Recombination and intraspecific polymorphism for the presence and absence of entire chromosomes in mitochondrial genomes

Abstract

Although mitochondrial genomes are typically thought of as single circular molecules, these genomes are fragmented into multiple chromosomes in many eukaryotes, raising intriguing questions about inheritance and (in)stability of mtDNA in such systems. A previous comparison of mitochondrial genomes from two different individuals of the angiosperm species Silene noctiflora found variation in the presence of entire mitochondrial chromosomes. Here, we expand on this work with a geographically diverse sampling of 25 S. noctiflora populations and the closely related species S. turkestanica and S. undulata. Using a combination of deep sequencing and PCR-based screening for the presence of 22 different mitochondrial chromosomes, we found extensive variation in the complement of chromosomes across individuals. Much of this variation could be attributed to recent chromosome loss events, suggesting that the massively expanded and fragmented mitochondrial genomes of S. noctiflora may have entered a phase of genome reduction in which they are losing entire chromosomes at a rapid rate. Sequence analysis of mitochondrial and plastid genomes revealed genealogical differences both between these organelles and within the mitochondrial genome, indicating a history of recombination. Evidence that recombination has generated novel combinations of alleles was more frequent between loci on different mitochondrial chromosomes than it was within chromosomes. Therefore, the fragmentation of mitochondrial genomes and the assortment of chromosomes during mitochondrial inheritance appears to have contributed to a history of sexual-like recombination in the mtDNA of this species.

Fig. 1

Phylogenetic relationships among S. turkestanica, S. undulata, and major lineages of S. noctiflora based on concatenated sequence alignments from a the mitochondrial genome and b the plastid genome. Bootstrap support values are shown for each node

Fig. 2

Phylogenetic relationships among S. turkestanica, S. undulata, and major lineages of S. noctiflora based on concatenated sequence alignments for each of 14 different chromosomes in the mitochondrial genome. Bootstrap support values greater than 70 are shown for each node. Chromosome labels refer to the numbering in the S. noctiflora BRP mitochondrial genome

Fig. 3

Presence/absence survey of 22 mitochondrial chromosomes from 25 different populations of S. noctiflora (Table S1) and the close relative S. undulata. For each chromosome, presence/absence was assessed with three different PCR markers in each chromosome. Dark gray shading indicates positive detection for all three markers; medium gray shading indicates positive detection for two of three markers; light gray shading indicates positive detection for only one of three markers. For three chromosomes, the analysis was performed with only two markers (BRP41-OSR45, OSR58, and BRP57). In these cases, dark gray shading indicates detection of both markers. The chromosomes are divided into four categories as described in the Methods. Note that one of the “OSR-specific” chromosomes (OSR46) was found to be present at low levels in BRP and other samples from that group (see Results). This chromosome is recorded as present, but it is also possible that it has been lost from the mitochondria and only retained as a numt

Fig. 4

Parsimony-based reconstruction of ancestral States for the presence (black) or absence (white) of each of the 22 sampled chromosomes across 25 S. noctiflora samples and the close relative S. undulata. a The constraint topology used for the analysis is based on concatenated sequence data from the mitochondrial genome (see Fig. 1a), and placement of the multiple OSR-like and BRP-like samples is their respective groups in based on the phylogenetic analysis in Figure S1. The remaining panels show the presence/absence states for b the five sampled chromosomes with high sequence divergence, c the five sampled chromosomes that are conserved between BRP and OSR and have no genes, d the six sampled “OSR-specific” chromosomes, and e the six sampled “BRP-specific” chromosomes

Fig. 5

Read depth across the 63 chromosomes of the S. noctiflora BRP reference mitochondrial genome based on Illumina sequencing of total-cellular DNA from S. noctiflora KEW 22121. Coverage estimates are based on a sliding window with a window size of 1000 bp and a step size of 500 bp. The plot was generated with the ggplot2 library in R. Reference chromosomes are ordered in decreasing size from chromosome 1 (191 kb) to chromosome 63 (65 kb) on the same x-axis scale, so coverage maps end before the end of the x-axis

Fig. 6

ddPCR analysis of copy number variation for two S. noctiflora mitochondrial chromosomes OSR46 (top) and OSR37-BRP37 (bottom) in seven S. noctiflora samples. Copy numbers are reported relative to two mitochondrial genes (nad2 and cox1). For each chromosome, three loci (ddPCR markers) were analyzed. The especially low copy number across all three markers for OSR46 in the BRP and BRP-like samples is consistent with the low observed sequencing coverage of this chromosome. The OSR37-BRP37 chromosome also exhibits a lower copy number than the reference markers in the BRP clade, but it more abundant than OSR46, and it was recovered in the original BRP assembly