Phylogenetic and Evolutionary Analysis of Mitochondrial Genomes in Phoebe

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We report the complete mitochondrial genome sequences of three species Phoebe zhennan , Phoebe bournei and Phoebe yaiensis , ranging in size from 807,952 to 865,014 base pairs. All three mitogenomes contain 40 conserved protein-coding genes. Comparative analysis identified abundant repetitive sequences, with P. zhennan showing the highest repeat content. RNA editing sites were highly conserved and predominantly increased encoded protein hydrophobicity. Synonymous codon usage favored A/T endings across all species, supporting closer phylogenetic affinity between P. yaiensis and P. zhennan . Chloroplast-derived sequences constituted 5.66–6.06% of the mitogenomes. Evolutionary analysis indicated widespread purifying selection, though nad6 and several other genes exhibited positive selection signals. Phylogenetic reconstruction confirmed Phoebe as monophyletic and sister to Cinnamomum . This study provides foundational mitogenomic resources for understanding evolutionary relationships within Phoebe and Lauraceae. Phoebe mitochondrial genome RNA editing codon usage phylogeny Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Plant mitochondrial genomes (mitogenomes) possess distinct evolutionary characteristics that render them particularly valuable for phylogenetic reconstruction [ 1 ]. Unlike other genomic compartments, mitogenomes combine slow sequence evolution with considerable structural plasticity, manifested through frequent genomic rearrangements and extensive intracellular gene transfer [ 2 , 3 ]. This structural dynamism underlies the remarkable size variation observed in plant mitogenomes, which range from approximately 66 kb in Viscum scurruloideum to over 11,000 kb in Silene conica [ 4 – 6 ]. Such substantial size disparities are primarily attributable to divergent repetitive DNA content and sequences acquired through intracellular transfer events [ 7 ]. Collectively, these attributes establish mitogenomes as powerful tools for resolving deep evolutionary relationships where conventional molecular markers offer limited phylogenetic resolution [ 8 , 9 ]. The Lauraceae family, a core group within the order Laurales, contains approximately 60 genera and 3,500 species [ 10 , 11 ]. Within this family, the genus Phoebe comprises about 100 species distributed across tropical and subtropical Asia [ 12 – 14 ]. umerous Phoebe species are prized for their high-quality timber, characterized by its fine texture, durability, and natural decay resistance, making it ideal for high-end furniture, woodworking, and traditional construction [ 15 , 16 ]. Ecologically, these species also play a significant role in the structure and function of subtropical forest ecosystems [ 17 ]. Despite their economic and ecological importance, the phylogenetic relationships within the genus Phoebe remain inadequately resolved [ 16 – 18 ]. Current classifications rely heavily on morphological traits that are prone to plasticity and convergent evolution [ 19 – 20 ]. While molecular systematics using plastid genomes has become a fundamental approach, genomic resources for Phoebe , particularly mitochondrial genomes, remain severely limited [ 21 – 23 ]. This gap significantly constrains our ability to investigate their evolutionary history and phylogenetic relationships, such as the complex relationship between P. zhennan and P. bournei , at a comprehensive genomic level [ 24 ]. In this study, we sequenced and assembled the complete mitogenomes of three Phoebe species, P. zhennan , P. bournei , and P. yaiensis . We conducted comparative genomic analyses to characterize their structural features, identify sequence variations, and infer selective pressures acting on protein-coding genes. Our findings provide foundational insights into the evolution of mitochondrial genomes within this valuable genus and the broader Lauraceae family. Materials and Methods Plant Materials and DNA Sequencing Fresh leaves of P. bournei , P. yaiensis , and P. zhennan were collected from their natural habitats in ChongQing. Total genomic DNA was extracted using a modified CTAB method [ 24 ]. DNA quality and concentration were assessed by agarose gel electrophoresis, NanoDrop spectrophotometry, and Qubit fluorometry. For each species, paired-end sequencing libraries were constructed and sequenced on the Illumina NovaSeq 6000 platform. Additionally, long-read sequencing was performed using the Oxford Nanopore PromethION platform to facilitate the assembly of complex mitochondrial regions. Mitochondrial Genome Assembly and Annotation Mitochondrial genomes were assembled using a hybrid strategy combining Illumina short reads and Nanopore long reads. Raw reads were quality-filtered with Fastp and GetOrganelle [ 25 ]. Filtered long reads were aligned to a reference set of plant mitochondrial core genes using Minimap2 [ 26 ], and candidate mitochondrial sequences were extracted. Subsequent assembly was performed using Canu for error correction [ 27 ], followed by hybrid assembly with Unicycler [ 28 ]. Final assembly graphs were visualized and manually verified using Bandage [ 29 ]. Gene annotation was conducted with GeSeq [ 30 ], using Cinnamomum camphora and related Lauraceae species as references [ 31 ]. tRNA genes were identified using tRNAscan-SE [ 32 ]. Circular maps of the mitochondrial genomes were generated using OGDRAW [ 33 ]. Analysis of Repetitive Sequences Simple sequence repeats (SSRs) were detected using MISA with default thresholds [ 34 ]. Tandem repeats were identified with Tandem Repeats Finder [ 35 ], and dispersed repeats (forward, palindromic, reverse, and complementary) were analyzed using BLASTN and REPuter [ 36 , 37 ]. The minimal length for dispersed repeats was set to 30 bp. The distribution and frequency of repeats were visualized with Circos [ 38 ]. Prediction of RNA Editing Sites Potential RNA editing sites in protein-coding genes (PCGs) were predicted using the online tool Deepred-Mt [ 39 ], which identifies C-to-U conversions based on sequence alignment and conservation. Only sites meeting a confidence threshold were retained. The impact of RNA editing on amino acid properties and codon position was analyzed. Codon Usage Analysis Relative Synonymous Codon Usage (RSCU) values were calculated for all mitochondrial PCGs using the CodonW [ 40 ]. Codon usage patterns were compared among the three Phoebe species to assess evolutionary constraints and translational preferences. Gene Transfer Analysis Homologous regions between mitochondrial and chloroplast genomes were identified using BLASTN with an E-value cutoff of 1e − 5 and sequence identity ≥ 70%. The total length and proportion of mitochondrial plastid DNA (MTPTs) fragments were calculated. Functional annotation of transferred genes was performed to assess their potential roles. Nucleotide Diversity and Selective Pressure Shared mitochondrial PCGs from three Phoebe species and four related Lauraceae taxa were aligned using MAFFT [ 41 ]. Nucleotide diversity (Pi) was calculated with DnaSP using a sliding window approach [ 42 ]. The nonsynonymous (Ka) and synonymous (Ks) substitution rates were estimated using KaKs_Calculator [ 43 ]. Genes with Ka/Ks > 1 were considered under positive selection. Phylogenetic Analysis A maximum likelihood phylogenetic tree was constructed using conserved mitochondrial PCGs from 18 angiosperm species, including the three Phoebe species and representatives from Lauraceae, and other families. Multiple sequence alignment was performed with MAFFT, and the best-fit substitution model (GTR + G) was selected using jModelTest [ 44 ]. The tree was built using maximum likelihood with 1000 bootstrap replicates to assess node support. Results Assembly and Characterization of Mitochondrial Genomes in Phoebe The complete mitochondrial genomes of P. bournei , P. yaiensis , and P. zhennan were assembled into circular mapping molecules with sizes of 865,014 bp, 830,727 bp, and 807,952 bp, respectively, displaying GC contents of 45.82%, 45.57%, and 46.03% (Fig. 1 ). These mitochondrial genomes fall within the moderate size range among reported Lauraceae species. Gene annotation identified a conserved set of 40 PCGs across the three species, which were classified into several functional categories (Table 1 ). These include ATP synthase (5 genes), cytochrome c biogenesis (4 genes), cytochrome c oxidase (3 genes), NADH dehydrogenase (9 genes), succinate dehydrogenase (2 genes), and the conserved matR gene. The total length of PCGs accounted for approximately 6.8–7.2% of each mitogenome. Notably, six genes ( ccmFn , rps3 , nad2 , nad4 , nad5 , and nad7 ) contained introns, with nad4 containing three introns and the others possessing single introns. The mitochondrial genomes also contained three ribosomal RNA genes ( rrn5 , rrn18 , rrn26 ) and a variable number of transfer RNA genes. Among the 24 tRNA genes identified across the three species, most were single-copy, except for trnM-CAT , which was duplicated in all three species. Interestingly, trnF-GAA and trnN-GTT were found to undergo RNA splicing before participating in protein translation. Comparative analysis revealed minor interspecific variations in tRNA gene content: trnA-TGC and trnI-GAT were absent in P. yaiensis and P. zhennan , trnF-AAA was missing in P. yaiensis , trnL-GAG was absent in P. bournei , and trnV-GAC was not detected in P. zhennan . Despite these variations, all three mitochondrial genomes maintained a complete set of tRNAs capable of recognizing all 20 standard amino acids. Table 1 Mitochondrial gene content across three Phoebe species Group of genes Gene name ATP synthase atp1 (1/1/1), atp4 (1/1/1), atp6 (1/1/1), atp8 (1/1/1), atp9 (2/2/2) Cytochrome c biogenesis ccmB (1/1/1), ccmC (1/1/1), ccmFc (1/1/1), ccmFn (1/1/1) Cytochrome c oxidase cox1 (1/1/1), cox2 (1/1/1), cox3 (1/1/1) NADH dehydrogenase nad1 (1/1/1), nad2 (1/1/1), nad3 (1/1/1), nad4 (1/1/1), nad4L (1/1/1), nad5 (1/1/1), nad6 (1/1/1), nad7 (1/1/1), nad9 (1/1/1) Ribosomal RNAs rrn18 (1/1/1), rrn26 (1/1/1), rrn5 (1/1/1) Succinate dehydrogenase sdh3 (1/1/1), sdh4 (1/1/1) Transfer RNAs trnA-TGC (0/0/1), trnC-GCA (1/1/1), trnD-GTC (1/1/1), trnE-TTC (1/1/1), trnF-AAA (0/1/1), trnF-GAA (1/1/1), trnG-GCC (1/1/1), trnH-GTG (1/1/1), trnI-GAT (0/0/1), trnK-CTT (1/1/1), trnK-TTT (1/1/1), trnL-CAA (1/1/1), trnL-GAG (1/1/0), trnM-CAT (1/1/1), trnN-GTT (1/1/1), trnP-TGG (1/1/1), trnQ-TTG (1/1/1), trnR-GCG (1/1/1), trnR-TCT (1/1/1), trnS-GCT (1/1/1), trnS-TGA (1/1/1), trnT-GGT (1/1/1), trnT-TGT (1/1/1), trnV-GAC (1/0/1), trnW-CCA (1/1/1), trnY-GTA (1/1/1) For each gene, presence is coded as 1 and absence as 0 in the ternary code corresponding to P. yaiensis , P. zhennan , and P. bournei . Comparative Analysis of Repetitive Sequences in Phoebe We characterized repetitive sequences in the three Phoebe mitogenomes, including SSRs, tandem repeats, and dispersed repeats. A comprehensive analysis of SSRs identified a total of 212 repeats across the three mitogenomes. Tetranucleotide repeats were the most abundant, accounting for 36.3% (n = 77) of the total, followed by mononucleotide (24.5%, n = 52), dinucleotide (22.6%, n = 48), trinucleotide (12.3%, n = 26), pentanucleotide (3.3%, n = 7), and hexanucleotide (0.9%, n = 2) repeats. The A/T motif was predominant among all SSR motifs, representing 26.4% (n = 56) of the total, with other notable motifs including AG/CT (8.5%, n = 18), GA/TC (8.0%, n = 17), and AAAG/CTTT (5.2%, n = 11) (Fig. 2 , Fig. 3 A). The distribution of SSRs gene pairs was visualized using Circos plots, which revealed that the P. zhennan mitogenome contained the highest number of SSR gene pairs (150), exceeding those in P. bournei (120 pairs) and P. yaiensis (100 pairs) (Fig. 2 , Fig. 3 A). The analysis of tandem repeat gene pairs revealed a parallel trend, with P. zhennan (50 pairs), P. bournei (40 pairs), and P. yaiensis (30 pairs) possessing the greatest to least abundance (Fig. 2 ). The prevalence of SSRs over tandem repeats across all three species suggests a fundamental role of SSRs in their genomic structure and evolutionary dynamics. The notably higher abundance of both repeat types in P. zhennan may indicate a history of more active duplication events or distinct evolutionary pressures shaping its mitochondrial genome. Analysis of the three mitogenomes revealed 276 dispersed repeats, which had an average length of 68 bp (Fig. 3 B–D). These were exclusively classified into 149 palindromic repeats and 127 forward repeats, with no reverse or complement repeats detected. Length distribution analysis showed that the majority (64.1%) of dispersed repeats ranged from 30 to 69 bp. Notably, within the 30–39 bp length category, both forward and palindromic repeats were equally represented (28 each, constituting 20.3% of the total dispersed repeats). Conserved RNA Editing Enhances Hydrophobicity of Mitochondrial Proteins in Phoebe Analysis of mitochondrial protein-coding genes revealed highly conserved RNA editing patterns across the three Phoebe species, with 623, 621, and 620 editing sites in P. bournei , P. yaiensis , and P. zhennan , respectively (Fig. 4 A). Gene-specific analysis showed that nad4 was the most heavily edited (68 sites), while ribosomal genes rpl2 and rps14 contained the fewest edits (2 sites) (Fig. 4 B). RNA editing predominantly targeted the second codon position (63.1%), followed by the first position (34.2%). A notable 2.7% of edits involved concurrent modifications at both first and second bases, enabling direct amino acid conversions such as proline to phenylalanine. At the protein level, 89.2% of editing events altered amino acid identity, with a strong bias toward hydrophobic residues. Leucine was the most frequent substitution (41.5%), mainly from serine (155 sites) and proline (138 sites). Critically, 93.5% of non-silent edits increased protein hydrophobicity, indicating a crucial role for RNA editing in refining mitochondrial protein structure in Phoebe species. Conserved Codon Usage Bias Reflects Evolutionary Relationships in Phoebe Analysis of codon usage patterns in the three Phoebe mitochondrial genomes revealed highly conserved synonymous codon preferences, with identical RSCU profiles between P. yaiensis and P. zhennan . All species exhibited a pronounced bias toward A/T-ending codons (Fig. 5 ), preferentially using UUA (Leu1, 1.40), GCU (Ala, 1.67), and CCU (Pro, 1.39) while avoiding C-ending codons such as CUG (Leu2, 0.55) and GCG (Ala, 0.47). Amino acid-specific patterns showed that UCU (Ser1, 1.37) was favored over AGC (Ser2, 0.54), while CGU (Arg, 1.28) and CGA (Arg, 1.33) were preferred over CGC (Arg, 0.63). The UGA codon functioned as tryptophan with strong underrepresentation (0.15). This conserved codon usage pattern suggests shared evolutionary constraints and indicates a closer phylogenetic relationship between P. yaiensis and P. zhennan . Predominance of Chloroplast-Derived DNA in Phoebe Sequence alignment identified numerous MTPTs between the organellar genomes of the three Phoebe species. A total of 73 to 83 homologous fragments were detected in each species, with a cumulative length representing 29.94–32.70% of the chloroplast genome but only 5.66–6.06% of the mitochondrial genome, indicating an asymmetric transfer bias (Fig. 6 ). Functional annotation revealed that these MTPTs contained multiple functional RNA genes. The transfer from chloroplast to mitochondrion was particularly notable, encompassing 4 tRNA genes and 30–34 rRNA genes across the species. Furthermore, specific homologous gene pairs, such as trnK-UUU/trnK-TTT and trnA-UGC/trnA-TGC , were conserved between the genomes, providing direct evidence for the interorganellar transfer of functional genetic material. These findings demonstrate widespread and biased DNA transfer from the chloroplast to the mitochondrial genome in Phoebe . Phylogenetic Analysis of Mitochondrial Genomes in Phoebe Our phylogenetic reconstruction based on mitochondrial PCGs from 18 species revealed strong nodal support (≥ 90) for most relationships within the mitochondrial gene tree (Fig. 7 ). Within the genus Phoebe , the three species formed a fully supported monophyletic group (100), with P. zhennan and P. yaiensis exhibiting the closest phylogenetic affinity. This Phoebe clade showed a strongly supported sister relationship with Cinnamomum species, while P. bournei was resolved as the basalmost lineage within the genus. The broader phylogenetic context clearly separated lauraceous species from monocot Poaceae and other eudicot lineages, with gymnosperms Ginkgo biloba and Pinus taeda consistently positioned as outgroups. Analysis of Evolutionary Rate and Selective Pressure To assess sequence variation, we estimated Pi for the mitochondrial PCGs that were conserved orthologous among the three Phoebe species and four related Lauraceae taxa. The analysis revealed substantial variation in Pi values across genes (Fig. 8 A). The nad6 gene exhibited the highest nucleotide diversity (0.0525), indicating pronounced sequence divergence. Other genes with notably high Pi values included atp9 (0.0263), ccmC (0.0257), cox2 (0.0252), and sdh3 (0.0249). In contrast, genes such as cox3 (0.0011), nad2 (0.0014), and nad1 (0.0021) displayed minimal diversity, suggesting strong evolutionary conservation and functional constraints. To evaluate selective pressures acting on the mitochondrial PCGs, the ratio of Ka/Ks was calculated for each gene (Fig. 8 B). The majority of PCGs exhibited Ka/Ks values below 1, indicating widespread purifying selection. Notable exceptions included the nad6 gene, which showed exceptionally high Ka/Ks values (ranging from 9.6 to 23), suggesting potential positive selection or relaxed functional constraints. Several other genes, including rps2 , rpl2 , ccmB , and ccmFC , also displayed elevated Ka/Ks ratios (> 3) in specific pairwise comparisons. Conversely, genes such as rps13 , cox3 , and mttB maintained Ka/Ks values near zero, reflecting strong functional conservation. These findings highlight heterogeneous evolutionary dynamics among mitochondrial genes and identify candidate markers, such as nad6 and ccmC , for future phylogenetic studies in Lauraceae. Discussion Structural and Compositional Features of Phoebe Mitogenomes Plant mitochondrial genomes are characterized by notable plasticity in both architectural organization and genetic composition [ 45 – 47 ]. Current assembly methodologies typically reconstruct these genomes as a single predominant circular molecule that incorporates the full complement of mitochondrial genes [ 48 , 49 ]. The mitochondrial genomes of the three Phoebe species exhibit considerable size variation, ranging from 807,952 to 865,014 bp. This range is consistent with the structural plasticity observed in other plant lineages, such as Ericales and Fagaceae [ 50 , 51 ]. A core set of 40 protein-coding genes, three rRNAs, and a variable number of tRNAs were identified, indicating functional conservation of mitochondrial genes despite extensive structural reorganization, a phenomenon also documented in related genera, including Cinnamomum and Quercus [ 52 , 53 ]. The presence of introns in genes such as nad4 and nad7 , along with interspecific variation in tRNA content, suggests a history of lineage-specific genomic rearrangements and gene loss/gain events. Notably, P. zhennan contained a higher abundance of SSRs and tandem repeats, which may reflect a more dynamic evolutionary trajectory or species-specific variation in DNA repair and recombination mechanisms, consistent with the role of repetitive elements in promoting structural diversification in plant mitogenomes [ 54 ]. Functional and Evolutionary Significance of RNA Editing and Codon Usage Patterns RNA editing was highly conserved across the three species, with each genome containing over 620 predicted editing sites. These C-to-U transitions significantly increase protein hydrophobicity through the introduction of leucine residues [ 55 ]. This pattern suggests an adaptive mechanism for optimizing membrane-associated proteins, aligning with findings in Primula and other angiosperms [ 56 – 58 ]. Synonymous codon usage was strongly biased toward A- and T-ending codons, a pattern commonly observed in plant mitochondrial genomes and likely influenced by both mutational bias and translational selection [ 59 – 61 ]. The identical RSCU profiles of P. yaiensis and P. zhennan further support their close phylogenetic relationship, consistent with patterns of codon conservation among congeneric species in Silene [ 62 – 64 ]. Interorganellar Gene Transfer and Evolutionary Constraints Substantial segments of plastid-derived DNA (MTPTs) were identified in all three mitogenomes, accounting for 5.66–6.06% of the total mitochondrial sequences. This finding aligns with previous reports of intracellular gene transfer in plants, including in Corydalis saxicola [ 65 , 66 ]. The preferential retention of functional tRNA and rRNA genes of plastid origin implies possible functional integration, as noted in other Lauraceae species [ 67 ]. The observed asymmetry in organellar DNA transfer, where plastid-derived sequences constitute a minor fraction of the mitochondrial genome despite their abundance in the plastome, may reflect differential selection pressures and structural stability between the two organelles [ 68 – 71 ]. Such interorganellar transfers are increasingly recognized as a major factor contributing to structural complexity and size variation in angiosperm mitogenomes [ 72 – 74 ]. Evolutionary Rates and Selective Pressures Pi and Ka/Ks ratios among mitochondrial protein-coding genes indicate heterogeneous evolutionary constraints [ 75 – 77 ]. The elevated Pi and Ka/Ks values for nad6 suggest that this gene may be under positive selection or subject to relaxed functional constraints, a pattern also reported in other plant groups where nad6 has been implicated in adaptive evolution [ 78 ]. In contrast, strong purifying selection was observed for cox3 , nad1 , and nad2 , underscoring their essential roles in oxidative phosphorylation and respiratory complex integrity. This pattern of evolutionary constraint is consistent with observations in Fagaceae and other perennial woody plants, where core energy-production genes are highly conserved [ 79 – 81 ]. Phylogenetic Inference and Taxonomic Implications Phylogenetic reconstruction based on mitochondrial protein-coding genes strongly supported the monophyly of Phoebe and a sister relationship with Cinnamomum , corroborating recent plastid-based phylogenies. This result underscores the utility of mitochondrial genomic data in resolving deep-level relationships within Lauraceae, despite their structural variability [ 82 ]. The close affinity between P. yaiensis and P. zhennan was further supported by shared codon usage and RNA editing profiles. These findings highlight the value of mitogenome as a complementary source of phylogenetic information, reinforcing conclusions drawn from nuclear and plastid genome [ 83 , 84 ]. Conclusion This study elucidates the structural and evolutionary dynamics of mitochondrial genomes in the genus Phoebe , highlighting the influence of RNA editing, MTPTs, and selective pressures on genomic diversity. The genomic resources and analytical insights presented here establish a foundation for future phylogenetic, comparative genomic, and conservation genetic studies in this economically and ecologically important genus, while contributing to a broader understanding of mitogenome evolution in woody plants. Declarations Authors ’ Contributions Conceptualization, X.Y., Z.W. and Q.D.; Methodology, Y.G., H.L., J.Z. and Z.W.; Software, F.H., X.H. and C.L.; Validation, M.L., D.C. and H.Z.; Formal Analysis, Z.W. and B.C.; Investigation, M.L., F.H., C.L., X.H., and D.C.; Resources, M.L., Y.G., H.L., J.Z. and Q.D.; Data Curation, X.Y., and Q.D.; Writing – Original Draft Preparation, Z.W.; Writing – Review & Editing, X.Y., P.P. and Q.D.; Visualization, X.Y. and Z.W.; Supervision, X.Y., P.P. and Q.D.; Project Administration, B.C., P.P. and Q.D.; Funding Acquisition, H.Z., B.C., and Q.D. All authors have read and agreed to the published version of the manuscript. Funding This research was funded by the Scientific and Technological Development of Forestry Research Projects in Chongqing (ZDXM2022-2 and YBXM2025-4). We thank all colleagues in our institution for technical assistance. Data availability The complete mitochondrial genomes of Phoebe zhennan , Phoebe bournei and Phoebe yaiensis are available at GenBank (https://www.ncbi.nlm.nih.gov/) with accession numbers PQ276137, PQ285648 and PQ285649. Ethics approval and consent to participate The methods involved in this study were carried out in compliance with local and national regulations. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Cole LW, Guo W, Mower JP, Palmer JD. High and Variable Rates of Repeat-Mediated Mitochondrial Genome Rearrangement in a Genus of Plants. Mol Biol Evol. 2018;35(11):2773–85. Sullivan AR, Eldfjell Y, Schiffthaler B, Delhomme N, Asp T, Hebelstrup KH, Keech O, Öberg L, Møller IM, Arvestad L, Street NR, Wang XR. The Mitogenome of Norway Spruce and a Reappraisal of Mitochondrial Recombination in Plants. Genome Biol Evol. 2020;12(1):3586–98. 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Jiang Z, Chen Y, Zhang X, Meng F, Chen J, Cheng X. Assembly and evolutionary analysis of the complete mitochondrial genome of Trichosanthes kirilowii , a traditional Chinese medicinal plant. PeerJ. 2024;12:e17747. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 18 May, 2026 Reviews received at journal 10 Dec, 2025 Reviewers agreed at journal 07 Dec, 2025 Reviewers invited by journal 27 Nov, 2025 Editor assigned by journal 26 Nov, 2025 Editor invited by journal 04 Nov, 2025 Submission checks completed at journal 04 Nov, 2025 First submitted to journal 04 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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17:10:36","extension":"html","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":188891,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7964358/v1/53fe86554fc6c5a65601d1e2.html"},{"id":97250366,"identity":"abcc7d01-3eb3-47e0-9d72-eed700585f59","added_by":"auto","created_at":"2025-12-02 13:14:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":842864,"visible":true,"origin":"","legend":"\u003cp\u003eCircular Maps and Gene Classification of the Three Phoebe Mitogenomes. Circular diagrams of the mitochondrial genomes of \u003cem\u003eP. yaiensis\u003c/em\u003e (A), \u003cem\u003eP. bournei\u003c/em\u003e (B), and \u003cem\u003eP. zhennan\u003c/em\u003e (C).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7964358/v1/a7a91c102071c08435197291.png"},{"id":97249595,"identity":"ad95ca66-7843-419e-a0e0-035d90716a79","added_by":"auto","created_at":"2025-12-02 13:12:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1013210,"visible":true,"origin":"","legend":"\u003cp\u003eCircos plot depicting tandems (red links) and SSRs (black links) gene pairs in the genomes of\u003cem\u003e P. bournei\u003c/em\u003e, \u003cem\u003eP. yaiensis\u003c/em\u003e, and \u003cem\u003eP. zhennan\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7964358/v1/62b770cf8a4ead7b02b8a372.png"},{"id":97249729,"identity":"8fd864f0-1900-49c2-a06c-d15aab5b76cb","added_by":"auto","created_at":"2025-12-02 13:13:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":124422,"visible":true,"origin":"","legend":"\u003cp\u003eRepeats in \u003cem\u003ePhoebe\u003c/em\u003e mitochondrial genomes. (A) Simple sequence repeats (SSRs) in \u003cem\u003eP. bournei\u003c/em\u003e, \u003cem\u003eP. yaiensis\u003c/em\u003e, and \u003cem\u003eP. zhennan\u003c/em\u003e mitogenomes. (B–D) Dispersed repeats in \u003cem\u003eP. bournei\u003c/em\u003e (B), \u003cem\u003eP. yaiensis\u003c/em\u003e (C), and \u003cem\u003eP. zhennan\u003c/em\u003e (D) mitogenomes, categorized by type and length.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7964358/v1/a96f171ad9c04b6c909dd898.png"},{"id":97250710,"identity":"140f8ac0-9d7a-4f63-b5a3-0fb11ebb9586","added_by":"auto","created_at":"2025-12-02 13:15:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":127604,"visible":true,"origin":"","legend":"\u003cp\u003eComparative analysis of RNA editing in three \u003cem\u003ePhoebe\u003c/em\u003e species. (A) Total number of RNA editing sites in each species. (B) Distribution of editing sites across protein-coding genes.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7964358/v1/d1b4f5db8f958731e4f4d6b3.png"},{"id":97184684,"identity":"8405400b-c14f-455f-937b-49375a10d18b","added_by":"auto","created_at":"2025-12-01 17:10:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":180954,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap visualization of RSCU values in (A) \u003cem\u003eP. bournei\u003c/em\u003e, (B) \u003cem\u003eP. yaiensis\u003c/em\u003e, and (C) \u003cem\u003eP. zhennan\u003c/em\u003emitochondrial genomes. The color gradient represents RSCU values, with red indicating preferred codons (RSCU \u0026gt; 1.0) and blue indicating under-represented codons (RSCU \u0026lt; 1.0).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7964358/v1/ee016076d246eefb9e51cfc3.png"},{"id":97184685,"identity":"f53099d1-81a0-491d-890e-7117fa3c9c06","added_by":"auto","created_at":"2025-12-01 17:10:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":368469,"visible":true,"origin":"","legend":"\u003cp\u003eInterorganellar gene transfer events in \u003cem\u003ePhoebe\u003c/em\u003e. Homologous sequences between the chloroplast (cp) and mitochondrial (mt) genomes were identified using BLASTN for \u003cem\u003eP. bournei \u003c/em\u003e(A), \u003cem\u003eP. yaiensis \u003c/em\u003e(B), and \u003cem\u003eP. zhennan \u003c/em\u003e(C).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7964358/v1/e7daf99e28cccc073d0f776d.png"},{"id":97184681,"identity":"26579289-8fb8-42f7-b5ec-e07f2e79d193","added_by":"auto","created_at":"2025-12-01 17:10:36","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":50041,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of \u003cem\u003ePhoebe\u003c/em\u003especies based on PCGs.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7964358/v1/e25656aaf2e1fee69805ee2b.png"},{"id":97249070,"identity":"e9db5c15-9b6e-425a-9617-6a485893cd75","added_by":"auto","created_at":"2025-12-02 13:10:10","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":186502,"visible":true,"origin":"","legend":"\u003cp\u003eNucleotide diversity (Pi) and Ka/Ks ratio of mitochondrial PCGs across three \u003cem\u003ePhoebe\u003c/em\u003e species and related Lauraceae. (A) Pi values of mitochondrial protein-coding genes. (B) Distribution of Ka/Ks ratios for all pairwise comparisons.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7964358/v1/eff9837a748daae53e2d8b6c.png"},{"id":97664527,"identity":"82d5a0ef-0c21-4891-82aa-69486d4a8e34","added_by":"auto","created_at":"2025-12-08 09:08:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4166223,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7964358/v1/94dde1d0-fc04-4aa3-a087-43cc5efe966e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Phylogenetic and Evolutionary Analysis of Mitochondrial Genomes in Phoebe","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlant mitochondrial genomes (mitogenomes) possess distinct evolutionary characteristics that render them particularly valuable for phylogenetic reconstruction [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Unlike other genomic compartments, mitogenomes combine slow sequence evolution with considerable structural plasticity, manifested through frequent genomic rearrangements and extensive intracellular gene transfer [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This structural dynamism underlies the remarkable size variation observed in plant mitogenomes, which range from approximately 66 kb in Viscum scurruloideum to over 11,000 kb in Silene conica [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Such substantial size disparities are primarily attributable to divergent repetitive DNA content and sequences acquired through intracellular transfer events [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Collectively, these attributes establish mitogenomes as powerful tools for resolving deep evolutionary relationships where conventional molecular markers offer limited phylogenetic resolution [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe Lauraceae family, a core group within the order Laurales, contains approximately 60 genera and 3,500 species [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Within this family, the genus \u003cem\u003ePhoebe\u003c/em\u003e comprises about 100 species distributed across tropical and subtropical Asia [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. umerous \u003cem\u003ePhoebe\u003c/em\u003e species are prized for their high-quality timber, characterized by its fine texture, durability, and natural decay resistance, making it ideal for high-end furniture, woodworking, and traditional construction [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Ecologically, these species also play a significant role in the structure and function of subtropical forest ecosystems [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDespite their economic and ecological importance, the phylogenetic relationships within the genus \u003cem\u003ePhoebe\u003c/em\u003e remain inadequately resolved [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Current classifications rely heavily on morphological traits that are prone to plasticity and convergent evolution [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. While molecular systematics using plastid genomes has become a fundamental approach, genomic resources for \u003cem\u003ePhoebe\u003c/em\u003e, particularly mitochondrial genomes, remain severely limited [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. This gap significantly constrains our ability to investigate their evolutionary history and phylogenetic relationships, such as the complex relationship between \u003cem\u003eP. zhennan\u003c/em\u003e and \u003cem\u003eP. bournei\u003c/em\u003e, at a comprehensive genomic level [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, we sequenced and assembled the complete mitogenomes of three \u003cem\u003ePhoebe\u003c/em\u003e species, \u003cem\u003eP. zhennan\u003c/em\u003e, \u003cem\u003eP. bournei\u003c/em\u003e, and \u003cem\u003eP. yaiensis\u003c/em\u003e. We conducted comparative genomic analyses to characterize their structural features, identify sequence variations, and infer selective pressures acting on protein-coding genes. Our findings provide foundational insights into the evolution of mitochondrial genomes within this valuable genus and the broader Lauraceae family.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePlant Materials and DNA Sequencing\u003c/h2\u003e\u003cp\u003eFresh leaves of \u003cem\u003eP. bournei\u003c/em\u003e, \u003cem\u003eP. yaiensis\u003c/em\u003e, and \u003cem\u003eP. zhennan\u003c/em\u003e were collected from their natural habitats in ChongQing. Total genomic DNA was extracted using a modified CTAB method [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. DNA quality and concentration were assessed by agarose gel electrophoresis, NanoDrop spectrophotometry, and Qubit fluorometry. For each species, paired-end sequencing libraries were constructed and sequenced on the Illumina NovaSeq 6000 platform. Additionally, long-read sequencing was performed using the Oxford Nanopore PromethION platform to facilitate the assembly of complex mitochondrial regions.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMitochondrial Genome Assembly and Annotation\u003c/h3\u003e\n\u003cp\u003eMitochondrial genomes were assembled using a hybrid strategy combining Illumina short reads and Nanopore long reads. Raw reads were quality-filtered with Fastp and GetOrganelle [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Filtered long reads were aligned to a reference set of plant mitochondrial core genes using Minimap2 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], and candidate mitochondrial sequences were extracted. Subsequent assembly was performed using Canu for error correction [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], followed by hybrid assembly with Unicycler [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Final assembly graphs were visualized and manually verified using Bandage [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eGene annotation was conducted with GeSeq [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], using \u003cem\u003eCinnamomum camphora\u003c/em\u003e and related Lauraceae species as references [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. tRNA genes were identified using tRNAscan-SE [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Circular maps of the mitochondrial genomes were generated using OGDRAW [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eAnalysis of Repetitive Sequences\u003c/h3\u003e\n\u003cp\u003eSimple sequence repeats (SSRs) were detected using MISA with default thresholds [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Tandem repeats were identified with Tandem Repeats Finder [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], and dispersed repeats (forward, palindromic, reverse, and complementary) were analyzed using BLASTN and REPuter [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The minimal length for dispersed repeats was set to 30 bp. The distribution and frequency of repeats were visualized with Circos [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003ePrediction of RNA Editing Sites\u003c/h3\u003e\n\u003cp\u003ePotential RNA editing sites in protein-coding genes (PCGs) were predicted using the online tool Deepred-Mt [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], which identifies C-to-U conversions based on sequence alignment and conservation. Only sites meeting a confidence threshold were retained. The impact of RNA editing on amino acid properties and codon position was analyzed.\u003c/p\u003e\n\u003ch3\u003eCodon Usage Analysis\u003c/h3\u003e\n\u003cp\u003eRelative Synonymous Codon Usage (RSCU) values were calculated for all mitochondrial PCGs using the CodonW [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Codon usage patterns were compared among the three \u003cem\u003ePhoebe\u003c/em\u003e species to assess evolutionary constraints and translational preferences.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eGene Transfer Analysis\u003c/h2\u003e\u003cp\u003eHomologous regions between mitochondrial and chloroplast genomes were identified using BLASTN with an E-value cutoff of 1e\u0026thinsp;\u0026minus;\u0026thinsp;5 and sequence identity\u0026thinsp;\u0026ge;\u0026thinsp;70%. The total length and proportion of mitochondrial plastid DNA (MTPTs) fragments were calculated. Functional annotation of transferred genes was performed to assess their potential roles.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eNucleotide Diversity and Selective Pressure\u003c/h3\u003e\n\u003cp\u003eShared mitochondrial PCGs from three \u003cem\u003ePhoebe\u003c/em\u003e species and four related Lauraceae taxa were aligned using MAFFT [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Nucleotide diversity (Pi) was calculated with DnaSP using a sliding window approach [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The nonsynonymous (Ka) and synonymous (Ks) substitution rates were estimated using KaKs_Calculator [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Genes with Ka/Ks\u0026thinsp;\u0026gt;\u0026thinsp;1 were considered under positive selection.\u003c/p\u003e\n\u003ch3\u003ePhylogenetic Analysis\u003c/h3\u003e\n\u003cp\u003eA maximum likelihood phylogenetic tree was constructed using conserved mitochondrial PCGs from 18 angiosperm species, including the three \u003cem\u003ePhoebe\u003c/em\u003e species and representatives from Lauraceae, and other families. Multiple sequence alignment was performed with MAFFT, and the best-fit substitution model (GTR\u0026thinsp;+\u0026thinsp;G) was selected using jModelTest [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The tree was built using maximum likelihood with 1000 bootstrap replicates to assess node support.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eAssembly and Characterization of Mitochondrial Genomes in\u003c/b\u003e \u003cb\u003ePhoebe\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe complete mitochondrial genomes of \u003cem\u003eP. bournei\u003c/em\u003e, \u003cem\u003eP. yaiensis\u003c/em\u003e, and \u003cem\u003eP. zhennan\u003c/em\u003e were assembled into circular mapping molecules with sizes of 865,014 bp, 830,727 bp, and 807,952 bp, respectively, displaying GC contents of 45.82%, 45.57%, and 46.03% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These mitochondrial genomes fall within the moderate size range among reported Lauraceae species.\u003c/p\u003e\u003cp\u003eGene annotation identified a conserved set of 40 PCGs across the three species, which were classified into several functional categories (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These include ATP synthase (5 genes), cytochrome c biogenesis (4 genes), cytochrome c oxidase (3 genes), NADH dehydrogenase (9 genes), succinate dehydrogenase (2 genes), and the conserved \u003cem\u003ematR\u003c/em\u003e gene. The total length of PCGs accounted for approximately 6.8\u0026ndash;7.2% of each mitogenome. Notably, six genes (\u003cem\u003eccmFn\u003c/em\u003e, \u003cem\u003erps3\u003c/em\u003e, \u003cem\u003enad2\u003c/em\u003e, \u003cem\u003enad4\u003c/em\u003e, \u003cem\u003enad5\u003c/em\u003e, and \u003cem\u003enad7\u003c/em\u003e) contained introns, with \u003cem\u003enad4\u003c/em\u003e containing three introns and the others possessing single introns.\u003c/p\u003e\u003cp\u003eThe mitochondrial genomes also contained three ribosomal RNA genes (\u003cem\u003errn5\u003c/em\u003e, \u003cem\u003errn18\u003c/em\u003e, \u003cem\u003errn26\u003c/em\u003e) and a variable number of transfer RNA genes. Among the 24 tRNA genes identified across the three species, most were single-copy, except for \u003cem\u003etrnM-CAT\u003c/em\u003e, which was duplicated in all three species. Interestingly, \u003cem\u003etrnF-GAA\u003c/em\u003e and \u003cem\u003etrnN-GTT\u003c/em\u003e were found to undergo RNA splicing before participating in protein translation. Comparative analysis revealed minor interspecific variations in tRNA gene content: \u003cem\u003etrnA-TGC\u003c/em\u003e and \u003cem\u003etrnI-GAT\u003c/em\u003e were absent in \u003cem\u003eP. yaiensis\u003c/em\u003e and \u003cem\u003eP. zhennan\u003c/em\u003e, \u003cem\u003etrnF-AAA\u003c/em\u003e was missing in \u003cem\u003eP. yaiensis\u003c/em\u003e, \u003cem\u003etrnL-GAG\u003c/em\u003e was absent in \u003cem\u003eP. bournei\u003c/em\u003e, and \u003cem\u003etrnV-GAC\u003c/em\u003e was not detected in \u003cem\u003eP. zhennan\u003c/em\u003e. Despite these variations, all three mitochondrial genomes maintained a complete set of tRNAs capable of recognizing all 20 standard amino acids.\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\u003eMitochondrial gene content across three \u003cem\u003ePhoebe\u003c/em\u003e species\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\u003eatp1 (1/1/1), atp4 (1/1/1), atp6 (1/1/1), atp8 (1/1/1), atp9 (2/2/2)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCytochrome c biogenesis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eccmB (1/1/1), ccmC (1/1/1), ccmFc (1/1/1), ccmFn (1/1/1)\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\u003ecox1 (1/1/1), cox2 (1/1/1), cox3 (1/1/1)\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\u003enad1 (1/1/1), nad2 (1/1/1), nad3 (1/1/1), nad4 (1/1/1), nad4L (1/1/1), nad5 (1/1/1), nad6 (1/1/1), nad7 (1/1/1), nad9 (1/1/1)\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\u003errn18 (1/1/1), rrn26 (1/1/1), rrn5 (1/1/1)\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\u003esdh3 (1/1/1), sdh4 (1/1/1)\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\u003etrnA-TGC (0/0/1), trnC-GCA (1/1/1), trnD-GTC (1/1/1), trnE-TTC (1/1/1), trnF-AAA (0/1/1), trnF-GAA (1/1/1), trnG-GCC (1/1/1), trnH-GTG (1/1/1), trnI-GAT (0/0/1), trnK-CTT (1/1/1), trnK-TTT (1/1/1), trnL-CAA (1/1/1), trnL-GAG (1/1/0), trnM-CAT (1/1/1), trnN-GTT (1/1/1), trnP-TGG (1/1/1), trnQ-TTG (1/1/1), trnR-GCG (1/1/1), trnR-TCT (1/1/1), trnS-GCT (1/1/1), trnS-TGA (1/1/1), trnT-GGT (1/1/1), trnT-TGT (1/1/1), trnV-GAC (1/0/1), trnW-CCA (1/1/1), trnY-GTA (1/1/1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eFor each gene, presence is coded as 1 and absence as 0 in the ternary code corresponding to \u003cem\u003eP. yaiensis\u003c/em\u003e, \u003cem\u003eP. zhennan\u003c/em\u003e, and \u003cem\u003eP. bournei\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eComparative Analysis of Repetitive Sequences in\u003c/b\u003e \u003cb\u003ePhoebe\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe characterized repetitive sequences in the three \u003cem\u003ePhoebe\u003c/em\u003e mitogenomes, including SSRs, tandem repeats, and dispersed repeats. A comprehensive analysis of SSRs identified a total of 212 repeats across the three mitogenomes. Tetranucleotide repeats were the most abundant, accounting for 36.3% (n\u0026thinsp;=\u0026thinsp;77) of the total, followed by mononucleotide (24.5%, n\u0026thinsp;=\u0026thinsp;52), dinucleotide (22.6%, n\u0026thinsp;=\u0026thinsp;48), trinucleotide (12.3%, n\u0026thinsp;=\u0026thinsp;26), pentanucleotide (3.3%, n\u0026thinsp;=\u0026thinsp;7), and hexanucleotide (0.9%, n\u0026thinsp;=\u0026thinsp;2) repeats. The A/T motif was predominant among all SSR motifs, representing 26.4% (n\u0026thinsp;=\u0026thinsp;56) of the total, with other notable motifs including AG/CT (8.5%, n\u0026thinsp;=\u0026thinsp;18), GA/TC (8.0%, n\u0026thinsp;=\u0026thinsp;17), and AAAG/CTTT (5.2%, n\u0026thinsp;=\u0026thinsp;11) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The distribution of SSRs gene pairs was visualized using Circos plots, which revealed that the \u003cem\u003eP. zhennan\u003c/em\u003e mitogenome contained the highest number of SSR gene pairs (150), exceeding those in \u003cem\u003eP. bournei\u003c/em\u003e (120 pairs) and \u003cem\u003eP. yaiensis\u003c/em\u003e (100 pairs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eThe analysis of tandem repeat gene pairs revealed a parallel trend, with \u003cem\u003eP. zhennan\u003c/em\u003e (50 pairs), \u003cem\u003eP. bournei\u003c/em\u003e (40 pairs), and \u003cem\u003eP. yaiensis\u003c/em\u003e (30 pairs) possessing the greatest to least abundance (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The prevalence of SSRs over tandem repeats across all three species suggests a fundamental role of SSRs in their genomic structure and evolutionary dynamics. The notably higher abundance of both repeat types in \u003cem\u003eP. zhennan\u003c/em\u003e may indicate a history of more active duplication events or distinct evolutionary pressures shaping its mitochondrial genome.\u003c/p\u003e\u003cp\u003eAnalysis of the three mitogenomes revealed 276 dispersed repeats, which had an average length of 68 bp (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u0026ndash;D). These were exclusively classified into 149 palindromic repeats and 127 forward repeats, with no reverse or complement repeats detected. Length distribution analysis showed that the majority (64.1%) of dispersed repeats ranged from 30 to 69 bp. Notably, within the 30\u0026ndash;39 bp length category, both forward and palindromic repeats were equally represented (28 each, constituting 20.3% of the total dispersed repeats).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eConserved RNA Editing Enhances Hydrophobicity of Mitochondrial Proteins in\u003c/b\u003e \u003cb\u003ePhoebe\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAnalysis of mitochondrial protein-coding genes revealed highly conserved RNA editing patterns across the three \u003cem\u003ePhoebe\u003c/em\u003e species, with 623, 621, and 620 editing sites in \u003cem\u003eP. bournei\u003c/em\u003e, \u003cem\u003eP. yaiensis\u003c/em\u003e, and \u003cem\u003eP. zhennan\u003c/em\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Gene-specific analysis showed that \u003cem\u003enad4\u003c/em\u003e was the most heavily edited (68 sites), while ribosomal genes \u003cem\u003erpl2\u003c/em\u003e and \u003cem\u003erps14\u003c/em\u003e contained the fewest edits (2 sites) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). RNA editing predominantly targeted the second codon position (63.1%), followed by the first position (34.2%). A notable 2.7% of edits involved concurrent modifications at both first and second bases, enabling direct amino acid conversions such as proline to phenylalanine. At the protein level, 89.2% of editing events altered amino acid identity, with a strong bias toward hydrophobic residues. Leucine was the most frequent substitution (41.5%), mainly from serine (155 sites) and proline (138 sites). Critically, 93.5% of non-silent edits increased protein hydrophobicity, indicating a crucial role for RNA editing in refining mitochondrial protein structure in \u003cem\u003ePhoebe\u003c/em\u003e species.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eConserved Codon Usage Bias Reflects Evolutionary Relationships in\u003c/b\u003e \u003cb\u003ePhoebe\u003c/b\u003e Analysis of codon usage patterns in the three \u003cem\u003ePhoebe\u003c/em\u003e mitochondrial genomes revealed highly conserved synonymous codon preferences, with identical RSCU profiles between \u003cem\u003eP. yaiensis\u003c/em\u003e and \u003cem\u003eP. zhennan\u003c/em\u003e. All species exhibited a pronounced bias toward A/T-ending codons (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), preferentially using UUA (Leu1, 1.40), GCU (Ala, 1.67), and CCU (Pro, 1.39) while avoiding C-ending codons such as CUG (Leu2, 0.55) and GCG (Ala, 0.47). Amino acid-specific patterns showed that UCU (Ser1, 1.37) was favored over AGC (Ser2, 0.54), while CGU (Arg, 1.28) and CGA (Arg, 1.33) were preferred over CGC (Arg, 0.63). The UGA codon functioned as tryptophan with strong underrepresentation (0.15). This conserved codon usage pattern suggests shared evolutionary constraints and indicates a closer phylogenetic relationship between \u003cem\u003eP. yaiensis\u003c/em\u003e and \u003cem\u003eP. zhennan\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePredominance of Chloroplast-Derived DNA in\u003c/b\u003e \u003cb\u003ePhoebe\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSequence alignment identified numerous MTPTs between the organellar genomes of the three \u003cem\u003ePhoebe\u003c/em\u003e species. A total of 73 to 83 homologous fragments were detected in each species, with a cumulative length representing 29.94\u0026ndash;32.70% of the chloroplast genome but only 5.66\u0026ndash;6.06% of the mitochondrial genome, indicating an asymmetric transfer bias (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Functional annotation revealed that these MTPTs contained multiple functional RNA genes. The transfer from chloroplast to mitochondrion was particularly notable, encompassing 4 tRNA genes and 30\u0026ndash;34 rRNA genes across the species. Furthermore, specific homologous gene pairs, such as \u003cem\u003etrnK-UUU/trnK-TTT\u003c/em\u003e and \u003cem\u003etrnA-UGC/trnA-TGC\u003c/em\u003e, were conserved between the genomes, providing direct evidence for the interorganellar transfer of functional genetic material. These findings demonstrate widespread and biased DNA transfer from the chloroplast to the mitochondrial genome in \u003cem\u003ePhoebe\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhylogenetic Analysis of Mitochondrial Genomes in\u003c/b\u003e \u003cb\u003ePhoebe\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOur phylogenetic reconstruction based on mitochondrial PCGs from 18 species revealed strong nodal support (\u0026ge;\u0026thinsp;90) for most relationships within the mitochondrial gene tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Within the genus \u003cem\u003ePhoebe\u003c/em\u003e, the three species formed a fully supported monophyletic group (100), with \u003cem\u003eP. zhennan\u003c/em\u003e and \u003cem\u003eP. yaiensis\u003c/em\u003e exhibiting the closest phylogenetic affinity. This \u003cem\u003ePhoebe\u003c/em\u003e clade showed a strongly supported sister relationship with \u003cem\u003eCinnamomum\u003c/em\u003e species, while \u003cem\u003eP. bournei\u003c/em\u003e was resolved as the basalmost lineage within the genus. The broader phylogenetic context clearly separated lauraceous species from monocot Poaceae and other eudicot lineages, with gymnosperms \u003cem\u003eGinkgo biloba\u003c/em\u003e and \u003cem\u003ePinus taeda\u003c/em\u003e consistently positioned as outgroups.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eAnalysis of Evolutionary Rate and Selective Pressure\u003c/h2\u003e\u003cp\u003eTo assess sequence variation, we estimated Pi for the mitochondrial PCGs that were conserved orthologous among the three \u003cem\u003ePhoebe\u003c/em\u003e species and four related Lauraceae taxa. The analysis revealed substantial variation in Pi values across genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). The \u003cem\u003enad6\u003c/em\u003e gene exhibited the highest nucleotide diversity (0.0525), indicating pronounced sequence divergence. Other genes with notably high Pi values included \u003cem\u003eatp9\u003c/em\u003e (0.0263), \u003cem\u003eccmC\u003c/em\u003e (0.0257), \u003cem\u003ecox2\u003c/em\u003e (0.0252), and \u003cem\u003esdh3\u003c/em\u003e (0.0249). In contrast, genes such as \u003cem\u003ecox3\u003c/em\u003e (0.0011), \u003cem\u003enad2\u003c/em\u003e (0.0014), and \u003cem\u003enad1\u003c/em\u003e (0.0021) displayed minimal diversity, suggesting strong evolutionary conservation and functional constraints.\u003c/p\u003e\u003cp\u003eTo evaluate selective pressures acting on the mitochondrial PCGs, the ratio of Ka/Ks was calculated for each gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). The majority of PCGs exhibited Ka/Ks values below 1, indicating widespread purifying selection. Notable exceptions included the \u003cem\u003enad6\u003c/em\u003e gene, which showed exceptionally high Ka/Ks values (ranging from 9.6 to 23), suggesting potential positive selection or relaxed functional constraints. Several other genes, including \u003cem\u003erps2\u003c/em\u003e, \u003cem\u003erpl2\u003c/em\u003e, \u003cem\u003eccmB\u003c/em\u003e, and \u003cem\u003eccmFC\u003c/em\u003e, also displayed elevated Ka/Ks ratios (\u0026gt;\u0026thinsp;3) in specific pairwise comparisons. Conversely, genes such as \u003cem\u003erps13\u003c/em\u003e, \u003cem\u003ecox3\u003c/em\u003e, and \u003cem\u003emttB\u003c/em\u003e maintained Ka/Ks values near zero, reflecting strong functional conservation. These findings highlight heterogeneous evolutionary dynamics among mitochondrial genes and identify candidate markers, such as \u003cem\u003enad6\u003c/em\u003e and \u003cem\u003eccmC\u003c/em\u003e, for future phylogenetic studies in Lauraceae.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cb\u003eStructural and Compositional Features of\u003c/b\u003e \u003cb\u003ePhoebe\u003c/b\u003e \u003cb\u003eMitogenomes\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePlant mitochondrial genomes are characterized by notable plasticity in both architectural organization and genetic composition [\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Current assembly methodologies typically reconstruct these genomes as a single predominant circular molecule that incorporates the full complement of mitochondrial genes [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The mitochondrial genomes of the three \u003cem\u003ePhoebe\u003c/em\u003e species exhibit considerable size variation, ranging from 807,952 to 865,014 bp. This range is consistent with the structural plasticity observed in other plant lineages, such as Ericales and Fagaceae [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. A core set of 40 protein-coding genes, three rRNAs, and a variable number of tRNAs were identified, indicating functional conservation of mitochondrial genes despite extensive structural reorganization, a phenomenon also documented in related genera, including \u003cem\u003eCinnamomum\u003c/em\u003e and \u003cem\u003eQuercus\u003c/em\u003e [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. The presence of introns in genes such as \u003cem\u003enad4\u003c/em\u003e and \u003cem\u003enad7\u003c/em\u003e, along with interspecific variation in tRNA content, suggests a history of lineage-specific genomic rearrangements and gene loss/gain events. Notably, \u003cem\u003eP. zhennan\u003c/em\u003e contained a higher abundance of SSRs and tandem repeats, which may reflect a more dynamic evolutionary trajectory or species-specific variation in DNA repair and recombination mechanisms, consistent with the role of repetitive elements in promoting structural diversification in plant mitogenomes [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eFunctional and Evolutionary Significance of RNA Editing and Codon Usage Patterns\u003c/h2\u003e\u003cp\u003eRNA editing was highly conserved across the three species, with each genome containing over 620 predicted editing sites. These C-to-U transitions significantly increase protein hydrophobicity through the introduction of leucine residues [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. This pattern suggests an adaptive mechanism for optimizing membrane-associated proteins, aligning with findings in \u003cem\u003ePrimula\u003c/em\u003e and other angiosperms [\u003cspan additionalcitationids=\"CR57\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Synonymous codon usage was strongly biased toward A- and T-ending codons, a pattern commonly observed in plant mitochondrial genomes and likely influenced by both mutational bias and translational selection [\u003cspan additionalcitationids=\"CR60\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. The identical RSCU profiles of \u003cem\u003eP. yaiensis\u003c/em\u003e and \u003cem\u003eP. zhennan\u003c/em\u003e further support their close phylogenetic relationship, consistent with patterns of codon conservation among congeneric species in \u003cem\u003eSilene\u003c/em\u003e [\u003cspan additionalcitationids=\"CR63\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eInterorganellar Gene Transfer and Evolutionary Constraints\u003c/h2\u003e\u003cp\u003eSubstantial segments of plastid-derived DNA (MTPTs) were identified in all three mitogenomes, accounting for 5.66\u0026ndash;6.06% of the total mitochondrial sequences. This finding aligns with previous reports of intracellular gene transfer in plants, including in \u003cem\u003eCorydalis saxicola\u003c/em\u003e [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. The preferential retention of functional tRNA and rRNA genes of plastid origin implies possible functional integration, as noted in other Lauraceae species [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. The observed asymmetry in organellar DNA transfer, where plastid-derived sequences constitute a minor fraction of the mitochondrial genome despite their abundance in the plastome, may reflect differential selection pressures and structural stability between the two organelles [\u003cspan additionalcitationids=\"CR69 CR70\" citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. Such interorganellar transfers are increasingly recognized as a major factor contributing to structural complexity and size variation in angiosperm mitogenomes [\u003cspan additionalcitationids=\"CR73\" citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eEvolutionary Rates and Selective Pressures\u003c/h2\u003e\u003cp\u003ePi and Ka/Ks ratios among mitochondrial protein-coding genes indicate heterogeneous evolutionary constraints [\u003cspan additionalcitationids=\"CR76\" citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. The elevated Pi and Ka/Ks values for \u003cem\u003enad6\u003c/em\u003e suggest that this gene may be under positive selection or subject to relaxed functional constraints, a pattern also reported in other plant groups where \u003cem\u003enad6\u003c/em\u003e has been implicated in adaptive evolution [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. In contrast, strong purifying selection was observed for \u003cem\u003ecox3\u003c/em\u003e, \u003cem\u003enad1\u003c/em\u003e, and \u003cem\u003enad2\u003c/em\u003e, underscoring their essential roles in oxidative phosphorylation and respiratory complex integrity. This pattern of evolutionary constraint is consistent with observations in Fagaceae and other perennial woody plants, where core energy-production genes are highly conserved [\u003cspan additionalcitationids=\"CR80\" citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003ePhylogenetic Inference and Taxonomic Implications\u003c/h2\u003e\u003cp\u003ePhylogenetic reconstruction based on mitochondrial protein-coding genes strongly supported the monophyly of \u003cem\u003ePhoebe\u003c/em\u003e and a sister relationship with \u003cem\u003eCinnamomum\u003c/em\u003e, corroborating recent plastid-based phylogenies. This result underscores the utility of mitochondrial genomic data in resolving deep-level relationships within Lauraceae, despite their structural variability [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. The close affinity between \u003cem\u003eP. yaiensis\u003c/em\u003e and \u003cem\u003eP. zhennan\u003c/em\u003e was further supported by shared codon usage and RNA editing profiles. These findings highlight the value of mitogenome as a complementary source of phylogenetic information, reinforcing conclusions drawn from nuclear and plastid genome [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study elucidates the structural and evolutionary dynamics of mitochondrial genomes in the genus \u003cem\u003ePhoebe\u003c/em\u003e, highlighting the influence of RNA editing, MTPTs, and selective pressures on genomic diversity. The genomic resources and analytical insights presented here establish a foundation for future phylogenetic, comparative genomic, and conservation genetic studies in this economically and ecologically important genus, while contributing to a broader understanding of mitogenome evolution in woody plants.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u003c/strong\u003e\u003cstrong\u003e\u0026rsquo;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConceptualization, X.Y., Z.W. and Q.D.; Methodology, Y.G., H.L., J.Z. and Z.W.; Software, F.H., X.H. and C.L.; Validation, M.L., D.C. and H.Z.; Formal Analysis, Z.W. and B.C.; Investigation, M.L., F.H., C.L., X.H., and D.C.; Resources, M.L., Y.G., H.L., J.Z. and Q.D.; Data Curation, X.Y., and Q.D.; Writing \u0026ndash; Original Draft Preparation, Z.W.; Writing \u0026ndash; Review \u0026amp; Editing, X.Y., P.P. and Q.D.; Visualization, X.Y. and Z.W.; Supervision, X.Y., P.P. and Q.D.; Project Administration, B.C., P.P. and Q.D.; Funding Acquisition, H.Z., B.C., and Q.D. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Scientific and Technological Development of Forestry Research Projects in Chongqing (ZDXM2022-2 and YBXM2025-4). We thank all colleagues in our institution for technical assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe complete mitochondrial genomes of \u003cem\u003ePhoebe zhennan\u003c/em\u003e, \u003cem\u003ePhoebe bournei\u003c/em\u003e and \u003cem\u003ePhoebe yaiensis\u003c/em\u003e are available at GenBank (https://www.ncbi.nlm.nih.gov/) with accession numbers PQ276137, PQ285648 and PQ285649.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe methods involved in this study were carried out in compliance with local and national regulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCole LW, Guo W, Mower JP, Palmer JD. 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PeerJ. 2024;12:e17747.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Phoebe, mitochondrial genome, RNA editing, codon usage, phylogeny","lastPublishedDoi":"10.21203/rs.3.rs-7964358/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7964358/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe genus \u003cem\u003ePhoebe\u003c/em\u003e represents ecologically and economically important members of the Lauraceae family, but their mitochondrial genomes remain largely uncharacterized. We report the complete mitochondrial genome sequences of three species \u003cem\u003ePhoebe zhennan\u003c/em\u003e, \u003cem\u003ePhoebe bournei\u003c/em\u003e and \u003cem\u003ePhoebe yaiensis\u003c/em\u003e, ranging in size from 807,952 to 865,014 base pairs. All three mitogenomes contain 40 conserved protein-coding genes. Comparative analysis identified abundant repetitive sequences, with \u003cem\u003eP. zhennan\u003c/em\u003e showing the highest repeat content. RNA editing sites were highly conserved and predominantly increased encoded protein hydrophobicity. Synonymous codon usage favored A/T endings across all species, supporting closer phylogenetic affinity between \u003cem\u003eP. yaiensis\u003c/em\u003e and \u003cem\u003eP. zhennan\u003c/em\u003e. Chloroplast-derived sequences constituted 5.66\u0026ndash;6.06% of the mitogenomes. Evolutionary analysis indicated widespread purifying selection, though \u003cem\u003enad6\u003c/em\u003e and several other genes exhibited positive selection signals. Phylogenetic reconstruction confirmed \u003cem\u003ePhoebe\u003c/em\u003e as monophyletic and sister to \u003cem\u003eCinnamomum\u003c/em\u003e. This study provides foundational mitogenomic resources for understanding evolutionary relationships within \u003cem\u003ePhoebe\u003c/em\u003e and Lauraceae.\u003c/p\u003e","manuscriptTitle":"Phylogenetic and Evolutionary Analysis of Mitochondrial Genomes in Phoebe","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-01 17:10:31","doi":"10.21203/rs.3.rs-7964358/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"244623165482756744230202132366539799147","date":"2026-05-18T15:44:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-10T13:42:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"311012807166802226634001340801679756385","date":"2025-12-07T10:03:56+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-27T12:08:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-26T13:22:10+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-04T09:20:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-04T08:23:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-11-04T08:17:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ddecad70-d535-4a13-ab32-02cc6f35437f","owner":[],"postedDate":"December 1st, 2025","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"244623165482756744230202132366539799147","date":"2026-05-18T15:44:14+00:00","index":97,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T17:10:31+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-01 17:10:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7964358","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7964358","identity":"rs-7964358","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}