Nucleotide Variation at the yellow Gene Region is not Reduced in Drosophila subobscura: A Study in Relation to Chromosomal Polymorphism

Agustí Munte, Montserrat Aguade and Carmen Segarra

Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
In contrast to Drosophila melanogaster and Drosophila simulans, the yellow (y) gene region of Drosophila subobscura is not located in a region with a strong reduction in recombination. In addition, this gene maps very close to the breakpoints of different inversions that segregate as polymorphic in natural populations of D. subobscura. Therefore, levels of variation at the y gene region in this species relative to those found in D. melanogaster and D. simulans may be affected not only by the change in the recombinational environment, but also by the presence of inversion polymorphism. To further investigate these aspects, an approximately 5.4-kb region of the A (=X) chromosome including the y gene was sequenced in 25 lines of D. subobscura and in the closely related species Drosophila madeirensis and Drosophila guanche. The D. subobscura lines studied differed in their A-chromosomal arrangements, Ast, A2, and A1. Unlike in D. melanogaster and D. simulans, levels of variation at the y gene region of D. subobscura are not reduced relative to those found at other genomic regions in the same species (rp49, Acp70A, and Acph-1). This result supports the effect of the change in the recombinational environment of a particular gene on the level of neutral variation. In addition, nucleotide variation is affected by chromosomal polymorphism. A strong genetic differentiation is detected between the A1 arrangement and either Ast or A2, but not between Ast and A2. This result is consistent with the location of the y gene relative to the breakpoints of inversions A1 and A2. In addition, the pattern of nucleotide polymorphism in Ast+A2 and A1 seems to point out that variation at the y gene region within these chromosomal classes is in the phase transient to equilibrium. The estimated ages of these arrangements assuming a star genealogy indicate that their origin cannot predate the D. madeirensis split. Therefore, the present results are consistent with a chromosomal phylogeny where Am1, which is an arrangement present in D. madeirensis but absent in current populations of D. subobscura, would be the ancestral arrangement.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
The yellow (y) gene of Drosophila was first cloned and sequenced in Drosophila melanogaster (Campuzano et al. 1985Citation ; Chia et al. 1986Citation ; Geyer, Spana, and Corces 1986Citation ). The gene is organized into two exons separated by a large intron and codes for a secreted protein that controls the pigmentation of larval and adult cuticular structures. Different cis-acting regulatory elements controlling the correct expression pattern in adults and larvae were identified in the gene 5'-flanking region and in the intron (Geyer and Corces 1987Citation ; Martin, Meng, and Chia 1989Citation ). In this species, the y gene is located at the tip of the X chromosome, where recombination is highly suppressed.

Nucleotide polymorphism in the y-achaete-scute (y-ac-sc) gene region has been extensively analyzed in natural populations of D. melanogaster and Drosophila simulans (Aguadé, Miyashita, and Langley 1989Citation ; Beech and Leigh Brown 1989Citation ; Eanes, Labate, and Ajioka 1989Citation ; Macpherson, Weir, and Leigh Brown 1990Citation ; Begun and Aquadro 1991, 1993, 1995Citation ; Martín-Campos et al. 1992Citation ). In most of these studies, a very strong reduction in the level of DNA variation was detected. Surveys of other genes and/or Drosophila species also revealed low levels of nucleotide variation in genomic regions with a strong reduction in recombination rate (see, e.g., Stephan and Langley 1989Citation ; Berry, Ajioka, and Kreitman 1991Citation ; Stephan and Mitchell 1992Citation ; Langley et al. 1993Citation ). As the low levels of polymorphism detected in regions with no (or reduced) recombination could not be explained by a lower mutation rate in these regions, two selective models have been proposed to explain the extremely low variation in these regions: the hitchhiking model and the background selection model. According to the hitchhiking model (Maynard Smith and Haigh 1974Citation ), a particular variant increasing in frequency due to positive selection will carry adjacent sites along, which will cause removal of variation at neutral linked sites when the selected variant is fixed. In contrast, the background selection model (Charlesworth, Morgan, and Charlesworth 1993Citation ) proposes negative selection acting against recessive deleterious mutations as the cause of reduced neutral variation. Assuming the same intensity of selection, the effect predicted by both models in reducing levels of neutral linked variation should be larger the lower the recombination rate.

In Drosophila subobscura, the y gene maps at section 2B of the X (=A) chromosome (Segarra et al. 1995Citation ). It is therefore separated from the centromere by more than one euchromatic section in standard chromosomes. Unlike in D. melanogaster and D. simulans, the recombination rate at the y gene region of D. subobscura should not be extremely reduced. The pattern of synonymous divergence between D. melanogaster and D. subobscura (Munté, Aguadé, and Segarra 1997Citation ), which is characterized by a high number of synonymous substitutions per synonymous site and by a much higher codon bias in the latter species, would support this difference in the recombination rate. This change in the recombinational environment of the y gene is also expected to cause a drastic difference in the level of nucleotide polymorphism between both species. In fact, an effect on nucleotide diversity has already been reported for other gene regions that differ in recombination rate in different species (Stephan et al. 1994Citation ; Schmid et al. 1999Citation ).

However, the y region maps very close to the proximal breakpoints of four inversions described in segment I (sections 1–7) of the A chromosome of D. subobscura: A1, A5, A6, and A7. The adaptive character of inversion polymorphism in Drosophila is well documented (reviewed by Powell 1997Citation , pp. 102–114). Thus, selective forces may contribute to the establishment of a newly arisen inversion. In this case, the increase in frequency of a new inversion may be a rapid process that can be envisaged as a partial selective sweep. As a consequence, nucleotide variation at loci associated with a particular inversion (as, for instance, those located near the breakpoints) may be null or very low even after the new inversion has reached its equilibrium frequency. Thereafter, these loci accumulate variation both by mutation and by genetic exchange with the preexisting arrangement. This period can be considered a transient phase that persists until nucleotide variation in the new arrangement reaches equilibrium. Therefore, the level of variation at the y gene region of D. subobscura will depend not only on its recombinational environment, but also on the age of the chromosomal lineages, and thus may be strongly affected by the history of the different polymorphic inversions in segment I.

To further investigate how the change in the recombinational environment and the presence of inversion polymorphism affect the level and pattern of variation in a particular gene region, nucleotide polymorphism at the y gene region was analyzed in a sample of two different chromosomal classes segregating for segment I of the A chromosome of D. subobscura and in the closely related species Drosophila madeirensis and Drosophila guanche. No reduction in the level of nucleotide polymorphism was detected in the y gene region compared with variation at other genomic regions analyzed in this species (Rozas and Aguadé 1994Citation ; Cirera and Aguadé 1998Citation ; Navarro-Sabaté, Aguadé, and Segarra 1999Citation ). Nucleotide variation was, however, affected by chromosomal polymorphism, as the two chromosomal classes were strongly differentiated. In addition, their pattern of variation seemed to reflect the strong bottleneck produced in the origin of the arrangements. According to this result, some inferences about the history and age of the chromosomal lineages have been made.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Fly Samples
Flies were collected in spring of 1995 in Riba-roja d'Ebre (Tarragona, Spain). Single sampled males or, alternatively, males from the progeny of isofemale lines were crossed with virgin females of the ch cu strain, which is homozygous for the Ast arrangement. After 5–6 days, males were individually frozen in liquid nitrogen and stored at -70°C. In order to determine the X chromosome gene arrangement of each male, a female larva of its progeny was dissected, and slides of the salivary glands were prepared by the standard orcein acetic-lactic acid procedure. Chromosomal arrangement frequencies in the population studied were determined after the analysis of 111 males. Twenty-five of these males were used for further analysis.

In Situ Hybridization
Salivary glands of third-instar larvae grown in uncrowded cultures were dissected and polytene chromosome slides were obtained according to Montgomery, Charlesworth, and Langley (1987)Citation . The recombinant phage {lambda}D.subRA4.1 (Munté, Aguadé, and Segarra 1997Citation ) that includes the second exon of the y gene of D. subobscura was used as probe. The probe was labeled by nick translation with biotin-16-dUTP. Prehybridization, hybridization, and detection conditions were as described in Segarra et al. (1995)Citation .

DNA Sequencing
Genomic DNA from individual males was purified according to Ashburner (1989Citation , pp. 106–107). The y gene region was subsequently amplified by the polymerase chain reaction (PCR). Amplification primers were designed on the available D. subobscura DNA sequence (Munté, Aguadé, and Segarra 1997Citation ). Although in some cases the complete region was amplified in one fragment or in three overlapping fragments, in most lines, the y gene region was PCR-amplified in two overlapping fragments using primers 5'-AACCACACGAACCACTCAACG-3' and 5'-CAAAAGTGGCCATTAGCTTGC-3' for one of the fragments and 5'-GTCATAAACCGTTCCACATGC-3' and 5'-GTGGTGTGGCTTTAAGAATTCC-3' for the other one. The optimal PCR conditions were as follows: 34 cycles of 94°C for 45 s, 57–58°C for 30 s, and 72°C for 3 min.

The amplified DNA was purified with the QIAquick PCR purification kit (Qiagen) and used as template for direct sequencing. Primers designed approximately every 300 nt on both strands of the sequence were used for this purpose. Sequencing reactions were performed with the ABI PRISM Dye Terminator Cycle Sequencing kit (Perkin Elmer) following the manufacturer's instructions. Sequences were run on an ABI PRISM 377 (Perkin Elmer) automated sequencer.

Sequences were assembled with Staden's (1982)Citation programs. Complete sequences were aligned manually or by using the CLUSTAL W program (Thompson, Higgins, and Gibson 1994Citation ) and edited with the MacClade program (Maddison and Maddison 1992Citation ). The DNA sequences obtained in this study were deposited in the EMBL database library with the accession numbers AJ289787–AJ289813.

Data Analysis
Nucleotide polymorphism was estimated as the number of segregating sites (S), the average number of pairwise differences (k), the average number of pairwise differences per site (or nucleotide diversity) ({pi}), and the heterozygosity per site expected in a population in mutation-drift equilibrium given the observed S value ({theta}, or Watterson's estimator; Watterson 1975Citation ). Under the neutral model, {pi} and {theta} have equal expectations in a stationary population.

Genetic differentiation between arrangements was estimated as the average number of nucleotide differences per site between arrangements (dxy) and as the number of net nucleotide differences per site (da) (eqs. 10.20 and 10.21 in Nei 1987Citation ). Putative genetic differentiation between gene arrangements was contrasted by the permutation test proposed by Hudson, Boos, and Kaplan (1992)Citation using the Ks estimator as a measure of differentiation. The statistical significance of the observed Ks value was determined by Monte Carlo simulations.

The number of shared polymorphic sites between arrangements (sites that segregate for the same variants in both arrangements) expected to have arisen independently in each arrangement by parallel mutation was estimated from the hypergeometric distribution, as proposed by Rozas and Aguadé (1994)Citation , under the assumption of no variation in mutation rate among sites. If the number of observed shared polymorphic sites is significantly higher than that expected, the existence of genetic exchange between arrangements can be inferred. This genetic exchange has to be explained mainly by gene conversion in regions like those located near the breakpoints of polymorphic inversions, where double crossover between arrangements is highly suppressed. Gene conversion tracts between arrangements were identified by the algorithm proposed by Betrán et al. (1997)Citation .

Linkage disequilibrium was analyzed between pairs of informative sites (sites where the less frequent variant is present at least twice in the sample). The {chi}2 test was used to detect significant linkage disequilibrium between pairs of informative sites. The minimum number of recombination events within each chromosomal class was inferred by the four-gamete test proposed by Hudson and Kaplan (1985)Citation .

The tests of neutrality proposed by Tajima (1989)Citation and Fu and Li (1993)Citation were applied to determine whether the pattern of polymorphism detected in the y gene region was concordant with neutral predictions. Tajima's test is based on the normalized difference between the observed average number of pairwise nucleotide differences (k) and its expectation ({theta} per sequence) in a population in mutation-drift equilibrium according to predictions of the neutral model. Fu and Li's F and F* statistics have the same basis, but for these statistics, the predicted {theta} per sequence is estimated from the number of mutations in the external branches of the gene genealogy. Fu and Li's D and D* statistics use the total number of mutations and the number of mutations in the internal branches of the gene genealogy to compare different estimates of {theta} per sequence. In the Fu and Li tests, the number of mutations in external branches is inferred from the number of singletons in the data set (D* and F* statistics) or from the information in an outgroup species (D and F statistics). Drosophila guanche was used as the outgroup species to estimate these last two statistics. Negative values of Tajima's D statistic indicate an excess of low-frequency variants in the data set, while negative values of the different Fu and Li statistics indicate an excess of unique polymorphisms.

The test proposed by Hudson, Kreitman, and Aguadé (1987)Citation was used to determine whether the levels of silent polymorphism relative to silent divergence present in the y gene region differed significantly from those detected at other genomic regions previously studied in D. subobscura. McDonald and Kreitman's (1991)Citation and McDonald's (1996)Citation tests were also applied (using D. guanche in the interspecific comparisons) to determine whether the y gene region was evolving according to neutral expectations. McDonald and Kreitman's (1991)Citation test examines whether the ratio of synonymous to nonsynonymous polymorphic sites in one species is equal to the ratio of synonymous to nonsynonymous fixed differences between species as expected under neutrality. McDonald's (1996)Citation test is based on the number of runs detected in a sample. A run is defined as a stretch of polymorphic (or fixed) sites limited at each end by a fixed (or polymorphic, respectively) site. Both directional and balancing selection cause a reduction in the number of runs detected in a sample. Monte Carlo simulations are performed to determine whether the number of runs observed is lower than that predicted under neutrality.

The DnaSP, version 3.4, program (Rozas and Rozas 1999Citation , personal communication) was used to perform most of the described analyses: nucleotide polymorphism and genetic differentiation estimates, detection of gene conversion tracts, linkage disequilibrium and recombination analyses, neutrality tests, and application of the hypergeometric distribution. This program also implements the coalescent algorithm proposed by Hudson (1983, 1990)Citation to perform computer simulations. The coalescent approach was used to infer the statistical significance (after 10,000 replicates) of Tajima's D and Fu and Li's D, F, D*, and F* statistics both under no recombination and assuming intermediate levels of recombination. The empirical distribution of the minimum number of recombination events expected for a particular recombination parameter (C = 3Nec for a sex-linked gene; where Ne is the effective size and c is the recombination rate between the most distant sites) was also inferred by this coalescent approach. The permtest (Hudson, Boos, and Kaplan 1992Citation ) and the DNAruns (McDonald 1996Citation ) programs were used to perform Monte Carlo simulations to contrast putative genetic differentiation between arrangements and to apply the runs test, respectively. Gene genealogies were reconstructed with the MEGA program (Kumar, Tamura, and Nei 1994Citation ).

The ages of the different gene arrangements were estimated assuming a star genealogy within the chromosomal class. This assumption considers that variation within each chromosomal class was null just after its origin and that current variation is in the phase transient to equilibrium. In this case, which can be considered a particular case of the expansion model (Slatkin and Hudson 1991Citation ; Rogers 1995Citation ), {pi} = 2{lambda}t (where {pi} is the average number of nucleotide differences per site within arrangement, t is the elapsed time since the origin of the arrangement, and {lambda} can be estimated from the rate of nucleotide substitutions per site, for instance, between D. subobscura and D. guanche). This approach may lead to an underestimate of the age of a gene arrangement if variation is at equilibrium, and to an overestimate if genetic exchange between arrangements is important.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Cytological Analysis and In Situ Hybridization
Three chromosomal arrangements were detected in Riba-roja: A2, Ast, and A1. The frequencies of these arrangements were 0.54, 0.36, and 0.1, respectively. When only the gene arrangement for segment I is considered, these arrangements can be grouped into two chromosomal classes: the A1 chromosomal class, which includes the A1 arrangement (I1+IIst), and the Ast+A2 chromosomal class, which includes arrangements Ast (Ist+IIst) and A2 (Ist+II2). Figure 1 shows the location of the y gene in D. subobscura, as determined by in situ hybridization on polytene chromosomes of homokaryotypes for the Ast and A1 gene arrangements, and its location in the Ast/A1 and Ast/A2 heterokaryotypes. Both the different locations of the gene in the Ast and A1 homokaryotypes and its location in the inversion loop of Ast/A1 heterokaryotypes confirm that the y gene region is included in inversion A1. In addition, in most nuclei of Ast/A1 heterokaryotypes, the hybridization signal appears in the asynaptic region of the inversion loop, which corroborates that y is located very close to the proximal breakpoint of this inversion. In contrast, and as predicted, y maps at quite a distance from the breakpoints of inversion A2.



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Fig. 1.—In situ hybridization of the y gene on Drosophila subobscura polytene chromosomes. a, Ast/Ast homozygotes. b, Ast/A2 heterozygotes. c, A1/A1 homozygotes. d, Ast/A1 heterozygotes. Hybridization signals are indicated with arrowheads. The two signals present in the Ast/A1 heterozygotes are due to the location of the gene in the asynapsed region of the inversion loop

 
Nucleotide Polymorphism
The y gene region was sequenced in 25 males: 10 were Ast, 10 were A1, and 5 were A2. Although this sample does not represent a random sample of the population, males were chosen at random within gene arrangement. The multiple alignment of the y gene region in the 25 lines studied included 5,630 sites. After excluding all sites with alignment gaps, a total of 229 nucleotide polymorphic sites were detected among 5,138 sites. Thirty-nine of the polymorphic sites were located in the coding region (1,704 sites), and the remaining 190 sites were in flanking regions and in the intron (3,434 sites). Figure 2 shows the information for these polymorphic sites using as reference the previously published D. subobscura sequence (Munté, Aguadé, and Segarra 1997Citation ). Three polymorphic sites segregated for three variants; thus, the minimum number of mutations in the data set was 232.



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Fig. 2.—Nucleotide polymorphic sites in the 25 lines studied from Riba-roja d'Ebre (Tarragona, Spain) after excluding all sites with alignment gaps. Numbering of sites is given using as reference the previously published sequence of Drosophila subobscura (D. sub.; EMBL accession number Y13909). Site 1 in the present alignment corresponds to site 1620 in the reference sequence. Numbers in boldface indicate polymorphic sites in the coding region. Polymorphic sites causing amino acid replacements are indicated by asterisks. Nucleotides identical to the reference sequence are indicated by dots. The identification number of each line includes its chromosomal arrangement after a slash. Gene conversion tracts transferred from the Ast+A2 chromosomal class to A1 or vice versa are shown by gray boxes. The last two rows of the alignment show the information for the sites detected as polymorphic in D. subobscura in the outgroup species, Drosophila madeirensis (D. mad.) and Drosophila guanche (D. gua.). Dashes in these two sequences indicate deletions

 
Apart from nucleotide polymorphisms, several polymorphic indels were detected in noncoding regions (data not shown). Indels were frequently generated by variation in the number of repetitions of short motifs. Some of these events were complex, since the motif repeated was not invariant. For instance, from site 1353, the motifs CCCCAGTCCGAG and CCCCACTCCGAG were differentially repeated in alternative orders in the studied lines. All sites with polymorphic indels were excluded in posterior analyses.

Genetic differentiation estimates between gene arrangements are summarized in table 1 . No fixed differences were detected between the Ast and the A2 arrangements, which, in addition, showed the highest number of shared polymorphic sites. These data are consistent with the lower dxy and da estimates between these arrangements relative to those found in the comparisons including the A1 arrangement. Therefore, A1 exhibits a much higher genetic differentiation versus either Ast or A2 than Ast versus the A2 arrangement.


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Table 1 Estimates of Genetic Differentiation Between Gene Arrangements

 
The permutation test proposed by Hudson, Boos, and Kaplan (1992)Citation was used to contrast putative significant genetic differentiation between gene arrangements. All comparisons including the A1 sample were highly significant (P = 0.000, after 1,000 computer replicates). In contrast, the comparison between Ast and A2 samples was not significant (P = 0.798, after 1,000 replicates). According to these results, Ast and A2 lines were grouped in a single chromosomal class (Ast+A2) in all posterior analyses, while A1 lines were analyzed independently. In fact, the Ast+A2 chromosomal class corresponds to the standard arrangement for segment I of the A chromosome.

There were 13 fixed differences (one of them nonsynonymous) between the A1 and Ast+A2 chromosomal classes. In addition, they shared 21 polymorphic sites (18 silent). The hypergeometric distribution was used to estimate the number of silent shared polymorphic sites expected to have arisen independently in each chromosomal class by parallel mutation. Eighty-one and 144 silent polymorphisms out of 3,863 silent sites were detected within the A1 and Ast+A2 chromosomal classes, respectively (table 2 ). According to these data, no more than five shared polymorphic sites are expected by chance (P > 0.05). Therefore, it can be inferred that genetic exchange between arrangements has contributed to the increase in the number of shared polymorphic sites. The algorithm proposed by Betrán et al. (1997)Citation was used to detect gene conversion tracts between the A1 and Ast+A2 chromosomal classes (fig. 2 ). The tract present in line 417RIB (from site 1261 to site 2789) was unusually long when compared to the other tracts detected in lines 211RIB (202 bp), 238RIB (107 bp), and 340RIB (13 bp). The two latter tracts included nonsynonymous variants, which caused the presence of the shared replacement polymorphic sites between arrangements (see below).


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Table 2 Estimates of Polymorphism Within Gene Arrangement

 
Levels of nucleotide polymorphism were estimated for both chromosomal classes independently (table 2 ). According to all estimates (S, k, {pi}, and {theta}), the A1 gene arrangement showed lower levels of variation when either all sites or only silent sites (noncoding sites plus synonymous sites in the coding region) were considered. Nucleotide diversity estimates ({pi}) were lower than {theta} estimates. As the estimate of {theta} depends only on the number of polymorphic sites while {pi} also depends on their frequency, the detected difference indicated an excess of low-frequency polymorphisms in the samples.

In the coding region, nucleotide diversity at all ({pi}), synonymous ({pi}s) and nonsynonymous ({pi}a) sites was also estimated for both chromosomal classes independently (table 3 ). In this case, {pi} and {pi}s values were also considerably higher in the Ast+A2 class than in A1; in contrast, {pi}a values in both chromosomal classes were much more similar.


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Table 3 Estimates of Polymorphism in the Coding Region Within Gene Arrangements

 
Linkage disequilibrium between pairs of parsimony-informative sites was analyzed. Nineteen out of 325 (5.8%) and 49 out of 1,326 (3.7%) comparisons were significant by the {chi}2 test within the A1 and Ast+A2 samples, respectively. However, the proportion of significant pairs was near the nominal 5% rejection probability. The minimum numbers of recombination events found in the Ast+A2 and A1 samples by the four-gamete test (Hudson and Kaplan 1985Citation ) were 29 and 15, respectively.

Replacement Polymorphism
Twelve replacement polymorphic sites were detected in the 25 lines studied, of which 7 were singletons (fig. 3 ). The first three polymorphic residues are located in the signal peptide of the preprotein and are thus not included in the mature protein. All amino acid differences that segregate as polymorphic in the mature protein are conservative except for the asparagine (N)/histidine (H) replacement at site 155 of the protein. When analyzing each chromosomal class independently, eight residues were polymorphic within Ast+A2, and six were polymorphic within A1. In addition, there was a fixed replacement substitution between chromosomal classes (at residue 451) and three amino acid polymorphisms shared by both chromosomal classes. However, the presence of a valine (V), a threonine (T), and an N at residues 163, 167, and 198, respectively, in the Ast 238RIB line can be explained by the gene conversion tract detected in this line (see above). Likewise, a gene conversion event can explain the presence in the A1 340RIB line of an I (isoleucine) and an A (alanine) at residues 163 and 167, respectively. Therefore, when taking into account gene conversion, it can be argued that the two chromosomal classes present three fixed differences and no shared amino acid polymorphisms. Among the three fixed differences between arrangements, the presence of a V at residue 163 in A1 and an A at residue 167 in Ast+A2 can be considered the derived states using parsimony criteria and the amino acid sequences of D. guanche and D. madeirensis.



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Fig. 3.—Replacement polymorphic sites at the YELLOW preprotein in the 25 lines studied from Riba-roja d'Ebre (Tarragona, Spain), using as reference the previously published sequence of Drosophila subobscura (D. sub.; EMBL accession number Y13909). The numbering at the top corresponds to nucleotide sites as in figure 2 . Numbers in boldface indicate amino acid residues in the preprotein. Lines are named as in figure 2 . Fixed differences relative to the Drosophila madeirensis (D. mad.) and Drosophila guanche (D. gua.) sequences are also shown in the last two rows of the alignment

 
Neutrality Tests
The tests of neutrality proposed by Tajima (1989)Citation and Fu and Li (1993)Citation were applied to determine whether the pattern of polymorphism detected in the y gene region was concordant with neutral predictions (table 4 ). All test statistics were negative in both the Ast+A2 and the A1 chromosomal classes, although values were consistently lower within Ast+A2. Table 4 also shows the probability of obtaining a lower value for the different test statistics than that observed (one-tailed test) under no recombination or assuming intermediate levels of recombination. The C value was increased gradually in different simulations until all test statistics became significant for a given chromosomal class. Within Ast+A2, all test statistics were significant (P < 0.05) for a recombination parameter of 4. In contrast, a higher recombination parameter, C = 12, has to be assumed to obtain probabilities lower than 0.05 for all statistics within A1. However, both levels of recombination can be considered conservative, given the minimum number of recombination events detected within each chromosomal class. The probability of detecting 29 or fewer recombination events within Ast+A2 is 1.000 for C = 4. Likely, a P = 1.000 is obtained when inferring the probability of detecting 15 or fewer recombination events within A1 assuming a recombination parameter of C = 14.


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Table 4 Tajima's and Fu and Li's Tests of Neutrality Within Gene Arrangement

 
Hudson, Kreitman, and Aguadé's (1987)Citation neutrality test was used to contrast whether the levels of silent polymorphism in D. subobscura and silent divergence between D. subobscura and D. guanche (or D. madeirensis) detected at the y gene region differed significantly from those found at other genomic regions previously studied in these species: rp49 (Rozas and Aguadé 1993, 1994Citation ), Acp70A (Cirera and Aguadé 1998Citation ) and Acph-1 (Navarro-Sabaté, Aguadé, and Segarra 1999Citation ). The analysis was performed independently for each chromosomal class, and D. guanche was used to compute divergence, except in the comparisons with the Acp70A gene, for which only the sequence of D. madeirensis was available. None of the tests were significant (results not shown). The highest deviation ({chi}2 = 1.842, df = 1, P = 0.175) was found in the comparison between y (A1 arrangement) and rp49 (O3+4 arrangement). These results indicate that the relative level of silent polymorphism and divergence in y is similar to that found in the other genomic regions.

McDonald (1996)Citation and McDonald and Kreitman (1991)Citation tests were also applied to the data from each chromosomal class, with D. guanche being used in the interspecific comparison to determine whether the y gene region was evolving neutrally. No departure from neutrality was detected in any of the tests performed.

Gene Genealogy
The genealogical relationships of the studied lines were inferred from information for the whole sequenced y gene region after excluding all sites with alignment gaps and applying the Jukes and Cantor (1969)Citation method to correct for multiple hits. Figure 4 shows the neighbor-joining tree obtained using D. madeirensis and D. guanche as outgroups. All lines grouped in two clusters in the gene genealogy. One cluster included all Ast and A2 lines that were mixed among them. The second cluster included all A1 lines. Bootstrap values for these two clusters (after 1,000 replicates) were 98% and 96%, respectively. However, in the A1 cluster, line 417RIB shows a somewhat anomalous branching. This line presents a long gene conversion tract (fig. 2 ) that may explain its position in the tree. In fact, when this line was subtracted from the data set, bootstrap values for the two chromosomal class clusters increased to 100 for Ast+A2 and 99 for A1. The clustering of the lines according to chromosomal class was also obtained using other genetic distances or when only information from the coding region was considered (results not shown).



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Fig. 4.—Gene genealogy of the 25 lines from Riba-roja d'Ebre (Tarragona, Spain), reconstructed by the neighbor-joining method (Saitou and Nei 1987Citation ) with the complete deletion option and the Jukes and Cantor (1969)Citation correction. Numbers at the nodes indicate the corresponding bootstrap percentages after 1,000 replicates. Lines are named as in figure 2

 
Ages of the Inversions
A star genealogy within each chromosomal class was assumed to estimate the ages of the inversions (table 5 ). Under this assumption, the nucleotide diversity observed within gene arrangement should have been accumulated by independent mutations. For this reason, sites included in gene conversion tracts were excluded from this analysis. Average silent divergence corrected according to Jukes and Cantor (1969)Citation between all the D. subobscura lines and D. guanche was used to estimate the rate of nucleotide substitutions in the y gene region assuming that both species diverged 1.8 MYA (Ramos-Onsins et al. 1998Citation ). This rate was 21.5 x 10-9 substitutions per site per year. A similar rate was obtained when silent divergence between D. subobscura and D. madeirensis, which diverged 0.6 MYA (Ramos-Onsins et al. 1998Citation ), was considered. This rate was used to estimate the ages of the chromosomal classes given the level of silent diversity within each arrangement. According to this approach, the Ast+A2 chromosomal class would have arisen some 175,000 years ago, and the A1 arrangement would have arisen approximately 126,000 years ago. Similar estimates were obtained when only data for the noncoding region were considered. However, the Ast+A2 estimate was somewhat lower (153,000 years).


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Table 5 Estimates of the Ages of the Ast+A2 and A1 Chromosomal Classes

 
These estimates would be overestimates if silent diversity estimates within an arrangement included variation that had not arisen by the independent accumulation of mutations. Despite this, analysis was performed after excluding all sites included in gene conversion tracts; the algorithm proposed by Betrán et al. (1997)Citation detects only part of the gene conversion tracts, and therefore the samples of each arrangement may still contain some variation introduced by gene conversion. In fact, four shared polymorphisms were detected in these samples, while the expected number according to the hypergeometric distribution was only 1.3. On the other hand, these estimates would be underestimates if the chromosomal classes were not in the phase transient to equilibrium, since when nucleotide polymorphism within an arrangement has reached equilibrium, the level of variation will be proportional to its effective size and will not increase with time.


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Levels of Nucleotide Diversity
Levels of nucleotide variation detected in the y-ac-sc region of D. melanogaster and D. simulans are, in general, extremely low. For instance, nucleotide diversity estimates for European and Japanese populations of D. melanogaster ranged from 0.00000 to 0.00035 (Martín-Campos et al. 1992Citation ). These estimates were based on restriction fragment length polymorphism surveys and thus refer to variation in the whole y-ac-sc region. The low level of variation in the y-ac-sc region in D. melanogaster and D. simulans could not be explained by a low neutral mutation rate in this region, as it would also affect interspecific divergence (Begun and Aquadro 1991Citation ; Martín-Campos et al. 1992Citation ). The corrected estimate of the number of synonymous substitutions per site (Ks) in the ac gene was similar to, and even higher than, the corresponding estimates for other genes also sequenced in the two species (Martín-Campos et al. 1992Citation ). The significant deviation from neutral expectations detected, at least in some populations, by the Hudson, Kreitman, and Aguadé (1987)Citation test would therefore confirm the nonneutral reduction of polymorphism detected in the y-ac-sc region in both D. melanogaster and D. simulans.

Nucleotide diversity estimates for the y-ac-sc region of D. melanogaster were over one order of magnitude lower than estimates for the y gene region in both the Ast+A2 and the A1 chromosomal classes of D. subobscura (table 2 ). The higher level of variation at the y gene region in D. subobscura could be due to a higher effective population size of this species relative to D. melanogaster and D. simulans. This factor should affect all regions of the genome. However, in D. subobscura, nucleotide diversity at y was not particularly reduced when compared with estimates for the other gene regions studied in this species (table 6 ). As the y, rp49, Acp70A, and Acph-1 gene regions studied included different fractions of noncoding versus coding region, comparison of {pi}silent estimates was better than direct comparison of nucleotide diversity at all sites ({pi}total). Nucleotide diversity estimates at y (Ast+A2) and rp49 (O3+4) were similar, and they were lower than at Acp70A and Acph-1 (O3+4). Regardless of the chromosomal class (Ast+A2 or A1), the ratio of silent polymorphism to divergence for the y region was similar to that for the rp49 (either O3+4 or Ost), Acp70A, or Acph-1 (either O3+4 or Ost) region, as indicated by the nonsignificant results of the Hudson, Kreitman, and Aguadé (1987)Citation tests. Unlike in D. melanogaster and D. simulans, the level of nucleotide polymorphism detected in the y gene region of D. subobscura would not be reduced relative to other genomic regions studied in this species. The location of the y gene in a region with no reduced recombination in D. subobscura would explain why this gene exhibited a normal level of variation in this species. In fact, the lack of linkage disequilibrium and the minimum number of recombination events detected within each chromosomal class indicated that recombination was important in homokaryotypes. This result further supported the idea that the low level of variation at y-ac-sc in D. melanogaster and D. simulans was due to its location in a region with a strong reduction in recombination and thus reinforced the effect of the recombination rate on neutral variation that, anyway, is rather well established and generally accepted for Drosophila (for reviews, see Begun and Aquadro 1992Citation ; Aguadé and Langley 1994Citation ; Aquadro, Begun, and Kindahl 1994Citation ).


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Table 6 Nucleotide Diversity in Different Genomic Regions Studied in Drosophila subobscura

 
Genetic Differentiation Between Arrangements
The study of levels of polymorphism in the y gene region in three different arrangements of the A chromosome of D. subobscura provides information about how the presence of inversions affects nucleotide variation. No significant genetic differentiation was detected at the y gene region between Ast and A2 chromosomes. In contrast, the A1 arrangement exhibits strong genetic differentiation when compared with either Ast or A2 arrangements. This difference in the level of genetic differentiation at y between these arrangements can be explained by the location of this gene relative to the breakpoints of inversions A1 and A2. Indeed, y maps approximately six sections apart from one of the breakpoints of the A2 inversion. Thus, recombination between y and the A2 inversion can occur frequently in Ast/A2 heterokaryotypes, despite the expected reduction in recombination in the proximal region of the chromosomes outside the inverted segment (Navarro et al. 1997Citation ). In contrast, y is located very close to one of the breakpoints of the A1 inversion, and, consequently, recombination at y in Ast/A1 and A2/A1 heterokaryotypes is highly suppressed. Therefore, y is a good marker of A1 versus either A2 or Ast but not of Ast versus A2.

The gene genealogy of the lines studied (fig. 4 ) also supports that conclusion. All A1 lines cluster together in the gene tree, which points out that the pattern of variation in this gene region still reflects and is consistent with the monophyletic origin of the A1 inversion. In Drosophila, the study of nucleotide variation at molecular markers closely linked to the breakpoints of particular inversions or at the breakpoints themselves has confirmed the monophyletic character of these inversions, as in the case of the In(3L) Payne inversion of D. melanogaster (Wesley and Eanes 1994Citation ), the Sex-Ratio arrangement of Drosophila pseudoobscura (Babcock and Anderson 1996Citation ), and the Ost and O3+4 arrangements of D. subobscura (Rozas and Aguadé 1993, 1994Citation ; Navarro-Sabaté, Aguadé, and Segarra 1999Citation ). This monophyletic character holds even for inversions whose origin seems to be mediated by transposable elements, such as inversion 2j of Drosophila buzzatii (Cáceres et al. 1999Citation ) or In(2L)t of D. melanogaster (Andolfatto, Wall, and Kreitman 1999Citation ). In contrast to the clustering of the A1 lines, lines Ast and A2 are intermixed in the gene genealogy. This result is concordant with the location of y relative to the breakpoints of inversion A2. Therefore, it does not question the monophyletic character of this inversion, but it indicates that recombination has erased the reliable history of these chromosomal arrangements.

Chromosomal Phylogeny
The chromosomal phylogeny of the A chromosome inversions is not well established. Four inversions have been described in segment I of the A chromosome relative to its Ast arrangement: A1, A5, A6, and A7 (Krimbas 1992Citation ). Figure 5a shows the relationships among these inversions. Krimbas and Loukas (1984)Citation proposed that according to the centrality criteria, either Ast or A5 may be the ancestral arrangement for segment I, although they favored the former alternative. The study of the closely related species D. guanche and D. madeirensis has not helped to clarify the phylogeny. In segment I, D. madeirensis differs from the Ast arrangement of D. subobscura by a single inversion that was named Am1 (1A/B-7C/D) (Papaceit and Prevosti 1991Citation ). On the other hand, segment I of D. guanche differs from that of D. subobscura by four overlapping inversions whose identities are not well established. Brehm and Krimbas (1990)Citation proposed that Am1 was one of these four inversions. If this assignment were correct, it would mean that the Am1 arrangement was present in the ancestral populations of D. subobscura before the split of the D. guanche and D. madeirensis lineages. As this arrangement has not been detected in current populations of D. subobscura, it has to be inferred that Am1 went extinct in the D. subobscura lineage after the split of D. madeirensis. Therefore, according to the different authors, either Am1 or Ast might be the ancestral arrangement for segment I of the A chromosome of D. subobscura. In addition, the putative ancestral character of A1 cannot be completely discarded a priori.



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Fig. 5.—a, Schematic representation of the previously described relationships of the inversions in segment I of the A chromosome of Drosophila subobscura. The Am1 inversion present in D. madeirensis and, putatively, in D. guanche is also included in the scheme. b, A modification of the previous scheme that indicates the relationships between the Am1, Ast, and A1 inversions of segment I as inferred from the present data

 
According to the monophyletic character of inversions, nucleotide variation in a recently arisen arrangement is expected to be zero. Thereafter, loci associated with the new arrangement accumulate variation gradually over generations. This phase of accumulation of variation persists until nucleotide variation at these loci reaches equilibrium. An excess of low-frequency polymorphisms would be expected in the transient phase, which in some cases could be reflected in significant negative Tajima and Fu and Li statistics. Both the Ast+A2 and the A1 chromosomal classes exhibit large negative values for these statistics, which become significant for moderate levels of recombination (table 4 ). Therefore, a star genealogy can be assumed to estimate the age of each chromosomal class (see also fig. 4 ). According to these estimates (table 5 ), it seems unlikely that either the Ast or the A1 arrangements for segment I predated the split of D. madeirensis that putatively occurred 0.6 MYA (Ramos-Onsins et al. 1998Citation ). From the relationship between Am1, Ast, and A1 depicted in figure 5a, it can be inferred that Ast would have originated from the Am1 arrangement and that A1 would have subsequently arisen on an Ast chromosome. Finally, if Am1 is indeed the ancestral arrangement, A6 and A5 have to be derived inversions that arose on a standard chromosome for segment I. Thus, present data on variation in the y gene region seem to support the phylogeny for segment I of the A chromosome shown in figure 5b.

The ancestral character of Am1 is also supported by estimates of the genetic distances between D. subobscura arrangements and between D. madeirensis and these arrangements. The silent dxy value between Ast+A2 and A1 was 0.0156. In contrast, silent divergence between D. madeirensis and these chromosomal classes was 0.0268 (for Ast+A2) and 0.0265 (for A1). The shortest distance found between the Ast+A2 and the A1 chromosomes indicates that differentiation between both chromosomal classes has accumulated over a shorter period of time than that contributing to the divergence between D. madeirensis and D. subobscura.

The gene genealogy depicted in figure 4 does not seem to reflect, however, the derived character of the A1 arrangement. If indeed the A1 arrangement arose on an Ast chromosome, one would expect a priori that all A1 lines branched from an Ast+A2 line. The observed clustering of the lines according to their arrangement for segment I indicates that both arrangements have accumulated enough fixed or nearly fixed differences to be clearly differentiated. Although this result is quite unexpected, genealogies where inverted chromosomes form a cluster highly differentiated from the ancestral standard chromosomes have also been detected for the In(3L)Payne and the In(2L)t inversions of D. melanogaster, with estimated ages of 360,000 and 160,000 years, respectively (Hasson and Eanes 1996Citation ; Andolfatto, Wall, and Kreitman 1999Citation ).

The proposed phylogeny in figure 5b is based on the assumption that variation in the Ast+A2 and the A1 chromosomal classes is in the phase transient to equilibrium. However, other evolutionary forces unrelated to the origin of an arrangement may cause a similar pattern of variation. For instance, a selective sweep in, or near, the y gene region within a particular chromosomal class might have erased all variation putatively accumulated in this gene region after the origin of the arrangement for segment I. In this case, the estimated age of a given inversion would correspond to the hitchhiking event. Therefore, if this pattern is caused by factors other than the origin of the arrangements, this phylogeny should be revised. Information at other loci located, for instance, near the other breakpoint of inversion A1 may provide valuable information in this respect.

Relative Effective Sizes of the Chromosomal Arrangements
Assuming that A1 arose on a standard chromosome for segment I, one would expect that the pattern of variation in this arrangement was more distant from equilibrium in A1 than in Ast+A2. In contrast, Tajima (1989)Citation and Fu and Li (1993)Citation tests indicate a stronger deviation from expectations of the neutral model in a population at equilibrium for Ast+A2 than for A1 (table 4 ). This result could be explained by assuming that the effective size of A1 is smaller than that of the standard arrangement for segment I. In fact, the expected time to reach the mutation-drift equilibrium for an X-linked locus is 3Ne generations, where Ne is the effective population size. Therefore, the number of generations needed to reach equilibrium would be lower for A1 than for Ast+A2 if the former chromosomal class had a lower effective size. The frequency of the A1 arrangement in the sampled population (10%) might be consistent with a smaller effective size of A1 relative to Ast+A2. However, the frequency of the A1 arrangement shows clinal variation in the Palearctic region, as it increases gradually from western to eastern Europe (Krimbas 1992Citation ). This increase in frequency of the A1 arrangement is accompanied by a decrease in frequency of the Ast+A2 chromosomal class. If migration between European populations were high enough, these populations would be genetically homogenous within arrangement. In this case, the effective sizes of the A1 and Ast+A2 chromosomal arrangements would depend on their overall frequencies in Europe, and thus they would be expected to be much more alike both between themselves and between populations. Therefore, the A1 arrangement might not have a lower effective size than Ast+A2 despite the lower frequency of the former arrangement in the sampled population. Only the study of nucleotide variation at the y gene region in other European populations along the established cline will help to ascertain the degree of genetic differentiation within arrangement across Europe.



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Fig. 2 (Continued)

 

    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
We thank J. Rozas for critical comments on the manuscript and for sharing his computer program DnaSP, version 3.4, where the coalescent algorithm under intermediate levels of recombination has been implemented. We also thank Serveis Científico-Tècnics, Universitat de Barcelona, for automated sequencing facilities. This work was supported by a predoctoral fellowship from Ministerio de Educación y Ciencia, Spain, to A.M. and by grants PB94-923 from Comisión Interdepartamental de Ciencia y Tecnología, Spain, and 1995SGR-577 from Comissió Interdepartamental de Recerca i Innovació Tecnològica, Catalonia, Spain, to M.A.


    Footnotes
 
Wolfgang Stephan, Reviewing Editor

1 Keywords: Drosophila subobscura, yellow gene nucleotide polymorphism X chromosome inversion polymorphism recombination rate Back

2 Address for correspondence and reprints: Carmen Segarra, Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, 08071 Barcelona, Spain. E-mail: carme{at}porthos.bio.ub.es Back


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 Introduction
 Materials and Methods
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Accepted for publication August 8, 2000.