Abstract
Toona is of a high economic value and has a great potential for industrial and medicinal utilization. Currently, the mitochondrial genome information of this genus is incomplete. Here we sequenced the mitochondrial genomes of T. rubriflora and T. microcarpa, and re-sequenced four varieties of T. ciliata. The mitochondrial genome of T. rubriflora was 653,710 bp in length, with a typical circular structure and the GC content of 45.42%. The mitochondrial genome of T. microcarpa was successfully assembled in two circulars, designated as chromosome 1 and chromosome 2, respectively. Chromosome 1 had 474,320 bp and its GC content was 45.37%. Chromosome 2 had 166,958 bp and its GC content was 46.64%. Comparative analyses of Toona mitochondrial genomes revealed that species were highly conserved in GC content, coding gene sequences and codon usage frequency. Mononucleotide repeats were the dominant type of genome repeats. Small differences existed among species in RNA editing sites, intracellular genome homology between mitochondrial and chloroplast genomes and between mitochondrial and nuclear genomes. Most protein coding genes (PCGs) were under purifying selection. Species in Toona were relatively recently divergent, and the varieties of T. ciliata were well genetically mixed. Overall, this study provided comprehensive information on Toona mitochondrial genomes, which could be used for species identification and molecular studies on Toona.
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Introduction
The Toona genus belongs to the Meliaceae family and is widely distributed in Asia and Oceania1. In China, the species of Toona are distributed in southern and southwestern regions as well as across northern regions. The genus is composed of four species in China: T. sinensis, T. ciliata, T. microcarpa, and T. rubriflora. Five varieties of T. ciliata are recognized according to leaf and flower characteristics, including T. ciliata var. ciliata, T. ciliata var. yunnanensis, T. ciliata var. henryi, T. ciliata var. sublaxiflora, and T. ciliata var. pubescens. Historically, the genus was misclassified as the Cedrela genus and officially recognized as a separate genus by M.J. Roemer in 1846. The misclassifications can also be indicated from multiple synonyms of a species. From Royal Botanic Gardens at Kew (http://www.kew.org/, accessed on September 9, 2024), there were eighteen synonyms for T. sinensis and the classified genera were Ailanthus, Cedrela, Mioptrila, Surenus, and Toona. There were twenty-one synonyms for T. ciliata and the classified genera were Cedrela, Surenus, Swietenia and Toona. There were eight synonyms for T. microcarpa and the classified genera were Cedrela, Surenus, Swietenia and Toona. This taxonomic complexity probably arose from large morphological variation within and between species. For example, inhabitants in western Yunnan historically considered the purple buds of T. sinensis as"T. microcarpa"and occasionally as"T. ciliata“or”T. ciliata var. pubescens". In studying the nutrient composition of young leaves of T. microcarpa under two different cultivation conditions, Luo et al. (2014) mistakenly identified the local purple buds of T. sinensis as “T. microcarpa”2. Other processes, such as interspecific hybridization3,4, may also lead to misclassification. A similar situation occurred in identifying varieties of T. ciliata based on the leaf and flower variations1. These varieties are sympatric or partially overlapping in geographical distribution and bring challenges to delimitate them. Thus, it is significant in taxonomy to elucidate the phylogenetic relationships among species in genus Toona and among varieties in T. ciliata.
Practically, the species of Toona are ideal materials for making furniture and interior decoration because of beautiful texture and excellent decay resistance5,6,7,8. They also have medicinal values in China9,10,11,12. T. sinensis has been cultivated in China for more than 2000 years. T. ciliata is not as widely distributed as T. sinensis in China but has a great potential for plantations. The species has been classified as an endangered species at Level II in China due to over logging and low nature regeneration13,14,15. Both T. microcarpa and T. rubriflora are more locally distributed. Although T. microcarpa was not on the list of rare and endangered plants in China, Zou et al. pointed out that it would be potentially endangered because global warming could influence the survival and reproduction of this species16. Thus, it is practically important to clarify their genetic divergence for conservation of Toona genetic resources.
Previous studies have examined genetic divergence among partial species. Wang et al. examined the phylogenetic relationship between T. sinensis and T. ciliata using nuclear genome sequences and estimated their divergent time at about 15.06 (6–25) Mya3,17,18. Xiao et al. used nuclear ribosomal DNA (nrDNA) ITS and mitochondrial DNA (mtDNA) markers to assess population variation in terms of the T. ciliata complex19. Both marker analyses showed the presence of geographic variation20,21.
Chloroplast genomes (cpDNA) were also applied to evaluating phylogenetic relationship among partial species in genus Toona. Three species (T. sinensis, T. ciliata, T. microcarpa) have comparable lengths of cpDNA sequences22. Xiao et al. examined phylogenetic relationships among T. sinensis and four varieties of T. ciliata19. Wang et al. used cpDNA markers to investigate geographic variation among populations in terms of the T. ciliata complex23. However, mitochondrial genome (mtDNA) has not been applied to assess phylogenetic relationships among species in genus Toona or among varieties of T. ciliata. Although mtDNA sequences of T. ciliata (NC_065060.1) and T. sinensis (NC_065061.1) were recorded in the literature, a comparison was only confined to these two species, without fully exploring the phylogeny of Toona24. It is well recognized that mtDNA is characterized by high variability in genomic structure, size, and rearrangement but conservative in coding sequences, which poses challenges for sequencing and assembly25. However, mtDNA is also used in comparative genomic analyses due to several key features, such as the high rate of evolution in animals or a lower mutation rate in plants compared with cpDNA, simple structure and maternal inheritance. Records showed that mtDNA was effective in elucidating phylogenetic connections between closely related species26,27. Here, we sequenced and assembled mitochondrial genomes of T. rubriflora and T. microcarpa and re-sequenced varieties of T. ciliata for assessing genomic variations among taxa in Toona.
The purposes of this study were (1) to compare the similarity and divergence in mitochondrial genome among species of genus Toona; (2) to analyze evolutionary mechanisms underlying species divergence; and (3) to reconstruct the phylogenetic relationships among species of genus Toona and among varieties of T. ciliata. The overall analyses provided complementary information to the existing taxonomic division of taxa and consolidated previous recommendations of genetic conservation on the T. ciliata complex from the perspective of mitochondrial genomes.
Results
Structural comparison and gene annotation
Substantial structural variation in mtDNA sequence occurred among species of Toona. For T. ciliata, we used the mtDNA sequence of individual NC_065060.1 of T. ciliata (GenBank Access NC_065060) as the reference sequence. The mtDNA sequences of twenty samples of T. ciliata could be categorized into two classes based on the coverage rate of sequencing reads against the reference sequence (Supplementary Table S1). Class A sequences had 100% coverage in alignment with the reference sequence. Small variation in the mtDNA sequences occurred among 16 class A samples. There were 119 single-nucleotide polymorphisms (SNPs) in total. Class B was represented by T. ciliata var. yunnanensis 1, with approximately 95% coverage in alignment with the reference sequence. Its mitochondrial genome was assembled in circular structure with 831,995 bp in length (Fig. 1A). The GC content was 45.40%, and the four base contents were 27.26% A, 27.33% T, 22.85% G, and 22.54% C (Table 1). There were 80 genes in total (Supplementary Table S2), including 35 PCGs, 42 transfer RNAs (tRNAs) and 3 ribosomal RNAs (rRNAs). Notably, T. ciliata var. yunnanensis 1 had a larger genome size compared to the other varieties of T. ciliata although comparable GC contents were observed. The mitochondrial genome of T. rubriflora was assembled in circular structure, with 653,710 bp in length (Fig. 1B). The GC content was 45.42%, and four base contents were comparable to those in mtDNA of T. ciliata var. yunnanensis 1 (Table 1). There were 70 genes in total (Supplementary Table S3), including 35 PCGs, 32 tRNAs, and 3 rRNAs (Table 2).
The mitochondrial genome maps of three species of Toona. (A) T. ciliata var. yunnanensis 1; (B) T. rubriflora; (C) T. microcarpa (chromosome 1); (D) T. microcarpa (chromosome 2).
The mitochondrial genome of T. microcarpa was successfully assembled in two circulars, designated as chromosome 1 and chromosome 2, respectively (Figs. 1C and 1D). Chromosome 1 had 474,320 bp and its GC content was 45.37%. The nucleotide compositions were 27.33% A, 27.30% T, 22.88% G and 22.50% C (Table 1). There were 44 genes in total (Supplementary Table S4), including 25 PCGs, 18 tRNAs and 1 rRNA. Chromosome 2 had 166,958 bp, where A, T, G, and C contents were 27.35%, 45, 27.02%, 22.79%, and 22.85%, respectively. The GC content was 46.64%. There were 26 genes in total, including 10 PCGs, 14 tRNAs and 2 rRNAs.
There were 35 PCGs and 3 rRNAs genes shared among species, while the number of tRNAs slightly varied from 32 to 42 genes across species. Except for T. sinensis, the protein-coding sequences for each species were 31,788 bp in length (Table 1). Among 35 PCGs, there were 19 genes encoding the proteins that were related to electron transport and ATP synthesis, including 9 for complex I (nad1, nad2, nad3, nad4, nad 4 l, nad5, nad6, nad7, and nad9), 2 for complex II (sdh3 and sdh4), 1 for complex III (cob), 3 for complex IV (cox1, cox2, cox3), and 5 for complex V (atp1, atp4, atp6, atp8, and atp9). Furthermore, there were 9 genes associated with ribosomal proteins (rpl2, rpl5, rpl10, rpl16, rps1, rps3, rps4, rps10 and rps12), 4 with cytochrome c biogenesis (ccmB, ccmC, ccmFN, and ccmFC), and 2 with other functions (matR and mttB). In chromosome 1 of T. microcarpa, there were 4 genes for complex I (nad3, nad 4 l, nad7, and nad9), 1 gene for complex II (sdh4), 1 gene for complex III (cob), 3 genes for complex IV (cox1, cox2, cox3), and 4 genes for complex V (atp1, atp4, atp6, and atp8). Additionally, there were 8 ribosomal protein genes (rpl2, rpl5, rpl10, rpl16, rps1, rps3, rps10, rps12), 2 genes involved in cytochrome c biogenesis (ccmC and ccmFC), and 2 other genes (matR and mttB). In chromosome 2 of T. microcarpa, there were 5 genes for complex I (nad1, nad2, nad4, nad5, and nad6), 1 gene for complex II (sdh3), 1 gene for complex V (atp9), 1 gene for ribosomal protein (rps4), and 2 genes responsible for cytochrome c biogenesis (ccmB and ccmFN).
Protein coding genes and codon usage
Comparison of PCGs along genome revealed that only the matR-nad3-rps12 and rps1-rpl16-rps3 gene fragments were fully conserved in all species (Fig. 2). The gene fragment atp8-cox3-sdh4-mttB in T. ciliata was entirely reversed, compared with the other species. Notably, T. ciliata var. yunnanensis 1 exhibited a high frequency of gene rearrangements (rps12-rpl2-nad7-sdh3 and rps4-nad6-nad4l-atp4-ccmC).
Structural comparison of protein-coding genes in mitochondrial genomes across species of Toona.
Most PCGs had ATG as the start codon except for four genes (rps10, rps4, nad1, nad 4 l) where ACG was the start codon. Gene rps10 possessed a non-typical stop codon CGA. Figure 3A shows the frequencies of different amino acids in PCGs in three newly sequenced species. The Leu was the most frequent amino acid, followed by Ser and Ile. Codons TTT (Phe), TTC (Phe) and ATT (Ile) were most common among 64 codons. Each amino acid had its preferred codon except for Met (ATG) and Trp (TGG) that had only one codon. All species had 30 optimal codons (RSCU > 1) except for the start codons ATG and TGG that had the RSCU values of 1 (Fig. 3B). The GCT codon had the highest RSCU value. The RSCU values for all NNA and NNT codons exceeded 1.00 except for GCA, ATA, and CTA (Supplementary Table S5).
Codon usage frequencies and RSCU values of amino acids in mitochondrial genomes of Toona. (A) Codon usage frequencies in protein-coding genes in T. ciliata var. yunnanensis 1, T. rubriflora and T. microcarpa; (B) A heatmap derived from RSCU value for each amino acid in each species of Toona.
Prediction of RNA editing sites
We used PREP (predictive RNA editors for plants) to predict RNA editing sites in 35 PCGs, with a cutoff value of 0.2. T. microcarpa had the most RNA editing sites (277), followed by T. ciliata (275) and T. sinensis (275), and T. rubriflora (272) and T. ciliata var. yunnanensis 1(272). There were 270 editing sites shared among species. All editing sites were changed from base C to T, and the transition from TCA to TTA was the most frequent (Supplementary Table S6). Gene ccmB had the highest frequency of RNA editing sites, whereas genes nad 4 l, rps1 and sdh4 had only one editing site. No editing events were detected in 9 genes (atp1, atp6, atp8, ccmFN, ccmFC, cox3, mttB, nad3 and rps12) (Fig. S1).
Most editing sites altered amino acids, encompassing 15 types of amino acid changes in total. A major proportion of these changes were the conversion of hydrophilic amino acids to hydrophobic amino acids (48.5%). The most frequent conversions were from Ser to Leu, Pro to Leu, and Ser to Phe. Additionally, a few genes had codons transformed into the stop codons (TAA, TAG, TGA). One common editing was that one of codons for glutamine in rpl16 was converted to a stop codon (CAG to TAG) and another codon for arginine in atp9 was converted to a stop codon (CGA to TGA) in all species. There was one editing site converting arginine to a stop codon in gene rps10 only in T. sinensis and T. ciliata var. yunnanensis 1.
Repeat sequences
Figure 4A shows the frequencies of different types of SSRs in mitochondrial genomes across species, including mononucleotides, dinucleotides, trinucleotides, tetranucleotides and hexanucleotides repeats. Notably, the pentanucleotide repeats were absent, and the tetranucleotide repeats were identified exclusively in T. ciliata. Mononucleotide repeats were most abundant. The (A)n and (T)n repeats were more than the (G)n and (C)n repeats. The (AT)n and (TA)n repeats were more than the (GA)n and (AG)n repeats (Supplementary Table S7). All species exhibited minor variations in repeat abundance.
Repeat sequences in mitochondrial genomes of Toona. (A) Simple sequence repeats; (B) Long sequence repeats.
Most long repeats were between 30 and 40 bp in length, and the longest was 162,009 bp in T. ciliata var. yunnanensis 1(Fig. 4B). T. ciliata var. yunnanensis 1 had the most repeats (212), while T. microcarpa had the fewest repeats (61 in chromosome 1 and 9 in chromosome 2). Among these repeat sequences, most of them were forward and palindromic repeats and did not contain complementary repeat sequences. Reverse repeats were exclusively identified in T. microcarpa (genome 2). T. ciliata var. yunnanensis 1 had the longest forward repeat sequence (30,433 bp) and palindromic repeat sequence (735 bp) compared to other species.
Intracellular homologous comparisons
Intracellular horizontal transfer was analyzed between chloroplast and mitochondrial genomes in species of Toona (excluding T. rubriflora) using BLASTn. Analysis of genomic homology indicated that T. ciliata var. yunnanensis 1 had the longest homologous sequences (29,582 bp in total), accounting for about 3.6% of its mitochondrial genome (Fig. 5A). The total homologous sequences between T. ciliata and T. sinensis were 24,065 bp and 22,588 bp in length, representing 3.8% and 3.3% of their respective mitochondrial genomes. T. microcarpa had the shortest length of homologous sequences (21,677 bp in total), accounting for 3.3% of its mitochondrial genome (Fig. S2). Furthermore, we identified PCGs, tRNA and rRNA genes that were transferred between chloroplast and mitochondrial genomes in both T. ciliata var. yunnanensis 1 and T. ciliata. Eight genes with 100% homologous sequences were identified in both mitochondrial and chloroplast genomes in T. ciliata var. yunnanensis 1 and T. ciliata, including 7 tRNA genes (trnM-CAT, trnS-GGA, trnW-CCA, trnD-GTC, trnI-GAT, trnH-GTG, and trnN-GTT) and 1 PCG (psbF).
Intracellular homologous comparisons. (A) Homology between chloroplast and mitochondrial genomes of T. ciliata var. yunnanensis 1. The mitochondrial genome is showed in blue and the chloroplast genome in green. The pink arcs represent homologous sequences; (B) Homology between the mitochondrial genome of T. ciliata var. yunnanensis 1 and the nuclear genome of T. ciliata. The homologous sequences were indicated in arcs in different colors.
We also analyzed homologous sequences of the mitochondrial genomes of T. ciliata and T. ciliata var. yunnanensis 1 with the nuclear genome of T. ciliata, respectively. The results revealed that the mitochondrial genome of T. ciliata var. yunnanensis 1 was partially homologous with nuclear genomes across 28 chromosomes of T. ciliata (Fig. 5B). A similar case was also identified in T. ciliata (Fig. S3).The most abundant homologous sequences occurred in the 24th nuclear chromosome, with 86 homologous fragments in T. ciliata var. yunnanensis 1 and 63 in T. ciliata, followed by the 16th nuclear chromosome (66 in T. ciliata var. yunnanensis 1 and 56 in T. ciliata), and the 23rd nuclear chromosome (56 in T. ciliata var. yunnanensis 1 and 43 in T. ciliata). There were additional 16 homologous fragments of more than 1,000 bp each in T. ciliata var. yunnanensis 1. The longest homologous fragment was 4,554 bp. The total length of homologous sequences was 195,805 bp in T. ciliata var. yunnanensis 1 (~ 23.5% of its mitochondrial genome) and 166,045 bp in T. ciliata (~ 24.3%).
Phylogeny and synteny analyses among taxa
Based on 35 PCG sequences, we constructed a phylogeny among species of Toona (Fig. 6A) where Melia azedarach was used as the outgroup. We estimated the species divergent times and 95% highest posterior density (HPD) based on two fossil calibration points using BEAST. The divergence time was 56.54 (39–74) Mya between Toona and M. azedarach. Within Toona, the minimum and maximum divergence times were approximately 4.42 Mya (0.04–14.41 Mya) and 21.99 Mya (5–39 Mya), respectively. T. ciliata (NC_065060.1) and T. ciliata var. yunnanensis 1 were clustered on one branch with a 100% supporting rate, consistent with the morphological classification. The Bayesian posterior probabilities for all clades were more than 99%, indicating a robust phylogenetic relationship constructed with mitochondrial PCG sequences. To elucidate the genetic similarity and differences among varieties of T. ciliata, we also conducted a phylogenetic relationship based on intergenic spacer (IGS) sequences. Figure 6B shows that T. sinensis, T. rubriflora, and T. microcarpa were distinctly separated into different branches, whereas the four varieties of T. ciliata were well mixed.
The phylogenetic relationship among species of Toona. (A) The phylogenetic relationship derived from protein-coding genes. M. azedarach was used as the outgroup. The point estimates and 95% highest posterior densities (HPDs) were indicated for divergent times; (B) The phylogenetic relationship among taxa of Toona based on IGS sequences. Twenty samples of T. ciliata were included. Maximum likelihood tree was constructed with 1000 samples by bootstrapping.
The collinearity analysis with mitochondrial genomes supported the phylogenetic relationships derived from PCG sequences among species of Toona (Fig. 7). The homologous collinearity was the highest between T. ciliata and T. microcarpa but generally low between M. azedarach and any species of Toona. These colinear fragments were short in size among species. One key feature was that the colinear fragments were arranged in quite different orders among species, indicating a genome-wide rearrangement occurred among them. The structure of mitochondrial genomes was not conserved among species.
Collinearity among species of Toona in mitochondrial genome. The bars in different colors are the mtDNA sequences of different species. The strips in grey stand for colinear homology and in pink for sequence inversions.
Test of positive and negative selection
Selection underlying 35 PCGs was detected in terms of non-synonymous (\(dN\)) and synonymous (\(dS\)) substitution rates based on the phylogeny of Toona (Fig. 6A). Likelihood ratio tests (LRTs) with the branching model indicated that the \(dN/dS\) ratio (ω) was essentially the same across species (p-values > 5%) (Supplementary Table S8). When each PCG was separately analyzed, sdh4 had the highest \(dS\) value whereas ccmB had the lowest \(dS\) value (Fig. 8). For non-synonymous substitution rate (\(dN\)), cox2 had the highest \(dN\) values, while atp8, atp9, and rps12 had the lowest \(dN\) values. Most PCGs had the ratio (ω) of less than 1, indicating that purifying selection occurred throughout their evolutionary history. Four genes (atp4, ccmFN, rps1, and rps10) had ω values of greater than 1 and were under positive selection during their evolution. Additionally, the posterior probability for each amino acid across various site types was calculated using the BEB (Bayes Empirical Bayes) method, and eleven sites were identified to be under positive selection (Supplementary Table S8).
The non-synonymous (\(dN\)) and synonymous (\(dS\)) substitution rates and their ratios (dN/dS) in 35 protein-coding genes in species of Toona.
Discussion
In this study, we provided evidence that species in genus Toona exhibited variation in mitochondrial genome structure (a high frequency of gene rearrangement) and genome size. The sequences of functional genes were conservative, like other plant mitochondrial genomes, such as comparable percentages of GC contents in Aglaia odorata (NC_084341.1) and M. azedarach (PP099859.1/PP099860.1). However, the proportions of coding sequences in Toona species were lower (3.81%−4.96%), compared with other angiosperms (9.54%)28, especially for T. ciliata var. yunnanensis 1 with only 3.81%. This observation reflected distinct evolutionary features among the Toona species since their common ancestors29.
T. microcarpa was assembled in two circulars, different from the other species of Toona that had a typical single circular. One circular of molecules (“master circle”) was an oversimplified representation of plant mitochondrial genomes30. It is recognized that unlike animal mtDNA, plant mitochondrial genome structure is quite variable, such as two mtDNA circulars in Neolamarckia cadamba and M. azedarach31,32, multi-chromosomal structures in Dendrobium33, linear structure in Lactuca sativa34 and branching structure in Quercus acutissima35. This phenomenon reflects the dynamic property of plant mitochondrial genomes that often undergo intracellular recombination and gene rearrangement. The presence of multiple repeat sequences provides the basis for forming ring-like structure and producing alternative conformations. For example, two pairs of repeat sequences in the mitochondrial genome of M. azedarach generate multiple conformations32. Chromosome 1 of T. macrocarpa (474,320 bp) had one long repeat sequence (> 1000 bp) and could likely have similar properties to a master circle. It was enriched with genes encoding complex III, complex IV, and ribosomal proteins, while chromosome 2 (166,958 bp) carried partial genes of complex I, complex V, and genes related to cytochrome c maturation. This gene distribution pattern could likely facilitate expression of some specific genes and affect the metabolic and physiological processes of T. microcarpa. The two-chromosome structure of T. microcarpa could enhance its adaptability and diversity in response to various environment changes and diverse habitats. This needs further verification in future study.
Besides the genome-wide rearrangement, a few other structural variations were observed in mitochondrial genomes of species of Toona. It is recognized that mitochondrial genes in angiosperms have the typical start codon ATG/GTG and the stop codon TAA/TAG/TGA36,37. PCGs of the mitochondrial genomes were quite variable among species of Toona and exhibited different start codons. This could be related to frequent RNA editing observed in plant mitochondrial genomes38,39. The relative usage of the synonymous codons was similar across Toona species. Amino acids Leu, Ser, and Ile were the most prevalent in Toona, which was consistent with patterns observed in many other plant species31,40. In Toona, the highest proportion of SSRs in the mitochondrial genome was the mononucleotides repeat. The high frequency of A/T in SSRs contributed to the overall abundance of AT observed in Toona, reflecting a common trend among land plants and suggesting a potentially parallel or convergent evolutionary trajectory in plant organelles41.
RNA editing is an important process to alter gene expression and primary protein structure and could play a significant role in plant adaptation42. All species of Toona generally exhibited comparable editing sites, implying parallel evolution in their mitochondrial genomes. In Toona, a strong preference of RNA editing sites (the conversion from TCA to TTA) was identified in mitochondrial genome. Such editing patterns were also reported in other plant species, such as Diospyros oleifera43. Evidence was also recorded in the literature to show the editing of start and stop codons. For instance, genes rps10, rps4, nad1, and nad 4 l exhibited ACG as the start codons, while CGA in rps10 was edited as the stop codon38,39. Most editing sites within encoded genes were located at the second position of the triplet codons. The biological significance of such RNA editing remains to be explored in genus Toona.
In angiosperms, the abundance of repeat sequences influences mitochondrial genome size44. This was recorded in the literature, showing that genomic size differences between closely related species were attributed to large sequence duplications45 or large insertions of exogenous DNA46,47. For example, Cucumis melo (2,740 kb) had larger mitochondrial genome size than Cucumis sativus (1,685 kb) due to abundant repeats sequences and a large proportion of DNA of nuclear origin48,49. Compared with T. ciliata (NC_065060.1), T. ciliata var. yunnanensis 1 had more repeat sequences and showed a tendency of genome expansion. The intracellular homologous analysis implicates that more horizontal transfers between mitochondrial and chloroplast genomes or between mitochondrial and nuclear genome are likely responsible for genome expansion in T. ciliata var. yunnanensis 1.
The divergent time among species of the genus Toona spanned from 4.42 to 21.99 Mya. This wide range could arise from the calibration point selection or the heterogeneity in mitochondrial genome mutation among sites. In this study, two fossil calibration points (the divergence times of M. azedarach-Toona and T. sinensis-T. ciliata) were used for molecular clock analysis. However, fossil record incompleteness or interpretive uncertainties likely contributed to the broad divergence time estimates. T. ciliata, a Class—II protected endangered species in China, has faced population bottlenecks or fragmentation due to over-exploitation and low natural regeneration. This would subsequently reduce its effective population size and the coalescent branch lengths and hence affect the estimation of divergent times. Moreover, the mitochondrial genomes of Toona had the highly rearranged structure but conservative coding sequences, which further compromised the accuracy of divergent time estimates.
Although mitochondrial genomes generally have lower mutation rates than chloroplast genomes in plant species50, our results provided evidence that the PCG sequences were effective to distinguish species of Toona. This was also demonstrated in N. cadamba31. Compared with the results derived from cpDNA sequences, three species (T. ciliata, T. sinensis and M. azedarach) were separated with mitochondrial genomes but the varieties of T. ciliata were not. The estimated divergent time between Toona and M. azedarach (56.535Mya) was longer in terms of mtDNA sequences than in terms of cpDNA sequences (48.02Mya)19. This was due to the lower mutation rate of plant mitochondrial genome. Besides, the high frequency of rearrangement could be used to design molecular markers (e.g., RFLP) for analyzing genetic variation, such as in Ficus carica and Symplocos laurina51,52. Integration of mitochondrial, chloroplast, and nuclear genomes can improve resolution to phylogeny within genus Toona. The chloroplast and nuclear genomic data are expected to more effective for clarifying species-level relationships due to the higher mutation rate. The mitochondrial sequences are suitable for insights into deep evolutionary divergences and ancestral lineage dynamics53. In the Orchidaceae family, the phylogenetic positions of subgroups such as the subtribe Epipogiinae, tribe Gastrodieae, and subfamily Tropidieae were poorly supported or excluded from analyses. However, these issues were effectively resolved through mitochondrial data analysis54. The present study confirmed the appropriateness of mitochondrial genomes for elucidating species of genus Toona.
Like the previous studies on T. ciliata or other species using chloroplast genomes20,55, a conflict phylogenetic relationship among varieties of T. ciliata was observed in terms of mitochondrial genome versus morphological traits. Both PCGs and IGS sequences could not distinguish varieties of T. ciliata, which also provided a biological basis to term the T. ciliata complex20. Since leaf and flower traits are predominantly controlled by nuclear genomes, such a conflict essentially reflects the cytonuclear conflict in taxonomic division, a common phenomenon in many plant phylogenies56,57. Besides the influences of the lower mutation rate, a few other processes could be involved in forming this conflict. One is the distinct genetic drift rates between nuclear and mitochondrial genomes58. In angiosperms, the genetic drift rate of the biparentally inherited nuclear genomes is a half of that of the maternally inherited mitochondrial genomes when male and female parents are equally abundant. The coalescent processes lead to reciprocal monophyly more swiftly in the mitochondrial genome compared with the nuclear genome59. Our phylogenetic relationship based IGS sequences probably reflected this situation (Fig. 6), when the divergent times are not long enough to reach the monophyletic phase. The difference between Fig. 6 of the present study and Fig. 4 of Xiao et al. for the varieties of T. ciliata mainly arose from lower mutation rates of mitochondrial genomes19.
On the other hand, lineage sorting driven solely by genetic drift is slow, allowing for ancestral variations are shared across thousands to millions of generations in descendant lineages after speciation. Lineage sorting is still incomplete among varieties of T. ciliata. Like some Quercus species, the mitochondrial gene tree is discordant with the classification based on leaf shape and fruit morphology due to incomplete lineage sorting and ancient hybridization60. Similar cases of cytonuclear discordance in phylogeny were reported in the literature55. Given recent species divergence of Toona, lineage sorting among varieties of T. ciliata remains in early stage. The earlier few polymorphic sites in mtDNA sequences inherited from the common ancestors remained unaltered in IGS regions (more likely neutral sites) in varieties of T. ciliata. Consequently, only a few mutations were accumulated since speciation from common ancestral populations. This could be expected in closely related lineages that are not reciprocally monophyletic61,62.
The second process is natural selection that impedes genomic divergence among taxa63. Almost all PCGs were under negative selection in mitochondrial genomes in Toona, like the case of chloroplast genomes19. There were no sequence variations in PCGs across varieties of Toona. However, adaptive selection could likely involve genetic divergence at genes associated with flower and leaf traits or other morphological traits, yielding morphological differentiation in T. ciliata. Alternative alleles were likely fixed due to selection in different varieties. This eventually leads to a conflict in phylogeny in terms of morphological traits versus the PCG sequences of mitochondrial genome, analogous to the findings in plant species of genus Crataegus64,65 and animal species Oophaga pumilio66.
One worthy point is that four PCGs (atp4, ccmFN, rps1, and rps10) were under positive selection (dN/dS > 1) at the species level. These genes participate in vital mitochondrial functions, including energy metabolism, the electron transport chain, and protein synthesis. Their adaptive evolution enhanced species divergence. Similar patterns of positive selection were observed in other plants. For instance, in the mitochondrial genome of the cave-endemic plant Primulina hunanensis41, positive selection signals were also detected in the atp4 and ccmFN genes, which are associated with energy metabolism adaptation under low-light conditions. Species of Toona are distributed across different climatic zones in China. Positive selection could optimize ATP synthesis efficiency for adaptation to different temperatures and energy demands in different habitats. Nevertheless, these functional genes were under purifying selection in varieties of T. ciliata, impeding evolutionary divergence between varieties.
The third process is gene introgression among varieties of T. ciliata that could not seriously affect phylogeny based on mitochondrial genomes. Gene flow through pollination does not disperse maternally inherited genes. Only seed flow contributes to mitochondrial gene flow. However, gene flow through natural hybridization could impede nuclear genomic divergence among varieties of T. ciliata67. These varieties are sympatric or partially overlap in geographical distribution in China and natural hybridization could not be excluded among them. This process could effectively affect genomic divergence at neutral sites. Differential selection at adaptive sites, especially for the sites associated with speciation, would not be seriously affected in the presence of gene flow, except for strong gene flow that can swamp locally adaptive genes68.
In general, this study provided comprehensive comparisons on mitochondrial genome structure of Toona and offered insights for identification and genetic research on the species of Toona. Owing to multiple properties of mitochondrial genomes, including uniparental inheritance, haploid, low mutation rates, a high frequency of recombination and gene rearrangement, these limit the power of elucidating species and recently diverging lineages. Natural hybridization could also reduce the power of identifying species and purifying selection impedes species divergence. This study confirmed the appropriateness for identifying species of Toona and the limitation for identifying varieties of T. ciliata. To address the unresolved phylogeny among varieties of T. ciliata, highly polymorphic markers or nuclear genome sequences are needed. Besides, future research could be expanded in following ways: (1) Improving sampling strategies by increasing the geographical coverage and number of populations, especially in the typical distribution areas of varieties, would help better understand genetic variation patterns; (2) Integrating quantitative analyses with transcriptomic data could provide new perspectives for clarifying taxonomic relationships in genus Toona, especially in the T. ciliata complex. These studies could further contribute to our understanding of the phylogeny and genetic diversity of genus Toona.
Methods
Plant material, DNA extraction and sequencing
Leaf samples of T. rubriflora and T. microcarpa were collected from healthy living plants in Fujian and Guangdong provinces, respectively (Table 3). Samples were then dried and stored in silica gel. From previous studies and literature of the varieties of T. ciliata67, we collected five samples of each variety (T. ciliata var. ciliata, T. ciliata var. yunnanensis, T. ciliata var. henryi, T. ciliata var. pubescens) for resequencing. Note that T. ciliata var. subaxaxflora was not found in natural forests and hence was not included in this study. In addition, two samples of M. azedarach were collected from South China Agricultural University (SCAU) as the outgroup. The specimen was stored for records in Guangdong Key Laboratory for Innovative Development and Utilization of Forest Plant Germplasm, SCAU, Guangdong Province, China. The use of plant leaves in this study complies with institutional guidelines. Collection of the plant specimen was permitted by the University.
The total genomic DNA of each sample was extracted by CTAB69, and the genomic DNA was used for library construction. For next-generation sequencing, the library was constructed with an insert length of 350 bp. It was sequenced on the Illumina Novaseq6000 platform (Illumina, USA). After library construction, the qPCR method and Agilent 2100 Bioanalyzer (Agilent Technologies, USA) were used for quality control. The qualified DNA libraries were sequenced using the Illumina Novaseq6000 (Illumina, USA) high-throughput sequencing platform with the PE150 (Pair-End 150) sequencing strategy. Raw sequencing data were filtered with fastp v.000.23.270 (-q 20 -l 150) to remove low-quality reads and adapters. The average Q20 and Q30 percentages were 98% and 94%, respectively, ensuring high-quality data for downstream analyses (Supplementary Table S9). For the third-generation sequencing, the libraries of 10 kb fragments were constructed using the PacBio RS II platform (Pacific Biosciences, PN 101–853-100). and sequenced on the PacBio Revio system. High-quality HiFi data were obtained by extracting reads with the quality value greater than Q20 from the CCS data.
Genome assembly and annotation
The next-generation sequencing (NGS) data were aligned to the reference mitochondrial genome using Bowtie2 (v.2.4.2)71. The alignment results were sorted with Samtools (v.1.8)72 to generate aligned BAM files. The assembled contig sequences were screened against related species’ mitochondrial and chloroplast genomes using BLASTn (E-value ≤ 1e-10) to identify putative mitochondrial sequences. To enhance accuracy, only paired-end alignments were retained, and the final mitochondrial genome sequence was refined using Pilon (v.1.23)73. The third-generation sequencing data were assembled using Canu (v.2.2)74 to obtain the genome sequence. The assembled sequences were compared to the NCBI nt database using Blastn (v.2.11.0 +)75 to extract mitochondrial genome sequences. Non-cyclic tig sequences were manually spliced into a circular arrangement based on overlaps and paired-end relationships from second-generation sequencing data. Following assembly, NGS and third-generation sequencing were remapped to the mitochondrial genome assembly to validate coverage uniformity and confirm structural accuracy.
The mitochondrial genome was initially annotated using GeSeq-web76. Protein sequences obtained from the annotation were compared to the NCBI nr database using Blastp (v.2.11.0 +) to adjust the positions of coding genes. The rRNA annotations were validated and corrected by comparing to the NCBI core nt database using Blastn (v.2.11.0 +). The tRNA secondary structures were generated using ARWEN (v.1.2)77, and any irregular tRNAs were further validated with tRNAscan-SE (v.2.0)78. The tRNAs with unreasonable lengths or incomplete structures were excluded from final annotation.
Codon usage and RNA editing analysis
The RSCU (relative synonymous codon usage) values of coding genes were calculated using MEGA 7.0.2679. No preference for an amino acid was indicated when RSCU was 1, while a preference for an amino acid was indicated when RSCU was greater than 1. Condon usage was represented through ggplot2 in R package. RNA editing sites were predicted for PCGs with online tool PREP suite (http://prep.unl.edu/).
Repeat and homologous sequence analysis
Simple sequence repeats (SSRs) were identified using MISA-web. Long repetitive sequences exceeding 30 bp were analyzed using REPuter80. The chloroplast genomes of T. sinensis (MF467522.1), T. microcarpa (MF467521.1), T. ciliata (OM135324.1), T. ciliata var. yunnanensis (OM135326.1) were downloaded from the NCBI database, respectively. The nuclear genome of T. ciliata was downloaded from the CNGB Nucleotide Sequence Archive (https://db.cngb.org/search/project/CNP0001985/) with accession number no. CNP0001985. The homologous sequences were analyzed using BLASTn with parameters (- e value 1e—5 -outfmt 6 -out output_file) and visualized using the Circos software package (version 0.67)81.
Positive and negative selection test
The non-synonymous (\(dN\)) and synonymous (\(dS\)) substitution rates and their ratios (dN/dS) were estimated for 35 PCGs in mitochondrial genome of Toona species using Codeml in the PAML package82,83. Branch models were used to estimate dN/dS for specific branches, and site models were applied to detect selection on PCGs. Likelihood ratio tests (LRTs) were conducted to compare alternative site models: M0(one ratio) vs. M1(neutral), M1(neutral) vs. M2(selection), and M7 (beta) vs. M8(beta and selection)83. Positively selective sites were identified using Bayesian posterior probabilities (BEB) with M2 and M8 models, with the probability greater than 0.95 indicating positive selection84.
Phylogeny and synteny analysis
To construct phylogenetic tree of the Toona genus, we aligned common PCGs using MAFFT (v7.490)85 and concatenated them into a “supergene” sequence set. The optimal substitution model (K3Pu + F + I) was determined using modelfinder in IQ-TREE (v1.5.6)86, and a maximum likelihood tree was constructed with IQ-TREE v1.5.6 (1000 bootstrap replicates). Additionally, intergenic spacer (IGS) sequences were aligned using MAFFT, concatenated, and used to build another maximum likelihood tree with IQ-TREE. Bayesian inference was performed with BEAST (v2.7.7)87 under the HKY model, using calibration times from Timetree (http://www.timetree.org/ accesss March 12, 2024) for divergence estimates. Four MCMC runs were executed for 20,000,000 iterations, with the ESS values greater than 200. For collinearity analysis, the mitochondrial genomes of Toona species were subjected to sequence alignment using BLASTn(- e value 1e—5 -outfmt 6 -out output_file). Individual colinear regions were identified using MCScanX88 and visualized in Tbtools89.
Conclusions
We sequenced the mitochondrial genomes of T. rubriflora and T. microcarpa, and re-sequenced four varieties of T. ciliata. A comprehensive analysis of Toona was conducted based on the mitochondrial genome, including codon preferences, repeat sequences, RNA editing events, genome homology and phylogenetic relationships. Comparative genomic analysis indicated the presence of substantial structural variation among species due to gene rearrangement. There were small variations in gene number, RNA editing sites, repeat sequences and genome homology. All species have comparable GC contents, conservative sequences of PCGs, codon occurrences, and RNA editing sites. There was no sequence divergence in PCGs among varieties of T. ciliata except for a few mutations in IGS regions. Phylogenetic analysis indicated that species of Toona were effectively identified in terms of PCG sequences. However, the varieties of T. ciliata were not well separated by both PCGs and IGS sequences, supporting the conservation strategy that considered the varieties of T. ciliata as a single conservation unit, termed as the T. ciliata complex. Most PCGs were predominantly under purifying selection across species. Species of Toona had about 4% mitochondrial genomes homologous with chloroplast genomes and about 24% homologous with nuclear genomes. Conflict phylogenies existed among varieties of T. ciliata in terms of mitochondrial sequences versus morphological (leaf and flower) traits. The overall results provided comprehensive information on Toona mitochondrial genomes, which could be used for species identification in genus Toona and conservation strategy for the T. ciliata complex.
Data availability
The datasets generated by this study are available in nucleotide database of GenBank (https://www.ncbi.nlm.nih.gov/nucleotide/) repository with accession numbers: Toona rubriflora (PQ287271.1) and Toona microcarpa (Chromosome 1: PQ318328.1, Chromosome 2: PQ318327.1). The re-sequencing data for the varieties of Toona ciliata were provided in the Supplementary Material.
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Acknowledgements
We appreciate Zhi-Jian Feng at South China Agricultural University, You-Pai Zeng at South China Botanical Garden for providing information of species, Dai-Quan Ye and Yong Li at Yangkou State Forest Farm of Fujian for kind help during our field sample collection.
Funding
The study was supported jointly by Guangdong Basic and Applied Basic Research Foundation (2023A1515012137), National Natural Science Foundation (32171819), and the funding from the South China Agricultural University (4400-K16013).
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L.Y.W. conducted the experiment, analyzed the data, and wrote the manuscript; L.Y.W., W.Z.Y., and H.Z.H. participated in field sampling and experiment; X.Y. and W.C. provided experimental supports; H.X.S. revised and finalized the manuscript. All authors read and approved the final manuscript.
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Lv, YW., Wang, ZY., He, ZH. et al. Structural and phylogenetic comparisons of the complete mitochondrial genomes among taxa in genus Toona. Sci Rep 15, 20296 (2025). https://doi.org/10.1038/s41598-025-06816-x
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DOI: https://doi.org/10.1038/s41598-025-06816-x
