* Grup de Biologia Evolutiva, Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Barcelona, Spain
Departamento de Ecología Genética y Evolución, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina
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Abstract |
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Key Words: Drosophila buzzatii inversion polymorphism nucleotide variability sequence-tagged sites
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Introduction |
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There is, however, little consensus about the extent or importance of genetic interactions in the evolutionary process after more than half a century of debate (Whitlock et al. 1995). On the other hand, the advent of molecular population genetics has provided compelling evidence that the level of nucleotide diversity is positively correlated with the regional rate of recombination (Begun and Aquadro 1992; Aguadé and Langley 1994). This pattern can be explained by variation-reducing selection on neutral variability in different recombination environments; namely, "selective sweep" events (Maynard Smith and Haigh 1974; Kaplan, Hudson, and Langley 1989) or recurrent selection against deleterious mutations ("background selection" [Charlesworth, Morgan, and Charlesworth 1993]). Conversely to the footprint left by these directional selection processes, the presence of an old balanced polymorphism will cause, in the long term, an increase of neutral diversity at closely linked sites (Hudson and Kaplan 1988; Hudson 1990). Because differences in recombination rates outside and within the chromosome segments covered by inversions are expected in heterokaryotypes (see Roberts 1976), the long-standing assumption of strong balancing selection acting on inversion polymorphisms can now be critically tested by analyzing patterns of nucleotide variation at molecular markers located in different regions relative to an inverted chromosomal fragment. To be more specific, enhanced levels of total nucleotide variability and differentiation between chromosome arrangements are expected at molecular markers closely linked to inversion breakpoints if the polymorphism has a long history of balancing selection (Strobeck 1983; Navarro, Barbadilla, and Ruiz 2000; Andolfatto, Depaulis, and Navarro 2001).
Previous studies of loci closely linked to inversion breakpoints in Drosophila reveal patterns of nucleotide variation consistent with reduced levels of gene flux (crossing over and/or gene conversion [see Navarro et al. 1997]) near breakpoints relative to loci located at longer distances (reviewed in Andolfatto, Depaulis, and Navarro 2001). The situation, however, is more complicated in the presence of a complex inversion system because of considerable gene flux between arrangements, as in the case of the rp49 gene region of chromosome O in D. subobscura (Rozas et al. 1999) or the Est-5 and Hsp83 genes on the right arm of the X chromosome in D. pseudoobscura, which segregates for the sex ratio inversion system (Kovacevic and Schaeffer 2000). By focusing on those works that have studied levels of nucleotide variation in chromosomal elements free of complex inversion systems (e.g., inversions In(3L)Payne and In(2L)t in D. melanogaster) another clear pattern emerges; namely, nucleotide diversity at markers located close to inversion breakpoints is substantially lower in the derived inverted chromosome relative to that found in the standard one (Wesley and Eanes 1994; Hasson and Eanes 1996; Andolfatto, Wall, and Kreitman 1999; Depaulis, Brazier, and Veuille 1999; Depaulis et al. 2000), and total nucleotide variability at those markers is generally lower than that found at loci far apart (>1,000 kb [see fig. 2 in Andolfatto, Depaulis, and Navarro 2000]). Overall, the data are inconsistent with these two inversions being an ancient polymorphism maintained by strong balancing selection. Theoretical work modeling inversions as balanced polymorphisms concludes that an increase of neutral diversity at sites closely linked to breakpoints is in fact expected, provided that balancing selection does not fluctuate with time and the age of inversions is greater than N generations, where N is the effective population size (Navarro, Barbadilla, and Ruiz 2000). The age of In(3L)Payne is approximately 0.36 Myr (Hasson and Eanes 1996) or approximately 1.2 N generations, assuming an effective population size of approximately (Kreitman 1983) and about 10 generations per year in D. melanogaster, and the age of In(2L)t is approximately 0.3N generations (Andolfatto, Wall, and Kreitman 1999). Therefore, it could be claimed that the life span of these inversions is shorter than the required time to achieve equilibrium.
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Materials and Methods |
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Sequence-Tagged Site (STS) Landmarks
Three anonymous regions previously mapped to the polytene chromosomes by in situ hybridization were chosen to study the effect of the polymorphic inversions on the level and distribution of nucleotide diversity. Going from distal to proximal ends on a 2st gene arrangement of the second chromosome, the mapping of the STSs was as follows (fig. 1): outside-distal 70.09.1sts on 2(C7e) and approximately 600 kb away (i.e., about 12 polytene chromosome bands and assuming approximately 50 kb per band as in D. hydei [see Laird 1973; Hartl et al. 1994]) from the distal breakpoint of inversion 2j; inside-middle 60.29.1sts, a randomly amplified polymorphic DNA fragment (RAPD) now converted to STS (see below) on 2(D3g) and approximately in the center of the chromosomal segment covered by inversion 2j (Laayouni, Santos, and Fontdevila 2000); and regD on 2(E4g-E5a) and very close (27 bp away) to the proximal breakpoint of inversion 2j (Cáceres et al. 1999). The first two STSs rendered significant "hits" with D. melanogaster nucleotide sequences located on the homologous Mueller/Sturtevant/Novitski chromosomal element E (= 2 in D. buzzatii and arm 3R in D. melanogaster [see Powell 1997, p. 307]) when using the Blast program (Altschul et al. 1997) in the GenBank database (http://www.ncbi.nlm.nih.gov/BLAST/). Thus, 70.09.1sts gives a match (a Blastn score of ) with sequence AE003725, and 60.29.1sts (a Blastn score of
) with sequence AE003727.
The 70.09.1sts locus shows a high similarity to a putative gene product described in D. melanogaster (GC5237, AE003725, Blastx score of ), and it seems to have at least one short exon of 75 bp (at positions 649 to 723). regD also shows consistent similarity to another gene product described in D. melanogaster (CG13617, AE003748 Blastx score of
) and seems to contain a short exon of 132 bp (at positions 286 to 417). However, given the small length of hypothetical coding regions in both sequences and uncertainties in the location of putative splicing sites, we did not analyze the number of synonymous and nonsynonymous substitutions.
DNA Isolation and Sequencing
DNA was extracted from three to five individuals of each isochromosomal line following Latorre, Moya, and Ayala (1986) after some modifications (see Laayouni, Santos, and Fontdevila 2000). The 70.09.1sts sequence previously reported (Laayouni, Santos, and Fontdevila 2000) (EMBL/GenBank accession number AF288346) was employed to design 20-nucleotide-long primers to be used in polymerase chain reactions (PCR) (see fig. 1 and below). Gel-purified (10 to 100 ng) RAPD 60.29.1 was directly cloned in pMOS Vector (Amersham Pharmacia, England), and its DNA sequence was determined on both strands by the dideoxynucleotide chain termination method (Sanger, Nicklen, and Coulson 1977) using an ALF sequencer (Pharmacia Biotech, Piscataway, N.J.). Amplification primers were designed after RAPD 60.29.1 was converted to STS 60.29.1sts (fig. 1). The proximal breakpoint regD sequence derived from a 2st gene arrangement (Cáceres et al. 1999) (EMBL/GenBank accession number AF162796) was used to design the primers regDL1 and regDR2 (see fig. 1) that amplified a fragment of approximately 650 bp. This fragment was then used as a probe for screening a D. buzzatii phage library (kindly provided by P. García). Plating of libraries, hybridization, and detection of positive clones were carried out following standard methods (Ausubel et al. 1998). Three positive clones were obtained, and their sequence was employed to design new primers for the amplification of a longer region towards the direction of the centromere. Figure 1 gives the sequences of the primers used for PCR amplifications and the total length of the regions investigated, as well as the strategies followed to determine overlaps between partial fragments.
PCR reactions were carried out in a final volume of 25µl, including 1x activity buffer (GIBCO BRL, Gaithersburg, Md.), 2 mM MgCl2 (3 mM in the case of 60.29.1sts), 200 µM of each dNTP (Boehringer Mannheim, Indianapolis, Ind.), 0.5 µM primer, template DNA (30 to 40 ng), and 0.8 units of Taq polymerase (GIBCO BRL). Amplifications were run in a MJ Research Inc. (Watertown, Mass.) thermocycler programmed as follows: a preliminary 5-min denaturation at 94°C; 35 cycles of 30 sec at 94°C (denaturation), 1.5 min at specific PCR annealing temperatures (primers 70091L1-R1, 70091L1-R2, and 70091L2-R1 at 60°C; 60091L1-R1at 53°C; 60291L1-R2 and 60291L2-R1 at 58°C; regDL1-R1 at 55°C; regDL1-R2 at 53°C; and regDL2-R1 at 58°C), and 1.5 min at 72°C (extension); and a final extension at 72°C for 5 min followed by storage at 4°C. Because of failures in the amplifications, two additional primers were used in some cases: 5'-AGTTCAACGCTCACACGTTT-3' instead of 60291L2, and 5'-GCCGTGGCATGTGTGTGTGT-3' instead of 60291R1. Electrophoresis was performed in 1.4% agarose gels (SeaKem), and in all cases, the fragments amplified with the most external primers were gel purified and reamplified with the appropriate internal primers (see fig. 1). These amplified products were also gel purified and directly sequenced on both strands.
D. koepferae sequences were obtained using the same primers and procedures as those previously described for D. buzzatii with minor modifications in PCR conditions. In the case of sequence 60.29.1sts, only one fragment (60291L1-R2 [see fig. 1 and below]) could be obtained.
Sequences collected in this study have been deposited into GenBank under accession numbers AY134753 to AY134842.
DNA Sequence Variation
A total of 2.6 kb were sequenced in each of the 29 D. buzzatii isochromosomal lines from Chumbicha. The multiple alignment of the three STSs analyzed (excluding insertion/deletion polymorphisms) included 943 sites for 70.09.1sts, 411 sites for 60.29.1sts, and 973 sites for regD. The difference between the approximately 948 bp for the total length of inside-middle 60.29.1sts and the shorter fragment used in the analyses (amplified with primers 60291L1-R2 [see fig. 1]) was due to technical difficulties when sequencing eight lines amplified for the overlapping second fragment (i.e., there is a gap of approximately 200 bp with many ambiguities that could not be resolved after several efforts). However, inspection of the complete fragment in the remaining 21 lines shows that molecular population estimates remain qualitatively the same (data not shown) and, hence, the shorter fragment can be considered as fully informative. Insertions/deletions (indels) were relatively frequent in all regions (D. koepferae generally had longer sequences), particularly in 60.29.1sts with eight indels between D. buzzatii and D. koepferae (four gaps in D. buzzatii ranging from 4 bp to 38 bp at sites 68 to 72, 96 to 97, 132 to 133, and 267 to 268 and four gaps in D. koepferae ranging from 2 bp to 16 bp at sites 178 to 179, 375 to 376, 380 to 382, and 97 to 112). Line chu31 (2j) presented a deletion of 3 bp, and chu74 (2st) presented two deletions of 4 bp and 28 bp, respectively. In 70.09.1sts 16 single indels were detected within D. buzzatii lines plus a large gap of 23 bp in D. koepferae (at sites 865 to 887). Finally, five single-base indels were observed in regD among D. buzzatii lines (one was only present in 2st), in addition to two gaps of 4 bp (at sites 476 to 478) and 28 bp (at sites 446 to 447) in D. buzzatii and a gap of 3 bp (at sites 727 to 730) in D. koepferae. Length polymorphisms within and between chromosomal arrangements were also relatively common in regD, with an indel of 4 bp (at sites 494 to 495) in 13 out of 15 2st chromosomes and in all 2j chromosomes (in which the indel was 2 bp longer) and an indel of 4 bp (at sites 691 to 692) present only in 2j chromosomes. Isochromosomal line chu40 (2st) had a deletion of 9 bp (at sites 648 to 653). Indels were excluded from further analyses.
Data Analyses
Sequences were multiply aligned using the default option of the program ClustalW (version 1.6) (Thompson, Higgins, and Gibson 1994) and thereafter aligned manually to minimize the number of differences. Sequences from line chu3 of D. buzzatii were used as reference in all cases. Nucleotide polymorphisms were estimated by commonly used measures of DNA variability as number of segregating nucleotide sites (S), nucleotide diversity () or average number of nucleotide differences per site (Nei 1987), and heterozygosity per site (
) expected under the infinite-site model at mutation-drift equilibrium given the observed S value (Watterson 1975).
Different neutrality tests were performed to determine whether the observed data conformed to the predictions of the neutral model of molecular evolution. Tajima's (1989) D statistic relies on intraspecific data and tests the null hypothesis that two estimates of the neutral mutation parameter, one derived from the average number of pairwise nucleotide differences and the other based on the number of segregating sites in the sample, are equal. Fu and Li (1993) proposed a set of tests that also rely on the comparison of different estimates of the mutation parameter. Their D statistic is based on the standardized difference between the total number of mutations and the number of mutations in the external branches of the genealogy, and their F statistic compares the standardized difference between the average number of pairwise differences and the number of mutations in external branches of the genealogy. An outgroup species is needed to estimate the number of mutations in external branches, and the sequences of D. koepferae were used for this purpose. Negative values of the tests indicate an excess of rare variants and are consistent with a selective sweep or with a recent population growth. On the other hand, positive values indicate an excess of intermediate frequency variants and are usually associated with stable population subdivision (either spatial or genetical). Fu's (1997) FS statistic tests the probability of having no fewer than the number of observed alleles in the sample given that . This statistic tends to be negative when there is an excess of recent mutations (or rare alleles) and may be used to better detect population growth, but it is not conservative against recombination. For increasing levels of recombination (see below) the behavior of statistical tests that use information of the mutation (segregating site) frequency is better than that for the FS statistic. Therefore, we have also used Ramos-Onsins and Rozas' (2002) R2 statistic, which is based on the difference between the number of singleton mutations and the average number of nucleotide differences. Lower values of R2 are expected under a scenario of recent population growth.
Intragenic recombination substantially affects the power of the statistical tests employed, and it is more realistic to assume because there is evidence of recombination in our data set. However, the use of estimators of the population recombination rate,
, from nucleotide polymorphism data (Hudson 1987) are problematic because they tend to be unreliable unless large stretches of sequence information are available from many individuals (Wall 2000). The analyses were performed using a lower bound Cm estimate of the recombination parameter (i.e., minimum number of detected recombination events [see Hudson and Kaplan 1985]) and an independent estimate (Cmap) based on the comparison of the physical and genetic maps, which represents our best guess of the true crossing-over rate. Thus, assuming that the size of the euchromatic portion for the second chromosome is approximately 28 Mb as in the homologous element E (chromosomal arm 3R) of D. melanogaster (Adams et al. 2000) and that the map length is approximately 138.5 cM (Schafer et al. 1993), an estimate for the recombination rate
per bp per generation (sex averaged) is obtained, assuming that recombination is approximately constant along this acrocentric chromosome in D. buzzatii (c.f. with the average value of
per bp per generation estimated in D. melanogaster females [see Lindsley and Zimm 1992]). Watterson's (1975) estimate of the neutral mutation parameter 4Nu is 0.0297 on average (table 1), and the neutral mutation rate in Drosophila is assumed to be
per site per generation (Andolfatto and Przeworski 2000). Therefore, the estimate for the effective population size in D. buzzatii is
, and
per bp per generation for 70.09.1sts, assuming that recombination is not inhibited in heterokaryotypes at the nonheterozygous distal portion of the second chromosome. On the other hand, we can expect a reduced level of recombination in heterokaryotypes at loci linked to the inversions (which seems to be the case for regD [see below]) and, therefore, the previous Cmap estimate has to be correspondingly multiplied by the expected frequency of homozygous individuals. Thus,
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The DnaSP version 3.98 software (Rozas and Rozas 1999) was used to obtain most estimates of nucleotide variability reported in this work, to perform neutrality tests, and to detect gene conversion tracts from the algorithm proposed by Betrán et al. (1997). Critical values for statistical tests were obtained by computer simulations (1,000 replicates) using the coalescent algorithm described in Hudson (1990) and implemented in DnaSP. In all cases, the most conservative estimate of the population recombination parameter (i.e., Cm versus Cmap) was used. Phylogenetic analyses employed to reconstruct the genealogies of the studied isochromosomal lines were conducted using MEGA version 2.1 (Kumar et al. 2001).
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Results |
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The algorithm proposed by Betrán et al. (1997) was used to detect gene conversion tracts among arrangements. The probabilities of a site being informative of a conversion event () were relatively high for regD between the ancestral and the two derived chromosomal arrangements (table 2). However, no gene conversion tracts were detected for this region. Four conversion tracts were identified for 70.09.1sts, but the one between 2st and 2j (line chu3) could correspond to a double crossover event because the length of the tract is unusually large (766 bp). The other three conversion tracts were identified between 2st and 2jz3 with an average length of 146 bp. Finally, for 60.29.1sts, three conversion tracts were identified between 2st-2jz3 and four were identified between 2j-2jz3, with an average length of 32 bp (figs. 2a and b).
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Linkage Disequilibrium and Genetic Differentiation
For each STS, linkage disequilibrium was analyzed for all pairs of informative polymorphic sites in the total data set as well as within each chromosomal class. In the proximal-breakpoint regD, 195 out of 780 (25%) comparisons showed a significant association by Fisher's exact test, and this number dropped to 91 (12%) after correcting for multiple contrasts. However, these percentages are not very informative, because in some cases no significant association can be detected even with extreme disequilibrium (Lewontin 1995). Therefore, linkage disequilibrium was also analyzed with the sign test on D (Lewontin 1995), first for the actual ordering of 40 informative sites in the whole sample (39 independent pair comparisons) and thereafter by recording the proportion of goodness-of-fit tests that rendered a statistically significant G-value after 200 random ordering permutations (including the actual ordering). Because linkage disequilibrium decays with physical distance due to intragenic recombination, we could expect that most random orderings would render a lower G statistic than the actual ordering (which was indeed the case [data not shown]). The observed number of negative Ds was always larger than expected (26 versus 19.03 for the actual ordering; ,
), and 68% of G-values were statistically significant. Thus, a significant excess of repulsion linkage in regD was detected, which may be partitioned into within-inversion and between-inversion components (Nei and Li 1973; Ruiz et al. 1991; Navarro et al. 1996). Within 2st, 20 independent pair comparisons are possible, and the observed number of negative Ds was slightly larger than expected in most random orderings (14 versus 11.25 for the actual ordering;
,
), with only 6% of the G-values being statistically significant. A similar result was found within the 2j arrangement with six independent pair comparisons, and, therefore, the conclusion is that the disequilibrium detected for regD is mostly due to the genetic differentiation between arrangements 2st and 2j (see below). Similarly, linkage disequilibria were also analyzed for the outside-distal 70.09.1sts, with 44 independent pair comparisons in the total data set (
for the actual ordering;
), and the inside-middle 60.29.1sts, with 27 independent pair comparisons in the total data set (
for the actual ordering;
). There was no evidence of linkage disequilibrium in the whole sample or within-inversion components for 70.09.1sts, but there was some excess of repulsion linkage for 60.29.1sts in the entire sample (not within chromosomal arrangements), with 14% of the G-values being significant after 200 random ordering permutations. In conclusion, statistically significant overall genetic associations between-inversions were mainly detected for regD.
The average number of nucleotide differences between sequences differing in chromosomal arrangement was 68.67 for 70.09.1sts (72.1% of the total number of mutations), 38.33 for 60.29.1sts (64.1%), and 48.67 for regD (96.7%), which give an estimate of the average number of nucleotide substitutions per site between arrangements (dXY) equal to 0.0198, 0.0338, and 0.0159, respectively (table 2). For the outside-distal and the inside-middle STSs no fixed differences between arrangements were observed, whereas the situation for the proximal-breakpoint regD was dramatically different, with 13 and 15 fixed differences between 2st-2j and 2st-2jz3, respectively, and 46 segregating sites in the ancestral 2st that were monomorphic in the derived arrangements. Significant differences among all arrangements were detected for 60.29.1sts and regD using KST as the test statistic but only between 2st and 2j for 70.09.1sts. Except for regD, however, the observed number of shared polymorphisms among arrangements is substantial (table 2). We have applied the hypergeometric distribution to estimate the probability that the number of shared polymorphisms between 2st-2j could be explained by independently arising parallel mutations (Rozas and Aguadé 1994). For 70.09.1sts, the expected number of shared polymorphisms would be less than or equal to five (cf. 33 shared mutations observed), and for 60.29.1sts it would be less than or equal to four (24 observed). There is, therefore, extensive genetic exchange between arrangements as previously pointed out.
Figures 2ac show neighbor-joining trees (Saitou and Nei 1987) for each STS analyzed. Genealogies of all lines were reconstructed using D. koepferae as the outgroup. In the regD tree (fig. 2c), sequences corresponding to the 2st arrangement form a unique group but with a weak bootstrap value (53%), whereas those corresponding to the derived 2j and 2jz3 arrangements form a monophyletic group with some subclustering for 2jz3. The high bootstrap value of the monophyletic cluster including all 2j and 2jz3 sequences corroborates the proposed unique origin of inversion 2j (Cáceres et al. 1999; see also Cáceres, Puig, and Ruiz 2001) and strongly argues for a monophyletic origin of inversion 2z3 on an ancestral 2j chromosome. This is further sustained by the fact that all fixed differences between 2st and 2j overlap with a fixed difference between 2st and 2jz3 (table 2), and, likewise, the distribution of indels between arrangements fully agrees with the distribution of fixed and shared polymorphisms.
An important concern regarding the regD tree in figure 2c is the mutational instability of the region close to the inversion breakpoint. Although such instability seems to be restricted to the transposable element sequences implicated in the origin of inversion 2j (Cáceres, Puig, and Ruiz 2001), we have tested the hypothesis of an accelerated substitution rate in this lineage. Relative-rate tests between lineages (using a regD phylogeny derived from maximum parsimony implemented in PAUP* 4.0b10 for Macintosh [Swofford 2000]) using RRTree version 1.1 program (Robinson et al. 1998; Robinson-Rechavi and Huchon 2000) suggest that standard (st) and inverted (j) lineages evolve at the same rate (Kimura's [1980] two-parameter model: mean , mean
,
). Therefore, mutational instability at the proximal breakpoint of inversion 2j seems to happen only at the inserted transposable element sequences since no increased rate of nucleotide change at regD is detected. Consistent with the higher level of polymorphism in 2st chromosomes, the tree is deeper in the branches connecting these sequences.
Neutrality Tests
The observed distributions of mutations between and within arrangements were contrasted with those predicted under the null hypothesis of selective neutrality. Tajima's (1989) D statistic was consistently negative and significantly so for the outside-distal marker 70.09.1sts in the total sample as well as within 2st gene arrangements, indicating a higher than expected number of low-frequency variants. An excess of unique polymorphisms was also confirmed for this marker with Fu and Li's (1993) tests. Interestingly, Fu's (1997) FS statistic was negative and statistically significant for all STSs when considering the 2st ancestral gene arrangement, suggesting that the excess of rare alleles may be the result of a population expansion or hitchhiking.
The possibility of an expansion event was further investigated by means of the R2 statistic described in Ramos-Onsins and Rozas (2002). They advocate its use for small sample sizes, particularly in the presence of recombination. The last row in table 1 gives the R2 estimates, which were always statistically significant in the subsample of 2st chromosomes. The null hypothesis of constant population size, however, cannot be rejected for the subsamples including the derived arrangements. Therefore, the most plausible conclusion seems to be that D. buzzatii has passed through a population expansion and that this event has likely preceded the last event of directional selection that swept nucleotide variation in the population of 2j chromosomes (see below).
A caveat to the above conclusion is that sample size affects the power to reject the constant size model (see figure 2 in Ramos-Onsins and Rozas 2002), and the number of lines used in this work for arrangement 2st was 15, whereas the number of lines for arrangement 2j was only 10. We have, therefore, generated independent subsamples by obtaining random combinations of 15 2st arrangements taken 10 at a time and calculated the R2 statistic for each subsample. The results indicate that R2 estimates remain statistically significant for 70.09.1sts and regD in most cases, although for the shorter fragment 60.29.1.sts most estimates felt in the 95% confidence limits but close to the lower limit and in some cases (15%) remained significant. Therefore, different sample sizes between 2st and 2j do not seem to account for the discrepancies we observed.
Evolutionary History of Inversion 2j
The age of inversion 2j can be estimated from the net number of nucleotide differences per site between 2st and 2j gene arrangements in regD. Given that the average number of nucleotide differences (dXY) between those arrangements is 0.0222 (table 2) and between D. buzzatii and D. koepferae the average number is 0.0601, the net number of nucleotide substitutions between 2st-2j is and between D. buzzatiiD. koepferae is
.
Using the Xdh sequences from D. buzzatii (GenBank accession numbers AF226958 to AF226959), D. koepferae (GenBank accession numbers AF226964 to AF226965), and D. hydei (GenBank accession numbers AF226974 to AF226975) reported in Rodríguez-Trelles, Alarcón, and Fontdevila (2000), rates of synonymous substitutions are and
. Divergence time between D. buzzatii and D. hydei is approximately 14.26 Myr (Russo, Takezaki, and Nei 1995), and, therefore, the divergence time between D. buzzatii and D. koepferae can be estimated as approximately 4.02 Myr. As a result, the age of inversion 2j can be estimated as approximately 1.16 Myr (in agreement with the figure reported by Cáceres, Puig, and Ruiz 2001).
Assuming that regD has diverged at a neutral rate, we can now obtain a mutation rate for this region from the net number of nucleotide substitutions between D. buzzatii and D. koepferae; namely, per site per year or about
per site per generation, assuming five generations per year in D. buzzatii. (Field data from a natural population in Spain point to a generation time in this species about two times longer than in D. melanogaster at the warmest months of the year [Quezada-Díaz et al. 1997; M. Santos unpublished observations].) This value is approximately twice the lower bound for the neutral mutation rate reported in Andolfatto and Przeworski (2000) and would render an
from the number of segregating variant sites at regD, which roughly corresponds to approximately 1.63 N generations for the age of inversion 2j. In other words, the second-chromosome polymorphism in D. buzzatii can be considered as to be a middle-aged polymorphism according to Andolfatto, Depaulis, and Navarro (2001). Finally, the amount of nucleotide diversity can be used to estimate the age of the sampled alleles for each chromosomal arrangement (Rozas et al. 1999), which yields approximately 0.79 Myr for 2st and approximately 0.27 Myr for 2j (or
0.38 N generations). It is interesting to note that in previous studies, coalescent times were grossly underestimated, probably due to the inclusion of a large number of alleles sampled in recently colonizing populations from Australia and Spain (Cáceres, Puig, and Ruiz 2001).
Our previous estimate of N from the net number of nucleotide substitutions between D. buzzatii and D. koepferae increases the Cmap estimates given in table 1 (see Materials and Methods) and, hence, are not a conservative assumption for Fu's (1997) FS test and may be not conservative for Ramos-Onsins and Rozas' (2002) R2 test. However, after using a corrected estimate for the recombination parameter, the statistical significance given in table 1 for FS and R2 values still remains. Therefore, the previous claim that D. buzzatii has passed through a population expansion seems to be sound.
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Discussion |
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Linkage disequilibria were almost absent at markers far apart from the 2j inversion breakpoints, but some genetic differentiation between 2st and 2j arrangements still remains, even at the outside-distal 70.09.1sts (table 2). This marker is located approximately 600 kb from the distal breakpoint and was used here in the belief that rates of exchange between arrangements were high enough as to make the nucleotide diversity of the marker independent of the evolutionary fate of inversions. Our wrong a priori conviction rested on the following reasoning: map distances in D. buzzatii for the second chromosome are about twice those estimated in D. melanogaster (see above), and theoretical models predict that recombination increases in the uninverted distal segment (Navarro et al. 1997). However, in accordance with empirical data from D. melanogaster (Andolfatto, Depaulis, and Navarro 2001), it seems that inversion effects on levels of nucleotide diversity may extend as far as 600 kb from breakpoints in D. buzzatii.
Patterns of divergence between chromosomal arrangements suggest that inversion 2j in D. buzzatii can be considered as a middle-aged polymorphism (1.63 N generations). However, a model of strong balancing selection that does not fluctuate with time (Navarro, Barbadilla, and Ruiz 2000) could be ruled out because patterns of nucleotide diversity place the age of the sampled 2j alleles at approximately 0.27 Myr (
0.38 N generations). As for the rare cosmopolitan 2jz3 and 2jq7 arrangements, whose frequencies are generally low in New World natural populations (Hasson et al. 1995), it could be the case that historical frequencies of inversion 2j have also remained quite low during most (
75%) of the time, and its rise in frequency, likely due to selection, has only occurred recently. This scenario is coherent with the reduced levels of nucleotide variation and the strong genetic differentiation between standard and inverted chromosomes (with no shared polymorphisms) observed at regD and also with the lower level of nucleotide variation in derived relative to ancestral chromosomes often found in Drosophila (Wesley and Eanes 1994; Hasson and Eanes 1996; Andolfatto, Wall, and Kreitman 1999; Depaulis et al. 2000). As previously pointed out, inversion 2z3 arose from a 2j chromosome and changed the location of regD relative to the inversion loops formed in heterokaryotypes. This relocation of regD would be expected to affect the level of genetic exchange between arrangements, but the most we can say is that gene flux seems to be absent despite the relatively high probability of detecting genetic exchange not only between the ancestral arrangement and 2j but also between 2st and the other derived arrangement 2jz3.
Under the infinite-site model with no recombination and no selection, the average number of pairwise differences within an allelic class (i.e., standard or inverted sequences at the breakpoints) is expected to be proportional to the frequency of the allelic class times the neutral mutation parameter, and the ratio of the sum of the average number of pairwise differences within allelic classes to that number in the total sample is expected to equal 1 (Innan and Tajima 1997, 1999). For regD, this ratio for 2st and 2j gene arrangements is 0.93, close to the neutral expectation but in the predicted direction (i.e., <1) if inversion 2j were maintained by balancing selection. On the other hand, Andolfatto, Depaulis, and Navarro (2001) have shown that total nucleotide diversity is lower at markers closer to inversion breakpoints, an "unexpected" result under neutrality. As can be seen in table 1, nucleotide diversity is 2.2 times larger for the inside-middle marker 60.29.1sts than for regD, in agreement with the previous findings. However, the net number of nucleotide substitutions between D. buzzatii and D. koepferae for 60.29.1sts is , which yields an estimate of the neutral mutation rate
per site per generation; that is, 2.1 times larger than that estimated for regD (see above) and consistent with the previous ratio between nucleotide diversities. Thus, the level of polymorphism within species does not seem to significantly differ from the divergence level between species, and heterogeneity in mutation rates seem to be sufficient to explain the different levels of nucleotide diversity observed. This conclusion is reinforced from the estimated synonymous nucleotide diversities and substitutions for locus Esterase-A (
,
) (Gómez and Hasson 2003), which maps on 2(D4f-h) and about seven polytene chromosome bands far from 60.29.1sts. Those values are, respectively, about 3.5 and 2.5 times larger than the corresponding estimates for regD. To summarize, the present results do not provide conclusive evidence for a putative long-term selective advantage of heterokaryotypes.
In the dynamics of inversion polymorphisms, selection may be operating at different levels, and several hypotheses have been proposed to explain the establishment of a given inversion in the population. Under a "genic selection" model, an inversion would be advantageous, provided it contains few or no deleterious alleles (Nei, Kojima, and Schaffer 1967; Santos 1986). However, deleterious mutations will accumulate in the inverted chromosome fragment and their frequencies eventually reach mutation-selection equilibrium. At this time, the inversion would become selectively neutral, and, therefore, it is obvious that other factors should be taken into account in order to explain the equilibrium frequencies of gene arrangements in natural populations of Drosophila. Data obtained for inversion In(3L)Payne in D. melanogaster showed no significant departure from neutral equilibrium (Wesley and Eanes 1994; Hasson and Eanes 1996; see also Innan and Tajima 1999) despite patterns suggesting selective sweeps. Similar patterns were observed in other species from the obscura group (Rozas and Aguadé 1990, 1993; Babcock and Anderson 1996). As stated by Andolfatto, Depaulis, and Navarro (2001), if selective sweeps are frequent in natural populations and differentially affect chromosome arrangements, how inversions remain polymorphic is unclear unless we assume that independently occurring selective sweeps cause a balanced polymorphism pattern. This explanation was put forward by Kirby and Stephan (1996) for small sequences of DNA where the absence of recombination is due to the small genetic distance, and it is known as the "traffic hypothesis."
While the hypothesis that the second-chromosome inversion polymorphism in D. buzzatii has a "long-lived" history of heterosis is not compatible with our observations, it is unclear what patterns of nucleotide diversity to expect from other types of balancing selection that can lead to shifts in inversion frequencies. Inversion polymorphisms have been classified in earlier works as "rigid" and "flexible" (Carson 1965; Sperlich and Pfriem 1986), somewhat suggesting that the frequency of polymorphic inversions may or may not remain approximately constant in natural populations. That shifts of second-chromosome arrangement frequencies in D. buzzatii occur in nature is strongly suggested by the latitudinal and altitudinal gradients observed in the native distribution range of the species (Hasson et al. 1995). If the antagonistic pleiotropic effects on fitness-related traits play an important role in the maintenance of the inversion polymorphism in nature (see above), it is not at all surprising that shifts in inversion frequencies occur depending on ecological gradients (for instance, arrangement 2j is almost fixed in Northern Monte localities of Argentina [see Hasson et al. 1995]). Theoretical models and computer simulations, in addition to further information from molecular markers inside inversion 2j and closer to the distal and proximal breakpoints than inside-middle 60.29.1sts, as well as far apart from the inversion breakpoint than our outside-distal marker, may help to better understand the dynamic role played by demographic history, selection, and rate of gene flux in shaping nucleotide variation connected to inversion polymorphisms.
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Wolfgang Stephan, Associate Editor
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