I. Contrasts Between the vermilion andforked Loci

We have surveyed three natural populations of Drosophila ananassae for restriction map variation at the forked c f ) and vermilion (v ) loci, using 6-cutter restriction enzymes. Both loci are located in the centromeric region of the X chromosome. Two major conclusions can be drawn from the data. First, we found strong evidence for population subdivision, i .e . , significant differences in the frequency distributions of polymorphisms and/or haplotypes between the Burma, India, and Brazil populations. Secondly, the pattern of DNA sequence variation between the two loci is unexpectedly different. The level of nucleotide variation in the v locus region is reduced (relative t o n , especially in the Burma population. Furthermore, in contrast to v , we found no insertions/deletions larger than 700 bp and no significant linkage disequilibrium at f. The genetic differentiation among subpopulations can readily be attributed to restricted migration as the predominant evolutionary force. According to population genetics theory, the differences in DNA polymorphisms between the two loci are in qualitative agreement with the hypothesis that recombination is reduced in the v locus region (“centromere effect”) but not at f. In order to test this hypothesis directly, we determined the cytogenetic positions of several loci in the centromeric region by in situ hybridization and found by comparison with the genetic map that recombination at v is indeed very low, much lower than at$ I N the 1980s, developments in DNA technology have allowed population geneticists to measure genetic variation in natural populations in new ways, either by sequencing (KREITMAN 1983) or by restriction mapping techniques (LANGLEY, MONTGOMERY and QUATTLEBAUM 1982; KREITMAN and A C U A D ~ 1986a). The initial efforts were primarily devoted to obtaining a rough estimate of the level of DNA polymorphism in Drosophila melanogaster, which turned out to be unexpectedly high, both in noncoding and coding regions (LANGLEY, MONTGOMERY and QUATTLEBAUM 1982; KREITMAN 1983; LEIGH BROWN 1983). Those studies led to investigations of more specific topics, such as the difference between restriction site and insertion variation in natural populations (AQUADRO et al. 1986), the level of DNA polymorphism in coding us. noncoding regions pertaining to the neutralist-selectionist controversy (KREITMAN and A G U A D ~ 1986b; HUDSON, KREITMAN and A G U A D ~ 1987) and the existence and range of non-random associations (MIYASHITA and LANGLEY 1988). In Drosophila, the great majority of the studies of DNA sequence variation focus on D. melanogaster. There are only a few exceptions: the recent investigations of AQUADRO, LADO and NOON (1988) comparing the rosy region of D. melanogaster with that of its sibling species Drosophila simulans and the study by Genetics 121: 89-99 (January, 1989) 89 SCHAEFFER, AQUADRO and ANDERSON (1987) on the Adh region in Drosophila pseudoobscura. Since it is important to determine the pattern of DNA polymorphism in Drosophila in general, we initiated investigations of DNA sequence variation in Drosophila ananassae. This species provides a potentially interesting comparison, because it is fairly distantly related to D. melanogaster [however, less distantly than D. pseudoobscura (PATTERSON and STONE 1952)J and because its geographic distribution differs from that of D. melanogaster. It is largely tropical (PATTERSON and STONE 1952), but has been found on all continents (MORIWAKI and TOBARI 19’75). The zoogeographical center of D. ananassae is in Southeast Asia. Of particular interest for this study is the fact that the X chromosome of D. ananassae is metacentric and that various genes located in the euchromatic middle portion of the X chromosome in D. melanogaster are in the centromeric region in D. ananassae, due to several rearrangements having been fixed in both species since their last common ancestor. The general question we ask in this paper is, how does recombination affect DNA polymorphism. We report here the results for two loci, forked and vermilion, located in the centromeric region of the X chromosome in D. ananassae. Both mutants (forked bristles and vermilion eye color) were first described 90 W. Stephan and C. H. Langley m v f Om Go mvfOmY 7 + + + + 8 m v f Om + + + + GI + + + + QQ x -(f / \ G2 + + + + 7 FIGURE 1 .-Crossing scheme for extracting the centromere region of the X chromosome in D. ananassae. The mutant chromosome carries the markers miniatvre (m), vermilion (u) , forked (f), O m ( l D ) (Om). In Go and GI, the same male has been used (indicated by an arrow). To confirm the isogenicity of a stock, we crossed a single female of the Gz generation to her sib brothers and screened the males of the next generation for evidence of recombination. by KIKKAWA in 1934 [See MORIWAKI and TOBARI (1975)l. We chose these loci, because we expected that both would exhibit the “centromere effect” (BEADLE 1932; MATHER 1939), ie., reduced levels of recombination. T h e present study follows the lines of our previous papers in which we explored some of the evolutionary consequences of reduced recombination in the centromere region, such as the accumulation of highly repeated DNA sequences (CHARLESWORTH, LANGLEY and STEPHAN 1986) and transposable elements (LANGLEY et al. 1988). MATERIALS AND METHODS Strains: Three natural populations of D. ananassae were used in this survey: a population from Burma collected by M. TODA in 1982 around Mandalay (19 isofemale lines), an Indian population from collections of F. HIHARA and 0. KITECAWA in 1981 around Hyderabad (20 lines), and a Brazilian population collected in 1986 by B. GOAI around Sao Paulo (2 1 lines). These stocks were kindly provided by Y . TOBARI. Since there are no balancers of the X chromosome of D. ananassae available at present, we used a chromosome carrying a dominant marker (Om(1D); HINTON 1984) to establish lines that are homozygous for the centromeric region of the X chromosome. The crossing scheme is depicted in Figure 1. Cloning of vermilion: A genomic DNA library was made from DNA from a ca; p x stock (HINTON 1984) as follows. 1.5 p g of ca; p x high-molecular weight DNA (see below) was partially digested with various amounts of MboI (ranging from 0.03 to 0.5 unit) for 15 min. The reactions were terminated by heating the samples up to 65” for 15 min. The digestion which yielded a high proportion of large fragments (>15 kb) was used to prepare the library. After treatment with 1 unit of alkaline phosphatase, the sample was phenol/chloroform extracted and ethanol precipitated, then resuspended in T E (10 mM Tris-HCI (pH 8.0)/1 mM EDTA) and ligated to 3 pg XEMBL4 DNA, digested with BamHI and SalI. The ligated DNA was packaged according to the instructions provided by the supplier (Stratagene). The library was screened using the 1.9-kb XhoI-Hind111 fragment [coordinates 1.1 to 3.0 of SEARLES and VOELKER (1986); see Figure 21 purified from the subclone Spv 8.7 (kindly provided by L. SEARLES). This fragment contains most of the 2.0-kb transcript of v from D. melanogaster. Plaque hybridization procedures are widely described (e.g., MANIATIS, FRITSCH and SAMBROOK 1982). In our case, hybridization was carried out at low stringency (35”), using 50% formamide. Furthermore, prior to putting the filters into prehybridization solution, they were treated in 0 . 1 ~ SSC, 1.0% SDS at 65” for 1 hr to reduce background. For the first screen we used nylon membranes (Du Pont), for the second nitrocellulose (Schleicher & Schuell). The final washes were done in 0.1 X SSC, 0.5% SDS at 35 ’. One of the positive clones (Xcpv8) was subcloned into the vector Bluescript KS M13+ (Stratagene) to remove the regions of repetitive DNA identified at both ends of the clone (Figure 2). Xcpv8 DNA was completely digested with BamHI and then partially with HindIII. The fragments were ligated to Bluescript KS M I3+, digested with BamHI and HindIII, as described by the supplier. The lengths of the resulting plasmids were measured by electrophoresis, using a supercoiled DNA plasmid ladder (BRL) as size marker. A subclone obtained in this way was used as a probe in our survey (coordinates -3.2 to +8.8; Figure 3), after comparing its restriction map with that of the original clone. Restriction map analysis: The restriction map of the v locus (Figure 2) was constructed, digesting the clones Xcpv4 and Xcpv8 with one or two restriction endonucleases and size-fractionating them on 1% agarose gels. We used the same enzymes as SEARLES and VOELKER (1986): BamHI, EcoRI, HindIII, SalI, and XhoI. Similarly, the restriction map of the f clone, Xc06, was obtained (data not shown). hco6 was a gift from S. HORI (Y . HATANO and S. HORI, unpublished data). Genomic DNA from each of the 60 lines was prepared as described by BINGHAM, LEVIS and RUBIN (1981). The DNA was digested with single restriction enzymes (BamHI, EcoRI, HindIII, PstI, PvuII, Sad, and SalI) and electrophoresed on 1% agarose gels. The DNA fragments were transferred to Zetaprobe-nylon membranes (Bio-Rad) by the usual Southern blotting technique. The filters were hybridized under standard conditions, using 50% formamide, then washed and autoradiographed (MANIATIS, FRITSCH and SAMBROOK 1982). Furthermore, for a standard restriction map, ca; p x genomic DNA was digested with single and pairs of restriction enzymes but otherwise prepared as the DNA from the natural populations. In situ hybridization: T o determine the cytogenetic location of several genes of interest for this study [including v andfurrowed (fi)], we prepared probes labeled with biotinylated-UTP (LANCER-SAFER, LEVINE and WARD 1982). Conditions for hybridization and detection are described in MONTGOMERY and LANGLEY (1983). The& clone from D. melanogaster, X320, was a gift from T. GORALSKI and K. CONRAD. Statistical procedures: The statistical significance of linkage disequilibrium was determined by the Fisher exact test. Furthermore, to test whether or not the observed frequencies of a polymorphism are significantly different between the three populations, we used Monte Carlo simulations on 2 x N-contingency tables, as suggested by LEWONTIN and FELSENSTEIN (1 965), and implemented in a program kindly provided by W. ENGELS. The levels of nucleotide variation were estimated using the methods of ENGELS (198 l ) , NEI and TAJIMA (1 98 1) and HUDSON (1 982).