*Department of Organismic and Evolutionary Biology, Harvard University;
Department of Biological Sciences, Stanford University;
Institute of Zoology, Chinese Academy of Sciences, Beijing, People's Republic of China; and
§School of Biological Sciences, University of East Anglia, Norwich, England
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Abstract |
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Introduction |
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Most acridid grasshoppers have larger genomes (5,95020,600 Mb) than other insects (988,900 Mb) and all mammals (1,4205,680 Mb), and P. pedestris has one of the largest of these (18,150 Mb) (genome sizes approximated from Rees, Shaw, and Wilkinson [1978]
, Rasch [1983],
Westerman, Barton, and Hewitt [1987]
, and Li [1997]
). What are the characteristics of DNA deletion in an organism with such a large genome? Is the rate at which nucleotide deletions accumulate in Podisma pseudogenes significantly lower than those observed in Drosophila, crickets, or mammals? Does the correlation between genome size and nucleotide deletion rates extend to comparisons between species without streamlined genomes? We addressed these questions by characterizing nucleotide deletions in the sister genera Podisma and Italopodisma across 58 different pseudogene DNA sequences and comparing them with the published data for other species. The genome size of Italopodisma sp. is not known, but we assumed it to be similar to that of P. pedestris. Several of the Podisma and Italopodisma pseudogenes studied arose prior to the divergence of these recently diverged sister genera.
The pseudogenes used in this study are recently arisen nuclear copies of mitochondrial genes (hereinafter referred to as "Numts", following the abbreviation of Lopez et al. [1994]
). Animal Numt sequences almost certainly lose their function when they escape the mitochondria (Gellissen and Michaelis 1987
). They are "dead on arrival." This makes them ideal for comparing rates of insertion and deletion (indel) mutation relative to rates of nuclear substitution mutation (Graur, Shauli, and Li 1989
; Petrov, Lozovskaya, and Hartl 1996
). Numt sequences evolve very differently from their paralogous mtDNA sequences, so nuclear mutations that arose since a Numt's loss of function can easily be distinguished from changes that arose in mitochondrial lineages. The Numts of Podisma and Italopodisma arose through many independent transfers to the nucleus (Bensasson, Zhang, and Hewitt 2000
), so the nuclear changes they accumulate probably arose independently in each Numt. The many pseudogenes studied here are paralogous, so sequence composition varies in a similar way across each one, which allows the study of sequence composition effects on mutation. Numts are widespread in eukaryotes and abundant in mammals and grasshoppers (Zhang and Hewitt 1996
), so this approach could be employed on other species.
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Materials and Methods |
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Total genomic DNA was extracted from each individual. Oligonucleotide PCR primers were designed with DNA sequences matched to that of L. migratoria (GenBank accession number X80245; Flook, Rowell, and Gellissen 1995
) in such a way that they would amplify a variable 643-bp portion of ND5 by annealing to stretches of sequence that are conserved among Orthopterans. These primers amplified paralogous ND5-like Numts, as well as mtDNA sequences, in all grasshoppers studied, although all PCR products appeared to be of the expected 688-bp size (643 bp plus primers). PCR products were cloned, and several (234) recombinant colonies were picked for each PCR product. The insert was amplified by PCR from each colony and sequenced from both strands. As the sequences used were not very divergent, they were aligned and checked by eye in MacClade, version 3.01 for Macintosh (Maddison and Maddison 1992
).
A DNA extraction that enriches for mtDNA was successfully employed on three frozen individuals (the two Italopodisma and one of the P. pedestris individuals). When the PCR primers were employed on mitochondrial enriched DNA, ND5 products were almost exclusively of one type in each individual, and this was assumed to be the mtDNA sequence. However, the mitochondrial enrichment protocol was not successful for the other P. pedestris individuals studied, probably because they were ethanol-preserved and their mitochondria were therefore less well preserved (Dowling et al. 1996
). For these other P. pedestris individuals, the mtDNA was assumed to be the type which contained no frameshift mutations and was present in high copy numbers.
Fifty of the 58 Numt sequences used were picked from PCR products that were amplified using a high-fidelity polymerase (Pfu polymerase). To confirm that the Pfu error was low enough for the purposes of this study, Pfu polymerase was used to amplify a PCR product from a colony with a known Numt insert instead of total genomic DNA. The product was cloned, 10 colonies were picked, and their inserts were sequenced from both strands. All 10 sequences were identical to the original insert sequence; therefore, misincorporations resulting from the techniques employed here were few. The remaining eight Numt sequences that were selected from Taq-amplified PCR products are labeled with a appended "t" (e.g., SpB5t). A total of two to three nucleotide substitutions and one indel would be expected to result from Taq errors in these eight sequences (estimated from the Taq error rates published in Kwiatowski et al. [1991]
). Three or four Taq misincorporations would not affect the outcome of this study.
Separating Nuclear from Mitochondrial Lineage Changes
All insertions and deletions were assumed to occur in the noncoding (Numt) lineages for the following reasons: (1) 65 of the 66 indels observed in this study would have generated a frameshift mutation and thus would probably be highly deleterious in the mitochondrial lineage; (2) no indels had arisen in the mtDNA sequences since their ancestral divergence, although the sequences represent acridids of three different subfamilies; and (3) no indels arising from somatic mutation of mtDNA molecules could be detected. To study somatic mutation in mtDNA, PCR products were amplified, cloned, and sequenced from the mitochondrial enriched DNA of three individuals: 11 colonies with mtDNA inserts were sequenced from one individual, 5 from another, and 4 from the third. All mtDNA sequences within individuals were identical. If there are any somatic mutations in mtDNA molecules, their frequency is too low to affect this study.
Thirty-one of the Italopodisma Numts belong to a closely related family of Numts (Bensasson, Zhang, and Hewitt 2000
). The nucleotide differences among these Numts are few. They differ from one another only by indels and one or two random point substitutions, but their majority-rule consensus sequence differs from the current mtDNA type by 13 point substitutions and no indels. It appears that the pseudogenes of this family are descended from the same mitochondrial sequence and that the differences among them arose in the nucleus. For a fuller discussion of this conclusion, see Bensasson, Zhang, and Hewitt (2000)
.
Each of the remaining 27 Italopodisma and Podisma Numts diverged at different times from the Italopodisma and Podisma mitochondrial lineages (fig. 1
). Some were 12% divergent from the current mtDNA sequences (at the base of the alignment in fig. 1
), while others differed by as little as one mutation (at the top of the alignment). Numt sequences are thought to resemble ancestral mtDNA sequences (Fukuda et al. 1985
; Hu and Thilly 1994
), and a comparison of the sequences in figure 1
would support this. This is reflected in the tendency of the more divergent Numt pseudogenes in figure 1
to have a greater similarity to the more distantly related mtDNA sequences (Par m, Sg, and Lm) than to the Podisma and Italopodisma mtDNA sequences (Fr Pp through Itsp).
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It is clear from the small proportion of variable sites (fig. 1 ) that if nucleotide substitutions are distributed randomly (as expected under nuclear evolution), then multiple hits are unlikely. Nucleotide differences which are shared by more than one pseudogene usually represent differences acquired in a common ancestor. Since most Numts diverged from each other before their ancestor arrived in the nucleus (Bensasson, Zhang, and Hewitt 2000
), shared differences probably arose in mitochondrial lineages (e.g., positions 3, 6, 9, 15, and 18 in fig. 1
). For this reason, only unique substitutions (occurring at sites which were identical in all other nuclear or mitochondrial sequences) were counted as mutations occurring in the nucleus (fig. 1
).
Of the 643 bp studied, there are 195 sites with variations in one of the mtDNA sequences or in two or more of the Numt sequences. Because only unique substitutions were counted as nuclear, any nuclear substitutions arising in these 195 sites were excluded. Therefore, the nuclear substitution frequency was estimated across what is effectively 448 bp.
This method for distinguishing nuclear and mitochondrial changes is similar in effect to a maximum-parsimony analysis. Maximum parsimony would assign all unique changes to the terminal branches of a tree, which would be occupied mostly by the dead-on-arrival lineages (Petrov, Lozovskaya, and Hartl 1996
). However, shared substitutions resulting from multiple hits could also be ascribed to nuclear evolution, because parsimony makes no distinction between active (constrained) and inactive (pseudogene) lineages. A parsimony analysis of our data would ascribe some changes to nuclear mutations that we ascribe to mtDNA mutations, so parsimony would overestimate the number of nuclear mutations relative to the analysis presented here. Because we predicted a low number of deletions relative to point substitutions in grasshoppers, the most conservative test of this prediction would be to underestimate the number of point substitutions, since this would overestimate the rate of DNA loss. For the purposes of this study, our analysis was consequently more conservative than a maximum-parsimony analysis.
Testing the Separation of Nuclear and Mitochondrial Changes
Insect mitochondrial changes are expected to show a greater transition-to-transversion bias than changes accumulated in the nucleus (Tamura 1992
; Moriyama and Powell 1996
; Petrov and Hartl 1999
). To test whether the separation of nuclear and mitochondrial changes was successful, we compared the transition-to-transversion bias observed for nuclear changes to that expected for mitochondria. For this comparison, we estimated mitochondrial transition-to-transversion bias by maximum likelihood for Podisma, Italopodisma, and closely related mitochondrial lineages (L. migratoria was not included in the analysis). The maximum-likelihood analysis of the mtDNA sequences of P. pedestris (five different sequences), Italopodisma, P. mikado, and S. gregaria was conducted with PAUP, version 4.0 (Swofford 1999). The settings used correspond to the Hasegawa, Kishino, and Yano (1985)
model with rate heterogeneity. Empirical nucleotide frequencies were used and the gamma distribution was estimated using four rate categories. The estimated transition-to-transversion ratio was 1.7 (kappa = 4.8). This shows a strong bias in favor of transition mutations, as expected for insect mtDNA sequence (Tamura 1992
).
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Results |
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A second, independent, test of whether these nucleotide substitutions have arisen in the nucleus was to examine the ratio of transitions to transversions. In insects, the observed bias in favor of transitions was much less under noncoding nuclear evolution than under mitochondrial evolution (Tamura 1992
; Moriyama and Powell 1996
; Petrov and Hartl 1999
). There were 18 transitions and 25 transversions. This ratio was not significantly different from 1:2, which is the ratio expected when there is no bias in favor of transitions (
2 test; P = 0.2, df = 1). On the other hand, 18:25 was significantly different from the 1.7:1 estimated for mtDNA sequences in these grasshoppers (
2 test; P = 0.004, df = 1). This result confirmed that the changes scored as nuclear in this study were characteristic of the type of mutation expected in the nucleus.
Similarly, the 126 changes scored as nuclear in the remaining data (27 Numts) showed no significant bias in their positions within codons (2 test; P = 0.9, df = 2). The numbers in the first, second, and third codon positions (47, 47, and 32, respectively) were very close to the numbers expected (45, 48, and 33). The expected numbers were based on a 1:1:1 ratio correcting for the fact that the numbers of first-, second-, and third-codon-position sites included in the analysis were 160, 172, and 116. In contrast, the sites of shared Numt differences that were excluded from the analysis (on the grounds that they occurred in mtDNA) were constrained by selection; the numbers for first, second, and third positions were 55, 42, and 98 (
2 test; P < 1 x 10-5, df = 2), suggesting that they were correctly assumed to be evolving under selective constraints in the mitochondria. The observed transition : transversion ratio among nuclear changes (45:81) also showed no significant bias (
2 test; P = 0.6, df = 1), but it was significantly different from the ratio estimated across mtDNA sequences only (
2 test; P < 1 x 10-9, df = 1). This result was also as expected for mutations arising in the nucleus.
Indel Hot Spots
Examination of Numt and mtDNA sequences revealed a 21-bp region (209229 bp from the start of the GenBank sequences used) with an unusual base composition. This region was very A+T-rich and featured a stretch of T's up to 10 bp long even in functional mtDNA. Forty of the 56 deletions observed, and 3 of the 9 insertions, occurred in this region. Both of these rates are much higher than would be expected by chance (Fisher's exact test; P = 3 x 10-11 and P = 1 x 10-5, respectively). Most of the pseudogenes used in this study arose independently of each other (Bensasson, Zhang, and Hewitt 2000
), so the high indel frequency observed in this region cannot be attributed to common ancestry. This region appears to be a major hot spot for indel mutations, but it does not have an elevated rate of point substitutions. Single-base repeats are well documented as indel hot spots (Levinson and Gutman 1987
).
A second hot spot was also discovered 137143 bp from the start of the GenBank sequences: of the 16 remaining deletions, four occurred within a stretch of six T's. This rate is significantly higher than would be expected by chance (Fisher's exact test; P = 0.002). Each of these four deletions arose in pseudogenes with separate mitochondrial origins (data not shown); therefore, they arose independently.
Close examination of Laupala and Drosophila sequences revealed no mononucleotide repeats of more than 5 bp for Drosophila and no regions with elevated indel frequencies. Laupala did have one unusual 9-bp region (three C's and six A's) at positions 131139. This was also the only region showing a significantly elevated indel frequency (7 of 65 indels occurred here) (Fisher's exact test; P = 0.0001). There were therefore major differences among the taxa studied in the sizes and strengths of hot spots. These differences appear to have arisen as a result of the differences in the lengths and compositions of the original sequence studied. For species comparability, we excluded hot spots from our analysis, except where otherwise specified, but we show a separate analysis that includes them in table 1 to illustrate how they could affect results.
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When the Italopodisma family of Numts was excluded, the numbers of indels observed in grasshoppers were counted across 616 bp of each Numt, but the numbers of point substitutions were counted across 448 bp. To adjust for this discrepancy when calculating the ratios of insertions or deletions relative to point substitutions, the total number of deletions observed was corrected to 5.8 (i.e., (8/616) x 448), and the number of insertions was corrected to 2.2 (i.e., (3/616) x 448). Such an adjustment inflates the variance of our estimate and therefore is conservative in hypothesis testing. The 95% confidence interval for the ratio of deletions to point substitutions was based on a maximum-likelihood analysis of the total numbers of deletions and the total numbers of substitutions observed. This analysis assumed that probabilities of mutations (deletions, insertions, or point substitutions) followed Poisson distributions and that mutations accumulated linearly with time.
The mean rate of DNA loss per point substitution was calculated as DNA loss = (deletion size x deletion ratio) - (insertion size x insertion ratio) using the mean indel sizes and ratios given in table 1 . To estimate the 95% confidence intervals for the rate of DNA loss, each of these four sample means was assumed to follow normal distributions, and the rates of DNA loss were calculated from means drawn randomly from these four distributions over 1,000 replicates.
The DNA Loss Observed for Grasshoppers Is Slower than That for Crickets or Drosophila
Podisma and Italopodisma appear to be losing nonessential DNA at a rate considerably slower than the rate of 0.34 bp per point substitution observed for Laupala crickets (P < 0.001) or 3.2 bp observed for Drosophila (P < 0.001) (table 1
). This effect can be mostly attributed to differences in the sizes of deletions. Podisma and Italopodisma accumulate significantly smaller deletions than Laupala (Wilcoxon test; P = 0.005) or Drosophila (Wilcoxon test; P < 0.001). More specifically, differences in deletion size appear to result from a difference in the frequency distributions of deletion sizes (fig. 2
). The proportion of deletions that are larger than 1 bp is significantly smaller in grasshoppers than in Laupala (G test; df = 1, P = 0.005) or Drosophila (G test; df = 1, P < 0.001).
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Discussion |
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The extent of bias appears to depend on the length of the hot spot sequence. The major hot spot (21 bp of unusual sequence) accounted for many more indels (43 out of 65) than the second grasshopper hot spot, with 6 bp of unusual sequence (4 of the remaining deletions), or the 9-bp Laupala hot spot (7 indels out of 66). This implies that if hot spot regions are included, species differences in the original hot spot sequence could lead to artifactual differences in deletion frequency.
Although in grasshoppers the overall rate of deletion is not significantly higher than the insertion rate (in table 1, the mean rate of DNA loss is not significantly greater than zero), in the hot spot regions studied it is (when hot spot regions are included, the mean rate of DNA loss is significantly greater than zero; P < 0.01). Newly arisen pseudogenes will lose DNA from these indel mutation hot spots, so the degree to which they remain hot spots may fall. Therefore, indel accumulation in hot spots is probably not linear in time; these hot spots may be more common in "young" pseudogenes.
Differences in deletion size rather than frequency account for the largest component of the species differences in the estimated rates of DNA loss. The indel hot spots discovered did not contain deletions differing in size from those observed in the rest of the grasshopper data (Wilcoxon test; P = 0.9). Even if the many indels that arose in the hot spots were included in our analysis, the estimated rate of DNA loss in grasshoppers would still be considerably (and significantly) lower than that observed in Drosophila, although not significantly different from that in crickets. The hot spot observed in the Laupala data is too mild to significantly affect estimates of DNA loss (table 1 ).
All other reported comparisons have shown that species with smaller genomes have higher rates of DNA loss (e.g., Graur, Shuali, and Li 1989
; Petrov, Lozovskaya, and Hartl 1996
; Ophir and Graur 1997
; Petrov and Hartl 1998
; Petrov et al. 2000
). With hot spots excluded, our data suggest that Laupala crickets (which have 10-fold less DNA than Podisma) lose nonessential DNA faster than P. pedestris grasshoppers. The low rate of DNA loss in Podisma is consistent with the reported pattern of DNA loss and suggests that the pattern may also apply to comparisons between species whose genomes are not as streamlined as that of Drosophila. The estimated rate of DNA loss in grasshoppers (less than 0.19 bp lost per nucleotide substitution) suggests that a site is less likely to be deleted than it is to change through a point substitution. In grasshoppers, therefore, the accumulation of point mutations is a more potent force for obscuring ancient pseudogenes than the accumulation of indels, while the reverse is true for Drosophila.
Petrov et al. (2000) showed that the frequencies of small deletions are similar between Laupala and Drosophila. Caenorhabditis elegans, with its small genome (86 Mb), shows a high proportion of large deletions, similar to those observed in Drosophila (176 Mb) and in contrast to those observed in humans (3,400 Mb) (Robertson 2000
). The data presented here also suggest that it is the sizes and frequencies of large (>1 bp) deletions that vary among species with different genome sizes (table 1
and fig. 2 ). In grasshoppers, no deletions were observed that were larger than 4 bp. Perhaps deletions are generated by more than one mechanism: one mechanism is relatively constant between species and generates predominantly small (single-nucleotide) deletions (e.g., as expected through slipped-strand mutation), while a second mechanism generates a broader range of deletion sizes (e.g., as expected through unequal sister chromatid exchange or unequal crossing over) and is more active in organisms with smaller genomes.
The 58 Numts analyzed here represent at least 40 kb of inserted DNA; in the time since they arose, they have sustained a net loss of only about 10 bp (0.025%) through indel accumulation. This rate may seem slow, but if applied across the colossal grasshopper genome, well over 4.6 Mb of DNA loss would be expected since the Numts used here arose ([169 point substitutions/40,000 bp Numt DNA] x 18,150,000,000 bp genome size x 0.06 bp mean DNA loss).
This and previous studies (e.g., Graur, Shuali, and Li 1989
; Petrov, Lozovskaya, and Hartl 1996
; Ophir and Graur 1997
; Petrov and Hartl 1998
; Petrov et al. 2000
) have demonstrated that relative rates of DNA loss differ across species. But what about absolute amounts of DNA loss? Surprisingly, it seems that absolute amounts of DNA loss are comparable in the species studied here. If grasshoppers have 100 times as much nonessential DNA as Drosophila and 10 times as much as Laupala (not unreasonable considering their differences in genome size), then indels will accumulate across 100 times as many sites as in Drosophila and across 10 times as many as in Laupala. For example, when each species has accumulated a million point substitutions across its genome, Drosophila will have lost 3.2 Mb of DNA, Laupala will have lost 3.4 Mb (0.34 x 10), and Podisma will have lost 6 Mb (0.06 x 100).
It is interesting that the significantly different rates of deletion per nucleotide site (brought about by different patterns of mutation) have arisen in different species of insects, while the absolute rate of DNA loss per genome remains on the same order of magnitude. Perhaps the absolute amounts of DNA gain due to large insertions (transposable elements, Numts, etc.) in these species are also similar, which would suggest that their genome sizes are near or at equilibrium. More information on the rate of DNA acquisition, and on other mechanisms of DNA loss, would be needed to evaluate this possibility.
Even if the genome sizes of the insects discussed here were presently at equilibrium, the observed differences in deletion rates may have played an important role in determining the present genome sizes. If the absolute rate of DNA gain remained constant, and if (for the sake of argument) there was no selection acting on genome size, a change in the relative rate of indel accumulation would drive a change in genome size until the same total amount of DNA was lost across the genome once more. For example, if grasshoppers were to acquire a 40-fold greater rate of DNA loss (the same relative rate of indel accumulation as Drosophila) in the absence of selection, and if the amount of DNA gain were to remain unchanged, the amount of nonessential DNA in grasshopper genomes would shrink. As genome size decreases, the absolute amount of DNA lost also decreases until it reaches its original value at an approximately 40-fold smaller genome size. In light of the extremely slow rate of DNA loss actually observed, it is not surprising that the genomes of Podisma and Italopodisma are gigantic.
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Acknowledgements |
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Footnotes |
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1 Abbreviations: indel, insertion or deletion; Numt, nuclear mitochondrial DNA.
2 Keywords: Acrididae
C-value paradox
Numt sequences
numtDNA
mtDNA
transposition
nuclear copies
horizontal gene transfer
dead-on-arrival (DOA) sequences
3 Address for correspondence and reprints: Douda Bensasson, Hartl Lab, Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts 02138. E-mail: dbensass{at}oeb.harvard.edu
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