Contrasting Histories of Three Gene Regions Associated ... · VC 805 St S Veracruz, Mexico VC 815 St S Veracruz, Mexico EM 10 In(3L)P F Veracruz, Mexico 178.7 In(3L)P F Maryland 709.6

Copyright 0 1996 by the Genetics Society of America

Contrasting Histories of Three Gene Regions Associated With In (3L)Payne of Drosophila melanogaster

Esteban Hasson' and Walter F. Eanes

Department of Ecology and Evolution, State University of New York, Stony Brook, New York 1 1 794 Manuscript received May 14, 1996

Accepted for publication September 9, 1996

ABSTRACT In the present report, we studied nucleotide variation in three gene regions of Drosophila melanogaster,

spanning >5 kb and showing different degrees of association with the cosmopolitan inversion In(3- L)Payne. The analysis of sequence variation in the regions surrounding the breakpoints and the heat shock 83 (Hsp83) gene locus, located close to the distal breakpoint, revealed the absence of shared polymorphisms and the presence of a number of fixed differences between arrangements, indicating absence of genetic exchange. In contrast, for the esterase-6 gene region, located in the center of the inversion, we observed the presence of shared polymorphisms between arrangements suggesting genetic exchange. In the regions close to the breakpoints, the common St arrangement is 10 times more polymorphic than inverted chromosomes. We propose that the lack of recombination between arrangements in these regions coupled with genetic hitchhiking is the best explanation for the low heterozygosity observed in inverted lines. Using the data for the breakpoints, we estimate that this inversion polymorphism is around 0.36 million yr old. Although it is widely accepted that inversions are examples of balanced polymorphisms, none of the current neutrality tests including our Monte Carlo simulations showed significant departure from neutral expectations.

I NVERSION polymorphisms in the genus Drosophila have long constituted a model system to study the

adaptive processes involved in the maintenance of genetic variation (DOBZHANSKY 1970). Associated with this study has been the perpetuation of assumptions surrounding the molecular or mutational origin, existence of monophyly, and role that reduced recombination plays in diversifjmg the variation forced into linked association with the inverted segment.

The general observations from early allozyme studies showed that electrophoretic alleles of loci located within or very close to the inverted segment exhibit strong nonrandom associations with the inversion (reviewed in ISRIMBAS and POWELL 1993). Initially, these associations were interpreted as the result of coadapted natural selection, however, further consideration dem- onstrated that these associations are also compatible with simple neutrality, i.e., different alleles become associated with inversions initially when the inversion originated, and these associations persist because of the low rate of recombination between arrangements (KRIMBAS

and POWELL 1993). To understand the extent to which an inversion is

evolving as a block, it is necessary to study sequence variation for genes near the inversion breakpoints as

Corresponding author: Walter F. Eanes, Department Ecology and Evo- lution, State University of New York, Stony Brook, NY 11794. E-mail: [email protected]

'Present address: GIBE, Departamento de Ciencias Biologicas, F a d - tad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria Pab. 11. (1428), Buenos Aires, Argentina.

Genetics 144: 1.565-1575 (December, 1996)

well as genes or regions in the central more freely recombining portion of the inversion. Molecular data from restriction fragment length polymorphism (RFLP) and DNA sequencing studies have shown the presence of shared polymorphisms between arrangements in Drosophila melanogaster (AGUADE 1988), D. subobscura (ROZAS and AGUADE 1990, 1993, 1994), and D. pseudoobscura (POPADIC et al. 1995), and these are consistent with some genetic exchange among arrangements. However, a study of restriction site variation of the amylase gene region, located within the break points of the naturally occupying inversions of D. pseudoobscura, still showed greater variation between rather than within inversions (AQUADRO et al. 1991). Likewise, a similar pattern also showing extensive differentiation between St and Sex-ratio Xchromosomes was observed for the Est-5 region (BABCOCK and ANDERSON 1996). These observations are consistent with both limited exchange of genetic content between arrangements, as well as sharing of variation within arrangements due to common history.

Inversion polymorphisms are generally accepted as examples of balanced polymorphisms (reviewed in KRIMBAS and POWELL 1993). One possible approach to resolve the role of natural selection in the maintenance of inversion polymorphisms is to study nucleotide variation in genomic regions tightly linked with the putative target of natural selection. This approach has allowed inferences to be made concerning the evolutionary histories of particular genes (e.g., -ITMAN and HUDSON 1991; EANES et al. 1993, 1996; HUDSON et al. 1994; KARO-

1566 E. Hasson and W. F. Eanes

TABLE 1

List of Drosophila melanogaster and D. sirnulam lines

Geographic Line Karyotype Est6 allele origin

Oregon R St S Laboratory Strain DPF 2 St S New York DPF 13 In(3L)P F New York DPF 30 St F New York DPF 46 St F New York DPF 62 St S New York DPF 77 St F New York DPF 82.1 In(3L)P F New York VC 805 St S Veracruz, Mexico VC 815 St S Veracruz, Mexico EM 10 In(3L)P F Veracruz, Mexico 178.7 In(3L)P F Maryland 709.6 In(3L)P F Maryland Mali 4.2 St F Mali, Africa Mali 4.4 In(3L)P s Mali, Africa Mali 10.2 In(3L)P S Mali, Africa D. sirnulans St S New York

TAM et al. 1995) because population genetic theory predicts that over time balanced polymorphisms should lead to the accumulation of genetic variation at tightly linked sites (HUDSON and KAPLAN 1988).

WESLEY and EANES (1994) first identified and characterized the sequence features associated with the pre- cise breakpoints of an inversion. Six and seven copies of the In(3L)Payne inversion and standard arrangements were sequenced respectively, and the inverted sequences were fixed for a number of differences, yet carried little within inversion polymorphism. In this study, we extend our molecular characterization of the breakpoints of the D. rnelunogaster cosmopolitan inversion In(3L)Payne to include two new gene regions. We present the homologous sequence at the breakpoints for D. simulans and extend the analysis to the heat-shock protein gene 83 (Hsp83), located very close to the distal breakpoint (at 63B, LINSDLEY and ZIMM 1992), and the esterase 6 (Est-6) locus located in the middle of the inverted segment (at 69A1-5, LINSDLEY and ZIMM 1992). We wish to address several questions. Are closely linked regions, as well as those inside the In(3L)Puyneinversion still monophyletic? How old is the inversion polymorphism? Is there evidence of genetic exchange between arrangements? And finally, does the analysis of sequence variation allow to infer the evolutionary history of this inversion?

MATERIALS AND METHODS

Fly samples: Sixteen D. rnelunoguster chromosomes, chosen to uncover a wide range of the species distribution, were included in the present study. The geographical origin, the inversion karyotype and the Est-6 genotype of each line are shown in Table 1.

PCR and sequencing: Three genomic regions were analyzed in the present study and their positions are shown in

+- In(3L)Payne "3

0

n

Hsp83 AIB 63B 63B8-11 69A1-5 7231-2

CID (1390 bp) (1175 bp) (17" bp) (1245 bp)

FIGURE 1. "Diagram of the left arm of the third chromofu- some of D. rnelanogaster showing the relative positions of the breakpoints of In(3L)Puyne (regions A-D), Hsp83, and Est-6.

Figure 1. These regions are (1) the genomic regions encompassing the break points (2420 bp) of In(3L)Puyne (63B8- 11 and 72E1-2 distal and proximal breakpoints in the cytological map of D. rnelunoguster, respectively) were amplified in three additional St D. rnelanogaster lines (DPF46, DPF62 and DPF77) and one line of D. simuluns. Primers employed to amplify and sequence these regions in D. sirnulans were the same as those reported in WESLEY and EANES (1994) with the addition of four new specific primers. (2) A fragment of the Hsp83 gene (at 63B) including part of the 5' untranslated (277 bp) and part of the coding region (1 113 bp) was amplified from nucleotide 968 to 2426 of the sequence published by BIACKMAN and MESELSON (1986), and sequenced using a set of eight primers. (3) A 1.7-kb fragment of the Est-6 gene (at 69A1- 5 ) , from nucleotide position 208 to 1930 of the sequence published by COLLET et ul. (1990), encompassing the entire coding region was amplified and sequenced using a set of 12 primers.

Sequencing procedures: The amplified products were ex- cised from 1% low-melting-point agarose gels after electro- phoresis and reamplified for production of single-stranded templates by the X-exonuclease procedure (HIGUCHI and HOCHMAN 1989). Single-stranded templates were sequenced using Sequenase version 2.0 (USB). In all cases, both strands were sequenced using sets of primers spaced 250 nucleotides. Sequences were manually aligned.

Statistical tests: In addition to simply characterizing the pattern of polymorphism within and between arrangements for these regions, we wish to examine the extent to which associated molecular variation is evolving in a nonneutral fashion, using the Wright-Fisher population model as a null hypothesis (KIMURA 1983). For each region, possible depar- tures from neutrality assuming the infinite sites neutral model (KIMUFU 1983) were tested by means of TAJIMA (1989), FU and LI (1993) and the HKA (HUDSON et al. 1987) tests. TAJI- MA'S test is based on the comparison between two estimators of the neutral parameter, 6' = 4Np. Under the null hypothesis of selective neutrality, estimates of 6' based on either the number of segregating sites S or the average number of painvise differences between alleles T , are expected to be statistically equal. The Fu and LI test is based on the comparison between the proportion of mutants in internal and external branches in a genealogy. The rationale of this test is based on the logic that old mutations should be located in internal branches while recent mutations should be in external branches. An external mutation is defined as a derived mutation (not shared with the outgroup species) found as a singleton. This number of singletons is compared to the total number of mutations present in the sample. In both tests, negative values of the test statistic D indicate past directional selection resulting in an excess of rare mutations, while positive values reflect potential effects of balancing selection resulting in an excess of mutations in intermediate frequency.

Recently SIMONSEN et al. (1995) have shown that TAJIMA'S critical D values are often too conservative, in particular at the upper tail of the distribution, and they have developed a simulation method for deriving less conservative confidence

Contrasting Histories and Inversions 1567

intervals. We also used SIMONSEN et aL’s (1995) Table 3 to test the significance of TAJIMA’S D.

We also use the HKA test (HUDSON et al. 1987), which is based on the prediction of the neutral theory that intraspecific polymorphism and interspecific divergence should be correlated (KIMURA 1983) across loci. Loci with high levels of polymorphism should display correspondingly high levels of interspecific divergence. Under selective neutrality, the ratio of intraspecific polymorphism and interspecific divergence for one locus should not differ significantly from ratio estimates obtained for other (presumably neutral) gene regions, and the HKA test formally tests this contrast. A locus showing an excess of polymorphism to divergence relative to the same ratios at other loci suggests the action of balancing selection at that locus. Conversely, deficiencies of polymorphism imply recent directional selection.

Last, we are interested in assessing the impact the inversion polymorphism may have imposed on the genealogical rela- tionships in our sample of alleles. The stochastic process reflected by the Wright-Fisher population model results in genealogies that may be characterized with respect to various statistical properties. For example, the distribution of times between coalescence events is expected to be approximately exponentially distributed (see HUDSON 1990 for review). Nat- ural selection has the effect of distorting the genealogy and the distribution of coalescence times away from the neutral expectations. A favored mutation that has recently increased rapidly in frequency will have the effect of producing a set of common lineages (those sharing the mutation) with little among-lineage sequence diversity relative to the overall variation among all lineages. The standard set of neutrality tests (e.g. TAJIMA, FU and LI, and HKA) may statistically detect such distortions, but more specific tests conditioned on unique mutation events in the genealogical history are often desired. Testing such mutation-specific hypotheses requires defining features of the genealogy expected to be affected by selection and then determining how often such features are observed in simulated replicates of the coalescence process. HUDSON (1990, 1993) first advocated this approach and subsequently used it to address the significance of the pattern of polymorphism associated with the Sod allozyme polymorphism in D. melanogaster (HUDSON et al. 1994). We have carried out a Monte Carlo simulation of the Wright-Fisher process to inves- tigate the significance of features of the sequence data with respect to the inversion polymorphism. The particular features, defined here a priori, are the number of silent polymorphisms associated with that subset of alleles bearing the inversion and the number of “fixed” mutations identified between the inverted and standard arrangements. Both would be expected to the affected by natural selection.

We have simulated the coalescent process using the logic and an adaptation of the computer algorithm introduced by HUDSON (1990). This is done operationally by generating a large number of replicate trees, each tree being a random topology of n lineages, where the time between coalescence events (nodes) is sampled from an exponential distribution. Each tree then has m polymorphic sites randomly distributed among its branches. Nodes are identified in the tree than possess the same number of descendants as the observed number of lineages possessing the specific defining mutation of interest (not all trees may satisfy this condition and these are dropped from the simulated sample). The number of polymorphisms associated with that subset of lineages, as well as the number fixed differences between subsets, is determined for each simulated tree and compared with the number of polymorphisms, or number of fixed differences, o b served in the real data. For example, if one has a real sample of n = 10 lines with an observed total of m = 46 silent polymorphisms and five lines possess the mutation of interest with no

silent polymorphisms segregating among them, it is possible to ask how often this disparity in polymorphism among subsets might be observed in a sample of “random” trees. As in HUDSON et al. (1994), the parameter 8 does not appear in the simulation. These simulations assume no recombination, recognizing that these will be, in the presence of recombination in the true sequences, conservative probabilities (HUD- SON et al. 1994). A sample of 10,000 replicate trees were generated in each test simulation.

RESULTS

Breakpoint regions: The three new alleles sequenced for the breakpoint regions added no new polymorphic variants to those already described by WESLEY and EANES (1994). These sequences along with the D. simulans sequence have been deposited under accession numbers U57372-U57387.

The region containing the breakpoints was divided by WESLEY and EANES (1994) in four subregions, A and B for the distal breakpoint and C and D for the proximal. Estimates of nucleotide diversity expressed as (3 (estimated from the number of segregating sites) or T, (the average pairwise number of differences between alleles per nucleotide) for each one of the four subregions are given in Table 2. In order to test whether these subregions show comparable levels of sequence polymorphism, we applied the homogeneity test pro- posed by KREITMAN and HUDSON (1991). This test com- pares observed and expected numbers of polymorphisms assuming a common heterozygosity parameter across regions. The test statistic is approximately x‘ distributed with k-1 degrees of freedom ( k is the number of regions being compared). This test failed to reject the null hypothesis of between-region homogeneity in levels of polymorphism (x’ = 3.1 df = 3, P = 0.38).

The inclusion of a single sequence for D. simulans permits a contrast of the level of polymorphism with divergence. In Table 2, divergence estimates expressed as the proportion of silent nucleotide differences between allele VC805 of D. melanogaster and the single D. simulans allele are shown. In addition, a large insertion/ deletion difference of 74 bp and several single base- pair insertion/deletions were detected. Overall, 102 single base differences were observed in the 2420 bp of the four regions, giving an overall divergence estimate of 4.2%. Although we may not partition this region into coding and noncoding function, this level of divergence is consistent with estimates of silent site and intron divergence for a number of gene regions (see HUDSON et al. 1994).

H$83 region: The summary of intraspecific variation for Hsp83 is presented in Table 3. Sequences are available under accession numbers U57459-U57473. Six and seven segregating sites were detected in the 277 bp of the 5‘ intron region and of the 11 13 bp (243.3 effective silent sites) coding region, respectively. Estimates of nucleotide variation for this locus in D. melanogaster are summarized in Table 2.

A novel feature present in the sample of 16 alleles


TABLE 2

Nucleotide heterozygosity and interspecific divergence between D. melanogaster and D. simulans, in the genomic regions spanning the breakpoints of Zn(3L)P and the H q 8 3 and &6 loci

Region Base pairs n,“ Bb ?rr Divergence ( d ) d / r

Hsp83 1390 520.3 0.0075 0.0067 0.071 10.6 Coding 1113 243.3 0.0087 0.008 0.058 7.3 Intron 277 277 0.0065 0.0056 0.076 13.5

Breakpoints 2420 - 0.0052 0.0058 0.042 7.2 A 880 - 0.0068 0.0085 0.036 4.2 B 473 - 0.0032 0.0023 0.053 23 C 484 - 0.0025 0.0034 0.027 7.9 D 583 - 0.0067 0.0064 0.055 8.6

Est-6 1682 464 0.0162 0.0192 0.110 5.7 Exons 1614

Silent - 396 0.0167 0.02 0.116 6 Replacement 1218 - 0.0027 0.0029 0.021 6.9 Noncoding 68 68 0.0132 0.013 0.073 5.4

n, is the effective number of silent sites (KREITMAN and HUDSON 1991). ‘ 8 is the estimator of 4Np determined from the number of segregating polymorphisms (see HUDSON et al.

1987). ?r is the “nucleotide diversity” (NEI 1987) or average number of nucleotide differences per silent site.

“ d is the average number of nucleotide differences per silent site between two species (see HUDSON et al. 1994).

was a deletion polymorphism of codon number 234, coding for lysine, in positions 1980-1982 (Table 3). This codon deletion is fixed in all Zn(3L)Payne chromosomes and present in all St chromosomes. Divergence estimates for Hsp83 were obtained by comparing the D. simulans sequence reported in BLACKMAN and MESEL- SON (1986) with allele VC805 of D. melanogmter. Thirty- seven sites, 23 in the intron and 14 in the coding region, were observed to differ, giving an estimate of divergence that is slightly larger for the intron (7.6%) than for the exon (5.8%).

E&6 region: All polymorphic nucleotide site variants observed in the D. melanogaster sample are listed in Ta- ble 4. Sequences are available under accession numbers U57475-U57488. Twenty-six synonymous and 11 nonsynonymous sites were polymorphic in the sample of 16 chromosomes. Six silent polymorphisms are new

variants that were not present in the nonrandom sample analyzed by COOKE and OAKESHOTT (1989). Six out of the 11 replacement polymorphisms at positions 315 (71 of COOKE and OAKESHOTT 1989), 1046 (802), 1048 (804), 1469 (1225), 1770 (1526), 1817 (1573) and 1851 (1607) were previously observed (COOKE and OAKES HOTT 1989), including the variant responsible for the Fast/Slow electrophoretic polymorphism (asparagine/ aspartic acid) at position 1016 (772). The four new amino acid variants predictable from the nucleotide sequence (observed at nt 273, 417, 603 and 1070) are listed in Table 4, and none of them theoretically changes the charge of the protein.

Estimates of heterozygosity based on the number of segregating sites and average number of painvise differences per site (T ) are given in Table 2. The distribution of nucleotide variation along the sequenced region of

TABLE 3

Polymorphic nucleotide sites in the Hsp83 gene locus in St and Zn(3L)P chromosomes

Position OR R DPF 2 DPF 30 DPF 46 DPF 62 DPF 77 VC 805 VC 815 Mali 4.2 DPF 13 DPF 82.1 EM 10 178.7 709. I Mali 4.4 Mali 10.2 ~

1061 C G G G G G G G G 1072 T C C C c c c C C 1078 T G 1092 T A 1127 T A A 1247 A G 1547 C T 1574 C T 1646 C T T 1688 G A A 1727 G A 1877 C A A A A A A A

2291 T C C C C C C C C C c C

St chromosomes are columns 2-10 and In(3L)P chromosomes are columns 11- 17.

. .

1990-92 AAG . - - _ ” - -

Contrasting Histories and Inversions

TABLE 4

Polymorphic nucleotide sites in the Es&6 gene locus in St and Zn(3L)P Drosophila melamgaster lines

1569

DPD DPF DPF DPF DPF VC VC Mali DPF DPF EM Mali Mali A. A. Position Or R 2 30 46 62 77 805 815 4 .2 13 82 10 178.7 709.1 4.4 10.2 DIFF"

238 G . A A A . 243 A . G G 273 T . C C C C . C I-T 315 C . T T T T . T T-*I 417 C . T T T T T T . T-1 425 C . T T T T T . 475 C . T T T T T . 562 A . G G G G G G . 603 G C . S-T 622 C . T T . . T T 835 T . C C C . C C 949 T . C C C c C c c c c c c C

1016 A . G G G G G G G G G . N-D 1042 C . T T T . 1046 A . G G G G G G G G G G . T-A 1048 C . A A 1070 T . C S-tP 1093 C . T T T T T T . T . 1102 C . T 1150 T . C C C . C 1153 A . G G G 1154 T . C

. G . C C . C .

1210 T . C C C C C . C 1378 G . A . 1469 T . G G L-v 1483 C . T T . 1600 G . A . A A . 1603 C . G G G G G 1612 T . C C 1618 C . T T 1649 T , A A . A 1690 A . G G G G G G G G G 1770 G .

G A R-tK

1817 T . G G G 1 8 5 1 G . A A A A A A

S-A A A A A A A A A

1864 G . A N-tS

A A

St lines are columns 2-10 and Zn(3L)P lines are columns 11 -17. a Corresponds to amino acid change from Oregon R. The Fand Sallozymes are the G (arginine) and A (aspartic acid) changes

at position 1016, respectively.

the gene is also shown in Table 2. As reported by KARo- TAM et a1 (1993) Est-6 is one of the most polymorphic genes in Drosophila.

The interspecific comparison between Est-6 allele VC805 of D. rnelanogaster and the D. simulans sequence published by KAROTAM et al. (1993) revealed 46 synonymous and 25 nonsynonymous differences in the exons, one difference in the 5' untranslated region and four in the intron. Estimates of interspecific divergence for the exon silent sites (12%), nonsilent sites ( 2 % ) and (7%) for the intron and 5' untranslated region are also presented in Table 2 . As it was pointed out by COOKE and OAKESHOTT (1989) the overall level of synonymous variation is higher among putative F alleles. The number of segregating synonymous sites in the sample of F alleles is 20 and 11 in S lines (Table 4), and the average number of painvise differences is 9.3 within the putative F alleles and 5.0 within S lines. The average number of nucleotide differences between F and S alleles is 6.7. Nine sites are segregating for polymorphisms in both

electrophoretic alleles suggesting extensive recombination between alleles. These results are similar to those reported by COOKE and OAKESHOTT (1989) although their sample was nonrandom. Fis ancestral to S as can also be deduced from the sequence comparisons between D. melanogaster and D. simulans (KAROTAM et al. 1995).

The distribution of nonsynonymous variants among electrophoretic alleles shows that five out of the re- maining 10 replacement polymorphisms are shared, although two are segregating only in S lines, and one in Flines. Site 1046, which is strongly associated with site 1016, and 1851 are both polymorphic in Slines for the variants that are fixed in F alleles (Table 4). No fixed nucleotide differences were observed between electrophoretic alleles (Table 4).

Nucleotide polymorphism associated with the St and Zn(3L)Puyne gene arrangements: Nucleotide heterozygosities specific with respect to the St and In(3L)Payne arrangements are summarized in Table 5 for the re-


TABLE 5

Comparisons of the levels of polymorphism (0 and ‘IT) within and between St and Zn(3L)P chromosomes for the three gene regions studied

Standard In(?L)P Between“

Region B 7l B 7r dst,ln

HspS? 0.0078 0.0065 0.0008 0.0009 0.009 Breakpoints 0.0058 0.0060 0.0003 0.0003 0.010

Silent” 0.0195 0.0162 0.0200 0.0200 0.017 Replacement 0.0034 0.0031 0.0016 0.0022 0.003

Tpi’ 0.0151 0.0135 0.0128 0.0128 0.014

Est-6

In the last column, the average number of pairwise differences per nucleotide between St and In(3L)P

“Calculated on the basis of the sum of the effective number of silent sites plus the base-pair number of

‘The unlinked triose phosphate isomerase locus, located in the right arm of chromosome 3, is included for

lines is given.

noncoding regions.

comparison (E. HASSON, I.-N. WANG, L.-W. ZENG, M. KREITMAN and W. F. EANES, unpublished results).

gions (A-D) encompassing the breakpoints, Hsp83 and Est-6. There are important differences between the regions closely associated to the breakpoints (regions A- D and Hsp83) and the Est-6 region, which is located in the middle of the inverted segment. For both the breakpoints and Hsp83, nucleotide variation is an order of magnitude higher in the sample of St chromosomes. There are eight “fixed” differences between St and In(3L)Payne lines (excluding the two deletions at the breakpoints). Seven correspond to single nucleotide substitutions, six in the breakpoint regions (WESLEY and EANES 1994) and one in Hsp83 (Table 3). There is also the deletion of an entire codon in Hsp83, which is fixed in Zn(3L)Payne lines. In addition, for the breakpoint regions and Hsp83, no shared polymorphisms were detected between arrangements, suggesting that genetic exchange is strongly suppressed in inversion heterozygotes for these regions. In contrast, the levels of silent and replacement polymorphism for Est-6 are similar within arrangements (Table 5), no fixed differences were detected, and several nucleotide polymorphisms are common to both St and In(3L)Payne chromosomes.

These patterns of variation are reflected in the gene- specific genealogies shown in Figures 2 and 3. In the neighbor-joining (NJ) tree (SAITOU and NEI 1987) generated from the pooled breakpoint and Hsp83 data, all Zn(3L)Payne chromosomes form a unique cluster of high bootstrap value. Consistent with the greatly dif- fering levels of polymorphism, the tree is deeper in the branches connecting St chromosomes. In contrast, the NJ tree generated using Est-6 synonymous and nonsynonymous nucleotide variation clearly shows not only that In(3L)Payne lines do not form a unique group, but that they also form three or more clusters, suggesting that there have been at least two genetic exchange events between arrangements, either through crossing over or gene conversion (Figure 3).

Tests of neutral models: All three regions were tested separately for deviation from neutral expectation

using the TAJIMA (1989) and Fu and LI (1993) tests as described in METHODS AND MATERIALS. All three regions failed to statistically reject the neutral models in either test. Further testing of TAJIMA’S model using the less conservative test developed by SIMONSEN et al. (1995) still failed to statistically reject neutrality. We carried out four HKA tests. Each region was contrasted with the 5’ Adh flanking region described in KRFLITMAN and HUDSON (1991) and no results were significantly significant. Finally, Est6 and Hsp83were contrasted against each other and the HKA test was not significant.

Our Monte Carlo simulations of the coalescence process were planned to examine two hypothesis associated with the distribution of polymorphisms within and between the two arrangements. Since the frequency of In(3L)Puyne varies globally from 0 to 40% (LEMEUNIER and AULARD 1993), we have constituted a “constructed random sample” (HUDSON et al. 1994) of all nine St sequences and two inverted sequences sampled at random. Our two inverted copies possess no polymorphisms, while there are 54 polymorphisms in the entire set of 11 sequences. There are seven differences “fixed” between the sets of arrangements. The first hypothesis asks what is the probability of observing 0 polymorphisms in the two copies, who exclusively share a common ancestor (the inversion), given there are 54 polymorphisms in the total sample of 11 sequences? This was a very common event in our sample of 10,000 trees. Second, we wish to ask what is the probability of observing seven or more fixed differences between the subsets of two and nine alleles, again conditioned on 54 total polymorphisms? Out of 10,000 simulated trees, 720, or 7.2%, showed this number of fixed differences between comparable subsets.

DISCUSSION

The data for the three regions differ sharply when interpreted in the context of inversion history. Nucleo-

Contrasting Histories and Inversions 1571

- "DPF-2

47- "DPF-30 82 - - *Or-R

35, "DPF-46

79 "VC-805

- *DPF-62 64

*DPF-77 96 FIGURE 2.-Neighbor-joining tree based on

the breakpoint region (A-D) and Hsp83 se- "vc-815 quence data for the nine St (*) and seven

In(3L)Payne (0) lines of D. mlanogaster and one *Ma-42 D. simulans line. Numbers indicate the boot-

strap percentages out of 500 bootstraps. The - ' ~ ~ - 1 0 scale bar denotes six nucleotide differences.

64 r' '178.7

- 'DPF-13

99 - 'DPF-82.1

87" '709.6

'Ma-4.4

'Ma-10.2

97

D. sim

tide variation associated with the regions flanking the breakpoints as well as the closely linked Hsp83 gene are consistent with a monophyletic origin for In(3L)Payne. First, as shown by WESLEY and EANES (1994), all inverted chromosomes have structurally identical breakpoints ( i e . , are broken between the same nucleotide pairs). The absence of any shared polymorphisms, and the number of fixed differences between arrangements, for regions closely linked to the breakpoints is consistent with all inverted chromosomes being derived from a single inversion event. Finally, in the NJ tree generated from variable sites at Hsp83 and breakpoints, all inverted chromosomes form a unique cluster that is obtained in 99% of the trees generated after 500 bootstraps.

When we focus our attention only on the pattern of nucleotide variation observed at the Est-6locus the issue of monophyly for In(3L)Payne is equivocal. The presence of nucleotide sites that are segregating for the same polymorphisms in both gene arrangements, and the absence of any fixed differences is consistent with either multiple origins of the inversion or extensive genetic exchange. In our NJ tree for the Est-6 region, the seven Zn(3L)Payne chromosomes form at least three

different clusters. Since St and In(3L)Payne arrangements share many silent and replacement Est-6 polymorphisms, and if we accept that inverted chromosomes are monophyletic based on the results for breakpoints and Hsp83, then it appears that genetic exchange has had sufficient time to distribute nucleotide variation between arrangements for the Est-6 region.

The large number of fixed differences between arrangements observed in the breakpoints and Hsp83 regions is inconsistent with a recent origin for the inversion. The average number of painvise differences between St and In(3L)Puyne lines is 27.7, compared to the average of 19.1 differences between St chromosomes.

The high diversity among In(3L)Payne lines for the Est-6 gene is also inconsistent with recent origin. Nucle- otide heterozygosity is nearly identical for silent and replacement variation in both arrangements. The average number of painvise differences are 7.9 (for silent variation) and 3.5 (for replacement variation) between St and inverted chromosomes, compared with 6.4 and 3.8 differences for St lines. This pattern of similarity is also seen across the same lines for the silent variation


*Or-R (S)

*DPF62(S)

30

I, lr O

Ma-4.4(S)

, I oMa-L0.2(S) FIGURE 3.-Neighbor-joining tree based

on the Est-6 sequence data for the for nine St (*) and seven Zn(3L)Puyne (0) lines D. melanogaster and one D. simulans line. Num- bers indicate the bootstrap percentages out of 500 bootstraps. The scale bar denotes six nucleotide differences.

9 8 r %PF-82.l(F)

4 '178.7(F)

D. sim

in the unlinked triose phosphate isomerase gene region (Hasson et al. unpublished results), which is located in the right arm of the third chromosome and segregates independently of the inversion.

A fundamental question concerns the age of the inversion. While there are a number of differences between arrangements, any estimate of age must be predi- cated on the recognition that some unknown level of divergence existed among standard chromosomes at the time Zn(3L)Payne originated. Contemporary divergence between arrangements must include the original level in addition to divergence accumulated since origination. Recognizing this, the addition to the original breakpoint data of the sequence for D. simulans allows (in the absence of any information on the functional classes of the breakpoint region) an estimate of the divergence of Zn(?Z,)Payne relative to the divergence between D. melanogasterand D. simulans. The average level of divergence among contemporary St chromosomes is 14.5 differences. Assuming this was also the average level of between chromosome differences at the time of origination, we should subtract this from the observed value of 23.6 differences between arrangements, resulting in a net of 9.1 accumulated differences. The average number of differences between D. rnelanogaster and D. Airnulansis 102, and again subtracting 14.5 differences as the assumed contribution from an ancestor of the two species (now resulting in a net 87.5 differences between species), we conclude that the age of Zn(3-

L)Payne is -10.4% of the time since the D. rnelanogaster and D. sirnulans split. Our own data on Hsp83, using net divergence as suggested by NEI (1987), results in an estimate of 4.0 million yr (Myr), which is fairly similar to the recent estimate, based on three loci, of 3.4 Myr (HEY and KLIMAN 1993). Therefore, based on the breakpoint sequence data we estimate the age of In(3L)Payne to be -0.364 Myr. It should also be noted that a similar analysis using the levels of polymorphism and divergence for Hsp83predicts an age that is only 3.8% of the time since species separation. Nevertheless, while In(3L)Payne is not a particularly old lineage with respect to time, it is old with respect to the sampled lineages.

Despite the apparent lack of association between Est- 6 variation and arrangement types, there is a cluster of three sequences (EM-10, DPF 82.1 and 178.7) that shows a high bootstrap value and is restricted to Zn(3- L)Payne. These three lines have in common two silent derived mutations (at nt 238 and 1042) not observed in the other inverted or St sequences. We speculate that these three lines are derived from the original Est-6 allele captured by the inversion, while others represent sequences exchanged with St. Nevertheless, other sequences as well as the former three possess an additional four polymorphisms seen only in the Zn(3Z)Payne lines.

Is the high level of polymorphism at Est-6 in Zn(3- L)Payne consistent with simple exchange? The rate of decay of original associations between neutral markers

Contrasting Histones and Inversions 1573

and inversions as well as the amount of Est-6 sequence variation seen in the sample of Zn(3L)Puyne is dependent on the rate of exchange in heterokaryotypes (ISHII and CHARLESWORTH 1977; NEI and LI 1980). Rates of double crossover and gene conversion per generation in Drosophila inversions vary from (ISHII and CHARLESWORTH 1980) to lop5 for a small inversion in the rosy region (CHOVNICK 1973). PAYNE (1924) specifi- cally examined the rate of recombination for several visible markers on the third left arm when in association with Zn(3L)Payne heterozygosity. The only informative mutation is hairy which, as with Est-6, is located in the central region of the inversion (at 66D15). Based on 27,550 meiotic products he concluded that the hairy gene was being exchanged at a rate of 1 X

The population genetics of divergence, convergence, and polymorphism associated with chromosomal arrangements is analogous to that of subdivided populations, with migration reflecting the rate of genetic exchange. There are intrinsic differences because there are population (or arrangement) specific levels of intra- arrangement recombination, and these are frequency dependent. Thus, we may envision the inverted population of chromosomes as a subpopulation of chromosomes with little within-arrangement recombination, while receiving migrants (as recombinants) from a larger population at a rate equal to the rate of exchange. Given the enormous sequence diversity at Est- 6 among the St chromosomes, each sequence can be operationally viewed as a unique allele entering the population of inverted chromosomes. This analogy to the infinite alleles model (KIMURA and CROW 1964), assuming N is the haploid population size of Zn(3- L)Puyne chromosomes and r is the heterozygous exchange rate of predicts a level of sequence ho- mozygosity at equilibrium of (2Nr + l)-’ = 0.048, assuming an effective population size of lo5 for the Zn(3L)Puyne chromosomes. Thus, for the Est-6 region, it would appear that the sequence diversity among Zn(3- L)Payne chromosomes is consistent with the observed levels of heterozygous exchange and reasonable population sizes.

This observation aside, the present data clearly dem- onstrate the ability of an inversion to strongly suppress crossing over and gene conversion in the regions surrounding the breakpoints. This is perhaps expected since both genetic processes depend on effective homo- logue pairing, which is likely to be restricted in the vicinity of the breakpoints. The absence of shared polymorphism in the breakpoints contrasts with the many sites segregating simultaneously in St and Zn(?L)Payne for the Est-6 region. These contrasting pictures suggest that double crossovers and gene conversion are effective mechanisms for shuffling genetic information between St and In(3L)Puyne in the Est-6 region, where effective pairing is permitted, since it is located in the center of the inverted segment (CHOVNICK 1973).

Nucleotide variation in the breakpoints, as well as in

the tightly linked Hsp83 region, is >10 times higher in the sample of St chromosomes. This feature can also be seen in the corresponding NJ tree, in which the branches leading to the node of Zn(?L)Payne chromosomes are substantially shallower than the branches connecting St chromosomes. Why are Zn(3L)Payne lines less variable than St in the regions surrounding the breakpoints? Given that the inversion appears to pre- date the oldest lineages in St lines, there would appear to have been sufficient time for the Zn(3L)Puyne chromosomes to have recovered polymorphism. Since recent origin is an unlikely explanation for the low diversity of inverted chromosomes, other explanations are necessary. The global frequency of Zn(3L)Payne in contemporary populations is -7% (LEMEUNIER and AUG ARD 1993). The lower heterozygosity might be a conse- quence of an historically smaller effective population size for Zn(3L)Puyne relative to the St chromosomes, which is a fundamental prediction under neutral theory (KIMURA 1983). Alternatively, within the population of Zn(3L)Payne chromosomes periodic adaptive selective “sweeps” (KAPLAN et ul. 1989) or background selection (CHARLESWORTH et ul. 1993; HUDSON and KAPLAN 1995) can also account for the low levels of variation in the regions located near the breakpoints in the inverted chromosomes.

In a population composed of these two arrangements, the opportunity for recombination will only be realized in homokaryotypes, thus relative levels of recombination will vary between arrangements de- pending upon population frequencies. The population of inverted chromosomes would be especially vulnera- ble to genetic hitchhiking, since an inversion with a population frequency of q = 0.07 will have a arrangement-specific population level recombination rate that is expected to be reduced -13.2-fold, relative to the more frequent St arrangement (proportions q and p of the inverted and standard chromosomes may engage in recombination respectively, and p / q = 13.2). Both explanations can account not only for the low level of variation within inverted chromosomes but also for the fixation of the codon deletion in the highly conserved gene Hsp83, a putative deleterious variant present only in the population of inverted chromosomes. Because relatively small population size and recombination-associated hitchhiking (with the accompanied effect of low- ered population size) both predict low levels of polymorphism, it is not possible to favor either to explain our data, although reduced population size is clearly sufficient to explain the difference in heterozygosities.

Finally, can the present data allow us to infer the evolutionary processes involved in the history of Zn(3- L)Puyne? That selection is the major force shaping inversion frequencies in nature appears to be quite likely (KRIMBAS and POWELL 1993). The most compelling evidence comes from surveys of inversion frequencies in natural populations. Clinal variation of inversion frequencies along environmental gradients is, in general,


interpreted as evidence of selective differences between arrangements, and Zn(3L)Puyne shows parallel latitudinal clines in different continents as well as reciprocating clines in the northern and southern hemispheres (KNIBB et al. 1981). Furthermore, these authors reviewed a pervasive and large evidence of seasonal variation.

An operational definition of a balanced polymorphism is one where life expectancy of the polymorphism would, if given sufficient time, exceed that expected for a neutral polymorphism. The effect of a balanced polymorphism is the elevation of heterozygosity at sites tightly linked to the target of selection (STRO- BECK 1983; KAPLAN et d . 1988). The size of the genomic region showing higher than expected levels of neutral variation is negatively correlated with the level of recombination. In the presence of a balanced polymorphism, this increased neutral variation is caused by the accumulation of differences between allelic classes (-ITMAN and HUDSON 1991). The older the polymorphism the higher the number of differences between allelic classes.

Is Zn(3L)Puyne a balanced polymorphism? The accumulation of unique differences between arrangements is a feature of the breakpoints, but the conventional neutrality tests (TAJIMA, FU and LI, and HKA) failed to reject the null hypothesis of selective neutrality in the regions analyzed, although the presence of seven fixed differences between arrangements approaches statistical significance ( P = 0.072) in our Monte Carlo simulation of the coalescence process. The low statistical power and insensitivity of the available neutrality tests is certainly problematic. This can be particularly true for the test of TAJIMA (see SIMONSEN et nZ. 1995). This aside, the best explanation is that there simply has not been enough time for the accumulation of a sufficient number of differences to become fixed between arrangements.

The authors thank to D. DYKHUIZEN and B. VERREILI for their useful comments on earlier versions of this manuscript. E.H. was partially supported by a CONICET (Argentina) Fellowship. This work was supported by National Science Foundation grant number DEB9318381 to W.F.E.

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