Sequence Differentiation Associated With an Inversion on the Neo-X Chromosome of Drosophila americana

Corresponding author: Bryant F. McAllister, 138 Biology Bldg., University of Iowa, Iowa City, IA 52242-1324., bryant-mcallister{at}uiowa.edu (E-mail)

Communicating editor: S. W. SCHAEFFER

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Sex chromosomes originate from pairs of autosomes that acquire controlling genes in the sex-determining cascade. Universal mechanisms apparently influence the evolution of sex chromosomes, because this chromosomal pair is characteristically heteromorphic in a broad range of organisms. To examine the pattern of initial differentiation between sex chromosomes, sequence analyses were performed on a pair of newly formed sex chromosomes in Drosophila americana. This species has neo-sex chromosomes as a result of a centromeric fusion between the X chromosome and an autosome. Sequences were analyzed from the Alcohol dehydrogenase (Adh), big brain (bib), and timeless (tim) gene regions, which represent separate positions along this pair of neo-sex chromosomes. In the northwestern range of the species, the bib and Adh regions exhibit significant sequence differentiation for neo-X chromosomes relative to neo-Y chromosomes from the same geographic region and other chromosomal populations of D. americana. Furthermore, a nucleotide site defining a common haplotype in bib is shown to be associated with a paracentric inversion [In(4)ab] on the neo-X chromosome, and this inversion suppresses recombination between neo-X and neo-Y chromosomes. These observations are consistent with the inversion acting as a recombination modifier that suppresses exchange between these neo-sex chromosomes, as predicted by models of sex chromosome evolution.

SEX chromosomes have arisen many times in diverse animal and plant taxa (GRAVES and SHETTY 2001 ; CHARLESWORTH 2002 ). In these independent lineages, similar characteristics are generally observed for the sex chromosome pair. Meiotic recombination in the heterogametic sex is limited between the sex chromosomes. Also, the sex-limited chromosome (the Y in male heterogamety) is genetically inert relative to its homolog (the X). These derived features of heteromorphic sex chromosomes evolve between homologous chromosomal pairs that have acquired the initial switch in the sex-determining cascade. Two processes demarcate the evolution of sex chromosomes: (i) restriction of recombination in the heterogametic sex and (ii) degeneration of gene function on the sex-limited chromosome (CHARLESWORTH 1991 ). The overall pattern of sex chromosome evolution has been empirically supported by sequence comparisons between orthologous genes on the human X and Y chromosomes (LAHN and PAGE 1999 ), although the interactions of forces acting in populations and driving these processes are not understood (RICE 1996 ; CHARLESWORTH and CHARLESWORTH 2000 ).

One of the major unresolved issues in studies of sex chromosome evolution is identifying the primary forces affecting the initial divergence between the sex chromosome pair. Sexual antagonism is considered a likely contributor to this process. Due to their patterns of segregation, new sex chromosomes may be subject to selective forces conducive to the accumulation of sexually antagonistic alleles, where genotypes are beneficial to one gender but detrimental to the other (RICE 1984 ; RICE and CHIPPINDALE 2001 ). Although sexually antagonistic genetic variation is apparently common (RICE 1992 ; GIBSON et al. 2002 ), its influence on newly evolved sex chromosomes has not been demonstrated. Sexually antagonistic alleles are considered critical for initiating divergence between incipient sex chromosomes, because they create a premium for reducing recombination between this chromosomal pair (BULL 1983 ; CHARLESWORTH 1991 ). In these regions of reduced recombination, the sex chromosome pair should be influenced asymmetrically by genetic drift (GORDO and CHARLESWORTH 2001 ), purifying selection (CHARLESWORTH et al. 1993 ; CHARLESWORTH 1996 ), and adaptive evolution (RICE 1987 ; ORR and KIM 1998 ), which may ultimately cause degeneration of gene function on the Y chromosome. Studies of relatively young pairs of sex chromosomes indicate that divergence proceeds rapidly following the cessation of recombination (ATANASSOV et al. 2001 ; BACHTROG and CHARLESWORTH 2002 ; BACHTROG 2003 ; MOORE et al. 2003 ). It is, therefore, critical to study incipient sex chromosomes to identify the mechanisms driving their initial differentiation.

Natural chromosomal translocations involving autosomes and the primary sex chromosomes provide ideal systems for studying mechanisms involved in sex chromosome evolution (STEINEMANN and STEINEMANN 1998 ; BACHTROG 2003 ). Populations of Drosophila americana exhibit such a translocation involving a centromeric fusion of the primary X chromosome (Muller element A) and the fourth chromosome (Fig 1A). Chromosomal pair 4 is autosomal in other species within the D. virilis species group, of which D. americana is a member (THROCKMORTON 1982 ); this chromosome is equivalent to Muller element B, which is chromosomal arm 2L in D. melanogaster (WHITING et al. 1989 ). The X-4 centromeric fusion is distributed along a latitudinal cline in the central United States and is present at high frequency in northern populations (MCALLISTER and CHARLESWORTH 1999 ; MCALLISTER 2001 , MCALLISTER 2002 ). Presence of the X-4 fusion in male flies causes sex-linked transmission of chromosomal pair 4. Male flies lack meiotic crossing over (KIKKAWA 1935 ), and the fourth chromosomal pair segregates as neo-sex chromosomes in populations fixed for X-4 fusion chromosomes (Fig 1A). Neo-X chromosomes consist of the fourth chromosomal arm of metacentric X chromosomes, and neo-Y chromosomes are unfused acrocentric fourth chromosomes.

View larger version (21K): In this window In a new window Download PPT slide   Figure 1. Karyotypic configurations and sampling localities for D. americana. (A) Possible karyotypic configurations due to polymorphism for the X-4 fusion, which is positively correlated with latitude. Each chromosomal element is labeled according to D. virilis group nomenclature, except for the unlabeled pair of dots (pair 6). Note the sex linkage of the fourth chromosomal pair in males with the X-4 fusion and the opportunity for recombination between fused and unfused fourth chromosomes in heterokaryotypic females. (B) Map of central U.S. representing collection localities of samples. Frequencies of the X-4 fusion at each locality are estimated as follows: NN/DN, 100%; G96, 98%; and LP97, 0%.

Populations of D. americana containing the derived X-4 fusion are not reproductively isolated from populations having the ancestral chromosomal arrangement, where the fourth chromosomal pair segregates as autosomes. Historically, these chromosomal forms have been identified respectively as D. a. americana and D. a. texana (THROCKMORTON 1982 ). Due to the widespread polymorphism for the X-4 fusion, which is the defining characteristic of D. a. americana, subspecific status is unjustified and is not used in this article (MCALLISTER 2002 ). Furthermore, genome-wide sequence divergence does not exist between the two karyotypic forms (HILTON and HEY 1996 , HILTON and HEY 1997 ; MCALLISTER and MCVEAN 2000 ), and no generalized population subdivision is evident in the geographic region exhibiting the X-4 chromosomal cline (MCALLISTER 2002 ). Unfused fourth chromosomes can, therefore, flow among populations and exist in northern latitudes as neo-Y chromosomes, which are sheltered from recombination; occur at intermediate latitudes in heterokaryotypic females, where recombination with X-4 fusion chromosomes is possible; or exist in southern latitudes as an autosomal pair (Fig 1A).

The pattern of differentiation between the neo-sex chromosomes remains unclear. Sequence analysis of Alcohol dehydrogenase (Adh) indicated previously that no differentiation is present between neo-X and neo-Y chromosomes collected in a single sample with a high frequency (98%) of the X-4 fusion. Low-level recombination between fused and unfused fourth chromosomes and/or recent origin of the X-4 fusion were proposed as reasons for the observed lack of differentiation (MCALLISTER and CHARLESWORTH 1999 ). However, a different view was conveyed from a survey of microsatellite variation that revealed two out of eight loci that were significantly differentiated between the neo-X and neo-Y chromosomes (SCHLOTTERER 2000 ). Sequence and microsatellite data have, therefore, yielded contrasting results concerning differentiation between the neo-X and neo-Y chromosomes of D. americana. Microsatellites also revealed differentiation (three out of eight loci) between the combined sample of fused and unfused fourth chromosomes from northern isolates (D. a. americana) compared to autosomal fourth chromosomes in southern isolates (D. a. texana). For sequence data, differentiation between northern and southern populations has been observed only for the fused1 locus, which is located near the centromere of the primary X chromosome (VIEIRA et al. 2001 ).

Due to the dynamic distribution of the X-4 fusion and the consequent geographically localized emergence of neo-X and neo-Y chromosomes in D. americana, this system holds great promise for identifying mechanisms that drive the initial divergence between pairs of sex chromosomes. In this study, sequence differentiation and diversity at Adh and two additional loci, big brain (bib) and timeless (tim), is examined across a broader sampling of chromosomes representing widespread D. americana populations. These sequence data reveal that differentiation on the fourth chromosome is localized within the chromosome and limited to a specific geographic region. The bib locus on neo-X chromosomes obtained from the northwestern range of this species is highly differentiated relative to neo-Y chromosomes from the same region and other chromosomal samples. A common haplotype responsible for this differentiation is associated with a derived paracentric inversion [In(4)ab] on the neo-X chromosome. The combination of the chromosomal fusion and the inversion suppresses recombination in the pericentromeric region between neo-X and neo-Y chromosomes, thus mediating the differentiation of the neo-X chromosome. These results demonstrate that the X chromosome is actively involved in the initial differentiation between this newly evolved pair of sex chromosomes.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

DNA sequencing:
Each of the genes analyzed has been localized previously on the fourth chromosome of the standard cytological map of D. virilis (GUBENKO and EVGEN'EV 1984 ); Adh is located proximally at subdivision 49B (NURMINSKY et al. 1996 ), bib is located close at subdivision 48E, and tim is located distally at subdivision 42E (MCALLISTER 2002 ). Each of these genes is located outside of commonly inverted regions on chromosome 4 of D. americana (HSU 1952 ). DNA sequences of Adh, bib, and tim were obtained for single chromosomes extracted from flies collected at four different geographic localities (Fig 1B). Samples from the G96 (41° 33.0' N; 87° 22.0' W) and LP97 (32° 42.7' N; 94° 40.0' W) localities are the same as those used by MCALLISTER and CHARLESWORTH 1999 . Additional samples were collected at the margin of the Niobrara River near Niobrara, NE (NN; 42° 44.9' N; 98° 2.6' W) in 1997 and 2000 and at the margin of the Platte River near Duncan, NE (DN; 41° 22.1' N; 97° 29.7' W) in 2000 and 2001. These wild-caught flies were used in laboratory crosses to obtain material for sequencing and were also used to establish isofemale lines.

It was necessary to determine the arrangement of the centromere for each fourth chromosome to identify sequences as being derived from neo-X or neo-Y chromosomes. For the G96 and LP97 samples, the DNA samples of MCALLISTER and CHARLESWORTH 1999 were used to obtain the bib and tim sequences. Microscopic examination of metaphase nuclei in larval ganglia confirmed the chromosomal organization for these samples. For the 1997 sample from Niobrara, NE (sequence identification NN97.#), isofemale lines were established from wild-caught females. Metaphase nuclei from at least six larvae were used to confirm the karyotype as having all X-4 fusion chromosomes in each line. An individual male from each NN97 line was crossed to a D. virilis strain (V46), so F₁ females contain a single X-4 fusion chromosome (neo-X) and F₁ males contain a single unfused fourth chromosome (neo-Y), each within a hybrid genetic background.

In lieu of performing karyotypic analyses of isofemale lines, molecular markers can be used to directly infer the organization of individual chromosomes as they are crossed into a hybrid background (MCALLISTER 2002 ). For the NN and DN samples collected in 2000 and 2001, which are represented in the sequence identifications as NN00.#, DN00.#, and DN01.#, sex-linked or autosomal transmission of each fourth chromosome was directly ascertained. Six male and six female progeny of individual wild-caught males or isofemale derived males were typed with four microsatellite loci on the fourth chromosome, GPDH, V68-62, V68-74, and V68-86.1 (SCHLOTTERER 2000 ). Microsatellite primers were 5' conjugated with fluorescein (Integrated DNA Technologies, Iowa City, IA), and resulting PCR products were electrophoresed in 6% polyacrylamide between borosilicate glass plates (CBS Scientific, Del Mar, CA) and visualized directly using a Fuji FLA-3000G laser scanner (Stamford, CT). Single F₁ progeny resulting from the wild-caught males or isofemale derived males crossed to the V46 strain of D. virilis were used to obtain sequencing template.

Sequences of an 884-bp region of Adh from the G96 and LP97 samples were obtained from GenBank accession nos. AF136656, AF136657, AF136658, AF136659, AF136660, AF136661, AF136662, AF136663, AF136664, AF136665, AF136666, AF136667, AF136668, AF136669, AF136670, AF136671, AF136672, AF136673, AF136674 and AF136680, AF136681, AF136682, AF136683, AF136684, AF136685, AF136686, AF136687, AF136688, AF136689, AF136690, AF136691, AF136692, AF136693, AF136694, AF136695, AF136696, AF136697, AF136698, AF136699, AF136700, AF136701, AF136702, AF136703, AF136704. The same methods as used previously (MCALLISTER and CHARLESWORTH 1999 ) were used to obtain Adh sequences from the NN and DN samples. The sequenced region corresponds to 7 bp upstream of the start codon and 11 bp upstream of the stop codon of Adh, which includes three exons and two introns (NURMINSKY et al. 1996 ). Species-specific primer pairs for amplification of D. americana alleles from F₁ hybrids with D. virilis were developed for bib and tim using the same strategy as MCALLISTER and MCVEAN 2000 . Sequences of an 943-bp region of bib were obtained by amplification [58° annealing temperature (AT)] with overlapping americana-specific primer pairs (5' tac gat ttc gga ctt gcg aa/5' ggt gta cag att ctg gcag, 5' gct caa tta cga cat gga ca/5' atg ctc tct gta cgc tgt tg). The sequenced region corresponds to contiguous partial sequences of intron 4 and exon 5 of the bib gene (BURRIS et al. 1998 ). Sequences of a 493-bp region from exon 2 of tim (MYERS et al. 1997 ; OUSLEY et al. 1998 ) were obtained by amplification (60° AT) with americana-specific primers (5' gat ccg aag agc acc aag ag/5' gtg gca tcg gtg ccc tca). To obtain sequences of single chromosomes, each region was amplified from DNA isolated from an F₁ hybrid resulting from a D. americana x D. virilis cross. DNA was obtained by either phenol/chloroform extraction (MCALLISTER and CHARLESWORTH 1999 ) or column purification (QIAGEN, Chatsworth, CA). Each sequenced chromosome was sampled independently from a natural population. Amplified products were column purified (QIAGEN) and sequenced directly using dye terminator chemistry with an ABI 377.

Sequence analyses:
In each of the samples from northern latitudes, sequences were classified as being obtained from neo-X or neo-Y chromosomes. These designations are based, respectively, on whether or not the fourth chromosome is fused with the X. Initial analyses of the data revealed that the NN and DN samples exhibited nearly identical patterns of sequence variation in cross-sample comparisons of neo-X and neo-Y chromosomes (see Table S1 in supplemental data at http://www.genetics.org/supplemental/). Sizes for the NN and DN samples are individually small, so these were combined as the Neb-X and Neb-Y samples for the analyses presented.

Standard measures of DNA sequence diversity, (TAJIMA 1983 ) and (WATTERSON 1975 ), were obtained using the SITES version 1.1 (HEY and WAKELEY 1997 ) and DnaSP version 3.97 (ROZAS and ROZAS 1999 ) computer packages. The frequency spectrum of polymorphisms in each sample was tested for departures from neutrality with the D_T (TAJIMA 1989 ), D_FL (FU and LI 1993 ), and H (FAY and WU 2000 ) statistics, and their significance relative to the standard neutral model was assessed by coalescent simulations conditioned on the numbers of sequences and segregating sites as implemented in DnaSP. Differentiation among samples (F_ST) was estimated using Equation 9 of HUDSON et al. 1992 with sample means weighted by the sample sizes. The P (F_ST 0) was assessed by 1000 Monte Carlo permutations of the samples using the total numbers of differences between sequences and ignoring gaps. Phylogenetic relationships were inferred by a heuristic search for the most parsimonious topology using PAUP* version 4.0b10 (SWOFFORD 2002 ).

Cytology:
Association between the common neo-X haplotype at the bib locus and In(4)ab was determined by combined genotype and chromosomal analysis of 15 isofemale lines. Six female and six male flies from each isofemale line were genotyped at bib to determine if the BbrPI⁺ allele was present or absent in lines derived from each of the localities used for the sequence analysis (Fig 1B). Primers Bibamf2 and Bibgenr2 (MCALLISTER 2002 ) were used to amplify (58° AT) bib from individual fly squash preparations. Each fly was squashed in 300 µl of a solution containing 10 mM Tris-Cl, 1 mM EDTA, 25 mM NaCl, and 200 µg/ml proteinase K; incubated for 1 hr at 37°; and incubated for 5 min at 95°. Digestion of the PCR product was obtained by adding 2.5 units of BbrPI (Roche, Indianapolis) [the isoschizomer PmlI (NEB) has also been used] and incubating for at least 1 hr at 37°. Digested PCR products were resolved on 1.5% agarose gels.

Polytene chromosomes were examined to identify the arrangement of the fourth chromosome in the isofemale lines. Standard methods for obtaining orcein-stained salivary gland preparations were used (KENNISON 2000 ). Breakpoints delineating the standard and inverted arrangements of chromosome 4 were compared with previously mapped inversion breakpoints (HSU 1952 ). For isofemale lines where only the BbrPI⁺ or the BbrPI^- allele was present among females, polytene chromosomes were prepared for larvae identified as female or male on the basis of gonad size. In northern lines (NN, DN, and G96) where the X-4 fusion was fixed in the lines, comparisons of polytene chromosomal arrangement between females (homokaryotypic for X-4 fusion) and males (heterokaryotypic for fused and unfused fourth chromosomes) revealed the individual arrangements of neo-X and neo-Y chromosomes. Two lines were segregating BbrPI alleles among female flies, so genotype identification of individual larvae was necessary. Following removal of salivary glands in Drosophila Ringer's solution, larval carcasses were used to obtain squash DNA preparations that were genotyped with BbrPI as described previously.

Recombination estimates:
Recombination relative to the centromere was measured at the Adh, bib, and tim loci in heterokaryotypic females. F₁ females were obtained from crosses between females from a ML97.4 strain and males from a NN97.9 strain. ML97.4 is homozygous for unfused X chromosomes and homozygous for the standard arrangement of the fourth chromosome. X chromosomes in the NN97.9 strain are fused with the fourth chromosome, and the X-4 fusion has the In(4)ab arrangement, whereas the unfused fourth has the standard arrangement. The karyotype of the resulting F₁ females consists of centromeric heterozygosity with a single X-4 fusion having unfused homologs and inversion heterozygosity with the X-4 fusion having the inverted arrangement and the unfused fourth having the standard arrangement.

Crossovers on the X and fourth chromosomes in these F₁ females redistribute alleles between the fused and unfused centromeres. To determine the arrangement of the centromere for potentially recombinant chromosomes, F₁ females were crossed to a strain (am-cd) homozygous for the cardinal (cd) mutation located on the fourth chromosome, and F₂ males were subsequently backcrossed to the V46 strain (cd/cd) of D. virilis. Sex-linked assortment of cd (wild-type females, cardinal males) in F₃ families indicates transmission of a fused X-4 chromosome by the F₁ female, and autosomal assortment of cd indicates transmission of unfused X and fourth chromosomes by the F₁ female. Each family contains wild-type F₃ females sharing a single chromosome transmitted through an F₁ female. Single F₃ females from 88 families with sex-linked and 85 families with autosomal fourth chromosomes were assayed. DNA was extracted from these flies using 96-column plates from QIAGEN. Genotypes at Adh, bib, and tim were assessed by restriction endonuclease digestion (Adh, MseI; bib, PmlI; tim, NlaIII) of PCR products obtained using the methods of MCALLISTER 2002 . This sample size has a >0.95 probability of observing at least one recombinant within an interval if the recombination rate is >1.8%.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Sequences at Adh, bib, and tim were assayed to examine patterns of nucleotide diversity and differentiation along the neo-sex chromosomal pair of D. americana. Samples were obtained from three localities at northern latitudes, and each of these samples (NN, DN, and G96) was characterized as having a high frequency of the X-4 fusion (Fig 1). A total of 10 independently isolated X chromosomes were examined for the NN sample, 12 were examined for the DN sample, and all were identified as X-4 fusion chromosomes. Although the frequency of the X-4 fusion is 100% for these two samples, these populations cannot be classified as fixed due to the small sample sizes. For example, the likelihood of not observing an unfused X chromosome is 30% if the frequency of the X-4 fusion in the population is 95%. The X-4 fusion was previously estimated as being present at 98% in the G96 sample and absent in the southern LP97 sample (MCALLISTER and CHARLESWORTH 1999 ). Overall, the populations examined in this study represent extreme frequencies of the X-4 fusion.

Sequence diversity within samples:
The sequence data are grouped to represent different geographic and chromosomal populations (Fig 1B). Neb-X and Neb-Y represent, respectively, samples of neo-X and neo-Y chromosomes from two separate localities within Nebraska. G96-X and G96-Y represent samples of neo-X and neo-Y chromosomes collected from a single locality near Gary, Indiana. A sample of autosomal fourth chromosomes is represented by the LP97 sample from northeast Texas. Total numbers of sequences obtained for each gene, measures of haplotype structure, numbers of silent (synonymous and intron) and nonsynonymous segregating sites, and estimates of nucleotide diversity are reported in Table 1. Variation in these gene regions occurs primarily at synonymous and intron sites. The few replacement substitutions appear evenly distributed among samples. Silent nucleotide diversity increases on the basis of distance relative to the centromere; Adh is closest to the centromere and average pairwise diversity () across all samples is 0.019, and pairwise diversity increases to 0.032 at bib and 0.046 at tim. Due to this consistent pattern along the chromosome, intergenic comparisons of variation using the Hudson-Kreitman-Aguadé test (HUDSON et al. 1987 ) are uninformative for quantifying deviations in levels of variation within samples.

Tests for sequence differentiation among populations:
One of the critical issues in examining the neo-sex chromosomes of D. americana is determining the extent to which these chromosomes have diverged. No fixed differences are present between samples in any of the pairwise comparisons of the Adh, bib, or tim sequences. Sequence differentiation (measured as a variant of F_ST, see MATERIALS AND METHODS) between the five different chromosomal samples was also examined. Significant differentiation is observed, but it is dependent upon the locus and the samples compared (Fig 2).

View larger version (19K): In this window In a new window Download PPT slide   Figure 2. Sequence differentiation among geographic and chromosomal samples of D. americana. Samples are represented by neo-X and neo-Y chromosomes from Nebraska (Neb) and Gary, Indiana (G96) and by autosomal fourth chromosomes (LP97). Values between samples represent estimates of FST, with the value of P (FST 0) indicated as follows: *P < 0.05, **P < 0.01, and ***P < 0.001. Comparisons between unfused fourth chromosomes are represented by thin lines, whereas comparisons involving X-4 fusion chromosomes are represented by thick lines. Differentiation between neo-X and neo-Y chromosomes within Neb and G96 are presented beside the sample designations.

Unfused fourth chromosomes reveal no evidence of general sequence differentiation among separate geographic populations of D. americana. All pairwise comparisons between the Neb-Y, G96-Y, and LP97 samples yield F_ST values that do not differ significantly from zero, an observation that is consistent for the Adh, bib, and tim sequences (Fig 2). The absence of differentiation among these geographically distinct populations indicates that gene flow sufficiently homogenizes neutral sequence variation among separated populations of D. americana. This result has been observed previously for the transformer gene region in comparison of the G96 and LP97 populations (MCALLISTER and MCVEAN 2000 ).

In contrast, a high level of differentiation (F_ST = 0.22) is observed between neo-X and neo-Y sequences at the bib locus for the Neb population (Fig 2). Differentiation at bib between these neo-sex chromosomes is due exclusively to the unique composition of the neo-X chromosome. Neb-X exhibits highly significant differentiation at the bib locus relative to neo-X and neo-Y (data not shown) chromosomes in the G96 sample and unfused fourth chromosomes in the LP97 sample (Fig 2). These comparisons demonstrate that the bib region in the Neb-X population is unique relative to all the other analyzed populations of D. americana, yet the chromosomes contained in the sample were obtained from flies originating from two distinct geographic localities separated by 160 km (Fig 1B). Differentiation is nonexistent between neo-X chromosomes from the NN and DN localities (F_ST = -0.02). This analysis reveals the unique sequence composition of the bib gene region among neo-X chromosomes in the northwestern range of D. americana.

For the three loci analyzed, differentiation of the neo-X chromosomes in the Neb population is greatest at the bib locus and limited to the pericentromeric region. Comparisons of the Adh sequences mirror the observations at bib, but the level of differentiation is less pronounced. Sequences of Adh exhibit significant differentiation between Neb-X and Neb-Y (Fig 2), but the level of differentiation at Adh is approximately one-third the level observed for bib. Differentiation in the Adh gene region is, however, also due to specific change within the Neb-X population. No significant differentiation is observed at the tim gene (Fig 2). The bib and Adh regions are both located in the proximal region of the fourth chromosome and tim is located near the distal end, so differentiation appears to be localized within these neo-X chromosomes.

Observed differentiation between samples of DNA sequences is inflated by low within-sample nucleotide diversity (CHARLESWORTH 1998 ). Low within-sample sequence diversity for the Neb-X sample at the bib locus relative to the other samples (Table 1) only partially accounts for its high level of differentiation. Measures of F_ST at bib are highest in comparisons of the Neb-X sample to unfused fourth chromosomes (Neb-Y, G96-Y, and LP97) and 50% less in comparisons with G96-X (Fig 2), although sequence diversity is similar within all of the samples except Neb-X (Table 1). Net nucleotide divergence between Neb-X and the unfused fourth chromosomes is estimated at 5.02 substitutions (without correction) over the entire bib region, and 30% of this divergence is contributed by unique variants among neo-X chromosomes. Sequence comparisons, including the bib sequence of D. virilis as an outgroup (BURRIS et al. 1998 ), reveal one derived nucleotide substitution (see site 206 in the supplemental data at http://www.genetics.org/supplemental/) at high frequency (13/15) and another (site 814 in the supplemental data) at intermediate frequency (6/15) in the Neb-X sample. Site 206 defines a group of closely related haplotypes (a haplogroup) and creates a BbrPI recognition site that was investigated in more detail (see below). Both of these substitutions are present at low frequencies (4/18 and 3/18, respectively) in the G96-X sample and absent from the 33 unfused fourth chromosomes. Therefore, reduced variation in the Neb-X sample is not the sole cause of its differentiation, because two unique recent substitutions have risen to high frequency among these neo-X chromosomes.

Evidence of nonneutral evolution:
A likely cause of the low and highly differentiated sequence variation observed at the bib locus for the Neb-X sample is through hitchhiking resulting from a selective sweep within this chromosomal region. Statistics summarizing the frequency spectra of mutations suggest that a selective sweep may have affected variation in the bib gene solely for the neo-X chromosomes in the Nebraska population. The D_T statistic (TAJIMA 1989 ) summarizes the overall frequency spectrum of polymorphic sites, the D_FL statistic (FU and LI 1993 ) quantifies the frequency of mutations that occur externally within the allelic genealogy, whereas the H statistic (FAY and WU 2000 ) summarizes the frequency spectrum of derived nucleotide substitutions. These statistics have their most extreme negative values for samples of bib sequences for Neb-X (Table 2), but none of the values are sufficient to reject the neutral model at the = 0.05 level upon application of a two-tailed test. Interestingly, under the same criterion, the D_T statistic rejects the neutral model for the individual bib samples of NN-X and DN-X (see Table S1, supplemental data at http://www.genetics.org/supplemental/).

Strong haplotype structure is another feature expected as a result of hitchhiking. The Neb-X sample is unique in having low numbers of haplotypes and haplotype diversities for the Adh and bib gene regions (Table 1). Methods have been proposed for testing the significance of these haplotype measures (DEPAULIS and VEUILLE 1998 ; DEPAULIS et al. 2001 ); however, an intragenic recombination parameter is necessary to account for the generally high values obtained for D. americana, and estimating this parameter for the Neb-X sample is problematic. Therefore, tests of statistical significance for these low haplotype measures were not performed.

Another method for examining haplotype structure is identifying relationships among sequences, and the bib gene region exhibits clearly resolvable structure upon phylogenetic analysis. All phylogenetic methods applied recovered a single clade containing 13 of the 15 sequences from the Neb-X sample (Fig 3). A single derived synapomorphic nucleotide substitution (position 206, supplemental data) within intron 4 of the bib gene is the defining feature of this neo-X haplogroup. The parsimony analysis inferred other nucleotide substitutions within the lineage leading to the group; however, these substitutions are homoplasies within the shortest topologies obtained in the analysis and have apparently been redistributed among haplotypes through intragenic recombination. Within the cluster, 4 sequences are contained in a basal haplotype and two distinct haplotypes are defined by a single derived substitution each, one containing 6 sequences and the other containing 2. The remaining haplotype in the cluster contains three derived substitutions. This haplogroup is not limited to the Neb-X sample, because four neo-X chromosomes from the G96 sample are also contained in the clade (Fig 3). Interestingly, strong phylogenetic structure for the Neb-X sample is limited to the bib gene region and does not extend to the Adh region. Phylogenetic analysis revealed minimal clustering of the seven Adh haplotypes in the Neb-X sample, because the haplotypes are contained in five separate clades (results not shown).

View larger version (27K): In this window In a new window Download PPT slide   Figure 3. Relationships among big brain sequences of D. americana. The neo-X haplogroup is labeled, and the two sequences from the Neb-X sample but not contained in the cluster are indicated with asterisks. Inferred relationships are based on maximum parsimony, with this tree being one of eight identified topologies with the smallest number of steps.

Association between BbrPI⁺ and In(4)ab:
A derived nucleotide substitution at site 206 in the sequenced region of bib defines the neo-X haplogroup and creates a recognition site for the restriction endonuclease BbrPI. Previous analyses indicate that a chromosomal rearrangement on the fourth chromosome [In(4)ab] consisting of a paracentric inversion within a paracentric inversion is present in northwestern populations of D. americana (HSU 1952 ). The proximal breakpoint of this inversion falls at the border of subdivisions 48C/D, which is distal to the location of bib (at 48E); therefore, bib is not located within the inverted region, but is close to the proximal breakpoint. Because of the proximity of bib to the inversion breakpoint and the apparent geographic similarity between the BbrPI⁺ haplotype and In(4)ab, their association was investigated by screening isofemale lines derived from the same collections as the sequenced samples.

Amplification and digestion of bib revealed lines without the BbrPI⁺ haplotype, lines segregating for the BbrPI⁺ haplotype, and lines fixed for the BbrPI⁺ haplotype on fused X-4 chromosomes. Two lines from the LP97 sample and three lines from the G96 sample were identified that had only BbrPI^- alleles present (Table 3). The LP97 lines have unfused X chromosomes and autosomal fourth chromosomes, whereas the G96 lines contained only X-4 fusion chromosomes. Upon analysis of polytene chromosomes, these lines were identified as having the standard virilis arrangement of the fourth chromosome (GUBENKO and EVGEN'EV 1984 ).

Digestion of bib with BbrPI identified eight lines having females homozygous for the BbrPI⁺ haplotype (Table 3). Four of the lines are derived from the NN locality, three from DN, and one from G96. Males in each of the lines are heterozygous (BbrPI⁺/BbrPI^-), owing to the presence of the X-4 fusion arrangement with the BbrPI⁺ allele and the unfused fourth chromosome with the BbrPI^- allele. Inversion heterozygosity on the fourth chromosome was evident in polytene chromosomes prepared from all of the gonad-identified male larvae (Fig 4). Two inversion loops were present and the breakpoints correspond with the previously described a and b inversions on chromosome 4 (HSU 1952 ); thus this inversion apparently corresponds to In(4)ab. Polytene chromosomes prepared from female larvae from the lines were homokaryotypic for the In(4)ab arrangement. Homozygosity for the inverted arrangement in females and heterozygosity for the inverted and standard arrangements in males implies that In(4)ab is on X-4 fusion chromosomes (neo-X). In each of these eight lines, an association is observed between X-4 fusion chromosomes having the BbrPI⁺ haplotype and In(4)ab, whereas unfused fourth chromosomes have BbrPI^- haplotypes and the standard arrangement.

View larger version (177K): In this window In a new window Download PPT slide   Figure 4. Loop formation in polytene chromosomes of St(4)/In(4)ab heterokaryotype. Previous in situ localization has been performed for Adh and bib, and these positions are indicated. Inversion breakpoints for In(4)a are clearly visible, and inversion breakpoints for In(4)b are visible in the inset.

Two lines from the G96 sample were identified as segregating BbrPI⁺ and BbrPI^- alleles among X-4 fusion chromosomes. Heterozygous (BbrPI⁺/BbrPI^-) and homozygous (BbrPI^-/BbrPI^-) flies were identified in both genders. Only X-4 fusion chromosomes are present in the lines. The bib genotypes and chromosomal organization reveal that an X-4 fusion chromosome with the BbrPI⁺ haplotype and one with a BbrPI^- haplotype are segregating in the line. Crosses have been used to demonstrate that homozygosity for the BbrPI⁺ haplotype is lethal within both lines (data not shown), thus explaining why this genotype is not observed in females. Due to the complex series of genotypes within these two lines, genotype identification was obtained on larval carcasses from which salivary glands were prepared. A total of eight larvae were analyzed for each line. Direct association was observed between homozygous BbrPI^-/BbrPI^- larvae (G96.12, two larvae; G96.45, five larvae) being homokaryotypic for the standard fourth chromosome arrangement and heterozygous BbrPI⁺/BbrPI^- larvae (G96.12, six larvae; G96.45, three larvae) being heterokaryotypic for In(4)ab/St(4).

Overall, comparison among these lines demonstrates the strong association (P < 0.001, Fisher's exact test) between the BbrPI⁺ haplotype and the In(4)ab arrangement (Table 3). Although the bib gene is not located within the inverted region, the association is complete within these lines, and these two derived changes are limited to X-4 fusion chromosomes.

Recombination in heterokaryotypes:
On the basis of the sequencing and the genotyping, it appears that the neo-X haplogroup is limited to X-4 fusion chromosomes. With the widespread polymorphism for the centromeric fusion of the X and fourth chromosomes, heterokaryotypic females with an X-4 fusion chromosome and unfused X and fourth chromosomes (Fig 1A) occur frequently in natural populations (MCALLISTER 2001 , MCALLISTER 2002 ). For example, 4% of females are expected to be heterozygous for the alternative centromeric arrangements in the G96 population. These heterokaryotypic females are a potential source of recombination between fused and unfused fourth chromosomes, and they provide the opportunity to redistribute alleles between neo-X and neo-Y chromosomes on the basis of linkage to the centromere. Inversions are known to profoundly suppress recombination in heterokaryotypes (DOBZHANSKY and EPLING 1948 ; COYNE et al. 1991 ); therefore, the pattern of recombination was investigated within chromosomal heterozygotes for the centromeric arrangement and In(4)ab.

Chromosomally heterozygous females were obtained in crosses between males of the NN97.9 line, which contains the X-4 fusion having the In(4)ab arrangement, and ML97.4, which contains unfused X chromosomes with the standard arrangement. Potentially recombinant chromosomes were assayed for their centromeric arrangement. A total of 88 X-4 fusion chromosomes and 85 unfused fourth chromosomes were genotyped by restriction endonuclease digestion at Adh, bib, and tim. Direct assessment of these meiotic products revealed no recombinants within the interval including the centromere, Adh, and bib, whereas 12 (6.9%) recombinants occurred in the interval between bib and tim. On the basis of this assessment, the combination of the centromeric fusion and the inversion has a profound effect in suppressing recombination between fused and unfused fourth chromosomes in heterokaryotypic females. Recombination in the pericentromeric region, including Adh and bib, is low (<2%) and potentially nonexistent. In contrast, the interval between Adh and bib has an estimated recombination rate of 6% for homokaryotypic females with unfused fourth chromosomes having standard arrangements (based on 748 products; B. F. MCALLISTER, unpublished data). These results demonstrate low recombination between neo-X chromosomes with the In(4)ab arrangement and unfused fourth chromosomes in the region between the centromere and the proximal inversion breakpoint and, presumably, throughout the entire inverted region, which spans 60% of this chromosomal arm.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Identification of the forces driving the initial divergence between pairs of sex chromosomes is necessary to fully understand the process of sex chromosome evolution. This study demonstrates that differentiation of the neo-X chromosome in D. americana is occurring, but it is localized within the chromosome and also limited to a particular geographic region. Neo-X chromosomes from two separate northwestern populations are significantly differentiated at bib and Adh relative to other chromosomal populations. The level of differentiation at bib is, however, about three times greater than that observed for Adh. Patterns of variation in the sequence data are influenced by strong haplotype structure among the neo-X chromosomes within the Nebraska population of D. americana. The bib sequences for the Neb-X sample are represented by a single haplogroup containing 13 of 15 chromosomes. This haplogroup, which is defined by a derived recognition site for BbrPI, is shown to be strongly associated with the derived paracentric inversion In(4)ab. Due to the measured effect of this inversion on suppressing recombination between the neo-sex chromosomes, natural selection may have increased the frequency of this arrangement, resulting in a selective sweep, which would have affected nucleotide variation on the fourth chromosome.

Directional selection on In(4)ab is a plausible cause of the patterns observed in the sequence data. The bib locus is closer than the Adh locus to the proximal breakpoint of In(4)ab, and tim is located distant from the distal inversion breakpoint. Hitchhiking is strongest near the target of a selective sweep, and as recombination increases relative to the focal point of a sweep, variation reaches the neutral equilibrium (STEPHAN et al. 1992 ; KIM and STEPHAN 2000 ). Therefore, if In(4)ab represents or contains the target of a sweep, the effects of hitchhiking should be strongest at bib relative to the other sequenced regions. Although the bib gene is located outside the inversion, hitchhiking throughout the region would have been accentuated by suppressed recombination around the proximal inversion breakpoint (NAVARRO et al. 1996 , NAVARRO et al. 2000 ).

Consistent with expectations of a selective sweep of In(4)ab, haplotype structure is greatest in the region containing bib, which is revealed by the preservation of the relationships among haplotypes. Numbers of haplotypes and haplotype diversities are also low at bib and at Adh in the Neb-X sample relative to other samples. Recombination events between X-4 fusion chromosomes with the In(4)ab arrangement and X-4 fusion chromosomes with the standard arrangement apparently disassociated a common haplotype in Adh relative to the inversion and increased the haplotype diversity in this region. It is important to note that suppressed recombination between inverted and standard chromosomes by itself is insufficient to explain the patterns in the bib and Adh regions for the Neb-X sample, because neutral patterns are observed for unfused fourth chromosomes derived from the same collections, and these are expected to have a much lower recombination rate (due to the absence of crossing over in males). Interestingly, the putative sweep of In(4)ab did not strongly affect the frequency spectrum of substitutions at bib and left no sign in the frequency spectrum at Adh, but it apparently had a profound effect on differentiation at these loci, which is consistent with the prediction that distribution of variation among subpopulations is highly sensitive to selective sweeps (HILTON et al. 1994 ). Patterns resulting from hitchhiking may be even stronger at loci within the inverted region, which should be examined in future studies.

Current data are unable to resolve if the In(4)ab arrangement is fixed and if it is completely associated with the bib haplogroup within northwestern populations of D. americana. Fixation in this case concerns only neo-X chromosomes, so males would be heterozygous for fourth chromosome arrangements, because of the presence of the standard arrangement on unfused fourth chromosomes. Two neo-X chromosomes in the northwestern samples had bib sequences not contained within the neo-X haplogroup (Fig 3). These represent either recombinant chromosomes with ancestral bib haplotypes and In(4)ab or ancestral chromosomes with ancestral bib haplotypes and the standard arrangement. The former result is consistent with fixation of In(4)ab on neo-X chromosomes, whereas the latter is consistent with a polymorphism. Unfortunately, no ancestral bib haplotypes are segregating in the isofemale lines derived from the northwestern collections, which would provide material for determining if X-4 fusion chromosomes with ancestral bib haplotypes and the standard arrangement are present in northwestern samples. The analysis of HSU 1952 suggests that the In(4)ab arrangement is still segregating in northwestern populations. Combined chromosomal and sequence analyses of northwestern samples are needed to resolve this issue.

The In(4)ab arrangement and bib haplogroup are clearly limited to X-4 fusion chromosomes; thus these represent derived features for a subset of neo-X chromosomes. These derived neo-X chromosomes are at high frequency in northwestern populations. If this geographic distribution represents a local adaptation, the derived variant apparently does not benefit homozygous males because a beneficial allele should spread across a barrier imposed by a low rate of exchange (PIALEK and BARTON 1997 ). The derived neo-X haplogroup currently exists at low frequency in more eastern populations of D. americana. In the G96 sample, this type of neo-X chromosome, identified by bib sequence, represents 20% of the X-4 fusion chromosomes. The bib haplogroup, measured by restriction enzyme digestion [B205 locus, Table 4 in MCALLISTER 2002 ], has also been identified on 29% of X-4 fusion chromosomes in a sample of D. americana collected near St. Louis. These data on the bib haplotype correspond nicely with chromosomal arrangements described in a previous study of different localities in the same region. BLIGHT 1955 analyzed 1863 X and fourth chromosomes, and the In(4)ab arrangement was observed on 19% of X-4 fusion chromosomes and was not observed on 217 unfused fourth chromosomes. This derived neo-X chromosome may be increasing in frequency in eastern populations or it may be maintained as a stable or neutral polymorphism. Comparison of the studies performed near St. Louis suggests that it may be increasing in frequency.

The combination of the In(4)ab arrangement and the X-4 centromeric fusion suppresses recombination in heterokaryotypic females. Direct ascertainment of 173 chromosomes transmitted through heterokaryotypic females yielded no recombinants in the interval between the centromere and bib. Interestingly, BLIGHT 1955 reported that a single recombinant chromosome was generated in the lab, but it is unclear how many meiotic products were examined. Suppression of recombination in the proximal region is therefore very strong, and it presumably occurs throughout the entire interval bounded by the centromere and the distal inversion breakpoint, which represents 60% of the fourth chromosome. This suppression of recombination apparently maintains In(4)ab, the bib haplogroup, and other associated variations on X-4 fusion chromosomes, even in populations where opportunities for exchange between fused and unfused fourth chromosomes exist. Studies of samples collected around the St. Louis area estimate the frequency of fused X chromosomes at 85% (BLIGHT 1955 ; MCALLISTER 2002 ), so the predicted frequency of heterokaryotypes for the centromeric fusion is 25%. These females present ample opportunities for exchange between fused and unfused fourth chromosomes. Presence of the In(4)ab arrangement on the X-4 fusion chromosome would be an effective suppressor recombination between neo-X and neo-Y chromosomes over much of their length.

Overall, the association of the neo-X haplogroup and In(4)ab, coupled with the sequence data, suggests that natural selection may have rapidly increased the frequency of the inversion as a suppressor of recombination on the neo-X chromosome of D. americana. This finding is important for studies of sex chromosome evolution, because it is consistent with the rapid emergence of a selective premium to suppress recombination between these neo-X and neo-Y chromosomes. An interesting aspect of this system is that the inversion arose on the neo-X chromosome, whereas a series of inversions on the mammalian Y chromosome apparently led to its successive isolation from the X (LAHN and PAGE 1999 ). Due to the dynamic nature of the neo-sex chromosome system in D. americana, the opportunity exists for examining the progressive fixation of this suppressor of recombination and identifying the underlying cause of the premium for suppressing recombination between evolving sex chromosomes. Theory predicts that antagonism drives the accumulation of recombination suppressors (BULL 1983 ). Experimental systems reveal an abundance of sexually antagonistic genetic variation (RICE 1992 ; GIBSON et al. 2002 ), and In(4)ab may protect a sexually antagonistic allele on the neo-X chromosome of D. americana. Experiments designed to identify sexually antagonistic variation on these neo-sex chromosomes of D. americana will provide a critical test of this hypothesis.

	FOOTNOTES

Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. AY340278, AY340279, AY340280, AY340281, AY340282, AY340283, AY340284, AY340285, AY340286, AY340287, AY340288, AY340289, AY340290, AY340291, AY340292, AY340293, AY340294, AY340295, AY340296, AY340297, AY340298, AY340299, AY340300, AY340301, AY340302, AY340303, AY340304, AY340305, AY340306, AY340307 (Adh), AY339897, AY339898, AY339899, AY339900, AY339901, AY339902, AY339903, AY339904, AY339905, AY339906, AY339907, AY339908, AY339909, AY339910, AY339911, AY339912, AY339913, AY339914, AY339915, AY339916, AY339917, AY339918, AY339919, AY339920, AY339921, AY339922, AY339923, AY339924, AY339925, AY339926, AY339927, AY339928, AY339929, AY339930, AY339931, AY339932, AY339933, AY339934, AY339935, AY339936, AY339937, AY339938, AY339939, AY339940, AY339941, AY339942, AY339943, AY339944, AY339945, AY339946, AY339947, AY339948, AY339949, AY339950, AY339951, AY339952, AY339953, AY339954, AY339955, AY339956, AY339957, AY339958, AY339959, AY339960, AY339961, AY339962, AY339963, AY339964, AY339965, AY339966, AY339967, AY339968, AY339969, AY339970, AY339971, AY339972 (bib), and AY340308, AY340309, AY340310, AY340311, AY340312, AY340313, AY340314, AY340315, AY340316, AY340317, AY340318, AY340319, AY340320, AY340321, AY340322, AY340323, AY340324, AY340325, AY340326, AY340327, AY340328, AY340329, AY340330, AY340331, AY340332, AY340333, AY340334, AY340335, AY340336, AY340337, AY340338, AY340339, AY340340, AY340341, AY340342, AY340343, AY340344, AY340345, AY340346, AY340347, AY340348, AY340349, AY340350, AY340351, AY340352, AY340353, AY340354, AY340355, AY340356, AY340357, AY340358, AY340359, AY340360, AY340361, AY340362, AY340363, AY340364, AY340365, AY340366, AY340367, AY340368, AY340369, AY340370, AY340371, AY340372, AY340373, AY340374, AY340375, AY340376, AY340377, AY340378, AY340379, AY340380, AY340381 (tim).

	ACKNOWLEDGMENTS

Thanks go to B. Charlesworth, D. Charlesworth, J. A. Marshall, S. Schaeffer, and anonymous reviewers for comments on previous versions of the manuscript. Technical assistance was provided by A. Long, E. Mancl, M. Murphy, and P. Mena. This article is based upon work supported by the National Science Foundation under grant nos. DEB-0075295/0228832.

Manuscript received May 8, 2003; Accepted for publication July 25, 2003.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

ATANASSOV, I., C. DELICHÉRE, D. A. FILATOV, D. CHARLESWORTH, and I. NEGRUTIU et al., 2001  Analysis and evolution of two functional Y-linked loci in a plant sex chromosome system. Mol. Biol. Evol. 18:2162-2168.[Abstract/Free Full Text]

BACHTROG, D., 2003  Adaptation shapes patterns of genome evolution on sexual and asexual chromosomes in Drosophila.. Nat. Genet. 34:215-219.[Medline]

BACHTROG, D. and B. CHARLESWORTH, 2002  Reduced adaptation of a non-recombining neo-Y chromosome. Nature 416:323-326.[Medline]

BLIGHT, W. C., 1955 A cytological study of linear populations of Drosophila americana near St. Louis, Missouri. Ph.D. Dissertation, Washington University, St. Louis.

BULL, J. J., 1983 Evolution of Sex Determining Mechanisms. Benjamin/Cummings, Menlo Park, CA.

BURRIS, P. A., Y. ZHANG, J. C. RUSCONI, and V. CORBIN, 1998  The pore-forming and cytoplasmic domains of the neurogenic gene product, BIG BRAIN, are conserved between Drosophila virilis and Drosophila melanogaster.. Gene 206:69-76.[Medline]

CHARLESWORTH, B., 1991  The evolution of sex chromosomes. Science 251:1030-1033.[Abstract/Free Full Text]

CHARLESWORTH, B., 1996  The evolution of chromosomal sex determination and dosage compensation. Curr. Biol. 6:149-162.[Medline]

CHARLESWORTH, B., 1998  Measures of divergence between populations and the effect of forces that reduce variability. Mol. Biol. Evol. 15:538-543.[Abstract]

CHARLESWORTH, B. and D. CHARLESWORTH, 2000  The degeneration of Y chromosomes. Philos. Trans. R. Soc. Lond. B 355:1563-1572.[Abstract/Free Full Text]

CHARLESWORTH, B., M. T. MORGAN, and D. CHARLESWORTH, 1993  The effect of deleterious mutations on neutral molecular variation. Genetics 134:1289-1303.[Abstract]

CHARLESWORTH, D., 2002  Plant sex determination and sex chromosomes. Heredity 88:94-101.[Medline]

COYNE, J. A., S. AULARD, and A. BERRY, 1991  Lack of underdominance in a naturally occurring pericentric inversion in Drosophila melanogaster and its implications for chromosome evolution. Genetics 129:791-802.[Abstract]

DEPAULIS, F. and M. VEUILLE, 1998  Neutrality tests based on the distribution of haplotypes under an infinite-site model. Mol. Biol. Evol. 15:1788-1790.[Medline]

DEPAULIS, F., S. MOUSSET, and M. VEUILLE, 2001  Haplotype tests using coalescent simulations conditional on the number of segregating sites. Mol. Biol. Evol. 18:1136-1138.[Free Full Text]

DOBZHANSKY, T. and C. EPLING, 1948  The suppression of crossing over in inversion heterozygotes of Drosophila pseudoobscura.. Proc. Natl. Acad. Sci. USA 34:137-141.[Free Full Text]

FAY, J. C. and C.-I WU, 2000  Hitchhiking under positive Darwinian selection. Genetics 155:1405-1413.[Abstract/Free Full Text]

FU, Y.-X. and W.-H. LI, 1993  Statistical tests of neutrality of mutations. Genetics 133:693-709.[Abstract]

GIBSON, J. R., A. K. CHIPPINDALE, and W. R. RICE, 2002  The X chromosome is a hot spot for sexually antagonistic fitness variation. Proc. R. Soc. Lond. Ser. B 269:499-505.[Medline]

GORDO, I. and B. CHARLESWORTH, 2001  The speed of Muller's ratchet with background selection, and the degeneration of Y chromosomes. Genet. Res. 78:149-161.[Medline]

GRAVES, J. A. M. and S. SHETTY, 2001  Sex from W to Z: evolution of vertebrate sex chromosomes and sex determining genes. J. Exp. Zool. 290:449-462.[Medline]

GUBENKO, I. S. and M. B. EVGEN'EV, 1984  Cytological and linkage maps of Drosophila virilis chromosomes. Genetica 65:127-139.

HEY, J. and J. WAKELEY, 1997  A coalescent estimator of the population recombination rate. Genetics 145:833-846.[Abstract]

HILTON, H. and J. HEY, 1996  DNA sequence variation at the period locus reveals the history of species and speciation events in the Drosophila virilis group. Genetics 144:1015-1025.[Abstract]

HILTON, H. and J. HEY, 1997  A multilocus view of speciation in the Drosophila virilis species group reveals complex histories and taxonomic conflicts. Genet. Res. 70:185-194.

HILTON, H., R. M. KLIMAN, and J. HEY, 1994  Using hitchhiking genes to study adaptation and divergence during speciation within the Drosophila melanogaster species complex. Evolution 48:1900-1913.

HSU, T. C., 1952  Chromosomal variation and evolution in the virilis group of Drosophila. Univ. Tex. Publ. 5204:35-72.

HUDSON, R. R., M. KREITMAN, and M. AGUADÉ, 1987  A test of neutral molecular evolution based on nucleotide data. Genetics 116:153-159.[Abstract/Free Full Text]

HUDSON, R. R., D. D. BOOS, and N. L. KAPLAN, 1992  A statistical test for detecting geographic subdivision. Mol. Biol. Evol. 9:138-151.[Abstract]

KENNISON, J. A., 2000 Preparation and analysis of polytene chromosomes, pp. 111–117 in Drosophila Protocols, edited by W. SULLIVAN, M. ASHBURNER and R. S. HAWLEY. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

KIKKAWA, H., 1935  Crossing-over in the male of Drosophila virilis.. Cytologia 6:190-194.

KIM, Y. and W. STEPHAN, 2000  Joint effects of genetic hitchhiking and background selection on neutral variation. Genetics 155:1415-1427.[Abstract/Free Full Text]

LAHN, B. T. and D. C. PAGE, 1999  Four evolutionary strata on the human X chromosome. Science 286:964-967.[Abstract/Free Full Text]

MCALLISTER, B. F., 2001  Genetic analysis of sex-chromosome arrangement in Drosophila americana: a laboratory exercise for undergraduate or advanced placement students. Dros. Inf. Serv. 84:227-234.

MCALLISTER, B. F., 2002  Chromosomal and allelic variation in Drosophila americana: selective maintenance of a chromosomal cline. Genome 45:13-21.[Medline]

MCALLISTER, B. F. and B. CHARLESWORTH, 1999  Reduced sequence variability on the neo-Y chromosome of Drosophila americana americana.. Genetics 153:221-233.[Abstract/Free Full Text]

MCALLISTER, B. F. and G. A. T. MCVEAN, 2000  Neutral evolution of the sex-determining gene transformer in Drosophila. Genetics 154:1711-1720.[Abstract/Free Full Text]

MOORE, R. C., O. KOZYREVA, S. LEVEL-HARDENACK, J. SIROKY, and R. HOBZA et al., 2003  Genetic and functional analysis of DD44, a sex-linked gene from the dioecious plant Silene latifolia, provides clues to early events in sex chromosome evolution. Genetics 163:321-334.[Abstract/Free Full Text]

MYERS, M. P., A. ROTHENFLUH, M. CHANG, and M. W. YOUNG, 1997  Comparison of chromosomal DNA composing timeless in Drosophila melanogaster and D. virilis suggests a new conserved structure for the TIMELESS protein. Nucleic Acids Res. 25:4710-4714.[Abstract/Free Full Text]

NAVARRO, A., E. BETRÁN, C. ZAPATA, and A. RUIZ, 1996  Dynamics of gametic disequilibria between loci linked to chromosome inversions: the recombination-redistributing effect of inversions. Genet. Res. 67:67-76.[Medline]

NAVARRO, A., A. BARBADILLA, and A. RUIZ, 2000  Effect of inversion polymorphism on the neutral variability of linked chromosomal regions in Drosophila. Genetics 155:685-698.[Abstract/Free Full Text]

NURMINSKY, D. I., E. N. MORIYAMA, E. R. LOZOVSKAYA, and D. L. HARTL, 1996  Molecular phylogeny and genome evolution in the Drosophila virilis species group: duplications of the Alcohol dehydrogenase gene. Mol. Biol. Evol. 13:132-149.[Abstract]

ORR, H. A. and Y. KIM, 1998  An adaptive hypothesis for the evolution of the Y chromosome. Genetics 150:1693-1698.[Abstract/Free Full Text]

OUSLEY, A., K. ZAFARULLAH, Y. CHEN, M. EMERSON, and L. HICKMAN et al., 1998  Conserved regions of the timeless (tim) clock gene in Drosophila analyzed through phylogenetic and functional studies. Genetics 148:815-825.[Abstract/Free Full Text]

PIÁLEK, J. and N. H. BARTON, 1997  The spread of an advantageous allele across a barrier: the effects of random drift and selection against heterozygotes. Genetics 145:493-504.[Abstract]

RICE, W. R., 1984  Sex chromosomes and the evolution of sexual dimorphism. Evolution 38:735-742.

RICE, W. R., 1987  Genetic hitchhiking and the evolution of reduced genetic activity of the Y sex chromosome. Genetics 116:161-167.[Abstract/Free Full Text]

RICE, W. R., 1992  Sexually antagonistic genes: experimental evidence. Science 256:1436-1439.[Abstract/Free Full Text]

RICE, W. R., 1996  Evolution of the Y sex chromosome in animals. Bioscience 46:331-343.

RICE, W. R. and A. K. CHIPPINDALE, 2001  Intersexual ontogenetic conflict. J. Evol. Biol. 14:685-693.

ROZAS, J. and R. ROZAS, 1999  DnaSP version 3: an integrated program for molecular population genetics and molecular evolution analysis. Bioinformatics 15:174-175.[Abstract/Free Full Text]

SCHLÖTTERER, C., 2000  Microsatellite analysis indicates genetic differentiation of the neo-sex chromosomes in Drosophila americana americana.. Heredity 85:610-616.[Medline]

STEINEMANN, M. and S. STEINEMANN, 1998  Enigma of Y chromosome degeneration: Neo-Y and Neo-X chromosomes of Drosophila miranda a model for sex chromosome evolution. Genetica 102(103):409-420.

STEPHAN, W., T. H. E. WIEHE, and M. W. ZENZ, 1992  The effect of strongly selected substitutions on neutral polymorphism: analytical results based on diffusion theory. Theor. Popul. Biol. 41:237-254.

SWOFFORD, D. L., 2002 Phylogenetic Analysis Using Parsimony* (*and Other Methods). Sinauer Associates, Sunderland, MA.

TAJIMA, F., 1983  Evolutionary relationship of DNA sequences in finite populations. Genetics 105:437-460.[Abstract/Free Full Text]

TAJIMA, F., 1989  Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123:585-595.[Abstract/Free Full Text]

THROCKMORTON, L. H., 1982 The virilis species group, pp. 227–296 in The Genetics and Biology of Drosophila, Vol. 3b, edited by M. ASHBURNER, J. L. CARSON and J. N. THOMPSON. Academic Press, New York.

VIEIRA, J., B. F. MCALLISTER, and B. CHARLESWORTH, 2001  Evidence for selection at the fused1 locus of Drosophila americana.. Genetics 158:279-290.[Abstract/Free Full Text]

WATTERSON, G. A., 1975  On the number of segregating sites in genetical models without recombination. Theor. Popul. Biol. 7:256-276.[Medline]

WHITING, J. H., JR., M. D. PLILEY, J. L. FARMER, and D. E. JEFFERY, 1989  In situ hybridization analysis of chromosomal homologies in Drosophila melanogaster and Drosophila virilis.. Genetics 122:99-109.[Abstract/Free Full Text]

This article has been cited by other articles:

				C. Lemaitre, M. D. V. Braga, C. Gautier, M.-F. Sagot, E. Tannier, and G. A. B. Marais Footprints of Inversions at Present and Past Pseudoautosomal Boundaries in Human Sex Chromosomes Gen Biol Evol, March 1, 2010; 2009(0): 56 - 66. [Abstract] [Full Text] [PDF]

				S. W. Schaeffer, A. Bhutkar, B. F. McAllister, M. Matsuda, L. M. Matzkin, P. M. O'Grady, C. Rohde, V. L. S. Valente, M. Aguade, W. W. Anderson, et al. Polytene Chromosomal Maps of 11 Drosophila Species: The Order of Genomic Scaffolds Inferred From Genetic and Physical Maps Genetics, July 1, 2008; 179(3): 1601 - 1655. [Abstract] [Full Text] [PDF]

				A. L. Evans, P. A. Mena, and B. F. McAllister Positive Selection Near an Inversion Breakpoint on the Neo-X Chromosome of Drosophila americana Genetics, November 1, 2007; 177(3): 1303 - 1319. [Abstract] [Full Text] [PDF]

				X. Maside and B. Charlesworth Patterns of Molecular Variation and Evolution in Drosophila americana and Its Relatives Genetics, August 1, 2007; 176(4): 2293 - 2305. [Abstract] [Full Text] [PDF]