instead of grouping them with \u003cem\u003eTriadenum\u003c/em\u003e, thereby corroborating plastome-based results (\u003cb\u003eSupplementary Fig.\u0026nbsp;8\u003c/b\u003e). Notably, the relationships among \u003cem\u003eThornea\u003c/em\u003e, \u003cem\u003eTriadenum\u003c/em\u003e, and the Trigynobrathys section appeared controversial in certain gene trees, particularly those constructed using \u003cem\u003erps\u003c/em\u003e, \u003cem\u003epet\u003c/em\u003e, and \u003cem\u003eycf\u003c/em\u003e genes. Collectively, both coalescent-based and concatenated tree methods support a scenario in which: The New World \u003cem\u003eHypericum\u003c/em\u003e clades (e.g., \u003cem\u003eThornea\u003c/em\u003e, \u003cem\u003eTriadenum\u003c/em\u003e, \u003cem\u003eTrigynobrathys\u003c/em\u003e) diverge early, While both New and Old-World clades (e.g., \u003cem\u003eAndrosaemum\u003c/em\u003e, \u003cem\u003eAscyria\u003c/em\u003e, \u003cem\u003eRoscyna\u003c/em\u003e, and the \u003cem\u003eHypericum\u003c/em\u003e core) form a monophyletic group sister to Vismieae. These results provide robust phylogenomic evidence for major taxonomic realignments within Hypericaceae and reinforce plastome structure and gene order as useful phylogenetic markers for resolving complex tribal and sectional relationships.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eElevated Substitution Rates in Hypericaceae\u003c/h2\u003e\u003cp\u003eAnalysis of substitution rates showed that plastid genes in Hypericeae experienced significantly elevated rates of molecular evolution in specific bordered disturbed genes among the tribe. Nonsynonymous (dN) and synonymous (dS) substitution rates were observed higher, respectively, than those observed in \u003cem\u003eCratoxylum\u003c/em\u003e. \u003cem\u003eH. ascyron\u003c/em\u003e exhibited especially high substitution rates across most plastid genes, with dN and dS values are greater than in \u003cem\u003eCratoxylum\u003c/em\u003e, respectively. The genes \u003cem\u003eaccD\u003c/em\u003e, \u003cem\u003eclpP\u003c/em\u003e, and \u003cem\u003ematK\u003c/em\u003e, which also underwent structural modifications, showed markedly accelerated substitution rates compared to those in \u003cem\u003eTristicha\u003c/em\u003e (\u003cb\u003eSupplementary Fig.\u0026nbsp;10\u0026ndash;15\u003c/b\u003e). Branch-site models and RELAX analyses further revealed that several genes in Hypericaceae underwent episodic positive selection (\u003cb\u003eSupplementary Table\u0026nbsp;2\u0026ndash;3\u003c/b\u003e). The genes \u003cem\u003eaccD\u003c/em\u003e, \u003cem\u003eclpP\u003c/em\u003e, \u003cem\u003ematK\u003c/em\u003e, \u003cem\u003ecemA\u003c/em\u003e, \u003cem\u003epsbN\u003c/em\u003e, \u003cem\u003erpl33\u003c/em\u003e, \u003cem\u003erps3\u003c/em\u003e, \u003cem\u003erps12\u003c/em\u003e, \u003cem\u003erpoA\u003c/em\u003e, and \u003cem\u003erpoC2\u003c/em\u003e showed elevated dN/dS ratios along branches leading to \u003cem\u003eHypericum\u003c/em\u003e, \u003cem\u003eCratoxylum\u003c/em\u003e, and \u003cem\u003eMarathrum\u003c/em\u003e. Likelihood ratio tests (LRTs) indicated significant positive selection (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Bonferroni-corrected) in branches leading to (\u003cem\u003eHypericum\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eCratoxylum\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eMarathrum\u003c/em\u003e) for \u003cem\u003eclpP\u003c/em\u003e, (\u003cem\u003eHypericum\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eCratoxylum\u003c/em\u003e) for \u003cem\u003eaccD\u003c/em\u003e, \u003cem\u003eclpP\u003c/em\u003e, and \u003cem\u003ematK\u003c/em\u003e, and at the \u003cem\u003eHypericum\u003c/em\u003e terminal branch for \u003cem\u003eaccD\u003c/em\u003e, \u003cem\u003eclpP\u003c/em\u003e, and \u003cem\u003ematK\u003c/em\u003e (\u003cb\u003eSupplementary Fig.\u0026nbsp;10\u0026ndash;16\u003c/b\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn a previous study, we reported that the plastid genome of \u003cem\u003eH. ascyron\u003c/em\u003e exhibited extensive structural disruption, including multiple large inversions, gene and intron loss, and significantly elevated substitution rates (Claude et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These features positioned \u003cem\u003eH. ascyron\u003c/em\u003e as one of the most highly rearranged plastomes among angiosperms and raised questions about whether such plastomes instability was species-specific or indicative of a broader evolutionary trend within Hypericaceae. The present study addresses this by expanding the taxonomic scope to include representatives from all three tribes of Hypericaceae (Hypericeae, Vismieae, and Cratoxyleae) and conducting comparative analyses with outgroups from Clusiaceae. Our results clearly demonstrate that the patterns observed in \u003cem\u003eH. ascyron\u003c/em\u003e are reflective of recurring evolutionary mechanisms operating across the family.\u003c/p\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003ePlastome Structural Dynamics and Gene Evolution in Hypericaceae\u003c/h2\u003e\u003cp\u003eStructural variation was widespread. Plastome sizes ranged from 138,163 bp (\u003cem\u003eH. patulum\u003c/em\u003e) to 176,170 bp (\u003cem\u003eH. madagascariensis\u003c/em\u003e), with much of this variation attributable to IR expansions and contractions. Members of the Vismieae tribe exhibited dramatic IR expansions exceeding 50 kb, leading to the duplication of over 20 genes. Conversely, significant IR contraction in \u003cem\u003eHypericeae\u003c/em\u003e resulted in the loss of multiple boundary-associated genes. These findings are paralleled in other lineages of Malpighiales, most notably in Passifloraceae, which exhibit IR loss and extensive structural reorganization (Cauz-Santos et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and Podostemaceae, which display compact, gene-reduced plastomes with high levels of rearrangement (Bedoya et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Such recurring patterns suggest that plastome instability may be an emergent trait in several clades of Malpighiales, especially those undergoing ecological specialization.\u003c/p\u003e\u003cp\u003eGene loss and pseudogenization were most pronounced in the Hypericeae tribe, where \u003cem\u003einf\u003c/em\u003eA, \u003cem\u003erpl\u003c/em\u003e23, \u003cem\u003erpl\u003c/em\u003e32, and \u003cem\u003erps\u003c/em\u003e16 were absent in all species, while \u003cem\u003erps\u003c/em\u003e7, \u003cem\u003eycf\u003c/em\u003e1, and \u003cem\u003eycf\u003c/em\u003e2 were pesduogenized in several lineages. These losses likely reflect functional transfers to the nuclear genome, a process previously documented in other angiosperm families such as \u003cem\u003eRanunculaceae\u003c/em\u003e and Passifloraceae (Park et al., 2015, Shrestha et al., 2020). For example, \u003cem\u003erpl\u003c/em\u003e32 was functionally replaced by a nuclear-encoded SOD fusion in \u003cem\u003ePopulus\u003c/em\u003e (Ueda et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), and \u003cem\u003erps\u003c/em\u003e16 loss occurred repeatedly with the emergence of dual-targeting mechanisms (Ueda et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In Geraniaceae, multiple plastid genes, including \u003cem\u003eclp\u003c/em\u003eP, underwent loss or pseudogenization, often associated with nuclear transfer or coevolutionary constraints (Weng et al., 2016). These examples collectively highlight that gene loss in plastid genomes is not stochastic but frequently accompanied by functional compensation and structural rearrangement (Wicke et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Jansen et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe consistent relocation of \u003cem\u003ematK\u003c/em\u003e into the IR region and its decoupling from the \u003cem\u003etrn\u003c/em\u003eK-UUU intron further suggest a functional repurposing under lineage-specific selective pressures. This inference is supported by our dN/dS analysis, which revealed elevated substitution rates in \u003cem\u003emat\u003c/em\u003eK across multiple branches in the Hypericeae tribe. Notably, in several species where \u003cem\u003ematK\u003c/em\u003e has relocated, we observed the presence of adjacent partial or large open reading frames (ORFs), a feature absents in closely related species where \u003cem\u003emat\u003c/em\u003eK remains embedded within the \u003cem\u003etrn\u003c/em\u003eK-UUU intron. These ORFs may represent truncated pseudogenes, novel gene fusions, or potentially co-evolving loci that have emerged in tandem with \u003cem\u003emat\u003c/em\u003eK displacement. Their consistent association with relocated \u003cem\u003ematK\u003c/em\u003e implies a possible structural or regulatory role, although further functional characterization is needed. The relocation of \u003cem\u003ematK\u003c/em\u003e or \u003cem\u003eaccD\u003c/em\u003e into IR regions, as seen in \u003cem\u003eHypericum\u003c/em\u003e species, may lead to structural disruption of neighboring intergenic regions and generation of lineage-specific ORFs, a phenomenon documented in other taxa (Guisinger et al., 2011b, Wicke et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Zhu et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Underlying this process is the presence of repeat elements that including palindromic and inverted repeats at IR junctions, which are known hotspots for homologous or illegitimate recombination, facilitating genomic rearrangements and the creation of novel ORFs (Kolodner and Tewari, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1979\u003c/span\u003e, Day and Madesis, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, Odahara et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eEvidence from previous studies reinforces the idea that \u003cem\u003ematK\u003c/em\u003e is subject to adaptive evolution. Hao et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) found that \u003cem\u003emat\u003c/em\u003eK evolves under positive selection across multiple angiosperm lineages, particularly in domains critical to its maturase function(Hao et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Furthermore, cases such as \u003cem\u003eEpifagus virginiana\u003c/em\u003e, \u003cem\u003eCuscuta\u003c/em\u003e, and leptosporangiate ferns demonstrate that \u003cem\u003ematK\u003c/em\u003e can function as a freestanding gene, independent of \u003cem\u003etrn\u003c/em\u003eK-UUU, and still retain its splicing role (Kuo et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Wicke et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). These findings align with our observations in Hypericaceae, suggesting that \u003cem\u003ematK\u003c/em\u003e relocation is not a random genomic event but rather a potentially adaptive response accompanied by secondary structural or functional changes.\u003c/p\u003e\u003cp\u003eIn addition, structural alterations in coding genes like \u003cem\u003eacc\u003c/em\u003eD, \u003cem\u003eclp\u003c/em\u003eP, and \u003cem\u003erpoC\u003c/em\u003e1 were associated with sequence divergence and loss of introns, a pattern consistent with accelerated substitution rates. These genes, essential for plastid function and organelle-nuclear communication, appear to be hotspots for adaptive evolution. Their modification, often via insertion or fragmentation, may be linked to selection acting on plastid performance in stress-prone habitats.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003ePhylogenetic Relationships and Tribal Evolution in Hypericaceae\u003c/h2\u003e\u003cp\u003ePhylogenomic reconstruction based on 67 plastid protein-coding genes yielded a well-resolved tree that supports the monophyly of the three recognized tribes of Hypericaceae: Hypericeae, Vismieae, and Cratoxyleae. Our analyses resolved Cratoxyleae as the earliest diverging lineage and sister to a clade comprising Vismieae and Hypericeae. This branching pattern provides strong support for tribal distinctions and clarifies deep evolutionary splits within the family, which were previously ambiguous in studies based on nuclear ITS and plastid intergenic spacers (Nurk and Blattner, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, N\u0026uuml;rk and Crockett, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Park and Kim, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCratoxyleae, with its conserved plastome structure and geographically restricted range in tropical Southeast Asia, likely represents an ancestral lineage within Hypericaceae. In contrast, Hypericeae and Vismieae, both show greater plastome rearrangements including IR expansion and gene loss that share a more recent common ancestor and reflect derived evolutionary trends. These patterns correspond with ecological transitions, as Hypericeae includes both tropical and temperate species, while \u003cem\u003eVismieae\u003c/em\u003e is largely tropical(Nurk and Blattner, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWithin Hypericeae, our plastome phylogeny revealed unexpected relationships. \u003cem\u003eH. laxum\u003c/em\u003e and \u003cem\u003eH. japonicum\u003c/em\u003e form a strongly supported clade with \u003cem\u003eTriadenum\u003c/em\u003e and \u003cem\u003eThornea\u003c/em\u003e, contradicting traditional genus-level assignments and indicating that these taxa may be better classified within a redefined \u003cem\u003eHypericum\u003c/em\u003e. This finding aligns with earlier ITS and low-copy nuclear marker studies suggesting the paraphyly of core \u003cem\u003eHypericum\u003c/em\u003e and potential instances of incomplete lineage sorting or ancient hybridization (Meseguer et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Norman, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Morphological similarities among these species may therefore reflect retained ancestral traits or convergent evolution rather than deep taxonomic splits(N\u0026uuml;rk et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMoreover, our results mirror geographic lineage structuring previously described in the genus. The \u003cem\u003eH. laxum\u0026ndash;Triadenum\u003c/em\u003e clade corresponds to the New World lineage (e.g., section \u003cem\u003eTrigynobrathys\u003c/em\u003e), while species such as \u003cem\u003eH. erectum\u003c/em\u003e and \u003cem\u003eH. patulum\u003c/em\u003e group with Old World clades like \u003cem\u003eAscyreia\u003c/em\u003e and \u003cem\u003eRoscyna\u003c/em\u003e. This East\u0026ndash;West divergence has been attributed to climatic shifts during the Oligocene\u0026ndash;Miocene, which drove altitudinal migrations, ecological niche shifts, and speciation events in \u003cem\u003eHypericum\u003c/em\u003e (N\u0026uuml;rk et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Meseguer et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Our genome-scale phylogeny supports this biogeographic scenario and suggests that these divergent events were accompanied by plastome evolution and structural remodeling.\u003c/p\u003e\u003cp\u003eOur study provides the most robust plastome-based framework to date for Hypericaceae. It not only affirms tribal relationships but also highlights hidden paraphyly within \u003cem\u003eHypericeae\u003c/em\u003e, signals the need for genus-level taxonomic revision, and corroborates ecological and geographic hypotheses about the group\u0026rsquo;s diversification. The concordance between plastome evolution, previous nuclear-based reconstructions, and biogeographic history reinforces a model of adaptive radiation in \u003cem\u003eHypericum\u003c/em\u003e driven by historical climate change and niche expansion across continents.\u003c/p\u003e\u003cp\u003eEnvironmental shifts may be a major driver of the plastome dynamism observed. N\u0026uuml;rk et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) demonstrated that cold-tolerant \u003cem\u003eHypericum\u003c/em\u003e species radiated rapidly in montane habitats such as the Andes and East Asia, likely in response to Oligocene\u0026ndash;Miocene climate changes. Structural genome evolution, including gene relocations, intron losses, and inversions may reflect adaptive responses to new ecological pressures, as similarly seen in \u003cem\u003eGeraniaceae\u003c/em\u003e and \u003cem\u003eSilene\u003c/em\u003e (Erixon and Oxelman, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Park et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Such plastome remodeling could facilitate transcriptional or regulatory flexibility under fluctuating environmental regimes.\u003c/p\u003e\u003cp\u003eIn summary, this study provides the first family-wide plastome-based phylogenomic assessment of Hypericaceae, revealing extensive structural dynamism, recurrent gene loss, and adaptive rearrangements across its three major tribes. By expanding the scope beyond the previously analyzed \u003cem\u003eH. ascyron\u003c/em\u003e, our data demonstrate that genome instability\u0026mdash;manifested as IR expansion/contraction, gene relocation, pseudogenization, and intron loss\u0026mdash;is not species-specific but a recurrent feature across the family, particularly within the \u003cem\u003eHypericeae\u003c/em\u003e and \u003cem\u003eVismieae\u003c/em\u003e lineages. The consistent relocation of \u003cem\u003emat\u003c/em\u003eK and its association with novel open reading frames, as well as elevated dN/dS ratios in \u003cem\u003emat\u003c/em\u003eK, \u003cem\u003eacc\u003c/em\u003eD, and \u003cem\u003eclp\u003c/em\u003eP, suggest that plastome remodeling is not a random consequence of genome decay, but rather reflects selective responses to ecological pressures. These structural changes are paralleled in other lineages of Malpighiales, such as Passifloraceae and Podostemaceae, where genome reconfiguration has also been linked to adaptation in specialized habitats. Phylogenetically, The placement of \u003cem\u003eTriadenum\u003c/em\u003e and \u003cem\u003eThornea\u003c/em\u003e within the \u003cem\u003eHypericum\u003c/em\u003e clade calls for taxonomic revision and reflects historical processes such as hybridization and incomplete lineage sorting. These findings are consistent with previous nuclear and morphological studies but now gain stronger support through genome-scale data. Biogeographically, our phylogeny reinforces the pattern of East\u0026ndash;West lineage divergence within \u003cem\u003eHypericum\u003c/em\u003e, supporting a scenario of rapid radiation in response to Miocene climate shifts and montane expansion. The convergence of genomic rearrangements with ecological transitions suggests that plastome evolution played a significant role in enabling the widespread distribution and diversification of \u003cem\u003eHypericum\u003c/em\u003e. Altogether, this study not only provides a resolved phylogenetic framework for Hypericaceae but also advances our understanding of how plastid genome structure, gene content, and selection intersect with environmental history. These findings lay the groundwork for future investigations into plastome\u0026ndash;nuclear interactions, functional consequences of gene loss, and the genomic basis of ecological adaptation in angiosperms.\u003c/p\u003e\u003c/div\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003ePlant material and Next generation sequencing:\u003c/h2\u003e\u003cp\u003eThis study analyzed the plastid genomes of 14 species across the families Hypericaceae and Clusiaceae, including eight species from the tribe Hypericeae, three from Vismieae, and two from \u003cem\u003eCratoxyleae\u003c/em\u003e. Additionally, two species from Clusiaceae served as outgroups. Plant material used in this study was obtained either as leaf tissue or DNA from the Kew DNA Bank. Wild-collected samples from Korea were collected from common, non-protected species and therefore did not require special permits under Korean biodiversity regulations. Formal identification of the species was performed by SeonJoo Park, and voucher information is provided in \u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e. Additional specimens were obtained from established DNA and herbaria, including the Missouri Botanical Garden, the Kew DNA Bank, and the University of Texas Herbarium, with voucher details available in \u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e.This study did not involve the collection of any species listed in the Convention on the International Trade in Endangered Species of Wild Fauna and Flora (CITES). Fresh leaves were used for several species, including \u003cem\u003eH. erectum\u003c/em\u003e Thunb., \u003cem\u003eH. patulum\u003c/em\u003e Thunb., \u003cem\u003eH. chejuense\u003c/em\u003e S.J.Park \u0026amp; K.J. Kim, \u003cem\u003eH. laxum\u003c/em\u003e Blume., \u003cem\u003eH. japonicum\u003c/em\u003e Thunp., and \u003cem\u003eTriadenum fauriei\u003c/em\u003e R.Keller, with genomic DNA extracted using the GeneAll Plant SV Mini Kit (GeneAll Biotechnology, Seoul, Korea). Herbarium specimens leaves such as \u003cem\u003eHarungana madagascariensis\u003c/em\u003e Lam. Ex Poir., \u003cem\u003eVismia baccifera\u003c/em\u003e (L.) Triana \u0026amp; Planch., \u003cem\u003eEliea articulata\u003c/em\u003e (Lam.), \u003cem\u003eThornea calcicola\u003c/em\u003e Standl. \u0026amp; Steyerm., \u003cem\u003eRheedia edulis\u003c/em\u003e (Seem.) Planch. \u0026amp; Triana., and \u003cem\u003eMoronobea riparia\u003c/em\u003e Planch. \u0026amp; Triana. were processed using a modified 3\u0026times; CTAB protocol with a 3-hour incubation, as described by Doyle and Doyle DNA extraction method (Allen et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). For \u003cem\u003eCratoxylum maingayi\u003c/em\u003e and \u003cem\u003eH. rubescens\u003c/em\u003e (Oliv.) Byng \u0026amp; Christenh., DNA was sourced from the Kew DNA Bank. High-throughput sequencing was carried out on the Illumina HiSeq 2500 platform (Illumina Inc., San Diego, CA), generating approximately 6 Gb of 150 bp paired-end reads per sample from 550 bp insert libraries.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eAssembly and genome size comparison\u003c/h2\u003e\u003cp\u003eChloroplast genome assembly was conducted using GetOrganelle v1.7.5.3 and Velvet v1.2.10, with k-mer sizes ranging from 97 to 147 optimized for genome coverage and accuracy (Jin et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Zerbino and Birney, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Assembly quality was evaluated by mapping paired-end reads to the assembled plastomes using Bowtie2 v2.2.6(Langmead and Salzberg, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The results were visually inspected and verified in Geneious R11.0.5 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.geneious.com\u003c/span\u003e\u003cspan address=\"https://www.geneious.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). To analyze genome size variation, 381 complete chloroplast genomes from the order Malpighiales were downloaded from the NCBI nucleotide database as of February 28, 2022. Genome size statistics and visualizations were generated in R v4.0.4 using the ggplot2 package(Wickham, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). All newly assembled plastome sequences have been deposited in the NCBI GenBank database under accession numbers [PQ010624\u0026ndash;PQ010637].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003ePlastid gene annotation and Genome rearrangement\u003c/h2\u003e\u003cp\u003eGene annotation was initially performed in Geneious R11.0.5 using \u003cem\u003eNicotiana tabacum\u003c/em\u003e as the reference genome(Shinozaki et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). Gene boundaries were verified by BLASTN searches using NCBI-BLAST\u0026thinsp;+\u0026thinsp;v2.7.1(Camacho et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Transfer RNA genes were annotated with tRNAscan-SE v2.0.3 (Chan and Lowe, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and ARAGORN v1.2.38 (Laslett and Canback, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), and circular genome maps were constructed using OGDraw v1.3.1.(Greiner et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Structural rearrangements were investigated by comparing species from each tribe of \u003cem\u003eHypericaceae\u003c/em\u003e with an outgroup species (\u003cem\u003eMesua ferrea\u003c/em\u003e L., Clusiaceae) using the progressive Mauve algorithm implemented in Geneious(Darling et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, Geneious).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003ePhylogenetic and substitution rate estimation\u003c/h2\u003e\u003cp\u003ePhylogenetic reconstruction was based on 67 plastid protein-coding genes extracted from 29 plastomes. Each gene was aligned using MAFFT v7.450 with the G-INS-i strategy and concatenated into a supermatrix(Katoh and Standley, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Phylogenetic inference was carried out using maximum likelihood in IQ-TREE v1.6.2 under the GTR\u0026thinsp;+\u0026thinsp;GAMMA\u0026thinsp;+\u0026thinsp;I model with 1,000 ultrafast bootstrap replicates(Minh et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and Bayesian inference using MrBayes v3.3.7a(Ronquist et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Coalescent-based species trees were generated using ASTRAL v5.7.8 with 67 gene trees derived from IQ-TREE, incorporating models selected by ModelFinder (Mirarab et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Additionally, nuclear ITS regions were retrieved using GetOrganelle and aligned with MUSCLE(Edgar, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). ITS based phylogenies were reconstructed using IQ-TREE with the GTR\u0026thinsp;+\u0026thinsp;GAMMA\u0026thinsp;+\u0026thinsp;I model.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eSelection analysis\u003c/h2\u003e\u003cp\u003eTo detect selection, nonsynonymous (dN) and synonymous (dS) substitution rates were estimated for each plastid gene using the CODEML program within the PAML v4.8 package under the F3\u0026times;4 codon frequency model(Yang, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Branch-site positive selection was tested using the adaptive Branch-Site Random Effects Likelihood (absREL) model in HyPhy v2.5 via the Datamonkey server, with Holm-Bonferroni correction for multiple testing(Pond et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). A total of 70 protein-coding genes were aligned using the Translation Align option in MAFFT, and likelihood ratio tests (LRTs) were conducted to evaluate variation in dN/dS ratios across branches. Visualization of substitution rates was performed in R v4.0.4 using ggplot2(Wickham, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). To further explore divergence in the inverted repeat (IR) regions, the \u003cem\u003eycf1\u003c/em\u003e and \u003cem\u003eycf2\u003c/em\u003e genes from 11 genera of Malpighiales, including a known pseudogene in \u003cem\u003ePassiflora edulis\u003c/em\u003e, were aligned using MAFFT. Phylogenetic trees were constructed using PhyML with 100 bootstrap replicates to assess evolutionary patterns associated with IR-specific gene dynamics(Guindon et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eContributions\u003c/h2\u003e\n\u003cp\u003eJCS performed the experiments, generated datasets and figures, and wrote the first draft of the manuscript. KTP contributed to the data assembled, performed analyses, and read/edited the manuscript. SJP contributed to project design and read/edited the manuscript. All authors read and approved the final draft of the manuscript.\u003c/p\u003e\n\u003ch2\u003eEthics declarations\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperimental study on the plant, including collection of the material, comply with institutional, national, and international guidelines.\u003c/p\u003e\n\u003ch2\u003eClinical trial number\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch2\u003eFunding:\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the 2024 Yeungnam University Grant (221A061009), South Korea.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eJCS performed the experiments, generated datasets and figures, and wrote the first draft of the manuscript. KTP contributed to the data assembled, performed analyses, and read/edited the manuscript. SJP contributed to project design and read/edited the manuscript. All authors read and approved the final draft of the manuscript.All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eAll newly generated plastome sequences have been deposited in GenBank under accession numbers [PQ010624\u0026ndash;PQ010637]. Supporting data, including alignment files, gene order maps, and phylogenetic trees, are available in Supplementary Figures and Tables.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eALLEN GC, FLORES-VERGARA MA, KRASNYANSKI S, KUMAR S, THOMPSON WF. A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. 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New Phytol. 2016;209:1747\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Plastome evolution, Hypericaceae, Hypericum, Genome rearrangements, matK relocation, IR expansion","lastPublishedDoi":"10.21203/rs.3.rs-7295767/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7295767/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eThe evolutionary history of the \u003cem\u003eHypericaceae\u003c/em\u003e Juss. family remains poorly understood despite previous phylogenomic efforts. A prior study on \u003cem\u003eHypericum ascyron\u003c/em\u003e revealed exceptional plastome rearrangements and gene loss events, prompting questions about whether such genomic patterns are unique to \u003cem\u003eHypericum\u003c/em\u003e or reflect broader evolutionary trends within the family.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eTo explore plastome evolution across \u003cem\u003eHypericaceae\u003c/em\u003e, we sequenced 14 complete chloroplast genomes representing seven genera from the three major tribes: \u003cem\u003eHypericeae\u003c/em\u003e, \u003cem\u003eVismieae\u003c/em\u003e, and \u003cem\u003eCratoxyleae\u003c/em\u003e, alongside two genera from the related family \u003cem\u003eClusiaceae\u003c/em\u003e. Comparative analyses revealed extensive variation in plastome structure and gene content, including lineage-specific rearrangements, inversions, and expansions of inverted repeat (IR) regions. Notably, species within \u003cem\u003eVismieae\u003c/em\u003e exhibited significantly expanded IR regions. In \u003cem\u003eHypericum\u003c/em\u003e, unique lineage-specific open reading frames (ORFs) were identified, with genes such as \u003cem\u003eaccD\u003c/em\u003e and \u003cem\u003ematK\u003c/em\u003e relocated into or near the IR regions, likely driven by repeat-mediated recombination. Multiple independent losses of genes (\u003cem\u003erpl23\u003c/em\u003e, \u003cem\u003erpl32\u003c/em\u003e, \u003cem\u003erps16\u003c/em\u003e, \u003cem\u003einfA\u003c/em\u003e, \u003cem\u003eycf1\u003c/em\u003e, \u003cem\u003eycf2\u003c/em\u003e) and introns were observed across the family, particularly in \u003cem\u003eHypericeae\u003c/em\u003e, often accompanying structural rearrangements. Additionally, \u003cem\u003ematK\u003c/em\u003e was translocated from its typical position within the \u003cem\u003etrnK-UUU\u003c/em\u003e intron into the IR region, a rare event in angiosperm plastomes. The protein-coding genes \u003cem\u003eaccD\u003c/em\u003e and \u003cem\u003eclpP\u003c/em\u003e also showed domain-disrupting expansions, potentially impacting their functional roles.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eOur results demonstrate that plastome evolution in \u003cem\u003eHypericaceae\u003c/em\u003e is highly dynamic, characterized by substantial structural plasticity, gene loss, and lineage-specific innovation. These findings provide new insights into plastome diversification across the family and lay the groundwork for further phylogenomic and evolutionary studies within Malpighiales.\u003c/p\u003e","manuscriptTitle":"Plastome Evolution at the Edge: Structural Rearrangements, IR Expansion, and Gene Flux in Hypericaceae (Malpighiales)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-08 19:01:45","doi":"10.21203/rs.3.rs-7295767/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-30T10:43:25+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-23T23:04:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-19T14:18:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-12T08:16:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"48860737158918288015684371841962822227","date":"2025-10-09T02:24:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"43340057295025018512163922909551530223","date":"2025-10-08T16:32:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"101678615860366697751422216685187067947","date":"2025-10-07T14:34:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"185047020094117447674555752781465635946","date":"2025-09-26T05:41:23+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-25T19:27:18+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-01T07:45:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-28T07:37:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-27T08:55:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-08-27T08:51:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8f7961a4-a372-4716-9488-1fa6a9b716ad","owner":[],"postedDate":"October 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-29T16:07:03+00:00","versionOfRecord":{"articleIdentity":"rs-7295767","link":"https://doi.org/10.1186/s12870-025-07888-7","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2025-12-23 15:57:48","publishedOnDateReadable":"December 23rd, 2025"},"versionCreatedAt":"2025-10-08 19:01:45","video":"","vorDoi":"10.1186/s12870-025-07888-7","vorDoiUrl":"https://doi.org/10.1186/s12870-025-07888-7","workflowStages":[]},"version":"v1","identity":"rs-7295767","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7295767","identity":"rs-7295767","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}