Department of Entomology, The Natural History Museum, London, United Kingdom; and Department of Biological Sciences, Imperial College London, Ascot, Berkshire, United Kingdom
Correspondence: E-mail: joap{at}nhm.ac.uk.
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Abstract |
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Key Words: numts population polymorphism nucleotide substitution rate Rivacindela ribosomal RNA secondary structure compensatory substitutions DNA taxonomy
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Introduction |
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When transferred to the nuclear genome, mitochondrial sequences become nonfunctional pseudogenes (Bensasson et al. 2001b) and can be detected due to the lower proportions of nonsynonymous substitutions in protein-coding genes and reduced transversion bias compared to their mitochondrial counterparts (e.g., Gellissen and Michaelis 1987; Sunnucks and Hales 1996; Bensasson, Zhang, and Hewitt 2000). Ribosomal RNA (rRNA) numts should be recognizable due to the disruption of their secondary structure, although this has not been confirmed in all cases (Olson and Yoder 2002). Numts have been found to exhibit a generally lower nucleotide substitution rate compared to the corresponding mtDNA sequences (Lopez et al. 1994; Arctander 1995; Dewoody, Chesser, and Baker 1999; Lu, Fu, and Zhang 2002). For example, nuclear rates in numts of mammals were up to six times lower than protein-coding mtDNA and almost equal to the slowly evolving small (rrnS) and large (rrnL) rRna subunits (Lopez et al. 1997). The absolute nucleotide substitution rates in numts were estimated to be 3 x 109 to 4 x 109 substitutions/site/year, in agreement with the well-documented rate in other nuclear pseudogenes (4 x 109; Li 1997) and hence clearly lower than the widely accepted mitochondrial rate of 2 x 108 to 2.5 x 108 substitutions/site/year (or 2%2.5% divergence per million years) (Hasegawa, Kishino, and Yano 1985; Brower 1994). Because of these findings, numts have been regarded as "molecular fossils," which maintain ancestral characteristics that are lost in the mtDNA lineages from which they originated (Olson and Yoder 2002; Woischnik and Moraes 2002). However, conclusions about the type and rate of sequence variation in numts can be problematic because of the difficulty in identifying sister lineages across taxa, for instance, when numt lineages from the human genome sequence were fitted to a tree inferred from the extant mtDNAs sequences of primates (Bensasson, Feldman, and Petrov 2003).
Similarly, precise knowledge about the relationships of mtDNA sequences and their corresponding nuclear paralogs is needed to establish the dynamics of their origination and loss in lineages of organisms. In most studies the point in time of the transposition event is inferred from evidence for gene duplication, i.e., from phylogenetic reconstruction of a common ancestor that gave rise to paralogous sequences. However, the transposed copy (numt) would be subject to processes of stochastic lineage sorting and hence at the moment of origination is not present in all individuals of a population. Due to the different modes of inheritance of mitochondrial and nuclear DNA, the distribution of the numt and its source mtDNA copy may differ substantially, giving rise to incongruence in the gene histories of both markers. The potentially short life span of numts and the similarly complex patterns of the fixation of secondary numt losses would lead to population polymorphisms. To date, only a few studies estimated the parameters of variation and persistence of numts in an explicit comparison with the corresponding mtDNA in a single lineage of organisms. These were based on a small number of taxa and minimal sampling for the purpose of establishing the common ancestor of mitochondrial and nuclear copies (Arctander 1995; Williams and Knowlton 2001; Lu, Fu, and Zhang 2002). As these studies have been carried out mostly on vertebrates, it also remains to be established if numts persist over similarly long periods in other organisms.
Here we analyze numt variation in a large radiation of closely related species of tiger beetles (Rivacindela) occupying the temporal salt lakes of interior Australia. mtDNA variation in this group has been sampled extensively to establish the extent and number of species ("DNA taxonomy") (Pons et al., unpublished data). In a subset of individuals, we encountered difficulties with DNA sequencing in one of three mtDNA regions, indicative of coamplification of a pseudogene. As these sequences can mislead the analysis of species trees and, in our case, the delimitation of species, we investigated the pattern of variation of these presumed numt sequences in detail. The analysis revealed an unexpectedly complex pattern of presence and absence within a narrow set of closely related populations, indicating a phylogenetic history of this element that was inconsistent with the mtDNA tree. Placing the numt sequences relative to their authentic mtDNA sequences was problematic due to the difference in the types and rates of character changes, but establishing sister relationships of the numt clade is a prerequisite for comparing tempo and mode of evolution in these elements and their mtDNA paralogs.
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Materials and Methods |
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Phylogenetic Analysis, Secondary Structure, and Nucleotide Substitution Rates
Alignment of mtDNA sequences was trivial due to the absence of indels. Numt sequences showed differences in length, but they could be aligned manually with unambiguous placement of indels. Assuming that multiple consecutive indels were the result of a single mutational event, gaps of multiple base pairs were recoded as binary characters according to Simmons and Ochotorena (2000) prior to parsimony analysis. The secondary structure prediction for the rrnL molecule was based on the previously described models for Cicindela dorsalis (Buckley et al. 2000) and Drosophila melanogaster (Gutell et al., unpublished; see http://www.rna.icmb-utexas.edu/). Parsimony tree searches were conducted in PAUP*4.0b10 (Swofford 2002) performing 1,000 random addition searches with Tree Bisection-Reconnection branch swapping and keeping 10 shortest trees per replicate. Shimodaira-Hasegawa (SH) tests were also performed in PAUP* with 1,000 RELL replicates.
Nucleotide composition bias between mtDNA and numt sequences of the rrnL gene was assessed with the 2 test of nucleotide homogeneity implemented in PAUP*. Because this statistical test does not take into account the phylogenetic relationships of sequences, the significance was also assessed using a null distribution from
2 statistics from simulations of nucleotide composition based on the tree and model of nucleotide evolution estimated from the real data (Foster 2004).
Maximum likelihood branch lengths were estimated on the topology of the shortest parsimony trees using the most appropriate model of nucleotide substitution selected by Modeltest 3.06 (Posada and Crandall 1998). Nucleotide substitution rates and node ages were determined using r8s 1.50 (Sanderson 2003), which estimates a substitution rate across the entire tree using a set of calibrated divergence times for all unfixed nodes but permits the application of separate rate parameters for different parts of the tree (local molecular clocks). Ages of focal nodes were estimated with six nodes constrained to known ages based on a calibrated mtDNA clock (Pons et al., unpublished data) with absolute ages obtained from biogeographic evidence (Barraclough and Vogler 2002; Pons et al. 2004).
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Results |
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Direct sequencing of the numt PCR product with the Riva16S primer resulted in clean sequences of 508510 bp, although in some cases readable electropherograms were only obtained after cloning in multicopy vectors. The analysis of clones revealed the presence of two alleles of different lengths in several individuals. Allelic sequences (found in multiple clones) were different by one substitution and one deletion of a single base in individual 9.4, one substitution and one long deletion of 26 bp in individual 39.2, or 12 nucleotide substitutions and a 1-bp deletion in individual 5.5. In addition, minor variants differing by one or two substitutions in singleton clones were encountered but were attributed to PCR or cloning errors and ignored. In total, the analysis of the numt-containing clade produced 29 different rrnL mtDNA haplotypes in the 84 individuals screened and 15 different numt alleles out of 50 individuals in this clade, which were positive for the numt. Most of the mtDNA haplotypes were limited to a single individual (19 haplotypes), seven were found in two or three individuals, and three haplotypes were widespread in 6, 13, and 31 individuals. Ten out of the 15 numt alleles were confined to a single individual, three alleles were found in a few individuals, and two other alleles were widely detected in 10 and 16 individuals (Table A1). The average pairwise divergence between mtDNA haplotypes was 0.46% and between numt alleles was 1.14%. The average divergence among mtDNA haplotypes and numts was 3.77%. The numt was not detectable in any of 27 representative populations selected from throughout the wider range of the genus Rivacindela, confirming its phylogenetically and geographically narrow distribution.
Analysis of Nucleotide Substitution Pattern and Secondary Structure
Patterns of nucleotide substitutions in stems and loops were compared in numt and authentic mtDNA haplotypes based on the hypothetical secondary structure of the rrnL sequence as shown in figure 2. Best fit was achieved when the large deletion present in all numt sequences was separated in two portions of 89 and 12 bp, affecting closely adjacent regions. In addition, three further deletions of 1 bp found in several individuals plus a long deletion of 26 bp (positions 6489) in allele 2 of individual 19.4 were mapped on the secondary structure model (fig. 2).
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Assuming that this tree is a good reflection of the mtDNA phylogeny, the analysis of rrnL sequences and their numts was performed again applying a backbone constraint to fit the topology from the combined mtDNA analysis. This search preserves all relationships inferred in the mtDNA analysis but permits the addition of new sequences anywhere into this topology. The search retrieved 24 trees of 237 steps, i.e., 10 steps longer and significantly worse than the unconstrained search (P = 0.013; SH test). However, it may still be accepted as a good hypothesis of the mtDNA phylogeny because it is based on the topology derived under the addition of 302 informative characters and 1,293 steps to the parsimony search. Surprisingly, in this tree the numt sequences were recovered in a position distant to Clade A (the expected position if a gene duplication in a common ancestor gave rise to the numt paralog). We also tested the fit of the numt Clade to several positions of the mtDNA tree by constraining it to be sister of alternative groups, including Clade A and several other major clades (not shown). These searches produced trees of 238 or 239 steps which were not significantly different from those with the simple backbone constraints only (not significant; SH test). Hence, the use of additional mtDNA genes could not unequivocally resolve the placement of the numt Clade, but a sister relationship with the numt containing mtDNA Clade A remains defensible. However, even this placement requires a complex scenario for the origin of the numt and its spread in populations as the numt containing individuals were not monophyletic based on mtDNA (both subclades A1 and A2 contain individuals with and without numt sequences).
Age and Nucleotide Substitution Rates of the Mitochondrial and Numt Sequences
Comparisons of evolutionary rates in mtDNA and numt sequences were attempted based on the inferred age and the corresponding nucleotide substitution rates for the origin of Clade A (mtDNA) and Clade P (numt). Absolute ages were obtained from an existing calibration based on biogeographic evidence (Material and Methods). Rates of nucleotide substitution for the rrnL sequences alone corresponded to 3.78 x 109 substitutions/site/year (0.378% divergence per million years) and were very similar to that estimated based on the topology obtained with the combined analysis of the three mtDNA regions (4.37 x 109). These values were about three to five times lower than the rates of cox1 (1.67 x 108), cob (2.11 x 108), or the three mtDNA regions combined (1.52 x 108). To calculate the rate of the numt Clade, two independent molecular clock estimates were applied separately to each DNA lineage. If constrained as sister to Clade A, the nucleotide substitution rate in the numt Clade was 1.66 x 108, i.e., about four times faster than the corresponding mtDNA lineage, based on the estimated age for both clades of 1.83 MYA. The estimated age for Clade A using rrnL sequences alone was very similar, 1.70 MYA.
Because of the uncertainty in the placement of the numt Clade, we also tested the age calibration under alternative topologies whereby the numt Clade was constrained to four different positions in the mtDNA tree. The age of Clade A and hence the nucleotide substitution rate of the mtDNA rrnL sequences was very similar under each topological constraint and deviated little from the above estimates of unconstrained searches of rrnL alone. However, the time of origin of the numt Clade varied greatly depending on its position on the tree. When constrained to a selection of other deep nodes in the mtDNA tree, the age of the numt Clade varied ranging from 3.02 to 3.96 MYA (corresponding to 8.96 x 109 to 6.85 x 109 substitutions/site/year). Based on an unconstrained search together with rrnL sequences only, the numt Clade was estimated to 2.22 MYA and a nucleotide substitution rate of 1.07 x 108, but the tree topology was highly inconsistent with the origin in a common ancestor with the numt-containing clade.
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Discussion |
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The presumed numts exhibited extensive deletions not present in functional copies of rrnL in other insects, their nucleotide substitution pattern suggests numerous noncompensatory changes in rRNA stem regions, and several individuals exhibited two alleles as expected for a single-copy Mendelian locus. Our findings argue against other possibilities to explain the presence of multiple copies of mtDNA genes, including contamination with parasitic or ingested organisms, laboratory artifacts, heteroplasmy, paternal leakage, and duplication of the mtDNA gene (Williams and Knowlton 2001). Although the evidence for a numt is strong, it proved remarkably difficult to determine their precise relationships with the mtDNA lineages and hence inferences of the hypothetical ancestor that gave rise to the numt. There were only 49 informative sites in the rrnL gene, plus 17 sites in numt sequences which, however, were invariant in the rrnL gene. The addition of many informative sites from other mtDNA regions provided a higher resolution of the tree but ultimately did not help to discriminate between several possible positions for the numt Clade. Numt sequences, as the product of an idiosyncratic integration of mtDNA into the nuclear genome, should be detectable as a gene duplication (the origin of a paralog) in the ancestor in which the transposition occurred. Therefore, the placement near Clade A, the only lineage containing this element, remains the most plausible scenario among several equally parsimonious possibilities.
Despite the uncertainties about precise sister relationships, the dense sample of orthologous numt sequences is a unique resource for studying the establishment of these elements in a clade. The most conspicuous finding of this analysis was that numt sequences were distributed inconsistently throughout Clade A1 and A2 and some populations were polymorphic for their presence. All phylogenetic analyses and the clock estimates agreed, however, that these sequences had a single origin which predates the age of the subgroups in Clades A1 and A2 lacking the numt. This finding might imply a secondary loss of the numts in several parts of Clade A, and perhaps its immediate mtDNA sister group which is only very slightly older (fig. 3b), or the difficulty of detecting these elements because of variation in primer-annealing sites. While the latter cannot be excluded, the rates of nucleotide substitutions and loss of base pairs are low (see below) and perhaps insufficient to account for the repeated loss of the element.
If secondary loss is not responsible for this pattern, other processes such as gene flow and the retention of ancestral polymorphisms could be invoked. Populations of Rivacindela are genetically highly structured throughout their range, and most populations were probably separated during the final stage of the drying of Australia some 4500,000 years ago (Pillans and Bourman 2001). The existing groups are largely the result of vicariance (Pons et al., unpublished data) and unlikely to undergo gene flow which could explain the observed numt distribution in Clades A1 and A2. Alternatively, lineage sorting after the initial transposition event could have affected the distribution of mtDNA and nuclear markers differently. The time of coalescence of numt sequences may exceed the age of recognizable mtDNA lineages, as expected for nuclear loci due to their greater effective population size. Only 1/2Ne of transposition events will be fixed (Sorenson and Fleischer 1996), and hence populations with intermediate frequencies of a numt should also be expected (Zischler et al. 1995; Bensasson, Feldman, and Petrov 2003) and in some cases have been observed (Thomas et al. 1996; Yuan et al. 1999; Ricchetti, Tekaia, and Dujon 2004).
The time calibration of the mtDNA tree was in good agreement with widely accepted estimates of mtDNA evolution (Hasegawa, Kishino, and Yano 1985; Brower 1994) and also showed the widely observed faster rates in protein-coding genes (Lopez et al. 1997). However, the age estimates for the numt Clade P were complicated and greatly dependent on the precise placement of this clade in the mtDNA tree. If set to be sister group to Clade A, the rates of nucleotide substitution estimated under a separate local clock were 1.66 x 108 substitutions/site/year and hence four to five times faster than previous estimates for numts (3 x 109 to 4 x 109; e.g., Lu, Fu, and Zhang 2002) and other nuclear pseudogenes (4 x 109; Li 1997). These values are lower when other phylogenetic positions of the numt Clade are considered but still exceed previous estimates by a factor of 1.53. These differences in estimates of neutral rates of nucleotide substitutions may be due to the methodological differences between the studies which lacked precise information about sister relationships, or they may suggest that rates are generally higher in insects. DNA-DNA hybridization studies have established that the average rate of mtDNA and single-copy nuclear DNA is similar in insects, in contrast to mammals where the mtDNA rate is about 510 times higher (Caccone, Amato, and Powell 1988; Sharp and Li 1989). Our results would corroborate these findings and perhaps indicate that the neutral rate in the nuclear chromosome of insects equals or even exceeds the mtDNA rate.
Aside from nucleotide substitutions, variation in numts is greatly affected by indels. We found six deletions in the Rivacindela numts of 189 bp in length but no insertions. This is in agreement with studies of neutral changes in Drosophila non-LTR retrotransposons where deletions outnumber insertions almost 9 to 1, and the average deletion size of 25 bp greatly exceeds the average insertion size of 2.8 bp (Petrov et al. 1998, and references therein). Deletions contribute greatly to the degradation of these pseudogenes and will lead to their eventual demise. The two long adjacent deletions found in all numts, and hence presumably fixed at the species level, must have arisen between 1.83 and 0.46 MYA in the calibrated tree (the internodes between origin of the numt clade and the inferred point of coalescence of the numt sequences). Thus, 101 of 890 bp were lost from the numt in a maximum time span of 1.37 MYA corresponding to a rate of nucleotide loss of 8.2 x 108 per site and year. Similar rates can be obtained based on a 26-bp plus two 1-bp deletions (28 of 789 lost in 0.46 MYA, equal to a minimum rate of 7.7 x 108) closer to the tips of the tree, although the latter deletions were not found in all individuals, and hence it remains possible that they will not ultimately be fixed in the lineage. If taken at face value, the rates of deletion in the numt Clade are about five times faster than the nucleotide substitution rate of 1.66 x 108 substitutions/site/year.
In comparisons between groups of organisms, the ratio of deletion rate to nucleotide substitution rate varies widely, ranging from 51 in Arabidopsis thaliana, to 4.25.9 in Caenorhabditis elegans, 4.5 in Drosophila, 34 in Strongylocentrotus purpuratus, to the much lower values of 0.13 in rodents and mammals, 0.34 in crickets, and 0.06 in grasshoppers (Petrov 2002b; Britten et al. 2003). This appears to be affected mostly by differences in the deletion spectrum size, rather than frequency at which deletions occur, and is related to genome size, as the rate of DNA loss and the number of numts (and other nuclear pseudogenes) present in the nucleus are correlated (Bensasson et al. 2001a, 2001b; Petrov 2002a; Gregory 2004). Species with small genomes such as D. melanogaster (0.2 pg) and C. elegans (0.1 pg) have higher rates of DNA loss and consequently harbor fewer numts and other pseudogenes than species with large genomes such as humans (3.5 pg) or grasshoppers (8.2 pg). The size distribution of the deletions we found here for a beetle is closer to that of Drosophila than grasshoppers and crickets. The low prevalence of numts in Rivacindela and many other groups of beetles (unpublished data) which have small genome sizes (0.50.6 pg, Gregory 2001) would confirm this trend of deletion rates and genome size. The fact that numt sequences have not been described in the Coleoptera previously may therefore indicate that they are truly rare, rather than having been overlooked. Numt sequences were found to be essentially absent from two fully sequenced genomes of Diptera, which also harbor few nuclear pseudogenes (Richly and Leister 2004). Although these findings are not fully conclusive because of the usual practice of genome sequencing projects to screen out mtDNA sequences, the low tendency to detect numts in holometabolan insects suggests their rarity compared to other insect groups with larger genomes.
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Conclusions |
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Appendix |
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Acknowledgements |
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Footnotes |
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