Two Divergent Species of the Virilis Group, Drosophila littoralis and Drosophila virilis, Share a Replacement Polymorphism at the fused Locus

Jorge VieiraGo

*Institute of Cell, Animal and Population Biology, University of Edinburgh;
{dagger}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. 1990Citation ), has undergone episodes of directional selection in three species of the virilis group of Drosophila (in D. virilis [Vieira and Charlesworth 1999, 2000Citation ], D. a. americana [Vieira, McAllister, and Charlesworth 2001Citation ], and D. montana [Vieira and Hoikkala 2001Citation ]), 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)Citation and Vieira and Hoikkala (2001)Citation . GenBank accession numbers are AF322533AF322576 (D. littoralis) and AF322524–AF322532 (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 1992Citation ) value is 0.003. The permutation test of Hudson, Boos, and Kaplan (1992)Citation 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.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1.—Drosophila littoralis haplotypes in Finnish populations. N is the sample size. Dots represent the same nucleotide as in the first sequence. Code is f for 5' flanking region sites, i for intron sites, s for synonymous sites, and r for replacement sites

 
For both D. ezoana and D. littoralis, the estimated level of silent site (5' noncoding flanking sites, synonymous and intron sites) polymorphism at fu is about 1.2%–1.3% (Watterson 1975Citation ; Nei 1987Citation , p. 256). Because the effective population size is positively correlated with the level of intraspecific polymorphism for neutral sites (Kimura 1983Citation , p. 43), it seems likely that D. ezoana and D. littoralis have a similar effective population size (Ne), of the order of 1,000,000 individuals, assuming a mutation rate of about 1–2 x 10-9 (Aquadro, Begun, and Kindahl 1994Citation ).

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 2000Citation ). The virilis group is at least 10 Myr old (Throckmorton 1982Citation ; Spicer 1992Citation ; Nurminsky et al. 1996Citation ), 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)Citation 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).



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 2.—Molecular phylogeny (unrooted Neighbor-Joining tree; distances were computed using the Kimura two-parameter model; bootstrap values higher than 50% are shown near each node; the computer software MEGA 1.02 was used [Kumar, Tamura, and Nei 1994Citation ]) based on Adh and Gpdh coding regions (Adh GenBank accession numbers are U26837U26839, U26841U26844, and U26846 [Nurminsky et al. 1996Citation ]; unpublished Adh sequences for D. novamexicana, D. littoralis, D. ezoana, and D. kanekoi were kindly provided by D. Nurminsky. The Gpdh accession numbers are D50087–D50091, D10697, AB19507, and AB19546–AB19550). Superimposed into these phylogenies is the information on which amino acids are present at fu at the same position where the D. littoralis aspartic acid-alanine replacement polymorphism is found and the sample size upon which the inference is based. For the americana complex where fu is duplicated, the information is for fu1. GenBank accession numbers for fu are AF322533AF322576 (D. littoralis), AF322524–AF322532 (D. ezoana), AY035186 (D. novamexicana 1031.8 from San Antonio, New Mexico), AY035187–AY035189 (three D. borealis wild males from Utah), AY035190 (D. flavomontana 0981.0 from Chester, Idaho), AY035191 (D. kanekoi 1540 from Sapporo, Japan), and AY035192 (D. lacicola 0991.0 from Saranac, New York). Drosophila lummei, D. virilis, D. americana, and D. montana fu accession numbers are listed in Vieira and Charlesworth (1999, 2000)Citation , Vieira, McAllister, and Charlesworth (2001)Citation , and Vieira and Hoikkala (2001)Citation . Strains are from the Bowling Green Drosophila Stock Center

 
There are therefore two possible explanations for the fu data shown in figure 2 . First, the ancestral population was monomorphic for GAT; this mutated to GCT and then to GCC in the virilis lineage, to AAT in the kanekoi-ezoana lineage, and to GCT in the littoralis lineage (a total of four mutations assuming that the root of the phylogeny is located between the virilis phylad [D. virilis, D. lummei, D. americana, and D. novamexicana] and the remaining species of the group). It should be noted that under this hypothesis the alanine mutation arose twice independently. In D. melanogaster, which belongs to another subgenus, at this amino acid site there is an aspartic acid (GAT) which is compatible with the assumption of the ancestral population being monomorphic for GAT.

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 2000Citation ) 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 1997Citation ), the maintenance of the aspartic acid–alanine 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 1993Citation ; Kelly 1997Citation ; Wall 1999Citation ), 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)Citation because when test statistics that assume no recombination are used for detecting selection in regions of normal recombination, their power is usually low (Wall 1999Citation ). 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 1985Citation ). 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 1987Citation ) 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 1983Citation ; Hudson and Kaplan 1988Citation ; Kaplan, Darden, and Hudson 1988Citation ; Nordborg 1997Citation ; Vieira and Charlesworth 2000Citation ). 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 1983Citation ; Hudson and Kaplan 1988Citation ; Kaplan, Darden, and Hudson 1988Citation ; Nordborg 1997Citation ; Vieira and Charlesworth 2000Citation ), 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 Back

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 Back

References

    Aquadro C. F., D. J. Begun, E. C. Kindahl, 1994 Selection, recombination and DNA polymorphism in Drosophila Pp. 46–56 in B. Golding, ed. Non-neutral evolution: theories and molecular data. Chapman and Hall, London

    Clark A. G., 1997 Neutral behavior of shared polymorphism Proc. Natl. Acad. Sci. USA 94:7730-7734[Abstract/Free Full Text]

    Filatov D. A., D. Charlesworth, 1999 DNA polymorphism, haplotype structure and balancing selection in the Leavenworthia PgiC locus Genetics 153:1423-1434[Abstract/Free Full Text]

    Fu Y. X., W. H. Li, 1993 Statistical tests of neutrality of mutations Genetics 133:693-709[Abstract/Free Full Text]

    Hudson R. R., 1987 Estimating the recombination parameter of a finite population model without selection Genet. Res 50:245-250[ISI][Medline]

    Hudson R. R., D. D. Boos, N. L. Kaplan, 1992 A statistical test for detecting geographic subdivision Mol. Biol. Evol 9:138-151[Abstract]

    Hudson R. R., N. L. Kaplan, 1985 Statistical properties of the number of recombination events in the history of a sample of DNA sequences Genetics 111:147-164[Abstract/Free Full Text]

    ———. 1988 The coalescent process in models with selection and recombination Genetics 120:831-840[Abstract/Free Full Text]

    Hudson R. R., M. Slatkin, W. P. Maddison, 1992 Estimation of levels of gene flow from DNA sequence data Genetics 132:583-589[Abstract/Free Full Text]

    Kaplan N. L., T. Darden, R. R. Hudson, 1988 The coalescent process in models with selection Genetics 120:819-829[Abstract/Free Full Text]

    Kelly J. K., 1997 A test of neutrality based on interlocus associations Genetics 146:1197-1206[Abstract/Free Full Text]

    Kimura M., 1983 The neutral allele theory of molecular evolution Cambridge University Press, Cambridge

    Kumar S., K. Tamura, M. Nei, 1994 MEGA: molecular evolutionary genetics analysis software for microcomputers Comput. Appl. Biosci 10:189-191[Abstract]

    Nei M., 1987 Molecular evolutionary genetics Columbia University Press, New York

    Nordborg M., 1997 Structured coalescent processes on different time scales Genetics 146:1501-1514[Abstract/Free Full Text]

    Nurminsky D. I., E. N. Moriyama, E. R. Lozovskaya, 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]

    Preat T., P. Thérond, C. Lamour-Isnard, B. Limbourg-Bouchon, H. Tricoire, I. Erk, M. C. Mariol, D. Busson, 1990 A putative serine/threonine protein kinase encoded by the segment-polarity fused gene of Drosophila Nature 347:87-89[ISI][Medline]

    Spicer G. S., 1992 Reevaluation of the phylogeny of the Drosophila virilis species group (Diptera: Drosophilidae) Ann. Entomol. Soc. Am 85:11-25[ISI]

    Strobeck C., 1983 Expected linkage disequilibrium for a neutral locus linked to a chromosomal arrangement Genetics 103:545-555[Abstract/Free Full Text]

    Throckmorton L. H., 1982 The virilis species group Pp. 227–296 in M. Ashburner, H. L. Carson, and J. N. Thompson, Jr., eds. The genetics and biology of Drosophila, Vol. 3b. Academic Press, New York

    Vieira J., B. Charlesworth, 1999 X chromosome DNA variation in Drosophila virilis Proc. R. Soc. Lond. B 266:1905-1912[ISI][Medline]

    ———. 2000 Evidence for selection at the fused locus of Drosophila virilis Genetics 155:1701-1709[Abstract/Free Full Text]

    Vieira J., A. Hoikkala, 2001 Variability levels, population size and structure of American and European D. montana populations Heredity 86:506-511[ISI][Medline]

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

    Wall J. D., 1999 Recombination and the power of statistical tests of neutrality Genet. Res 74:65-79[ISI]

    Watterson G. A., 1975 On the number of segregating sites in genetical models without recombination Theor. Popul. Biol 7:256-275[ISI][Medline]

Accepted for publication December 12, 2001.