Illinois Natural History Survey, Champaign, Illinois
Correspondence: E-mail: kjohnson{at}inhs.uiuc.edu.
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Abstract |
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Key Words: deletion bias introns evolution
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Introduction |
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Resolving this debate will require an understanding of the underlying mutation rates and substitution properties of insertion and deletion events (indels) in introns. Studies of nuclear pseudogenes and their functional counterparts in humans and mice have revealed that deletions outnumber insertions by about 2.7 to 1 (Ophir and Graur 1997). Similarly, deletions were estimated to outnumber insertions by 2 to 1 in nuclear pseudogenes and spacer regions in primates (Saitou and Ueda 1994). In Drosophila, polymorphic sites indicate that deletions outnumber insertions by 1.35 to 1 (Comeron and Kreitman 2000). Polymorphisms are presumably a closer reflection of the underlying mutation bias, because selection is relatively stronger over evolutionary timescales such that deleterious mutations may be eliminated (Lynch 2002). Both mutation and selection interact to determine the substitution rates of insertions and deletions over evolutionary timescales. Recent theoretical work has suggested that there are reasons why insertion events in introns might be deleterious relative to deletion events, including reduced transcription efficiency and decreased splicing accuracy (Lynch 2002). However, it is unclear how these selection biases might interplay with underlying mutation biases.
A detailed examination of the substitution pattern of indels in introns over evolutionary timescales has not been conducted. Prychitko and Moore (2003) examined molecular evolution in the ß-fibrinogen intron 7 across 28 species of birds. Although they did report a deletion bias, it was not quantified. Mapping indels over a phylogeny provides polarity information, which allows identification of indels as either insertions or deletions. In the present study, I reconstruct a phylogeny for pigeons and doves (Aves: Columbiformes) using mitochondrial protein coding and nuclear intron DNA sequences. I then reconstruct the history of indel substitutions for the ß-fibrinogen intron 7 over the phylogeny, using parsimony. This reconstruction provides an assessment of the relative frequency and size distribution of indel events over evolutionary timescales. In addition, this study provides an assessment of the degree of convergence (or homoplasy) of intron indels.
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Materials and Methods |
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Phylogenies were reconstructed from the combined sequence data using both parsimony and Bayesian maximum likelihood searches. FIB7 indels were initially treated as missing data in the phylogenetic analysis, such that they could be independently reconstructed over the tree in subsequent analyses. In parsimony analyses, 100 random addition replicate searches with all characters unweighted and unordered were conducted using PAUP* (Swofford 2001). To assess the sensitivity of this tree to character resampling (Felsenstein 1985), 1,000 bootstrap replicate searches were conducted. Bayesian maximum likelihood analyses were conducted using MrBayes (Huelsenbeck 2001) with a GTR + I + G model. Posterior probabilities for various nodes in this tree were calculated sampling trees every 1,000 generations from a chain of 2 million generations and ignoring the first 500,000 burn-in trees. Two runs were performed and the average posterior probability is reported.
Using the alignment of ß-fibrinogen intron 7, the evolutionary timing of insertion and deletion events within the ingroup (Columbiformes) was evaluated with respect to the base position of the aligned sequences. Indels involving the same position and number of base pairs were considered homologous. With MacClade (Maddison and Maddison 1992), parsimony was used to reconstruct the number of indel events, and to determine whether these events were insertions or deletions. For each indel, all most parsimonious optimizations were examined. A string of T repeats at aligned positions 559565 was not included in this analysis because of difficulty in evaluating the homology of various T indels.
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Results |
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Deletions outnumbered insertions 44 to 7 (or 43 to 8 in an alternate equally parsimonious reconstruction). The proportion of indels that were deletions (86.3%) was significantly greater than chance (Sign test: P < 0.0001) and also significantly greater than 73% (Wilcoxon signed rank test, P < 0.0005), which is the highest bias previously reported for nuclear deletion events (Ophir and Graur 1997). The size of intron indels ranged from 1 to 167 base pairs (fig. 2). The mean insertion length (3.9 bp) was smaller than the mean deletion length (12.2 bp), but this difference was not significant (t-test, P = 0.48).
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Discussion |
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The size of indels was variable, but it did not differ significantly between insertions and deletions. Large insertions and deletions occur in a single mutational step, and there is no evidence of intermediates for these large indels. The prevalence of repeat elements in indel regions, especially for insertions, supports the slipped-strand mispairing mechanism for the origin of insertion events (Levinson and Gutman 1987). This mechanism may be an important process in intron regions, and not just in regions where repeats are already prevalent (Kelchner 2000).
Given the strong bias toward deletion substitutions, what keeps the introns from disappearing over evolutionary time and intron size relatively stable (Waltari and Edwards 2002)? It may be that once introns get below a certain size, mutations that knock out the splice sites become more likely (Lynch 2002). Over evolutionary time, such indirect selection may counteract the bias toward deletion substitutions in introns. Much more work is needed on the mutation properties of nuclear introns and the fate of these mutations over evolutionary time.
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Acknowledgements |
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Footnotes |
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Literature Cited |
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