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. 2023 Aug 21;15(9):evad155. doi: 10.1093/gbe/evad155

The Mitochondrial Genome of the Holoparasitic Plant Thonningia sanguinea Provides Insights into the Evolution of the Multichromosomal Structure

Shuaixi Zhou 1, Neng Wei 2, Matthias Jost 3, Stefan Wanke 4, Mathew Rees 5,6, Ying Liu 7, Renchao Zhou 8,✉
Editor: Daniel Sloan
PMCID: PMC10476698  PMID: 37603455

Abstract

Multichromosomal mitochondrial genome (mitogenome) structures have repeatedly evolved in many lineages of angiosperms. However, the underlying mechanism remains unclear. The mitogenomes of three genera of Balanophoraceae, namely Lophophytum, Ombrophytum, and Rhopalocnemis, have already been sequenced and assembled, all showing a highly multichromosomal structure, albeit with different genome and chromosome sizes. It is expected that characterization of additional lineages of this family may expand the knowledge of mitogenome diversity and provide insights into the evolution of the plant mitogenome structure and size. Here, we assembled and characterized the mitogenome of Thonningia sanguinea, which, together with Balanophora, forms a clade sister to the clade comprising Lophophytum, Ombrophytum, and Rhopalocnemis. The mitogenome of T. sanguinea possesses a multichromosomal structure of 18 circular chromosomes of 8.7–19.2 kb, with a total size of 246,247 bp. There are very limited shared regions and poor chromosomal correspondence between T. sanguinea and other Balanophoraceae species, suggesting frequent rearrangements and rapid sequence turnover. Numerous medium- and small-sized repeats were identified in the T. sanguinea mitogenome; however, no repeat-mediated recombination was detected, which was verified by Illumina reads mapping and PCR experiments. Intraspecific mitogenome variations in T. sanguinea are mostly insertions and deletions, some of which can lead to degradation of perfect repeats in one or two accessions. Based on the mitogenome features of T. sanguinea, we propose a mechanism to explain the evolution of a multichromosomal mitogenome from a master circle, which involves mutation in organellar DNA replication, recombination and repair genes, decrease of recombination, and repeat degradation.

Keywords: DNA-RRR genes, holoparasites, master circle, mitogenome, parasitic plants, recombination

Significance.

Quite a few angiosperms have evolved the multichromosomal mitogenome structure; however, how the multichromosomal structure has evolved from a single circular chromosome remains unclear. We characterized a multichromosomal mitogenome of a parasitic plant, Thonningia sanguinea, and found that all repeats in the genome have no recombination activity. We propose a mechanism to explain the formation of a multichromosomal structure through repeat degradation and the resultant decrease and eventually cessation of repeat-mediated recombination.

Introduction

Although plant mitochondrial DNA exists in many forms, that is, circular, linear, and branched molecules in vivo (Bendich 1993, 2007; Gualberto et al. 2014; Oldenburg and Bendich 2015; Smith and Keeling 2015), most angiosperm mitochondrial genomes (mitogenomes) have been assembled into a single circular chromosome (master circle) (reviewed in Wu, Liao et al. 2020). These master-circle mitogenomes usually contain many medium- (100–1,000 bp) and/or large-sized (>1,000 bp) repeats that can mediate recombination and form alternative, interconvertible conformations of multiple smaller subgenomic circles (Sloan 2013). This kind of mitogenomes is also called multipartite mitogenomes (Sloan 2013). In plants, the multichromosomal mitogenome structure was first identified in Cucumis sativus with three chromosomes of 1,554, 84, and 45 kb, without any shared medium or large repeats between the largest chromosome and each of the two smaller chromosomes (Alverson et al. 2011). Since then, multichromosomal mitogenomes have been characterized in multiple angiosperm lineages (Sloan et al. 2012; Rice et al. 2013; Shearman et al. 2016; Sanchez-Puerta et al. 2017; Wang et al. 2019; Roulet et al. 2020; Yu et al. 2022; Li et al. 2023; Yang et al. 2023). While some multichromosomal mitogenomes contain only a handful of chromosomes (2–10), others are highly multichromosomal and consist of many chromosomes (>10). For example, there are at least 128 chromosomes in the mitogenome of Silene conica (Sloan et al. 2012). Sizes of angiosperm mitogenomes with a multichromosomal structure vary greatly, from the largest one, 11.3 Mb in S. conica (Sloan et al. 2012), to the smallest one, 130.7 kb in Rhopalocnemis phalloides (Yu et al. 2022). Among angiosperms, Viscum scurruloideum has the smallest mitogenome (65.9 kb) but its mitogenome structure has not been well resolved (Skippington et al. 2015). Sizes of chromosomes within a multichromosomal mitogenome also vary greatly, from 69.2-fold in Silene vulgaris (Sloan et al. 2012) to only 1.6-fold in R. phalloides (Yu et al. 2022). Most of the highly multichromosomal mitogenomes consist of relatively large (>10 kb) or a combination of relatively large and small chromosomes (<10 kb) (Sloan et al. 2012; Rice et al. 2013; Shearman et al. 2016; Sanchez-Puerta et al. 2017; Wang et al. 2019; Roulet et al. 2020; Yu et al. 2022; Li et al. 2023; Yang et al. 2023), while the mitogenome of R. phalloides is entirely made up of small chromosomes (Yu et al. 2022). So far, the mechanism underlying the evolution of multichromosomal mitogenomes remains unclear.

The family Balanophoraceae, belonging to Santalales, consists of 16 genera and 42 species distributed in tropical regions (Kuijt 1969). All members of this family are holoparasites, with a highly reduced plant architecture. To date, mitogenomes of three genera from a single lineage of this family, namely, Lophophytum, Ombrophytum, and Rhopalocnemis, have been published (Sanchez-Puerta et al. 2017; Roulet et al. 2020; Yu et al. 2022), all showing a highly multichromosomal structure, albeit with different genome and chromosomal sizes. The mitogenomes of Ombrophytum subterraneum and Lophophytum mirabile are more similar to each other in structure and size: the former consists of 54 circular chromosomes ranging from 4,900 to 27,184 bp with a total length of 713,777 bp (Roulet et al. 2020), and the latter comprises 60 circular chromosomes ranging from 7,177 to 45,414 bp with a total length of 821,906 bp (Sanchez-Puerta et al. 2017). The mitogenome of R. phalloides on the other hand is composed of 21 all-minicircular chromosomes of only 4–8 kb with a total length of 130,713 bp (Yu et al. 2022). This minicircular chromosome structure has been verified using Southern blot experiment (Yu et al. 2022). All 21 mitochondrial chromosomes of R. phalloides have a shared conserved region (CR) of 896 bp (Yu et al. 2022), and theoretically, these chromosomes can be merged into a big master circle via CR-mediated recombination. With such different multicircular chromosomal structures and mitogenome sizes in a single lineage of Balanophoraceae, it is expected that characterization of other lineages of this family may expand the knowledge of mitogenome diversity and provide insights into the evolution of the plant mitogenome structure and size.

Thonningia, another genus of Balanophoraceae, together with Balanophora, belongs to another clade sister to the clade comprising Lophophytum, Ombrophytum, and Rhopalocnemis (Ceriotti et al. 2021; Kim et al. 2023). Thonningia contains a single species, T. sanguinea Vahl., which is distributed in tropical Africa (Burkill 1985). In this study, we sequenced three accessions of T. sanguinea using an Illumina platform and assembled their mitogenomes to identify the mitogenome features of this species, assess intraspecific mitogenome variation, and infer the evolution of the multichromosomal mitogenome structure in Balanophoraceae, and more generally in plants.

Results

Mitogenome Structure, Gene, and Intron Contents

The three accessions of T. sanguinea (T01, T02, and T03) were sampled from Angola. For each of the three accessions of T. sanguinea, the mitogenome was assembled into 18 circular-mapping chromosomes with only slight variations in sequence among them (see the section below and supplementary table S1, Supplementary Material online). Here, we describe in detail the mitogenome features of one accession, T01 (fig. 1; supplementary table S1, Supplementary Material online). Sequencing depths of the 18 chromosomes of T01 range from 226.4 to 417.2×. GC (guanine and cytosine) contents of these chromosomes vary from 41.9% to 46.9%, with an overall mitogenome GC content of 45.0%, a typical value for most angiosperms. The total mitogenome size is 246,247 bp, with the longest and the shortest chromosomes being 19,213 bp and 8,726 bp, respectively.

Fig. 1.

Fig. 1.

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Mitogenome map of T. sanguinea (accession T01). Mitogenome size of this accession is 246,247 bp. Intron-containing genes were marked with an asterisk.

Thirty-five unique protein coding genes (PCGs), 3 rRNA genes, and 11 unique tRNA genes were identified for the T01 mitogenome (fig. 1). All chromosomes encode 1–6 genes each, with the exception of Chr13, where no mitochondrial genes could be identified. Although Chr13 has no identifiable genes, 1,274-bp sequences on this chromosome are shared with mitogenomes of Malania oleifera, L. mirabile, or O. subterraneum. All 24 core PCGs conserved in seed plants were recovered. The additional 11 PCGs consist of 10 ribosomal protein genes (rpl2, rpl5, rpl10, rpl16, rps3, rps4, rps10, rps12, rps14, and rps19) and one succinate dehydrogenase gene (sdh4). Thirty-two of the 35 PCGs each exist in just one chromosome, while exons of the three remaining PCGs, nad1, nad2, and nad5, are distributed on two or three chromosomes. We found multiple gene copies, distributed across multiple chromosomes for two tRNA genes. Three slightly different copies of trnfM-CAU were identified on Chr7, Chr9, and Chr15, respectively. Two identical copies of trnN-GUU are present on Chr2 and Chr14. The T01 mitogenome contains 19 cis-spliced introns and five trans-spliced introns (nad1_i1, nad1_i3, nad2_i2, nad5_i2, and nad5_i3), with the total and average lengths of cis-spliced group II introns being 26,056 bp and 1371.4 bp, respectively. Coding sequences (including PCGs, tRNAs, and rRNAs) and cis-spliced introns account for 14.5% and 11.0% of the genome, respectively (supplementary table S1, Supplementary Material online). Compared with other species of Santalales, T. sanguinea and three other Balanophoraceae species (Lophophytum, Ombrophytum, and Rhopalocnemis) exhibit a shorter average intron size (fig. 2; supplementary table S2, Supplementary Material online).

Fig. 2.

Fig. 2.

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Evolution of mitogenome size, structure, and cis-spliced group II intron size in Santalales. (A) A schematic diagram showing three mechanisms of intron loss. (B) Mitogenome size, structure, and cis-spliced group II intron size evolution. Autotrophic, hemiparasitic, and holoparasitic species are shown in hollow circle, semi-solid circle, and solid circle, respectively. Events of intron loss are marked on the branches. cox1_i1 is not shown here because of its complex evolutionary history. *The mitogenome structure of these species has not been resolved yet. Phylogenetic relationships in Santalales and Balanophoraceae are based on Su et al. (2015) and Ceriotti et al. (2021).

Sequence Content Comparison between T. Sanguinea and Its Relatives

Mitogenome sequences were compared between T. sanguinea and three other Balanophoraceae species (Lophophytum, Ombrophytum, and Rhopalocnemis), as well as a hemiparasite, M. oleifera from Olacaceae (supplementary fig. S1, Supplementary Material online). Surprisingly, T. sanguinea has more shared mitochondrial regions with M. oleifera than the three other Balanophoraceae. Specifically, a total of 66,824 bp in the T. sanguinea mitogenome (27.1% of the genome) is shared with 83,152 bp in the M. oleifera mitogenome (15.8%) and much longer sequences in the latter are due to intragenomic duplications. In contrast, 50,591 bp (20.5%) and 44,861 bp (18.2%) in T. sanguinea are shared with 55,136 bp and 57,044 bp in L. mirabile (6.7%) and O. subterraneum (8.0%), respectively. Although R. phalloides is very similar to T. sanguinea in mitochondrial chromosome number and relatively even chromosome size, only 32,307 bp in T. sanguinea (13.1%) is shared with 33,341 bp in R. phalloides (25.5%) and there is no one to one correspondence in sequence between chromosomes of the two species. Moreover, most regions in T. sanguinea shared with the R. phalloides mitogenome belong to genic regions (93.9%), which are also shared with other mitogenomes, while 13,744 bp (20.6%) is shared with only the mitogenome of M. oleifera, but not to the more closely related species (supplementary fig. S1, Supplementary Material online). For intergenic regions, only 1,964 bp in T. sanguinea is shared with the R. phalloides mitogenome, while 15,308 bp in T. sanguinea is shared with the M. oleifera mitogenome, suggesting rapid sequence turnover and/or loss in the intergenic regions of the R. phalloides mitogenome. In addition, the distribution of shared sequences on the chromosomes revealed that frequent mitogenome rearrangements have happened among these species (supplementary fig. S1, Supplementary Material online).

Repeat Content and Repeat-Mediated Recombination Analysis

Nine hundred and twenty-four unique repeat units, ranging from 17 to 328 bp, were detected in the T. sanguinea (T01) mitogenome (supplementary fig. S2 and table S3, Supplementary Material online), including six repeats ≥ 100 bp in size (100–328 bp), 25 repeats with a size of 50–100 bp, and 893 small repeats < 50 bp in size. Copy numbers of these repeat units range from 2 to 35, with an average of 4.4 copies per repeat unit. For all 4,042 repeat copies, 51, 610, 32, 7, and 3,342 were located in exons, introns, rRNAs, tRNAs, and intergenic regions, respectively. Twenty-five tandem repeats, with motif sizes and copy numbers ranging from 5 to 35 bp and from 1.9 to 14.8, respectively, were detected (supplementary table S4, Supplementary Material online). In addition, 51 microsatellites with a motif of 2–4 bp were identified (supplementary table S5, Supplementary Material online). Taken together, the aggregate length of repetitive sequences sums up to 20,089 bp, covering 8.2% of the mitogenome (supplementary table S6, Supplementary Material online).

The six largest repeat units (R1–R6 with a length of 100–328 bp) each have two copies. R1, R2, R3, R4, and R5 are shared by Chr4 and Chr9, Chr4 and Chr10, Chr5 and Chr8, Chr2 and Chr14, and Chr5 and Chr9, respectively, while both copies of R6 have a large overlap of 67 bp on Chr2. Assuming that recombination is mediated by these five largest repeats, two larger circular molecules can be generated: one is 30,193 bp by merging Ch2 and Chr14 via R4 and the other is 72,213 bp by merging Ch4, Chr5, Chr8, Chr9, and Chr10 via R1, R2, R3, and R5 (fig. 3A). For the latter case, various smaller circular molecules can be generated by merging two, three, or four chromosomes. However, it is surprising that no repeat-mediated recombination was detected for any of the five largest repeats. Specifically, for the four largest repeat units (R1–R4), no PCR products were detected for the hypothesized recombination conformations (fig. 3B and 3C; supplementary fig. S3, Supplementary Material online), indicating the absence of recombination mediated by these repeats. For 10 smaller two-copied repeats with a repeat unit size ranging from 51 to 120 bp including R5, no reads spanned over the hypothesized recombination conformations, based on read mapping.

Fig. 3.

Fig. 3.

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Absence of repeat-mediated recombination in the T. sanguinea mitogenome verified by PCR experiments. Only PCR results for one repeat pair were shown here. See supplementary figure S3, Supplementary Material online, for the PCR results for other repeat pairs. (A) The hypothesized repeat-mediated, interchromosomal recombination. Dashed lines between repeats indicate the positions and directions of hypothesized recombination. (B) The positions of PCR primers used to amplify the reference and hypothesized conformations for R1–R4. (C) PCR amplification of the reference and hypothesized recombination conformations for R3. Dashed rectangles mark the PCR products of the reference conformations, and no expected PCR products were detected for the hypothesized recombination conformations. “+” and “−” in parentheses of panel C indicate the presence of the expected PCR product and the absence of the PCR product due to no repeat-mediated recombination, respectively.

Intraspecific Mitogenome Sequence Variation among the Three Accessions of T. Sanguinea

As shown in supplementary table S1, Supplementary Material online, the mitogenomes of the three accessions exhibit very minor variation, with T02 and T03 being much more similar to each other. Mitogenome sizes of T02 and T03 (246,807 bp and 246,812 bp, respectively) are slightly larger than that of T01 (246,247 bp). The three mitogenomes also share identical gene and intron contents. In total, 72 single nucleotide polymorphisms (SNPs), 166 insertions/deletions (InDels) of 1–335 bp, and 15 microinversions of 2–4 bp were detected among the mitogenomes of the three accessions (table 1; supplementary table S6 and fig. S4, Supplementary Material online). The mitogenomes of the three accessions have a very low level of nucleotide diversity (π = 7.3 × 10−3). There are no SNPs and only four InDels on Chr2, Chr7, and Chr14 between the mitogenomes of T02 and T03, while all 72 SNPs, 166 InDels, and 15 microinversions exist between T01 and T02/T03. No SNPs and InDels were found in PCGs, and only one 2-bp microinversion was found in cox3 between T01 and T02/03, which causes changes of two consecutive amino acids. Ninety-nine of the 166 InDels belong to copy number variation of short sequences, most likely originating from replication slippage (Viguera et al. 2001).

Table 1.

Sequence Variations between Mitogenomes of Three Thonningia sanguinea accessions

Chromosome # of SNPs # of InDels # of microinversions # of InDels between T01 and T02/T03 # of InDels between T02 and T03
Chr1 4 8 1 8 0
Chr2 3 13 2 13 2
Chr3 3 6 0 6 0
Chr4 6 4 2 4 0
Chr5 3 9 0 9 0
Chr6 2 5 0 5 0
Chr7 5 7 0 7 1
Chr8 2 7 2 7 0
Chr9 5 12 0 12 0
Chr10 0 8 2 8 0
Chr11 7 7 0 7 0
Chr12 7 9 1 9 0
Chr13 1 8 0 8 0
Chr14 1 9 1 9 1
Chr15 7 16 0 16 0
Chr16 1 9 0 9 0
Chr17 11 10 2 10 0
Chr18 4 19 2 19 0
Total 72 166 15 166 4
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Some intraspecific variations, when occurring within repeats, lead to lower similarity between repeat sequences on different chromosomes in one or two accessions. Here, we show three examples in which InDels affect sequence similarity between repeats on different chromosomes. First, a relatively large, imperfect repeat (95/96 bp) was shared between Chr10 and Chr13 in both T02 and T03 but an 11-bp insertion in the corresponding region on Chr13 of T01 divides it into two smaller repeats of 39 and 55 bp for T01 (fig. 4A). Thus, the large repeat in T02 and T03 is no longer present in T01. Another nearly perfect repeat (298 bp, 98.7% identity) is shared between Chr2 and Chr14 in T01, and a 14-copy microsatellite repeat unit of “CTTCC” is embedded in this repeat for both chromosomes. Whereas, copy numbers of this microsatellite unit are 20 on Chr2 and 10 on Chr14 of T02 and 22 on Chr2 and 9 on Chr14 of T03 (fig. 4B). Repeats between Chr2 and Chr14 are much shorter in both T02 and T03. The third one involves a nearly perfect tandem repeat with a motif size of 28 bp, which has one copy each on Chr2 and Chr7 of T01, three copies on Chr2 and six copies on Chr7 of T02, and four copies on Chr2 and six copies on Chr7 of T03 (fig. 4C). Such changes in copy number result in only a 28-bp repeat pair retained between Chr2 and Chr7 in T01. Reduced similarity between repeat pairs in the three examples may cause a lower, if any, chance for recombination between different chromosomes in one or two accessions.

Fig. 4.

Fig. 4.

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Examples of intraspecific variation in three accessions of T. sanguinea that can cause repeat degradation. (A) A repeat shared by Chr10 and Chr13, in which an 11-bp insertion occurred on Chr13 of T01; (B) a repeat shared by Chr2 and Chr14, in which copy numbers of a 5-bp microsatellite vary between Chr2 and Chr14 of T02 and T03; (C) a repeat shared by Chr2 and Chr7, in which copy numbers of a 28-bp motif vary between Chr2 and Chr7 of T02 and T03. In this case, a substitution from T to A makes the first copy of the 28-bp motif on Chr2 imperfect.

Discussion

Evolution of Mitochondrial Intron Size in Santalales

Mitochondrial introns have experienced complex evolution in plants (Mower 2020). There are three mechanisms accounting for the loss of a cis-spliced intron: direct merge of flanking exons, loss of intron-containing gene, and shift to trans-spliced intron (Guo et al. 2020). All three mechanisms can be applied to Santalales, as shown in figure 2. In holoparasitic Balanophoraceae examined in this study, no further loss of mitochondrial introns was observed in T. sanguinea and R. phalloides, while cox2_i1 is lost in both L. mirabile and O. subterraneum and cox2_i2 is lost only in L. mirabile. Thus, the intron contents of T. sanguinea and R. phalloides could represent a more ancestral state in this family.

Among the nine species of Santalales with available mitogenomes, Erythropalum scandens, R. phalloides, and T. sanguinea have the same and the most introns, while the Tolypanthus–Helicanthes–Viscum clade has experienced a complicated progress of intron loss via all three mechanisms, in which V. scurruloideum has lost 17 introns mainly because of gene loss, while Tolypanthus maclurei, and Helicanthus elastica have experienced shared loss of cox2_i2, nad2_i3, rpl2_i1, and rps3_i1 and differential loss of introns of some nad genes through all three mechanisms (fig. 2; supplementary table S2, Supplementary Material online). M. oleifera has lost cox1_i1, and L. mirabile and M. oleifera have lost cox2_i2 independently, while both O. subterraneum and L. mirabile have lost cox2_i1, which most likely happened in their common ancestor. Among the nine species, the autotrophic species E. scandens has a larger average intron size than the hemiparasitic and holoparasitic species (supplementary table S2, Supplementary Material online). Among the four species of Balanophoraceae, the Rhopalocnemis–Lophophytum–Ombrophytum clade has experienced a further decrease in average intron size compared with T. sanguinea (supplementary table S2, Supplementary Material online), especially for R. phalloides. It is noteworthy that the L. mirabile and O. subterraneum mitogenomes are larger than those of many autotrophic angiosperms despite reduced average intron size.

However, intron size decrease is not a pervasive phenomenon in parasitic plants, for example, no signal of intron size decrease has been found in the holoparasitic Aeginetia indica (Orobanchaceae, Lamiales) and the hemiparasitic Cuscuta campestris (Convolvulaceae, Solanales) (supplementary table S2, Supplementary Material online). Whether intron size evolution is associated with the parasitic lifestyle cannot be inferred at present, as only a limited number of mitogenome sequences for parasitic plants are available. Moreover, the size of cox1_i1 does not decrease in the hemiparasitic and holoparasitic species of Santalales, which may be caused by its special evolutionary history: cox1_i1 has experienced frequent horizontal gene transfers in angiosperms (Sanchez-Puerta et al. 2008).

Evolution of Mitogenome Size and Structure in Balanophoraceae

Most angiosperms have a mitogenome size of 300–600 kb (reviewed in Gualberto and Newton 2017) and two early-diverging Santalales species, E. scandens and M. oleifera, have their mitogenome sizes of 305,320 bp (GenBank accession numbers: MZ430522 and MZ430523) and 527,575 bp (Luo et al. 2020), respectively. It is a reasonable inference that the mitogenome size of the common ancestor of Balanophoraceae may be also around this range. Thus, it seems that the T. sanguinea mitogenome mostly approaches the ancestral size. While the L. mirabile and O. subterraneum mitogenomes (822 and 713 kb) experienced an expansion and the R. phalloides mitogenome (131 kb) experienced a shrinkage in size. The size increase of the O. subterraneum and L. mirabile mitogenomes results from the incorporation of a large quantity of foreign sequences (Sanchez-Puerta et al. 2019; Roulet et al. 2020), while the much smaller R. phalloides mitogenome is caused by a substantial decrease of intron size and intergenic regions (Yu et al. 2022). Given that shared regions between T. sanguinea and L. mirabile/O. subterraneum are much more than those between T. sanguinea and R. phalloides, we can infer that R. phalloides has experienced rapid mitogenome sequence turnover and/or loss.

Among angiosperms with a highly multichromosomal mitogenome structure, T. sanguinea and R. phalloides have the smallest mitogenome sizes and the first and third evenest chromosome sizes, with 2.2- and 1.6-fold size changes in the two species, respectively, while 2.0–69.2-fold chromosome size changes are found in other highly multichromosomal mitogenomes (supplementary table S7, Supplementary Material online). A big difference between mitogenomes of the two species is that R. phalloides has a shared CR among its chromosomes while T. sanguinea has not. Because mitogenomes of all other plants don’t have such a CR, evolution of this CR should represent a derived trait. The mitogenomes of L. mirabile and O. subterraneum consist of considerably more mitochondrial chromosomes than those of T. sanguinea and R. phalloides, and this likely results from further fragmentation of ancestral chromosomes.

It is well recognized that repeats and repeat-mediated recombination are common in angiosperm mitogenomes (Alverson et al. 2011; Cole et al. 2018; Springer et al. 2019). A “master circle” molecule can form an alternative multipartite conformation via repeat-mediated recombination and vice versa (Sloan 2013). Large repeats (>1,000 bp) usually have higher recombination activity (Maréchal and Brisson 2010; Mower et al. 2012; Guo et al. 2016), and recombination mediated by medium-sized repeats (100–1,000 bp) and small-sized repeats (<100 bp) is also not uncommon in plants (Skippington et al. 2015; Zhong et al. 2022; Yang et al. 2023). However, despite the presence of multiple medium-sized repeats and abundant small-sized repeats in the T. sanguinea mitogenome, we could not detect any signal of recombination mediated by these repeats, unlike frequent repeat-mediated recombination observed in most angiosperm mitogenomes. No repeat-mediated recombination has also been found in other plants with a multichromosomal mitogenome structure, for example, sugarcane (Shearman et al. 2016).

InDels Explain Most Intraspecific Mitogenome Variations

For most of the studied plants to date, only a single mitogenome from a single accession is available. Therefore, intraspecific mitogenome variation could not yet be evaluated. Here, we examined intraspecific variation by characterizing three accessions of T. sanguinea. Very few variations were found between T02 and T03, while many more variations were detected between T01 and T02. Similar interaccession divergence of the same three accessions was found in their plastomes (Kim et al. 2023). The majority (65.6%) of intraspecific variations are InDels, different from some other angiosperms including Fragaria, Pyrus, Arabidopsis thaliana, and Tylosema esculentum, in which SNPs are more prevalent than InDels (Wu, Waneka et al. 2020; Fan et al. 2022; Sun et al. 2022; Li and Cullis 2023). Among the 15 microinversions in the mitogenomes of the three accessions, 13 can form a palindrome with their flanking sequences. This kind of variation has also been observed in strawberry mitogenomes (Fan et al. 2022). Notably, more than half (51.7%) of intraspecific mitogenome variations of T. sanguinea result from copy number variations of tandem repeats. As shown in Results, variation in the copy number of these tandem repeats can result in lower sequence similarity. When these tandem repeats are embedded in other larger repeats, their copy number variation can have the potential to influence the level of repeat-mediated recombination.

In contrast, patterns of intraspecific polymorphism are very different in another plant, Silene noctiflora, which has a highly multichromosomal mitogenome and exhibits extensive variation in the presence/absence of the entire mitochondrial chromosomes among different accessions (Wu et al. 2015; Wu and Sloan 2019). The main reason for this contrasting difference might be that there are a large number of “empty chromosomes” in S. noctiflora with only one “empty chromosome” (Chr13) in T. sanguinea. Although Chr13 of T. sanguinea has no identifiable genes, 1,274 bp sequences on this chromosome are shared with mitogenomes of M. oleifera, L. mirabile, or O. subterraneum, suggesting the possible existence of functional elements on this chromosome. Thus, it seems that functional constraints may prevent the loss of mitochondrial chromosomes.

Evolution of Multichromosomal Mitogenomes: A Proposed Mechanism

Multichromosomal mitogenome structures have evolved in many lineages of angiosperms, including all four species of Balanophoraceae characterized so far (supplementary table S7, Supplementary Material online). Here, we propose a mechanism to explain the evolutionary process of a multichromosomal mitogenome from a master circle mitogenome.

  1. The ancestral mitogenome of angiosperms most likely has a master circle structure, and the presence of relatively large repeats can mediate recombination and thus lead to the formation of alternative multicircular molecules (multipartite mitogenome structure; fig. 5A). This is supported by the fact that over 90% of all characterized angiosperm mitogenomes, including basal angiosperms like Nymphaea and Liriodendron, possess a single master circle structure (Richardson et al. 2013; Dong et al. 2018; Wu, Liao et al. 2020a). One exception is Amborella trichopoda, which maintains a multichromosomal structure resulting largely from horizontal gene transfer of other plants (Rice et al. 2013). Repeats and repeat-mediated recombination have been observed in the mitogenome of Nymphaea colorata (Nymphaeales) (Dong et al. 2018).

  2. The multicircular subgenomic structure, with the advantage of rapid replication of smaller molecules (Shao et al. 2009), becomes predominant in some lineages, likely those in need of rapid DNA replication (fig. 5B).

  3. Recombination decreases, most likely caused by loss or dysfunction of nuclear-encoded, organellar DNA replication, recombination, and repair (DNA-RRR) genes due to mutations (Worth et al. 1994; Calmann and Marinus 2004; Shedge et al. 2007; Maréchal et al. 2009; Cappadocia et al. 2010; Maréchal and Brisson 2010; Carrie and Small 2013) (fig. 5C).

  4. When the capability of homologous recombination–based DNA repair decreases, accumulation of mutations in the repeats shared by different subgenomic molecules will reduce sequence similarity between repeat pairs and thus further reduce the levels of repeat-mediated recombination (fig. 5D). Elevated substitution rates of mitochondrial genes in two Silene species, S. conica and S. noctiflora (Sloan et al. 2012), which have a low level of recombination, are consistent with this prediction. For the accessions of T. sanguinea, mutations in their mitogenomes include InDels and copy number variation of tandem repeats. As the recombination activity is usually positively associated with repeat length (e.g., Skippington et al. 2015; Yang et al. 2023), recombination is further suppressed.

  5. Finally, further accumulation of mutations between repeat pairs results in the cessation of recombination and the formation of the multichromosomal structure (fig. 5E). The mitogenomes of T. sanguinea, O. subterraneum, and sugarcane appear to be at this stage. In O. subterraneum, there is no evidence for recombination for repeats < 800 bp (no available information of recombination for two repeats > 800 bp) (Roulet et al. 2020), and in sugarcane, no recombination was found between the two chromosomes (Shearman et al. 2016). Lophophytum seems to approach this stage, with only 11 of 55 repeats of 150–700 bp showing evidence of recombination (Sanchez-Puerta et al. 2017).

Fig. 5.

Fig. 5.

Open in a new tab

A proposed mechanism to explain the evolution of the multichromosomal mitogenome structure. (A) Ancestral master-circle mitogenome, with an alternative multicircular subgenomic structure formed by repeat-mediated recombination; (B) multicircular subgenomic structure becomes predominant with the advantage of rapid mitochondrial DNA replication; (C) recombination decreases due to the loss or dysfunction of organellar DNA-RRR genes; (D) with reduced recombination, repeats begin to degrade by accumulation of various mutations like indels; (E) repeats continue to degrade, and recombination eventually ceases with continuous degradation of repeats, which leads to the formation of the multichromosomal structure.

However, the mitogenome structure of R. phalloides is a special case, because this species has evolved a new unique 896-bp CR shared by all its chromosomes, which can mediate recombination among these chromosomes (Yu et al. 2022). Because this kind of mitogenome structure has not been observed in other angiosperms, it represents a derived state. It is highly likely that R. phalloides had evolved a multichromosomal mitogenome before the emergence of the CR shared by all its chromosomes. Moreover, this mechanism may be applied to other eukaryotic lineages, including some animals and fungi, which have also evolved a multichromosomal mitogenome structure (reviewed in Wu, Liao et al. 2020). Interestingly, that multichromosomal mitogenome structure is much more frequently observed in holoparasitic plants (Bellot et al. 2016; Sanchez-Puerta et al. 2017; Roulet et al. 2020; Yu et al. 2022; Zhang et al. 2022; Mitrastemon yamamotoi and Sapria himalayana [our unpublished data]) than non-holoparasitic plants, in conjunction with dramatic loss of nuclear genes (including RRR genes) in the genomes of holoparasitic plants (Sun et al. 2018; Vogel et al. 2018; Cai et al. 2021; Xu et al. 2022; Yu et al. 2022), which is consistent with this proposed mechanism.

Material and Methods

Plant Sampling and DNA Sequencing

The three accessions of T. sanguinea (T01, T02, and T03) were collected from Uige, Angola. Sampling details, DNA extraction, and Illumina sequencing can be found in Kim et al. (2023); 11.2, 8.6, and 11.5 Gb Illumina paired-end reads of 150 bp were obtained for the three accessions.

Mitogenome Assembly and Annotation

Raw Illumina reads were filtered using Trimmomatic v0.39 (Bolger et al. 2014) with the following parameters: SLIDINGWINDOW:5:20 LEADING:5 TRAILING:5 MINLEN:50. Clean reads of each sample were then de novo assembled using GetOrganelle v1.7.5.1 (Jin et al. 2020) with parameters -R set to 15 and -k set to 127. The original assemblies were visualized with Bandage v0.8.1 (Wick et al. 2015), and mitochondrial contigs were chosen based on their sequencing depths and high sequence similarity to the mitogenome of M. oleifera (NCBI accession number: MT902145) assessed by Blastn implemented in Bandage. All selected contigs were circular, either in a single circle or within a multiple-circle network in which these circles are connected together by shared repeats. These repeats don’t mediate recombination (see Results), and sequences of all the individual circles were extracted and used as their chromosome sequences. The chromosome numbers of T01 were sorted by chromosome size, and those of T02 and T03 were given based on chromosome sequence similarity to chromosomes of T01. Sequencing depth of the chromosomes was calculated by mapping the corresponding Illumina reads to the mitogenome using BWA-mem (Li and Durbin 2010). Mitochondrial genes were annotated using Geseq (Tillich et al. 2017) with the mitogenome of M. oleifera, E. scandens, and Liriodendron tulipifera (NCBI accession numbers: MT902145, MZ430522–MZ430523, and KC821969, respectively) as references. The annotation was manually corrected and then plotted with OGDRAW (Lohse et al. 2007). Intron size information of other species, including three additional species (Lophophytum, Ombrophytum, and Rhopalocnemis) from Balanophoraceae, five additional species (V. scurruloideum, H. elastica, T. maclurei, M. oleifera, and E. scandens) from other families of Santalales, and an outgroup (Vitis vinifera), were extracted from GenBank and used for comparison (supplementary table S2, Supplementary Material online; note that some of the original annotations were corrected). Phylogenetic relationships in Santalales and Balanophoraceae were adopted from Su et al. (2015) and Ceriotti et al. (2021).

Repeat Identification and Repeat-Mediated Recombination Analysis

Repeats were identified in the T01 mitogenome by ROUSfinder2.py (Wynn and Christensen 2019) with the parameter -m set to 17. The identified repeats were divided into three categories based on their sizes: <50 bp, 50–100 bp, and ≥100 bp. Tandem repeats (≥5 bp motif) were identified using Tandem Repeats Finder (Benson 1999) with default parameters, and microsatellites with motif sizes of 2, 3, and 4 bp were identified using MISA-web (Thiel et al. 2003; Beier et al. 2017) with the minimum copy number set to 5, 4, and 3, respectively. To determine if there is repeat-mediated recombination for the four repeat pairs with repeat units > 150 bp, primers were designed to amplify the repeats and at least 100 bp flanking sequences of the reference conformations and the putative recombined conformations (supplementary table S8, Supplementary Material online). For ten relatively large two-copied repeats with repeat units < 150 bp, Illumina reads were mapped to the reference and the putative recombination conformations to determine whether there is repeat-mediated recombination. The reference and putative recombination conformations include the repeats themselves and at least 10 bp of flanking sequences.

Sequence Comparison between T. Sanguinea and Its Relatives

Shared regions between T. sanguinea and three other species of Balanophoraceae (Lophophytum, Ombrophytum, and Rhopalocnemis) as well as M. oleifera were identified with Blastn (Altschul et al. 1990) with the parameter e-value set to 1e−5 and -perc_identity set to 85. Hits ≥ 50 bp in length were recorded. Shared regions between these species were plotted by the karyotype graph module in JCVI (Tang et al. 2008).

Analysis of Intraspecific Variation

Each mitochondrial chromosome of the three accessions was aligned with ClustalW (Thompson et al. 1994). SNPs, InDels, and microinversions were recorded with Geneious Prime v2022.0.2 (https://www.geneious.com). For InDels, we also checked whether there are copy number variations of tandem repeats.

Supplementary Material

Supplementary data are available at Genome Biology and Evolution online (http://www.gbe.oxfordjournals.org/).

Supplementary Material

evad155_Supplementary_Data
Click here for additional data file. (16.5MB, zip)

Acknowledgements

This work was supported by the National Natural Science Foundation of China (grant numbers 32170217 and 31811530297) and Guangzhou Collaborative Innovation Center on S&T of Ecology and Landscape (grant number 202206010058).

Contributor Information

Shuaixi Zhou, State Key Laboratory of Biocontrol and Guangdong Provincial Key Laboratory of Plant Resources, Sun Yat-Sen University, Guangzhou, Guangdong, China.

Neng Wei, Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan, Hubei, China.

Matthias Jost, Institut für Botanik, Technische Universität Dresden, Dresden, Germany.

Stefan Wanke, Institut für Botanik, Technische Universität Dresden, Dresden, Germany.

Mathew Rees, School of GeoSciences, University of Edinburgh, Edinburgh, United Kingdom; Royal Botanic Garden, Edinburgh, United Kingdom.

Ying Liu, State Key Laboratory of Biocontrol and Guangdong Provincial Key Laboratory of Plant Resources, Sun Yat-Sen University, Guangzhou, Guangdong, China.

Renchao Zhou, State Key Laboratory of Biocontrol and Guangdong Provincial Key Laboratory of Plant Resources, Sun Yat-Sen University, Guangzhou, Guangdong, China.

Author Contributions

R.Z. carried out the investigation and offered plant samples and experiment design. S.Z. carried out data analysis. N.W., M.J., S.W., and M.R. contributed to plant sampling and data analysis. S.Z. and R.Z. wrote the main manuscript text. S.Z. and Y.L. prepared the figures and tables. All authors reviewed and revised the manuscript.

Data Availability

Illumina raw sequence reads of three individuals of T. sanguinea have been deposit in NCBI SRA database (https://www.ncbi.nlm.nih.gov/sra, Illumina reads accession numbers: SRR25131790, SRR25131789, and SRR25131788). Mitogenome sequences and annotations of T01 accession of T. sanguinea are available in NCBI GenBank (https://www.ncbi.nlm.nih.gov/genbank, accession numbers: OR233148–OR233165).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

evad155_Supplementary_Data
Click here for additional data file. (16.5MB, zip)

Data Availability Statement

Illumina raw sequence reads of three individuals of T. sanguinea have been deposit in NCBI SRA database (https://www.ncbi.nlm.nih.gov/sra, Illumina reads accession numbers: SRR25131790, SRR25131789, and SRR25131788). Mitogenome sequences and annotations of T01 accession of T. sanguinea are available in NCBI GenBank (https://www.ncbi.nlm.nih.gov/genbank, accession numbers: OR233148–OR233165).

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