Why does mitochondrial dna evolve faster

Alternatively, these genes may have remained intact but been subject to reduced functional constraint or inefficient selection, resulting in the accumulation of mutations that alter their function. Although positive and relaxed selection have not been examined in mt-RRR genes from angiosperms with elevated mt mutation rates, Zhang et al.

Here, we used transcriptomic data from 12 Silene species with variable mt mutation rates Fig. Overall, we find evidence of positive selection in these nuclear genes, which we argue is likely due to increased mtDNA mutation rates. Moreover, we find little evidence for relaxed selection, suggesting positive selection is the predominant driver of accelerated evolution in N-mt genes of fast Silene species.

We discuss this finding in light of mitonuclear interactions and possible causes of elevated mitochondrial mutation rates in Silene. Note that S. Moreover, there may have been independent accelerations in the lineage leading to S. This is evidenced by low bootstrap support 0. Photos are from greenhouse-reared individuals except for S. For S. After growing for 5 months, RNA was extracted from rosette leaves from a single individual of each species as described above. A Ribo-Zero Plant Leaf rRNA Removal Kit Illumina was used during library preparation in order to capture organellar transcripts, which are not consistently poly-adenylated, for future studies.

Reads were used as delivered i. In addition to these new resources, we made use of previously generated Silene transcriptomes. We also used transcriptomes for 19 geographically diverse populations of S. In some analyses, we also made use of the Beta vulgaris genome Dohm et al. In total, we examined 14 species Fig. Proteins that make up complex II succinate dehydrogenase; SDH serve as a control for mitonuclear interactions in this gene class, as all complex II proteins are nuclear-encoded in Silene Sloan et al.

N-mt ribosomal proteins form multisubunit complexes with mtDNA-encoded ribosomal proteins and rRNAs and are responsible for mt translation. We used the Silene ribosomal protein sequences from Sloan et al. We focused on RRR genes that were targeted solely to the mitochondria, or those that were dual-targeted to both mitochondria and plastids.

N ote. BLAST 2. After manual curation and preliminary tree building in MEGA to ensure all sequences were orthologous, the alignment was trimmed according to the longest ORF. Finally, TargetP Emanuelsson et al. The MK test uses counts of synonymous and nonsynonymous substitutions both within species polymorphism and between species divergence to test for positive selection McDonald and Kreitman Note that the D and P terms in these expressions represent simple counts.

We distinguish these from the d N and d S values described previously, which are defined on a per site basis. These two outgroup species were chosen to represent two different scales of interspecific divergence: within S. Direction of Selection DoS values were calculated following Stoletzki and Eyre-Walker , with positive DoS values indicative of positive selection. We also report MK-associated statistics from a set of ribosomal proteins targeted to the cytosol, which act as a specific control for the mt-targeted ribosomal proteins.

Fifty randomly chosen, single-copy nuclear genes were also investigated as described previously Rockenbach et al. To categorize positive selection on an entire class of genes, we summed counts for genes belonging to each gene class.

Because summing counts across contingency tables can introduce statistical bias, we also calculated NI TG , which is an unbiased estimator of NI Stoletzki and Eyre-Walker In addition to MK tests, we calculated several other population genetic metrics to assess if and how selection was currently acting on these genes.

These tests do not explicitly test for positive selection but rather for a deviation from neutrality by taking the SFS into account. Using the DH program with default settings Zeng et al. These tests were performed twice for each gene and dataset: once for the sequences used in MK analyses when S.

Note that even though many of these tests do not employ an outgroup, the results for both gene sets are presented as a comparison with MK tests. A smaller gene set was used for the S. In the main text we report uncorrected P values, although all statistics, including P values corrected for multiple comparisons via Bonferroni correction, are reported in supplementary data file S1 , Supplementary Material online.

We also performed the same analyses on four other previously described datasets: OXPHOS genes, mt-targeted ribosomal proteins, cytosolic-targeted ribosomal proteins, and the set of 50 randomly chosen, single-copy nuclear genes.

For each gene, custom Perl scripts were used to extract orthologs from the 14 species we examined 12 Silene species, Agrostemma githago , and Beta vulgaris. Briefly, sequences were extracted and aligned from Silene and outgroup transcriptomes as above.

Reference branches were then selected to include all terminal branches leading to species with slowly evolving mtDNA Beta , Agrostemma , S. Any internal branches that led exclusively to one type of branch were designed as that type, while internal braches giving rise to both types of branches were designated as reference branches e. In addition to individual gene analyses, RELAX analyses were performed after concatenating sequences within each gene class as above. However, this pattern was not found in genes encoding cytosolic-targeted ribosomal proteins Sloan et al.

Here, we used slightly different methods to reanalyze these gene sets in a standardized fashion, and the results confirm our previous analyses. Comparisons between Silene species with fast S. Numbers in parentheses indicate the number of genes and nucleotides analyzed in each gene set. Silene mt-RRR genes were identified and analyzed here for the first time supplementary table S3 , Supplementary Material online.

They showed a similar pattern as the other N-mt genes Fig. Importantly, this trend was not driven by outlier genes, because when genes were analyzed individually, all mt-RRR genes showed the same pattern supplementary fig. S1 , Supplementary Material online. When performing MK tests using the 19 populations of S.

S2 , Supplementary Material online, Table 1. This pattern is a signature of positive selection McDonald and Kreitman Similar, although nonsignificant, results were obtained when the closer relative S. Mt-targeted and cytosolic-targeted ribosomal proteins both showed a significant excess of nonsynonymous divergence when S.

For mt- but not cytosolic-targeted ribosomal proteins, a significant excess of nonsynonymous divergence was also found when using S. For mt-RRR genes, a significant excess of nonsynonymous divergence was found when using either S. As described previously Rockenbach et al. All gene sets include nuclear-encoded genes N-mt genes that code for proteins targeted to the mitochondria, except cytosolic-targeted ribosomal proteins and a set of 50 randomly chosen nuclear genes, which act as controls for mitonuclear interactions.

Subunits in OXPHOS complex II act as another type of control, as they are targeted to the mitochondria, but do not interact directly with any mt-encoded subunits. Neutrality indices NI above or below 1 indicated by dashed lines suggest purifying or positive selection, respectively. Note that the wide variation in mt-targeted ribosomal proteins is due to a single gene RPS11 with an abnormally low NI 0. We also calculated several metrics that examine the SFS.

S3—S6 , Supplementary Material online. Colors and boxplots as in Figure 3 , except larger points represent values for concatenated sequences within a gene set. OXPHOS subunits that interact with mt-encoded subunits, mt-targeted ribosomal proteins, and mt-targeted RRR genes exhibited k values in RELAX analyses that were generally clustered around 1, and genes that were individually significant were roughly evenly split between k values greater than and less than 1 Fig.

In addition, the distribution of k values was similar between N-mt genes and the set of 50 random nuclear genes Fig. These results indicate that neither relaxed nor intensified selection is acting in any consistent way on these sets of genes in fast compared with slow Silene species.

All gene sets are for nuclear-encoded genes N-mt genes that code for proteins targeted to the mitochondria, except a set of 50 randomly chosen nuclear genes, which act as a control for mitonuclear interactions. Points indicate individual loci, while horizontal lines indicate values for concatenated sequences in each gene set. When all genes within a dataset were concatenated, there were significant signatures of relaxed selection in fast Silene species for some gene sets, but overall these effects were limited Fig.

S7 , Supplementary Material online. There are several potential reasons why this artifact may have been produced see Discussion. One possibility for why some Silene species have undergone an acceleration in mtDNA mutation rate and structural rearrangements in the mitochondrial genome is that mt-RRR genes are absent or have altered functionality in these species.

Of the mt-RRR genes that have been characterized in other angiosperms supplementary table S3 , Supplementary Material online , we were able to identify homologs for all of them in Silene species with both fast and slowly evolving mtDNA. None of these genes had obvious frameshift mutations, premature stop codons, or other hallmarks of pseudogenization or loss of function.

After examining genomic and transcriptomic resources from Nicotiana tabacum and Oryza sativa , we found that these species also have two or more mt-DNA polymerases, but that they each represent independent duplications and are not orthologous to POL1A and POL1B of Arabidopsis supplementary fig. S8 , Supplementary Material online.

Therefore, the difference in mt-DNA polymerase copy number appears to be the result of independent duplications in other lineages rather than a loss in Silene. Similarly, two endonucleases NTH1 , plastid-targeted; NTH2 , mt-targeted in Arabidopsis appear to be Arabidopsis -specific duplicates, and only a single, mt-targeted NTH gene was found in Silene and analyzed here supplementary fig.

Some mt-RRR genes were excluded from phylogenetic and population genetic analyses because of difficulties in their assemblies. However, the detection of transcript fragments indicates that RDR is present in Silene , albeit expressed at low levels or suffering from assembly problems. Silene appears to have undergone a lineage-specific duplication for OSB1 supplementary fig. S9 , Supplementary Material online , leading to multiple distinct OSB1 -like sequences, the assembly of which is further complicated by large tandem duplications of the PDF domain Zaegel et al.

Many of these sequences contained predicted mt-targeting peptides. These OSB1 duplicates were found in both slow and fast Silene , but were not analyzed here because of paralogy and assembly concerns among the OSB1 -like duplicates.

Overall, fast Silene appear to have a complete, functional contingent of mt-RRR genes and show no obvious deviations in gene content from slow Silene. Mitonuclear interactions have been theorized to have important implications for human health, diversification of eukaryotes, and major transitions in the evolution of life Burton and Barreto ; Dowling ; Havird et al.

One of the underlying assumptions of these arguments is that genes encoded by the nuclear genome are under positive selection to compensate for slightly deleterious mitochondrial mutations, which may escape selection owing to the uniparental and haploid nature of mtDNA Lynch ; Lynch and Blanchard While several independent studies support this scenario Osada and Akashi ; Barreto and Burton ; Gong et al.

Here, we present three lines of evidence that positive selection is playing a significant role in shaping the evolution of N-mt genes in Silene with fast mtDNA evolution. This pattern could be caused by positive or relaxed selection. Because this pattern was not observed in a set of randomly chosen nuclear genes that do not interact with mitochondria or plastids Fig. Second, we used population genetic data to show an abundance of nonsynonymous divergence compared to polymorphism in S.

This was not observed for the set of 50 random nuclear genes, again excluding any demographic cause of this pattern. Moreover, these effects remained significant in many of the N-mt gene sets examined whether using S. This implies positive selection on N-mt genes took place across at least two time scales because S. Moreover, because a much lower number of homologous sequences were identified in S.

There was no more evidence for relaxed selection in N-mt genes in fast Silene than in the set of 50 random nuclear genes. However, our interpretation of the RELAX data is complicated by the fact that we found a potential artifact when analyzing the concatenated mt-RRR dataset which exhibited the opposite direction of effect than each gene analyzed individually.

It is not clear what caused this anomalous result. One potential explanation is that large amounts of missing data in the concatenated mt-RRR alignment i. Another possibility is that incomplete lineage sorting or introgression of mt-RRR genes may have also influenced results of concatenation analyses where all genes were forced into a single phylogeny. A relatively low number of species included in our analyses 14, whereas the median number used on the RELAX webserver is 19 may have also reduced the power to detect changes in selection using RELAX.

Overall, N-mt genes showed evidence of positive selection. Although this pattern was largely consistent with our predictions under the nuclear compensation hypothesis, there were some unexpected results.

Nevertheless, it is possible that even though complex II does not contain any mt-encoded subunits, it may experience indirect effects associated with having to interact with mitochondrial import and assembly proteins, which may be under positive selection themselves. A similar result was observed when comparing mt- and cytosolic-targeted ribosomal proteins, as MK tests produced evidence of positive selection for both.

When they were available, fossil calibration points were extracted from the publications. The absolute mutation rate was computed as the average rate across genes. Generation times were approximated using age at sexual maturity or number of generations per year.

For vertebrates, we mostly relied on the AnAge database version 13, Tacutu etal. For nonvertebrates, generation times were obtained from the literature and, in rare cases, from web-sites see supplementary table S2 , Supplementary Material online for details.

In most cases, no information was available for the very species of our data sets and we used the median value of a taxonomical group including our species mostly at the family level. For example, for a data set composed of Trioceros Chameleo species Ceccarelli etal. All the statistical analyses were performed using R R Core Team Variables were log transformed.

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