Report on the complete mitochondrial genome of the critically endangered and endemic Lauraceae plant Syndiclis anlungensis in China

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However, the complete mitochondrial genome of this species has not yet been systematically described, hindering our understanding of the genetic diversity and evolutionary relationships of mitochondrial genomes within the genus Syndiclis . Result This study combined Illumina and Oxford Nanopore sequencing technologies to complete the sequencing, assembly, and annotation of the mitochondrial genome of S. anlungensis . The mitochondrial genome of S. anlungensis has a total length of 2,370,899 bp, comprising 26 core protein-coding genes (PCGs), 18 variable PCGs, and 55 tRNA genes, exhibiting a multipartite substructure mediated by 3 direct repeats. Analysis revealed that the genome contains 703 simple sequence repeats (SSRs), 204 tandem repeats, and 1,993 pairs of dispersed repeats. Among the mitochondrial PCGs, 93.1% of high-frequency codons end with A/T. A total of 755 RNA editing sites were identified, with 357 sites (47.28%) resulting in amino acid residue changes from hydrophilic to hydrophobic and 69 sites (9.14%) showing hydrophobic-to-hydrophilic shifts. Ka/Ks analysis indicated that genes such as ccmFc and rpl16 are under positive selection. Additionally, 62 homologous fragments (totaling 67,900 bp) were identified between the mitochondrial and chloroplast genomes, accounting for approximately 2.8639% of the mitochondrial genome length. Phylogenetic analysis of the mitochondrial genome placed S. anlungensis at the basal position within Lauraceae, while chloroplast genome-based phylogeny revealed S. marlipoensis as the closest relative to S. anlungensis . Conclusions This study presents the first comprehensive decoding of the mitochondrial genome of S. anlungensis , unveiling its features of frequent recombination, repeat sequence expansion, and adaptive evolution. These findings provide critical data for understanding the evolutionary mechanisms of mitochondrial genomes in the genus Syndiclis , while establishing a molecular foundation for the conservation of its genetic resources and the development of population restoration strategies. Syndiclis anlungensis Mitochondrial genome Horizontal transfer Phylogenetic analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Syndiclis anlungensis belongs to the genus Syndiclis (Subtribe Beilschmiediineae, Lauraceae) and is listed as Critically Endangered (CR) in the China Biodiversity Red List—Volume of Higher Plants (2013) [ 1 ]. Since the 1980s, the species was presumed extinct due to the absence of documented sightings during prolonged field surveys, until its rediscovery in 2015 when living wild individuals were identified [ 2 ]. Currently, only two mature trees with trunk diameters of 20–40 cm exist globally, distributed in Wangyuan Village and Polao Village, Dushan Town, Anlong County, Guizhou Province. Studies indicate that S. anlungensis faces survival challenges such as weak natural regeneration capacity and poor environmental adaptability [ 3 ]. The extreme rarity of the remaining population and its limited regeneration heighten the risk of severe genetic diversity loss. From an evolutionary genetic perspective, long-term isolation in small populations predisposes the species to genetic bottleneck effects, leading to the accumulation of deleterious alleles and reduced adaptive potential [ 4 ]. Although the rediscovery of living individuals offers hope for species survival, severely restricted gene flow may exacerbate inbreeding depression, further diminishing the species’ resilience to environmental fluctuations. In this context, in-depth characterization of its genetic system—particularly the evolutionary dynamics of its genome—has become crucial for assessing species viability and formulating science-based conservation strategies. In genomic research, current studies on S. anlungensis remain limited, with existing work focusing solely on chloroplast genome phylogeny and codon usage bias analysis, while its complete mitochondrial genome remains unexplored [ 5 , 6 ]. This critical gap substantially hinders investigations into the species’ evolutionary adaptation mechanisms and the development of science-based conservation approaches. The mitochondrion, one of the most vital plant organelles, is fundamentally linked to respiratory metabolism and energy synthesis [ 7 ]. As the second-largest genome in plants, the mitochondrial genome encodes key subunits of respiratory chain complexes such as cytochrome c oxidase and NADH dehydrogenase, which are indispensable for respiration, electron transport systems, and energy conversion [ 8 ]. Although mitochondrial protein-coding genes (PCGs) exhibit slower evolutionary rates, their functional variations and RNA editing have been demonstrated to significantly influence plant stress resistance and reproductive adaptation [ 9 – 12 ]. For S. anlungensis , investigating mitochondrial genome mutations and evolution will not only clarify its evolutionary dynamics but also provide essential molecular insights for assessing its environmental stress response capabilities. Notably, plant mitochondrial genomes exhibit extraordinary plasticity: frequent recombination generates diverse structural configurations, including master circular molecules, subgenomic circular forms, and multipartite architectures [ 13 – 14 ]. Their sizes span two orders of magnitude in angiosperms—from approximately 222 Kbp in Brassica napus to 11.3 Mbp in Silene conica —and reach up to 11.7 Mbp in the gymnosperm Larix sibirica [ 15 – 17 ]. During long-term evolution, mitochondria continuously integrate chloroplast-derived DNA fragments through asymmetric intracellular DNA transfer mechanisms. These integrated fragments (ranging from tens to thousands of base pairs) often retain intact chloroplast genes or gene clusters, accumulating varying degrees of recombination and mutations during mitochondrial genome evolution, thereby serving as critical repositories of genetic and evolutionary information [ 18 ]. Such highly dynamic genetic features endow mitochondrial genomes with rich taxonomic signals, offering unique advantages for resolving genetic and evolutionary challenges in S. anlungensis and its closely related species. This study employed a combination of Illumina and Oxford Nanopore sequencing technologies to achieve the first sequencing and assembly of the mitochondrial genome of S. anlungensis . Comprehensive analyses were conducted on its compositional structure, codon usage bias, repetitive sequences, RNA editing sites, and chloroplast-derived homologous sequences to characterize the genomic architecture. Furthermore, Ka/Ks analysis elucidated the evolutionary mechanisms of mitochondrial protein-coding genes (PCGs) in S. anlungensis and its close relatives under environmental stress. Phylogenetic trees were reconstructed using mitochondrial and chloroplast PCG datasets from S. anlungensis and other higher plants, respectively, resolving the species’ phylogenetic position. These findings not only unveil the genetic characteristics and evolutionary dynamics of the mitochondrial genome in S. anlungensis but also provide foundational molecular data for its genetic improvement. 2. Materials and methods 2.1. Material collection, DNA extraction and sequencing The specimen of S. anlungensis was collected from its native habitat in Wangyuan Village, Dushan Town, Anlong County, Guizhou Province, China (geographic coordinates: E105°36′55.08″, N25°15′51.98″). The taxonomic identification was conducted by Dr. Chenghua Yang (Guizhou Academy of Forestry). The voucher specimen (collection number: GB2024132) is deposited in the Herbarium of Guizhou Academy of Forestry. Healthy, disease-free young leaves were collected from the mid-canopy of the tree. Immediately after field sampling, leaf tissues were flash-frozen in liquid nitrogen and stored long-term at − 80°C. For DNA extraction, frozen leaf tissues were pulverized using liquid nitrogen grinding, followed by a modified CTAB protocol [ 19 ] to obtain high-purity mitochondrial genomic DNA. The mitochondrial genome was sequenced using a hybrid approach combining Illumina short-read and Oxford Nanopore long-read technologies. For Illumina sequencing, DNA quality and concentration were assessed via 1% agarose gel electrophoresis (Fig. S1 A) and NanoDrop 2000. Qualified samples underwent ultrasonic fragmentation, followed by purification, end repair, adapter ligation, and PCR amplification to construct sequencing libraries. Libraries were sequenced on the Illumina NovaSeq 6000 platform (Illumina, San Diego, USA), generating 150 bp paired-end reads [ 20 ]. After quality control with fastp (v0.23.4) [ 21 ], 67,670,917 clean reads were retained, with a GC content of 40.79%, and Q20/Q30 values of 97.55% and 92.90%, respectively. For Oxford Nanopore sequencing, genomic DNA was randomly sheared, and large fragments (> 10 kb) were enriched using a magnetic bead-based size selection system. Target fragments were gel-purified (Fig. S1 B), then subjected to DNA damage repair, end-blunting, and 3′-dA-tailing. Libraries were prepared with the SQK-LSK109 kit and sequenced on the PromethION platform via single-molecule real-time (SMRT) technology [ 22 ]. Raw data were filtered using Filtlong (v0.24). 2.2 Assembly and annotation of the mitochondrial genome First, the raw third-generation sequencing data was aligned to the plant mitochondrial gene database using minimap2 (v2.1). Next, sequences with alignment lengths exceeding 50 bp were selected as candidate aligned sequences. Sequences containing multiple core genes and exhibiting high coverage completeness were prioritized as initial seed sequences. Subsequently, minimap2 was used to align the raw sequencing data to these seed sequences, and sequences with overlaps greater than 1 kb and similarity exceeding 70% were incorporated into the seed sequences [ 23 ]. The obtained third-generation data were then error-corrected using Canu ( https://github.com/marbl/canu ), a third-generation assembly tool [ 24 ]. Illumina second-generation sequencing data were aligned to the corrected sequences using Bowtie2 (v2.3.5.1). A preliminary assembly was performed by integrating the second-generation data and corrected third-generation data using Unicycler (v0.4.8) with default parameters [ 25 ], and the assembly results were visualized and manually adjusted using Bandage (v0.8.1). Considering the unique multi-circular/non-circular structural features of mitochondrial genomes, the corrected third-generation data were realigned to the contigs generated by Unicycler. The final high-integrity mitochondrial genome assembly was achieved by manually determining branch orientations. The annotation of mitochondria is conducted using the following steps: Protein-coding genes and rRNA genes are aligned with publicly available reference plant mitochondrial sequences using the Basic Local Alignment Search Tool-Nucleotide (BLASTN, https://blast.ncbi.nlm.nih.gov/ ) [ 26 ]. Manual adjustments are then performed using the closely related species C. camphora (GenBank ID: NC_086632) as the reference genome to ensure the accuracy and reliability of the alignment. Additionally, tRNA genes are annotated using tRNAscan-SE ( http://lowelab.ucsc.edu/tRNAscan-SE/ ) [ 27 ]. Open Reading Frame (ORF) annotation is carried out using the Open Reading Frame Finder ( https://www.ncbi.nlm.nih.gov/orffinder/ ) [ 28 ], with a minimum length threshold of 102 bp to exclude redundant sequences and those overlapping with known genes. Sequences longer than 300 bp are annotated by aligning against the nr database. The mitochondrial genome map is constructed using OGDRAW ( https://chlorobox.mpimp-golm.mpg.de/OGDraw.html ) [ 29 ]. 2.3 Repetitive sequence analysis and RNA editing site prediction The identification of simple sequence repeats (SSRs) was performed using MISA (v1.0) ( https://webblast.ipk-gatersleben.de/misa/ ) [ 30 , 31 ], while tandem repeats were detected using Tandem Repeats Finder (TRF, v4.09) ( http://tandem.bu.edu/trf/trf.submit.options.html ) [ 32 ]. Dispersed repeats were identified via BLASTN (v2.10.1; parameters: -word_size 7, -evalue 1e-5) for homology-based analysis [ 33 ]. Redundant sequences and previously identified tandem repeat regions were excluded during the analysis. All repeat data were visualized using Circos (v0.69-5) [ 34 ]. RNA editing sites in Anlong camphora were predicted using the PREPACT3 online platform ( http://www.prepact.de/ ) with a significance threshold of 0.001 [ 35 ]. Finally, statistical analysis and visualization (histograms and pie charts) were conducted using Microsoft Excel 2021. 2.4 Codon usage bias and chloroplast homologous sequence analysis Protein-coding sequences were extracted using Phylosuit (v1.22) with default parameters [ 36 ]. The relative synonymous codon usage (RSCU) of mitochondrial genome protein-coding genes was subsequently calculated using MEGA (v7.0) [ 37 ]. Chloroplast genome sequences from the same sample were also extracted, and homologous sequences between the chloroplast and mitochondrial genomes were identified using BLAST with a similarity threshold of 70% and an E-value cutoff of 1e-5. To visually represent homologous fragments between the chloroplast and mitochondrial genomes, Circos (v0.69-5) was employed for visualization [ 38 ]. 2.5 Caculations of Pi (nucleic acid diversity) and Ka/Ks (non-synonymous substitutions/synonymous substitutions) Mitochondrial genome data for three Lauraceae species: Caryodaphnopsis henryi (NC_088584), Cinnamomum camphora (NC_086632), and Cinnamomum chekiangense (NC_082065), were downloaded from the NCBI Database ( http://www.ncbi.nlm.nih.gov/genome/organelle/ ). These mitochondrial genomes were aligned using MAFFT (v7.427) [ 39 ]. Nucleotide diversity (Pi) values for shared genes were calculated with DnaSP (v6.12.03) [ 40 ]. Additionally, the aligned gene sequences were subjected to BLAST analysis using MAFFT, and the resulting BLAST output files were imported into KaKs_Calculator to estimate the nonsynonymous substitution rate (Ka), synonymous substitution rate (Ks), and the Ka/Ks ratio [ 41 ]. 2.6 Phylogenetic Analysis To investigate the phylogenetic relationships of S. anlungensis , we conducted phylogenetic analyses of its mitochondrial and chloroplast genomes. For the mitochondrial genome, 39 complete mitochondrial genome sequences were downloaded from the NCBI database ( http://www.ncbi.nlm.nih.gov/genome/organelle/ ), including 10 magnoliids, 23 eudicots, 5 monocots, and 1 gymnosperm ( Ginkgo biloba ) as the outgroup. Conserved protein-coding genes shared between these species and S. anlungensis were extracted using TBtools software [ 42 ]. Multiple sequence alignment of coding sequences (CDS) from these 40 mitochondrial genomes was performed using MAFFT (v7.427) [ 39 ]. The maximum likelihood phylogenetic tree was constructed with RAxML (v8.2.10) ( https://cme.h-its.org/exelixis/software.html ) under the GTRGAMMA model, with the bootstrap value set to 1000 [ 43 ]. For chloroplast genome phylogenetic analysis, 24 chloroplast genomes of Lauraceae species (including S. anlungensis ) were downloaded from NCBI, comprising 6 species from the genus Syndiclis , 17 species from its closely related genera Beilschmiedia and Sinopora , with Machilus rehderi as the outgroup. The maximum likelihood tree based on chloroplast protein-coding genes (PCGs) was constructed using the same analytical methods as for the mitochondrial genome. All trees were visualized using ITOL software (v4.0) [ 44 ]. 3. Results 3.1. Assembly and annotation of the S. anlungensis mitochondrial genome This study successfully resolved the mitochondrial genome structure of S. anlungensis using a hybrid assembly strategy. Results revealed that its mitochondrial DNA exhibits typical branched characteristics and can form multi-subtype dynamic structures through recombination mediated by three sets of direct repeat sequences. Through systematic analysis with Unicycler software, we constructed the main circular molecules as two linear chromosomes: Chr1 (2,135,163 bp, GC = 46.13%) was assembled along the topological path contig2 - contig3 - contig11 - LR12 - contig1 - contig9 - contig14 - contig8 - LR15 - contig5 - LR12 - contig4 - contig6 - LR15 - contig13, while Chr2 (235,736 bp, GC = 46.47%) adopted a linear structure of contig7 - LR14 - contig10 (Fig. 1 ). Genome coverage analysis demonstrated complete sequencing read coverage across all assembled regions, verifying the continuity of assembly results (Fig. S2 ). Annotation results identified 26 core protein-coding genes in the S. anlungensis mitochondrial genome, including the atp 9 and na d4L genes with two copy loci each, and one pseudogene ( mat R). Additionally, 18 variable protein-coding genes were detected, with only rps19 showing a single copy event (Fig. 2 , Table 1 ). For non-coding components, the genome contains 3 ribosomal RNA (rRNA) genes and 55 transfer RNA (tRNA) genes. Among tRNA genes, nine types were annotated twice: trn A-TGC, trn E-TTC, trn F-GAA, trn G-GCC, trn H-GTG, trn I-GAT, trn P-TGG, trn R-TCT, and trn V-GAC; trn T-TGT and trn Y-GTA were annotated three times; trn L-CAA and trn N-GTT four times; while trn M-CAT showed the highest annotation frequency with 10 sites. In the mitochondrial genome of S. anlungensis , 13 genes possess one intron each ( ccm Fc, rpl 2, rps 10, rps 3, trn A-TGC (2), trn I-GAT (2), trn Q-CTG, trnR-TCT (2), trn T-TGT (3)), one gene containstwo introns ( cox 2), one gene contains three introns ( nad 4), and four genes have four introns ( nad 1, nad 2, nad 5, and nad 7). Table 1 List of genes in the mitochondrial genome of S. anlungensis . Group of genes Gene name ATP synthase atp 1, atp 4, atp 6, atp 8, atp 9(2) Cytohrome c biogenesis ccm B, ccm C, ccm Fc*, ccm Fn Ubichinol cytochrome c reductase cob Cytochrome c oxidase cox 1, cox 2**, cox 3 Maturases # mat R Transport membrance protein mtt B NADH dehydrogenase nad 1****, nad 2****, nad 3, nad 4,*** nad 4L(2), nad 5****, nad 6, nad 7,**** nad 9 Ribosomal proteins (LSU) rpl 10, rpl 16, rpl 2*, rpl 5 Ribosomal proteins (SSU) rps 1, rps 10*, rps 11, rps 12, rps 13, rps 14, rps 19(2), rps 2, rps 3*, rps 4, rps 7 Succinate dehydrogenase sdh 3, sdh 4, Ribosomal RNAs rrn 18, rrn 26, rrn 5 Transfer RNAs trn A-TGC*(2), trn C-GCA, trn D-GTC, trn E-TTC(2), trn F-GAA(2), trn G-GCC(2), trn G-GCC*, trn H-GTG(2), trn I-GAT*(2), trn K-TTT, trn L-CAA(4), trn M-CAT(10), trn N-GTT(4), trn P-TGG(2), trn Q-CTG*, trn Q-TTG, trn R-ACG, trn R-TCT, trn R-TCT*(2), trn S-GCT, trn S-GGA, trn S-TGA, trn T-TGT, trn T-TGT*(3), trn V-GAC(2), trn W-CCA, trn Y-GTA(3) Note: Numbers after gene names are the number of copies. Genes preceded by the # symbol represent pseudogenes. The number of * symbols after a gene indicates the number of introns it contains. 3.2. Different configurations of the S. anlungensis mitochondrial genome In the mitochondrial genome of S. anlungensis , three pairs of direct repeat sequences mediate high-frequency recombination, designated as LR12, LR14, and LR15, indicated by yellow rectangles in Fig. 1 . Among these, LR14 mediates recombination between contighLR1 and contighLR2 across domains (Fig. 1 ). For LR14, the sequence configuration in the assembled mitochondrial genome is contig9→LR14→contig8 and contig7→LR14→contig10 (42%). After recombination, the sequence configuration changes to contig9→LR14→contig10 and contig7→LR14→contig8 (58%). Both LR12 and LR15 repeat pairs are located in distinct segments of contighLR1. In the assembled mitochondrial genome, their configurations are contig5→LR12→contig4 and contig11→LR12→contig1 (50%), and contig6→LR15→contig13 and contig8→LR15→contig5 (45%), respectively. Post-recombination, the configurations shift to contig5→LR12→contig1 and contig11→LR12→contig1 (50%), and contig6→LR15→contig5 and contig8→LR15→contig13 (55%). Since LR12, LR14, and LR15 are all direct repeats (rather than inverted repeats), their mediated recombination processes only generate sequence replacement rearrangements without causing DNA segment inversions. Additionally, the mitochondrial genome of S. anlungensis contains four branch points not overlapping repeat regions: specifically, the 5' ends of contig11 and contig6, and the 3' end of contig10 can simultaneously connect to two contigs. The 3' end of contig2 can linearly link to the 5' end of contig3 or form a closed structure by connecting to its own 5' end. The above hypothesis is corroborated by the coverage validation map aligned with long-read assembly results, confirming the existence of potential substructures in different configurations of the S. anlungensis mitochondrial genome (Fig. S2 ). 3.3. Analysis of repeat sequences The results revealed three types of repetitive sequences in the mitochondrial genome of S. anlungensis : simple sequence repeats (SSRs), tandem repeats, and dispersed repeats. Among the 703 SSRs identified, 629 were located on Chr 1 and 74 on Chr 2. Specifically, mono-nucleotide SSRs (mono-SSRs) were detected in Chr1 (142) and Chr2 (19), di-SSRs (116 and 19), tri-SSRs (78 and 9), tetra-SSRs (252 and 22), penta-SSRs (32 and 4), and hexa-SSRs (9 in Chr1 and 1 in Chr2), highlighting the rarity of hexa-SSRs (Fig. 3 ; Table S1 A). Although tetra-SSRs were the most abundant in both Chr1 and Chr2, their distribution patterns differed: in Chr1, the total number of tetra-SSRs (252) was nearly equal to the combined count of mono- and di-SSRs (258), whereas in Chr2, tetra-SSRs (22) slightly outnumbered mono-SSRs (19) by only three. A total of 204 tandem repeats were identified in the mitochondrial genome, with lengths varying significantly. The longest tandem repeat (133 bp, copy number = 1.9) was located in Chr1, while the shortest (34 bp, copy number = 17) was found in Chr2 (Table S1 B). Additionally, 1,993 pairs of dispersed repeats were identified, spanning a cumulative length of 241,810 bp (10.19% of the total genome length). Many repeats spanned Chr1 and Chr2, including 161 sequences copied from Chr1 to Chr2 and 28 from Chr2 to Chr1 (Table S1 C). Notably, three highly conserved dispersed repeat sequences (length > 1,000 bp, similarity = 99.96%), all classified as direct repeats, were implicated in mitochondrial genome recombination. Two of these repeats were intrachromosomal (Chr1), while the third functioned as an interchromosomal repeat element spanning homologous regions between Chr1 and Chr2. 3.4. Prediction of RNA editing sites This study predicted RNA editing sites in 40 protein-coding genes (PCGs) of the mitochondrial genome of S. anlungensis , identifying 755 editing sites. These editing events triggered 17 types of amino acid substitutions: H(His)→Y(Tyr), R(Arg)→C(Cys), T(Thr)→I(Ile), T(Thr)→M(Met), R(Arg)→W(Trp), S(Ser)→L(Leu), S(Ser)→F(Phe), P(Pro)→S(Ser), P(Pro)→L(Leu), P(Pro)→F(Phe), L(Leu)→F(Phe), A(Ala)→V(Val), Q(Gln)→X, and R(Arg)→X (“X” represents stop codons). Among these, S→L substitutions were the most frequent (168 sites, 22.25%), while T→M substitutions were the least common (10 sites, 1.32%) (Fig. 4A). Analysis of amino acid physicochemical properties revealed that 357 sites (47.28%) caused shifts from hydrophilic to hydrophobic residues, 69 sites (9.14%) exhibited hydrophobic-to-hydrophilic polarity changes, and 235 sites (31.13%) showed no alteration in hydrophobicity (Table S2 A; Fig. 4B). Among the five stop codon formation events, two types were observed: CGA (R) → TGA (X) and CAA (Q) → TAA (X). The CGA→TGA changes occurred in the final codons of ccm Fc and rps10, while the CAA→TAA substitutions were detected in atp 6 and rps 11, with an additional instance in the 13th codon of rpl16, resulting in premature termination of mRNA translation (Table S2 B; Fig. 4C). At the gene expression level, nad 4 exhibited the highest RNA editing frequency (65 events), followed by nad 5 (45 events) and ccm Fn (44 events). In contrast, ribosomal protein genes rps 1, rps 11, and rps 7 showed significantly reduced editing activity, each containing only three editing sites. 3.5 PCGs codon usage analysis The results showed that the mitochondrial genome contains 10,804 amino acid-encoding codons, covering all 20 amino acid types and corresponding to 64 codon variants (Fig. 5 ; Table S3 ). The most frequent codon was AUU (354 occurrences, 3.27%). Among the 20 amino acids, Ser (serine) exhibited the highest codon usage (1,066 codons, 9.87%), followed by Leu (leucine) with 1,014 codons (9.19%). Ter (stop codons) had the lowest count, with only 40 codons (1.42%). All PCGs used ATG (or ACG) as start codons, while stop codons included TAA, TAG, and TGA. Notably, only TAA had a relative synonymous codon usage (RSCU) value greater than 1. Amino acid usage was dominated by Arg (arginine), Leu, and Ser, while Met (methionine) and Trp (tryptophan) showed relatively low frequencies. RSCU analysis of the 64 codons in the mitochondrial PCGs revealed 29 codons with underrepresentation (RSCU 1). Among codons with RSCU > 1, Ala (GCU) and His (CAU) displayed the highest RSCU values (1.6505 and 1.6071, respectively). Conversely, Phe (UUU), Thr (ACC), Ter (UGA), Ala (GCA), Ser (UCC), Val (GUG), and Ser (AGU) showed weak codon bias (RSCU < 1.1). Met (AUG) and Trp (UGG), each encoded by a single codon, had RSCU values of 1. Notably, 93.103% of high-frequency codons ended with A or T, while only 6.897% terminated with C or G, indicating a strong preference for NNA/NNU codon endings in the mitochondrial genome of S. anlungensis . 3.6 Mitochondrial plastid DNAs (MTPTs) in the mitochondrial genome In plant cells, gene fragment transfer between mitochondria and chloroplasts is common. Comparative analysis of the genomes of these two organelles revealed 62 homologous fragments between the mitochondrial and chloroplast genomes of S. anlungensis , including 19 fragments exceeding 1,000 bp in length (Fig. 6 ; Table S4 ). Among these, MTPT14-61 are located on Chr1, while MTPT1-13 are on Chr2. These fragments span a total length of 679,00 bp, accounting for approximately 2.8639% of the mitochondrial genome. The longest fragments, MTPT1 and MTPT14 (7,937 bp each), are located at positions 188,069–195,997 bp on Chr2 and 1,379,727–1,387,655 bp on Chr1, respectively. Both sequences correspond to positions 138,772–146,708 bp in the chloroplast DNA. In contrast, MTPT12 is the shortest fragment (32 bp), located at 116,329–116,360 bp on Chr2. Annotation results indicate that these fragments originate from chloroplast protein-coding genes, rRNA genes, tRNA genes, and intergenic regions. However, all chloroplast-derived protein-coding genes inserted into the mitochondrial genome underwent pseudogenization or loss. Among rRNA genes, only partial fragments of rrn 26 (partial: 2.58%) and rrn 18 (partial: 38.97%) were identified in MTPT10 and MTPT40, respectively. A total of 18 tRNA genes were distributed across 20 homologous sequences: trn A-TGC, trn I-GAT, trn V-GAC, trn L-CAA, trn M-CAT, trn H-GTG, trn T-TGT, trn S-GGA, trn R-ACG, trn N-GTT, trn F-GAA, trn R-TCT, trn G-GCC, trn E-TTC, trn Y-GTA, trn D-GTC, trn W-CCA, and trn P-TGG. Some tRNA genes were present in multiple MTPTs, including trn A-TGC, trn I-GAT, trn V-GAC, trn L-CAA, trn M-CAT, and trn N-GTT. 3.7. Analysis of Pi (nucleotide diversity) and Ka/Ks (non-synonymous substitutions/ synonymous substitutions) To investigate the evolutionary rates of mitochondrial genes between S. anlungensis and related species, we calculated nucleotide diversity (Pi) values for 43 mitochondrial genes across four Lauraceae species, including S. anlungensis (Table S5 ). The results revealed high Pi values for rps 109 (0.02709), sdh 3 (0.02626), nad 6 (0.0235), and atp 6 (0.01852), suggesting elevated genetic variability in these regions. Although rrn26 exhibited a relatively low Pi value (0.00808), its long sequence length (3,777 bp) resulted in the highest number of mutation sites (57) among all mitochondrial genes. Notably, rrn 5 showed no detectable mutations (Pi = 0), reflecting its extreme conservation. To further explore the influence of environmental pressures on mitochondrial PCG mutations in these species, we performed Ka/Ks analysis and identified 38 genes with valid Ka/Ks values. Comparative results indicated that most genes had Ka/Ks ratios 1), including ccmFc , cob , rpl 16, and rpl 2. Notably, ccm Fc ( S. anlungensis vs. C. henryi (NC_088584): 2.98482) and rpl16 (S. anlungensis vs. C. chekiangense (NC_082065): 2.76801) displayed particularly high Ka/Ks ratios, suggesting these genes may have played pivotal roles in the adaptive evolution of S. anlungensis . 3.8. Phylogenetic analysis The phylogenetic tree constructed based on mitochondrial genome CDS (Fig. 8 ) demonstrated that most nodes exhibited bootstrap values (BS) exceeding 80%, indicating high reliability of the phylogenetic relationships revealed by the coding sequences of 24 conserved mitochondrial PCGs. S. anlungensis clustered closely with three other Lauraceae species within the Lauraceae Clade (BS = 100). Additionally, Lauraceae plants formed the Magnoliids Clade (BS = 100) alongside magnoliid species including Magnolia biondii , Chimonanthus praecox , and Hernandia nymphaeifolia , with Hernandia nymphaeifolia showing the closest phylogenetic relationship to Lauraceae species (BS = 100). The phylogenetic tree based on chloroplast PCGs (Fig. 9 ) revealed that species from the genera Beilschmiedia , Sinopora , and Syndiclis separated from Machilus rehderi and formed an independent branch, which received strong support (BS = 96). Within this grouping, S. anlungensis clustered in the same branch as other Syndiclis species and Sinopora hongkongensis (BS = 100), exhibiting a closer phylogenetic relationship to S. marlipoensis . 4.Discussion The mitochondrial genomes of plants undergo frequent recombination during evolution, resulting in their diverse and complex structures [ 45 – 48 ]. As one of the most primitive extant angiosperm groups, studies on the mitochondrial genomes of Lauraceae species provide critical insights into the recombination mechanisms of early angiosperm mitochondria [ 49 ]. Previous reports on the mitochondrial genome structures of Lauraceae plants have identified two main types: the typical circular structures observed in C. chekiangense and C. henryi , and the repeat-mediated branched structures found in C. camphora [ 50 – 52 ]. In the mitochondrial genome of S. anlungensis , three out of its seven branch points were mediated by direct repeat sequences, generating diverse DNA substructures. However, the remaining four branch points were located outside repeat regions, leading to irregular configurations that could not be directly assembled into circular molecules. This result parallels the mitochondrial genome assembly of Abelmoschus esculentus [ 53 ]. This phenomenon may arise because during evolution, when specific mitochondrial DNA in S. anlungensis incurred damage or breaks, repair pathways such as non-homologous end joining (NHEJ) or microhomology-mediated repair (MHMR) formed contig connections distinct from native mitochondrial DNA [ 54 ]. These repaired sequences, like native mitochondrial DNA, underwent replication and accumulation within mitochondria. In terms of genome-scale dynamic evolution, the mitochondrial genome of S. anlungensis exhibits significant expansion. Its DNA length reaches 2,370,899 bp, far exceeding those of three other Lauraceae species: C. camphora (900,894 bp), C. chekiangense (750,457 bp), and C. henryi (1,168,029 bp) [ 50 – 52 ]. Repeat sequence analysis suggests that this marked genome size disparity may be closely linked to the expansion of dispersed repeats (including tandem repeats). For instance, C. camphora ’s mitochondrial genome contains 1,421 pairs of dispersed repeats (including tandem repeats), totaling 63,004 bp. In contrast, S. anlungensis harbors 2,197 pairs of dispersed repeats (including tandem repeats) spanning 246,080 bp—nearly fourfold greater in total length. Although the difference in identifiable repeat sequence length accounts for only a minor proportion (~ 14.4%) of the total genome size disparity, the pronounced expansion of dispersed repeats in S. anlungensis implies that frequently recombined and mutated sequences escaping standard repeat detection may play a dominant role in genome expansion [ 55 ]. Notably, the difference in SSR counts between S. anlungensis and C. camphor a (703 vs. 697) is insignificant, suggesting minimal contribution of SSRs to overall genome size expansion. In addition to DNA length variation, the expansion of repetitive sequences also drives interspecific differentiation in mitochondrial gene copy numbers among Lauraceae plants. Specifically, the mitochondrial PCGs of C. camphora contain a dual-copy gene nad 6 and a multi-copy gene sdh 3, with copy number variations detected in two tRNA genes: trn E-TTC (2 copies) and trn P-TGG (3 copies). In C. chekiangense , only atp 6 exhibits dual-copy status, while the tRNA genes trn M-CAT and trn P-TGG both show 3 copies. No mitochondrial PCG or tRNA gene copy phenomena were observed in C. henryi [ 50 – 52 ]. In this study, S. anlungensis displayed PCG duplication events involving dual-copy core genes ( nad 4L, atp 9) and a dual-copy variable gene ( rps 19). Its tRNA genes exhibited an extreme amplification pattern: 14 tRNA genes (e.g., trn A-TGC, trn E-TTC, trn F-GAA, trn M-CAT, and trn Y-GTA) showed copy number variations, with trn M-CAT reaching up to 10 copies. These copy number differences may reflect molecular strategies through which distinct Lauraceae species adapt to specialized ecological niches via gene dosage effects during evolution. However, the specific driving mechanisms require further validation through population genetics and epigenetic regulation studies [ 56 ]. Notably, the mat R gene shows pseudogenization in C. camphora , C. chekiangense , and S. anlungensis , while remaining intact in C. henryi . Given the critical role of mat R-encoded maturase-related proteins in mitochondrial biosynthesis [ 57 ], its functional loss may be closely associated with functional substitution or regulatory network restructuring during mitochondrial genome evolution in Cinnamomum and Syndiclis species. RNA editing typically converts specific cytidines (C) in the primary transcripts of mitochondrial PCGs into uridines (U) in mature mRNAs, creating discrepancies between the genomic sequence and the mature RNA. Predictions of RNA editing sites in the mitochondrial PCGs of S. anlungensis revealed that RNA editing corrects non-initiator ACG codons at the start positions of certain mitochondrial PCGs ( nad 1, nad 4L, rps 10, and cox 1) into AUG translation initiation codons. This process ensures proper mRNA translation of these genes [ 58 – 59 ]. Additionally, RNA editing often alters the hydrophilic-hydrophobic properties of encoded amino acids. Studies have confirmed that this regulatory mechanism is not only critical for maintaining the stability of protein secondary structures and functions but also enhances plant adaptability to abiotic stresses such as drought [ 60 – 62 ]. This study identified 357 RNA editing sites (47.28%) in the mitochondrial PCGs of S. anlungensis that shift amino acid residues from hydrophilic to hydrophobic, and 69 sites (9.14%) that reverse this trend. Given the species-specific nature of mitochondrial RNA editing sites, these results provide crucial molecular data for investigating the functional roles and adaptive evolution of RNA editing in S. anlungensis and the genus Syndiclis . Furthermore, RNA editing in certain mitochondrial PCGs, such as atp 9, is essential for normal pollen development. In tobacco and rice, failure to edit codons at positions 28, 45, 64, and 71(CTT (L) → TTT (F), TCA (S) → TTA (L), CCA (P) → CTA (L), TCA (S) → TTA (L)) of this gene leads to pollen abortion [ 63 ]. In S. anlungensis , the codon types at these RNA editing sites in the atp 9 gene align completely with those in the aforementioned species. This finding may offer a theoretical foundation for developing male sterility hybridization techniques in this species. The study of codon usage bias represents a critical topic in molecular biology and genomics, with its significance spanning multiple aspects, including gene expression regulation and evolutionary mechanism elucidation. This research identified 33 high-frequency codons (RSCU > 1) in the mitochondrial PCGs of S. anlungensis , revealing that over 90% of these codons terminate with A/T. Their types align closely with previous reports on mitochondrial genomes in angiosperms, suggesting the evolutionary conservation of this genetic feature in plants [ 45 – 48 , 50 – 52 ]. Earlier studies proposed that these highly used codons typically match abundant intracellular tRNAs, with their biological significance lying in enhancing translation rates, reducing ribosome stalling, and optimizing protein synthesis efficiency [ 64 ]. Notably, certain low-frequency codons (e.g., GGC, GAC, GCG, and CAG) distributed across gene sequences may also play regulatory roles in mRNA translation. This is attributed to their ability to slow elongation speed, thereby decreasing ribosome congestion at the 3’ end, promoting uniform ribosome distribution along mRNA strands, and preventing translation interruptions caused by spontaneous collisions or stalling [ 65 ]. Frequent horizontal gene transfer events occur between chloroplast DNA and mitochondrial DNA. Studies suggest that the integration of chloroplast DNA fragments confers evolutionary advantages: early plant mitochondrial genomes, lacking a complete tRNA gene system, required continuous integration of chloroplast DNA fragments to acquire functional tRNAs, thereby partially refining their gene expression mechanisms [ 66 – 67 ]. This evolutionary process dates back at least to the common ancestor of extant gymnosperms and angiosperms (~ 300 million years ago, 300 Mya). Notably, the trn V(UAC)- trn M(CAU)- atp E- atp B- rbc L gene cluster—the oldest mitochondrial DNA-integrated fragment—exhibits striking distribution differences between the two organellar DNAs in S. anlungensis : the corresponding chloroplast DNA region (56,815–62,093 bp) displays high continuity, containing only two small spacer regions (107 bp between trn M-atpE and 11 bp between atp B- rbc L). In contrast, the mitochondrial DNA homologous fragments are structurally rearranged and distributed across three discrete regions: trn M-CAT (513,460–513,382 bp), atp E/ atp B (1,280,675-1,282,768 bp), and rbc L (796,948–793,978 bp) [ 18 ]. This spatial heterogeneity vividly illustrates the dynamic evolutionary features of mitochondrial genomes and further supports the hypothesis proposed by Richly and Leister (2004): “Primary insertions of organellar DNAs are large and then diverge and fragment over evolutionary time” [ 68 ]. The Ka/Ks analysis serves as a pivotal tool for deciphering the evolutionary drivers of genes. By quantifying the ratio of nonsynonymous substitution rates (Ka) to synonymous substitution rates (Ks), it precisely distinguishes the effects of purifying selection, neutral evolution, and positive selection on the accumulation of genetic mutations [ 69 ]. In this study, Ka/Ks analysis revealed that mitochondrial protein-coding genes (PCGs) in Lauraceae plants are predominantly under strong purifying selection, indicating functional conservation, consistent with previous studies in angiosperms. Notably, specific genes such as ccm Fc, cob , and rpl 2 exhibited significant positive selection signals (Ka/Ks > 1), with nonsynonymous substitution rates markedly exceeding theoretical expectations under neutral evolutionary models. This evolutionary pattern suggests that these genes may have undergone adaptive evolution during phylogeny, potentially linked to critical biological processes such as ecological niche adaptation strategies, mitochondrial energy metabolism network restructuring, or reproductive system specialization [ 8 ]. To further elucidate their evolutionary drivers, future studies should integrate functional genomics experiments and cross-species comparative genomics analyses to explore the functional relevance of these genes and their biological effects in species-specific adaptive evolution. Consistent with the APG classification system, the phylogenetic tree constructed based on mitochondrial CDS strongly supports the division between magnoliids, eudicots, and monocots, while molecular biological evidence confirms the close phylogenetic relationship between S. anlungensis and three Lauraceae species: C. camphora , C. chekiangense , and C. henryi . Furthermore, aligning with previous studies, Cinnamomum and Caryodaphnopsis species within Lauraceae exhibit closer phylogenetic affinities. S. anlungensis occupies the basal position of this clade, likely because it belongs to the Cryptocaryeae lineage - the earliest diverged evolutionary branch in Lauraceae, independent from the lineages containing Cinnamomum and Caryodaphnopsis [ 70 ]. Phylogenetic analysis based on chloroplast PCGs supports the distinct status of the genus Syndiclis within Lauraceae, revealing S. anlungensis as most closely related to S. marlipoensis within this genus. Notably, previous morphological studies proposing the merger of Sinopora with Syndiclis (based on semicircular stomata, fine leaf venation without free vein endings, small terminal buds, and large globose fruits) gain new molecular support from this study [ 71 – 73 ]. Our phylogenetic results demonstrate that S. hongkongensis of Sinopora is nested within the Syndiclis clade and forms a sister relationship with S. chinensis of Syndiclis , providing fresh evidence for this taxonomic hypothesis. 5. Conclusion This study presents the first complete mitochondrial genome of critically endangered S. anlungensis , revealing a dynamic 2,370,899 bp structure shaped by recombination via three direct repeat pairs (R12/R14/R15). It comprises 26 core and 18 variable protein-coding genes (PCGs), 55 tRNAs, and abundant repeats (703 SSRs, 204 tandem, 1,993 dispersed). RNA editing altered 47.28% of 755 sites to hydrophobic amino acids, potentially enhancing protein stability. Positive selection (Ka/Ks) in ccm Fc and rpl 16 suggests adaptive evolution. Mitochondrial-chloroplast genome comparisons identified 62 homologous fragments (67,900 bp). Phylogenetically, mitochondrial CDS places S. anlungensis basally within Lauraceae, while chloroplast PCGs indicate closest affinity to S. marlipoensis . These findings elucidate recombination mechanisms, repeat expansion, and adaptive traits, offering critical insights for conservation strategies, population restoration, and advancing Lauraceae mitochondrial evolution studies. Abbreviations PCGs Protein-coding genes mtDNA Mitochondrial genome cpDNA Chloroplast genome Ka/Ks Non-synonymous/synonymous mutation ratio RSCU Relative synonymous codon usage MTPT Mitochondrial plastid DNA sequence tRNA Transfer RNA rRNA Ribosomal RNA SSR simple sequence repeat Pi nucleotide diversity BS bootstrap support value PP posterior probabilities. Declarations Declaration of competing interest The authors declare that they have no competing or conflicting interests. Ethics approval and consent to participate All materials used in this study comply with international and national legal standards. The collected species material does not pose a threat to other species, and the collection of the species is recognized by the relevant authorities. Consent for publication Not applicable. Competing interests All authors declare that they have no competing interests. Funding This research was supported by the Guizhou Provincial Science and Technology Program Project(Qiankehejichu-ZK[2022] General 240), Guizhou Provincial Forestry Research Project (QianlinKehe-J[2019] 11), and the National Natural Science Foundation of China (Grant No. 32400179). Author Contribution L.H and X.X: Conceptualization, L.H and D.Z.J: Writing - original draft, Data curation, Formal analysis, Software. L.H&Z.L: Funding acquisition, Resources, Review & editing, Investigation. Y.B.Y: Investigation, Methodology. R.C: Supervision, Visualization. Z.L: Methodology, Validation. Acknowledgement We thank the editors and the anonymous reviewers for their insightful comments and suggestions on the manuscript. 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Sangaré A, Weil JH, Grienenberger JM, Fauron C, Lonsdale D. Localization and organization of tRNA genes on the mitochondrial genomes of fertile and male sterile lines of maize. Mol Gen Genet. 1990;223(2):224–32. https://doi.org/10.1007/BF00265058 . Richly E, Leister D. NUMTs in sequenced eukaryotic genomes. Mol Biol Evol. 2004;21(6):1081–4. https://doi.org/10.1093/molbev/msh110 . Zhang Z, Li J, Zhao XQ, Wang J, Wong GK, Yu J. KaKs_Calculator: calculating Ka and Ks through model selection and model averaging. Genomics Proteom Bioinf. 2006;4(4):259–63. https://doi.org/10.1016/S1672-0229(07)60007-2 . Song Y, Yu QF, Zhang D, Chen LG, Tan YH, Zhu W, Su HL, Yao X, Liu C, Corlett RT. New insights into the phylogenetic relationships within the Lauraceae from mitogenomes. BMC Biol. 2024;22(1):241. https://doi.org/10.1186/s12915-024-02040-7 . Li H, Liu B, Davis CC, Yang Y. Plastome phylogenomics, systematics, and divergence time estimation of the Beilschmiedia group (Lauraceae). Mol Phylogenet Evol. 2020;151:106901. https://doi.org/10.1016/j.ympev.2020.106901 . Yang Y, Zhang L. Venation pattern of Syndiclis Hook.f. and its related genera. J Trop Subtrop Bot. 2010;18(6):643–9. https://doi.org/10.3969/j.issn.1005-3395.2010.06.008 . Yang Y, Zhang L, Liu B, van der Werff H. Leaf cuticular anatomy and taxonomy of Syndiclis (Lauraceae) and its allies. Syst Bot. 2012;37(4):861–78. https://doi.org/10.1600/036364412X656518 . Additional Declarations No competing interests reported. Supplementary Files TableS1.RepeatsofthemitochondrialgenomeinSyndiclisanlungensis.xlsx TableS2.PredictionofRNAeditingsitesintheSyndiclisanlungensismitochondrialgenome.xlsx TableS3.Codonpreferenceinformation.xlsx TableS4.ListforthechloroplasttomitochondrialsequencestransferofSyndiclisanlungensis.xlsx TableS5.NucleotidediversitymitochondrialgenomesofS.anlungensisC.henryiC.camphoraandC.chekiangense.xlsx TableS6.KaKsratiosofconservedPCGsinthemitochondrialgenomesofS.anlungensisC.henryiC.camphoraandC.chekiangense.xlsx FigS1.GelsandBlotsimagesofsequencingresults.pdf Fig.S2Thecoveragevalidationmapalignedtotheassemblyresultsoflongreadsspecificallyfilteringsequenceswithoverlapsgreaterthan5k..pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6835863","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":478447917,"identity":"66f29794-033e-430d-b886-47f3cdb25d37","order_by":0,"name":"Lang Huang","email":"","orcid":"","institution":"Guizhou Academy of Forestry","correspondingAuthor":false,"prefix":"","firstName":"Lang","middleName":"","lastName":"Huang","suffix":""},{"id":478447919,"identity":"3098200b-68ed-4b5a-a9f4-51d526cf5dd4","order_by":1,"name":"Dongzhen Jiang","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Dongzhen","middleName":"","lastName":"Jiang","suffix":""},{"id":478447920,"identity":"489c7900-fe40-42c0-a1f6-dccceb871f04","order_by":2,"name":"Yanbing Yang","email":"","orcid":"","institution":"Guizhou Academy of Forestry","correspondingAuthor":false,"prefix":"","firstName":"Yanbing","middleName":"","lastName":"Yang","suffix":""},{"id":478447922,"identity":"19abbd79-08dd-4828-a404-407801a9d45c","order_by":3,"name":"Rui Chen","email":"","orcid":"","institution":"Guizhou Academy of Forestry","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Chen","suffix":""},{"id":478447925,"identity":"1b997c79-e801-4dae-a3f7-e4187a4f1152","order_by":4,"name":"Zhi Li","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Zhi","middleName":"","lastName":"Li","suffix":""},{"id":478447926,"identity":"84c9eb94-9b11-4c81-a3d4-5009a90f894a","order_by":5,"name":"Lei Zhou","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Zhou","suffix":""},{"id":478447928,"identity":"4c0e7827-d66a-4e1e-9a99-cb2f16244577","order_by":6,"name":"Xu Xiao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIiWNgGAWjYDACZjApIcfP3thw4IOBjR2xWiyMJXsOHzw4oyAtmVi7KhI33EhLPszz4RBjAyG1BseZHz7mbZNI3HDmjMFhG4MDzAzsh49uwKdFspnN2HBmm4TxzOM9BodzDO7wMfCkpd3Ap4WfmcFM4mObhGwfyJYcg2fMDBI8Zni1sDGzf5NIbJNgbLiRY3DYwuAwYwMhLfzMPGBbFCfcSEs4zECMFslmnmLDGeckQIF84GCPQVoyGyG/GJw/vvExT1kdKCqbP/z4Y2PHz374GF4tWHxHmvJRMApGwSgYBdgAAPGfTcjS+zMOAAAAAElFTkSuQmCC","orcid":"","institution":"Guizhou University","correspondingAuthor":true,"prefix":"","firstName":"Xu","middleName":"","lastName":"Xiao","suffix":""}],"badges":[],"createdAt":"2025-06-06 09:53:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6835863/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6835863/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85808619,"identity":"87384ecc-50b5-42b1-868a-8dedc97306b5","added_by":"auto","created_at":"2025-07-02 03:04:55","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":7622548,"visible":true,"origin":"","legend":"\u003cp\u003eAssembly graph of the \u003cem\u003eS. anlungensis\u003c/em\u003e \u003cem\u003emitochondrial genome\u003c/em\u003e. Segments are designated as C (contig) /R (LR)1–15 based on size ranking. Among these, only segments 12, 14, and 15 are identified as repeat regions. Long-read sequencing data confirmed all segment connections, revealing a branched genomic architecture. The possible structures formed by high frequency rearrangements mediated by three long repeats were drew\u003c/p\u003e","description":"","filename":"Fig.1.AssemblygraphoftheSyndiclisanlungensismitogenome.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6835863/v1/399084822a31a5fe7e7994a4.jpg"},{"id":85808620,"identity":"177a2977-7d4b-4ef8-9081-53e173614a6a","added_by":"auto","created_at":"2025-07-02 03:04:55","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2303662,"visible":true,"origin":"","legend":"\u003cp\u003eCircular map of the mitochondrial genome of \u003cem\u003eS. anlungensis.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.2.CircularmapofthemitochondrialgenomeofSyndiclisanlun.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6835863/v1/db3433ce86f2c3a4f3ece35f.jpg"},{"id":85808675,"identity":"c8b2dab0-c98e-495a-80fb-9730446074ce","added_by":"auto","created_at":"2025-07-02 03:04:56","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":10113959,"visible":true,"origin":"","legend":"\u003cp\u003eRepeat analysis of the mitochondrial genome in \u003cem\u003eS. anlungensis\u003c/em\u003e. The arc represents Chr1 (turquoise) and Chr2 (purple). The ticks inner circles are SSR (Blue) and tandem repeat (red). The turquoise ribbons represents dispersed repeat\u003c/p\u003e","description":"","filename":"Fig.3.RepeatanalysisofthemitochondrialgenomeinSyndiclisanlungensis.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6835863/v1/cbb55bb94f6f87832aaadf54.jpg"},{"id":85808622,"identity":"df6adf1f-0b32-42b5-97a4-2e6ec3650d75","added_by":"auto","created_at":"2025-07-02 03:04:55","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":652650,"visible":true,"origin":"","legend":"\u003cp\u003ePrediction of RNA editing sites in the \u003cem\u003eS. anlungensis\u003c/em\u003e mitochondrial genome. (A: Characterization of RNA-editing sites; B: Proportion of different RNA-editing types; C: Numbers of RNA-editing sites in the mtDNA)\u003c/p\u003e","description":"","filename":"Fig.4.PredictionofRNAeditingsitesintheSyndiclisanlungensismitochondrialgenome.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6835863/v1/44a4de6dd93e29bfe964bcad.jpg"},{"id":85808629,"identity":"5aaa2b4b-0664-4e3e-ac74-4fd8d76bc6ca","added_by":"auto","created_at":"2025-07-02 03:04:55","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":769554,"visible":true,"origin":"","legend":"\u003cp\u003eRelative synonymous codon usage in the \u003cem\u003eS. anlungensis\u003c/em\u003e mitochondrial genome.\u003c/p\u003e","description":"","filename":"Fig.5.RelativesynonymouscodonusageintheSyndiclisanlungensismitochondrialgenome.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6835863/v1/ba34b9ed52725bd1fbd09024.jpg"},{"id":85808678,"identity":"a2b59a19-ff42-4f20-b100-0870f2662970","added_by":"auto","created_at":"2025-07-02 03:04:56","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2944021,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic for the chloroplast-to-mitochondrial sequence transfer of \u003cem\u003eS. anlungensis\u003c/em\u003e. The turquoise arc represents Chr1. The blue arc represents Chr2. The green arc represents chloroplast DNA. The homologous fragments are indicated by the connecting ribbons between the turquoise (blue) and green arcs.\u003c/p\u003e","description":"","filename":"Fig.7.SchematicforthechloroplasttomitochondrialsequencetransferofSyndiclisanlungensis.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6835863/v1/ea552a79a3596df52b98c88a.jpg"},{"id":85809238,"identity":"3a1e6ff8-80c1-459e-83a8-759f691d8d51","added_by":"auto","created_at":"2025-07-02 03:12:55","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":340226,"visible":true,"origin":"","legend":"\u003cp\u003eKa/Ks ratios of 38 PCGs in the \u003cem\u003emitochondrial genome\u003c/em\u003eof \u003cem\u003eS. anlungensis\u003c/em\u003e, \u003cem\u003eC. henryi\u003c/em\u003e, \u003cem\u003eC. camphora\u003c/em\u003e, and \u003cem\u003eC. chekiangense\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Fig.6KaKsratiosof38PCGsinthemitogenomeofS.anlungensisC.henryiC.camphoraandC.chekiangense.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6835863/v1/abe535b8e18ce3dd44f87275.jpg"},{"id":85809371,"identity":"aeb8c4b2-e3f4-4930-a960-56e552fdf0db","added_by":"auto","created_at":"2025-07-02 03:20:55","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":420211,"visible":true,"origin":"","legend":"\u003cp\u003eConstruction of a phylogenetic tree based on mitochondrial CDS\u003c/p\u003e","description":"","filename":"Fig.8ConstructionofaphylogenetictreebasedonmitochondrialCDS.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6835863/v1/6cfe54d3713171380ff5ba6f.jpg"},{"id":85808648,"identity":"8511df18-f8b9-4b60-b272-4657c4c8f273","added_by":"auto","created_at":"2025-07-02 03:04:56","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":588788,"visible":true,"origin":"","legend":"\u003cp\u003eConstruction of a phylogenetic tree based on chloroplast PCG\u003c/p\u003e","description":"","filename":"Fig.9ConstructionofaphylogenetictreebasedonchloroplastPCG.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6835863/v1/d22ba7219b761d4a201a0abe.jpg"},{"id":88392155,"identity":"14ec0d4a-4e51-4bf6-8b28-eeb97754af87","added_by":"auto","created_at":"2025-08-06 04:47:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":26959038,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6835863/v1/94c63a26-6ad1-4886-a25b-4a753ac312af.pdf"},{"id":85808643,"identity":"35b9d99e-4817-4858-87ae-791364082c06","added_by":"auto","created_at":"2025-07-02 03:04:55","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":185165,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.RepeatsofthemitochondrialgenomeinSyndiclisanlungensis.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6835863/v1/550ac255b11fb21b43e16a5b.xlsx"},{"id":85808618,"identity":"c1d226e3-2962-44d7-8b8d-ce81c1a46a80","added_by":"auto","created_at":"2025-07-02 03:04:54","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":44723,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.PredictionofRNAeditingsitesintheSyndiclisanlungensismitochondrialgenome.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6835863/v1/ccab64907f038e624bb98b84.xlsx"},{"id":85808621,"identity":"e8a0509e-c691-4d09-987f-ecf00abf2059","added_by":"auto","created_at":"2025-07-02 03:04:55","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":12271,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.Codonpreferenceinformation.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6835863/v1/abf698ab473901cadbe680b1.xlsx"},{"id":85808628,"identity":"f91e2175-09b1-4653-9dbe-32d6a5e5f443","added_by":"auto","created_at":"2025-07-02 03:04:55","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":15412,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.ListforthechloroplasttomitochondrialsequencestransferofSyndiclisanlungensis.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6835863/v1/4f753e17480d8a4f9d0daa37.xlsx"},{"id":85808644,"identity":"23738ff7-335e-4428-b643-2c63840cfb00","added_by":"auto","created_at":"2025-07-02 03:04:55","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":11681,"visible":true,"origin":"","legend":"","description":"","filename":"TableS5.NucleotidediversitymitochondrialgenomesofS.anlungensisC.henryiC.camphoraandC.chekiangense.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6835863/v1/353c7d5f4b23a773b14e9f9f.xlsx"},{"id":85808666,"identity":"4ea9d3e8-12a8-4dac-b45e-8a484995df06","added_by":"auto","created_at":"2025-07-02 03:04:56","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":13595,"visible":true,"origin":"","legend":"","description":"","filename":"TableS6.KaKsratiosofconservedPCGsinthemitochondrialgenomesofS.anlungensisC.henryiC.camphoraandC.chekiangense.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6835863/v1/676a8c3f84d1ede7b78868a7.xlsx"},{"id":85808661,"identity":"02ed9212-7714-4538-858e-743af0ddb544","added_by":"auto","created_at":"2025-07-02 03:04:56","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":524026,"visible":true,"origin":"","legend":"","description":"","filename":"FigS1.GelsandBlotsimagesofsequencingresults.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6835863/v1/1c10fe89d3c87bf4df86a50d.pdf"},{"id":85808664,"identity":"2abdf758-cf9b-463f-ad2b-fe644714bac8","added_by":"auto","created_at":"2025-07-02 03:04:56","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":1914135,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.S2Thecoveragevalidationmapalignedtotheassemblyresultsoflongreadsspecificallyfilteringsequenceswithoverlapsgreaterthan5k..pdf","url":"https://assets-eu.researchsquare.com/files/rs-6835863/v1/5f6b8dad22994af1e55ff859.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Report on the complete mitochondrial genome of the critically endangered and endemic Lauraceae plant Syndiclis anlungensis in China","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cem\u003eSyndiclis anlungensis\u003c/em\u003e belongs to the genus \u003cem\u003eSyndiclis\u003c/em\u003e (Subtribe Beilschmiediineae, Lauraceae) and is listed as Critically Endangered (CR) in the \u003cem\u003eChina Biodiversity Red List\u0026mdash;Volume of Higher Plants (2013)\u003c/em\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Since the 1980s, the species was presumed extinct due to the absence of documented sightings during prolonged field surveys, until its rediscovery in 2015 when living wild individuals were identified [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Currently, only two mature trees with trunk diameters of 20\u0026ndash;40 cm exist globally, distributed in Wangyuan Village and Polao Village, Dushan Town, Anlong County, Guizhou Province. Studies indicate that \u003cem\u003eS. anlungensis\u003c/em\u003e faces survival challenges such as weak natural regeneration capacity and poor environmental adaptability [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The extreme rarity of the remaining population and its limited regeneration heighten the risk of severe genetic diversity loss. From an evolutionary genetic perspective, long-term isolation in small populations predisposes the species to genetic bottleneck effects, leading to the accumulation of deleterious alleles and reduced adaptive potential [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Although the rediscovery of living individuals offers hope for species survival, severely restricted gene flow may exacerbate inbreeding depression, further diminishing the species\u0026rsquo; resilience to environmental fluctuations. In this context, in-depth characterization of its genetic system\u0026mdash;particularly the evolutionary dynamics of its genome\u0026mdash;has become crucial for assessing species viability and formulating science-based conservation strategies. In genomic research, current studies on \u003cem\u003eS. anlungensis\u003c/em\u003e remain limited, with existing work focusing solely on chloroplast genome phylogeny and codon usage bias analysis, while its complete mitochondrial genome remains unexplored [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This critical gap substantially hinders investigations into the species\u0026rsquo; evolutionary adaptation mechanisms and the development of science-based conservation approaches.\u003c/p\u003e \u003cp\u003eThe mitochondrion, one of the most vital plant organelles, is fundamentally linked to respiratory metabolism and energy synthesis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. As the second-largest genome in plants, the mitochondrial genome encodes key subunits of respiratory chain complexes such as cytochrome c oxidase and NADH dehydrogenase, which are indispensable for respiration, electron transport systems, and energy conversion [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Although mitochondrial protein-coding genes (PCGs) exhibit slower evolutionary rates, their functional variations and RNA editing have been demonstrated to significantly influence plant stress resistance and reproductive adaptation [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. For \u003cem\u003eS. anlungensis\u003c/em\u003e, investigating mitochondrial genome mutations and evolution will not only clarify its evolutionary dynamics but also provide essential molecular insights for assessing its environmental stress response capabilities.\u003c/p\u003e \u003cp\u003eNotably, plant mitochondrial genomes exhibit extraordinary plasticity: frequent recombination generates diverse structural configurations, including master circular molecules, subgenomic circular forms, and multipartite architectures [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Their sizes span two orders of magnitude in angiosperms\u0026mdash;from approximately 222 Kbp in \u003cem\u003eBrassica napus\u003c/em\u003e to 11.3 Mbp in \u003cem\u003eSilene conica\u003c/em\u003e\u0026mdash;and reach up to 11.7 Mbp in the gymnosperm \u003cem\u003eLarix sibirica\u003c/em\u003e [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. During long-term evolution, mitochondria continuously integrate chloroplast-derived DNA fragments through asymmetric intracellular DNA transfer mechanisms. These integrated fragments (ranging from tens to thousands of base pairs) often retain intact chloroplast genes or gene clusters, accumulating varying degrees of recombination and mutations during mitochondrial genome evolution, thereby serving as critical repositories of genetic and evolutionary information [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Such highly dynamic genetic features endow mitochondrial genomes with rich taxonomic signals, offering unique advantages for resolving genetic and evolutionary challenges in \u003cem\u003eS. anlungensis\u003c/em\u003e and its closely related species.\u003c/p\u003e \u003cp\u003eThis study employed a combination of Illumina and Oxford Nanopore sequencing technologies to achieve the first sequencing and assembly of the mitochondrial genome of \u003cem\u003eS. anlungensis\u003c/em\u003e. Comprehensive analyses were conducted on its compositional structure, codon usage bias, repetitive sequences, RNA editing sites, and chloroplast-derived homologous sequences to characterize the genomic architecture. Furthermore, Ka/Ks analysis elucidated the evolutionary mechanisms of mitochondrial protein-coding genes (PCGs) in \u003cem\u003eS. anlungensis\u003c/em\u003e and its close relatives under environmental stress. Phylogenetic trees were reconstructed using mitochondrial and chloroplast PCG datasets from \u003cem\u003eS. anlungensis\u003c/em\u003e and other higher plants, respectively, resolving the species\u0026rsquo; phylogenetic position. These findings not only unveil the genetic characteristics and evolutionary dynamics of the mitochondrial genome in \u003cem\u003eS. anlungensis\u003c/em\u003e but also provide foundational molecular data for its genetic improvement.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Material collection, DNA extraction and sequencing\u003c/h2\u003e \u003cp\u003eThe specimen of \u003cem\u003eS. anlungensis\u003c/em\u003e was collected from its native habitat in Wangyuan Village, Dushan Town, Anlong County, Guizhou Province, China (geographic coordinates: E105\u0026deg;36\u0026prime;55.08\u0026Prime;, N25\u0026deg;15\u0026prime;51.98\u0026Prime;). The taxonomic identification was conducted by Dr. Chenghua Yang (Guizhou Academy of Forestry). The voucher specimen (collection number: GB2024132) is deposited in the Herbarium of Guizhou Academy of Forestry. Healthy, disease-free young leaves were collected from the mid-canopy of the tree. Immediately after field sampling, leaf tissues were flash-frozen in liquid nitrogen and stored long-term at \u0026minus;\u0026thinsp;80\u0026deg;C. For DNA extraction, frozen leaf tissues were pulverized using liquid nitrogen grinding, followed by a modified CTAB protocol [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] to obtain high-purity mitochondrial genomic DNA.\u003c/p\u003e \u003cp\u003eThe mitochondrial genome was sequenced using a hybrid approach combining Illumina short-read and Oxford Nanopore long-read technologies. For Illumina sequencing, DNA quality and concentration were assessed via 1% agarose gel electrophoresis (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA) and NanoDrop 2000. Qualified samples underwent ultrasonic fragmentation, followed by purification, end repair, adapter ligation, and PCR amplification to construct sequencing libraries. Libraries were sequenced on the Illumina NovaSeq 6000 platform (Illumina, San Diego, USA), generating 150 bp paired-end reads [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. After quality control with fastp (v0.23.4) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], 67,670,917 clean reads were retained, with a GC content of 40.79%, and Q20/Q30 values of 97.55% and 92.90%, respectively.\u003c/p\u003e \u003cp\u003eFor Oxford Nanopore sequencing, genomic DNA was randomly sheared, and large fragments (\u0026gt;\u0026thinsp;10 kb) were enriched using a magnetic bead-based size selection system. Target fragments were gel-purified (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB), then subjected to DNA damage repair, end-blunting, and 3\u0026prime;-dA-tailing. Libraries were prepared with the SQK-LSK109 kit and sequenced on the PromethION platform via single-molecule real-time (SMRT) technology [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Raw data were filtered using Filtlong (v0.24).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Assembly and annotation of the mitochondrial genome\u003c/h2\u003e \u003cp\u003eFirst, the raw third-generation sequencing data was aligned to the plant mitochondrial gene database using minimap2 (v2.1). Next, sequences with alignment lengths exceeding 50 bp were selected as candidate aligned sequences. Sequences containing multiple core genes and exhibiting high coverage completeness were prioritized as initial seed sequences. Subsequently, minimap2 was used to align the raw sequencing data to these seed sequences, and sequences with overlaps greater than 1 kb and similarity exceeding 70% were incorporated into the seed sequences [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The obtained third-generation data were then error-corrected using Canu (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/marbl/canu\u003c/span\u003e\u003cspan address=\"https://github.com/marbl/canu\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), a third-generation assembly tool [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Illumina second-generation sequencing data were aligned to the corrected sequences using Bowtie2 (v2.3.5.1). A preliminary assembly was performed by integrating the second-generation data and corrected third-generation data using Unicycler (v0.4.8) with default parameters [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and the assembly results were visualized and manually adjusted using Bandage (v0.8.1). Considering the unique multi-circular/non-circular structural features of mitochondrial genomes, the corrected third-generation data were realigned to the contigs generated by Unicycler. The final high-integrity mitochondrial genome assembly was achieved by manually determining branch orientations.\u003c/p\u003e \u003cp\u003eThe annotation of mitochondria is conducted using the following steps: Protein-coding genes and rRNA genes are aligned with publicly available reference plant mitochondrial sequences using the Basic Local Alignment Search Tool-Nucleotide (BLASTN, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Manual adjustments are then performed using the closely related species \u003cem\u003eC. camphora\u003c/em\u003e (GenBank ID: NC_086632) as the reference genome to ensure the accuracy and reliability of the alignment. Additionally, tRNA genes are annotated using tRNAscan-SE (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://lowelab.ucsc.edu/tRNAscan-SE/\u003c/span\u003e\u003cspan address=\"http://lowelab.ucsc.edu/tRNAscan-SE/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Open Reading Frame (ORF) annotation is carried out using the Open Reading Frame Finder (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/orffinder/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/orffinder/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], with a minimum length threshold of 102 bp to exclude redundant sequences and those overlapping with known genes. Sequences longer than 300 bp are annotated by aligning against the nr database. The mitochondrial genome map is constructed using OGDRAW (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://chlorobox.mpimp-golm.mpg.de/OGDraw.html\u003c/span\u003e\u003cspan address=\"https://chlorobox.mpimp-golm.mpg.de/OGDraw.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Repetitive sequence analysis and RNA editing site prediction\u003c/h2\u003e \u003cp\u003eThe identification of simple sequence repeats (SSRs) was performed using MISA (v1.0) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://webblast.ipk-gatersleben.de/misa/\u003c/span\u003e\u003cspan address=\"https://webblast.ipk-gatersleben.de/misa/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], while tandem repeats were detected using Tandem Repeats Finder (TRF, v4.09) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://tandem.bu.edu/trf/trf.submit.options.html\u003c/span\u003e\u003cspan address=\"http://tandem.bu.edu/trf/trf.submit.options.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Dispersed repeats were identified via BLASTN (v2.10.1; parameters: -word_size 7, -evalue 1e-5) for homology-based analysis [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Redundant sequences and previously identified tandem repeat regions were excluded during the analysis. All repeat data were visualized using Circos (v0.69-5) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRNA editing sites in Anlong camphora were predicted using the PREPACT3 online platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.prepact.de/\u003c/span\u003e\u003cspan address=\"http://www.prepact.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with a significance threshold of 0.001 [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Finally, statistical analysis and visualization (histograms and pie charts) were conducted using Microsoft Excel 2021.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Codon usage bias and chloroplast homologous sequence analysis\u003c/h2\u003e \u003cp\u003eProtein-coding sequences were extracted using Phylosuit (v1.22) with default parameters [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The relative synonymous codon usage (RSCU) of mitochondrial genome protein-coding genes was subsequently calculated using MEGA (v7.0) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Chloroplast genome sequences from the same sample were also extracted, and homologous sequences between the chloroplast and mitochondrial genomes were identified using BLAST with a similarity threshold of 70% and an E-value cutoff of 1e-5. To visually represent homologous fragments between the chloroplast and mitochondrial genomes, Circos (v0.69-5) was employed for visualization [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Caculations of Pi (nucleic acid diversity) and Ka/Ks (non-synonymous substitutions/synonymous substitutions)\u003c/h2\u003e \u003cp\u003eMitochondrial genome data for three Lauraceae species: \u003cem\u003eCaryodaphnopsis henryi\u003c/em\u003e (NC_088584), \u003cem\u003eCinnamomum camphora\u003c/em\u003e (NC_086632), and \u003cem\u003eCinnamomum chekiangense\u003c/em\u003e (NC_082065), were downloaded from the NCBI Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/genome/organelle/\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/genome/organelle/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). These mitochondrial genomes were aligned using MAFFT (v7.427) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Nucleotide diversity (Pi) values for shared genes were calculated with DnaSP (v6.12.03) [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Additionally, the aligned gene sequences were subjected to BLAST analysis using MAFFT, and the resulting BLAST output files were imported into KaKs_Calculator to estimate the nonsynonymous substitution rate (Ka), synonymous substitution rate (Ks), and the Ka/Ks ratio [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Phylogenetic Analysis\u003c/h2\u003e \u003cp\u003eTo investigate the phylogenetic relationships of \u003cem\u003eS. anlungensis\u003c/em\u003e, we conducted phylogenetic analyses of its mitochondrial and chloroplast genomes. For the mitochondrial genome, 39 complete mitochondrial genome sequences were downloaded from the NCBI database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/genome/organelle/\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/genome/organelle/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), including 10 magnoliids, 23 eudicots, 5 monocots, and 1 gymnosperm (\u003cem\u003eGinkgo biloba\u003c/em\u003e ) as the outgroup. Conserved protein-coding genes shared between these species and \u003cem\u003eS. anlungensis\u003c/em\u003e were extracted using TBtools software [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Multiple sequence alignment of coding sequences (CDS) from these 40 mitochondrial genomes was performed using MAFFT (v7.427) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The maximum likelihood phylogenetic tree was constructed with RAxML (v8.2.10) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cme.h-its.org/exelixis/software.html\u003c/span\u003e\u003cspan address=\"https://cme.h-its.org/exelixis/software.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) under the GTRGAMMA model, with the bootstrap value set to 1000 [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor chloroplast genome phylogenetic analysis, 24 chloroplast genomes of Lauraceae species (including \u003cem\u003eS. anlungensis\u003c/em\u003e ) were downloaded from NCBI, comprising 6 species from the genus \u003cem\u003eSyndiclis\u003c/em\u003e, 17 species from its closely related genera \u003cem\u003eBeilschmiedia\u003c/em\u003e and \u003cem\u003eSinopora\u003c/em\u003e, with \u003cem\u003eMachilus rehderi\u003c/em\u003e as the outgroup. The maximum likelihood tree based on chloroplast protein-coding genes (PCGs) was constructed using the same analytical methods as for the mitochondrial genome. All trees were visualized using ITOL software (v4.0) [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Assembly and annotation of the \u003cem\u003eS. anlungensis\u003c/em\u003e mitochondrial genome\u003c/h2\u003e \u003cp\u003eThis study successfully resolved the mitochondrial genome structure of \u003cem\u003eS. anlungensis\u003c/em\u003e using a hybrid assembly strategy. Results revealed that its mitochondrial DNA exhibits typical branched characteristics and can form multi-subtype dynamic structures through recombination mediated by three sets of direct repeat sequences. Through systematic analysis with Unicycler software, we constructed the main circular molecules as two linear chromosomes: Chr1 (2,135,163 bp, GC\u0026thinsp;=\u0026thinsp;46.13%) was assembled along the topological path contig2 - contig3 - contig11 - LR12 - contig1 - contig9 - contig14 - contig8 - LR15 - contig5 - LR12 - contig4 - contig6 - LR15 - contig13, while Chr2 (235,736 bp, GC\u0026thinsp;=\u0026thinsp;46.47%) adopted a linear structure of contig7 - LR14 - contig10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Genome coverage analysis demonstrated complete sequencing read coverage across all assembled regions, verifying the continuity of assembly results (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAnnotation results identified 26 core protein-coding genes in the \u003cem\u003eS. anlungensis\u003c/em\u003e mitochondrial genome, including the \u003cem\u003eatp\u003c/em\u003e9 and \u003cem\u003ena\u003c/em\u003ed4L genes with two copy loci each, and one pseudogene (\u003cem\u003emat\u003c/em\u003eR). Additionally, 18 variable protein-coding genes were detected, with only rps19 showing a single copy event (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For non-coding components, the genome contains 3 ribosomal RNA (rRNA) genes and 55 transfer RNA (tRNA) genes. Among tRNA genes, nine types were annotated twice: \u003cem\u003etrn\u003c/em\u003eA-TGC, \u003cem\u003etrn\u003c/em\u003eE-TTC, \u003cem\u003etrn\u003c/em\u003eF-GAA, \u003cem\u003etrn\u003c/em\u003eG-GCC, \u003cem\u003etrn\u003c/em\u003eH-GTG, \u003cem\u003etrn\u003c/em\u003eI-GAT, \u003cem\u003etrn\u003c/em\u003eP-TGG, \u003cem\u003etrn\u003c/em\u003eR-TCT, and \u003cem\u003etrn\u003c/em\u003eV-GAC; \u003cem\u003etrn\u003c/em\u003eT-TGT and \u003cem\u003etrn\u003c/em\u003eY-GTA were annotated three times; \u003cem\u003etrn\u003c/em\u003eL-CAA and \u003cem\u003etrn\u003c/em\u003eN-GTT four times; while \u003cem\u003etrn\u003c/em\u003eM-CAT showed the highest annotation frequency with 10 sites.\u003c/p\u003e \u003cp\u003eIn the mitochondrial genome of \u003cem\u003eS. anlungensis\u003c/em\u003e, 13 genes possess one intron each (\u003cem\u003eccm\u003c/em\u003eFc, \u003cem\u003erpl\u003c/em\u003e2, \u003cem\u003erps\u003c/em\u003e10, \u003cem\u003erps\u003c/em\u003e3, \u003cem\u003etrn\u003c/em\u003eA-TGC (2), \u003cem\u003etrn\u003c/em\u003eI-GAT (2), \u003cem\u003etrn\u003c/em\u003eQ-CTG, trnR-TCT (2), \u003cem\u003etrn\u003c/em\u003eT-TGT (3)), one gene containstwo introns (\u003cem\u003ecox\u003c/em\u003e2), one gene contains three introns (\u003cem\u003enad\u003c/em\u003e4), and four genes have four introns (\u003cem\u003enad\u003c/em\u003e1, \u003cem\u003enad\u003c/em\u003e2, \u003cem\u003enad\u003c/em\u003e5, and \u003cem\u003enad\u003c/em\u003e7).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eList of genes in the mitochondrial genome of \u003cem\u003eS. anlungensis\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup of genes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGene name\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eATP synthase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eatp\u003c/em\u003e1, \u003cem\u003eatp\u003c/em\u003e4, \u003cem\u003eatp\u003c/em\u003e6, \u003cem\u003eatp\u003c/em\u003e8, \u003cem\u003eatp\u003c/em\u003e9(2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCytohrome c biogenesis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eccm\u003c/em\u003eB, \u003cem\u003eccm\u003c/em\u003eC, \u003cem\u003eccm\u003c/em\u003eFc*, \u003cem\u003eccm\u003c/em\u003eFn\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUbichinol cytochrome c reductase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ecob\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCytochrome c oxidase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ecox\u003c/em\u003e1, \u003cem\u003ecox\u003c/em\u003e2**, \u003cem\u003ecox\u003c/em\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaturases\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e#\u003cem\u003emat\u003c/em\u003eR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTransport membrance protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003emtt\u003c/em\u003eB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNADH dehydrogenase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003enad\u003c/em\u003e1****, \u003cem\u003enad\u003c/em\u003e2****, \u003cem\u003enad\u003c/em\u003e3, \u003cem\u003enad\u003c/em\u003e4,*** \u003cem\u003enad\u003c/em\u003e4L(2), \u003cem\u003enad\u003c/em\u003e5****, \u003cem\u003enad\u003c/em\u003e6, \u003cem\u003enad\u003c/em\u003e7,**** \u003cem\u003enad\u003c/em\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRibosomal proteins (LSU)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003erpl\u003c/em\u003e10, \u003cem\u003erpl\u003c/em\u003e16, \u003cem\u003erpl\u003c/em\u003e2*, \u003cem\u003erpl\u003c/em\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRibosomal proteins (SSU)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003erps\u003c/em\u003e1, \u003cem\u003erps\u003c/em\u003e10*, \u003cem\u003erps\u003c/em\u003e11, \u003cem\u003erps\u003c/em\u003e12, \u003cem\u003erps\u003c/em\u003e13, \u003cem\u003erps\u003c/em\u003e14, \u003cem\u003erps\u003c/em\u003e19(2), \u003cem\u003erps\u003c/em\u003e2, \u003cem\u003erps\u003c/em\u003e3*, \u003cem\u003erps\u003c/em\u003e4, \u003cem\u003erps\u003c/em\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSuccinate dehydrogenase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003esdh\u003c/em\u003e3, \u003cem\u003esdh\u003c/em\u003e4,\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRibosomal RNAs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003errn\u003c/em\u003e18, \u003cem\u003errn\u003c/em\u003e26, \u003cem\u003errn\u003c/em\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTransfer RNAs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003etrn\u003c/em\u003eA-TGC*(2), \u003cem\u003etrn\u003c/em\u003eC-GCA, \u003cem\u003etrn\u003c/em\u003eD-GTC, \u003cem\u003etrn\u003c/em\u003eE-TTC(2), \u003cem\u003etrn\u003c/em\u003eF-GAA(2), \u003cem\u003etrn\u003c/em\u003eG-GCC(2), \u003cem\u003etrn\u003c/em\u003eG-GCC*, \u003cem\u003etrn\u003c/em\u003eH-GTG(2), \u003cem\u003etrn\u003c/em\u003eI-GAT*(2), \u003cem\u003etrn\u003c/em\u003eK-TTT, \u003cem\u003etrn\u003c/em\u003eL-CAA(4), \u003cem\u003etrn\u003c/em\u003eM-CAT(10), \u003cem\u003etrn\u003c/em\u003eN-GTT(4), \u003cem\u003etrn\u003c/em\u003eP-TGG(2), \u003cem\u003etrn\u003c/em\u003eQ-CTG*, \u003cem\u003etrn\u003c/em\u003eQ-TTG, \u003cem\u003etrn\u003c/em\u003eR-ACG, \u003cem\u003etrn\u003c/em\u003eR-TCT, \u003cem\u003etrn\u003c/em\u003eR-TCT*(2), \u003cem\u003etrn\u003c/em\u003eS-GCT, \u003cem\u003etrn\u003c/em\u003eS-GGA, \u003cem\u003etrn\u003c/em\u003eS-TGA, \u003cem\u003etrn\u003c/em\u003eT-TGT, \u003cem\u003etrn\u003c/em\u003eT-TGT*(3), \u003cem\u003etrn\u003c/em\u003eV-GAC(2), \u003cem\u003etrn\u003c/em\u003eW-CCA, \u003cem\u003etrn\u003c/em\u003eY-GTA(3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003eNote: Numbers after gene names are the number of copies. Genes preceded by the # symbol represent pseudogenes. The number of * symbols after a gene indicates the number of introns it contains.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Different configurations of the \u003cem\u003eS. anlungensis\u003c/em\u003e mitochondrial genome\u003c/h2\u003e \u003cp\u003eIn the mitochondrial genome of \u003cem\u003eS. anlungensis\u003c/em\u003e, three pairs of direct repeat sequences mediate high-frequency recombination, designated as LR12, LR14, and LR15, indicated by yellow rectangles in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Among these, LR14 mediates recombination between contighLR1 and contighLR2 across domains (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For LR14, the sequence configuration in the assembled mitochondrial genome is contig9\u0026rarr;LR14\u0026rarr;contig8 and contig7\u0026rarr;LR14\u0026rarr;contig10 (42%). After recombination, the sequence configuration changes to contig9\u0026rarr;LR14\u0026rarr;contig10 and contig7\u0026rarr;LR14\u0026rarr;contig8 (58%). Both LR12 and LR15 repeat pairs are located in distinct segments of contighLR1. In the assembled mitochondrial genome, their configurations are contig5\u0026rarr;LR12\u0026rarr;contig4 and contig11\u0026rarr;LR12\u0026rarr;contig1 (50%), and contig6\u0026rarr;LR15\u0026rarr;contig13 and contig8\u0026rarr;LR15\u0026rarr;contig5 (45%), respectively. Post-recombination, the configurations shift to contig5\u0026rarr;LR12\u0026rarr;contig1 and contig11\u0026rarr;LR12\u0026rarr;contig1 (50%), and contig6\u0026rarr;LR15\u0026rarr;contig5 and contig8\u0026rarr;LR15\u0026rarr;contig13 (55%). Since LR12, LR14, and LR15 are all direct repeats (rather than inverted repeats), their mediated recombination processes only generate sequence replacement rearrangements without causing DNA segment inversions. Additionally, the mitochondrial genome of \u003cem\u003eS. anlungensis\u003c/em\u003e contains four branch points not overlapping repeat regions: specifically, the 5' ends of contig11 and contig6, and the 3' end of contig10 can simultaneously connect to two contigs. The 3' end of contig2 can linearly link to the 5' end of contig3 or form a closed structure by connecting to its own 5' end. The above hypothesis is corroborated by the coverage validation map aligned with long-read assembly results, confirming the existence of potential substructures in different configurations of the \u003cem\u003eS. anlungensis\u003c/em\u003e mitochondrial genome (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Analysis of repeat sequences\u003c/h2\u003e \u003cp\u003eThe results revealed three types of repetitive sequences in the mitochondrial genome of \u003cem\u003eS. anlungensis\u003c/em\u003e: simple sequence repeats (SSRs), tandem repeats, and dispersed repeats. Among the 703 SSRs identified, 629 were located on Chr 1 and 74 on Chr 2. Specifically, mono-nucleotide SSRs (mono-SSRs) were detected in Chr1 (142) and Chr2 (19), di-SSRs (116 and 19), tri-SSRs (78 and 9), tetra-SSRs (252 and 22), penta-SSRs (32 and 4), and hexa-SSRs (9 in Chr1 and 1 in Chr2), highlighting the rarity of hexa-SSRs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Although tetra-SSRs were the most abundant in both Chr1 and Chr2, their distribution patterns differed: in Chr1, the total number of tetra-SSRs (252) was nearly equal to the combined count of mono- and di-SSRs (258), whereas in Chr2, tetra-SSRs (22) slightly outnumbered mono-SSRs (19) by only three.\u003c/p\u003e \u003cp\u003eA total of 204 tandem repeats were identified in the mitochondrial genome, with lengths varying significantly. The longest tandem repeat (133 bp, copy number\u0026thinsp;=\u0026thinsp;1.9) was located in Chr1, while the shortest (34 bp, copy number\u0026thinsp;=\u0026thinsp;17) was found in Chr2 (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). Additionally, 1,993 pairs of dispersed repeats were identified, spanning a cumulative length of 241,810 bp (10.19% of the total genome length). Many repeats spanned Chr1 and Chr2, including 161 sequences copied from Chr1 to Chr2 and 28 from Chr2 to Chr1 (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). Notably, three highly conserved dispersed repeat sequences (length\u0026thinsp;\u0026gt;\u0026thinsp;1,000 bp, similarity\u0026thinsp;=\u0026thinsp;99.96%), all classified as direct repeats, were implicated in mitochondrial genome recombination. Two of these repeats were intrachromosomal (Chr1), while the third functioned as an interchromosomal repeat element spanning homologous regions between Chr1 and Chr2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Prediction of RNA editing sites\u003c/h2\u003e \u003cp\u003eThis study predicted RNA editing sites in 40 protein-coding genes (PCGs) of the mitochondrial genome of \u003cem\u003eS. anlungensis\u003c/em\u003e, identifying 755 editing sites. These editing events triggered 17 types of amino acid substitutions: H(His)\u0026rarr;Y(Tyr), R(Arg)\u0026rarr;C(Cys), T(Thr)\u0026rarr;I(Ile), T(Thr)\u0026rarr;M(Met), R(Arg)\u0026rarr;W(Trp), S(Ser)\u0026rarr;L(Leu), S(Ser)\u0026rarr;F(Phe), P(Pro)\u0026rarr;S(Ser), P(Pro)\u0026rarr;L(Leu), P(Pro)\u0026rarr;F(Phe), L(Leu)\u0026rarr;F(Phe), A(Ala)\u0026rarr;V(Val), Q(Gln)\u0026rarr;X, and R(Arg)\u0026rarr;X (\u0026ldquo;X\u0026rdquo; represents stop codons). Among these, S\u0026rarr;L substitutions were the most frequent (168 sites, 22.25%), while T\u0026rarr;M substitutions were the least common (10 sites, 1.32%) (Fig.\u0026nbsp;4A).\u003c/p\u003e \u003cp\u003eAnalysis of amino acid physicochemical properties revealed that 357 sites (47.28%) caused shifts from hydrophilic to hydrophobic residues, 69 sites (9.14%) exhibited hydrophobic-to-hydrophilic polarity changes, and 235 sites (31.13%) showed no alteration in hydrophobicity (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA; Fig.\u0026nbsp;4B). Among the five stop codon formation events, two types were observed: CGA (R) \u0026rarr; TGA (X) and CAA (Q) \u0026rarr; TAA (X). The CGA\u0026rarr;TGA changes occurred in the final codons of \u003cem\u003eccm\u003c/em\u003eFc and rps10, while the CAA\u0026rarr;TAA substitutions were detected in \u003cem\u003eatp\u003c/em\u003e6 and \u003cem\u003erps\u003c/em\u003e11, with an additional instance in the 13th codon of rpl16, resulting in premature termination of mRNA translation (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB; Fig.\u0026nbsp;4C). At the gene expression level, \u003cem\u003enad\u003c/em\u003e4 exhibited the highest RNA editing frequency (65 events), followed by \u003cem\u003enad\u003c/em\u003e5 (45 events) and \u003cem\u003eccm\u003c/em\u003eFn (44 events). In contrast, ribosomal protein genes \u003cem\u003erps\u003c/em\u003e1, \u003cem\u003erps\u003c/em\u003e11, and \u003cem\u003erps\u003c/em\u003e7 showed significantly reduced editing activity, each containing only three editing sites.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.5 PCGs codon usage analysis\u003c/h2\u003e \u003cp\u003eThe results showed that the mitochondrial genome contains 10,804 amino acid-encoding codons, covering all 20 amino acid types and corresponding to 64 codon variants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e; Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). The most frequent codon was AUU (354 occurrences, 3.27%). Among the 20 amino acids, Ser (serine) exhibited the highest codon usage (1,066 codons, 9.87%), followed by Leu (leucine) with 1,014 codons (9.19%). Ter (stop codons) had the lowest count, with only 40 codons (1.42%). All PCGs used ATG (or ACG) as start codons, while stop codons included TAA, TAG, and TGA. Notably, only TAA had a relative synonymous codon usage (RSCU) value greater than 1. Amino acid usage was dominated by Arg (arginine), Leu, and Ser, while Met (methionine) and Trp (tryptophan) showed relatively low frequencies. RSCU analysis of the 64 codons in the mitochondrial PCGs revealed 29 codons with underrepresentation (RSCU\u0026thinsp;\u0026lt;\u0026thinsp;1) and 33 codons with overrepresentation (RSCU\u0026thinsp;\u0026gt;\u0026thinsp;1). Among codons with RSCU\u0026thinsp;\u0026gt;\u0026thinsp;1, Ala (GCU) and His (CAU) displayed the highest RSCU values (1.6505 and 1.6071, respectively). Conversely, Phe (UUU), Thr (ACC), Ter (UGA), Ala (GCA), Ser (UCC), Val (GUG), and Ser (AGU) showed weak codon bias (RSCU\u0026thinsp;\u0026lt;\u0026thinsp;1.1). Met (AUG) and Trp (UGG), each encoded by a single codon, had RSCU values of 1. Notably, 93.103% of high-frequency codons ended with A or T, while only 6.897% terminated with C or G, indicating a strong preference for NNA/NNU codon endings in the mitochondrial genome of \u003cem\u003eS. anlungensis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Mitochondrial plastid DNAs (MTPTs) in the mitochondrial genome\u003c/h2\u003e \u003cp\u003eIn plant cells, gene fragment transfer between mitochondria and chloroplasts is common. Comparative analysis of the genomes of these two organelles revealed 62 homologous fragments between the mitochondrial and chloroplast genomes of \u003cem\u003eS. anlungensis\u003c/em\u003e, including 19 fragments exceeding 1,000 bp in length (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e; Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). Among these, MTPT14-61 are located on Chr1, while MTPT1-13 are on Chr2. These fragments span a total length of 679,00 bp, accounting for approximately 2.8639% of the mitochondrial genome. The longest fragments, MTPT1 and MTPT14 (7,937 bp each), are located at positions 188,069\u0026ndash;195,997 bp on Chr2 and 1,379,727\u0026ndash;1,387,655 bp on Chr1, respectively. Both sequences correspond to positions 138,772\u0026ndash;146,708 bp in the chloroplast DNA. In contrast, MTPT12 is the shortest fragment (32 bp), located at 116,329\u0026ndash;116,360 bp on Chr2.\u003c/p\u003e \u003cp\u003eAnnotation results indicate that these fragments originate from chloroplast protein-coding genes, rRNA genes, tRNA genes, and intergenic regions. However, all chloroplast-derived protein-coding genes inserted into the mitochondrial genome underwent pseudogenization or loss. Among rRNA genes, only partial fragments of \u003cem\u003errn\u003c/em\u003e26 (partial: 2.58%) and \u003cem\u003errn\u003c/em\u003e18 (partial: 38.97%) were identified in MTPT10 and MTPT40, respectively. A total of 18 tRNA genes were distributed across 20 homologous sequences: \u003cem\u003etrn\u003c/em\u003eA-TGC, \u003cem\u003etrn\u003c/em\u003eI-GAT, \u003cem\u003etrn\u003c/em\u003eV-GAC, \u003cem\u003etrn\u003c/em\u003eL-CAA, \u003cem\u003etrn\u003c/em\u003eM-CAT, \u003cem\u003etrn\u003c/em\u003eH-GTG, \u003cem\u003etrn\u003c/em\u003eT-TGT, \u003cem\u003etrn\u003c/em\u003eS-GGA, \u003cem\u003etrn\u003c/em\u003eR-ACG, \u003cem\u003etrn\u003c/em\u003eN-GTT, \u003cem\u003etrn\u003c/em\u003eF-GAA, \u003cem\u003etrn\u003c/em\u003eR-TCT, \u003cem\u003etrn\u003c/em\u003eG-GCC, \u003cem\u003etrn\u003c/em\u003eE-TTC, \u003cem\u003etrn\u003c/em\u003eY-GTA, \u003cem\u003etrn\u003c/em\u003eD-GTC, \u003cem\u003etrn\u003c/em\u003eW-CCA, and \u003cem\u003etrn\u003c/em\u003eP-TGG. Some tRNA genes were present in multiple MTPTs, including \u003cem\u003etrn\u003c/em\u003eA-TGC, \u003cem\u003etrn\u003c/em\u003eI-GAT, \u003cem\u003etrn\u003c/em\u003eV-GAC, \u003cem\u003etrn\u003c/em\u003eL-CAA, \u003cem\u003etrn\u003c/em\u003eM-CAT, and \u003cem\u003etrn\u003c/em\u003eN-GTT.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Analysis of Pi (nucleotide diversity) and Ka/Ks (non-synonymous substitutions/ synonymous substitutions)\u003c/h2\u003e \u003cp\u003eTo investigate the evolutionary rates of mitochondrial genes between \u003cem\u003eS. anlungensis\u003c/em\u003e and related species, we calculated nucleotide diversity (Pi) values for 43 mitochondrial genes across four Lauraceae species, including \u003cem\u003eS. anlungensis\u003c/em\u003e (Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). The results revealed high Pi values for \u003cem\u003erps\u003c/em\u003e109 (0.02709), \u003cem\u003esdh\u003c/em\u003e3 (0.02626), \u003cem\u003enad\u003c/em\u003e6 (0.0235), and \u003cem\u003eatp\u003c/em\u003e6 (0.01852), suggesting elevated genetic variability in these regions. Although rrn26 exhibited a relatively low Pi value (0.00808), its long sequence length (3,777 bp) resulted in the highest number of mutation sites (57) among all mitochondrial genes. Notably, \u003cem\u003errn\u003c/em\u003e5 showed no detectable mutations (Pi\u0026thinsp;=\u0026thinsp;0), reflecting its extreme conservation.\u003c/p\u003e \u003cp\u003eTo further explore the influence of environmental pressures on mitochondrial PCG mutations in these species, we performed Ka/Ks analysis and identified 38 genes with valid Ka/Ks values. Comparative results indicated that most genes had Ka/Ks ratios\u0026thinsp;\u0026lt;\u0026thinsp;1, implying their mutations were predominantly shaped by purifying selection (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003e; Table \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e). However, a few PCGs exhibited signals of positive selection (Ka/Ks\u0026thinsp;\u0026gt;\u0026thinsp;1), including \u003cem\u003eccmFc\u003c/em\u003e, \u003cem\u003ecob\u003c/em\u003e, \u003cem\u003erpl\u003c/em\u003e16, and \u003cem\u003erpl\u003c/em\u003e2. Notably, \u003cem\u003eccm\u003c/em\u003eFc (\u003cem\u003eS. anlungensis\u003c/em\u003e vs. \u003cem\u003eC. henryi\u003c/em\u003e (NC_088584): 2.98482) and rpl16 (S. anlungensis vs. \u003cem\u003eC. chekiangense\u003c/em\u003e (NC_082065): 2.76801) displayed particularly high Ka/Ks ratios, suggesting these genes may have played pivotal roles in the adaptive evolution of \u003cem\u003eS. anlungensis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.8. Phylogenetic analysis\u003c/h2\u003e \u003cp\u003eThe phylogenetic tree constructed based on mitochondrial genome CDS (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003e) demonstrated that most nodes exhibited bootstrap values (BS) exceeding 80%, indicating high reliability of the phylogenetic relationships revealed by the coding sequences of 24 conserved mitochondrial PCGs. \u003cem\u003eS. anlungensis\u003c/em\u003e clustered closely with three other Lauraceae species within the Lauraceae Clade (BS\u0026thinsp;=\u0026thinsp;100). Additionally, Lauraceae plants formed the Magnoliids Clade (BS\u0026thinsp;=\u0026thinsp;100) alongside magnoliid species including \u003cem\u003eMagnolia biondii\u003c/em\u003e, \u003cem\u003eChimonanthus praecox\u003c/em\u003e, and \u003cem\u003eHernandia nymphaeifolia\u003c/em\u003e, with \u003cem\u003eHernandia nymphaeifolia\u003c/em\u003e showing the closest phylogenetic relationship to Lauraceae species (BS\u0026thinsp;=\u0026thinsp;100).\u003c/p\u003e \u003cp\u003eThe phylogenetic tree based on chloroplast PCGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003e) revealed that species from the genera \u003cem\u003eBeilschmiedia\u003c/em\u003e, \u003cem\u003eSinopora\u003c/em\u003e, and \u003cem\u003eSyndiclis\u003c/em\u003e separated from \u003cem\u003eMachilus rehderi\u003c/em\u003e and formed an independent branch, which received strong support (BS\u0026thinsp;=\u0026thinsp;96). Within this grouping, \u003cem\u003eS. anlungensis\u003c/em\u003e clustered in the same branch as other \u003cem\u003eSyndiclis\u003c/em\u003e species and \u003cem\u003eSinopora hongkongensis\u003c/em\u003e (BS\u0026thinsp;=\u0026thinsp;100), exhibiting a closer phylogenetic relationship to \u003cem\u003eS. marlipoensis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4.Discussion","content":"\u003cp\u003eThe mitochondrial genomes of plants undergo frequent recombination during evolution, resulting in their diverse and complex structures [\u003cspan additionalcitationids=\"CR46 CR47\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. As one of the most primitive extant angiosperm groups, studies on the mitochondrial genomes of Lauraceae species provide critical insights into the recombination mechanisms of early angiosperm mitochondria [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Previous reports on the mitochondrial genome structures of Lauraceae plants have identified two main types: the typical circular structures observed in \u003cem\u003eC. chekiangense\u003c/em\u003e and \u003cem\u003eC. henryi\u003c/em\u003e, and the repeat-mediated branched structures found in \u003cem\u003eC. camphora\u003c/em\u003e [\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. In the mitochondrial genome of \u003cem\u003eS. anlungensis\u003c/em\u003e, three out of its seven branch points were mediated by direct repeat sequences, generating diverse DNA substructures. However, the remaining four branch points were located outside repeat regions, leading to irregular configurations that could not be directly assembled into circular molecules. This result parallels the mitochondrial genome assembly of \u003cem\u003eAbelmoschus esculentus\u003c/em\u003e [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. This phenomenon may arise because during evolution, when specific mitochondrial DNA in \u003cem\u003eS. anlungensis\u003c/em\u003e incurred damage or breaks, repair pathways such as non-homologous end joining (NHEJ) or microhomology-mediated repair (MHMR) formed contig connections distinct from native mitochondrial DNA [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. These repaired sequences, like native mitochondrial DNA, underwent replication and accumulation within mitochondria.\u003c/p\u003e \u003cp\u003eIn terms of genome-scale dynamic evolution, the mitochondrial genome of \u003cem\u003eS. anlungensis\u003c/em\u003e exhibits significant expansion. Its DNA length reaches 2,370,899 bp, far exceeding those of three other Lauraceae species: \u003cem\u003eC. camphora\u003c/em\u003e (900,894 bp), \u003cem\u003eC. chekiangense\u003c/em\u003e (750,457 bp), and \u003cem\u003eC. henryi\u003c/em\u003e (1,168,029 bp) [\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Repeat sequence analysis suggests that this marked genome size disparity may be closely linked to the expansion of dispersed repeats (including tandem repeats). For instance, \u003cem\u003eC. camphora\u003c/em\u003e\u0026rsquo;s mitochondrial genome contains 1,421 pairs of dispersed repeats (including tandem repeats), totaling 63,004 bp. In contrast, \u003cem\u003eS. anlungensis\u003c/em\u003e harbors 2,197 pairs of dispersed repeats (including tandem repeats) spanning 246,080 bp\u0026mdash;nearly fourfold greater in total length. Although the difference in identifiable repeat sequence length accounts for only a minor proportion (~\u0026thinsp;14.4%) of the total genome size disparity, the pronounced expansion of dispersed repeats in \u003cem\u003eS. anlungensis\u003c/em\u003e implies that frequently recombined and mutated sequences escaping standard repeat detection may play a dominant role in genome expansion [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Notably, the difference in SSR counts between \u003cem\u003eS. anlungensis\u003c/em\u003e and \u003cem\u003eC. camphor\u003c/em\u003ea (703 vs. 697) is insignificant, suggesting minimal contribution of SSRs to overall genome size expansion.\u003c/p\u003e \u003cp\u003eIn addition to DNA length variation, the expansion of repetitive sequences also drives interspecific differentiation in mitochondrial gene copy numbers among Lauraceae plants. Specifically, the mitochondrial PCGs of \u003cem\u003eC. camphora\u003c/em\u003e contain a dual-copy gene \u003cem\u003enad\u003c/em\u003e6 and a multi-copy gene \u003cem\u003esdh\u003c/em\u003e3, with copy number variations detected in two tRNA genes: \u003cem\u003etrn\u003c/em\u003eE-TTC (2 copies) and \u003cem\u003etrn\u003c/em\u003eP-TGG (3 copies). In \u003cem\u003eC. chekiangense\u003c/em\u003e, only \u003cem\u003eatp\u003c/em\u003e6 exhibits dual-copy status, while the tRNA genes \u003cem\u003etrn\u003c/em\u003eM-CAT and \u003cem\u003etrn\u003c/em\u003eP-TGG both show 3 copies. No mitochondrial PCG or tRNA gene copy phenomena were observed in \u003cem\u003eC. henryi\u003c/em\u003e [\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. In this study, \u003cem\u003eS. anlungensis\u003c/em\u003e displayed PCG duplication events involving dual-copy core genes (\u003cem\u003enad\u003c/em\u003e4L, \u003cem\u003eatp\u003c/em\u003e9) and a dual-copy variable gene (\u003cem\u003erps\u003c/em\u003e19). Its tRNA genes exhibited an extreme amplification pattern: 14 tRNA genes (e.g., \u003cem\u003etrn\u003c/em\u003eA-TGC, \u003cem\u003etrn\u003c/em\u003eE-TTC, \u003cem\u003etrn\u003c/em\u003eF-GAA, \u003cem\u003etrn\u003c/em\u003eM-CAT, and \u003cem\u003etrn\u003c/em\u003eY-GTA) showed copy number variations, with \u003cem\u003etrn\u003c/em\u003eM-CAT reaching up to 10 copies. These copy number differences may reflect molecular strategies through which distinct Lauraceae species adapt to specialized ecological niches via gene dosage effects during evolution. However, the specific driving mechanisms require further validation through population genetics and epigenetic regulation studies [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Notably, the \u003cem\u003emat\u003c/em\u003eR gene shows pseudogenization in \u003cem\u003eC. camphora\u003c/em\u003e, \u003cem\u003eC. chekiangense\u003c/em\u003e, and \u003cem\u003eS. anlungensis\u003c/em\u003e, while remaining intact in \u003cem\u003eC. henryi\u003c/em\u003e. Given the critical role of \u003cem\u003emat\u003c/em\u003eR-encoded maturase-related proteins in mitochondrial biosynthesis [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], its functional loss may be closely associated with functional substitution or regulatory network restructuring during mitochondrial genome evolution in \u003cem\u003eCinnamomum\u003c/em\u003e and \u003cem\u003eSyndiclis\u003c/em\u003e species.\u003c/p\u003e \u003cp\u003eRNA editing typically converts specific cytidines (C) in the primary transcripts of mitochondrial PCGs into uridines (U) in mature mRNAs, creating discrepancies between the genomic sequence and the mature RNA. Predictions of RNA editing sites in the mitochondrial PCGs of \u003cem\u003eS. anlungensis\u003c/em\u003e revealed that RNA editing corrects non-initiator ACG codons at the start positions of certain mitochondrial PCGs (\u003cem\u003enad\u003c/em\u003e1, \u003cem\u003enad\u003c/em\u003e4L, \u003cem\u003erps\u003c/em\u003e10, and \u003cem\u003ecox\u003c/em\u003e1) into AUG translation initiation codons. This process ensures proper mRNA translation of these genes [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Additionally, RNA editing often alters the hydrophilic-hydrophobic properties of encoded amino acids. Studies have confirmed that this regulatory mechanism is not only critical for maintaining the stability of protein secondary structures and functions but also enhances plant adaptability to abiotic stresses such as drought [\u003cspan additionalcitationids=\"CR61\" citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. This study identified 357 RNA editing sites (47.28%) in the mitochondrial PCGs of \u003cem\u003eS. anlungensis\u003c/em\u003e that shift amino acid residues from hydrophilic to hydrophobic, and 69 sites (9.14%) that reverse this trend. Given the species-specific nature of mitochondrial RNA editing sites, these results provide crucial molecular data for investigating the functional roles and adaptive evolution of RNA editing in \u003cem\u003eS. anlungensis\u003c/em\u003e and the genus \u003cem\u003eSyndiclis\u003c/em\u003e. Furthermore, RNA editing in certain mitochondrial PCGs, such as \u003cem\u003eatp\u003c/em\u003e9, is essential for normal pollen development. In tobacco and rice, failure to edit codons at positions 28, 45, 64, and 71(CTT (L) \u0026rarr; TTT (F), TCA (S) \u0026rarr; TTA (L), CCA (P) \u0026rarr; CTA (L), TCA (S) \u0026rarr; TTA (L)) of this gene leads to pollen abortion [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. In \u003cem\u003eS. anlungensis\u003c/em\u003e, the codon types at these RNA editing sites in the \u003cem\u003eatp\u003c/em\u003e9 gene align completely with those in the aforementioned species. This finding may offer a theoretical foundation for developing male sterility hybridization techniques in this species.\u003c/p\u003e \u003cp\u003eThe study of codon usage bias represents a critical topic in molecular biology and genomics, with its significance spanning multiple aspects, including gene expression regulation and evolutionary mechanism elucidation. This research identified 33 high-frequency codons (RSCU\u0026thinsp;\u0026gt;\u0026thinsp;1) in the mitochondrial PCGs of \u003cem\u003eS. anlungensis\u003c/em\u003e, revealing that over 90% of these codons terminate with A/T. Their types align closely with previous reports on mitochondrial genomes in angiosperms, suggesting the evolutionary conservation of this genetic feature in plants [\u003cspan additionalcitationids=\"CR46 CR47\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Earlier studies proposed that these highly used codons typically match abundant intracellular tRNAs, with their biological significance lying in enhancing translation rates, reducing ribosome stalling, and optimizing protein synthesis efficiency [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Notably, certain low-frequency codons (e.g., GGC, GAC, GCG, and CAG) distributed across gene sequences may also play regulatory roles in mRNA translation. This is attributed to their ability to slow elongation speed, thereby decreasing ribosome congestion at the 3\u0026rsquo; end, promoting uniform ribosome distribution along mRNA strands, and preventing translation interruptions caused by spontaneous collisions or stalling [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFrequent horizontal gene transfer events occur between chloroplast DNA and mitochondrial DNA. Studies suggest that the integration of chloroplast DNA fragments confers evolutionary advantages: early plant mitochondrial genomes, lacking a complete tRNA gene system, required continuous integration of chloroplast DNA fragments to acquire functional tRNAs, thereby partially refining their gene expression mechanisms [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. This evolutionary process dates back at least to the common ancestor of extant gymnosperms and angiosperms (~\u0026thinsp;300\u0026nbsp;million years ago, 300 Mya). Notably, the \u003cem\u003etrn\u003c/em\u003eV(UAC)-\u003cem\u003etrn\u003c/em\u003eM(CAU)-\u003cem\u003eatp\u003c/em\u003eE-\u003cem\u003eatp\u003c/em\u003eB-\u003cem\u003erbc\u003c/em\u003eL gene cluster\u0026mdash;the oldest mitochondrial DNA-integrated fragment\u0026mdash;exhibits striking distribution differences between the two organellar DNAs in \u003cem\u003eS. anlungensis\u003c/em\u003e: the corresponding chloroplast DNA region (56,815\u0026ndash;62,093 bp) displays high continuity, containing only two small spacer regions (107 bp between \u003cem\u003etrn\u003c/em\u003eM-atpE and 11 bp between \u003cem\u003eatp\u003c/em\u003eB-\u003cem\u003erbc\u003c/em\u003eL). In contrast, the mitochondrial DNA homologous fragments are structurally rearranged and distributed across three discrete regions: \u003cem\u003etrn\u003c/em\u003eM-CAT (513,460\u0026ndash;513,382 bp), \u003cem\u003eatp\u003c/em\u003eE/\u003cem\u003eatp\u003c/em\u003eB (1,280,675-1,282,768 bp), and \u003cem\u003erbc\u003c/em\u003eL (796,948\u0026ndash;793,978 bp) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This spatial heterogeneity vividly illustrates the dynamic evolutionary features of mitochondrial genomes and further supports the hypothesis proposed by Richly and Leister (2004): \u0026ldquo;Primary insertions of organellar DNAs are large and then diverge and fragment over evolutionary time\u0026rdquo; [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe Ka/Ks analysis serves as a pivotal tool for deciphering the evolutionary drivers of genes. By quantifying the ratio of nonsynonymous substitution rates (Ka) to synonymous substitution rates (Ks), it precisely distinguishes the effects of purifying selection, neutral evolution, and positive selection on the accumulation of genetic mutations [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. In this study, Ka/Ks analysis revealed that mitochondrial protein-coding genes (PCGs) in Lauraceae plants are predominantly under strong purifying selection, indicating functional conservation, consistent with previous studies in angiosperms. Notably, specific genes such as \u003cem\u003eccm\u003c/em\u003eFc, \u003cem\u003ecob\u003c/em\u003e, and \u003cem\u003erpl\u003c/em\u003e2 exhibited significant positive selection signals (Ka/Ks\u0026thinsp;\u0026gt;\u0026thinsp;1), with nonsynonymous substitution rates markedly exceeding theoretical expectations under neutral evolutionary models. This evolutionary pattern suggests that these genes may have undergone adaptive evolution during phylogeny, potentially linked to critical biological processes such as ecological niche adaptation strategies, mitochondrial energy metabolism network restructuring, or reproductive system specialization [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. To further elucidate their evolutionary drivers, future studies should integrate functional genomics experiments and cross-species comparative genomics analyses to explore the functional relevance of these genes and their biological effects in species-specific adaptive evolution.\u003c/p\u003e \u003cp\u003eConsistent with the APG classification system, the phylogenetic tree constructed based on mitochondrial CDS strongly supports the division between magnoliids, eudicots, and monocots, while molecular biological evidence confirms the close phylogenetic relationship between \u003cem\u003eS. anlungensis\u003c/em\u003e and three Lauraceae species: \u003cem\u003eC. camphora\u003c/em\u003e, \u003cem\u003eC. chekiangense\u003c/em\u003e, and \u003cem\u003eC. henryi\u003c/em\u003e. Furthermore, aligning with previous studies, \u003cem\u003eCinnamomum\u003c/em\u003e and \u003cem\u003eCaryodaphnopsis\u003c/em\u003e species within Lauraceae exhibit closer phylogenetic affinities. \u003cem\u003eS. anlungensis\u003c/em\u003e occupies the basal position of this clade, likely because it belongs to the Cryptocaryeae lineage - the earliest diverged evolutionary branch in Lauraceae, independent from the lineages containing \u003cem\u003eCinnamomum\u003c/em\u003e and \u003cem\u003eCaryodaphnopsis\u003c/em\u003e [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. Phylogenetic analysis based on chloroplast PCGs supports the distinct status of the genus \u003cem\u003eSyndiclis\u003c/em\u003e within Lauraceae, revealing \u003cem\u003eS. anlungensis\u003c/em\u003e as most closely related to \u003cem\u003eS. marlipoensis\u003c/em\u003e within this genus. Notably, previous morphological studies proposing the merger of \u003cem\u003eSinopora\u003c/em\u003e with \u003cem\u003eSyndiclis\u003c/em\u003e (based on semicircular stomata, fine leaf venation without free vein endings, small terminal buds, and large globose fruits) gain new molecular support from this study [\u003cspan additionalcitationids=\"CR72\" citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Our phylogenetic results demonstrate that \u003cem\u003eS. hongkongensis\u003c/em\u003e of \u003cem\u003eSinopora\u003c/em\u003e is nested within the \u003cem\u003eSyndiclis\u003c/em\u003e clade and forms a sister relationship with \u003cem\u003eS. chinensis\u003c/em\u003e of \u003cem\u003eSyndiclis\u003c/em\u003e, providing fresh evidence for this taxonomic hypothesis.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study presents the first complete mitochondrial genome of critically endangered \u003cem\u003eS. anlungensis\u003c/em\u003e, revealing a dynamic 2,370,899 bp structure shaped by recombination via three direct repeat pairs (R12/R14/R15). It comprises 26 core and 18 variable protein-coding genes (PCGs), 55 tRNAs, and abundant repeats (703 SSRs, 204 tandem, 1,993 dispersed). RNA editing altered 47.28% of 755 sites to hydrophobic amino acids, potentially enhancing protein stability. Positive selection (Ka/Ks) in \u003cem\u003eccm\u003c/em\u003eFc and \u003cem\u003erpl\u003c/em\u003e16 suggests adaptive evolution. Mitochondrial-chloroplast genome comparisons identified 62 homologous fragments (67,900 bp). Phylogenetically, mitochondrial CDS places \u003cem\u003eS. anlungensis\u003c/em\u003e basally within Lauraceae, while chloroplast PCGs indicate closest affinity to \u003cem\u003eS. marlipoensis\u003c/em\u003e. These findings elucidate recombination mechanisms, repeat expansion, and adaptive traits, offering critical insights for conservation strategies, population restoration, and advancing Lauraceae mitochondrial evolution studies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCGs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProtein-coding genes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003emtDNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMitochondrial genome\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ecpDNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eChloroplast genome\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKa/Ks\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNon-synonymous/synonymous mutation ratio\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRSCU\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRelative synonymous codon usage\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMTPT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMitochondrial plastid DNA sequence\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003etRNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTransfer RNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003erRNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRibosomal RNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSSR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esimple sequence repeat\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePi\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enucleotide diversity\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebootstrap support value\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eposterior probabilities.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing or conflicting interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll materials used in this study comply with international and national legal standards. The collected species material does not pose a threat to other species, and the collection of the species is recognized by the relevant authorities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis research was supported by the Guizhou Provincial Science and Technology Program Project(Qiankehejichu-ZK[2022] General 240), Guizhou Provincial Forestry Research Project (QianlinKehe-J[2019] 11), and the National Natural Science Foundation of China (Grant No. 32400179).\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eL.H and X.X: Conceptualization, L.H and D.Z.J: Writing - original draft, Data curation, Formal analysis, Software. L.H\u0026amp;Z.L: Funding acquisition, Resources, Review \u0026amp; editing, Investigation. Y.B.Y: Investigation, Methodology. R.C: Supervision, Visualization. Z.L: Methodology, Validation.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eWe thank the editors and the anonymous reviewers for their insightful comments and suggestions on the manuscript.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe mitochondrial genome sequences supporting the conclusions of this article are available in GenBank (https://www.ncbi.nlm.nih.gov/) with accession numbers: PV744311 and PV744312.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMinistry of Ecology and Environment of the People\u0026rsquo;s Republic of China and Chinese Academy of Sciences. 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Syst Bot. 2012;37(4):861\u0026ndash;78. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1600/036364412X656518\u003c/span\u003e\u003cspan address=\"10.1600/036364412X656518\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Syndiclis anlungensis, Mitochondrial genome, Horizontal transfer, Phylogenetic analysis","lastPublishedDoi":"10.21203/rs.3.rs-6835863/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6835863/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e \u003cem\u003eSyndiclis anlungensis\u003c/em\u003e is a critically endangered (CR) species belonging to the genus \u003cem\u003eSyndiclis\u003c/em\u003e in the family Lauraceae. However, the complete mitochondrial genome of this species has not yet been systematically described, hindering our understanding of the genetic diversity and evolutionary relationships of mitochondrial genomes within the genus \u003cem\u003eSyndiclis\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eResult\u003c/h2\u003e \u003cp\u003eThis study combined Illumina and Oxford Nanopore sequencing technologies to complete the sequencing, assembly, and annotation of the mitochondrial genome of \u003cem\u003eS. anlungensis\u003c/em\u003e. The mitochondrial genome of \u003cem\u003eS. anlungensis\u003c/em\u003e has a total length of 2,370,899 bp, comprising 26 core protein-coding genes (PCGs), 18 variable PCGs, and 55 tRNA genes, exhibiting a multipartite substructure mediated by 3 direct repeats. Analysis revealed that the genome contains 703 simple sequence repeats (SSRs), 204 tandem repeats, and 1,993 pairs of dispersed repeats. Among the mitochondrial PCGs, 93.1% of high-frequency codons end with A/T. A total of 755 RNA editing sites were identified, with 357 sites (47.28%) resulting in amino acid residue changes from hydrophilic to hydrophobic and 69 sites (9.14%) showing hydrophobic-to-hydrophilic shifts. Ka/Ks analysis indicated that genes such as \u003cem\u003eccmFc\u003c/em\u003e and \u003cem\u003erpl16\u003c/em\u003e are under positive selection. Additionally, 62 homologous fragments (totaling 67,900 bp) were identified between the mitochondrial and chloroplast genomes, accounting for approximately 2.8639% of the mitochondrial genome length. Phylogenetic analysis of the mitochondrial genome placed \u003cem\u003eS. anlungensis\u003c/em\u003e at the basal position within Lauraceae, while chloroplast genome-based phylogeny revealed \u003cem\u003eS. marlipoensis\u003c/em\u003e as the closest relative to \u003cem\u003eS. anlungensis\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThis study presents the first comprehensive decoding of the mitochondrial genome of \u003cem\u003eS. anlungensis\u003c/em\u003e, unveiling its features of frequent recombination, repeat sequence expansion, and adaptive evolution. These findings provide critical data for understanding the evolutionary mechanisms of mitochondrial genomes in the genus \u003cem\u003eSyndiclis\u003c/em\u003e, while establishing a molecular foundation for the conservation of its genetic resources and the development of population restoration strategies.\u003c/p\u003e","manuscriptTitle":"Report on the complete mitochondrial genome of the critically endangered and endemic Lauraceae plant Syndiclis anlungensis in China","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-02 03:04:49","doi":"10.21203/rs.3.rs-6835863/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"419dd85b-932d-4881-afb8-18b923d0defc","owner":[],"postedDate":"July 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-06T04:38:50+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-02 03:04:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6835863","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6835863","identity":"rs-6835863","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}