*Department Biologie IIEvolutionsbiologie, Universität München, Germany;
Department of Biology, University of Rochester, New York
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Abstract |
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Introduction |
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In addition to its crucial role in the determination of anterior-posterior polarity in the fruit fly, two aspects of bcd make it an interesting candidate for evolutionary analysis. First, recent studies in Drosophila and closely related insects suggest that bcd, a derived Hox3 homologue, may be unique in insect developmental systems in terms of function and evolutionary history (Stauber, Jäckle, and Schmidt-Ott 1999
). Numerous laboratories have consistently failed in attempts to isolate bcd from insects other than Cyclorrhaphan flies, despite the usual ease in cloning homologues of other developmental genes from distantly related species (Stauber, Jäckle, and Schmidt-Ott 1999
). One possibility is that bcd is a rapidly evolving homeobox gene, and failure to clone it is the result of technical difficulties (Schröder and Sander 1993
; Patel 2000
). However, when bcd was cloned from a basal Cyclorrhaphan fly (Megaselia abdita), it was found to be most closely related to the Megaselia zerknüllt (zen) gene, suggesting that bcd may be the result of a gene duplication and diversification, leading to a novel regulatory protein (Stauber, Jäckle, and Schmidt-Ott 1999
). Most recently, a single Hox3 homologue (more similar to D. melanogaster zen than bcd) having expression patterns characteristic of both zen and bcd was identified in three non-Cyclorrhaphan flies (Stauber, Prell, and Schmidt-Ott 2002
). Finally, Schaeffer et al. (2000)
found functional redundancy between Bicoid and the terminal system's role in thorax development, supporting the recent evolution of an anterior morphogenetic center comprised of both Bicoid and the terminal system. The second interesting feature of bcd is the presence of a large, conserved secondary structure in the 3' UTR. This makes the bcd gene a good candidate for studying compensatory evolution and the relationship between RNA secondary structure and patterns of standing variation in natural populations (Chen et al. 1999
).
Despite these intriguing observations, a population-level analysis has until now not been performed on bcd. In this study, DNA sequence variation was examined for a 4-kb region of the bcd gene, including a portion of the 5' UTR, the entire coding region, and the 3' UTR, for 25 D. melanogaster isofemale lines from Lake Kariba, Zimbabwe. This population was chosen because it has previously been shown to harbor more than twice the amount of genetic variation and lower levels of linkage disequilibrium than non-African populations of D. melanogaster (Begun and Aquadro 1993
, 1995a,
1995b
). Thus, Zimbabwe likely represents an ancestral population closer to mutation-drift equilibrium, enabling the selective forces determining DNA sequence variation to be more easily elucidated (David and Capy 1988
; Begun and Aquadro 1995b
). The goals of this study are (1) to test whether there is evidence for natural selection attributable to the relatively recent origin of bcd in the Dipteran lineage, and (2) to test whether the pattern of variation in the 3' UTR is consistent with the presence of a large conserved mRNA secondary structure.
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Materials and Methods |
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DNA Extraction, PCR Amplification, and Direct Sequencing of the bcd Alleles
Genomic DNA was extracted from homozygous whole flies with the DNeasy tissue kit (Qiagen). Oligonucleotides for amplification and direct sequencing were designed based on a previously published D. melanogaster bcd sequence (GenBank accession number X07870). These primers were used in PCR reactions to amplify a 4-kb region of bcd, comprising 450 bp of 5' flanking region, the entire coding region, and 1 kb of 3' flanking region. PCR products were purified with QIAquick columns (Qiagen), and both strands were subsequently sequenced using primers spaced 400 bp apart. Sequencing was performed on an ABI377 automated sequencer with the Dye Terminator chemistry (Perkin-Elmer). The homologous region of D. simulans was amplified using the PCR primers designed for D. melanogaster, and new D. simulans primers were designed based on the available D. simulans sequence and used if the D. melanogaster primers failed in the sequencing reactions because of mismatches. The D. melanogaster sequences are deposited in GenBank as a population set with accession numbers AF46662145, and the accession number of the D. simulans sequence is AF465792. The coordinates according to the reference sequence (GenBank accession X07870) are used throughout this paper.
Sequence Analysis
Sequences were assembled and aligned with the SeqEd program (Perkin-Elmer), and all variable sites were checked manually and verified in both strands. The bcd gene of D. melanogaster is alternatively spliced at intron 2 (positions 22162270, 55 bp; or positions 22162255, 40 bp). Because smaller introns (<51 bp) are usually spliced less efficiently (Mount et al. 1992
), the assignment of coding and noncoding regions are according to the major transcript; i.e., positions 22562270 are regarded as noncoding region. The homologous region of D. simulans was aligned to the D. melanogaster bcd sequence, and gaps in the alignment were not used in the sequence analysis. After the sequence alignment, the coding and noncoding regions of D. simulans bcd were assigned according to the D. melanogaster sequence. The DnaSP program version 3.50 (Rozas J and Rozas R 1999
) was used for most intraspecific and interspecific analyses. Nucleotide diversity,
, was estimated according to Watterson (1975)
and
according to Nei (1987, p. 256)
. Nucleotide divergence,
, between D. melanogaster and both D. simulans and D. pseudoobscura (GenBank accession number X55735) was estimated according to Nei (1987, p. 65)
.
The following neutrality tests were performed using the program DnaSP (Rozas J and Rozas R 1999
): The HKA test (Hudson, Kreitman, and Aguadé 1987
), Tajima's (1989)
test, and the McDonald and Kreitman (1991)
test. The probabilities for the McDonald and Kreitman (1991)
test were obtained by both the two-tailed Fisher's exact test and the G-test. To detect heterogeneity in the ratio of polymorphism to divergence in the region surveyed, the program DNA slider (McDonald 1996
, 1998
) was used.
Coalescent simulations for obtaining the probabilities of the number of haplotypes and haplotype diversity (Depaulis and Veuille 1998
) were performed using the program DnaSP (Rozas J and Rozas R 1999
), and the haplotype test of Hudson et al. (1994)
was performed using a program written by J. Braverman (kindly provided by J. Parsch). The test of Hudson et al. (1994)
determines the probability of observing a subset of alleles of size i with j or fewer segregating sites, given an overall sample of n alleles with S segregating sites. The recombination parameter, R, was estimated by three methods, including two based on polymorphism data (Hudson 1987
; Hey and Wakeley 1997
) and one determined from experimental laboratory crosses (Comeron, Kreitman, and Aguadé 1999
). The program of Comeron, Kreitman, and Aguadé (1999)
(kindly provided by J. Comeron) estimates recombination rates in D. melanogaster based on cytological map position using polynomial curves (Kliman and Hey 1993
) as a function of the amount of DNA in each chromosomal division versus the change in cytological map position (Sorsa 1988
). For the coalescent simulations, 10,000 replicates were performed for each test.
Inversion Analysis
Larvae were grown in yeast-rich corn meal-sugar media at 18°C without larval crowding. Late third instar larvae were dissected in a drop of insect Ringer's solution. Both salivary glands were placed under a coverslip with a drop of lacto-aceto-orcein stain. After 5 min, the glands were squashed, and polytene chromosomes were analyzed under a phase contrast microscope at 100x. Inversion break points were determined according to the photographic map of Lefevre (1976)
.
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Results |
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A second test of neutrality, the HKA test (Hudson, Kreitman, and Aguadé 1987
), examines the prediction of the neutral mutation hypothesis that levels of intraspecific polymorphism are positively correlated with levels of interspecific divergence. We tested six possible pairwise comparisons using 5' UTR, intron 1, intron 3, and the 3' UTR. Though none of these tests were significant, comparisons between the 5' and 3' UTR (P = 0.065) and between intron 3 and the 3' UTR (P = 0.053) approach significance (table 2
). Comparisons between both the 5' UTR and intron 3 with intron 1 also give small P values of around 0.1. These may suggest lower levels of polymorphism in 5' UTR and intron 3 when compared with the two other reference noncoding loci and heterogeneity in the ratio of polymorphism to divergence along the bcd DNA sequence. However, the HKA test was applied somewhat post hoc, so the results should be interpreted with caution.
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Another noteworthy observation is the distribution of both replacement fixed differences and polymorphisms along the Bicoid protein (fig. 2
). All six replacement polymorphisms and five of six replacement fixed differences cluster within one of two regions of the protein. The first region (amino acids 249332, corresponding to positions 27653025 of the reference sequence) contains a cluster of three polymorphisms and four fixed differences and overlaps opa-like repeats and portions of the protein with no known function (i.e., linker or hinge regions) (Seeger and Kaufman 1990
). The second region (amino acids 433455, corresponding to positions 38393907 of the reference sequence) contains a cluster of three polymorphisms and one fixed difference and overlaps a region that may contain an RNA recognition motif (Seeger and Kaufman 1990
). In an interspecific comparison of the D. melanogaster sequence with D. pseudoobscura, a sliding window analysis of divergence at replacement sites revealed a peak of divergence in this region (fig. 3
), and the ratio of replacement to silent substitutions,
a/
s, was nearly four times greater (
a/
s = 0.554) than the value for the entire coding region (
a/
s = 0.139).
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Given the pattern of linkage disequilibrium and significant structuring of haplotypes, we further investigated our data in the context of two possible explanations: associations with polymorphic inversions or RNA secondary structure (or both). First, because D. melanogaster is known to be polymorphic for well over 300 inversions (Das and Singh 1991
; Lemeunier and Aulard 1992
), four of which are cosmopolitan and reach appreciable frequencies, we investigated whether certain haplotype classes (i.e., H1 through H4) may be associated with chromosomal inversion types. Though the cosmopolitan inversion In(3R)P (breakpoints 89C to 96A) was found segregating at approximately 14%, there is no association between this inversion and haplotype classes, and no inversions associated with the cytological interval containing bcd (84A5) were detected.
Second, we analyzed linkage disequilibrium with respect to the large, conserved secondary structural element in the 3' UTR (Macdonald 1990
). This structure plays an essential role in the localization of bcd mRNA and has been well characterized by both mutational (Ferrandon et al. 1997
; Macdonald and Kerr 1998
) and phylogenetic analyses (Parsch, Braverman, and Stephan 2000
). However, there seems to be no obvious association between the observed haplotype structure and the predicted mRNA secondary structure in the 3' UTR (further discussed subsequently).
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Discussion |
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Evidence for Relaxed Purifying Selection
A significant excess of replacement polymorphisms is usually interpreted as a relaxation of selective constraints. The effect of purifying selection may be weaker on some amino acid replacement mutations than others, and slightly deleterious polymorphisms may persist at low frequencies within a population for a period of time because of genetic drift but are unlikely to either rise in frequency or become fixed (Kimura 1983, p. 44
; Ohta 1992
). Under this scenario, slightly deleterious mutations contribute more to intraspecific polymorphism than to interspecific fixed differences (Kimura 1983
; Ohta 1992
). Several other studies have reported similar cases of an excess of intraspecific replacement polymorphism in mitochondrial DNA (Ballard and Kreitman 1994
; Nachman, Boyer, and Aquadro 1994
; Nachman et al. 1996
; Rand and Kann 1996
; Wise, Sraml, and Easteal 1998
) and in one case a nuclear gene, Pgm (Verrelli and Eanes 2000
, 2001
), and interpretation has ranged from that of slightly deleterious mutations to positive selection.
At least some of our observed amino acid polymorphisms at the bcd locus appear to be slightly deleterious. All the polymorphisms are at relatively low frequency (4%16%), three of which are singletons; all of them are derived. Three of these polymorphisms also cause drastic changes in amino acid property (table 4
). It is usually hard to imagine that replacement polymorphisms, especially those that drastically change encoded amino acid properties, are only slightly deleterious. Therefore, we investigated the protein regions where the replacement polymorphisms are found. The three found in exon 3 are located within an opa-like repeat region and regions with no known function (possibly linker or hinge regions in the polypeptide chain) (Seeger and Kaufman 1990
). Therefore, despite the changes in amino acid property, their effect could be minimal because of the functional insignificance of their location. The other three replacement polymorphisms are located in exon 4, within a region containing a putative, but ill-characterized RNA recognition motif (Seeger and Kaufman 1990
). Sliding window analysis of divergence between D. melanogaster and D. pseudoobscura shows that this region overlaps with a peak in both divergence and the replacement to silent substitution ratio (
a/
s = 0.554) (fig. 3
).
a/
s ratios are usually kept low by purifying selection (
a/
s = 0.139 for the entire coding region). The rise in
a/
s ratio may suggest that this region is under less constraint than other parts of the molecule, and the replacement polymorphisms (sites 3839, 3899, and 3905) found here are caused by a relaxation of purifying selection.
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If bcd arose through tandem duplication from zen, then it would initially have a function very similar to that of zen. This would allow deleterious mutations to rise in frequency without being eliminated by purifying selection. At the same time, positively selected mutations may also arise, go to fixation, and thus give bcd new functions. However, the functions of bcd and zen may still be similar enough that adaptive sweeps and relaxed selection in localized regions of the gene occur simultaneously. In the following, we discuss the evidence that, in addition to the relaxed purifying selection, positive selection has also occurred at bcd or at near sites. This evidence is for the most part based on the other two observations, namely the extensive haplotype structure and the apparent heterogeneity in the polymorphism-to-divergence ratio along the gene.
Evidence for Positive Selection
First, we observed extensive linkage disequilibria and distinct haplotype structure in our sample of bcd alleles, and positive natural selection is likely involved in maintaining this structure. We found a significantly smaller number of haplotypes than the neutral expectation (P = 0.032) by the Depaulis and Veuille (1998)
test of haplotypes. Coalescent simulations with the most conservative value of the recombination parameter, R, gave the range for the number of haplotypes as [13, 22], and we observed 13 haplotypes in our sample. It appears that the variants at the polymorphic sites structure into a few haplotypes. Because the Zimbabwe population typically exhibits less linkage disequilibria than non-African populations and is thought to be a panmictic population close to mutation-drift equilibrium (Begun and Aquadro 1993
), demographic and bottleneck effects should be minimal. Thus, it seems likely that balancing selection or partial selective sweeps of haplotypes are contributing to this pattern. In the latter model, variants are positively selected for, but fail to go to fixation because of "traffic" with haplotypes where selection is acting on other sites (Kirby and Stephan 1996
).
In particular, the H4 haplotype appears to be the target of positive selection operating at or near the bcd locus. This haplotype has a frequency of 28% and has no within-class variation. By applying a statistical test for high-frequency haplotypes (Hudson et al. 1994
), our results show that there is too little variation within the H4 class, given the level of variation in the rest of the sample, and cannot be explained by a neutral equilibrium model of mutation and drift. This pattern suggests that this haplotype has arisen recently and is being pulled to high frequency because of directional selection at or near the region we surveyed.
A second phenomenon possibly attributable to positive selection is the heterogeneity in the ratio of polymorphism to divergence along the bcd region. The /
ratio is high in the intron 1 region (0.0949), whereas it is low in the coding region (0.0025) (table 1 ), which is suggestive of balancing selection on sites within intron 1 or a selective sweep (or both) that occurred in the region of exon 2 to exon 4. Comparisons between gene regions involving intron 1 by the HKA test approach significance (table 2 ). In addition, the run's test (McDonald 1996
, 1998
), a measure of heterogeneity, produces a marginally significant result (P
0.05). However, the results of these tests alone do not put forth convincing evidence for such underlying selective mechanisms. It is difficult to distinguish this pattern of heterogeneity from merely neutral fluctuations of polymorphisms along the gene region (Kim and Stephan 2002
).
Haplotype Structure and mRNA Secondary Structure in the bcd 3' UTR
The cis-acting sequences necessary for the localization of bcd mRNA fall within a large, phylogenetically conserved and well-characterized secondary structural element in the 3' UTR (Macdonald and Struhl 1988
; Macdonald 1990
; Seeger and Kaufman 1990
; Ferrandon et al. 1997
; Macdonald and Kerr 1998
; Parsch, Braverman, and Stephan 2000
). We investigated whether the observed linkage disequilibrium and haplotype structure is related to the maintenance of the mRNA secondary structure in the 3' UTR.
Of the 40 nucleotide polymorphisms observed within the entire bcd gene region, nine fall within the region of the localization signal in the 3' UTR, and only two (A4350C and G4612A) are located within the pairing regions supported by both phylogenetic study (Parsch, Braverman, and Stephan 2000
) and mutational analysis (Ferrandon et al. 1997
; Macdonald and Kerr 1998
). Both these substitutions cause mismatches in the original Watson-Crick pair of the mRNA secondary structure. No covariations (compensatory mutations) with the pairing regions were observed in our sample of the Zimbabwe population. This is in qualitative agreement with the theoretical results developed under a two-locus, two-allele, reversible mutation-compensatory model (Innan and Stephan 2001
). According to the predictions of this model, the populations spend most of the time in the first stage of waiting for a successful double mutant to appear in the population. The second stage of fixing the successful double mutant in the population is much shorter than the first stage (Innan and Stephan 2001
). Their simulations showed that almost no linkage disequilibrium caused by compensatory interactions is expected during the first stage, so it is unlikely to observe much linkage disequilibrium or covariations caused by epistatic selection on mRNA secondary structures. Thus, the strong haplotype pattern and linkage disequilibria of our data set cannot be explained by epistatic selection on the bcd 3' UTR.
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Conclusion |
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Acknowledgements |
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Footnotes |
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1 Contributed equally to this work
Keywords: replacement polymorphism
haplotype structure
gene duplication
biciod
Drosophila melanogaster
Address for correspondence and reprints: Wolfgang Stephan, Department Biologie IIEvolutionsbiologie, Universität München, Luisenstr. 14, 80333 München, Germany. stephan{at}zi.biologie.uni-muenchen.de
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