*Institute of Cell, Animal and Population Biology, University of Edinburgh;
Departamento de Genética Molecular, Instituto de Biologia Molecular e Celular, Porto, Portugal
Recent work suggests that fused (fu), a segment-polarity gene that encodes a putative serine-threonine kinase (Preat et al. 1990
), has undergone episodes of directional selection in three species of the virilis group of Drosophila (in D. virilis [Vieira and Charlesworth 1999, 2000
], D. a. americana [Vieira, McAllister, and Charlesworth 2001
], and D. montana [Vieira and Hoikkala 2001
]), although the putative target(s) of selection are different. Here I have analyzed levels and patterns of nucleotide variability at fu for two other species of the virilis group, D. littoralis and D. ezoana. Two Finnish D. littoralis populations (24 individuals from Savonlinna and 20 from Kuopio) and one D. ezoana Finnish population (9 individuals from Oulanka) were analyzed. All individuals are males caught in the field during summer 1999 (D. ezoana) and summer 2000 (D. littoralis) by A. Hoikkala and S. Lakovaara. Sequencing of both strands of a PCR product obtained from the genomic DNA of a single male and analysis of DNA polymorphism were performed as described in Vieira and Charlesworth (1999, 2000)
and Vieira and Hoikkala (2001)
. GenBank accession numbers are AF322533AF322576 (D. littoralis) and AF322524AF322532 (D. ezoana). The fu region analyzed (478 bp) includes the first and a region of the second exon of this gene, the first intron, and a small region of the 5' noncoding flanking region. There is no evidence for differentiation between the D. littoralis Kuopio and Savonlinna populations. There are no fixed nucleotide differences, and nine out of the 12 segregating sites are shared between the two populations (fig. 1
). The average level of silent site divergence between the two populations is about 1%, and the FST (Hudson, Slatkin, and Maddison 1992
) value is 0.003. The permutation test of Hudson, Boos, and Kaplan (1992)
did not reveal significant isolation (P > 0.05) between the two populations. This is to be expected because the two populations are only about 100 km apart. In what follows, I have therefore pooled the two samples.
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In the D. ezoana fu region analyzed there are no replacement polymorphisms, but in D. littoralis there is a replacement of an aspartic acid (GAT) by an alanine (GCT) which is present at a frequency of about 8% in the Kuopio population and 20% in the Savonlinna population (14% in the combined sample). This replacement polymorphism is located at the same position where the common D. virilis aspartic acid (GAT)-alanine (GCC) replacement polymorphism is found (Vieira and Charlesworth 2000
). The virilis group is at least 10 Myr old (Throckmorton 1982
; Spicer 1992
; Nurminsky et al. 1996
), and despite the relatively low bootstrap values associated with some nodes, the unrooted Adh-Gpdh phylogeny shown in figure 2
is largely congruent with Spicer's (1992)
consensus phylogeny based on developmental, morphological, chromosomal, behavioral, and biochemical data sets and thus likely reflects species relationships. There is no evidence for recent gene flow between species of the virilis phylad and D. littoralis because in contrast with what is observed, there should be fixed nucleotide differences between the D. littoralis alanine and aspartic acid haplotypes and significant linkage disequilibrium in this region (data not shown).
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Second, the ancestral population was polymorphic for GAT and GCT (or GCC). The GCT (or GCC) mutation was then lost in the montana lineage. The GAT to AAT mutation is fixed in the ezoana-kanekoi lineage. In the virilis lineage, the polymorphism persists for at least 10 Myr because of selection (Vieira and Charlesworth 2000
) and mutates to GCC (or GCT). The alanine mutation is then lost at the fu locus in most species of this lineage. In the littoralis lineage, the GAT and GCT (or GCC) polymorphism also persists for 10 Myr (where it may have mutated to GCT). Only two mutational steps (assuming that the root of the phylogeny is located between the virilis phylad and the remaining species of the group) but also several allelic losses are thus required. Because only 5% of the ancestral neutral polymorphisms between a pair of species are expected to be retained after 3.8 Ne generations (where Ne is the effective population size) after their separation (Clark 1997
), the maintenance of the aspartic acidalanine polymorphism in the littoralis lineage is unlikely, unless it has been maintained by balancing selection. No significant deviations from neutrality were, however, detected for D. littoralis using four statistical tests of departure from neutrality (Fu and Li 1993
; Kelly 1997
; Wall 1999
), when C was assumed to be lower than 0.010. Recombination was included in the tests using the methods described in detail in Filatov and Charlesworth (1999)
because when test statistics that assume no recombination are used for detecting selection in regions of normal recombination, their power is usually low (Wall 1999
). The value of C used is compatible with that inferred from the fu sequence data. In the history of the D. littoralis sample a minimum of two recombination events has been inferred (Hudson and Kaplan 1985
). An estimate of the level of recombination between adjacent sites (C = 3Nec for an X-linked locus, where c is the population average recombination frequency per nucleotide site and Ne is the effective population size for X-linked loci; Hudson 1987
) is 0.013.
Furthermore, if balancing selection has been maintaining the D. littoralis aspartic acid-alanine replacement polymorphism for a relatively long period of time, there should be a window of enhanced variability and linkage disequilibria near this site (Strobeck 1983
; Hudson and Kaplan 1988
; Kaplan, Darden, and Hudson 1988
; Nordborg 1997
; Vieira and Charlesworth 2000
). There are, however, no fixed nucleotide differences between the D. littoralis alanine and aspartic acid haplotypes, and the average level of silent site divergence between these two haplotypes (0.0107) is similar to the average level of polymorphism for the aspartic acid haplotype (0.0123). There is also no significant linkage disequilibrium at the D. littoralis fu gene between the aspartic acid-alanine replacement polymorphism and surrounding polymorphic sites (data not shown), although this could simply reflect the relatively low frequency of the alanine haplotype or high recombination rates at the D. littoralis fu gene. There should also not be a deficiency of polymorphism levels within haplotypes carrying one of the two selectively maintained alleles (Strobeck 1983
; Hudson and Kaplan 1988
; Kaplan, Darden, and Hudson 1988
; Nordborg 1997
; Vieira and Charlesworth 2000
), but the six D. littoralis alanine sequences are identical (fig. 1 ). Because of the small sample size of the alanine haplotype (N = 6), there is, however, no statistical power to determine whether the observed lack of polymorphism within the alanine haplotype is incompatible with a simple neutral scenario.
In conclusion, there is no evidence for the D. littoralis aspartic acid-alanine polymorphism being a case of balancing selection. Therefore, the neutral double mutation hypothesis seems more likely, although because of the low frequency of the alanine haplotype much larger D. littoralis samples are required in order to fully address this issue.
Acknowledgements
I thank A. Hoikkala and S. Lakovaara for collecting and identifying the D. littoralis and D. ezoana males. I thank D. Nurminsky for providing unpublished Adh sequences and D. Filatov for providing computer software. I also thank B. Charlesworth and C. P. Vieira for helpful comments on the work. Until December 2000, J.V. was supported by the Fundação para a Ciencia e Tecnologia (PRAXIS XXI/BPD/14120/97).
Footnotes
Adam Eyre-Walker, Reviewing Editor
Keywords: Drosophila littoralis
Drosophila ezoana
fused gene
selection
DNA sequence variation
Address for correspondence and reprints: Jorge Vieira, Departamento de Genética Molecular, Instituto de Biologia Molecular e Celular, Rua do Campo Alegre 823, Porto 4150-180, Portugal. jbvieira{at}ibmc.up.pt
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