Jia-Xin Yu
Rui-Zhu Bai
Ya-Ping Chen1,3
Xiao-Lei Ma
Ferhat Celep
Chun-Lei Xiang- 1State Key Laboratory of Phytochemistry and Natural Medicines, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China
- 2College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China
- 3Yunnan Key Laboratory of Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China
- 4Department of Biology, Faculty of Engineering and Natural Sciences, Kırıkkale University, Kırıkkale, Türkiye
Hypericum is the largest genus in Hypericaceae and is widely recognized for its medicinal importance. To investigate the evolutionary dynamics of the genus, we assembled and annotated the first mitochondrial genomes of H. ascyron (411,519 bp) and H. perforatum (485,128 bp). Both genomes were recovered as circular structures and contained 30 conserved protein-coding genes (PCGs). Phylogenetic analyses based on 29 mitochondrial PCGs resolved Hypericaceae as sister to Podostemaceae, forming a distinct clade closely related to Calophyllaceae. Notably, we detected topological incongruence between mitochondrial and plastid phylogenies for H. hirsutum, H. pulchrum, and H. perforatum, indicating inter-organellar conflict within the genus. In addition, 316–324 RNA editing sites were predicted, many of which resulted in non-synonymous codon changes. These newly generated mitogenomes represent valuable genomic resources and provide new insights into the genetic diversity and evolutionary complexity of Hypericum.
1 Introduction
Mitochondria are essential energy-producing organelles in eukaryotic cells, generating adenosine triphosphate (ATP) through oxidative phosphorylation and contributing to a wide range of fundamental cellular processes (Møller et al., 2021; Borcherding and Brestoff, 2023; Monzel et al., 2023). The mitochondrial genome (mitogenome) retains genetic evidence of the organelle’s evolutionary origin and encodes several core functional proteins (Sloan et al., 2012; Roger et al., 2017; Wang et al., 2024). Plant mitogenomes exhibit remarkable variation in size, ranging from 66 kb in the parasitic plant Viscum scurruloideum Barlow (Santalaceae) to as large as 18.99 Mb in Cathaya argyrophylla Chun & Kuang (Pinaceae) (Skippington et al., 2015; Huang et al., 2025). Their structure organization is highly complex and dynamic; although often depicted as circular molecules, plant mitogenomes can also occur in linear or branched forms (Smith and Keeling, 2015; Kozik et al., 2019). Such structural diversity is largely driven by repeat-mediated recombination, which generates multiple alternative genomic conformations. For example, in Morus L., large repeats promote the coexistence of multiple mitogenome structures, resulting in pronounced structural heterogeneity (Liu et al., 2024a). Similarly, extensive alternative conformations and reticulated structures have been reported in Gossypium L. (Kong et al., 2025 ). and Sorghum Moench (Zhang et al., 2023). In addition, cytosine (C) to uracil (U) RNA editing is a widespread post-transcriptional modification in plant mitochondria (Small et al., 2020). Despite extensive variation in genome size and structure, protein-coding genes (PCGs) within plant mitogenomes remain generally conserved (Gualberto and Newton, 2017; Hu et al., 2025).
Obtaining pure mitochondria is technically challenging due to persistent plastid contamination and tissue-specific metabolites (Boussardon et al., 2020; Luo et al., 2020). Consequently, most plant mitogenomes are typically assembled from total genomic DNA, which often leads to the co-assembly of nuclear and plastid sequences (Li et al., 2025a; Ni et al., 2025; Shen et al., 2025). This process is further complicated by frequent intracellular gene transfer (IGT), resulting in the formation of nuclear mitochondrial transferred fragments (NUMTs) and mitochondrial plastid transferred fragments (MTPTs) (Wang et al., 2024). Third-generation sequencing platforms such as PacBio HiFi and Oxford Nanopore have therefore become indispensable for resolving complex repetitive regions and structural rearrangements in plant mitogenomes (Lian et al., 2024; Almeida and Marques, 2025).
Hypericum L. (Malpighiales), a diverse genus of herbs, shrubs, and small trees, comprises approximately 500 species, making it the largest genus in Hypericaceae (Nürk et al., 2013; APG IV, 2016). The genus is distributed globally, with major centers of diversity in Eurasia and the Andean region of South America (Nürk and Blattner, 2010; Meseguer et al., 2013; Nürk et al., 2013). In China, 75 species and 9 subspecies have been documented (Robson, 2012, 2016; Bai et al., 2023, 2025). Owing to their long history of medicinal use, several species of Hypericum are widely recognized for their pharmacological properties. Among them, H. ascyron L. and H. perforatum L. (Saint John’s wort) exhibits diverse pharmacological activities, including antitumor, antidepressant, anti-inflammatory, antiproliferative, antimicrobial, neuroprotective and antioxidant activities (Zhang et al., 2020; Vincent et al., 2021; Kapoor et al., 2023; Liu et al., 2024b). These species also serve as ingredients in cosmeceutical formulations and food products (Jarzębski et al., 2020; Jakubczyk et al., 2021; Silva et al., 2021).
Previous phylogenetic studies of Hypericum have relied primarily on nuclear ribosomal ITS and plastid markers (Meseguer et al., 2013; Nürk et al., 2013, 2015). Although Ruhfel et al. (2011) included a mitochondrial gene (matR) in phylogeny reconstruction of Hypericum within the clusioid clade, using combined plastid and mitochondrial sequences, single-gene mitochondrial data provide limited insight into mitogenome evolution. Phylogenetic incongruence among nuclear, plastid and mitochondrial genomes is common in land plants, and may arise from processes such as incomplete lineage sorting (ILS) and hybridization, or analytical artifacts (Cox, 2018; Sousa et al., 2020). Recent mitogenome studies in large genera such as Dendrobium Sw. (Orchidaceae) and Quercus L. (Fagaceae) have revealed substantial cytonuclear discordance (Wang et al., 2023; Song et al., 2025). Given their low nucleotide substitution rates (Chen et al., 2017) and uniparental inheritance (Hu et al., 2025), plant mitogenomes provide an independent line of evidence for testing phylogenetic conflict. Furthermore, mitochondrial phylogenomics has yielded new insights into deep angiosperm relationships (Xue et al., 2022). As a species-rich and widely distributed lineage, Hypericum may likewise exhibit complex evolutionary patterns, underscoring the need for comprehensive mitogenome analyses.
In this study, we assembled and characterized the complete mitochondrial genomes of H. ascyron and H. perforatum using PacBio high-fidelity (HiFi) long-read sequencing. By integrating these newly generated mitogenomes with four additional Hypericum mitogenomes obtained from the Darwin Tree of Life (DToL) Project (The Darwin Tree of Life Project Consortium, 2022), we performed the first comparative mitogenomic analysis of this genus, with a particular focus on RNA editing, genome collinearity, and phylogenetic relationships. In addition, we constructed a mitochondrial phylogenetic framework for Malpighiales based on conserved mitochondrial PCGs. Collectively, our results provide foundational mitochondrial genomic resources for Hypericum, improve understanding of its evolutionary history within Malpighiales, and offer new perspectives for future breeding and evolutionary studies.
2 Materials and methods
2.1 Plant materials and sequencing
Hypericum ascyron and H. perforatum were cultivated in the greenhouse of the Kunming Institute of Botany, Chinese Academy of Sciences. The plants were originally transplanted from Anlong County (24°58′ E, 105°35′ N) and Fenggang County (27°39′ E, 107°43′ N), Guizhou Province, China, respectively. Fresh young leaves were collected, immediately frozen in liquid nitrogen and stored at –80°C.
Total genomic DNA was extracted using the modified cetyltrimethylammonium bromide (CTAB) protocol (Doyle and Doyle, 1987). The standard HiFi sequencing libraries were prepared with the SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences), and sequencing was performed on the PacBio Revio platform by Wuhan Frasergen Bioinformatics Co., Ltd. (Wuhan, China). For each species, approximately 10 Gb of PacBio HiFi data were generated, with total read numbers ranging from 516,416 to 632,999 and mean read lengths of 17,234 to 19,075 bp (Supplementary Table S1).
2.2 Genome assembly and annotation
Initial mitochondrial genome assemblies were generated using four PacBio HiFi read using four assemblers: HiMT v.1.1.0 (Tang et al., 2025), Oatk v.1.0 (Zhou et al., 2025), PMAT2 v.2.1.5 (Han et al., 2025) and TIPPo v.2.4 (Xian et al., 2025). These assemblers employ complementary strategies, including read filtering (HiMT, TIPPo) and graph-based resolution (Oatk, PMAT2). Parameters were set as follows: HiMT (default), Oatk (-k 1001 -c 50 -m embryophyta_mito.fam), TIPPo (-p hifi --trf), and PMAT2 (“autoMito” mode with estimates of 700 Mb for H. perforatum and 500 Mb for H. ascyron).
Plastid genomes of H. ascyron and H. perforatum were also assembled from HiFi reads using Oatk v.1.0, with the specialized “embryophyta_pltd.fam” gene profile. Assembly graphs were visualized and examined using Bandage v.0.8.1 (Wick et al., 2015). Based on structural comparative assessment of assembly and contiguity metrics (Supplementary Table S2), the HiMT-derived assemblies were selected as the final mitogenomes. These assemblies were subsequently validated for continuity and completeness. Assembly completeness was evaluated with the HiMT “assess” module by detecting conserved mitochondrial genes.
Raw HiFi reads were mapped to the draft mitogenomes using minimap2 v.2.28-r1209 (Li, 2021) with the “-ax map-hifi” parameters to evaluate continuity. Coverage depth was calculated with samtools coverage v.1.15.1 (Li et al., 2009) and bamtocov v.2.2.0 (Birolo and Telatin, 2022) and visualized using custom python script.
Mitogenomes and plastomes of H. androsaemum L., H. hircinum L., H. hirsutum L. and H. pulchrum L. were retrieved as unannotated assemblies from the Darwin Tree of Life (DToL) Project (The Darwin Tree of Life Project Consortium, 2022); the corresponding accession numbers are provided in Supplementary Tables S3 and S4. Mitochondrial genomes were annotated using the online tool PMGA (Li et al., 2025b), with the “29 Mitogenomes” database, whereas plastomes were annotated using PGA v.2.0 (Zhang et al., 2025). All annotation were manually curated in Geneious Prime v.2025.0.2 (Kearse et al., 2012) using closely related mitogenomes (e.g., Terniopsis yongtaiensis X.X.Su, Miao Zhang & B.Hua Chen OR818323, Calophyllum soulattri Burm.f. NC_079842) and plastomes (e.g., Cratoxylum arborescens (Vahl) Blume NC_062807, Vismia macrophylla Kunth PX719231). Circular genome maps were generated with OGDRAW v.1.3.1 (Greiner et al., 2019), and trans-splicing gene structures were visualized using PMGmap (Zhang et al., 2024c).
2.3 Repeat sequences and codon usage bias analysis
Three types of repeat sequences were identified: simple sequence repeats (SSRs), dispersed repeats, and tandem repeats. SSRs were detected with MISA v.2.1 (Beier et al., 2017) using ‘1-10 2-5 3-4 4-3 5-3 6-3’ thresholds. Dispersed repeats were identified with REPuter (Kurtz, 2001) using parameters ‘-c -f -p -r -l 30 -h 3 -best 5000’. Tandem repeats were detected using Tandem Repeats Finder v.4.09 (Benson, 1999) with default settings. The distribution and frequency of repeat sequences were visualized in R using ggplot2 (Wickham, 2016) and circlize (Gu et al., 2014) package.
Protein-coding genes (PCGs) were extracted using PhyloSuite v.1.2.3 (Xiang et al., 2023) and relative synonymous codon usage (RSCU) values were calculated using CodonW v.1.4.2 (https://codonw.sourceforge.net/).
2.4 Identification of intracellular transferred sequences
Homologous regions between mitochondrial and plastid genomes were identified using BLASTN v.2.16.0 (Camacho et al., 2009) with an E-value cutoff of 1e-5. Only alignments with a minimum length of 30 bp and a sequence identity of at least 70% were retained for subsequent analyses. The identified mitochondrial plastid transferred fragments (MTPTs) were visualized using the R package circlize.
2.5 Comparative analysis of six Hypericum mitochondrial genomes
Six Hypericum mitogenomes (two newly assembled and four from DToL) were compared. Gene copy number variation was visualized using heatmaps. RNA editing sites were predicted from PCGs using Deepred-Mt (Edera et al., 2021) with default settings, retaining sites with prediction probability values ≥ 0.9. Results were plotted using ggplot2 package.
Mitogenomes of Calophyllum soulattri (NC_079842) and Terniopsis yongtaiensis (OR818323), and plastomes of Cratoxylum arborescens (NC_062807) and Vismia macrophylla (PX719231) were retrieved from National Center for Biotechnology Information (NCBI) (Supplementary Tables S3-S4), and used as outgroups. A total of 30 mitochondrial PCGs and 71 plastid PCGs shared among taxa were aligned with MAFFT v.7.505 (Katoh and Standley, 2013) using the ‘--auto’ strategy and codon alignment mode. Gaps were removed with trimAl v.1.2rev57 (Capella-Gutiérrez et al., 2009) using “-automated1”, and concatenation was performed in Phylosuite (Xiang et al., 2023).
Phylogenetic trees were reconstructed using RAxML-NG v.1.2.2 (Kozlov et al., 2019) under the best-fit substitution models determined by ModelFinder v.2.2.0 (Kalyaanamoorthy et al., 2017) based on the Akaike Information Criterion (AIC): “GTR+F+G4” for mitogenomes and “GTR+F+I+G4” for plastomes. Maximum likelihood (ML) analyses used the “--all --bs-trees 5000” parameters, phylogenetic trees were visualized and edited using Figtree v.1.4.5 (http://tree.bio.ed.ac.uk/software/figtree/).
Pairwise synteny analysis among Hypericum mitogenomes was conducted using the “GetTwoGenomeSyn.pl” script of NGenomeSyn v.1.41 (He et al., 2023) with integrated minimap2. Homologous regions longer than 5,000 bp were retained as conserved colinear blocks for visualization and analysis.
2.6 Phylogenetic analysis for Malpighiales
To determine the phylogenetic position of Hypericum within Malpighiales, 19 published mitogenomes from seven families (Calophyllaceae, Clusiaceae, Euphorbiaceae, Passifloraceae, Podostemaceae, Rhizophoraceae and Salicaceae) were retrieved from NCBI. Five species from Celastrales and Sapindales were selected as outgroups (Supplementary Table S3). Twenty-nine shared mitochondrial PCGs were extracted in PhyloSuite v.1.2.3 (Xiang et al., 2023) and analyzed using the same ML method described above under the best-fit model “GTR+F+I+G4”.
Bayesian inference (BI) analyses were conducted in MrBayes v.3.2.7a (Ronquist et al., 2012) using two independent runs of 2000000 generations, sampling every 1,000 generations. The first 25% of samples were discarded as burn-in, and convergence was confirmed by an average standard deviation of split frequencies < 0.01.
3 Results
3.1 Genome assembly and general features of H. ascyron and H. perforatum
Using approximately 9.8–10.9 Gb of Pacbio HiFi long reads (Supplementary Table S1), we assembled the mitochondrial genomes of H. ascyron and H. perforatum with four assemblers. Although minor differences in genome topology and contiguity were observed (Supplementary Figure S1; Supplementary Table S2), these discrepancies were largely attributed to differences in how long repeat sequences were resolved by respective algorithms.
As shown in Supplementary Figure S1, the assemblies of H. perforatum generated by HiMT, Oatk, and TIPPo were highly consistent, each producing two contigs that formed a typical circular mitogenome. In contrast, all assemblers produced a single circular molecule for H. ascyron, with one or two repeat regions (3,302–5,233 bp) inferred from doubled coverage depth relative to unique contigs. Owing to its superior contiguity and well-resolved assembly graph topology, the HiMT assembly was selected as the final mitochondrial genome for both species (Figures 1A, B).
Figure 1. Mitogenome assembly graphs and annotation maps of H. ascyron and H. perforatum. (A) Assembly graph of the H. ascyron mitogenome. (B) Assembly graph of the H. perforatum mitogenome. (C) Annotated circular map of the H. ascyron mitogenome. (D) Annotated circular map of the H. perforatum mitogenome. Genes belonging to different functional categories are indicated by distinct colors. The inner circle represents the GC content (%).
Mapping back the HiFi reads confirmed consistent coverage depth, i.e. 456.47× for H. ascyron, 470.17× for H. perforatum Chr1, and 950.57× for H. perforatum Chr2 (Supplementary Figure S2). The mitogenome lengths were 411,519 bp for H. ascyron and 485,128 bp for H. perforatum. The latter comprises two circular chromosomes of 204,492 bp (Chr1) and 280,636 bp (Chr2) (Supplementary Table S1). The plastomes of H. ascyron and H. perforatum were 163,564 bp and 139,734 bp in length, respectively. Both exhibited a typical quadripartite structure consisting of a large single-copy (LSC) region and a small single-copy (SSC) region, separated by a pair of inverted repeat (IR) regions (Supplementary Figure S3).
The length of Hypericum mitogenomes ranged from 411,519 bp to 494,351 bp with GC contents of 43.92–44.28% (Supplementary Table S5). Gene content showed moderate variation among species, with 30 unique PCGs and 17–21 tRNAs identified (Table 1). Several mitochondrial genes contained multiple introns. Specifically, nad1, nad2, and nad5 were trans-spliced (Supplementary Figure S4), whereas ccmFC, nad4, nad7 and rps3 underwent cis-splicing. Plastid genome lengths ranged from 139,437 to 165,518 bp, with GC content of 37.3–37.7%, and encoded 75–79 PCGs, 34–35 tRNAs, and 8 rRNAs (Supplementary Table S6).
Table 1. Gene composition of the six Hypericum mitogenomes.
3.2 Repeat sequences in the mitogenomes of H. ascyron and H. perforatum
Three classes of repetitive sequences were identified: simple sequence repeats (SSRs), tandem repeats, and dispersed repeats. Their distribution varied markedly across the genomes (Figure 2A). SSRs were broadly and evenly distributed, whereas tandem repeats occurred less frequently. Dispersed repeats were the most abundant, dominated by short-distance events, with only a few long-distance pairs. Summary statistics for all repeat categories are presented in Supplementary Table S7 and Figure 2B.
Figure 2. Repeated sequences identified in the H. ascyron and H. perforatum mitogenomes. (A) Distribution of repetitive elements. The innermost circle shows dispersed repeats, the middle circle displays tandem repeats (red bars), and the outermost circle depicts SSRs (blue bars). The scale on the outermost circle is marked at 20-kb intervals. (B) Frequency of SSRs, tandem repeats, and dispersed repeats. The x-axis represents repeat types, and the y-axis indicates the number of repeats detected.
We detected 122 SSRs in H. ascyron and 153 in H. perforatum, totaling 1,630 bp (0.40%) and 2,117 bp (0.44%), respectively. These SSRs ranged from mono- to hexameric motifs, with mono-, di-, and tetrameric most prevalent. Hexameric SSRs were absent from H. perforatum but occurred five times in H. ascyron. Both species contained 43 tandem repeats; however, H. perforatum had a greater total tandem repeat length (2,616 bp) than H. ascyron (2,213 bp).
Dispersed repeats were classified as forward, palindromic, reverse, and complementary (Kurtz, 2001). Forward and palindromic repeats predominated in both species. Hypericum ascyron exhibited more dispersed repeats (267) than H. perforatum (206), corresponding 5.70% and 2.95% of the mitogenome, respectively. Both species contained one reverse repeat and no complementary repeats.
3.3 Codon usage bias analysis
Codon usage was analyzed across 31 and 33 mitochondrial PCGs from H. ascyron and H. perforatum, respectively (Figure 3). The genes used all 64 possible codons, representing 20 amino acids. A total of 32 codons showed relative synonymous codon usage (RSCU) values ≥ 1 in both species, indicating preferential codon usage (Supplementary Tables S8-S9). Three stop codons (UAA, UGA, and UAG) were present. Among all codons, GCU (Ala) was the most frequently used, whereas CAC (His) was the least. Leucine (Leu) was the most abundant amino acid encoded.
Figure 3. Relative synonymous codon usage (RSCU) patterns in mitochondrial protein-coding genes of (A) H. ascyron and (B) H. perforatum. The x-axis represents amino acids, the y-axis indicates RSCU value. Codons encoding the same amino acid are displayed as histogram in different colors. Amino acid usage values are indicated above the corresponding bars.
3.4 Identification of mitochondrial plastid transferred fragments
We detected 22 plastid-derived fragments (Ha_MTPT 1–22) in H. ascyron and 25 (Hp_MTPT 1–25) in H. perforatum (Figure 4). MTPTs ranged from 39 bp to 6,337 bp and accounted for 5.9% of the H. ascyron and 4.6% in H. perforatum (Supplementary Tables S10-S11). Twelve MTPTs overlapped coding regions, collectively containing seven intact plastid tRNA genes and one partial rrn18 sequence. The longest MTPT in H. ascyron (Ha_MTPT-1) included a full-length of trnI-CAU gene, which was also present in the second-longest MTPT of H. perforatum (Hp_MTPT-1; positions 113,185–115,949 bp on chr 1).
Figure 4. Homologous sequences identified between the plastomes and mitogenomes of (A) H. ascyron and (B) H. perforatum. Dark green and light green circles represent the plastome and mitogenome, respectively. The inner arcs show the homologous DNA fragments, with corresponding annotations shown along each arc.
3.5 Comparative mitogenomic analysis among Hypericum species
Comparisons among six Hypericum mitogenomes revealed variation in gene copy number (Figure 5A). All species shared a core set of 50 genes (30 PCGs, 17 tRNAs, and 3 rRNAs). Most PCGs were single-copy, but several genes showed species-specific duplications. trnM-CAU and rps12 were duplicated in all species, atp1, rpl5, and rps4 were duplicated only in H. hircinum, and cox3 and trnC-GCA were duplicated in H. androsaemum and H. ascyron, respectively. trnE-UUC and trnI-CAU had three copies in H. ascyron and H. perforatum, but one or two copies in other species. Several tRNAs (trnH-GUG, trnR-UCU, trnT-UGU, and trnfM-CAU) were absent from some mitogenomes (Figure 5A).
Figure 5. Integrated comparative analyses of six Hypericum mitogenomes, including four retrieved from the Darwin Tree of Life project. (A) Distribution patterns of mitochondrial PCGs, rRNA, and tRNA genes across Hypericum species. Color intensity corresponds to gene copy number. (B) Maximum-likelihood (ML) phylogenies of six Hypericum species inferred from 71 shared plastid genes (left) and 30 shared mitochondrial PCGs (right). Numbers at nodes represent bootstrap support values. Topological conflicts between the mitochondrial and plastid trees are highlighted with green squares. (C) Distribution of C-to-U RNA editing sites in mitochondrial PCGs among the six Hypericum species. The x-axis denotes gene names, and the y-axis indicates the number of RNA editing sites. (D) Colinear blocks among the six Hypericum mitogenomes. Rectangles represent the mitogenomes of each species.
Phylogenetic relationships inferred from shared mitochondrial and plastid PCGs consistently recovered Hypericum as monophyletic (Figure 5B; ML-BS = 100%). Both datasets supported three clades: H. androsaemum + H. hircinum, H. ascyron, and H. perforatum + H. hirsutum + H. pulchrum. While both phylogenies placed H. androsaemum + H. hircinum as sister to all remaining species, they differed in resolving relationships among H. hirsutum, H. pulchrum, and H. perforatum (Figure 5B). The plastome-based phylogeny resolved H. hirsutum + H. pulchrum as a clade, whereas mitogenome data grouped H. pulchrum with H. perforatum (Figure 5B).
C-to-U RNA editing predictions across 28 shared PCGs identified 316–324 C-to-U editing sites per species (probability ≥ 0.9) (Figure 5C). mttB contained the most editing sites (30), followed by ccmC, ccmFN, nad2 and nad4 (each > 20). Editing profiles were largely conserved except for ccmC (five additional sites in H. pulchrum) and cox3, which was edited only in H. pulchrum. Of all predicted edits, 28–30 were synonymous, while the remainder were non-synonymous, corresponding to 11 types of amino acid changes (Supplementary Tables S12-S17). Most edits occurred at the second position (186–192), followed by the first (107–112) and the third (20–24).
Synteny analysis of homologous blocks > 5,000 bp revealed extensive rearrangements among species (Figure 5D). The largest conserved collinear block occurred between H. androsaemum Chr2 and H. hircinum Chr1, and the largest inverted block was found between H. pulchrum Chr1 and H. perforatum Chr1 (Supplementary Table S18).
3.6 Phylogenetic analysis of Hypericum within Malpighiales
To clarify the evolutionary placement of Hypericum within Malpighiales, we reconstructed phylogenies using 29 conserved mitogenome PCGs from six Hypericum species and 24 representatives of Malpighiales, Celastrales and Sapindales. Both Maximum likelihood (ML) and Bayesian inference (BI) analyses strongly supported the monophyly of Malpighiales (Figure 6), which comprised three major clades: Rhizophoraceae + Euphorbiaceae, Passifloraceae + Salicaceae, and Hypericaceae + Calophyllaceae + Podostemaceae + Clusiaceae (Figure 6). Within the latter clade, Clusiaceae formed a sister group to the lineage including Calophyllaceae, Hypericaceae, and Podostemaceae, with Hypericaceae and Podostemaceae recovered as sister families.
Figure 6. Phylogenetic tree of Hypericum and 19 additional Malpighiales species reconstructed from 29 conserved mitochondrial PCGs. Numbers at nodes indicate the bootstrap support values (ML-BS) and posterior probabilities (BI-PP). Different colors denote distinct taxonomic groups.
4 Discussion
4.1 Structural and size variation of Hypericum mitogenomes
Plant mitochondrial genomes (mitogenomes) are characterized by exceptional structural complexity, which is largely driven by extensive repeat sequences and frequent recombination events (Yurina and Odintsova, 2016). In this study, we obtained complete and well-supported mitochondrial genome assemblies for Hypericum ascyron and H. perforatum by integrating multiple assembly strategies. Depth-of-coverage assessment and the HiMT “assess” module indicated that all genomic regions in the final assemblies were robustly supported (Supplementary Figure S2). Both mitogenomes contained the full set of 24 core mitochondrial protein-coding genes (PCGs) (Mower et al., 2012; Ni et al., 2025; Tang et al., 2025; Table 1), consistent with findings in other Malpighiales species such as Calophyllum soulattri (Cadorna et al., 2024) and Terniopsis yongtaiensis (Zhang et al., 2024b), and suggesting a relatively stable mitochondrial gene repertoire in this order compared with the extensive gene loss observed in Viscum L. (Petersen et al., 2015; Skippington et al., 2015).
As of November 2025, mitochondrial genomes have been reported for only about a dozen species within Malpighiales, revealing substantial size variation ranging from 371,235 bp in Garcinia mangostana L. (Wee et al., 2022 ). to 1,402,206 bp in Hevea camargoana Pires (Niu et al., 2024). Pronounced mitogenome size variation has also been documented in other angiosperm genera, such as Dendrobium Sw. (Soe et al., 2025). Despite this broad size range, the number of PCGs remains highly conserved, ranging from 29 in G. mangostana to 36 in Banisteriopsis caapi (Spruce ex Griseb.) C.V.Morton (Chavarro-Mesa et al., 2024) and Viola diffusa Ging (Zhang et al., 2024a). In this study, the mitogenomes of H. ascyron (411,519 bp) and H. perforatum (485,128 bp) fall within the lower-to-medium range of Malpighiales and are comparable to the closely related Terniopsis yongtaiensis (426,928 bp; Zhang et al., 2024b), but significantly larger than those of Calophyllum soulattri (378,262 bp; Cadorna et al., 2024) and G. mangostana (371,235 bp).
Although both Hypericum mitogenomes were assembled as circular molecules, previously deposited Hypericum assemblies from the DToL project remain as fragmented scaffolds (1–4 contigs; Supplementary Table S5), suggesting that multichromosomal structures may also occur within the genus (Wu et al., 2022). GC contents were highly conserved between the two species (43.9–44.2%) and closely matched other Malpighiales, such as Hevea Aubl. (44.13–44.24%) and C. soulattri (43.97%), highlighting stability in nucleotide composition despite considerable structural dynamics (Gualberto and Newton, 2017).
The ~74 kb size difference between H. ascyron and H. perforatum did not correspond to differences in unique gene content, supporting the widely accepted hypothesis that mitogenome expansion in angiosperms is primarily driven by increases in non-coding sequences, including repeats and foreign sequences acquired through intracellular gene transfer (MTPTs/NUMTs) or horizontal gene transfer (Chen et al., 2017; Wu et al., 2022). For example, size variation in Cucurbitaceae mitogenomes (390 kb to 2.9 Mb) is largely attributable to the accumulation of short dispersed repeats and plastid sequences (Alverson et al., 2010), whereas the extraordinary expansion in Amborella trichopoda Baill. mitogenome is primarily driven by extensive horizontal gene transfer (Rice et al., 2013). In Hypericum, the observed size divergence is most likely due to differential accumulation of intergenic spacer sequences, a process that enables rapid structural plasticity without impairing essential mitochondrial respiratory functions (Gualberto and Newton, 2017).
4.2 Contribution of repetitive sequences and MTPTs to mitogenome expansion
Repetitive sequences in plant mitogenomes are key drives of homologous recombination, contributing to genome evolution and extensive structural rearrangements (Maréchal and Brisson, 2010; Gualberto and Newton, 2017). Repeat-mediated recombination can generate highly dynamic mitochondrial conformations (Cole et al., 2018; Zhong et al., 2022), including circular forms in Melia azedarach L. (Hao et al., 2024), linear molecules in Haematoxylum campechianum L. (Shen et al., 2025), or multichromosomal structures in Lilium tsingtauense Gilg (Qu et al., 2024). In the H. ascyron mitogenome, we identified a putative recombinationally active repeat that likely mediates genomic reconfiguration, a phenomenon also reported in Morus notabilis C.K.Schneid (Liu et al., 2024a).
Both H. ascyron and H. perforatum contained three categories of repetitive sequences, SSRs, tandem repeats, and dispersed repeats, widely distributed across their mitogenomes. Among the SSRs, six motif types were detected, with monomeric repeats being the most abundant. This pattern mirrors observation in Terniopsis yongtaiensis (Zhang et al., 2024b) and Hevea species (Niu et al., 2024). Dispersed repeats were dominated by forward and palindromic types, consistent with those in closely related Malpighiales such as T. yongtaiensis, Calophyllum soulattri (Cadorna et al., 2024), and Viola diffusa (Zhang et al., 2024a), suggesting that repeat proliferation in this order may be shaped by conserved evolutionary constraints.
A total of 22 and 25 MTPTs were identified in H. ascyron and H. perforatum, respectively, accounting for 24,248 bp (5.9%) and 22,228 bp (4.6%) of their mitogenomes. These proportions are comparable to Calophyllum soulattri (4.6%), but lower than in Terniopsis yongtaiensis (14.6%), and higher than in Garcinia mangostana (1.7%). Such variation reflects lineage-specific dynamics of plastid-to-mitochondrial DNA transfer, a widespread hallmark of angiosperm mitogenomes (Chen et al., 2025). Notably, H. ascyron incorporated multiple plastid-derived tRNAs, including trnC-GCA, trnD-GUC, trnI-CAU, trnM-CAU, trnS-GGA, trnV-GAC, and trnW-CCA, as well as a partial rrn18 gene. In contrast, H. perforatum lacked trnC-GCA but uniquely retained trnH-GUG. Most MTPTs originated from the plastome large single-copy (LSC) region, consistent with observations in T. yongtaiensis. Furthermore, two shared “hotspots” (trnD-GUC and psaB-psaA) were detected across H. ascyron, H. perforatum, and T. yongtaiensis, indicating that plastid DNA integration may be influenced by sequence-specific biases (Wang et al., 2017; Nhat Nam et al., 2024). The presence of uncharacterized MTPTs further suggests potential functional innovation (Wang et al., 2012). Collectively, these findings highlight the important contribution of MTPTs to mitogenome expansion and structural diversification within Hypericum.
4.3 Distribution patterns and functional significance of mitochondrial RNA editing
RNA editing is an essential post-transcriptional mechanism in plant mitochondria, restoring conserved codons and ensuring proper mitochondrial protein function (Small et al., 2020; Knoop, 2023). In angiosperms, cytosine-to-uracil (C-to-U) conversions predominate, although uracil-to-cytosine (U-to-C) editing is widespread in other plant lineages, including hornworts, lycophytes, and ferns (Kwok van der Giezen et al., 2025). While the editing mechanism is broadly conserved, the number and distribution of RNA editing sites vary markedly among taxa, with most edits occurring at the first and second codon positions (Covello and Gray, 1993).
In Hypericum, 316–324 RNA editing sites were predicted, exhibiting a strong positional bias toward the fist (109–112) and second (187–192) codon positions, with relatively fewer at the third (20–23). This pattern is consistent with other angiosperms and reflects the primary role of RNA editing in correcting non-synonymous mutations (Edera et al., 2018). Among examined PCGs, ccmC, mttB, nad2, and nad4 harbored the highest number of editing sites, similar to observations in Terniopsis yongtaiensis (ccmB, ccmC, ccmFn, nad2, nad4). The nad4 gene contained the largest number of editing sites in Hypericum (28), comparable to related Malpighiales such as T. yongtaiensis (25 sites), Calophyllum soulattri (35 sites), and Garcinia mangostana (35 sites), suggesting evolutionary conservation in editing intensity among key respiratory genes.
Despite these overall similarities, noticeable interspecific differences were observed. For example, the ccmC gene in H. pulchrum contained 29 editing sites, compared with 24–25 in other Hypericum species, a trend also reported in other plant lineages (i.e., Quercus L.; Song et al., 2025). Editing sites in cox3 were predicted exclusively in H. pulchrum, reminiscent of lineage-specific editing loss observed in Amaryllidaceae and Iridaceae, likely attributable to recombination and reverse transcription involving edited transcripts (Lopez et al., 2007).
RNA editing also contributes to the creation of functional start and stop codons (Small et al., 2020; Hao et al., 2021). In Hypericum, this included C-to-U conversions such as the edited start codon of nad1 (ACG→ATG) and the edited stop codon of ccmFC (CGA→TGA). U-to-C editing can perform equivalent functions o in other plant lineages, particularly in early vascular plants (Kwok van der Giezen et al., 2025). Similar regulatory roles have been reported in Azolla Lam. and Salvinia Ség. (Li et al., 2018), underscoring the fundamental importance of RNA editing in mitochondrial gene expression.
4.4 Phylogenetic relationships and inter-organellar discordance
Phylogenetic analysis based on shared mitochondrial PCGs from 30 species across Malpighiales, Celastrales and Sapindales recovered a close relationship between Hypericaceae and Podostemaceae, consistent with established taxonomic frameworks (Wurdack and Davis, 2009; Xi et al., 2012; APG IV, 2016). However, discrepancies emerged between mitochondrial and plastid phylogenies within Hypericum. In the plastid tree, H. hirsutum and H. pulchrum formed a well-supported clade, in agreement with previous plastid and nrITS-based studies (Meseguer et al., 2013; Nürk et al., 2013). In contrast, the mitochondrial phylogeny placed H. pulchrum closer to H. perforatum. Morphological characters support the plastid/nuclear topology: both H. hirsutum and H. pulchrum belong to sect. Taeniocarpium and share traits such as sepals with gland-fringed margins (Robson, 2010), whereas H. perforatum has fewer or no intramarginal black glands (Li and Robson, 2007). These observations indicate that mitochondrial topology alone does not fully reflect the species relationships inferred from morphology and plastid/nuclear markers.
Similar inter-organellar discordance has been reported in other angiosperm lineages, including Brassicaceae (Dominicus et al., 2025), Dalbergia odorifera T.C.Chen (Hong et al., 2021), Gossypium L. (Kong et al., 2025), and Potentilla L. (Xue et al., 2024). Such conflicts are often attributed to hybridization, incomplete lineage sorting, horizontal gene transfer, or differences in substitution rates (Lin et al., 2025). Although plant mitochondrial genomes evolve slowly, with synonymous substitution rates approximately one-third and one-sixteenth those of plastid and nuclear genomes, respectively (Drouin et al., 2008; Smith and Keeling, 2015), their conservative nature makes them valuable for resolving deep evolutionary relationships (Qiu et al., 2010). Moreover, mitogenomic features such as RNA editing profiles, repeat content, and GC composition can provide independent phylogenetic signals (Nie et al., 2025). Integrating mitogenomic, plastid, and nuclear data thus enables a more comprehensive understanding of the complex evolutionary history of Hypericum.
5 Conclusions
This study reports the complete mitochondrial genomes of Hypericum ascyron and H. perforatum and presents the first comparative mitogenomic analysis within the genus. Although both species retain a conserved set of mitochondrial protein-coding genes, they exhibit notable variation in genome size, largely driven by differences in intergenic spacer sequences and plastid-derived DNA insertions. The identified repetitive sequences and MTPTs provide new insights into the structural dynamism of Hypericum mitogenomes. Extensive RNA editing events were also detected, including species-specific patterns that may contribute to post-transcriptional regulation and adaptive evolution. Phylogenetic analyses based on mitochondrial genes corroborated the close relationship between Hypericaceae and Podostemaceae within Malpighiales, while revealing discordance between mitochondrial and plastid phylogenies among Hypericum species. Together, these findings provide valuable genomic resources and establish a foundation for further investigations into mitochondrial genome evolution, structural variation, and functional adaptation in Hypericum and related lineages.
Data availability statement
The raw sequencing data generated in this study have been deposited in the Genome Sequence Archive at the National Genomics Data Center (NGDC), China National Center for Bioinformation (CNCB), Beijing Institute of Genomics, Chinese Academy of Sciences, under accession number CRA035721 (https://ngdc.cncb.ac.cn/gsa/) (CNCB-NGDC Members and Partners, 2025). Additionally, the data reported in this study have also been deposited in the GenBase (Bu et al., 2024) at NGDC/CNCB under accession number C_AA132761.1–C_AA132762.1 and C_AA133240.1–C_AA133242.1 (https://ngdc.cncb.ac.cn/genbase). Annotations of the mitochondrial and plastid genomes for the other four Hypericum species from the Darwin Tree of Life (DToL) project are available in Figshare (https://doi.org/10.6084/m9.figshare.31045846).
Author contributions
C-LX: Conceptualization, Funding acquisition, Resources, Supervision, Writing – original draft, Writing – review & editing. J-XY: Formal Analysis, Methodology, Software, Writing – original draft. R-ZB: Data curation, Formal Analysis, Methodology, Resources, Writing – review & editing. Y-PC: Data curation, Resources, Writing – review & editing. X-LM: Methodology, Writing – review & editing. FC: Writing – review & editing.
Funding
The author(s) declared that financial support was received for this work and/or its publication. This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (grant XDB1230000); the National Natural Science Foundation of China (grant no. 32370221); the Strategic Priority Research Program of Kunming Institute of Botany, Chinese Academy of Sciences (grant NO. KIB202403); Yunnan Revitalization Talent Support Program “Innovation Team” Project (202305AS350014).
Acknowledgments
We are grateful to Dr. Bryan Drew for reading the manuscript, improving the English, and providing constructive comments.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declared that generative AI was not used in the creation of this manuscript.
Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.
Publisher’s note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2026.1763082/full#supplementary-material
References
Almeida, C. and Marques, A. (2025). Giant mitogenomes in Rhynchospora are a result of nuclear gene and retrotransposon insertions in intergenic spaces. Ann. Bot. 136, 397–405. doi: 10.1093/aob/mcaf098
Alverson, A. J., Wei, X. X., Rice, D. W., Stern, D. B., Barry, K., and Palmer, J. D. (2010). Insights into the evolution of mitochondrial genome size from complete sequences of Citrullus lanatus and Cucurbita pepo (Cucurbitaceae). Mol. Biol. Evol. 27, 1436–1448. doi: 10.1093/molbev/msq029
APG IV (2016). An update of the angiosperm phylogeny group classification for the orders and families of flowering plants: APG IV. Bot. J. Linn. Soc 181, 1–20. doi: 10.1111/boj.12385
Bai, R. Z., He, Z. L., Celep, F., Wang, L., and Xiang, C. L. (2025). Hypericum chishuiense (Hypericaceae, sect. Ascyreia), a new St. John’s wort species from Guizhou, China. Turk. J. Bot. 49, 133–142. doi: 10.55730/1300-008X.2848
Bai, R. Z., Zhao, F., Cai, J., Shen, S. K., and Xiang, C. L. (2023). Hypericum podocarpoides N. Robson (Hypericaceae): A newly recorded species from China. Plant Sci. J. 41, 1–6. doi: 10.11913/PSJ.2095-0837.22090
Beier, S., Thiel, T., Münch, T., Scholz, U., and Mascher, M. (2017). MISA-web: a web server for microsatellite prediction. Bioinformatics 33, 2583–2585. doi: 10.1093/bioinformatics/btx198
Benson, G. (1999). Tandem repeats finder: A program to analyze DNA sequences. Nucleic Acids Res. 27, 573–580. doi: 10.1093/nar/27.2.573
Birolo, G. and Telatin, A. (2022). BamToCov: An efficient toolkit for sequence coverage calculations. Bioinformatics 38, 2617–2618. doi: 10.1093/bioinformatics/btac125
Borcherding, N. and Brestoff, J. R. (2023). The power and potential of mitochondria transfer. Nature 623, 283–291. doi: 10.1038/s41586-023-06537-z
Boussardon, C., Przybyla-Toscano, J., Carrie, C., and Keech, O. (2020). Tissue-specific isolation of Arabidopsis/plant mitochondria – IMTACT (isolation of mitochondria tagged in specific cell types). Plant J. 103, 459–473. doi: 10.1111/tpj.14723
Bu, C. F., Zheng, X. C., Zhao, X. T., Xu, T. Y., Bai, X., Jia, Y. K., et al. (2024). GenBase: A nucleotide sequence database. Genom. Proteomics Bioinf. 22, qzae047. doi: 10.1093/gpbjnl/qzae047
Cadorna, C. A. E., Pahayo, D. G., and Rey, J. D. (2024). The first mitochondrial genome of Calophyllum soulattri burm.f. Sci. Rep. 14, 5112–5123. doi: 10.1038/s41598-024-55016-6
Camacho, C., Coulouris, G., Avagyan, V., Ma, N., Papadopoulos, J., Bealer, K., et al. (2009). BLAST+: architecture and applications. BMC Bioinf. 10, 421–430. doi: 10.1186/1471-2105-10-421
Capella-Gutiérrez, S., Silla-Martínez, J. M., and Gabaldón, T. (2009). trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25, 1972–1973. doi: 10.1093/bioinformatics/btp348
Chavarro-Mesa, E., Almeida, J. V. D. A., Silva, S. R., Lopes, S. S., Barbosa, J. B. F., Oliveira, D., et al. (2024). The mitogenomic landscape of Banisteriopsis caapi (Malpighiaceae), the sacred liana used for ayahuasca preparation. Genet. Mol. Biol. 47, e20230301. doi: 10.1590/1678-4685-GMB-2023-0301
Chen, L., Yan, R. R., Yang, C. Y., Ling, L. Z., Bai, X. X., Ren, Q. F., et al. (2025). Mitochondrial genome analysis of the endangered Oreocharis esquirolii: Insights into evolutionary adaptation and conservation. BMC Plant Biol. 25, 827–840. doi: 10.1186/s12870-025-06838-7
Chen, Z. W., Zhao, N., Li, S. S., Grover, C. E., Nie, H. S., Wendel, J. F., et al. (2017). Plant mitochondrial genome evolution and cytoplasmic male sterility. Crit. Rev. Plant Sci. 36, 55–69. doi: 10.1080/07352689.2017.1327762
CNCB-NGDC Members and Partners (2025). Database resources of the national genomics data center, China national center for bioinformation in 2025. Nucleic Acids Res. 53, D30–D44. doi: 10.1093/nar/gkae978
Cole, L. W., Guo, W., Mower, J. P., and Palmer, J. D. (2018). High and variable rates of repeat-mediated mitochondrial genome rearrangement in a genus of plants. Mol. Biol. Evol. 35, 2773–2785. doi: 10.1093/molbev/msy176
Covello, P. and Gray, M. W. (1993). On the evolution of RNA editing. Trends Genet. 9, 265–268. doi: 10.1016/0168-9525(93)90011-6
Cox, C. J. (2018). Land plant molecular phylogenetics: A review with comments on evaluating incongruence among phylogenies. Crit. Rev. Plant Sci. 37, 113–127. doi: 10.1080/07352689.2018.1482443
Dominicus, L. J. M. A., Al-Shehbaz, I. A., German, D. A., Mummenhoff, K., Hay, N. M., Lysák, M. A., et al. (2025). Mitoplastomic discordance in Brassicaceae phylogenomics confirms the complex evolutionary history of the family. Ann. Bot., mcaf084. doi: 10.1093/aob/mcaf084
Doyle, J. J. and Doyle, J. L. (1987). A rapid DNA isolation procedure for small amounts of fresh leaf tissue. Phytochem. Bull. 19, 11–15.
Drouin, G., Daoud, H., and Xia, J. N. (2008). Relative rates of synonymous substitutions in the mitochondrial, chloroplast and nuclear genomes of seed plants. Mol. Phylogenet. Evol. 49, 827–831. doi: 10.1016/j.ympev.2008.09.009
Edera, A. A., Gandini, C. L., and Sanchez-Puerta, M. V. (2018). Towards a comprehensive picture of C-to-U RNA editing sites in angiosperm mitochondria. Plant Mol. Biol. 97, 215–231. doi: 10.1007/s11103-018-0734-9
Edera, A. A., Small, I., Milone, D. H., and Sanchez-Puerta, M. V. (2021). Deepred-Mt: Deep representation learning for predicting C-to-U RNA editing in plant mitochondria. Comput. Biol. Med. 136, 104682. doi: 10.1016/j.compbiomed.2021.104682
Greiner, S., Lehwark, P., and Bock, R. (2019). OrganellarGenomeDRAW (OGDRAW) version 1.3.1: expanded toolkit for the graphical visualization of organellar genomes. Nucleic Acids Res. 47, W59–W64. doi: 10.1093/nar/gkz238
Gu, Z. G., Gu, L., Eils, R., Schlesner, M., and Brors, B. (2014). circlize implements and enhances circular visualization in R. Bioinformatics 30, 2811–2812. doi: 10.1093/bioinformatics/btu393
Gualberto, J. M. and Newton, K. J. (2017). Plant mitochondrial genomes: dynamics and mechanisms of mutation. Annu. Rev. Plant Biol. 68, 225–252. doi: 10.1146/annurev-arplant-043015-112232
Han, F. C., Bi, C. W., Chen, Y. C., Dai, X. G., Wang, Z. F., Wu, H. T., et al. (2025). PMAT2: An efficient graphical assembly toolkit for comprehensive organellar genomes. iMeta 4, e70064. doi: 10.1002/imt2.70064
Hao, W., Liu, G. X., Wang, W. P., Shen, W., Zhao, Y. P., Sun, J. L., et al. (2021). RNA editing and its roles in plant organelles. Front. Genet. 12, 757109. doi: 10.3389/fgene.2021.757109
Hao, Z. G., Zhang, Z. P., Jiang, J., Pan, L., Zhang, J. N., Cui, X. F., et al. (2024). Complete mitochondrial genome of Melia azedarach L., reveals two conformations generated by the repeat sequence mediated recombination. BMC Plant Biol. 24, 645–656. doi: 10.1186/s12870-024-05319-7
He, W. M., Yang, J., Jing, Y., Xu, L., Yu, K., and Fang, X. D. (2023). NGenomeSyn: an easy-to-use and flexible tool for publication-ready visualization of syntenic relationships across multiple genomes. Bioinformatics 39, btad121. doi: 10.1093/bioinformatics/btad121
Hong, Z., Liao, X. Z., Ye, Y. J., Zhang, N. N., Yang, Z. J., Zhu, W. D., et al. (2021). A complete mitochondrial genome for fragrant Chinese rosewood (Dalbergia odorifera, Fabaceae) with comparative analyses of genome structure and intergenomic sequence transfers. BMC Genomics 22, 672–685. doi: 10.1186/s12864-021-07967-7
Hu, S. Y., Shi, G., Yang, C. A., Van De Peer, Y., Li, Z., and Xue, J. Y. (2025). Comprehensive sampling of mitochondrial genomes substantiates the Neoproterozoic origin of land plants. Plant Commun. 6, 101497. doi: 10.1016/j.xplc.2025.101497
Huang, K. R., Xu, W. B., Hu, H. L., Jiang, X. L., Sun, L., Zhao, W. Y., et al. (2025). Super-large record-breaking mitochondrial genome of Cathaya argyrophylla in Pinaceae. Front. Plant Sci. 16, 1556332. doi: 10.3389/fpls.2025.1556332
Jakubczyk, A., Kiersnowska, K., Ömeroğlu, B., Gawlik-Dziki, U., Tutaj, K., Rybczyńska-Tkaczyk, K., et al. (2021). The influence of Hypericum perforatum L. Addition to wheat cookies on their antioxidant, anti-metabolic syndrome, and antimicrobial properties. Foods 10, 1379–1394. doi: 10.3390/foods10061379
Jarzębski, M., Smułek, W., Baranowska, H. M., Masewicz, Ł., Kobus-Cisowska, J., Ligaj, M., et al. (2020). Characterization of St. John’s wort (Hypericum perforatum L.) and the impact of filtration process on bioactive extracts incorporated into carbohydrate-based hydrogels. Food Hydrocolloids 104, 105748. doi: 10.1016/j.foodhyd.2020.105748
Kalyaanamoorthy, S., Minh, B. Q., Wong, T. K. F., von Haeseler, A., and Jermiin, L. S. (2017). ModelFinder: Fast model selection for accurate phylogenetic estimates. Nat. Methods 14, 587–589. doi: 10.1038/nmeth.4285
Kapoor, S., Chandel, R., Kaur, R., Kumar, S., Kumar, R., Janghu, S., et al. (2023). The flower of Hypericum perforatum L.: A traditional source of bioactives for new food and pharmaceutical applications. Biochem. Syst. Ecol. 110, 104702. doi: 10.1016/j.bse.2023.104702
Katoh, K. and Standley, D. M. (2013). MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780. doi: 10.1093/molbev/mst010
Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., et al. (2012). Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28, 1647–1649. doi: 10.1093/bioinformatics/bts199
Knoop, V. (2023). C-to-U and U-to-C: RNA editing in plant organelles and beyond. J. Exp. Bot. 74, 2273–2294. doi: 10.1093/jxb/erac488
Kong, J. L., Wang, J., Nie, L. Y., Tembrock, L. R., Zou, C. S., Kan, S. L., et al. (2025). Evolutionary dynamics of mitochondrial genomes and intracellular transfers among diploid and allopolyploid cotton species. BMC Biol. 23, 9–30. doi: 10.1186/s12915-025-02115-z
Kozik, A., Rowan, B. A., Lavelle, D., Berke, L., Schranz, M. E., Michelmore, R. W., et al. (2019). The alternative reality of plant mitochondrial DNA: One ring does not rule them all. PloS Genet. 15, e1008373. doi: 10.1371/journal.pgen.1008373
Kozlov, A. M., Darriba, D., Flouri, T., Morel, B., and Stamatakis, A. (2019). RAxML-NG: a fast, scalable and user-friendly tool for maximum likelihood phylogenetic inference. Bioinformatics 35, 4453–4455. doi: 10.1093/bioinformatics/btz305
Kurtz, S. (2001). REPuter: the manifold applications of repeat analysis on a genomic scale. Nucleic Acids Res. 29, 4633–4642. doi: 10.1093/nar/29.22.4633
Kwok van der Giezen, F. M., McDowell, R., Duncan, O. W., Zumkeller, S., Colas des Francs-Small, C., and Small, I. (2025). High conservation of translation-enabling RNA editing sites in hyper-editing ferns implies they are not selectively neutral. Mol. Biol. Evol. 42, msaf241. doi: 10.1093/molbev/msaf241
Li, H. (2021). New strategies to improve minimap2 alignment accuracy. Bioinformatics 37, 4572–4574. doi: 10.1093/bioinformatics/btab705
Li, F.-W., Brouwer, P., Carretero-Paulet, L., Cheng, S. F., De Vries, J., Delaux, P.-M., et al. (2018). Fern genomes elucidate land plant evolution and cyanobacterial symbioses. Nat. Plants 4, 460–472. doi: 10.1038/s41477-018-0188-8
Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., et al. (2009). The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079. doi: 10.1093/bioinformatics/btp352
Li, J. L., Ni, Y., Lu, Q. Q., Chen, H. M., and Liu, C. (2025b). PMGA: A plant mitochondrial genome annotator. Plant Commun. 6, 101191. doi: 10.1016/j.xplc.2024.101191
Li, X. W. and Robson, N. K. B. (2007). “Hypericum,” in Flora of China. Eds. Wu, Z. Y. and Raven, P. H. (Beijing: Science Press & Missouri Botanical Garden Press), 2–35.
Li, D. L., Zhou, Y. Y., Fu, C. L., Pan, X. Z., Liang, Y. P., Fang, Z. Z., et al. (2025a). Assembly and comparative analysis of the first complete mitochondrial genome of Camellia sinensis var. assamica ‘Hainan Dayezhong’, endemic to hainan province, China. BMC Plant Biol. 25, 1304–1329. doi: 10.1186/s12870-025-07349-1
Lian, Q., Zhang, S., Wu, Z. Q., Zhang, C. Z., and Negrão, S. (2024). Assembly and comparative analysis of the mitochondrial genome in diploid potatoes. Plant Cell Rep. 43, 249–261. doi: 10.1007/s00299-024-03326-4
Lin, D. L., Shao, B. Y., Gao, Z. Y., Li, J. W., Li, Z. H., Li, T. Y., et al. (2025). Phylogenomics of angiosperms based on mitochondrial genes: insights into deep node relationships. BMC Biol. 23, 45–61. doi: 10.1186/s12915-025-02135-9
Liu, L. X., Long, Q., Lv, W., Qian, J. Y., Egan, A. N., Shi, Y., et al. (2024a). Long repeat sequences mediated multiple mitogenome conformations of mulberries (Morus spp.), an important economic plant in China. Genom. Commun. 1, e005. doi: 10.48130/gcomm-0024-0005
Liu, M. H., Zhou, Y. M., Rui, X. X., Ye, Z., Zheng, L. Y., Zang, H., et al. (2024b). A review of traditional applications, geographic distribution, botanical characterization, phytochemistry, and pharmacology of Hypericum ascyron L. Horticulturae 10, 555–596. doi: 10.3390/horticulturae10060555
Lopez, L., Picardi, E., and Quagliariello, C. (2007). RNA editing has been lost in the mitochondrial cox3 and rps13 mRNAs in Asparagales. Biochimie 89, 159–167. doi: 10.1016/j.biochi.2006.09.011
Luo, L., He, Y., Xu, Q., Lyu, W., Yan, J., Xin, P., et al. (2020). Rapid and specific isolation of intact mitochondria from Arabidopsis leaves. J. Genet. Genomics 47, 65–68. doi: 10.1016/j.jgg.2020.01.001
Maréchal, A. and Brisson, N. (2010). Recombination and the maintenance of plant organelle genome stability. New Phytol. 186, 299–317. doi: 10.1111/j.1469-8137.2010.03195.x
Meseguer, A. S., Aldasoro, J. J., and Sanmartín, I. (2013). Bayesian inference of phylogeny, morphology and range evolution reveals a complex evolutionary history in St. John’s wort (Hypericum). Mol. Phylogenet. Evol. 67, 379–403. doi: 10.1016/j.ympev.2013.02.007
Møller, I. M., Rasmusson, A. G., and Van Aken, O. (2021). Plant mitochondria – past, present and future. Plant J. 108, 912–959. doi: 10.1111/tpj.15495
Monzel, A. S., Enríquez, J. A., and Picard, M. (2023). Multifaceted mitochondria: Moving mitochondrial science beyond function and dysfunction. Nat. Metab. 5, 546–562. doi: 10.1038/s42255-023-00783-1
Mower, J. P., Sloan, D. B., and Alverson, A. J. (2012). “Plant mitochondrial genome diversity: The genomics revolution,” in Plant genome diversity volume 1: plant genomes, their residents, and their evolutionary dynamics. Eds. Wendel, J. F., Greilhuber, J., Dolezel, J., and Leitch, I. J. (Springer Vienna, Vienna), 123–144. doi: 10.1007/978-3-7091-1130-7_9
Nhat Nam, N., Pham Anh Thi, N., and Do, H. D. K. (2024). New insights into the diversity of mitochondrial plastid DNA. Genome Biol. Evol. 16, evae184. doi: 10.1093/gbe/evae184
Ni, Y., Li, J. L., Tan, Y. H., Shen, G. A., and Liu, C. (2025). Advance in the assembly of the plant mitochondrial genomes using high-throughput DNA sequencing data of total cellular DNAs. Plant Biotechnol. J. 23, 4944–4965. doi: 10.1111/pbi.70249
Nie, L., Wang, J., Huang, L., Kong, J., Nie, B., Tembrock, L. R., et al. (2025). Dissecting mitogenomic conflict to illuminate angiosperm deep phylogeny: Sequence and architectural evidence. Plant Divers., S2468265925001738. doi: 10.1016/j.pld.2025.10.003
Niu, Y. F., Gao, C. W., and Liu, J. (2024). Mitochondrial genome variation and intergenomic sequence transfers in Hevea species. Front. Plant Sci. 15, 1234643. doi: 10.3389/fpls.2024.1234643
Nürk, N. and Blattner, F. (2010). Cladistic analysis of morphological characters in Hypericum (Hypericaceae). Taxon 59, 1495–1507. doi: 10.2307/20774044
Nürk, N. M., Madriñán, S., Carine, M. A., Chase, M. W., and Blattner, F. R. (2013). Molecular phylogenetics and morphological evolution of St. John’s wort (Hypericum; Hypericaceae). Mol. Phylogenet. Evol. 66, 1–16. doi: 10.1016/j.ympev.2012.08.022
Nürk, N. M., Uribe-Convers, S., Gehrke, B., Tank, D. C., and Blattner, F. R. (2015). Oligocene niche shift, miocene diversification – cold tolerance and accelerated speciation rates in the St. John’s worts (Hypericum, Hypericaceae). BMC Evol. Biol. 15, 80–93. doi: 10.1186/s12862-015-0359-4
Petersen, G., Cuenca, A., Møller, I. M., and Seberg, O. (2015). Massive gene loss in mistletoe (Viscum, Viscaceae) mitochondria. Sci. Rep. 5, 17588–17594. doi: 10.1038/srep17588
Qiu, Y. L., Li, L. B., Wang, B., Xue, J. Y., Hendry, T. A., Li, R. Q., et al. (2010). Angiosperm phylogeny inferred from sequences of four mitochondrial genes. J. Syst. Evol. 48, 391–425. doi: 10.1111/j.1759-6831.2010.00097.x
Qu, K., Chen, Y., Liu, D., Guo, H. L., Xu, T., Jing, Q., et al. (2024). Comprehensive analysis of the complete mitochondrial genome of Lilium tsingtauense reveals a novel multichromosome structure. Plant Cell Rep. 43, 150–165. doi: 10.1007/s00299-024-03232-9
Rice, D. W., Alverson, A. J., Richardson, A. O., Young, G. J., Sanchez-Puerta, M. V., Munzinger, J., et al. (2013). Horizontal transfer of entire genomes via mitochondrial fusion in the angiosperm Amborella. Science 342, 1468–1473. doi: 10.1126/science.1246275
Robson, N. K. B. (2010). Studies in the genus hypericum L (Hypericaceae) 5(2). Sections 17. Hirtella to 19. Coridium. Phytotaxa 4, 127–258. doi: 10.11646/phytotaxa.4.1.3
Robson, N. K. B. (2012). Studies in the genus Hypericum L. (Hypericaceae) 9. Addenda, corrigenda, keys, lists and general discussion. Phytotaxa 72, 1–111. doi: 10.11646/phytotaxa.72.1.1
Robson, N. K. B. (2016). And then came molecular phylogenetics—Reactions to a monographic study of Hypericum (Hypericaceae). Phytotaxa 255, 181–198. doi: 10.11646/phytotaxa.255.3.1
Roger, A. J., Muñoz-Gómez, S. A., and Kamikawa, R. (2017). The origin and diversification of mitochondria. Curr. Biol. 27, R1177–R1192. doi: 10.1016/j.cub.2017.09.015
Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D. L., Darling, A., Höhna, S., et al. (2012). MrBayes 3.2: efficient bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 61, 539–542. doi: 10.1093/sysbio/sys029
Ruhfel, B. R., Bittrich, V., Bove, C. P., Gustafsson, M. H. G., Philbrick, C. T., Rutishauser, R., et al. (2011). Phylogeny of the clusioid clade (Malpighiales): evidence from the plastid and mitochondrial genomes. Am. J. Bot. 98, 306–325. doi: 10.3732/ajb.1000354
Shen, S. Y., Xie, Y. N., Hu, Z. J., Wu, W. Q., Li, Y., Shen, W. W., et al. (2025). Complete mitochondrial and chloroplast genomes of Haematoxylum campechianum L. reveal insights into gene transfer and RNA editing events. Ind. Crops Prod. 236, 122050. doi: 10.1016/j.indcrop.2025.122050
Silva, A. R., Taofiq, O., Ferreira, I. C. F. R., and Barros, L. (2021). Hypericum genus cosmeceutical application – A decade comprehensive review on its multifunctional biological properties. Ind. Crops Prod. 159, 113053. doi: 10.1016/j.indcrop.2020.113053
Skippington, E., Barkman, T. J., Rice, D. W., and Palmer, J. D. (2015). Miniaturized mitogenome of the parasitic plant Viscum scurruloideum is extremely divergent and dynamic and has lost all nad genes. Proc. Natl. Acad. Sci. 112, E3515–E3524. doi: 10.1073/pnas.1504491112
Sloan, D. B., Alverson, A. J., Chuckalovcak, J. P., Wu, M., McCauley, D. E., Palmer, J. D., et al. (2012). Rapid evolution of enormous, multichromosomal genomes in flowering plant mitochondria with exceptionally high mutation rates. PloS Biol. 10, e1001241. doi: 10.1371/journal.pbio.1001241
Small, I. D., Schallenberg-Rüdinger, M., Takenaka, M., Mireau, H., and Ostersetzer-Biran, O. (2020). Plant organellar RNA editing: What 30 years of research has revealed. Plant J. 101, 1040–1056. doi: 10.1111/tpj.14578
Smith, D. R. and Keeling, P. J. (2015). Mitochondrial and plastid genome architecture: Reoccurring themes, but significant differences at the extremes. Proc. Natl. Acad. Sci. 112, 10177–10184. doi: 10.1073/pnas.1422049112
Soe, T., Kong, J. L., Xu, B., Kan, J. H., Tembrock, L. R., Nie, L. Y., et al. (2025). Mitochondrial genomes of four Dendrobium species (Orchidaceae): a comprehensive analysis of structural diversity, synteny, and plastid-derived sequences. Genom. Commun. 2, e018. doi: 10.48130/gcomm-0025-0018
Song, Y., Pan, S. J., Chen, B., Xiao, Z. T., Huang, K. R., Li, H., et al. (2025). Structural variations and phylogenetic implications of mitochondrial genomes in oaks. Ind. Crops Prod. 235, 121817. doi: 10.1016/j.indcrop.2025.121817
Sousa, F., Civáň, P., Brazão, J., Foster, P. G., and Cox, C. J. (2020). The mitochondrial phylogeny of land plants shows support for Setaphyta under composition-heterogeneous substitution models. PeerJ 8, e8995. doi: 10.7717/peerj.8995
Tang, S. Y., Liang, Y. Y., Wu, F. Q., Wu, Y., Li, J. W., Lin, L., et al. (2025). HiMT: An integrative toolkit for assembling organelle genomes using HiFi reads. Plant Commun. 6, 101467. doi: 10.1016/j.xplc.2025.101467
The Darwin Tree of Life Project Consortium, Blaxter, M., Mieszkowska, N., Di Palma, F., Holland, P., Durbin, R., et al. (2022). Sequence locally, think globally: The Darwin Tree of Life Project. Proc. Natl. Acad. Sci. 119, e2115642118. doi: 10.1073/pnas.2115642118
Vincent, O. M., Nguta, J. M., Mitema, E. S., Musila, F. M., Nyak, D. M., Mohammed, A. H., et al. (2021). Ethnopharmacology, pharmacological activities, and chemistry of the Hypericum genus. J. Phytopharm. 10, 105–113. doi: 10.31254/phyto.2021.10206
Wang, X. C., Chen, H. M., Yang, D., and Liu, C. (2017). Diversity of mitochondrial plastid DNAs (MTPTs) in seed plants. Mitochondrial DNA A 29, 635–642. doi: 10.1080/24701394.2017.1334772
Wang, M. T., Hou, Z. Y., Li, C., Yang, J. P., Niu, Z. T., Xue, Q. Y., et al. (2023). Rapid structural evolution of Dendrobium mitogenomes and mito-nuclear phylogeny discordances in Dendrobium (Orchidaceae). J. Syst. Evol. 61, 790–805. doi: 10.1111/jse.12912
Wang, J., Kan, S. L., Liao, X. Z., Zhou, J. W., Tembrock, L. R., Daniell, H., et al. (2024). Plant organellar genomes: much done, much more to do. Trends Plant Sci. 29, 754–769. doi: 10.1016/j.tplants.2023.12.014
Wang, D., Rousseau-Gueutin, M., and Timmis, J. N. (2012). Plastid sequences contribute to some plant mitochondrial genes. Mol. Biol. Evol. 29, 1707–1711. doi: 10.1093/molbev/mss016
Wee, C. C., Nor Muhammad, N. A., Subbiah, V. K., Arita, M. S. O. R., Nakamura, Y., and Goh, H. H. (2022). Mitochondrial genome of Garcinia mangostana L. variety Mesta. Sci. Rep. 12, 9480–9488. doi: 10.1038/s41598-022-13706-z
Wick, R. R., Schultz, M. B., Zobel, J., and Holt, K. E. (2015). Bandage: interactive visualization of de novo genome assemblies. Bioinformatics 31, 3350–3352. doi: 10.1093/bioinformatics/btv383
Wickham, H. (2016). ggplot2: elegant graphics for data analysis (New York: Springer-Verlag). Available online at: https://ggplot2.tidyverse.org (Accessed September 18, 2025).
Wu, Z. Q., Liao, X. Z., Zhang, X. N., Tembrock, L. R., and Broz, A. (2022). Genomic architectural variation of plant mitochondria—a review of multichromosomal structuring. J. Syst. Evol. 60, 160–168. doi: 10.1111/jse.12655
Wurdack, K. J. and Davis, C. C. (2009). Malpighiales phylogenetics: Gaining ground on one of the most recalcitrant clades in the angiosperm tree of life. Am. J. Bot. 96, 1551–1570. doi: 10.3732/ajb.0800207
Xi, Z. X., Ruhfel, B. R., Schaefer, H., Amorim, A. M., Sugumaran, M., Wurdack, K. J., et al. (2012). Phylogenomics and a posteriori data partitioning resolve the cretaceous angiosperm radiation Malpighiales. Proc. Natl. Acad. Sci. 109, 17519–17524. doi: 10.1073/pnas.1205818109
Xian, W. F., Bezrukov, I., Bao, Z. G., Vorbrugg, S., Gautam, A., and Weigel, D. (2025). TIPPo: A user-friendly tool for de novo assembly of organellar genomes with high-fidelity data. Mol. Biol. Evol. 42, msae247. doi: 10.1093/molbev/msae247
Xiang, C. Y., Gao, F. L., Jakovlić, I., Lei, H. P., Hu, Y., Zhang, H., et al. (2023). Using PhyloSuite for molecular phylogeny and tree-based analyses. iMeta 2, e87. doi: 10.1002/imt2.87
Xue, J. Y., Dong, S. S., Wang, M. Q., Song, T. Q., Zhou, G. C., Li, Z., et al. (2022). Mitochondrial genes from 18 angiosperms fill sampling gaps for phylogenomic inferences of the early diversification of flowering plants. J. Syst. Evol. 60, 773–788. doi: 10.1111/jse.12708
Xue, T. T., Janssens, S. B., Liu, B. B., and Yu, S. X. (2024). Phylogenomic conflict analyses of the plastid and mitochondrial genomes via deep genome skimming highlight their independent evolutionary histories: A case study in the cinquefoil genus Potentilla sensu lato (Potentilleae, Rosaceae). Mol. Phylogenet. Evol. 190, 107956. doi: 10.1016/j.ympev.2023.107956
Yurina, N. P. and Odintsova, M. S. (2016). Mitochondrial genome structure of photosynthetic eukaryotes. Biochem. (Moscow) 81, 101–113. doi: 10.1134/S0006297916020048
Zhang, X. Y., Chen, H. M., Ni, Y., Wu, B., Li, J. L., Burzyński, A., et al. (2024c). Plant mitochondrial genome map (PMGmap): a software tool for the comprehensive visualization of coding, noncoding and genome features of plant mitochondrial genomes. Mol. Ecol. Resour. 24, e13952. doi: 10.1111/1755-0998.13952
Zhang, R. F., Ji, Y. Y., Zhang, X. B., Kennelly, E. J., and Long, C. L. (2020). Ethnopharmacology of Hypericum species in China: A comprehensive review on ethnobotany, phytochemistry and pharmacology. J. Ethnopharmacol. 254, 112686. doi: 10.1016/j.jep.2020.112686
Zhang, C. S., Rasool, A., Qi, H. L., Zou, X., Wang, Y. M., Wang, Y. H., et al. (2024a). Comprehensive analysis of the first complete mitogenome and plastome of a traditional Chinese medicine Viola diffusa. BMC Genomics 25, 1162–1176. doi: 10.1186/s12864-024-11086-4
Zhang, N. N., Stull, G. W., Zhang, X. J., Fan, S. J., Yi, T. S., and Qu, X. J. (2025). PlastidHub: An integrated analysis platform for plastid phylogenomics and comparative genomics. Plant Divers. 47, 544–560. doi: 10.1016/j.pld.2025.05.005
Zhang, S., Wang, J., He, W. C., Kan, S. L., Liao, X. Z., Jordan, D. R., et al. (2023). Variation in mitogenome structural conformation in wild and cultivated lineages of sorghum corresponds with domestication history and plastome evolution. BMC Plant Biol. 23, 91–108. doi: 10.1186/s12870-023-04104-2
Zhang, M., Zhang, X. H., Huang, Y. L., Chen, Z. X., and Chen, B. H. (2024b). Comparative mitochondrial genomics of Terniopsis yongtaiensis in Malpighiales: Structural, sequential, and phylogenetic perspectives. BMC Genomics 25, 853–871. doi: 10.1186/s12864-024-10765-6
Zhong, Y., Yu, R. X., Chen, J. F., Liu, Y., and Zhou, R. C. (2022). Highly active repeat-mediated recombination in the mitogenome of the holoparasitic plant Aeginetia indica. Front. Plant Sci. 13, 988368. doi: 10.3389/fpls.2022.988368
Keywords: genomic evolution, Hypericum, inter-organellar conflict, Malpighiales, mitochondrial genome, phylogenomics
Citation: Yu J-X, Bai R-Z, Chen Y-P, Ma X-L, Celep F and Xiang C-L (2026) Newly assembled mitochondrial genomes of Hypericum (Hypericaceae) provide insights into phylogenetic relationships and inter-organellar conflict. Front. Plant Sci. 17:1763082. doi: 10.3389/fpls.2026.1763082
Received: 08 December 2025; Accepted: 15 January 2026; Revised: 15 January 2026;
Published: 04 February 2026.
Edited by:
Zhiqiang Wu, Chinese Academy of Agricultural Sciences, ChinaReviewed by:
Jie Wang, Murdoch University, AustraliaShenglong Kan, Shandong University, Weihai, China
Copyright © 2026 Yu, Bai, Chen, Ma, Celep and Xiang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Chun-Lei Xiang, eGlhbmdjaHVubGVpQG1haWwua2liLmFjLmNu
†These authors have contributed equally to this work