* Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain
Departamento de Parasitología, Ecología y Genética, Facultad de Farmacia/Biología, Universidad de La Laguna, La Laguna, Tenerife, Spain
Correspondence: E-mail address: aguade{at}porthos.bio.ub.es.
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Abstract |
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Key Words: weak selection codon bias effective population size Drosophila guanche Drosophila subobscura
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Introduction |
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In Drosophila, the mutational process is biased toward A/T, with an expected A+T content at equilibrium of about 65% (Petrov and Hartl 1999). The mutational input could thus explain the A+T content of introns (Moriyama and Hartl 1993; Hey and Kliman 2002) but not the codon bias detected in this genus. Indeed, preferred codons (i.e., those found at high frequencies in highly biased genes) end in C or G in Drosophila melanogaster and in other Drosophila species (Akashi 1995; Kreitman and Antezana 2000). In addition, a significant positive correlation between the G+C content at third positions of fourfold degenerate codons and that of the adjacent introns was only detected for low bias genes (Kliman and Hey 1994). These observations would allow precluding that the mutational process is mainly responsible for codon bias, at least in Drosophila.
Several observations on synonymous variation in Drosophila are consistent with predictions of the mutation-selection-drift equilibrium model and would thus favor the role of weak selection in maintaining codon bias: (1) significant associations between codon bias and both the recombination rate and the length of the coding region were detected by the analysis of a large set of genes in D. melanogaster (Kliman and Hey 1993; Comeron, Kreitman, and Aguadé 1999; Duret and Mouchiroud 1999; Hey and Kliman 2002); (2) a parallel increase in codon bias and the rate of recombination was noticed in interspecies comparisons of particular genes that have changed their recombinational environment (Munté, Aguadé, and Segarra 1997, 2001; Takano-Shimizu 1999); (3) the analysis of synonymous polymorphism and divergence in small sets of genes revealed differences between preferred and unpreferred changes either in the ratio of polymorphism to divergence or in the frequency spectrum of derived variants (Akashi 1995, 1999; Akashi and Schaeffer 1997; Kliman 1999; Llopart and Aguadé 2000; Begun 2002); and (4) analysis of synonymous divergence between two species differing in their effective population size (Ne) showed an excess of fixed unpreferred mutations in the lineage with lower Ne (Llopart and Aguadé 1999).
According to the nearly neutral model of molecular evolution (Ohta and Kimura 1971; Ohta 1972), the fate of weakly selected mutations is expected to differ in species with strong differences in effective population size. A direct approach to contrast the nearly neutral character of synonymous mutations would consist of comparing the distribution of preferred and unpreferred polymorphic variants between a pair of related species with similar generation times and strong (and sequence variation independent) support for important differences in their effective population size. Given the difficulties associated with assessing effective population size, pairs of species consisting of one continental species with a rather large distribution area and another species endemic to a volcanic island would be among the best candidates for such an approach. D. subobscura and D. guanche would constitute such a pair. Indeed, D. subobscura is widespread and abundant in the Palearctic region, and D. guanche is restricted to some isolated gorges of the Tenerife Island (Canary Archipelago, Spain). The endemic character of D. guanche, its association with the relict tertiary flora of the island, and the lack of important climatic fluctuation in the archipelago over evolutionary time would support the long-term low effective population size of D. guanche. Furthermore, the divergence time between both species, although short at the evolutionary time scale, is long enough (about 1.8 to 2.8 MYA, Ramos-Onsins et al. 1998) to minimize the effect of shared ancestral polymorphisms.
Herein, we report the survey of nucleotide polymorphism in the RpII215 gene region of D. guanche, aiming to contrast the nearly neutral character of synonymous mutations and thus the role of weak selection in maintaining codon bias. This gene is located in a region of high recombination (section 10A of the X chromosome, Segarra and Aguadé 1992), and it has a long coding region (5,667 nt, 1,889 codons). Both characteristics allow predicting the detection of a sufficiently high number of synonymous polymorphic variants in a species with a putative low effective population size. In addition, the study of a single gene has an additional advantage over studies in which several genes are jointly analyzed: it eliminates the overdispersion of the selection coefficients caused by differences in codon bias among genes. Most importantly, previous data on the divergence of this gene between D. subobscura and D. guanche (Llopart and Aguadé 2000) revealed an excess of unpreferred differences fixed in the D. guanche lineage, which would be consistent with its long-term reduced effective size if unpreferred mutations were under weak negative selection. Synonymous polymorphism in D. guanche should also reflect the reduced effective size of this species. Indeed, if weak selection were relaxed in the D. guanche lineage, there should be an excess of unpreferred mutations segregating in D. guanche relative to D. subobscura. In addition, these mutations would segregate at higher frequencies in the insular than in the continental species. The pattern of synonymous polymorphism detected in the RpII215 gene of D. guanche conforms to these expectations and indicates that most synonymous mutations in the D. guanche lineage behave as neutral. Therefore, present data show that selection is relaxed in the D. guanche lineage and strongly support the nearly neutral character of synonymous mutations.
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Materials and Methods |
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DNA Sequencing Strategy
Genomic DNA from a single male of each isofemale line was extracted using a standard small-scale procedure (Ashburner 1989). An approximately 6.9-kb DNA fragment that includes the RpII215 gene and its flanking regions was PCR amplified by means of three overlapping fragments. Oligonucleotides for PCR amplification and sequencing were designed on the available sequences of D. guanche and D. subobscura (Llopart and Aguadé 1999, 2000). After purification of PCR products with Microcon®-PCR Filter Columns (Millipore), both strands were completely sequenced using internal primers. An inverse-PCR and primer-walking strategy was used to extend the D. pseudoobscura sequence. Sequencing reactions were carried out with Abi Prism® BigDyeTM Terminators version 2.0 Cycle Sequencing Kit (Applied Biosystems) following manufacturer's instructions. The sequencing reaction products were separated, after ethanol precipitation, on an Abi Prism 377 automated DNA sequencer (PerkinElmer), and sequences were assembled with the SeqEd version 1.03 program (Hagemann and Kwan 1997).
Newly reported DNA sequences from D. guanche have been deposited in the EMBL/GenBank Database under accession numbers AJ547806 and AJ548510 to AJ548532. The accession number of the partial sequence of D. pseudoobscura is AJ544770.
Data Analyses
DNA sequences were multiply aligned using the ClustalW program (Thompson, Higgins, and Gibson 1994) and further edited with the MacClade version 3.06 program (Maddison and Maddison 1992). Sites with alignment gaps were excluded from the analyses. The DnaSP version 3.98 program (Rozas and Rozas 1999) was used to perform most analyses. Genetic differentiation between populations was tested using the Ks* and Snn statistics (Hudson, Boos, and Kaplan 1992; Hudson 2000); statistical significance was obtained by the permutation test (10,000 replicates). The population recombination parameter R = 2Ner (where r is the recombination rate per generation between the most distant sites) was estimated from the minimum number of recombination events found in the sample, or RM (Hudson and Kaplan 1985). The minimum and maximum values of R compatible with the observed RM value (at the 5% level), RL and RU, were estimated following the method of Rozas et al. (2001). The global measures of linkage disequilibrium ZnS (Kelly 1997) and ZA (Rozas et al. 2001) were also obtained.
The gene genealogy was reconstructed by the neighbor-joining method (Saitou and Nei 1987), as implemented in the MEGA program (Kumar et al. 2001). The ancestral state of each variable site in the 24 D. guanche sequences and in the 11 previously reported D. subobscura sequences (Llopart and Aguadé 2000) was inferred using the RpII215 sequence of D. pseudoobscura (Llopart and Aguadé 1999; this study) and, occasionally (in six out of 130 cases), that of D. melanogaster (Jokerst et al. 1989) as the outgroup. Variable sites for which phylogenetic reconstruction did not result in a single most-parsimonious tree (i.e., ambiguous sites) were excluded from the analyses. Polarized synonymous changes were classified as preferred (from unpreferred to preferred codons) or unpreferred (from preferred to unpreferred codons) according to the D. melanogaster codon preferences (Akashi 1995). Although D. pseudoobscura is phylogenetically closer to D. subobscura than is D. melanogaster, we used the codon preferences of this latter species rather than those of D. pseudoobscura (Akashi and Schaeffer 1997), because they are based on a larger set of genes. Additionally, Kreitman and Antezana (2000) showed that codon preferences are similar in D. subobscura and D. melanogaster. Only changes from preferred to unpreferred codons and from unpreferred to preferred codons were analyzed. For the comparative analysis between species, previously published data on polymorphism in D. subobscura and divergence from D. guanche (Llopart and Aguadé 1999, 2000) were reanalyzed, since the D. pseudoobscura codon preferences had been used in those studies.
The numbers of preferred and unpreferred changes expected from the mutational process (i.e., under a strictly neutral model) were obtained using the pattern of nucleotide mutation detected in unconstrained DNA sequences of Drosophila (Petrov and Hartl 1999). The mutational matrix was applied to the particular codon composition of the RpII215 sequence inferred for the ancestor of D. guanche and D. subobscura.
Tajima's test of neutrality (Tajima 1989), which is based on the frequency spectrum of polymorphisms, was applied to the frequency distributions of the different classes of derived variants (preferred and unpreferred synonymous changes, and changes in noncoding regions). The Mann-Whitney U test (z value from the fdMWU test, Akashi 1999) was applied to compare polymorphism frequency distributions using five frequency intervals of equal size.
The scaled selection coefficient ( = Nes, where s is the selection coefficient) for the different kinds of changes was estimated using two different approaches based on the Poisson random field (PRF) theory (Sawyer and Hartl 1992), which assumes that the number of changes is an independent Poisson random variable. The first approach uses information on the frequency distribution (fd) of derived polymorphic variants (Hartl, Moriyama, and Sawyer 1994; Akashi and Schaeffer 1997). The PRFMLE software (Hartl, Moriyama, and Sawyer 1994) was used to estimate the
values, their confidence intervals, and the statistical significance against the null hypothesis of no selection (
= 0). The second approach uses information on the ratio of polymorphism to divergence (rpd) and requires comparison with a neutrally evolving region (Sawyer and Hartl 1992; Akashi 1995). In this case, confidence intervals of
were obtained by parametric bootstrap (10,000 replicates); for each computer replicate, the numbers of preferred, unpreferred, and noncoding changes (both polymorphic and fixed) were randomly obtained from a Poisson distribution with the number of changes observed in each class as the mean.
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Results |
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Preferred and Unpreferred Changes in the D. guanche and D. subobscura Lineages
The 24 sequences of D. guanche and the 11 previously reported sequences of D. subobscura (Llopart and Aguadé 2000) were used to obtain the numbers of fixed and polymorphic changes separately in each lineage (table 2). The number of synonymous changes fixed in the D. guanche lineage largely exceeds the number of those fixed in the D. subobscura lineage, which is reflected in the gene genealogy shown in figure 2. The 130 (out of 142) synonymous changes (either polymorphic within species or fixed between species) that could be unambiguously polarized were subsequently classified according to the D. melanogaster codon usage preferences (Akashi 1995). There were 104 preferred or unpreferred changes, five changes from preferred to preferred codons, and 21 changes from unpreferred to unpreferred codons. Only preferred and unpreferred changes were considered in further analyses.
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Discussion |
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The pattern of synonymous variation in D. guanche contrasts with that detected in D. subobscura (Llopart and Aguadé 1999, 2000). As revealed in the present study, the frequency distribution of unpreferred polymorphic mutations differs significantly between species, which clearly indicates a different behavior of these mutations in both species. In fact, only in D. subobscura, there is an excess of unpreferred mutations segregating at low frequency, as reflected by the significantly negative value of Tajima's D statistic (Llopart and Aguadé 2000; present study). The contrasting results between D. guanche and D. subobscura can be explained according to the nearly neutral model of molecular evolution. Indeed, assuming that selection coefficients against unpreferred mutations are similar in both species, the detected differences are consistent with the much lower effective population size of D. guanche relative to D. subobscura. In D. guanche, a large fraction of unpreferred mutations would behave as neutral, as supported by the two estimated values of the scaled selection coefficient that do not differ significantly from zero (table 5). In contrast, in D. subobscura, selection against these mutations would be more effective, as indicated by the significantly negative fd estimate of the scaled selection coefficient. It could be argued that selection coefficients on synonymous mutations at RpII215 were smaller in D. guanche than in D. subobscura. Given the lower population size of the insular species, this alternative explanation seems rather unlikely.
Estimates of the scaled selection coefficient could be biased if the free recombination assumption of the PRF model (Sawyer and Hartl 1992) were severely violated. No major effect is expected in D. guanche since estimates of the recombination parameter and measures of linkage disequilibrium indicate that the rate of recombination at the RpII215 region is relatively high. Although in D. subobscura the RpII215 gene is also located in a region of high recombination, the presence of two chromosomal arrangements (Ast and A2) affecting this region might constitute a rather important departure from the free recombination assumption. Nevertheless, similar estimates of the scaled selection coefficients were obtained using all D. subobscura sequences (Nes = 4.33) and only sequences from the putatively ancestral Ast arrangement (Nes = 4.77). Furthermore, the magnitude (in absolute value) of these estimates in D. subobscura, although rather high for nearly neutral mutations, would be in good agreement with previous estimates reported for D. pseudoobscura (Nes = 4.6 for the Adh-Adhr genes) and D. simulans (Nes = 2.1, data from eight genes) (Akashi and Schaeffer 1997).
As an alternative to weak selection acting on synonymous variation, demographic factors affecting differentially the two species might also explain their different pattern of unpreferred mutations. Thus, a population expansion in D. subobscura but not in D. guanche might account for the observed results. Indeed, a skew towards rare variants and therefore negative Tajima's D values are expected after population expansions. However, demographic factors should affect similarly different kinds of nucleotide variation. Although both noncoding and unpreferred mutations in D. subobscura show negative Tajima's D values, this is not the case for preferred mutations. This differential behavior of preferred mutations in D. subobscura is therefore not consistent with demographic hypotheses.
The results obtained in the present study are thus consistent with an evolutionary scenario where codon bias was actively maintained by natural selection before the species split. The population size would have remained large in the continental species D. subobscura and, thus, codon bias was maintained by natural selection. The small population size of D. guanche would have caused, on the contrary, a relaxation of natural selection and, consequently, an important fraction of synonymous mutations that were slightly deleterious in the ancestral lineage (and in D. subobscura) would behave as neutral in the endemic species. This shift in the selective behavior of synonymous mutations would cause a progressive reduction of codon bias in D. guanche. Indeed, codon bias would move slowly to a new mutation-selection-drift equilibrium, given the minor contribution of weakly selected mutations.
Under strict neutrality, the expected heterozygosity () in a stationary population at mutation-drift equilibrium is equal to 4Neu (3Neu for X-linked genes), where u is the mutation rate. According to this prediction, the expected heterozygosity should differ substantially between D. subobscura and D. guanche. Estimated nucleotide diversity in this gene is about twofold lower in D. guanche than in D. subobscura. This difference is qualitatively consistent with the lower effective population size of D. guanche relative to D. subobscura, but it is quantitatively much smaller than expected for neutral variation, given the putative large disparity in effective sizes. However, if both species effective sizes were much more similar, it would be difficult to explain, for instance, the significantly different numbers of unpreferred and preferred synonymous changes fixed in the two lineages or the significantly different distribution of unpreferred polymorphic mutations in the two species.
In addition, a lack of correlation between heterozygosity and effective size has been extensively reported (Lewontin 1974), which has led to the development of population genetic models that account for this discrepancy. Thus, the pseudohitchhiking model (Gillespie 2000, 2001), which considers the effect of strongly selected mutations on linked neutral variation, can explain the insensitivity of heterozygosity to changes in the population size and may even predict a reduction of variation with increasing population size. On the other hand, variation in the RpII215 gene region is mainly due to synonymous mutations. In D. subobscura, unlike in D. guanche, synonymous mutations (and probably also mutations at noncoding sites) are weakly selected and thus not strictly neutral. Since weak selection acting in the maintenance of codon bias reduces the sojourn time of these mutations in the population, the expected level of standing synonymous variation in the RpII215 gene would be lower in D. subobscura than in D. guanche. In addition, the interference model predicts a reduction of intraspecific variation due to linkage between mutations under weak selection (McVean and Charlesworth 2000; Comeron and Kreitman 2002). As this kind of selection is much more efficient in D. subobscura, the effect of interference should be stronger in this species than in D. guanche. However, both the pseudohitchhiking and the interference effects are based on linked selection, which is expected to be less efficient in regions of high recombination, such as the RpII215 region. Thus, the mere twofold reduction of variation in D. guanche relative to D. subobscura might be explained by the differential behavior of synonymous mutations in the two species and, likely to a minor extent, by the pseudohitchhiking and interference effects.
In conclusion, the comparative study of nucleotide variation at RpII215 in D. subobscura and D. guanche clearly shows a different behavior of synonymous mutations in these species. This contrasting behavior would be caused by differences in their effective population size, assuming that synonymous mutations are under weak selection. Present results provide strong evidence, therefore, for the role of weak selection in the maintenance of codon bias. They also prove that the D. subobscura and D. guanche species pair is a good model to further analyze the action of weak selection and, more specifically, how the effective population size affects the level and pattern of nucleotide variation.
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Acknowledgements |
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Footnotes |
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