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Published online before print
November 7, 2007, 10.1101/gr.6376807 Genome Res. 17:1888-1897, 2007 ©2007 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/07 $5.00 OPEN ACCESS ARTICLE
12 Drosophila Genomes/Letter Sequence variation within the rRNA gene loci of 12 Drosophila speciesUniversity of Rochester, Department of Biology, Rochester, New York 14627, USA
Concerted evolution maintains at near identity the hundreds of tandemly arrayed ribosomal RNA (rRNA) genes and their spacers present in any eukaryote. Few comprehensive attempts have been made to directly measure the identity between the rDNA units. We used the original sequencing reads (trace archives) available through the whole-genome shotgun sequencing projects of 12 Drosophila species to locate the sequence variants within the 7.8–8.2 kb transcribed portions of the rDNA units. Three to 18 variants were identified in >3% of the total rDNA units from 11 species. Species where the rDNA units are present on multiple chromosomes exhibited only minor increases in sequence variation. Variants were 10–20 times more abundant in the noncoding compared with the coding regions of the rDNA unit. Within the coding regions, variants were three to eight times more abundant in the expansion compared with the conserved core regions. The distribution of variants was largely consistent with models of concerted evolution in which there is uniform recombination across the transcribed portion of the unit with the frequency of standing variants dependent upon the selection pressure to preserve that sequence. However, the 28S gene was found to contain fewer variants than the 18S gene despite evolving 2.5-fold faster. We postulate that the fewer variants in the 28S gene is due to localized gene conversion or DNA repair triggered by the activity of retrotransposable elements that are specialized for insertion into the 28S genes of these species.
Eukaryotic ribosomal RNAs (rRNA) are encoded by hundreds of gene copies organized in tandem arrays (the rDNA loci) (Long and Dawid 1980  ; Hillis and Dixon 1991). Each unit within the array contains one copy of three major rRNA genes: 18S, 28S, and 5.8S (Fig. 1). The different copies of the rDNA units have high-sequence identity within species, but differ between species, a phenomenon called concerted evolution. While the sequence of the rRNA genes evolves slowly, the internal transcribed spacers (ITS1 and ITS2), the external transcribed spacer (ETS), and the intergenic spacer (IGS) evolve rapidly.
Most models for the concerted evolution of the rDNA locus suggest that frequent recombination events involving unequal crossovers and gene conversions result in the high-sequence identity between units (Ohta 1980 ). In this process, a mutation originating in one unit is increased or decreased in number by recombination until it is either eliminated or present in all units. Substitutions in the coding regions are subject to selection for rRNA function and most are eliminated, while substitutions in the noncoding areas are under less selective pressure and are more often fixed by stochastic recombination events. Thus, concerted evolution is a dynamic balance between mutation, selection, and recombination.
Many studies have been conducted to follow the concerted evolution of the rDNA units in Drosophila melanogaster (Coen et al. 1982
Evolution of the rDNA loci in Drosophila, as in many animals, is also influenced by retrotransposable elements, R1 and R2, which insert into specific sites of the 28S rRNA gene (Jakubczak et al. 1991 A detailed view of the level and distribution of the sequence variation within the rDNA units of an organism would provide critical insights into the concerted evolution process. However, the dearth of distinguishing details between repeats has made this difficult. Even with the "complete genome sequences" now available for many species, the rDNA loci have not been assembled or the level of sequence variation quantified. While whole-genome shotgun sequencing does not provide a means to assemble rDNA loci, it does provide a wealth of information on the nucleotide variation that exists. In this report, the rDNA sequences from the original unassembled sequencing reads are used to characterize for the first time the nucleotide sequence variation that exists in the rDNA units of 11 Drosophila species. These data provide valuable insights into both the efficiency and the mechanism of the concerted evolution.
Comparison of the consensus rDNA unit among species Consensus sequences for the transcribed portion of each rDNA unit were first assembled (Supplemental Fig. 1a–l). The small 5.8S and 2S genes were highly conserved with only the first position of the 5.8S gene having undergone a substitution in species of the melanogaster group. The 18S gene in all species was 1995 bp in length, while the 28S gene varied in length from 3948 to 3976 bp. The 18S and 28S rRNA sequences can be subdivided into the slower evolving "core regions," which include the active sites, substrate binding sites, and contact points between subunits, and the "expansion regions," which vary in sequence and account for most of the length differences among eukaryotic and prokaryotic rRNA genes (Clark et al. 1984 ; Hassouna et al. 1984). Figure 2 plots the sequence differences among the 12 species (vertical lines). The expansion regions (white boxes) contained most of the nucleotide differences and all but one of the indels found between Drosophila species, with most length variation in expansion regions D7a and D12. Most changes occurred in single-stranded or loop regions of the proposed secondary structure (Hancock and Dover 1988).
To calculate the rate at which the core and expansion regions accumulated nucleotide substitutions, the sequence divergences of these regions were determined for various species pairs. These divergences are plotted in Figure 3 as a function of the time estimates since separation of the species. The expansion regions of the 18S and 28S genes diverged about 15 times faster than their respective core regions. Both the core and expansion regions of the 28S gene diverged 2.5 times faster than the 18S gene.
The transcriptional start site marking the beginning of the ETS has been determined for D. melanogaster and Drosophila virilis (Long et al. 1981 ; Murtif and Rae 1985). Because in these species the ETS starts near the last subrepeat in the IGS, the first nucleotide downstream of the last tandem subrepeat in the IGS was arbitrarily defined as the ETS boundary for the remaining species. The length of the ETS as well as the ITS1 and ITS2 regions in each species are shown in Figure 4. Regions within these spacers with >75% nucleotide identity to the corresponding D. melanogaster sequence are indicated by the thicker horizontal lines. Extensive sequence identities were only found between members of the melanogaster species subgroup (melanogaster, simulans, sechellia, yakuba, and erecta). However, as was noted previously by Schlötterer et al. (1994) the 3' end of ITS1 and 5' end of ITS2 are more conserved, showing sequence identity across all species. Sequence conservation in these two regions has been suggested to be a result of secondary structures needed for processing of the primary RNA transcript.
Simplified diagrams of the tandem subrepeat organization of the IGS regions in each of the 12 species are shown in Figure 5. These assembled IGS regions are not consensus sequences, because individual rDNA units within the same species differ in the numbers of each subrepeat (Coen et al. 1982 ; Polanco et al. 1998, 2000; Averbeck and Eickbush 2005). The only region of the IGS not organized into tandem repeats is the region immediately downstream of the 28S gene. An example of the primary sequence of each subrepeat and of the nonrepeated regions can be found in Supplemental Table 1.
A previous study of the IGS from four Drosophila species found the subrepeats and unique regions to change rapidly in length and sequence between species (Tautz et al. 1987 ). Our data confirm and extend these observations. The IGS regions are composed of from one to six subrepeats, with most species containing two or three subrepeats. Subrepeat lengths varied from only 6 nucleotides (Drosophila pseudoobscura) to almost 500 nucleotides (Drosophila grimshawi). All subrepeats were AT rich (median 71% AT), with neighboring subrepeats often sharing sequence motifs (asterisks in Fig. 5). While not diagrammed in Figure 5 for simplicity, the boundaries between different subrepeats often contained partial repeats, chimeras of adjacent repeats, or short nonrepeated sequences. These boundary sequences suggest that occasional recombination between subrepeats gives rise to the rapid changes in repeat lengths seen between species.
The only IGS feature commonly found between species was that the subrepeats closest to the ETS were in most cases 225–267 bp in length. The 240-bp repeat in D. melanogaster contains a partial copy of the promoter sequences associated with transcription of the rDNA unit (Kohorn and Rae 1983
Sequence variation within the rDNA units of each species
Our ability to identify rDNA variants differed between the 12 Drosophila species for a number of reasons. First, the level and type of error may differ because different vectors were used and the sequencing was conducted at six centers. Second, the fold coverage of the shotgun sequencing varied from threefold (simulans, persimilis, sechellia) to 12-fold (melanogaster, pseudoobscura). Third, the number and location of the rDNA loci is not known for most species (Lohe and Roberts 2000
The following approach allowed us to test for the level at which variants in the rDNA locus could be reliably scored in the different species (see also Methods). The 7.8–8.2 kb consensus transcriptional rDNA unit was divided into 525-bp regions for screening the trace archives. Consecutive BLAST searches were spaced at 500-bp intervals to allow 25 bp of overlap between searches. Approximately 250 sequence traces were selected at random from the BLAST results for each screen. For a selected trace to be used, it had to contain the entire query sequence and no more than two undetermined positions (N) in the query region. This approach equally sampled all rDNA sequences, because no preference was given to those traces with greater sequence identity to the query. The initial
Our approach was based on the assumption that the trace reads of each sequencing project representatively sampled the rDNA units. While biases are sometimes encountered in the cloning of DNA fragments, the following arguments suggest that such biases were not present. First, in all species but D. pseudoobscura, the number of trace reads from the rDNA locus was generally consistent with the fold-sequencing coverage and an estimated 100–400 rDNA units. In some species, a reduction in the number of traces corresponding to the 3' ends of the transcription unit was detected and was assumed to be due to the greater instability of clones containing tandemly repeated IGS sequences. Second, in D. melanogaster we attempted to determine whether specific rDNA units were over- or under-represented in the trace archive by monitoring the number of reads corresponding to specific R2 5' junctions on the X chromosome. R2 insertions with identifiable 5' junctions have previously been shown to be predominately single copy (Perèz-Gonzalèz and Eickbush 2001 Initial analyses were conducted with the sequence reads obtained from melanogaster, simulans, virilis, willistoni, and yakuba. These analyses indicated that variants identified in only two traces from the original collection of 250 were often difficult to confirm, because insufficient numbers of total traces are present to allow resolution between sequencing errors and the stochastic recovery of traces from one orientation. Greater reliability was obtained by limiting our analyses to variant sites present in at least three trace reads in the original collection of 250 traces. Using this approach, from 17 (Drosophila yakuba) to 44 (D. melanogaster) variant nucleotide positions were detected in the rDNA unit of the five species (Table 1).
Models for the concerted evolution of the rDNA locus suggest that selective pressure to maintain a specific structure for the rRNA should eliminate many substitutions in the coding regions of the locus (Ohta 1980 ). Thus, one would expect to see lower levels of standing nucleotide variation in the coding regions compared with the noncoding regions of the rDNA unit. The distribution of the variants detected in the coding and noncoding regions of the transcription units of each species is shown in Table 1. To correct for the length of each region, the total number of variants within the coding and noncoding regions of all five species were divided by their lengths to give the number of variant positions per kilobase. The variants have been divided into two frequency classes: those variants that are present in <5% of the units (low frequency) and those variants present in >5% of the units (high frequency). This separation yielded a striking difference in the distribution of variants. The coding and noncoding regions had similar levels of variants present in the low-frequency class (3.4 and 3.6 variants/kilobase, respectively), but for the high-frequency variants, the noncoding regions had 12 times the number of variants present in the coding region (2.59 and 0.21 variants/kilobase, respectively). This distribution suggests that selection can prevent many variants in the coding region to expand beyond 5% of the rDNA units in the genome.
A larger number of low-frequency variants in each species could be scored by initially sampling more than 250 traces and using programs to monitor sequence reliability. However, this report concerns only those variants present in the rDNA units at frequencies in which the effects of selection could be measured. Thus, for the remaining Drosophila species we scored only those variants present eight or more times in the initial set of
Distribution of sequence variants within the rDNA units
A summary diagram of the locations and frequencies of the 103 variants detected in the 11 species is presented in Figure 6. Variants in the 300 bp flanking the 5' and 3' ends of the transcription unit are also shown in Figure 6. However, because these flanking areas are part of tandem subrepeats, it is not possible to distinguish between mutations arising in the subrepeats and the scrambling of extant variation within the subrepeats. Thus, our subsequent discussions will not include these IGS region variants.
Figure 6 suggests that a larger number of variants are present in the noncoding regions of the rDNA unit. The nature of the variants detected in the coding and noncoding regions also differed. Nearly 40% of the variants detected in the noncoding regions were indels typically 1–10 bp in length. Only 20% of the variants in the coding regions of the unit were indels, and their distribution was not random. Of the seven indels within the genes (circled letters in Fig. 6), one was at the R1 insertion site within the 28S gene, one was located 4 bp upstream of the R2 insertion site (at 33 bp, this deletion represented the largest indel detected), and two other indels were in the general area of the R1 and R2 sites. This clustering of indels within the 28S gene near the R1 and R2 insertion sites suggests that they were generated by the repair of DNA cleavages produced by the site-specific endonucleases encoded by these elements (see Discussion).
The mean number of variants/kilobase for each coding and noncoding region of the rDNA among the 11 species was plotted in Figure 7A. By evaluating the pooled data from all 11 species, we were unable to detect differences in the level of variation among the noncoding regions, ETS, ITS1, and ITS2 (P = 0.80,
The association between the abundance of variants in a region of the rDNA and the level of selective pressure could also be detected within the coding regions of the genes. Comparison of the rDNA sequences from each species revealed that the expansion regions of the 18S and 28S genes diverged 15 times faster than the core regions (see Fig. 3). Consistent with their faster rate of evolution, significantly more variants were present in the expansion regions compared with the core regions (P = 0.003,  2 = 8.57, d.f. = 1). As shown in Table 3, variants in the expansion regions of the 18S and 28S genes were two- and threefold higher than the core regions, respectively. If one factors in the frequency of each variant, then variants in the expansion regions are nearly three times more abundant than the core regions in the 18S genes and nearly nine times more abundant in the 28S genes. As will be discussed below, this association between the abundance of variants within a species and the rate of divergence between species was violated in only one comparison. The 28S rRNA gene, which diverges 2.5 times more rapidly than the 18S gene (Fig. 2) has lower than the expected level of variants (P < 0.0001, 2 = 17.9, d.f. = 1).
Patterns of nucleotide change between species The assembled consensus sequences of the rDNA units of 12 species revealed that the expansion regions of the 18S and 28S genes diverged 15-fold faster than the core regions, and the 28S gene diverged 2.5-fold faster than the 18S gene. The former is consistent with the higher rate of substitutions for the expansion regions in primates (Gonzalez et al. 1985 ), Xenopus (Ajuh et al. 1991), and diverse plants (Kuzoff et al. 1998). In the last study, the 26S gene was also found to diverge two times faster than the 18S gene. Thus, the relative rates of sequence change for the rRNA genes are similar across many eukaryotic taxa. The expansion regions of the Drosophila genes are composed of A-T-rich simple sequences that could presumably undergo rapid segmental changes (Hancock and Dover 1988); however, only expansion regions D7A and D12 of the 28S genes varied significantly.
In contrast to the slowly evolving rRNA genes, the noncoding regions of the Drosophila rDNA transcription unit changed rapidly in sequence. As previously noted (Schlötterer et al. 1994
A single population of rDNA units in each species
The uniformity of all rDNA units was somewhat surprising given that a significant fraction of the rDNA units in all Drosophila species are disrupted by R1 and R2 insertions (Lathe et al. 1995
The low levels of variants detected in all species studied here suggest that the abundant R1 and R2 elements present within the rDNA loci are not significantly disrupting the concerted evolution of the individual units. This finding is consistent with previous suggestions that R1 and R2 inserted units are rapidly lost from the rDNA locus, and the elements maintain their presence only by active retrotransposition (Jakubczak et al. 1992
Another factor that might affect the degree of concerted evolution of the rDNA units is the distribution of the rDNA units within the genome. Studies in several organisms have suggested that there is greater sequence homogeneity among units from the same locus compared with the units from loci on different chromosomes (Schlötterer and Tautz 1994
However, two species did suggest that variants might be differentially fixed in different rDNA loci. As shown in Figure 6D, D. ananassae had a series of variants present in the ITS regions with frequencies near 30%, and D. mojavensis had variants present in the ETS, ITS, and 28S gene with frequencies near 40% of the units. These two species account for nine of the 14 total variants detected with frequencies >25%. Because the rDNA units in D. ananassae are on the Y and 4th chromosomes (Roy et al. 2005
Distribution of variants across the rDNA unit and the mechanism of concerted evolution
A number of different studies have suggested that only a small fraction of the 200–250 rRNA units present in most D. melanogaster strains are utilized. Deletion studies of the rDNA loci suggested that only 35–60 units are needed for normal viability in the laboratory (Ritossa 1968
Can the data in this report help to reveal the relative contributions of unequal crossovers and gene conversion to the concerted evolution of the rDNA locus? Unequal crossovers in the rDNA loci of Drosophila appear to account for the two- to fourfold differences in number of rDNA units associated with individuals of a population (Lyckegaard and Clark 1991
The only unexpected feature of the distribution of variants in the rDNA loci of Drosophila was the different levels in the 18S and 28S genes. Because the rate of divergence of the 28S gene is 2.5-fold faster than that of the 18S gene (Fig. 3), the 28S gene appears to be under less sequence constraint, and thus selection against new substitutions should be less than for the 18S gene. However, fewer variants per kilobase were found in the 28S gene than in the 18S gene. This lower level of variants in the 28S gene was observed in both the core and expansion regions of the genes (Table 3). As just discussed, fewer variants within the 28S genes could be the result of localized gene conversions. However, the presence of R1 and R2 insertions in nearly one-half of the 28S genes of these species would be expected to inhibit recombination within the 28S gene, and we have found no evidence for gene-conversion-like events at the 5' or 3' ends of the R1 and R2 elements (Eickbush and Eickbush 1995
Finally, our data differ considerably from a similar analysis recently reported for the rDNA loci of five fungal species (Ganley and Kobayashi 2007
Species and databases The trace archives at GenBank, containing the original, unassembled sequencing reads generated by whole-genome shotgun (WGS) sequencing were used in this report. The Drosophila species analyzed were ananassae, erecta, grimshawi, melanogaster, mojavensis, persimilis, pseudoobscura, sechellia, simulans (white 501 strain), virilis, willistoni, and yakuba (http://www.ncbi.nlm.nih.gov/blast/mmtrace.shtml). All nucleotide sequences for this report were obtained from these trace archives by Mega BLAST (Zhang et al. 2000 ).
Assembling consensus rDNA sequences
Because of the subrepeat structure and greater sequence variation, starting at the 3' end of the 28S gene, at least 50 sequences were selected from each BLAST search and assembled using ClustalX (Thompson et al. 1997
Identification of sequence variation within each species All putative sequence variants were confirmed by BLAST search using query sequences incorporating each change. Queries were 100-bp long (longer if necessary to encompass multiple linked variants) and matches had to be 100% identical in sequence. Cases where the initially identified sequence change occurred predominantly on sequencing reads of one orientation were assumed to be recurrent sequencing error and were excluded from the analysis. To be kept in the data set, there had to be at least two identical reads of 100 bp in each orientation with each orientation constituting at least 20% of the reads. All variants detected in at least three traces of the original set of 250 were tested in five species (melanogaster, simulans, virilis, willistoni, and yakuba). Based on the findings from these species, only variants present in a least eight traces in the initial set of 250 were tested in ananassae, erecta, grimshawi, mojavensis, persimilis, and sechellia. Variants in the rDNA units of D. pseudoobscura were not determined because rDNA traces were greatly under-represented in the database.
Statistical tests for the analysis of the distribution and frequency of variants were conducted using
Support for this work was provided by the National Science Foundation (MCB-0544071) (T.H.E.) and a Caspari Fellowship from the University of Rochester (D.E.S.). We thank Michael Eickbush for his programming prowess, and the following sequencing centers for making the trace sequences publicly available prior to publication: Genome Sequencing Center, Washington University (D. simulans, D. yakuba); Broad Institute (D. sechellia, D. persimilis); Agencourt Bioscience Corporation (D. erecta, D. ananassae, D. mojavensis, D. virilis, D. grimshawi); J. Craig Venter Institute (D. willistoni). Finally, we thank Xian Zhang, Danna Eickbush, and Bill Burke for their suggestions and comments on the manuscript.
1 Corresponding author.
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Abstract
Results
250 sequences were aligned and putative variants identified. Variants present in multiple traces were confirmed by BLAST searches using shorter (100 bp) query sequences incorporating each putative variant (see Methods). 
