Organelle genome assembly and analysis of Euonymus chloranthoides, insights into an endemic and endangered species

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Euonymus chloranthoides is a species within the family Celastraceae, yet its organellar genomes have remained uncharacterized. In this study, we assembled, annotated, and comprehensively analyzed the organellar genomes of this species. Results The chloroplast genome of E. chloranthoides is 157,967 bp in length, exhibits a typical quadripartite structure, encodes 125 genes, and is characterized by a low abundance and diversity of repeat sequences. In stark contrast, its mitochondrial genome is large and complex, with a size of 968,269 bp, assembled into six contigs forming an intricate structure. Comparative analyses further revealed that the chloroplast genome possesses a stronger and more widespread codon usage bias than the mitochondrial genome. Moreover, we predicted 508 C-to-U RNA editing sites in mitochondrial protein-coding genes, the majority of which result in non-synonymous changes. Eleven short mitochondrial plastid DNAs (MTPTs) were detected between the two organelles, four of which contain intact tRNA genes that were likely transferred from the chloroplast genome. We also identified 18 repeat pairs that may mediate homologous recombination in the mitochondrial genome. Among these, three long repeat pairs showed recombination frequencies close to 50%, while the recombination rates of the remaining short repeat pairs were less than or close to 1%. Conclusions This study presents the first complete characterization of both organellar genomes in E. chloranthoides , revealing their distinctly different evolutionary landscapes. The findings provide a valuable genomic resource and fundamental insights for comparative and evolutionary genomics of organelles within the family Celastraceae. Euonymus chloranthoides Plastid genome Mitochondrial genome RNA editing Phylogenetic analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background The genus Euonymus (Celastraceae) is an important group comprising approximately 200 species of shrubs, woody lianas, and small trees. These plants hold significant ecological, ornamental, and economic value worldwide. Euonymus species are widely distributed across temperate and subtropical regions of the Northern Hemisphere, with a center of diversity in East Asia (particularly China, Japan, and Korea) [ 1 ]. They are also found in Europe, North America, North Africa, and Australia. China is one of the primary distribution areas for this genus, harboring over 90 species. Owing to their diverse morphologies, evergreen or brilliantly colored autumn foliage, and vividly colored fruits, Euonymus plants are extensively used in landscaping, commonly serving as hedges, ground covers, potted ornamental plants [ 2 ], or specimen trees in gardens. Popular cultivated species include E. japonicus , E. fortunei , E. europaeus , and E. alatus . Previous studies have shown that plants of the genus Euonymus , particularly E. alatus (winged euonymus), possess rich phytochemical diversity and notable medicinal potential [ 3 ]. More than 128 compounds have been identified from their tissues, primarily falling into structural classes such as flavonoids, terpenoids, steroids, alkaloids, cardenolides, and lignans [ 4 , 5 ]. Substantial in vitro and in vivo experimental evidence confirms that extracts from these plants and specific pure compounds (e.g., quercetin, kaempferol) exhibit clear anti-diabetic activity [ 6 , 7 ]. Their mechanisms of action involve regulating insulin secretion and sensitivity, acting on the PPARγ pathway [ 8 ], and inhibiting key targets like aldose reductase (AR) and protein tyrosine phosphatase 1B (PTP1B) [ 9 ]. This systematic research on their chemical constituents and pharmacological activities provides a modern scientific basis for the traditional use of these plants in treating diabetes and its complications and establishes an important foundation for their potential as sources of novel drug leads. Plant cells contain two semi-autonomous organellar genomes: the chloroplast genome and the mitochondrial genome. These are genetic systems independent of the nuclear genome, residing in the cytoplasm and governing core life processes such as photosynthesis and respiration, respectively [ 10 ]. The chloroplast genome is typically circular, structurally relatively conserved, and exhibits a characteristic quadripartite structure, containing approximately 110–130 genes. Its small size, high copy number, uniparental inheritance pattern, and relatively slow evolutionary rate make it an ideal tool for studying plant phylogenetics, species identification, and phylogeography [ 11 , 12 ]. In contrast, the plant mitochondrial genome displays remarkable diversity, complexity, and dynamism: its size can range from 66 kb [ 13 ]to 19 Mb [ 14 ] it often contains large amounts of non-coding DNA and repetitive sequences, and it undergoes frequent recombination. This leads to highly variable structures, with possible coexistence of circular, linear, or branched isomers. This structural plasticity is one of the most distinctive features of plant mitochondrial genomes [ 15 ]. Research on organellar genomes holds significant value across multiple dimensions. In the field of evolutionary biology, they serve as crucial evidence for revealing species origins, divergence times, and phylogenetic relationships among taxa [ 16 ]. At the level of comparative and functional genomics, analyzing their gene content, sequence variation, RNA editing, codon usage bias, and horizontal gene transfer enables deep insights into the mechanisms of genomic structure and functional evolution [ 17 ]. In population genetics and conservation biology, organellar genetic markers are effective tools for assessing species genetic diversity, historical population dynamics, gene flow, and identifying evolutionarily significant units [ 18 ]. Furthermore, due to the sequence conservation of the chloroplast genome, it has become the “gold standard” for DNA barcoding and species identification [ 19 ]. Euonymus chloranthoides Y.C. Yang ( https://www.worldfloraonline.org/taxon/wfo-0000681671 ) is an evergreen shrub in the family Celastraceae, reaching about 1 meter in height. It is a species endemic to China, currently known only from Sichuan Province or Chongqing Municipality, where it grows in forests and woodlands at altitudes of 300–400 meters. Since its first description in the 1940s, collection records have been scarce, and it is considered a rare species. Currently, there is a severe lack of research on E. chloranthoides , particularly in genomics. There are no reported genomic resources for this species. In this study, we sampled E. chloranthoides and performed genome sequencing. Based on Oxford Nanopore long-read data, we completed the assembly and annotation of the complete organellar genomes for this species. Our results will generate the first complete organellar genomic resources for E. chloranthoides , laying an indispensable data foundation for subsequent research involving molecular marker development, population genetics, phylogenetics, or functional genomics. Methods Sampling and voucher Deposition A sample (plant part) of E. chloranthoides was collected from Jinyun Mountain, Beibei District, Chongqing, China (coordinates: 108.329181, 30.897192). The specimen was identified by Professor Jie Yu based on descriptions in the Flora of China . A voucher specimen was prepared following standard procedures, assigned the voucher number HWA202501953, and deposited in the Herbarium of the College of Horticulture and Landscape Architecture, Southwest University. The curator responsible for the management of these voucher specimens at the herbarium is Shiyao Liu ( [email protected] ). DNA extraction and sequencing For whole-genome sequencing, total genomic DNA was extracted using the CTAB method [20]. DNA libraries with an insert size of 350 bp were constructed using the NEBNext® Ultra™ DNA Library Prep Kit (Illumina, USA) [21] and sequenced on the Illumina HiSeq X Ten platform to generate PE150 reads at Nanjing Jisi Huiyuan Biotechnology Co., Ltd. (Nanjing, China). Approximately 10.77 Gb of raw data were generated per sample. Raw sequencing reads were processed with Trimmomatic [22] to obtain clean reads by removing low-quality sequences, applying the following filtering criteria: reads with more than 50% of bases having a Phred quality score < 19, or containing over 5% N bases, were discarded. The same DNA sample was also subjected to long-read sequencing. Library preparation for Oxford Nanopore Technology sequencing was performed following the manufacturer's standard protocol, conducted by Nanjing Jisi Huiyuan Biotechnology Co., Ltd. company (Nanjing, China). Sequencing yielded a total of 10.31 Gb of long-read data, comprising 709,002 sequences. The longest read length was 211,993 bp, with an average read length of 14,546.5 bp. Nanjing Jisi Huiyuan Biotechnology Co., Ltd. company (Nanjing, China) The plastome was assembled using the GetOrganelle pipeline (v1.7.3) [23] with default parameters: “-R 15 -k 21,45,65,85,105 -F embplant_pt”. Circular plastome sequences were successfully obtained. Annotation of the chloroplast genome was performed using CPGAVAS2 with a custom reference database, using the chloroplast genome of the congeneric species E. sachalinensis (accession no. OL770079.1) as the reference. The circular genome map was drawn using OGDRAW [24], with font sizes adjusted during image editing. Visualization of the chloroplast genome annotation was conducted using the CPGView software [25]. Mitochondrial genome assembly was initially performed using the Flye tool for de novo assembly of all long-read data. A BLAST database was constructed from the assembly results using makeblastdb. The mitochondrial genome of a closely related species, E. alatus (MW009108.1), was then used as a query to identify contigs containing mitochondrial sequences. The extracted contigs served as reference sequences for mapping the long reads. Reads with an aligned length exceeding 5000 bp were retained, resulting in 15,345 potential mitochondrial long-read sequences. These sequences were reassembled using Flye, yielding the complete mitochondrial genome sequence. Completeness was assessed based on the presence of all known core mitochondrial genes and the assembly forming a closed, connected structure without dead ends. The assembly was error-corrected using the tools Fmlrc2 (v0.1.7), Ropebwt2 (vr187), and Msbwt (v0.2.9). Annotation of the mitochondrial genome was performed using the PMGA [26] tool ( http://www.1kmpg.cn/pmga/ ) with a reference database of 319 mitochondrial genomes, which is effective for annotating splice sites and trans-splicing genes [27]. Mitochondrial tRNAs were annotated using tRNAscan-SE (v.2.0.11) [28], and rRNAs were annotated using BLASTN (v2.16.0) [29]. The mitochondrial genome map was visualized using OGDRAW [24]. Codon usage analysis Protein-coding sequences (CDS) were extracted from the genome using PhyloSuite (v1.1.16) [30]. Codon usage bias analysis for the mitochondrial protein-coding genes (PCGs) was performed using MEGA (v7.0) [31], and the Relative Synonymous Codon Usage (RSCU) values were calculated. Repeat element analysis Repeat sequences, including microsatellites (SSRs), tandem repeats, and dispersed repeats, were identified using MISA (v2.1) [32], TRF (v4.09) [33], and ROUSFinder [34], respectively. The identified repeat units of SSRs included mono-, di-, tri-, tetra-, penta-, and hexanucleotides with minimum repeat thresholds of 10, 5, 4, 3, 3, and 3, respectively. The results were visualized using Excel (2021) and the Circos package (v0.69.9) [35]. Sequence migration analysis between organelle genomes Homologous sequences between the chloroplast and mitochondrial genomes of E. chloranthoides were identified using BLASTN (v2.16.0) [29]. Specifically, a BLAST database was created for the mitochondrial genome sequence using makeblastdb. The chloroplast genome sequence was then used as the query with the command: blastn -query cpDNA.fasta -db mtDNA.fasta -word_size 7 -evalue 1e-10 -outfmt 6. The results were visualized using the Circos package (v0.69.9) [35]. Identification of homologous recombination in the mitogenome The homologous repetitive sequences in the mitogenome sequences are identified using the ROUSFinder software describe as above. For the forward repetitive sequences and palindromic repetitive sequences identified by ROUSFinder, we extract the sequences of these repetitive sequences and their upstream and downstream regions of 2,000 bp each based on the positions of the two repetitive units of each repetitive sequence. These extracted sequences are used as reference sequences for configuration 1 (p1-1, and p1-2). Furthermore, we simulated homologous recombination by swapping the sequences of the downstream 2,000 bp regions of the two repetitive units, and the obtained sequences are used as reference sequences for configuration 2 (p2-1, and p2-2). The raw sequencing data in fasta format is used to construct the database with the command line: makeblastdb -in raw_data.fasta -out raw_data.fasta -dbtype nucl. Here, raw_data.fasta represents the raw sequencing data. The reference sequences for configuration 1 and configuration 2 generated above are used as query sequences for querying. The command line is: blastn -query reference.fasta -db raw_data.fasta -word_size 7 -evalue 1e-10 -out blast_out.txt -outfmt 6 -num_threads 10 -max_hsps 1 -max_target_seqs 10000000. For each repetitive sequences, only long-reads that completely span the repetitive sequence and its flanking 2,000 bp regions are selected. These long-reads are considered to support the respective configuration. Finally, the number of long-reads supporting different configurations is counted. Phylogenetic Analysis Mitochondrial genome sequences of closely related species were downloaded from GenBank. Tetraena mongolica (NC_086675.1) and Tribulus terrestris (MK431825.1) were selected as outgroups. Using PhyloSuite (v1.1.16) [30], annotation files in GenBank format were processed to extract mitochondrial genes shared among all species. A total of 25 shared mitochondrial genes were obtained: atp1 , atp4 , atp6 , atp8 , atp9 , ccmB , ccmC , ccmFC , ccmFN , cob , cox1 , cox2 , cox3 , matR , mttB , nad1 , nad2 , nad3 , nad4 , nad4L , nad5 , nad6 , nad7 , nad9 , and rps12 . These genes were individually aligned using MAFFT (v7.525) [36], and the aligned sequences were concatenated into a single data matrix for phylogenetic analysis. A maximum likelihood (ML) phylogenetic tree was constructed using IQ-TREE2 (v2.3.6) [37] with the parameters “--alrt 1000 -B 1000”. The GTR+F+I+R2 model was selected as the best-fit substitution model, and bootstrap values were set to 1000. The resulting tree was visualized using the iTOL software (v6) [38]. Prediction of RNA editing sites The sequences of all 36 annotated mitochondrial protein-coding genes (PCGs) from this species were used as input to predict C-to-U RNA editing sites using Deepred-mt [39]. This tool employs a convolutional neural network (CNN) model, offering higher accuracy compared to previous prediction tools. All predictions with a probability score greater than 0.9 were retained. Results Organellar genome structure and content The assembly of the organellar genomes of E. chloranthoides is represented by graphical fragment assembly (Figure 1). The chloroplast genome assembly initially consisted of three contigs: a pair of inverted repeats (IRs) connecting two single-copy regions, which can be further converted into a typical quadripartite structure (Figure 2A), where the two single-copy regions are linked by IRa and IRb, respectively. The mitochondrial genome assembly comprises six contigs, interconnected to form a complex network (Figure 1B). To validate the connections (linkages) between these contigs, long reads were mapped onto the linkage sequences. The results confirmed that all linkages are supported by long reads, verifying the accuracy of the assembly. The number of supporting long reads for each linkage is labeled in Figure 1B, and the original mapping results are presented in Table S1. As shown in Table 1, the complete chloroplast genome is 157,967 bp in length. The large single-copy (LSC) region is 86,430 bp with a GC content of 34.98%, and the small single-copy (SSC) region is 18,677 bp with a GC content of 31.46%. The two repeat units of the inverted repeat (IR) region, IRa and IRb, are each 26,640 bp long with a GC content of 42.63%, which is higher than that of the LSC and SSC regions. The complete mitochondrial genome is 968,269 bp in length, consisting of six contigs designated as contig1 to contig6. Their lengths are 337,294 bp, 195,308 bp, 161,205 bp, 162,092 bp, 97,731 bp, and 14,639 bp, respectively, with GC contents ranging from 44.18% to 45.31%. To assess assembly quality, short reads were mapped to the assembled chloroplast genome. The sequencing depth coverage profile is shown in Figure S1, with an average depth exceeding 2,500×. Correspondingly, long reads were mapped to the six mitochondrial contigs, yielding an average coverage depth of approximately 200× (Figure S2). The results show no regions of abrupt coverage drop, and all sequences are supported by high-depth reads, confirming the reliability of the assemblies. Annotation of the E. chloranthoides chloroplast genome identified 81 unique protein-coding genes (PCGs) (Table 2). Among these, nine genes ( ndhB, rpl2, rpl23, rps12, rps19, rps7, ycf15, ycf2, ycf68 ) are duplicated, resulting in a total of 90 PCGs. Four rRNA genes ( rrn4.5, rrn5, rrn16, rrn23 ) were annotated, all of which are duplicated, totaling eight rRNA genes. Thirty unique tRNA genes were identified, seven of which are duplicated, resulting in 37 tRNA genes. Thus, the chloroplast genome encodes a total of 125 genes. A map of the E. chloranthoides chloroplast genome is presented in Figure 2 A. Annotation of the mitochondrial genome identified 36 unique PCGs (Table 3). Two of these ( atp8 and rps19 ) are duplicated, yielding a total of 38 PCGs. Three rRNA genes ( rrn5, rrn18, rrn26 ) were annotated, all present in single copies. Seventeen unique tRNA genes were identified; five are duplicated ( trnE-UUC, trnF-GAA, trnN-GUU, trnS-GCU, trnS-UGA ) and one ( trnfM-CAU ) is present in three copies, resulting in 24 tRNA genes. Consequently, the mitochondrial genome encodes a total of 65 genes. A map of the E. chloranthoides mitochondrial genome is presented in Figure 2B. Table 1 Organelle genome assembly statistics of E. chloranthoides . Organelle Contigs Length (bp) GC content (%) Chloroplast genome LSC SSC IRa IRb Total 86,430 18,677 26,640 26,640 157,967 34.98 31.46 42.63 42.63 37.16 Mitochondrial genome Contig 1 Contig 2 Contig 3 Contig 4 Contig 5 Contig 6 Total 337,294 195,308 161,205 162,092 97,731 14,639 968,269 44.93 45.31 45.16 44.79 45.03 44.18 45.02 Table 2 Genes encoded by the chloroplast genome of E. chloranthoides . Group of genes Name of genes Subunits of ATP synthase atpA , atpB , atpE , atpF , atpH , atpI Subunits of NADH-dehydrogenase ndhA , ndhB (×2), ndhC , ndhD , ndhE , ndhF , ndhG , ndhH , ndhI , ndhJ , ndhK Subunits of cytochrome b/f complex petA , petB , petD , petG , petL , petN Subunits of photosystem I psaA , psaB , psaC , psaI , psaJ Subunits of photosystem II psbA , psbB , psbC , psbD , psbE , psbF , psbH , psbI , psbJ , psbK , psbL , psbM , psbN , psbT , psbZ , ycf3 Large subunit of ribosome rpl14 , rpl16 , rpl2 (×2), rpl20 , rpl22 , rpl23 (×2), rpl32 , rpl33 , rpl36 Small subunit of ribosome rps11 , rps12 (×2), rps14 , rps15 , rps16 , rps18 , rps19 (×2), rps2 , rps3 , rps4 , rps7 (×2), rps8 DNA dependent RNA polymerase rpoA , rpoB , rpoC1 , rpoC2 Subunit of rubisco rbcL c-type cytochrom synthesis gene ccsA Envelop membrane protein cemA Maturase matK Protease clpP Subunit of Acetyl-CoA-carboxylase accD Translational initiation factor infA Conserved open reading frames ycf1 , ycf15 (×2), ycf2 (×2), ycf4 , ycf68 (×2) Ribosomal RNA rrn4.5 (×2), rrn5 (×2), rrn16 (×2), rrn23 (×2) Continue Table 2 Genes encoded by the chloroplast genome of E. chloranthoides . Group of genes Name of genes Transfer RNA trnA-UGC (×2), trnC-GCA , trnD-GUC , trnE-UUC , trnF-GAA , trnfM-CAU , trnG-GCC , trnG-UCC , trnH-GUG , trnI-CAU (×2), trnI-GAU (×2), trnK-UUU , trnL-CAA (×2), trnL-UAA , trnL-UAG , trnM-CAU , trnN-GUU (×2), trnP-UGG , trnQ-UUG , trnR-ACG (×2), trnR-UCU , trnS-GCU , trnS-GGA , trnS-UGA , trnT-GGU , trnT-UGU , trnV-GAC (×2), trnV-UAC , trnW-CCA , trnY-GUA Note. (×2) means this gene has two copies. Table 3 Genes encoded by the mitochondrial genome of E. chloranthoides . Group of genes Name of genes ATP synthase atp1 , atp4 , atp6 , atp8 (×2), atp9 NADH dehydrogenase nad1 , nad2 , nad3 , nad4 , nad4L , nad5 , nad6 , nad7 , nad9 Cytochrome b cob Cytochrome c biogenesis ccmB , ccmC , ccmFC , ccmFN Cytochrome c oxidase cox1 , cox2 , cox3 Maturases matR Protein transport subunit mttB Ribosomal protein large subunit rpl2 , rpl5 , rpl10 , rpl16 Ribosomal protein small subunit rps1 , rps3 , rps4 , rps10 , rps12 , rps13 , rps19 (×2) Succinate dehydrogenase sdh4 Ribosomal RNA rrn5 , rrn18 , rrn26 Transfer RNA trnC-GCA , trnD-GUC , trnE-UUC (×2), trnF-GAA (×2), trnfM-CAU (×3), trnG-GCC , trnH-GUG , trnI-CAU , trnK-UUU , trnM-CAU , trnN-GUU (×2), trnP-UGG , trnQ-UUG , trnS-GCU (×2), trnS-UGA (×2), trnW-CCA , trnY-GUA Note. (×2) means this gene has two copies, (×3) means this gene has three copies. Analysis of repeat elements in organellar genomes We analyzed three types of repeat elements in both organellar genomes: SSRs, tandem repeats, and dispersed repeats. The results are shown in Figure 3. In the chloroplast genome, only eight dispersed repeats were identified (Table S2). With the exception of those in the IR region, the lengths of other dispersed repeats were all below 100 bp. In contrast, 65 tandem repeats were found (Table S3). Finally, 109 SSRs were identified (Table S4). Figure 3A illustrates the distribution of these repeats across the chloroplast genome. Figure 3C shows the number of different SSR types identified. Monometric SSRs were the most abundant (75, accounting for 68.81% of the total). In the mitochondrial genome, 225 dispersed repeats were identified. The largest dispersed repeat was 10,259 bp in length (R1, Table S2), followed by repeats of 3,500 bp (R2) and 1,562 bp (R3). Seventeen dispersed repeats exceeded 100 bp in length. Forty-six tandem repeats were identified (Table S3), fewer than the 65 found in the chloroplast genome. Finally, 322 SSRs were detected. The counts for mono-, di-, tri-, tetra-, penta-, and hexameric SSRs were 103, 63, 35, 98, 17, and 6, respectively. Although monomeric SSRs repeats remained the most frequent type, they constituted only 31.99% of the total, indicating a higher proportion of multi-nucleotide repeat units compared to the chloroplast genome. Notably, tetrameric SSRs were abundant (98, accounting for 30.43% of total SSRs). Figure 3B illustrates the distribution of repeats across the mitochondrial genome, and Figure 3D shows the counts of different SSR types identified. Codon usage bias Due to the degeneracy of the genetic code, a single amino acid can be encoded by multiple synonymous codons. The Relative Synonymous Codon Usage (RSCU) value for the codons corresponding to each amino acid was calculated to assess potential usage bias. Typically, an RSCU value greater than 1 indicates a preference for that codon. For the protein-coding genes (PCGs) in the chloroplast genome, all amino acids except Met and Trp (which are each encoded by a single codon) exhibited varying degrees of preference for specific codons (Figure 4A). For instance, the RSCU value for the UUA codon of Leu was 1.88, for the GCU codon of Ala was 1.79, and for the AGA codon of Arg was 1.78 (Table S5). A chi-square test further revealed that the codon preferences for 18 amino acids were extremely significant, with only the stop codon preference being statistically significant (Table S6). For the PCGs in the mitochondrial genome, Met and Trp are likewise encoded by single codons. Beyond these, the codon preferences observed for other amino acids were weaker than those in the chloroplast PCGs (Figure 4B). For example, the highest RSCU value was 1.59 for the GCU codon of Ala, which is lower than the corresponding value (1.79) in the chloroplast. This was followed by an RSCU value of 1.52 for the UAU codon of Tyr, and 1.54 for the CAA codon of Gln (Table S5). The chi-square test indicated that codon preferences for only 15 amino acids reached an extremely significant level. The preferences for Cys, Phe, and Val were statistically significant, while the preference for the stop codon was not significant (Table S6). Prediction of RNA editing sites Considering that RNA editing events are rare in chloroplast-encoded PCGs, here we did not analyze the RNA editing sites in plastidial PCGs. We predicted potential C-to-U RNA editing sites for the mitochondrial PCGs. The results indicated that all 36 unique PCGs possess C-to-U RNA editing sites, with a total of 508 sites predicted (Table S7). Among these, only 28 sites were synonymous edits that did not alter the amino acid. A substantial majority of 480 sites (94.49%) resulted in amino acid changes. The most frequent change was from Ser to Leu (114 sites), followed by Pro to Leu (106 sites), and then Ser to Phe (66 sites) (Figure 5A). We also noted two edits that introduced stop codons: Gln-to-End and Arg-to-End. These changes occurred at the termination codon positions of the atp6 (CAA to UAA) and ccmFC (CGA to UGA) genes, respectively, indicating that the stop codons for these transcripts are acquired via post-transcriptional C-to-U modification. Regarding the distribution across individual PCGs, we observed that three genes contained over 30 editing sites: nad4 (39 sites), ccmB (37 sites), and nad7 (34 sites). Additionally, ccmC and mttB each had 30 sites (Figure 5B). In contrast, rpl2 had the fewest, with only one C-to-U RNA editing site. Analysis of sequence transfer between organelles We subsequently investigated sequence transfer between the two organelles of E. chloranthoides . Using BLASTn, we identified only 11 homologous sequences (Mitochondrial Plastid DNA Sequences, MTPTs) shared between the two genomes (Table S8). Most of these MTPTs were short; only MTPT2 exceeded 1 kb in length (1068 bp), while the lengths of the remaining fragments ranged from 38 to 332 bp. Figure 6 illustrates the relative genomic locations of these 11 MTPTs. Their combined length is merely about 2.7 kb, suggesting that large-scale sequence transfer has not occurred frequently between these two organellar genomes. Although the transferred fragments were few and short, we found that some contained intact genes. These were exclusively tRNA genes: trnW-CCA on MTPT1, trnN-GUU on MTPT10, trnD-GUC on MTPT11, and trnI-CAU on MTPT12. These tRNA genes were likely transferred from the plastid genome to the mitochondrial genome. Analysis of mitochondrial genome recombination Given the high sequencing coverage (~200×) of our long-read data, we leveraged the advantage of long-reads to identify potential homologous recombination events. The results are presented in Table 4, where a total of 18 repeat pairs were identified as potentially mediating homologous recombination. The three longest repeats (R1, 10,259 bp; R2, 3,500 bp; R3, 1,562 bp) exhibited active recombination features. Both of their possible linkage conformations received similarly high support from the sequencing data (Conformation 1 accounted for 51.22%, 55.25%, and 45.03%, respectively). This suggests frequent homologous recombination exchange between these long repeats, which may lead to the co-existence of two major isomeric structures in the mitochondrial genome across different cells or individuals at appreciable frequencies. These long repeats represent a primary source of structural dynamics in the mitochondrial genome. The remaining 15 shorter repeats (lengths ranging from 70 bp to 430 bp) displayed a distinctly different pattern. Among these, for the vast majority (e.g., R4, R5, R6, etc.), Conformation 1 was overwhelmingly dominant (support rate >95%, mostly >99%), while Conformation 2 was supported by very few reads (typically only 1-3 reads), accounting for less than 1%. This strongly indicates that homologous recombination events between these short repeats occur at an extremely low frequency, or only in a very small minority of mitochondrial molecules. The structural variations they mediate are likely rare types and do not represent the major forms present in the population. The original mapping results of the long-read data to the sequences of different conformations are shown in Table S9. Table 4 Number of long-reads supporting the different configurations of E. chloranthoides mitogenome. Repeat ID Repeat length (bp) p1-1 p1-2 p2-1 p2-2 Reads support configuration 1 Percentage of Conformation 1 (%) Reads support configuration2 Percentage of Conformation 2 (%) R1 10,259 35 28 28 32 63 51.22 60 48.78 R2 3,500 57 64 50 48 121 55.25 98 44.75 R3 1,562 122 23 116 61 145 45.03 177 54.97 R4 430 175 173 9 7 348 95.60 16 4.40 R5 276 158 199 4 1 357 98.62 5 1.38 R6 235 130 126 0 2 256 99.22 2 0.78 R8 213 184 199 0 2 383 99.48 2 0.52 R12 168 163 164 0 2 327 99.39 2 0.61 R14 117 198 185 1 0 383 99.74 1 0.26 R17 110 187 203 2 1 390 99.24 3 0.76 R18 98 183 219 0 1 402 99.75 1 0.25 R22 93 165 167 0 2 332 99.4 2 0.6 R23 84 241 169 2 1 410 99.27 3 0.73 R33 71 172 168 2 0 340 99.42 2 0.58 R34 70 188 204 0 2 392 99.49 2 0.51 R38 64 184 240 0 1 424 99.76 1 0.24 R53 52 242 170 2 1 412 99.28 3 0.72 R54 52 172 170 2 0 342 99.42 2 0.58 Phylogenetic analysis Considering that phylogenetic analyses based on chloroplast genomes have been extensively studied in numerous previous publications, we performed phylogenetic analysis here based solely on mitochondrial PCGs. Due to the scarcity of usable mitochondrial genome data in public databases, our analysis was conducted at the order level. We downloaded an additional 26 mitochondrial genome datasets from public databases (Table S10) and combined them with our newly sequenced one, resulting in a total of 27 species. We identified 25 common protein-coding genes (see the legend of Figure 7). Our phylogenetic tree (Figure 7 ) shows no conflict with previous studies based on the APG IV system [40]. The two species from the genus E. clustered together as a monophyletic clade with 100% bootstrap support. Discussion The chloroplast genomes of plants in the family Celastraceae have been extensively studied, with over 100 resources currently available [ 41 – 44 ]. In contrast, research on the mitochondrial genomes of this family remains in a very preliminary stage, and data on complete or nearly complete mitochondrial genomes that have been published are extremely limited. To date, only three Celastraceae species with published, complete mitochondrial genome drafts can be retrieved from the GenBank database, all of which are results from recent years (2021–2025). They are: Tripterygium wilfordii (720,306 bp) [ 45 ], E. alatus (1,045,106 bp) [ 46 ], and E. europaeus (approximately 1.47 Mb, belonging to the Darwin Tree of Life Project, https://www.darwintreeoflife.org/ ). These published genomes exhibit substantial size variation, suggesting that the mitochondrial genomes of plants in this family may possess a high degree of dynamism and complexity. Furthermore, mitochondrial sequences of a few other species (such as Parnassia faberi and Parnassia rhombipetala ) have been uploaded to the database but remain unreported. Moreover, their assembly results are highly fragmented (consisting of 7 to 11 independent contigs), which further reflects the challenges in achieving complete assembly of the mitochondrial genomes for this group. In this study, a comprehensive comparative analysis of the organellar genomes of E. chloranthoides was conducted. Marked differences were observed between its chloroplast and mitochondrial genomes in terms of genomic structure, gene content, and repeat composition, reflecting their distinct evolutionary trajectories. The chloroplast genome of E. chloranthoides possesses a typical quadripartite structure, encodes 125 genes, and is dominated by short SSRs and tandem repeats. This aligns with the highly conserved nature of chloroplast genomes in most higher plants [ 47 ]. In contrast, the mitochondrial genome exhibits characteristically large, complex, and dynamic features: it is approximately six times larger than the chloroplast genome, and its assembly into multiple contigs forming a network structure suggests potential active recombination. The genome is interspersed with numerous long fragment repeats and contains a more diverse array of SSR types, particularly tetranucleotide repeats. Recombination analysis of the dispersed repeats in the mitochondria revealed that up to 18 repeat pairs may be involved in mediating homologous recombination. These results indicate that the mitochondrial genome has greater potential for recombination, sequence plasticity, and tolerance for structural variation [ 48 – 50 ]. This fundamental difference likely stems from their distinct mutation rates [ 51 ], DNA repair mechanisms [ 52 ], and functional selective pressures. The chloroplast genome tends towards structural stability, whereas the mitochondrial genome may operate under an evolutionary mode that permits greater sequence expansion and structural remodeling [ 49 ]. We also found differences in codon usage bias between the chloroplast and mitochondrial genomes of E. chloranthoides . Chloroplast-encoded genes exhibited stronger and more widespread codon preference. The degree of preference for several amino acids (e.g., UUA of Leu, RSCU = 1.88) was significantly higher than that in the mitochondria (highest being GCU of Ala, RSCU = 1.59). A chi-square test further supported this trend: codon preferences for 18 amino acids reached an “extremely significant” level in the chloroplast PCGs, compared to only 15 in the mitochondrial PCGs. Moreover, the preference for stop codon usage in the mitochondrial PCGs was not significant. These differences likely reflect the varying selective pressures and functional constraints experienced by the two organelles during evolution. The strong codon bias in the chloroplast genome is generally consistent with the hypothesis of translational efficiency optimization for highly expressed genes [ 53 , 54 ]. During photosynthesis, the chloroplast requires the rapid and copious synthesis of photosynthesis-related proteins. A strong codon preference facilitates the recruitment of more abundant corresponding tRNAs, thereby enhancing translation rates [ 55 ]. In contrast, the weaker codon preference in the mitochondrial PCGs may be related to its higher mutation rate, more relaxed selective pressure, or more complex post-transcriptional regulation, such as post-transcriptional RNA editing [ 56 ]. The non-significant preference for stop codons in the mitochondria might also suggest differences in its translation termination mechanism compared to the chloroplast. We predicted up to 508 C-to-U RNA editing sites in mitochondrial PCGs, with 94.49% causing amino acid changes. This indicates that post-transcriptional modification plays a crucial role in mitochondrial gene expression in E. chloranthoides . The extremely high proportion of non-synonymous edits strongly suggests its core function is to restore conserved amino acid residues by repairing transcripts, a common mechanism in plant mitochondria for maintaining protein function [ 57 – 59 ]. Editing sites were concentrated in specific genes (e.g., nad4 , ccmB , and nad7 ), consistent with previous studies indicating that respiratory chain complex subunits (especially nad genes of Complex I) and genes involved in cytochrome c maturation ( ccm genes) are often hotspots for RNA editing [ 60 ]. The creation of stop codons for the atp6 and ccmFC genes via editing is a typical case of post-transcriptional stop codon generation, adding a layer of regulatory dimension to mitochondrial gene translation [ 61 ]. Furthermore, we identified only 11 short MTPT fragments, with a total length of merely about 2.7 kb, indicating that large-scale, recent DNA transfer from the chloroplast to the mitochondria is infrequent in E. chloranthoides . This limited transfer is consistent with general observations in many angiosperms [ 62 – 64 ]. However, the discovery of four intact tRNA genes within these transferred fragments is noteworthy. The chloroplast genome encodes 30 types of tRNA genes, constituting a complete tRNA transport system. These genes are highly conserved and widely present in the chloroplast genomes of other species. Therefore, they are more likely to have been transferred from the plastid genome to the mitochondrial genome. Only 17 types of tRNA genes (including those transferred from the chloroplast) were identified in the mitochondrial genome. The intact retention of these plastid-derived tRNA genes in the mitochondrial genome suggests they may have been selectively maintained due to functional necessity, possibly to compensate for deficiencies in the mitochondrial tRNA pool or to adapt to the specific codon usage bias of the mitochondrial genome [ 65 ]. Analysis of homologous recombination mediated by dispersed repeats, based on long-read sequencing data, revealed a dynamic landscape of the mitochondrial genome. The three longest repeat sequences exceeding 1.5 kb (R1, R2, R3) exhibited active recombination features, with both possible linkage conformations coexisting at appreciable frequencies (45–55%). This strongly indicates that these long repeats are primary sites for homologous recombination, leading to a significant proportion of structural isomers within the mitochondrial genome population and constituting the core of its structural plasticity [ 66 ]. In contrast, the vast majority of shorter repeats (70–430 bp) showed a highly stable state, with one conformation overwhelmingly dominant (> 95% support) [ 67 ]. Although numerous, the recombination potential of these shorter repeats is likely greatly suppressed, possibly due to physical distance or intracellular molecular mechanisms, thus playing a primary role in maintaining the overall structural stability of the genome [ 68 , 69 ]. Conclusions In summary, this study provides a comprehensive genomic resource for E. chloranthoides by presenting and analyzing its complete chloroplast and mitochondrial genomes. The comparative framework reveals differences in their genome structure, stability, and evolutionary mechanisms. The chloroplast genome adheres to a conserved, stable model typical of angiosperms. Conversely, the mitochondrial genome exhibits a dynamic and complex state, characterized by its large size, active recombination mediated by long repeats, and a heavy dependence on pervasive RNA editing for proper gene function. Collectively, these results highlight the divergent evolutionary strategies and selective pressures operating on the two co-resident genomes within a plant cell. This work lays the groundwork for future functional studies and broader comparative genomic investigations within the Celastraceae family. Abbreviations SSR Simple sequence repeat ML Maximum-likelihood NCBI National Center for Biotechnology Information PCGs Protein-coding gene sequences WGS Whole-genome sequencing LSC Large single copy SSC Small single copy IR Inverted repeat RSCU Relative Synonymous Codon Usage Ctg contig MTPTs Mitochondrial Plastid sequences Declarations Author Contributions: J.Y. and X.Y. conceived and designed the research; C. C.(co-first authors) wrote the manuscript; J. L. analyzed the data; B.L.carried out the comparative analysis;. All authors read and approved the manuscript. Funding: This research was funded by the Chongqing Ecology and Environment Bureau, grant number 2026-024. The funders were not involved in the study design, data collection and analysis, decision to publish, or manuscript preparation. Data Availability Statement: The plastome sequences have been deposited in GenBank with accession number: PV948728.1; The mitochondrial genome sequences have been deposited in GenBank with accession number: PZ168290, PZ168291, PZ168292, PZ168293, PZ168294, and PZ168295, respectively. Acknowledgments: The authors would like to express their sincere gratitude to all colleagues who provided valuable comments and suggestions for improving this manuscript. Conflicts of Interest: The authors declare that they have no competing interests. References Jamshed S, Kim JH. Disjunction and Vicariance Between East and West Asia: A Case Study on Euonymus sect. Uniloculares Based on Plastid Genome Analysis. Front Plant Sci. 2022;13:825209. 10.3389/fpls.2022.825209 . Zeng P, Guo Z, Cao X, Xiao X, Liu Y, Shi L. 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PLoS ONE. 2011;6(1):e16404. 10.1371/journal.pone.0016404 . Brieba LG. Structure-Function Analysis Reveals the Singularity of Plant Mitochondrial DNA Replication Components: A Mosaic and Redundant System. Plants (Basel Switzerland). 2019;8(12). 10.3390/plants8120533 . Gualberto JM, Newton KJ. Plant Mitochondrial Genomes: Dynamics and Mechanisms of Mutation. Annu Rev Plant Biol. 2017;68:225–52. 10.1146/annurev-arplant-043015-112232 . Additional Declarations No competing interests reported. Supplementary Files TableS1S10.xlsx FigureS1S2.docx 6NCBI.zip Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 29 Apr, 2026 Editor assigned by journal 28 Apr, 2026 Submission checks completed at journal 28 Apr, 2026 First submitted to journal 27 Apr, 2026 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-9546695","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":635270309,"identity":"82a5994e-e22b-4335-b000-60bb1c7331bd","order_by":0,"name":"Xiaoyu Yang","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyu","middleName":"","lastName":"Yang","suffix":""},{"id":635270311,"identity":"11d1c3a7-b32f-4c26-bbe0-a1b9031eaa23","order_by":1,"name":"Can Chen","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Can","middleName":"","lastName":"Chen","suffix":""},{"id":635270313,"identity":"a27e083c-4f84-455a-a85c-2b5443463396","order_by":2,"name":"Jingling Li","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Jingling","middleName":"","lastName":"Li","suffix":""},{"id":635270316,"identity":"32c826ff-bf83-415e-a895-0af956768374","order_by":3,"name":"Botong Lv","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Botong","middleName":"","lastName":"Lv","suffix":""},{"id":635270321,"identity":"44553ed2-1ab5-47d1-92ea-8f4438a0c8b9","order_by":4,"name":"Jie Yu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYBACgwMMCQcYDGzk+NkbwAKMDcRpKUgzluw5QKQWSbCKD4cTN9xIIFILv0TCwwMfDA4bM9x8/kyah8FGdsMB5mcP8Glhk0hIODjDIF2OcXaOGVBLmvGGA2zmBoS0HOYxsDZmls5hu83DAHThAR42CYJa/hgwJ7ZJHn8G1PKfSC0MBs6JPRIMZkAtB4jQwvMg4WCPQZqxBE+O+c85BsnGMw+zmeHXwp6T/OHHHxs5++PHHxu8qbCT7Tve/AyvFgYGngQkDiiomPGrBwL2AwSVjIJRMApGwQgHAJc1SylpqigsAAAAAElFTkSuQmCC","orcid":"","institution":"Southwest University","correspondingAuthor":true,"prefix":"","firstName":"Jie","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2026-04-28 00:53:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9546695/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9546695/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108806357,"identity":"176a223c-dffe-43c9-80df-68742a7a868e","added_by":"auto","created_at":"2026-05-08 15:28:22","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":202733,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical fragment assembly of the organellar genomes of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003echloranthoides\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. All colored bars represent assembled contigs. Links between contigs indicate that they can be merged to form longer sequences. Each contig is labeled with its name, length, and coverage depth. (a) Graphical fragment assembly of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003echloranthoides\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e chloroplast genome. The left panel shows the initial output from GetOrganelle, consisting of three contigs (LSC, SSC, and one IR region). The IR region shows approximately twice the coverage depth of the single-copy regions. By duplicating the IR, the classic quadripartite chloroplast genome structure (right panel) is obtained. (b) Graphical fragment assembly of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. chloranthoides\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mitochondrial genome. This graph was generated by Flye and comprises six contigs (ctg1–ctg6). The links between contigs are labeled with the number of long reads supporting each connection.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9546695/v1/bc179fab3421980874de3e9e.jpeg"},{"id":108782412,"identity":"e05fde5a-03ad-4747-8a1c-b339ff56b52e","added_by":"auto","created_at":"2026-05-08 10:29:36","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":428052,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe organelle genome map of the of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003echloranthoides\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. Genomic features on transcriptionally clockwise and counter-clockwise strands are drawn on the inside and outside of the circle, respectively. Genes belonging to different functional groups are color-coded. (a) The chloroplast genome map of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003echloranthoides\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. (b) The mitochondrial genome map of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003echloranthoides\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9546695/v1/9050488fc36eccd55ea7d1ae.jpeg"},{"id":108806943,"identity":"aca57259-1608-4815-99d2-47ce6a65bf8b","added_by":"auto","created_at":"2026-05-08 15:29:41","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":404067,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe three types of repetitive elements identified on the organelle genome of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003echloranthoides\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. (a) (b) shows the schematic diagram of the distribution of identified repeat elements on the chloroplast genome and mitochondrial genome. The outermost circle represents the genome size, with a scale marked in 10 kb units. The adjacent inner circle shows the distribution of SSRs across the genome. The next inner circle displays the distribution of tandem repeats. The innermost circle, where repeats are linked by ribbons, illustrates the distribution of dispersed repeats. Among these, purple indicates palindromic repeats, and orange indicates forward repeats. (c) (d) shows the identified SSRs on the chloroplast genome and mitochondrial genome of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003echloranthoides\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. The different colored blocks represent distinct SSR types.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9546695/v1/b7a35291b500af294cd185b2.jpeg"},{"id":108782414,"identity":"b2744137-545f-4e1e-bd78-88754b0cff50","added_by":"auto","created_at":"2026-05-08 10:29:36","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":373866,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePreference of codon usage in PCGs of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003echloranthoides\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e organelle genome. The RSCU value for each codon of a given amino acid was calculated to assess whether a usage bias exists. Typically, an RSCU value greater than 1 indicates a preference for that codon. (a) RSCU value of plastidial PCGs; (b) RSCU value of mitochondrial PCGs.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9546695/v1/d398ba3aa2a3e5c1dad53e7f.jpeg"},{"id":108807700,"identity":"90a1e089-3718-4ae2-8df2-47a1860d04c2","added_by":"auto","created_at":"2026-05-08 15:31:13","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":350096,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacteristics of the RNA editing sites identified in mitochondrial PCGs of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003echloranthoides\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. (a) Statistics of amino acid changes before and after RNA editing. The horizontal axis represents the types of amino acid changes, and the vertical axis indicates the frequency of each change pattern. (b) Number of predicted C-to-U RNA editing sites on each PCG. The vertical axis shows the number of RNA editing sites identified in PCGs; the horizontal axis shows the name of PCGs.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9546695/v1/23bd483c971797fdaf275361.jpeg"},{"id":108807678,"identity":"199a5df7-3e47-455c-9624-fc1a32980089","added_by":"auto","created_at":"2026-05-08 15:31:08","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":302366,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic representation of the distribution of MTPTs among the organelle genome of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003echloranthoides\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. The MTPTs on the chloroplast IR regions were counted only once. The MTPT names labeled near the ribbons.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9546695/v1/fa8b26de8675b403aeb5ad18.jpeg"},{"id":108782417,"identity":"a9b10c2c-ba0c-4b5e-ade3-08bf03d14fa7","added_by":"auto","created_at":"2026-05-08 10:29:37","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":319255,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe phylogenetic relationships of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003echloranthoides\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. The tree was constructed based on the nucleotide sequences of 25 conserved mitochondrial PCGs, including: \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eatp1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eatp4\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eatp6\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eatp8\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eatp9\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eccmB\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eccmC\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eccmFC\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eccmFN\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ecob\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ecox1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ecox2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e,\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ecox3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ematR\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003emttB\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003enad1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003enad2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003enad3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003enad4\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003enad4L\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003enad5\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003enad6\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003enad7\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003enad9\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, r\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eps12\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. We used Maximum Likelihood (ML) method to reconstruct the phylogenetic tree. The ML topology is indicated with ML bootstrap support values. Tetraena mongolica (NC_086675.1) and Tribulus terrestris (MK431825.1) were used as outgroups. The tree shown in the upper-left corner preserves the original branch lengths; its topology is corresponds to that of the main phylogenetic tree displayed in the lower-right corner.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9546695/v1/a4547cbb69be8cbaf9e79f46.jpeg"},{"id":108810028,"identity":"e7cd256b-703f-41e5-944e-786cc99fb7a6","added_by":"auto","created_at":"2026-05-08 15:56:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3018118,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9546695/v1/21308595-c18e-4924-ac3c-4b372e40f3b9.pdf"},{"id":108782410,"identity":"125aacf2-678a-4537-a17b-ae2b2a6ad966","added_by":"auto","created_at":"2026-05-08 10:29:36","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":710705,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1S10.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9546695/v1/72461587b4eccd0986f485e7.xlsx"},{"id":108807738,"identity":"2bf5a91d-b898-419d-94bc-fafe013096bb","added_by":"auto","created_at":"2026-05-08 15:31:25","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1732873,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1S2.docx","url":"https://assets-eu.researchsquare.com/files/rs-9546695/v1/e2a38f82b8d1e6c9f352158b.docx"},{"id":108806428,"identity":"c092f9d9-d31a-4fb8-a29a-f5fa8c0334a4","added_by":"auto","created_at":"2026-05-08 15:28:33","extension":"zip","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1982012,"visible":true,"origin":"","legend":"","description":"","filename":"6NCBI.zip","url":"https://assets-eu.researchsquare.com/files/rs-9546695/v1/7345380d5d38d904ecad0ee5.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Organelle genome assembly and analysis of Euonymus chloranthoides, insights into an endemic and endangered species","fulltext":[{"header":"Background","content":"\u003cp\u003eThe genus \u003cem\u003eEuonymus\u003c/em\u003e (Celastraceae) is an important group comprising approximately 200 species of shrubs, woody lianas, and small trees. These plants hold significant ecological, ornamental, and economic value worldwide. \u003cem\u003eEuonymus\u003c/em\u003e species are widely distributed across temperate and subtropical regions of the Northern Hemisphere, with a center of diversity in East Asia (particularly China, Japan, and Korea) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. They are also found in Europe, North America, North Africa, and Australia. China is one of the primary distribution areas for this genus, harboring over 90 species. Owing to their diverse morphologies, evergreen or brilliantly colored autumn foliage, and vividly colored fruits, \u003cem\u003eEuonymus\u003c/em\u003e plants are extensively used in landscaping, commonly serving as hedges, ground covers, potted ornamental plants [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], or specimen trees in gardens. Popular cultivated species include \u003cem\u003eE. japonicus\u003c/em\u003e, \u003cem\u003eE. fortunei\u003c/em\u003e, \u003cem\u003eE. europaeus\u003c/em\u003e, and \u003cem\u003eE. alatus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003ePrevious studies have shown that plants of the genus \u003cem\u003eEuonymus\u003c/em\u003e, particularly \u003cem\u003eE. alatus\u003c/em\u003e (winged euonymus), possess rich phytochemical diversity and notable medicinal potential [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. More than 128 compounds have been identified from their tissues, primarily falling into structural classes such as flavonoids, terpenoids, steroids, alkaloids, cardenolides, and lignans [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Substantial in vitro and in vivo experimental evidence confirms that extracts from these plants and specific pure compounds (e.g., quercetin, kaempferol) exhibit clear anti-diabetic activity [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Their mechanisms of action involve regulating insulin secretion and sensitivity, acting on the PPARγ pathway [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and inhibiting key targets like aldose reductase (AR) and protein tyrosine phosphatase 1B (PTP1B) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This systematic research on their chemical constituents and pharmacological activities provides a modern scientific basis for the traditional use of these plants in treating diabetes and its complications and establishes an important foundation for their potential as sources of novel drug leads.\u003c/p\u003e \u003cp\u003ePlant cells contain two semi-autonomous organellar genomes: the chloroplast genome and the mitochondrial genome. These are genetic systems independent of the nuclear genome, residing in the cytoplasm and governing core life processes such as photosynthesis and respiration, respectively [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The chloroplast genome is typically circular, structurally relatively conserved, and exhibits a characteristic quadripartite structure, containing approximately 110\u0026ndash;130 genes. Its small size, high copy number, uniparental inheritance pattern, and relatively slow evolutionary rate make it an ideal tool for studying plant phylogenetics, species identification, and phylogeography [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In contrast, the plant mitochondrial genome displays remarkable diversity, complexity, and dynamism: its size can range from 66 kb [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]to 19 Mb [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] it often contains large amounts of non-coding DNA and repetitive sequences, and it undergoes frequent recombination. This leads to highly variable structures, with possible coexistence of circular, linear, or branched isomers. This structural plasticity is one of the most distinctive features of plant mitochondrial genomes [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eResearch on organellar genomes holds significant value across multiple dimensions. In the field of evolutionary biology, they serve as crucial evidence for revealing species origins, divergence times, and phylogenetic relationships among taxa [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. At the level of comparative and functional genomics, analyzing their gene content, sequence variation, RNA editing, codon usage bias, and horizontal gene transfer enables deep insights into the mechanisms of genomic structure and functional evolution [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In population genetics and conservation biology, organellar genetic markers are effective tools for assessing species genetic diversity, historical population dynamics, gene flow, and identifying evolutionarily significant units [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Furthermore, due to the sequence conservation of the chloroplast genome, it has become the \u0026ldquo;gold standard\u0026rdquo; for DNA barcoding and species identification [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eEuonymus chloranthoides\u003c/em\u003e Y.C. Yang (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.worldfloraonline.org/taxon/wfo-0000681671\u003c/span\u003e\u003cspan address=\"https://www.worldfloraonline.org/taxon/wfo-0000681671\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) is an evergreen shrub in the family Celastraceae, reaching about 1 meter in height. It is a species endemic to China, currently known only from Sichuan Province or Chongqing Municipality, where it grows in forests and woodlands at altitudes of 300\u0026ndash;400 meters. Since its first description in the 1940s, collection records have been scarce, and it is considered a rare species. Currently, there is a severe lack of research on \u003cem\u003eE. chloranthoides\u003c/em\u003e, particularly in genomics. There are no reported genomic resources for this species.\u003c/p\u003e \u003cp\u003eIn this study, we sampled \u003cem\u003eE. chloranthoides\u003c/em\u003e and performed genome sequencing. Based on Oxford Nanopore long-read data, we completed the assembly and annotation of the complete organellar genomes for this species. Our results will generate the first complete organellar genomic resources for \u003cem\u003eE. chloranthoides\u003c/em\u003e, laying an indispensable data foundation for subsequent research involving molecular marker development, population genetics, phylogenetics, or functional genomics.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eSampling and voucher Deposition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA sample (plant part) of \u003cem\u003eE. chloranthoides\u0026nbsp;\u003c/em\u003ewas collected from\u0026nbsp;Jinyun Mountain, Beibei District, Chongqing, China (coordinates: 108.329181, 30.897192). The specimen was identified by Professor Jie Yu based on descriptions in the \u003cem\u003eFlora of China\u003c/em\u003e. A voucher specimen was prepared following standard procedures, assigned the voucher number HWA202501953, and deposited in the Herbarium of the College of Horticulture and Landscape Architecture, Southwest University. The curator responsible for the management of these voucher specimens at the herbarium is Shiyao Liu ([email protected]).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA extraction and sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor whole-genome sequencing, total genomic DNA was extracted using the CTAB method [20]. DNA libraries with an insert size of 350 bp were constructed using the NEBNext\u0026reg; Ultra\u0026trade; DNA Library Prep Kit (Illumina, USA) [21] and sequenced on the Illumina HiSeq X Ten platform to generate PE150 reads at Nanjing Jisi Huiyuan Biotechnology Co., Ltd. (Nanjing, China). Approximately 10.77 Gb of raw data were generated per sample. Raw sequencing reads were processed with Trimmomatic [22] to obtain clean reads by removing low-quality sequences, applying the following filtering criteria: reads with more than 50% of bases having a Phred quality score \u0026lt; 19, or containing over 5% N bases, were discarded.\u003c/p\u003e\n\u003cp\u003eThe same DNA sample was also subjected to long-read sequencing. Library preparation for Oxford Nanopore Technology sequencing was performed following the manufacturer\u0026apos;s standard protocol, conducted by Nanjing Jisi Huiyuan Biotechnology Co., Ltd. company (Nanjing, China). Sequencing yielded a total of 10.31 Gb of long-read data, comprising 709,002 sequences. The longest read length was 211,993 bp, with an average read length of 14,546.5 bp.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNanjing Jisi Huiyuan Biotechnology Co., Ltd. company (Nanjing, China)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe plastome was assembled using the GetOrganelle pipeline (v1.7.3) [23] with default parameters: \u0026ldquo;-R 15 -k 21,45,65,85,105 -F embplant_pt\u0026rdquo;. Circular plastome sequences were successfully obtained. Annotation of the chloroplast genome was performed using CPGAVAS2 with a custom reference database, using the chloroplast genome of the congeneric species \u003cem\u003eE. sachalinensis\u0026nbsp;\u003c/em\u003e(accession no. OL770079.1) as the reference. The circular genome map was drawn using OGDRAW [24], with font sizes adjusted during image editing. Visualization of the chloroplast genome annotation was conducted using the CPGView software [25].\u003c/p\u003e\n\u003cp\u003eMitochondrial genome assembly was initially performed using the Flye tool for de novo assembly of all long-read data. A BLAST database was constructed from the assembly results using makeblastdb. The mitochondrial genome of a closely related species, \u003cem\u003eE. alatus\u0026nbsp;\u003c/em\u003e(MW009108.1), was then used as a query to identify contigs containing mitochondrial sequences. The extracted contigs served as reference sequences for mapping the long reads. Reads with an aligned length exceeding 5000 bp were retained, resulting in 15,345 potential mitochondrial long-read sequences. These sequences were reassembled using Flye, yielding the complete mitochondrial genome sequence. Completeness was assessed based on the presence of all known core mitochondrial genes and the assembly forming a closed, connected structure without dead ends. The assembly was error-corrected using the tools Fmlrc2 (v0.1.7), Ropebwt2 (vr187), and Msbwt (v0.2.9). Annotation of the mitochondrial genome was performed using the PMGA [26] tool (\u003ca href=\"http://www.1kmpg.cn/pmga/\" target=\"_blank\"\u003ehttp://www.1kmpg.cn/pmga/\u003c/a\u003e) with a reference database of 319 mitochondrial genomes, which is effective for annotating splice sites and trans-splicing genes\u0026nbsp;[27]. Mitochondrial tRNAs were annotated using tRNAscan-SE (v.2.0.11)\u0026nbsp;[28], and rRNAs were annotated using BLASTN (v2.16.0)\u0026nbsp;[29]. The mitochondrial genome map was visualized using OGDRAW\u0026nbsp;[24].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCodon usage analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein-coding sequences (CDS) were extracted from the genome using PhyloSuite (v1.1.16) [30]. Codon usage bias analysis for the mitochondrial protein-coding genes (PCGs) was performed using MEGA (v7.0) [31], and the Relative Synonymous Codon Usage (RSCU) values were calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRepeat element analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepeat sequences, including microsatellites (SSRs), tandem repeats, and dispersed repeats, were identified using MISA (v2.1) [32], TRF (v4.09) [33], and ROUSFinder [34], respectively. The identified repeat units of SSRs included mono-, di-, tri-, tetra-, penta-, and hexanucleotides with minimum repeat thresholds of 10, 5, 4, 3, 3, and 3, respectively. The results were visualized using Excel (2021) and the Circos package (v0.69.9) [35].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSequence migration analysis between organelle genomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHomologous sequences between the chloroplast and mitochondrial genomes of E. chloranthoides were identified using BLASTN (v2.16.0) [29]. Specifically, a BLAST database was created for the mitochondrial genome sequence using makeblastdb. The chloroplast genome sequence was then used as the query with the command: blastn -query cpDNA.fasta -db mtDNA.fasta -word_size 7 -evalue 1e-10 -outfmt 6. The results were visualized using the Circos package (v0.69.9) [35].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of homologous recombination in the mitogenome\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe homologous repetitive sequences in the mitogenome sequences are identified using the ROUSFinder software describe as above. For the forward repetitive sequences and palindromic repetitive sequences identified by ROUSFinder, we extract the sequences of these repetitive sequences and their upstream and downstream regions of 2,000 bp each based on the positions of the two repetitive units of each repetitive sequence. These extracted sequences are used as reference sequences for configuration 1 (p1-1, and p1-2). Furthermore, we simulated homologous recombination by swapping the sequences of the downstream 2,000 bp regions of the two repetitive units, and the obtained sequences are used as reference sequences for configuration 2 (p2-1, and p2-2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe raw sequencing data in fasta format is used to construct the database with the command line: makeblastdb -in raw_data.fasta -out raw_data.fasta -dbtype nucl. Here, raw_data.fasta represents the raw sequencing data. The reference sequences for configuration 1 and configuration 2 generated above are used as query sequences for querying. The command line is: blastn -query reference.fasta -db raw_data.fasta -word_size 7 -evalue 1e-10 -out blast_out.txt -outfmt 6 -num_threads 10 -max_hsps 1 -max_target_seqs 10000000. For each repetitive sequences, only long-reads that completely span the repetitive sequence and its flanking 2,000 bp regions are selected. These long-reads are considered to support the respective configuration. Finally, the number of long-reads supporting different configurations is counted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMitochondrial genome sequences of closely related species were downloaded from GenBank. \u003cem\u003eTetraena mongolica\u0026nbsp;\u003c/em\u003e(NC_086675.1) and \u003cem\u003eTribulus terrestris\u0026nbsp;\u003c/em\u003e(MK431825.1) were selected as outgroups. Using PhyloSuite (v1.1.16) [30], annotation files in GenBank format were processed to extract mitochondrial genes shared among all species. A total of 25 shared mitochondrial genes were obtained: \u003cem\u003eatp1\u003c/em\u003e, \u003cem\u003eatp4\u003c/em\u003e, \u003cem\u003eatp6\u003c/em\u003e, \u003cem\u003eatp8\u003c/em\u003e, \u003cem\u003eatp9\u003c/em\u003e, \u003cem\u003eccmB\u003c/em\u003e, \u003cem\u003eccmC\u003c/em\u003e, \u003cem\u003eccmFC\u003c/em\u003e, \u003cem\u003eccmFN\u003c/em\u003e, \u003cem\u003ecob\u003c/em\u003e, \u003cem\u003ecox1\u003c/em\u003e, \u003cem\u003ecox2\u003c/em\u003e, \u003cem\u003ecox3\u003c/em\u003e, \u003cem\u003ematR\u003c/em\u003e, \u003cem\u003emttB\u003c/em\u003e, \u003cem\u003enad1\u003c/em\u003e, \u003cem\u003enad2\u003c/em\u003e, \u003cem\u003enad3\u003c/em\u003e, \u003cem\u003enad4\u003c/em\u003e, \u003cem\u003enad4L\u003c/em\u003e, \u003cem\u003enad5\u003c/em\u003e, \u003cem\u003enad6\u003c/em\u003e, \u003cem\u003enad7\u003c/em\u003e, \u003cem\u003enad9\u003c/em\u003e, and \u003cem\u003erps12\u003c/em\u003e. These genes were individually aligned using MAFFT (v7.525) [36], and the aligned sequences were concatenated into a single data matrix for phylogenetic analysis. A maximum likelihood (ML) phylogenetic tree was constructed using IQ-TREE2 (v2.3.6) [37] with the parameters \u0026ldquo;--alrt 1000 -B 1000\u0026rdquo;. The GTR+F+I+R2 model was selected as the best-fit substitution model, and bootstrap values were set to 1000. The resulting tree was visualized using the iTOL software (v6) [38].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrediction of RNA editing sites\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sequences of all 36 annotated mitochondrial protein-coding genes (PCGs) from this species were used as input to predict C-to-U RNA editing sites using Deepred-mt [39]. This tool employs a convolutional neural network (CNN) model, offering higher accuracy compared to previous prediction tools. All predictions with a probability score greater than 0.9 were retained.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eOrganellar genome structure and content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe assembly of the organellar genomes of \u003cem\u003eE.\u003c/em\u003e \u003cem\u003echloranthoides\u003c/em\u003e is represented by graphical fragment assembly (Figure 1). The chloroplast genome assembly initially consisted of three contigs: a pair of inverted repeats (IRs) connecting two single-copy regions, which can be further converted into a typical quadripartite structure (Figure 2A), where the two single-copy regions are linked by IRa and IRb, respectively. The mitochondrial genome assembly comprises six contigs, interconnected to form a complex network (Figure 1B). To validate the connections (linkages) between these contigs, long reads were mapped onto the linkage sequences. The results confirmed that all linkages are supported by long reads, verifying the accuracy of the assembly. The number of supporting long reads for each linkage is labeled in Figure 1B, and the original mapping results are presented in Table S1.\u003c/p\u003e\n\u003cp\u003eAs shown in\u0026nbsp;Table 1, the complete chloroplast genome is 157,967 bp in length. The large single-copy (LSC) region is 86,430 bp with a GC content of 34.98%, and the small single-copy (SSC) region is 18,677 bp with a GC content of 31.46%. The two repeat units of the inverted repeat (IR) region, IRa and IRb, are each 26,640 bp long with a GC content of 42.63%, which is higher than that of the LSC and SSC regions.\u003c/p\u003e\n\u003cp\u003eThe complete mitochondrial genome is 968,269 bp in length, consisting of six contigs designated as contig1 to contig6. Their lengths are 337,294 bp, 195,308 bp, 161,205 bp, 162,092 bp, 97,731 bp, and 14,639 bp, respectively, with GC contents ranging from 44.18% to 45.31%.\u003c/p\u003e\n\u003cp\u003eTo assess assembly quality, short reads were mapped to the assembled chloroplast genome. The sequencing depth coverage profile is shown in Figure S1, with an average depth exceeding 2,500\u0026times;. Correspondingly, long reads were mapped to the six mitochondrial contigs, yielding an average coverage depth of approximately 200\u0026times; (Figure S2). The results show no regions of abrupt coverage drop, and all sequences are supported by high-depth reads, confirming the reliability of the assemblies.\u003c/p\u003e\n\u003cp\u003eAnnotation of the \u003cem\u003eE.\u003c/em\u003e \u003cem\u003echloranthoides\u003c/em\u003e chloroplast genome identified 81 unique protein-coding genes (PCGs) (Table 2). Among these, nine genes (\u003cem\u003endhB, rpl2, rpl23, rps12, rps19, rps7, ycf15, ycf2, ycf68\u003c/em\u003e) are duplicated, resulting in a total of 90 PCGs. Four rRNA genes (\u003cem\u003errn4.5, rrn5, rrn16, rrn23\u003c/em\u003e) were annotated, all of which are duplicated, totaling eight rRNA genes. Thirty unique tRNA genes were identified, seven of which are duplicated, resulting in 37 tRNA genes. Thus, the chloroplast genome encodes a total of 125 genes. A map of the \u003cem\u003eE.\u003c/em\u003e \u003cem\u003echloranthoides\u003c/em\u003e chloroplast genome is presented in Figure \u003ca href=\"#tu2\"\u003e2\u003c/a\u003eA.\u003c/p\u003e\n\u003cp\u003eAnnotation of the mitochondrial genome identified 36 unique PCGs (Table 3). Two of these (\u003cem\u003eatp8\u003c/em\u003e and \u003cem\u003erps19\u003c/em\u003e) are duplicated, yielding a total of 38 PCGs. Three rRNA genes (\u003cem\u003errn5, rrn18, rrn26\u003c/em\u003e) were annotated, all present in single copies. Seventeen unique tRNA genes were identified; five are duplicated (\u003cem\u003etrnE-UUC, trnF-GAA, trnN-GUU, trnS-GCU, trnS-UGA\u003c/em\u003e) and one (\u003cem\u003etrnfM-CAU\u003c/em\u003e) is present in three copies, resulting in 24 tRNA genes. Consequently, the mitochondrial genome encodes a total of 65 genes. A map of the \u003cem\u003eE.\u003c/em\u003e \u003cem\u003echloranthoides\u003c/em\u003e mitochondrial genome is presented in Figure 2B.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Organelle genome assembly statistics of \u003cem\u003eE.\u003c/em\u003e \u003cem\u003echloranthoides\u003c/em\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOrganelle\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eContigs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLength (bp)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGC content (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eChloroplast genome\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003eLSC\u003c/p\u003e\n \u003cp\u003eSSC\u003c/p\u003e\n \u003cp\u003eIRa\u003c/p\u003e\n \u003cp\u003eIRb\u003c/p\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e86,430\u003c/p\u003e\n \u003cp\u003e18,677\u003c/p\u003e\n \u003cp\u003e26,640\u003c/p\u003e\n \u003cp\u003e26,640\u003c/p\u003e\n \u003cp\u003e157,967\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003e34.98\u003c/p\u003e\n \u003cp\u003e31.46\u003c/p\u003e\n \u003cp\u003e42.63\u003c/p\u003e\n \u003cp\u003e42.63\u003c/p\u003e\n \u003cp\u003e37.16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eMitochondrial genome\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003eContig 1\u003c/p\u003e\n \u003cp\u003eContig 2\u003c/p\u003e\n \u003cp\u003eContig 3\u003c/p\u003e\n \u003cp\u003eContig 4\u003c/p\u003e\n \u003cp\u003eContig 5\u003c/p\u003e\n \u003cp\u003eContig 6\u003c/p\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e337,294\u003c/p\u003e\n \u003cp\u003e195,308\u003c/p\u003e\n \u003cp\u003e161,205\u003c/p\u003e\n \u003cp\u003e162,092\u003c/p\u003e\n \u003cp\u003e97,731\u003c/p\u003e\n \u003cp\u003e14,639\u003c/p\u003e\n \u003cp\u003e968,269\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003e44.93\u003c/p\u003e\n \u003cp\u003e45.31\u003c/p\u003e\n \u003cp\u003e45.16\u003c/p\u003e\n \u003cp\u003e44.79\u003c/p\u003e\n \u003cp\u003e45.03\u003c/p\u003e\n \u003cp\u003e44.18\u003c/p\u003e\n \u003cp\u003e45.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Genes encoded by the chloroplast genome of \u003cem\u003eE.\u003c/em\u003e \u003cem\u003echloranthoides\u003c/em\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" colspan=\"2\" valign=\"top\" style=\"width: 27px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup of genes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"2\" valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eName of genes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003eSubunits of ATP synthase \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"3\" valign=\"top\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cem\u003eatpA\u003c/em\u003e, \u003cem\u003eatpB\u003c/em\u003e, \u003cem\u003eatpE\u003c/em\u003e, \u003cem\u003eatpF\u003c/em\u003e, \u003cem\u003eatpH\u003c/em\u003e, \u003cem\u003eatpI\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" colspan=\"3\" valign=\"top\" style=\"width: 30px;\"\u003e\n \u003cp\u003eSubunits of NADH-dehydrogenase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cem\u003endhA\u003c/em\u003e, \u003cem\u003endhB\u003c/em\u003e (\u0026times;2), \u003cem\u003endhC\u003c/em\u003e, \u003cem\u003endhD\u003c/em\u003e, \u003cem\u003endhE\u003c/em\u003e, \u003cem\u003endhF\u003c/em\u003e, \u003cem\u003endhG\u003c/em\u003e, \u003cem\u003endhH\u003c/em\u003e, \u003cem\u003endhI\u003c/em\u003e, \u003cem\u003endhJ\u003c/em\u003e, \u003cem\u003endhK\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" colspan=\"3\" valign=\"top\" style=\"width: 30px;\"\u003e\n \u003cp\u003eSubunits of cytochrome b/f complex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cem\u003epetA\u003c/em\u003e, \u003cem\u003epetB\u003c/em\u003e, \u003cem\u003epetD\u003c/em\u003e, \u003cem\u003epetG\u003c/em\u003e, \u003cem\u003epetL\u003c/em\u003e, \u003cem\u003epetN\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" colspan=\"3\" valign=\"top\" style=\"width: 30px;\"\u003e\n \u003cp\u003eSubunits of photosystem I\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cem\u003epsaA\u003c/em\u003e, \u003cem\u003epsaB\u003c/em\u003e, \u003cem\u003epsaC\u003c/em\u003e, \u003cem\u003epsaI\u003c/em\u003e, \u003cem\u003epsaJ\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" colspan=\"3\" valign=\"top\" style=\"width: 30px;\"\u003e\n \u003cp\u003eSubunits of photosystem II\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cem\u003epsbA\u003c/em\u003e, \u003cem\u003epsbB\u003c/em\u003e, \u003cem\u003epsbC\u003c/em\u003e, \u003cem\u003epsbD\u003c/em\u003e, \u003cem\u003epsbE\u003c/em\u003e, \u003cem\u003epsbF\u003c/em\u003e, \u003cem\u003epsbH\u003c/em\u003e, \u003cem\u003epsbI\u003c/em\u003e, \u003cem\u003epsbJ\u003c/em\u003e, \u003cem\u003epsbK\u003c/em\u003e, \u003cem\u003epsbL\u003c/em\u003e, \u003cem\u003epsbM\u003c/em\u003e, \u003cem\u003epsbN\u003c/em\u003e, \u003cem\u003epsbT\u003c/em\u003e, \u003cem\u003epsbZ\u003c/em\u003e, \u003cem\u003eycf3\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" colspan=\"3\" valign=\"top\" style=\"width: 30px;\"\u003e\n \u003cp\u003eLarge subunit of ribosome\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cem\u003erpl14\u003c/em\u003e, \u003cem\u003erpl16\u003c/em\u003e, \u003cem\u003erpl2\u0026nbsp;\u003c/em\u003e(\u0026times;2), \u003cem\u003erpl20\u003c/em\u003e, \u003cem\u003erpl22\u003c/em\u003e, \u003cem\u003erpl23\u0026nbsp;\u003c/em\u003e(\u0026times;2), \u003cem\u003erpl32\u003c/em\u003e, \u003cem\u003erpl33\u003c/em\u003e, \u003cem\u003erpl36\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" colspan=\"3\" valign=\"top\" style=\"width: 30px;\"\u003e\n \u003cp\u003eSmall subunit of ribosome\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cem\u003erps11\u003c/em\u003e, \u003cem\u003erps12\u0026nbsp;\u003c/em\u003e(\u0026times;2), \u003cem\u003erps14\u003c/em\u003e, \u003cem\u003erps15\u003c/em\u003e, \u003cem\u003erps16\u003c/em\u003e, \u003cem\u003erps18\u003c/em\u003e, \u003cem\u003erps19\u0026nbsp;\u003c/em\u003e(\u0026times;2), \u003cem\u003erps2\u003c/em\u003e, \u003cem\u003erps3\u003c/em\u003e, \u003cem\u003erps4\u003c/em\u003e, \u003cem\u003erps7\u0026nbsp;\u003c/em\u003e(\u0026times;2), \u003cem\u003erps8\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" colspan=\"3\" valign=\"top\" style=\"width: 30px;\"\u003e\n \u003cp\u003eDNA dependent RNA polymerase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cem\u003erpoA\u003c/em\u003e, \u003cem\u003erpoB\u003c/em\u003e, \u003cem\u003erpoC1\u003c/em\u003e, \u003cem\u003erpoC2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" colspan=\"3\" valign=\"top\" style=\"width: 30px;\"\u003e\n \u003cp\u003eSubunit of rubisco\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cem\u003erbcL\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" colspan=\"3\" valign=\"top\" style=\"width: 30px;\"\u003e\n \u003cp\u003ec-type cytochrom synthesis gene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cem\u003eccsA\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" colspan=\"3\" valign=\"top\" style=\"width: 30px;\"\u003e\n \u003cp\u003eEnvelop membrane protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cem\u003ecemA\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" colspan=\"3\" valign=\"top\" style=\"width: 30px;\"\u003e\n \u003cp\u003eMaturase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cem\u003ematK\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" colspan=\"3\" valign=\"top\" style=\"width: 30px;\"\u003e\n \u003cp\u003eProtease\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cem\u003eclpP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" colspan=\"3\" valign=\"top\" style=\"width: 30px;\"\u003e\n \u003cp\u003eSubunit of Acetyl-CoA-carboxylase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cem\u003eaccD\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" colspan=\"3\" valign=\"top\" style=\"width: 30px;\"\u003e\n \u003cp\u003eTranslational initiation factor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cem\u003einfA\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" colspan=\"3\" valign=\"top\" style=\"width: 30px;\"\u003e\n \u003cp\u003eConserved open reading frames\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cem\u003eycf1\u003c/em\u003e, \u003cem\u003eycf15\u0026nbsp;\u003c/em\u003e(\u0026times;2), \u003cem\u003eycf2\u0026nbsp;\u003c/em\u003e(\u0026times;2), \u003cem\u003eycf4\u003c/em\u003e, \u003cem\u003eycf68\u0026nbsp;\u003c/em\u003e(\u0026times;2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" colspan=\"3\" valign=\"top\" style=\"width: 30px;\"\u003e\n \u003cp\u003eRibosomal RNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cem\u003errn4.5\u0026nbsp;\u003c/em\u003e(\u0026times;2),\u003cem\u003e\u0026nbsp;rrn5\u0026nbsp;\u003c/em\u003e(\u0026times;2), \u003cem\u003errn16\u0026nbsp;\u003c/em\u003e(\u0026times;2), \u003cem\u003errn23\u0026nbsp;\u003c/em\u003e(\u0026times;2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eContinue Table 2 Genes encoded by the chloroplast genome of \u003cem\u003eE.\u003c/em\u003e \u003cem\u003echloranthoides\u003c/em\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 27px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup of genes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"2\" valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eName of genes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" colspan=\"2\" valign=\"top\" style=\"width: 30px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eTransfer RNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cem\u003etrnA-UGC\u0026nbsp;\u003c/em\u003e(\u0026times;2),\u003cem\u003e\u0026nbsp;trnC-GCA\u003c/em\u003e,\u003cem\u003e\u0026nbsp;trnD-GUC\u003c/em\u003e,\u003cem\u003e\u0026nbsp;trnE-UUC\u003c/em\u003e,\u003cem\u003e\u0026nbsp;trnF-GAA\u003c/em\u003e,\u003cem\u003e\u0026nbsp;trnfM-CAU\u003c/em\u003e,\u003cem\u003e\u0026nbsp;trnG-GCC\u003c/em\u003e,\u003cem\u003e\u0026nbsp;trnG-UCC\u003c/em\u003e,\u003cem\u003e\u0026nbsp;trnH-GUG\u003c/em\u003e,\u003cem\u003e\u0026nbsp;trnI-CAU\u0026nbsp;\u003c/em\u003e(\u0026times;2),\u003cem\u003e\u0026nbsp;trnI-GAU\u0026nbsp;\u003c/em\u003e(\u0026times;2),\u003cem\u003e\u0026nbsp;trnK-UUU\u003c/em\u003e,\u003cem\u003e\u0026nbsp;trnL-CAA\u0026nbsp;\u003c/em\u003e(\u0026times;2),\u003cem\u003e\u0026nbsp;trnL-UAA\u003c/em\u003e,\u003cem\u003e\u0026nbsp;trnL-UAG\u003c/em\u003e,\u003cem\u003e\u0026nbsp;trnM-CAU\u003c/em\u003e,\u003cem\u003e\u0026nbsp;trnN-GUU\u0026nbsp;\u003c/em\u003e(\u0026times;2),\u003cem\u003e\u0026nbsp;trnP-UGG\u003c/em\u003e,\u003cem\u003e\u0026nbsp;trnQ-UUG\u003c/em\u003e,\u003cem\u003e\u0026nbsp;trnR-ACG\u0026nbsp;\u003c/em\u003e(\u0026times;2),\u003cem\u003e\u0026nbsp;trnR-UCU\u003c/em\u003e,\u003cem\u003e\u0026nbsp;trnS-GCU\u003c/em\u003e,\u003cem\u003e\u0026nbsp;trnS-GGA\u003c/em\u003e, \u003cem\u003etrnS-UGA\u003c/em\u003e,\u003cem\u003e\u0026nbsp;trnT-GGU\u003c/em\u003e,\u003cem\u003e\u0026nbsp;trnT-UGU\u003c/em\u003e, \u003cem\u003etrnV-GAC\u0026nbsp;\u003c/em\u003e(\u0026times;2),\u003cem\u003e\u0026nbsp;trnV-UAC\u003c/em\u003e,\u003cem\u003e\u0026nbsp;trnW-CCA\u003c/em\u003e,\u003cem\u003e\u0026nbsp;trnY-GUA\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote. (\u0026times;2) means this gene has two copies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Genes encoded by the mitochondrial genome of \u003cem\u003eE.\u003c/em\u003e \u003cem\u003echloranthoides\u003c/em\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 34px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup of genes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eName of genes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 34px;\"\u003e\n \u003cp\u003eATP synthase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cem\u003eatp1\u003c/em\u003e, \u003cem\u003eatp4\u003c/em\u003e, \u003cem\u003eatp6\u003c/em\u003e, \u003cem\u003eatp8\u003c/em\u003e (\u0026times;2), \u003cem\u003eatp9\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 34px;\"\u003e\n \u003cp\u003eNADH dehydrogenase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cem\u003enad1\u003c/em\u003e, \u003cem\u003enad2\u003c/em\u003e, \u003cem\u003enad3\u003c/em\u003e, \u003cem\u003enad4\u003c/em\u003e, \u003cem\u003enad4L\u003c/em\u003e, \u003cem\u003enad5\u003c/em\u003e, \u003cem\u003enad6\u003c/em\u003e, \u003cem\u003enad7\u003c/em\u003e, \u003cem\u003enad9\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 34px;\"\u003e\n \u003cp\u003eCytochrome \u003cem\u003eb\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cem\u003ecob\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 34px;\"\u003e\n \u003cp\u003eCytochrome \u003cem\u003ec\u003c/em\u003e biogenesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cem\u003eccmB\u003c/em\u003e, \u003cem\u003eccmC\u003c/em\u003e, \u003cem\u003eccmFC\u003c/em\u003e, \u003cem\u003eccmFN\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 34px;\"\u003e\n \u003cp\u003eCytochrome \u003cem\u003ec\u003c/em\u003e oxidase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cem\u003ecox1\u003c/em\u003e, \u003cem\u003ecox2\u003c/em\u003e, \u003cem\u003ecox3\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 34px;\"\u003e\n \u003cp\u003eMaturases\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cem\u003ematR\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 34px;\"\u003e\n \u003cp\u003eProtein transport subunit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cem\u003emttB\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 34px;\"\u003e\n \u003cp\u003eRibosomal protein large subunit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cem\u003erpl2\u003c/em\u003e, \u003cem\u003erpl5\u003c/em\u003e, \u003cem\u003erpl10\u003c/em\u003e, \u003cem\u003erpl16\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 34px;\"\u003e\n \u003cp\u003eRibosomal protein small subunit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cem\u003erps1\u003c/em\u003e, \u003cem\u003erps3\u003c/em\u003e, \u003cem\u003erps4\u003c/em\u003e, \u003cem\u003erps10\u003c/em\u003e, \u003cem\u003erps12\u003c/em\u003e, \u003cem\u003erps13\u003c/em\u003e, \u003cem\u003erps19\u003c/em\u003e (\u0026times;2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 34px;\"\u003e\n \u003cp\u003eSuccinate dehydrogenase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cem\u003esdh4\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 34px;\"\u003e\n \u003cp\u003eRibosomal RNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cem\u003errn5\u003c/em\u003e, \u003cem\u003errn18\u003c/em\u003e, \u003cem\u003errn26\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 34px;\"\u003e\n \u003cp\u003eTransfer RNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cem\u003etrnC-GCA\u003c/em\u003e, \u003cem\u003etrnD-GUC\u003c/em\u003e, \u003cem\u003etrnE-UUC\u003c/em\u003e (\u0026times;2), \u003cem\u003etrnF-GAA\u003c/em\u003e (\u0026times;2), \u003cem\u003etrnfM-CAU\u003c/em\u003e (\u0026times;3), \u003cem\u003etrnG-GCC\u003c/em\u003e, \u003cem\u003etrnH-GUG\u003c/em\u003e, \u003cem\u003etrnI-CAU\u003c/em\u003e, \u003cem\u003etrnK-UUU\u003c/em\u003e, \u003cem\u003etrnM-CAU\u003c/em\u003e, \u003cem\u003etrnN-GUU\u003c/em\u003e (\u0026times;2), \u003cem\u003etrnP-UGG\u003c/em\u003e, \u003cem\u003etrnQ-UUG\u003c/em\u003e, \u003cem\u003etrnS-GCU\u003c/em\u003e (\u0026times;2), \u003cem\u003etrnS-UGA\u003c/em\u003e (\u0026times;2), \u003cem\u003etrnW-CCA\u003c/em\u003e, \u003cem\u003etrnY-GUA\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote. (\u0026times;2) means this gene has two copies, (\u0026times;3) means this gene has three copies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of repeat elements in organellar genomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe analyzed three types of repeat elements in both organellar genomes: SSRs, tandem repeats, and dispersed repeats. The results are shown in\u0026nbsp;Figure 3.\u003c/p\u003e\n\u003cp\u003eIn the chloroplast genome, only eight dispersed repeats were identified (Table S2). With the exception of those in the IR region, the lengths of other dispersed repeats were all below 100 bp. In contrast, 65 tandem repeats were found (Table S3). Finally, 109 SSRs were identified (Table S4).\u0026nbsp;Figure 3A illustrates the distribution of these repeats across the chloroplast genome.\u0026nbsp;Figure 3C shows the number of different SSR types identified. Monometric SSRs were the most abundant (75, accounting for 68.81% of the total).\u003c/p\u003e\n\u003cp\u003eIn the mitochondrial genome, 225 dispersed repeats were identified. The largest dispersed repeat was 10,259 bp in length (R1, Table S2), followed by repeats of 3,500 bp (R2) and 1,562 bp (R3). Seventeen dispersed repeats exceeded 100 bp in length. Forty-six tandem repeats were identified (Table S3), fewer than the 65 found in the chloroplast genome. Finally, 322 SSRs were detected. The counts for mono-, di-, tri-, tetra-, penta-, and hexameric SSRs were 103, 63, 35, 98, 17, and 6, respectively. Although monomeric SSRs repeats remained the most frequent type, they constituted only 31.99% of the total, indicating a higher proportion of multi-nucleotide repeat units compared to the chloroplast genome. Notably, tetrameric SSRs were abundant (98, accounting for 30.43% of total SSRs).\u0026nbsp;Figure 3B illustrates the distribution of repeats across the mitochondrial genome, and\u0026nbsp;Figure 3D shows the counts of different SSR types identified.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCodon usage bias\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDue to the degeneracy of the genetic code, a single amino acid can be encoded by multiple synonymous codons. The Relative Synonymous Codon Usage (RSCU) value for the codons corresponding to each amino acid was calculated to assess potential usage bias. Typically, an RSCU value greater than 1 indicates a preference for that codon.\u003c/p\u003e\n\u003cp\u003eFor the protein-coding genes (PCGs) in the chloroplast genome, all amino acids except Met and Trp (which are each encoded by a single codon) exhibited varying degrees of preference for specific codons (Figure 4A). For instance, the RSCU value for the UUA codon of Leu was 1.88, for the GCU codon of Ala was 1.79, and for the AGA codon of Arg was 1.78 (Table S5). A chi-square test further revealed that the codon preferences for 18 amino acids were extremely significant, with only the stop codon preference being statistically significant (Table S6).\u003c/p\u003e\n\u003cp\u003eFor the PCGs in the mitochondrial genome, Met and Trp are likewise encoded by single codons. Beyond these, the codon preferences observed for other amino acids were weaker than those in the chloroplast PCGs (Figure 4B). For example, the highest RSCU value was 1.59 for the GCU codon of Ala, which is lower than the corresponding value (1.79) in the chloroplast. This was followed by an RSCU value of 1.52 for the UAU codon of Tyr, and 1.54 for the CAA codon of Gln (Table S5). The chi-square test indicated that codon preferences for only 15 amino acids reached an extremely significant level. The preferences for Cys, Phe, and Val were statistically significant, while the preference for the stop codon was not significant (Table S6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrediction of RNA editing sites\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsidering that RNA editing events are rare in chloroplast-encoded PCGs, here we did not analyze the RNA editing sites in plastidial PCGs. We predicted potential C-to-U RNA editing sites for the mitochondrial PCGs. The results indicated that all 36 unique PCGs possess C-to-U RNA editing sites, with a total of 508 sites predicted (Table S7). Among these, only 28 sites were synonymous edits that did not alter the amino acid. A substantial majority of 480 sites (94.49%) resulted in amino acid changes. The most frequent change was from Ser to Leu (114 sites), followed by Pro to Leu (106 sites), and then Ser to Phe (66 sites) (Figure 5A). We also noted two edits that introduced stop codons: Gln-to-End and Arg-to-End. These changes occurred at the termination codon positions of the atp6 (CAA to UAA) and ccmFC (CGA to UGA) genes, respectively, indicating that the stop codons for these transcripts are acquired via post-transcriptional C-to-U modification.\u003c/p\u003e\n\u003cp\u003eRegarding the distribution across individual PCGs, we observed that three genes contained over 30 editing sites: \u003cem\u003enad4\u003c/em\u003e (39 sites), \u003cem\u003eccmB\u003c/em\u003e (37 sites), and \u003cem\u003enad7\u003c/em\u003e (34 sites). Additionally, \u003cem\u003eccmC\u003c/em\u003e and \u003cem\u003emttB\u003c/em\u003e each had 30 sites (Figure 5B). In contrast, \u003cem\u003erpl2\u003c/em\u003e had the fewest, with only one C-to-U RNA editing site.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of sequence transfer between organelles\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe subsequently investigated sequence transfer between the two organelles of \u003cem\u003eE.\u003c/em\u003e \u003cem\u003echloranthoides\u003c/em\u003e. Using BLASTn, we identified only 11 homologous sequences (Mitochondrial Plastid DNA Sequences, MTPTs) shared between the two genomes (Table S8). Most of these MTPTs were short; only MTPT2 exceeded 1 kb in length (1068 bp), while the lengths of the remaining fragments ranged from 38 to 332 bp.\u0026nbsp;Figure 6\u0026nbsp;illustrates the relative genomic locations of these 11 MTPTs. Their combined length is merely about 2.7 kb, suggesting that large-scale sequence transfer has not occurred frequently between these two organellar genomes.\u003c/p\u003e\n\u003cp\u003eAlthough the transferred fragments were few and short, we found that some contained intact genes. These were exclusively tRNA genes: \u003cem\u003etrnW-CCA\u003c/em\u003e on MTPT1,\u003cem\u003e\u0026nbsp;trnN-GUU\u003c/em\u003e on MTPT10,\u003cem\u003e\u0026nbsp;trnD-GUC\u003c/em\u003e on MTPT11, and \u003cem\u003etrnI-CAU\u003c/em\u003e on MTPT12. These tRNA genes were likely transferred from the plastid genome to the mitochondrial genome.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of mitochondrial genome recombination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the high sequencing coverage (~200\u0026times;) of our long-read data, we leveraged the advantage of long-reads to identify potential homologous recombination events. The results are presented in\u0026nbsp;Table 4, where a total of 18 repeat pairs were identified as potentially mediating homologous recombination. The three longest repeats (R1, 10,259 bp; R2, 3,500 bp; R3, 1,562 bp) exhibited active recombination features. Both of their possible linkage conformations received similarly high support from the sequencing data (Conformation 1 accounted for 51.22%, 55.25%, and 45.03%, respectively). This suggests frequent homologous recombination exchange between these long repeats, which may lead to the co-existence of two major isomeric structures in the mitochondrial genome across different cells or individuals at appreciable frequencies. These long repeats represent a primary source of structural dynamics in the mitochondrial genome.\u003c/p\u003e\n\u003cp\u003eThe remaining 15 shorter repeats (lengths ranging from 70 bp to 430 bp) displayed a distinctly different pattern. Among these, for the vast majority (e.g., R4, R5, R6, etc.), Conformation 1 was overwhelmingly dominant (support rate \u0026gt;95%, mostly \u0026gt;99%), while Conformation 2 was supported by very few reads (typically only 1-3 reads), accounting for less than 1%. This strongly indicates that homologous recombination events between these short repeats occur at an extremely low frequency, or only in a very small minority of mitochondrial molecules. The structural variations they mediate are likely rare types and do not represent the major forms present in the population. The original mapping results of the long-read data to the sequences of different conformations are shown in Table S9.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Number of long-reads supporting the different configurations of \u003cem\u003eE.\u003c/em\u003e \u003cem\u003echloranthoides\u003c/em\u003e mitogenome.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"690\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRepeat ID\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRepeat length (bp)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 53px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ep1-1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 42px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ep1-2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 42px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ep2-1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 42px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ep2-2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eReads support configuration 1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 100px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePercentage of Conformation 1 (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eReads support configuration2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 100px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePercentage of Conformation 2 (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 57px;\"\u003e\n \u003cp\u003eR1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e10,259\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 53px;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 42px;\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 42px;\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 42px;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 94px;\"\u003e\n \u003cp\u003e63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 100px;\"\u003e\n \u003cp\u003e51.22\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 104px;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 100px;\"\u003e\n \u003cp\u003e48.78\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 57px;\"\u003e\n \u003cp\u003eR2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e3,500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 53px;\"\u003e\n \u003cp\u003e57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 42px;\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 42px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 42px;\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 94px;\"\u003e\n \u003cp\u003e121\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 100px;\"\u003e\n \u003cp\u003e55.25\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 104px;\"\u003e\n \u003cp\u003e98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 100px;\"\u003e\n \u003cp\u003e44.75\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 57px;\"\u003e\n \u003cp\u003eR3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e1,562\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 53px;\"\u003e\n \u003cp\u003e122\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 42px;\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 42px;\"\u003e\n \u003cp\u003e116\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 42px;\"\u003e\n \u003cp\u003e61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 94px;\"\u003e\n \u003cp\u003e145\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 100px;\"\u003e\n \u003cp\u003e45.03\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 104px;\"\u003e\n \u003cp\u003e177\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 100px;\"\u003e\n \u003cp\u003e54.97\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 57px;\"\u003e\n \u003cp\u003eR4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e430\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 53px;\"\u003e\n \u003cp\u003e175\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 42px;\"\u003e\n \u003cp\u003e173\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 42px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 42px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 94px;\"\u003e\n \u003cp\u003e348\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 100px;\"\u003e\n \u003cp\u003e95.60\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 104px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 100px;\"\u003e\n \u003cp\u003e4.40\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 57px;\"\u003e\n \u003cp\u003eR5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e276\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 53px;\"\u003e\n \u003cp\u003e158\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 42px;\"\u003e\n \u003cp\u003e199\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 42px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 42px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 94px;\"\u003e\n \u003cp\u003e357\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 100px;\"\u003e\n \u003cp\u003e98.62\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 104px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 100px;\"\u003e\n \u003cp\u003e1.38\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 57px;\"\u003e\n \u003cp\u003eR6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e235\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 53px;\"\u003e\n \u003cp\u003e130\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 42px;\"\u003e\n \u003cp\u003e126\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 42px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 42px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 94px;\"\u003e\n \u003cp\u003e256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 100px;\"\u003e\n \u003cp\u003e99.22\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 104px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.78\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 57px;\"\u003e\n \u003cp\u003eR8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e213\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 53px;\"\u003e\n \u003cp\u003e184\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 42px;\"\u003e\n \u003cp\u003e199\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 42px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 42px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 94px;\"\u003e\n \u003cp\u003e383\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 100px;\"\u003e\n \u003cp\u003e99.48\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 104px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.52\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003eR12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e168\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 53px;\"\u003e\n \u003cp\u003e163\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e164\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003e327\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 100px;\"\u003e\n \u003cp\u003e99.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 104px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.61\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003eR14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e117\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 53px;\"\u003e\n \u003cp\u003e198\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e185\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003e383\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 100px;\"\u003e\n \u003cp\u003e99.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 104px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003eR17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e110\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 53px;\"\u003e\n \u003cp\u003e187\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e203\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003e390\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 100px;\"\u003e\n \u003cp\u003e99.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 104px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003eR18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 53px;\"\u003e\n \u003cp\u003e183\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e219\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003e402\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 100px;\"\u003e\n \u003cp\u003e99.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 104px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003eR22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 53px;\"\u003e\n \u003cp\u003e165\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e167\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003e332\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 100px;\"\u003e\n \u003cp\u003e99.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 104px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003eR23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 53px;\"\u003e\n \u003cp\u003e241\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e169\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003e410\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 100px;\"\u003e\n \u003cp\u003e99.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 104px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.73\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003eR33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 53px;\"\u003e\n \u003cp\u003e172\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e168\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003e340\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 100px;\"\u003e\n \u003cp\u003e99.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 104px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.58\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003eR34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 53px;\"\u003e\n \u003cp\u003e188\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e204\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003e392\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 100px;\"\u003e\n \u003cp\u003e99.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 104px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003eR38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 53px;\"\u003e\n \u003cp\u003e184\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e240\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003e424\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 100px;\"\u003e\n \u003cp\u003e99.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 104px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003eR53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 53px;\"\u003e\n \u003cp\u003e242\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e170\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003e412\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 100px;\"\u003e\n \u003cp\u003e99.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 104px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.72\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 57px;\"\u003e\n \u003cp\u003eR54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 53px;\"\u003e\n \u003cp\u003e172\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e170\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 42px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003e342\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 100px;\"\u003e\n \u003cp\u003e99.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 104px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.58\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsidering that phylogenetic analyses based on chloroplast genomes have been extensively studied in numerous previous publications, we performed phylogenetic analysis here based solely on mitochondrial PCGs. Due to the scarcity of usable mitochondrial genome data in public databases, our analysis was conducted at the order level. We downloaded an additional 26 mitochondrial genome datasets from public databases (Table S10) and combined them with our newly sequenced one, resulting in a total of 27 species. We identified 25 common protein-coding genes (see the legend of Figure 7). Our phylogenetic tree (Figure \u003ca href=\"#tu7\"\u003e7\u003c/a\u003e) shows no conflict with previous studies based on the APG IV system [40]. The two species from the genus \u003cem\u003eE.\u003c/em\u003e clustered\u003cem\u003e\u0026nbsp;\u003c/em\u003etogether as a monophyletic clade with 100% bootstrap support.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe chloroplast genomes of plants in the family Celastraceae have been extensively studied, with over 100 resources currently available [\u003cspan additionalcitationids=\"CR42 CR43\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In contrast, research on the mitochondrial genomes of this family remains in a very preliminary stage, and data on complete or nearly complete mitochondrial genomes that have been published are extremely limited. To date, only three Celastraceae species with published, complete mitochondrial genome drafts can be retrieved from the GenBank database, all of which are results from recent years (2021\u0026ndash;2025). They are: \u003cem\u003eTripterygium wilfordii\u003c/em\u003e (720,306 bp) [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], \u003cem\u003eE. alatus\u003c/em\u003e (1,045,106 bp) [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], and \u003cem\u003eE. europaeus\u003c/em\u003e (approximately 1.47 Mb, belonging to the Darwin Tree of Life Project, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.darwintreeoflife.org/\u003c/span\u003e\u003cspan address=\"https://www.darwintreeoflife.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). These published genomes exhibit substantial size variation, suggesting that the mitochondrial genomes of plants in this family may possess a high degree of dynamism and complexity. Furthermore, mitochondrial sequences of a few other species (such as \u003cem\u003eParnassia faberi\u003c/em\u003e and \u003cem\u003eParnassia rhombipetala\u003c/em\u003e) have been uploaded to the database but remain unreported. Moreover, their assembly results are highly fragmented (consisting of 7 to 11 independent contigs), which further reflects the challenges in achieving complete assembly of the mitochondrial genomes for this group.\u003c/p\u003e \u003cp\u003eIn this study, a comprehensive comparative analysis of the organellar genomes of \u003cem\u003eE. chloranthoides\u003c/em\u003e was conducted. Marked differences were observed between its chloroplast and mitochondrial genomes in terms of genomic structure, gene content, and repeat composition, reflecting their distinct evolutionary trajectories. The chloroplast genome of \u003cem\u003eE. chloranthoides\u003c/em\u003e possesses a typical quadripartite structure, encodes 125 genes, and is dominated by short SSRs and tandem repeats. This aligns with the highly conserved nature of chloroplast genomes in most higher plants [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In contrast, the mitochondrial genome exhibits characteristically large, complex, and dynamic features: it is approximately six times larger than the chloroplast genome, and its assembly into multiple contigs forming a network structure suggests potential active recombination. The genome is interspersed with numerous long fragment repeats and contains a more diverse array of SSR types, particularly tetranucleotide repeats. Recombination analysis of the dispersed repeats in the mitochondria revealed that up to 18 repeat pairs may be involved in mediating homologous recombination. These results indicate that the mitochondrial genome has greater potential for recombination, sequence plasticity, and tolerance for structural variation [\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. This fundamental difference likely stems from their distinct mutation rates [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], DNA repair mechanisms [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], and functional selective pressures. The chloroplast genome tends towards structural stability, whereas the mitochondrial genome may operate under an evolutionary mode that permits greater sequence expansion and structural remodeling [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe also found differences in codon usage bias between the chloroplast and mitochondrial genomes of \u003cem\u003eE. chloranthoides\u003c/em\u003e. Chloroplast-encoded genes exhibited stronger and more widespread codon preference. The degree of preference for several amino acids (e.g., UUA of Leu, RSCU\u0026thinsp;=\u0026thinsp;1.88) was significantly higher than that in the mitochondria (highest being GCU of Ala, RSCU\u0026thinsp;=\u0026thinsp;1.59). A chi-square test further supported this trend: codon preferences for 18 amino acids reached an \u0026ldquo;extremely significant\u0026rdquo; level in the chloroplast PCGs, compared to only 15 in the mitochondrial PCGs. Moreover, the preference for stop codon usage in the mitochondrial PCGs was not significant. These differences likely reflect the varying selective pressures and functional constraints experienced by the two organelles during evolution. The strong codon bias in the chloroplast genome is generally consistent with the hypothesis of translational efficiency optimization for highly expressed genes [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. During photosynthesis, the chloroplast requires the rapid and copious synthesis of photosynthesis-related proteins. A strong codon preference facilitates the recruitment of more abundant corresponding tRNAs, thereby enhancing translation rates [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn contrast, the weaker codon preference in the mitochondrial PCGs may be related to its higher mutation rate, more relaxed selective pressure, or more complex post-transcriptional regulation, such as post-transcriptional RNA editing [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. The non-significant preference for stop codons in the mitochondria might also suggest differences in its translation termination mechanism compared to the chloroplast. We predicted up to 508 C-to-U RNA editing sites in mitochondrial PCGs, with 94.49% causing amino acid changes. This indicates that post-transcriptional modification plays a crucial role in mitochondrial gene expression in \u003cem\u003eE. chloranthoides\u003c/em\u003e. The extremely high proportion of non-synonymous edits strongly suggests its core function is to restore conserved amino acid residues by repairing transcripts, a common mechanism in plant mitochondria for maintaining protein function [\u003cspan additionalcitationids=\"CR58\" citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Editing sites were concentrated in specific genes (e.g., \u003cem\u003enad4\u003c/em\u003e, \u003cem\u003eccmB\u003c/em\u003e, and \u003cem\u003enad7\u003c/em\u003e), consistent with previous studies indicating that respiratory chain complex subunits (especially \u003cem\u003enad\u003c/em\u003e genes of Complex I) and genes involved in cytochrome c maturation (\u003cem\u003eccm\u003c/em\u003e genes) are often hotspots for RNA editing [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. The creation of stop codons for the \u003cem\u003eatp6\u003c/em\u003e and \u003cem\u003eccmFC\u003c/em\u003e genes via editing is a typical case of post-transcriptional stop codon generation, adding a layer of regulatory dimension to mitochondrial gene translation [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, we identified only 11 short MTPT fragments, with a total length of merely about 2.7 kb, indicating that large-scale, recent DNA transfer from the chloroplast to the mitochondria is infrequent in \u003cem\u003eE. chloranthoides\u003c/em\u003e. This limited transfer is consistent with general observations in many angiosperms [\u003cspan additionalcitationids=\"CR63\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. However, the discovery of four intact tRNA genes within these transferred fragments is noteworthy. The chloroplast genome encodes 30 types of tRNA genes, constituting a complete tRNA transport system. These genes are highly conserved and widely present in the chloroplast genomes of other species. Therefore, they are more likely to have been transferred from the plastid genome to the mitochondrial genome. Only 17 types of tRNA genes (including those transferred from the chloroplast) were identified in the mitochondrial genome. The intact retention of these plastid-derived tRNA genes in the mitochondrial genome suggests they may have been selectively maintained due to functional necessity, possibly to compensate for deficiencies in the mitochondrial tRNA pool or to adapt to the specific codon usage bias of the mitochondrial genome [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnalysis of homologous recombination mediated by dispersed repeats, based on long-read sequencing data, revealed a dynamic landscape of the mitochondrial genome. The three longest repeat sequences exceeding 1.5 kb (R1, R2, R3) exhibited active recombination features, with both possible linkage conformations coexisting at appreciable frequencies (45\u0026ndash;55%). This strongly indicates that these long repeats are primary sites for homologous recombination, leading to a significant proportion of structural isomers within the mitochondrial genome population and constituting the core of its structural plasticity [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. In contrast, the vast majority of shorter repeats (70\u0026ndash;430 bp) showed a highly stable state, with one conformation overwhelmingly dominant (\u0026gt;\u0026thinsp;95% support) [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Although numerous, the recombination potential of these shorter repeats is likely greatly suppressed, possibly due to physical distance or intracellular molecular mechanisms, thus playing a primary role in maintaining the overall structural stability of the genome [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, this study provides a comprehensive genomic resource for \u003cem\u003eE. chloranthoides\u003c/em\u003e by presenting and analyzing its complete chloroplast and mitochondrial genomes. The comparative framework reveals differences in their genome structure, stability, and evolutionary mechanisms. The chloroplast genome adheres to a conserved, stable model typical of angiosperms. Conversely, the mitochondrial genome exhibits a dynamic and complex state, characterized by its large size, active recombination mediated by long repeats, and a heavy dependence on pervasive RNA editing for proper gene function. Collectively, these results highlight the divergent evolutionary strategies and selective pressures operating on the two co-resident genomes within a plant cell. This work lays the groundwork for future functional studies and broader comparative genomic investigations within the Celastraceae family.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"524\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eSSR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eSimple sequence repeat\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eML\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eMaximum-likelihood\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eNCBI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eNational Center for Biotechnology Information\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003ePCGs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eProtein-coding gene sequences\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eWGS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eWhole-genome sequencing\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eLSC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eLarge single copy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eSSC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eSmall single copy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eIR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eInverted repeat\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eRSCU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eRelative Synonymous Codon Usage\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eCtg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003econtig\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eMTPTs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eMitochondrial Plastid sequences\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e J.Y. and X.Y. conceived and designed the research; C. C.(co-first authors) wrote the manuscript; J. L. analyzed the data; B.L.carried out the comparative analysis;. All authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This research was funded by the Chongqing Ecology and Environment Bureau, grant number 2026-024. The funders were not involved in the study design, data collection and analysis, decision to publish, or manuscript preparation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e The plastome sequences have been deposited in GenBank with accession number: PV948728.1; The mitochondrial genome sequences have been deposited in GenBank with accession number:\u0026nbsp;PZ168290, PZ168291, PZ168292, PZ168293, PZ168294, and PZ168295, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e The authors would like to express their sincere gratitude to all colleagues who provided valuable comments and suggestions for improving this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e The authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJamshed S, Kim JH. 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Annu Rev Plant Biol. 2017;68:225\u0026ndash;52. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1146/annurev-arplant-043015-112232\u003c/span\u003e\u003cspan address=\"10.1146/annurev-arplant-043015-112232\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Euonymus chloranthoides, Plastid genome, Mitochondrial genome, RNA editing, Phylogenetic analysis","lastPublishedDoi":"10.21203/rs.3.rs-9546695/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9546695/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePlant organellar genomes (chloroplasts and mitochondria) are valuable resources for understanding plant evolution, phylogenetics, and genetic diversity. \u003cem\u003eEuonymus chloranthoides\u003c/em\u003e is a species within the family Celastraceae, yet its organellar genomes have remained uncharacterized. In this study, we assembled, annotated, and comprehensively analyzed the organellar genomes of this species.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe chloroplast genome of \u003cem\u003eE. chloranthoides\u003c/em\u003e is 157,967 bp in length, exhibits a typical quadripartite structure, encodes 125 genes, and is characterized by a low abundance and diversity of repeat sequences. In stark contrast, its mitochondrial genome is large and complex, with a size of 968,269 bp, assembled into six contigs forming an intricate structure. Comparative analyses further revealed that the chloroplast genome possesses a stronger and more widespread codon usage bias than the mitochondrial genome. Moreover, we predicted 508 C-to-U RNA editing sites in mitochondrial protein-coding genes, the majority of which result in non-synonymous changes. Eleven short mitochondrial plastid DNAs (MTPTs) were detected between the two organelles, four of which contain intact tRNA genes that were likely transferred from the chloroplast genome. We also identified 18 repeat pairs that may mediate homologous recombination in the mitochondrial genome. Among these, three long repeat pairs showed recombination frequencies close to 50%, while the recombination rates of the remaining short repeat pairs were less than or close to 1%.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThis study presents the first complete characterization of both organellar genomes in \u003cem\u003eE. chloranthoides\u003c/em\u003e, revealing their distinctly different evolutionary landscapes. The findings provide a valuable genomic resource and fundamental insights for comparative and evolutionary genomics of organelles within the family Celastraceae.\u003c/p\u003e","manuscriptTitle":"Organelle genome assembly and analysis of Euonymus chloranthoides, insights into an endemic and endangered species","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-08 10:29:30","doi":"10.21203/rs.3.rs-9546695/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-04-29T10:05:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-28T09:30:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-28T09:30:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2026-04-28T00:38:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e4ff553b-6a40-4a57-8e9a-dec0767f6601","owner":[],"postedDate":"May 8th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewersInvited","content":"8","date":"2026-04-29T10:05:11+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-08T10:29:30+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-08 10:29:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9546695","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9546695","identity":"rs-9546695","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}