Comparative Molecular Population Genetics of the Xdh Locus in the Cactophilic Sibling Species Drosophila buzzatii and D. koepferae

Romina Piccinali*, Montserrat Aguadé{dagger} and Esteban Hasson*

* Departamento de Ecología, Genética y Evolución, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Buenos Aires, Argentina
{dagger} Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain

Correspondence: E-mail: rpicci{at}bg.fcen.uba.ar.


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
The Xdh (rosy) gene is one of the best studied in the Drosophila genus from an evolutionary viewpoint. Here we analyze nucleotide variation in a 1875-bp fragment of the second exon of Xdh in Argentinian populations of the cactophilic D. buzzatii and its sibling D. koepferae. The major electrophoretic alleles of D. buzzatii not only lack diagnostic amino acids in the region studied but also differ on average from each other by four to 13 amino acid changes. Our data also suggest that D. buzzatii populations belonging to different phytogeographic regions are not genetically differentiated, whereas D. koepferae exhibits a significant pattern of population structure. The Xdh region studied is twice as polymorphic in D. buzzatii as in D. koepferae. Differences in historical population size or in recombinational environment between species could account for the differences in the level of nucleotide variation. In both species, the Xdh region exhibits a great number of singletons, which significantly departs from the frequency spectrum expected under neutrality for nonsynonymous sites and also for synonymous sites in D. buzzatii. These excesses of singletons could be the signature of a recent population expansion in D. buzzatii, whereas they may be simply explained as the result of negative selection in D. koepferae.

Key Words: Xdh • Cactophilic Drosophila • population structure • population expansion • negative selection


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
The Xdh (rosy) gene, which encodes an enzyme (xanthine dehydrogenase, or XDH) of the molybdopterin oxidoreductase family (Chovnic, Gelbart, and McCarron 1977; Wooton et al. 1991), is among the best-studied genic regions of Drosophila from an evolutionary viewpoint. Population genetic surveys in D. melanogaster and D. pseudoobscura have shown that XDH is polymorphic for several electrophoretic alleles (Buchanon and Johnson 1983; Keith et al. 1985). Four-cutter restriction analyses revealed that this gene is also highly polymorphic at the nucleotide level in D. simulans (Aquadro, Lado, and Noon 1988) and D. pseudoobscura (Riley, Hallas, and Lewontin 1989). Moreover, a sequence survey in the latter species revealed that more than 3% of the sites are variable and that the level and pattern of nucleotide variation is compatible with neutral expectations (Riley, Kaplan, and Veuille 1992). More recent evolutionary studies on Xdh in Drosophila have focused on the distribution of synonymous substitutions along its coding region (Comeron and Aguadé 1996), intron evolution (Tarrío, Rodríguez-Trelles, and Ayala 1998; Rodríguez-Trelles, Tarrío, and Ayala 2000), its utility as a phylogenetic marker (Rodríguez-Trelles, Tarrío, and Ayala 1999; Rodríguez-Trelles, Alarcón, and Fontdevila 2000), and the evolution of base composition (Begun and Whitley 2002).

Here we investigate patterns of nucleotide variation in the Xdh locus in the pair of cactophilic sibling species, D. buzzatii and D. koepferae. These taxa belong to the buzzatii cluster of the D. buzzatii complex (Ruiz and Wasserman 1993), which is part of the D. mulleri subgroup of the D. repleta group (Drosophila subgenus). They have an overlapping distribution in the arid zones of southern South America (Fontdevila et al. 1988; Hasson, Naveira, and Fontdevila 1992; Fanara, Fontdevila, and Hasson 1999), although D. buzzatii has a wider geographical range than D. koepferae, which is mainly restricted to the eastern slopes of the Andes Mountains (Fontdevila et al. 1988).

These two species breed and feed on the decaying tissues of several cactus species. D. buzzatii is mainly associated to the necrotic cladodes of prickly pears (Opuntia genus), and the rotting stems of columnar cacti of the genera Cereus and Trichocereus are the preferred breeding and feeding substrates of D. koepferae (Hasson, Naveira, and Fontdevila 1992; Fanara, Fontdevila, and Hasson 1999).

A recent survey of XDH electrophoretic variation in D. buzzatii populations of Argentina (Rodríguez et al. 2000) revealed the presence of four alleles whose frequencies vary clinally along latitudinal and altitudinal gradients and a striking pattern of population structure among phytogeographical regions. In addition, XDH alleles exhibited some degree of linkage disequilibrium with the inversion polymorphism of the second chromosome (Rodríguez et al. 2001), although this locus is located outside the segments rearranged by inversions and far away from their breakpoints (Ranz, Segarra, and Ruiz 1997). It was thus proposed that natural selection might be playing a significant role in shaping XDH allele frequency variation in D. buzzatii (Rodríguez et al. 2000).

Our analysis of Xdh nucleotide variation in natural populations of D. buzzatii and its sibling D. koepferae addresses the following questions: (1) Are XDH electrophoretic alleles of D. buzzatii homogeneous at the DNA level? (2) Are populations of D. buzzatii and D. koepferae genetically differentiated for the Xdh gene? (3) Is nucleotide variation at Xdh compatible with neutral expectations? (4) Can the evolutionary forces that shaped nucleotide DNA variability at the Xdh region in D. buzzatii and D. koepferae be inferred from levels and patterns of variation?


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Drosophila Lines
The 20 D. buzzatii lines isogenic for the second chromosome utilized in the present study were a subset of those used by Gómez and Hasson (2003). This subset was chosen to construct a representative sample of the three more common inversions of the second chromosome, st, j, and jz3 (Knibb and Barker 1988; Hasson et al. 1995). In fact, inversion frequencies in this sample were not significantly different ({chi}2 = 1.806, P = 0.595) from global estimations from previous surveys (Rodríguez et al. 2000). However, they were randomly chosen with respect to XDH electrophoretic alleles and collection site. A brief description of the sampling localities is summarized in table 1.


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Table 1 Geographical Situation, Cactus Species, and Phytogeographic Region of the Localities Sampled for the Present Study.

 
The 18 D. koepferae lines were collected at eight localities of Argentina (table 1) either in Autumn 1997 or in Autumn 1999. In all populations, wild females were trapped by means of net sweeping on fermented banana baits, carried to the laboratory, and placed in individual vials. After four generations, some progeny individuals were individually frozen at –80°C and preserved until DNA was extracted. Neither the inversion karyotype nor the XDH protein phenotype was assayed for the D. koepferae flies employed in the present survey.

DNA Sequencing
Genomic DNA was extracted from individual flies using the Puregene kit following the manufacturer's instructions (GENTRA Systems). A fragment of the second exon of Xdh was PCR amplified using three primers that were designed from conserved regions between D. melanogaster, D. subobscura, and D. pseudoobscura sequences (GenBank accession numbers Y00308, Y08237, and M33977, respectively). A 2,147-bp fragment was obtained with primers m2 (5'-CAGTGCGGCTTCTGCACGCC-3') and m-2 (5'-GACCAACCGGCATTGTTGTA-3'), and because of the low amount of DNA obtained, it was used as template for a second amplification of a smaller fragment using primers m2 and m-1 (5'-GTCTCCAGATAGAAGTGCTC-3'). This yielded a 1,875-bp fragment, homologous to the D. melanogaster sequence of Xdh between positions 2682 and 4563 (Keith et al. 1987).

Amplifications were all performed according to the following PCR profile: one cycle of 1 min at 94°C, 35 cycles of denaturation (1 min at 94°C), annealing (1 min at 55°C) and extension (1.5 min at 72°C), and a final extension step of 3.5 min at 72°C.

DNA fragments were purified with Qiaquick columns (Qiagen) and used as templates for direct sequencing of both strands with specific primers spaced 300 to 400 bp. Lines were manually sequenced using fmol DNA sequencing system (Promega) or with the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit version 2.0 (Perkin Elmer) in a Perkin Elmer ABI PRISM 3700 DNA Analyzer automated sequencer.

Because D. koepferae flies were not isogenic, several heterozygous sites were detected per individual. A nucleotide position was classified as heterozygous if two overlapping signals were consistently observed at the same site in the chromatograms of two or three sequencing reactions. Data were treated as genotypic with unknown gametic phase in further analyses using the software Arlequin, or, when ambiguous sites were not allowed by the program used (DnaSP and MEGA), two sequences per individual were created by distributing the segregating sites at random between each of them. Partial and complete sequences for each line were manually aligned or using the ClustalW program (Higgins, Thompson, and Gibson 1994). Newly reported sequences are available at GenBank under accession numbers AY219187 to AY219224.

Genetic Differentiation
Genetic differentiation among electrophoretic alleles and chromosomal arrangements in D. buzzatii, as well as patterns of geographic population structure in both species, were analyzed by means of statistics KST* (Hudson, Boos, and Kaplan 1992), FST (Weir and Cockerham 1984), Snn (Hudson 2000), and analysis of molecular variance (AMOVA [Excoffier, Smouse, and Quattro 1992]) over total, nonsynonymous, and synonymous variation. AMOVA is a hierarchical analysis of molecular variance, which also allows defining {Phi}-statistics analogous to F-statistics. In the AMOVA, sequences sharing the same chromosomal arrangement and sampling locality were considered as populations in the comparisons among chromosomal arrangements, whereas higher order groupings were constructed with all populations carrying the same inversion. For the analysis of geographic population structure, populations (sites of collection) were grouped according to the phytogeographic region to which they belong (table 1). The significance of all test statistics was obtained by 10,000 permutations of haplotypes (or genotypes) between chromosomes, alleles, populations, or regions.

Nucleotide Diversity
Nucleotide diversity was estimated as the observed number of segregating sites (S), the observed average number of pairwise differences per gene (k) and per site ({pi}), and Watterson's estimator ({theta}W).

Neutrality Tests
The tests of Tajima (1989) and Fu and Li (1993) were applied to determine whether intraspecific allele frequency distributions were in agreement with neutral predictions. Both the HKA (Hudson, Kreitman, and Aguadé 1987) and the MK (McDonald and Kreitman 1991) tests were used to examine whether intraspecific polymorphism and interspecific divergence are correlated, as predicted by the neutral theory. The reference locus in the HKA test involving D. buzzatii was the STS 70.09.1sts, an anonymous marker unlinked to the inversion system (Laayouni et al. 2003). A similar analysis could not be conducted in D. koepferae because of the lack of information on nucleotide polymorphism data for other genes in this species. The Fay and Wu H test (Fay and Wu 2000), devised to detect positive selection, was also employed. Finally, the R2 statistic (Ramos-Onsins and Rozas 2002), based on the difference between the number of singleton mutations and the average number of pairwise nucleotide differences, was applied to synonymous variation in both species to investigate changes in population size.

In all tests based on the comparison of polymorphism and divergence, all sequences of the other species were used as outgroups (i.e., D. koepferae for D. buzzatii and vice versa). Xdh sequences of other members of the D. buzzatii cluster (Rodríguez-Trelles, Alarcón, and Fontdevila 2000) were not used because (1) available sequences only partially overlap (~700 bp) the sequences reported herein, and (2) the phylogenetic relationships within the cluster are still controversial (Durando et al. 2000; Rodríguez-Trelles, Alarcón, and Fontdevila 2000; Manfrin, De Brito, and Sene 2001). Significance of tests was calculated using coalescent simulations (10,000 replicates) based on a Monte Carlo process (Hudson 1990) and assuming the most conservative option of no recombination.

The DnaSP (versions 3.53 and 3.97) software (Rozas and Rozas 1999) was used to obtain estimates of nucleotide variation and genetic differentiation and for neutrality tests. The MEGA version 2.01 program (Kumar et al. 2001) was utilized for the estimation of some descriptive statistics. The Arlequin version 2.001 program (Schneider, Roessli, and Excoffier 2000) was employed for the AMOVAs.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Electrophoretic Alleles
The average number of amino acid differences among sequences within previously defined electrophoretic alleles of D. buzzatii was 7.04 ± 1.42 (5.33 ± 1.98 for XDHa, 5.50 ± 1.42 for XDHb, 13.33 ± 3.17 for XDHc, and 4.00 ± 2.04 for XDHd), which was very similar to the average between allelic classes (7.54 ± 2.92). Moreover, this trend remained unchanged even after excluding allele XDHc, which showed the highest intra allelic heterogeneity (within alleles: 4.95 ± 1.26; between alleles: 4.88 ± 0.21). The major electrophoretic classes not only lack diagnostic amino acid differences in the region sequenced but also seem to be heterogeneous groups that on average differ from each other by four to 13 amino acid changes. Genetic differentiation among electrophoretic alleles was tested by means of the KST* and Snn statistics for total, nonsynonymous, and synonymous variation, but neither global nor pairwise comparisons were significant (results not shown).

Genetic Differentiation Among Arrangements in D. buzzatii
Because D. buzzatii lines were sampled according to chromosomal arrangements, and because a certain degree of linkage disequilibrium between XDH electrophoretic alleles and inversions was previously reported (Rodríguez et al. 2001), we investigated the extent to which inversion arrangements are genetically differentiated at the DNA sequence level. AMOVA, KST*, and Snn tests failed to reveal nucleotide differentiation among second chromosome arrangements for total, nonsynonymous, and synonymous variation in the Xdh region (results not shown).

Geographic Population Structure
Because one of the assumptions of neutrality tests is that populations are not subdivided, and given that our samples consisted of sequences from different geographic origins, we decided to investigate patterns of population subdivision in both species. The AMOVA showed that differentiation among populations within and among regions for total, nonsynonymous, and synonymous variation was nonsignificant in D. buzzatii (table 2). In addition, the within-population component of variance was not significant, pointing out that sequences sampled in the same population tend to be as similar as those belonging to different populations. This result is also in line with the idea that the genetic makeup of populations is very similar.


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Table 2 Results of Analysis of Molecular Variance (AMOVA) for Total, Nonsynonymous, and Synonymous Variation.

 
The lack of a significant population structure in D. buzzatii was confirmed by both KST* and Snn statistics (results not shown). Conversely, the AMOVAs showed that variation among phytogeographic regions was significant in D. koepferae and accounted for 24% of total variation. Similar results were obtained when only synonymous variation was considered, whereas the component of variation among regions was not significant for nonsynonymous sites (table 2). Moreover, differentiation among populations within phytogeographic regions was nonsignificant for the three types of variation, giving support to the a priori pooling of populations.

To determine which set of populations was responsible for the detected pattern of population structure in D. koepferae, FST (Weir and Cockerham 1984) values were calculated for total and synonymous variation between pairs of regions (table 3). This statistic was chosen rather than KST* or Snn because it is based on genotypic instead of haplotypic information. Prepuna populations were significantly differentiated from those of the remaining regions, and Espinal populations were significantly differentiated from Northern Monte populations. However only comparisons between Prepuna and Southern Monte and between Prepuna and Espinal remained significant after adjusting P values for multiple comparisons using Bonferroni criteria (Weir 1996, pp. 133–134). We also decided to perform a Mantel test to search for signs of isolation by distance, but the correlation between geographic and genetic distance was not significant (r = 0.643, P = 0.167).


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Table 3 Pairwise FST Among Phytogeographical Regions for Total (Below Diagonal) and Synonymous (Above Diagonal) Variation in D. koepferae.

 
Because the pattern of population subdivision in D. koepferae precludes any pooling of populations belonging to different regions, the latter were analyzed separately in subsequent analyses. Contrarily, given the lack of genetic differentiation among chromosomal arrangements and among populations in D. buzzatii, all sequences were treated as samples drawn from a single population in further tests.

Nucleotide Diversity
D. buzzatii and D. koepferae Xdh sequences contained approximately 1,423 nonsynonymous and 452 synonymous sites. The numbers of segregating sites were different between species: 140 variable sites were observed in D. buzzatii, whereas only 76 variable sites were observed in D. koepferae (figs. 1 and 2). Nonsynonymous variation accounted for 36% to 39% of polymorphism in both species.



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FIG. 1. Variable nucleotide sites in D. buzzatii. A dot indicates the same nucleotide as in the reference sequence, an asterisk indicates a nonsynonymous site, and the symbol # indicates the presence of synonymous and nonsynonymous mutations at that same site. Arr refers to the inversion carried by each isogenic chromosome, and All refers to the XDH electrophoretic allele. Question marks indicate missing data. Position number 1 corresponds to position number 2682 in D. melanogaster (Keith et al. 1987)

 


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FIG. 2. Variable sites in D. koepferae. A dot indicates the same nucleotide, an asterisk indicates a nonsynonymous site, and the symbol # indicates the presence of synonymous and nonsynonymous mutations at that same site. Position number 1 corresponds to position number 2682 in D. melanogaster (Keith et al. 1987). Following the IUBMB single-letter code, K, M, R, S, W, and Y mean G/T, A/C, A/G, C/G, A/T and C/T, respectively

 
The levels of total and synonymous variation were twofold lower in D. koepferae than in D. buzzatii, and the difference was even more pronounced for nonsynonymous variation (table 4). In particular, the Prepuna sample was the least polymorphic, with an average of only 3.6 pairwise differences between sequences. Nucleotide variability increased in Southern populations (Northern Monte, Southern Monte, and Espinal), in which the number of pairwise differences reached values three times higher. These data are in line with previous studies of inversion (Hasson 1988) and allozyme polymorphisms (Sanchez 1986), which also revealed lower levels of variation in populations of the Prepuna.


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Table 4 Estimates of Nucleotide Heterozygosity and Diversity for Total, Synonymous, and Nonsynonymous Sites for Xdh in D. buzzatii and D. koepferae.

 
Singletons constituted 70% to 100% of the total number of segregating sites for both nonsynonymous and synonymous sites in D. buzzatii and in D. koepferae of Prepuna and Espinal and for nonsynonymous sites in Northern Monte. However, a remarkably lower proportion of singletons was found in Southern Monte for nonsynonymous (38%) and synonymous (14%) sites and in Northern Monte for synonymous sites (47%).

Interestingly, there were 13 shared polymorphisms between species, two at nonsynonymous sites (positions 1348 and 1609), 10 at synonymous sites (positions 270, 1017, 1132, 1347, 1350, 1485, 1488, 1500, 1512, and 1797), and one in a site with two synonymous and one nonsynonymous variant (position 1720). Transpecific polymorphisms were ubiquitous in D. koepferae but reached the highest frequency in Prepuna (figs. 1 and 2).

Neutrality and Population Size Tests
Tajima's D and Fu and Li's D and F statistics were negative and highly significant for total, nonsynonymous, and synonymous variation in D. buzzatii (table 5). On the other hand, these tests were negative but only significant for nonsynonymous variation in the entire D. koepferae sample. The trend toward negative values for these statistics for nonsynonymous variation was concordant in three phytogeographic regions, and the values were significantly lower than zero in Prepuna and Espinal. For synonymous variation, only Tajima's D statistic was significantly lower than zero in Prepuna. The HKA, MK, and Fay and Wu tests were not significant in either species (results not shown). Finally, the R2 statistic was significant for synonymous variation in D. buzzatii (R2 = 0.06, P < 0.01) and in Espinal (R2 = 0.109, P < 0.05).


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Table 5 Results of Tajima's D and Fu and Li's D and F Statistics.

 
Because the presence of shared polymorphisms between species may affect estimates of nucleotide polymorphism and interespecific divergence, we decided to rerun all the tests removing the shared polymorphic sites. The results for all the neutrality tests performed were similar to those obtained considering all segregating sites (results not shown).


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
XDH Electrophoretic Alleles in D. buzzatii
Electrophoretic surveys led to the identification of four clearly different XDH electromorphs in natural populations of D. buzzatii. However, our present survey of Xdh sequence variation failed to reveal consistent differences between electrophoretic alleles in a region that comprises more than half the length of the coding region and is one of the most variable parts of the gene (Riley, Kaplan, and Veuille 1992). Moreover, there is a complete lack of diagnostic amino acids in the studied region, which precludes the inference of electrophoretic classes on the basis of sequence variation. Two alternative explanations can account for this observation. The first is that mutations responsible for charge differences among electrophoretic alleles lay outside the region sequenced. The second is that electrophoretic alleles are actually heterogeneous classes resulting from different amino acid changes that confer the same net charge to the protein. Actually, a high percentage of the nonsynonymous substitutions detected are charge nonconservative changes, suggesting that charge convergence may be frequent in Xdh. This seems to be a common observation, because similar results have been reported in other loci such as esterase-5B (Est-5B) in D. pseudoobscura (Veuille and King 1995), allele PGI-65 of the phosphoglucose isomerase locus in Gryllus pennsylvanicus (Katz and Harrison 1997), allele ACPH-1054 of the acid phosphatase-1 locus in D. subobscura (Navarro-Sabaté, Aguadé, and Segarra 1999), and the Slow allele of the phosphoglucomutase locus in D. melanogaster (Verrelli and Eanes 2000). Thus, charge convergence among XDH electrophoretic alleles seems to be a plausible explanation for the absence of genetic differentiation among chromosomal arrangements and among geographic regions in the Xdh sequence, in contrast with the patterns reported in an electrophoretic survey of the protein (Rodríguez et al. 2000, 2001).

Levels of Nucleotide Variation and Shared Polymorphisms
Estimates of heterozygosity and nucleotide diversity in the Xdh gene in D. buzzatii were similar to those reported for D. pseudoobscura (Riley, Kaplan, and Veuille 1992), and twice larger than in its sibling D. koepferae, for both synonymous and nonsynonymous sites. One possible explanation for this difference may be that the historical effective population size in D. buzzatii has been larger than in D. koepferae, given that under neutrality, the heterozygosity is expected to be proportional to the effective population size. A similar interpretation has been proposed to account for the 1.5 to 2 times higher molecular diversity observed in coding regions of D. simulans as compared with its sibling D. melanogaster (reviewed in Andolfatto [2001]).

Another factor that can affect levels of nucleotide variation is the recombinational environment in which a gene is embedded. Recombination can have a significant effect on polymorphism and is a potential explanation for the heterogeneity observed in levels of nucleotide variation among genes (reviewed in Powell [1997]). Paracentric inversions affect levels of recombination in the vicinity of breakpoints both in inverted and noninverted regions, because of the inhibition of chiasmata by asynapsis in inversion heterokaryotypes (Roberts 1976; Coyne et al. 1991, 1993). In addition, theoretical work has demonstrated that inversions not only reduce but also redistribute recombination, generating complex patterns of recombination and gene flux all along the chromosome (Navarro et al. 1997). Moreover, a recent review (Andolfatto, Depaulis, and Navarro 2001) has shown that the presence of an inversion can affect nucleotide variation in genes located as far as 1,000 kb away from the breakpoints within an inverted segment.

Interestingly, the distance of Xdh to the breakpoints of polymorphic inversions is quite different in D. buzzatii and D. koepferae as inferred from the cytological position of the gene at 2B3c in the polytene map of the repleta group (Ranz, Segarra, and Ruiz 1997). In D. buzzatii, Xdh is more than 40 polytene bands away from the breakpoints of inversions j and jz3, that is, more than 2 Mb if we assume that each band is equivalent to 50 kb as in D. hydei (Laird 1973; Hartl et al. 1994), another species of the repleta group. In D. koepferae, Xdh is about seven bands (~350 kb) and three bands (~150 kb) away from the proximal and distal breakpoints of inversions l9 and k9, respectively.

Although the conclusions drawn in Navarro et al. (1997) are applicable to single inversion systems and must be taken with caution when applied to complex systems such as those present in D. buzzatii and D. koepferae, we can ask whether the recombinational environment of the Xdh gene could differ between these species. Two lines of evidence suggest that the Xdh gene is indeed located in a region where recombination is reduced in D. koepferae relative to D. buzzatii. First, inversions l9 and k9 are never found in association, even though the intervening segment is about 10 polytene bands long. Moreover, inversion l9 is in linkage disequilibrium with inversions m9 and n9 that partially overlap the region rearranged by k9. Second, the cytological region where Xdh maps almost always appears asynaptic in l9m9/k9 or l9m9n9/k9 heterokaryotypes, which are very common in natural populations (Hasson 1988, unpublished data). Surveys of variation in other genes or regions mapping between the breakpoints of l9 and k9, that is, Hsc 70-2 and M(3)99D (Ranz, Segarra, and Ruiz 1997) and STSs 60.03.2, 60.03.5, 80.132 and 70.20.2 (Laayouni, Santos, and Fontdevila 2000), would be needed to establish whether the different levels of nucleotide variation in the Xdh region in D. koepferae and D. buzzatii are at least partly caused by the differential effect of inversions in the two species.

The presence of shared polymorphisms between these two well-defined species is an intriguing observation. Several factors have been proposed to explain the occurrence of transpecific polymorphisms: recurrent mutation, persistence of neutral ancestral variants in populations of large size, balancing selection, and ancient or recent interspecific gene flow (Clark 1997; Filatov and Charlesworth 1999; Kliman et al. 2000; Khadem et al. 2001; Machado et al. 2002; Ramos-Onsins et al. 2004). We applied the hypergeometric distribution (Clark 1997) to test whether the shared polymorphisms observed could be explained by recurrent mutation alone. For this analysis we used the 10 shared polymorphisms that segregate at third codon positions because they exhibit a low substitution rate heterogeneity along Xdh (Rodríguez-Trelles, Alarcón, and Fontdevila 2000). The low probability of observing 10 shared polymorphisms by chance alone (P = 0.046) indicates that other forces rather than parallel mutation are responsible for the presence of transpecific polymorphisms.

Theoretical work has shown that the expected time for the loss of a shared ancestral neutral polymorphism is 1.7 Ne generations (Clark 1997). We could also exclude the persistence of ancestral variants, given that the available information suggests that D. buzzatii and D. koepferae diverged more than 4 MYA (Gómez and Hasson 2003). Indeed, this time would be sufficient for the loss of most shared ancestral neutral polymorphisms. The third possible explanation for transpecific polymorphisms is balancing selection. However, neutrality tests did not reveal any sign of the presence of balanced polymorphisms along the region sequenced (see below). Finally, ancient or recent interespecific gene flow can also account for the presence of shared polymorphisms. Although available evidence indicates that D. buzzatii and D. koepferae are reproductively isolated by ecological factors (Fanara, Fontdevila, and Hasson 1999) and sterility of male hybrid progeny (Naveira and Fontdevila 1986), introgression could take place via fertile hybrid females. Moreover, the observation of shared segregating sites in the Est-A gene (Gómez and Hasson 2003) strongly argues in favor of gene flow as a suitable explanation for the presence of transpecific polymorphisms.

Excess of Singletons in D. buzzatii and D. koepferae
Neutrality tests based on intraspecific data (Tajima's D and Fu and Li's D and F statistics) suggest that nucleotide variation in the Xdh region is not in mutation-drift equilibrium. The negative values of these statistics point to an overall excess of rare variants at nonsynonymous sites in both species and at synonymous sites in D. buzzatii. The pattern observed in D. koepferae is relatively easy to explain because the action of negative selection on amino acid variation is expected to differentially affect synonymous and nonsynonymous sites within the same gene region, keeping deleterious amino acid variants at low frequency and having little effect on synonymous mutations (Smirnova et al. 2001; Hahn, Rausher, and Cunningham 2002). This hypothesis is supported by theoretical work showing that Tajima's D and Fu and Li's D and F statistics can be altered by purifying selection when selection targets are directly examined (Przeworski, Charlesworth, and Wall 1999) but have less power when the analysis focuses on linked neutral variation (Charlesworth, Charlesworth, and Morgan 1995; Przeworski, Charlesworth, and Wall 1999). Moreover, our results are in agreement with the conclusion of Riley, Kaplan, and Veuille (1992) that XDH amino acid variation in D. pseudoobscura is not completely neutral, because the number of nonsynonymous mutations is 10 times lower than expected if selection were acting with similar intensity than on synonymous variation. More recently, Begun and Whitley (2002) analyzed data of 22 species of Drosophila for this gene region and concluded that a significant fraction of XDH amino acid variants are slightly deleterious.

Unfortunately, there is not a unique explanation for the departures from neutrality observed in D. buzzatii, because the excess of singletons was observed at both nonsynonymous and synonymous sites. Population subdivision, background selection, selective sweeps over the examined region or near it, and population expansions could account for the observed patterns of nucleotide variation in this species.

Concerning population subdivision, several lines of evidence seem to indicate that D. buzzatii populations are not geographically structured at the DNA level for the Xdh gene region. Specific tests of population structure (AMOVA, KST*, and Snn) as well as neighbor-joining trees constructed with silent and replacement variation (results not shown) did not reveal any sign of population subdivision. However, we should consider the possibility that our findings might result from an undetected population subdivision, given the low number of sequences sampled per deme. This point has been treated in detail by Ptak and Pzeworski (2002), who compared the results of Tajima's D statistic in surveys of human populations according to the number of individuals and sampling localities.

However, recent theoretical work in this field has shown that there is a simple coalescent process under several models of population subdivision (e.g., metapopulations and general finite island models [Wakeley 2001; Wakeley and Aliacar 2001]). Under these models, if only one sequence is sampled per deme, it was shown that the behavior of a sample during most of the coalescent process is similar to that of a nonsubdivided population but with an effective population size that depends on the number and sizes of demes and migration rates (Wakeley 2001; Wakeley and Aliacar 2001). Moreover, when the number of demes is large, the distribution of Tajima's D statistic in samples consisting of one sequence per deme is expected to be identical to that of a panmictic population (Wakeley et al. 2004). We tested the hypothesis that the observed excess of external mutations in our D. buzzatii sample may be an artifact of the low sample size per deme and a nondetected population structure using two resampling strategies (Ramos-Onsins personal communication) and comparing the distributions of Tajima's D statistic and Fu and Li's D and F tests with those calculated with the entire data set. The first resampling strategy, which takes into consideration the possible sampling structure of the data set, consisted of drawing smaller samples of all possible combinations (648) of one sequence from each one of the nine demes. The second consisted of subsamples of nine sequences taken at random from the total data set, (i.e., not considering the sampling structure). These analyses confirmed the general trend of both Tajima's D and Fu and Li's D and F statistics toward negative values detected for the entire data set and showed that the mean values obtained with both types of resampling strategies were almost identical (considering structure: Tajima's D statistic = –1.18, Fu and Li's D statistic = –1.53, Fu and Li's F statistic = –1.76; not considering structure: Tajima's D statistic = –1.16, Fu and Li's D statistic = –1.56, Fu and Li's F statistic = –1.79). These results suggest that even though our sampling strategy imposes a limit to the power of detection of differentiation among demes, another factor in addition to an undetected population structure may be the cause of the observed excess of singletons. Finally, we can invoke the general trends observed in previous surveys of variation in D. buzzatii that have shown that populations are only differentiated for inversions and certain allozyme loci (Rodríguez et al. 2000), whereas for other markers such as mtDNA and the nuclear locus Est-A among population differentiation is nonsignificant (Rossi et al. 1996; Gómez and Hasson 2003).

Background selection is not expected to cause dramatic shifts in the frequency spectrum (Charlesworth, Charlesworth, and Morgan 1995; Przeworski, Charlesworth, and Wall 1999), but an excess of rare variants can be observed if the species is recovering from a recent selective sweep caused by the fixation of an adaptive variant in the examined region or near it (Braverman et al. 1995). However, background selection can be rejected on grounds of the results of tests of the neutral model. The HKA test, which can detect local reductions in the levels of DNA variation in comparison with a neutral standard locus, the MK test, which has the advantage of being able to detect positive selection under the presence of a strong selective constraint (McDonald and Kreitman 1991; Fay and Wu 2001), and Fay and Wu's H test, which can detect an excess of high frequency derived variants in the frequency spectrum (Fay and Wu 2000), were not significant. However, it is important to note that nonsignificant results do not completely rule out the action of natural selection (Simonsen, Churchill, and Aquadro 1995; Wayne and Simonsen 1998).

Finally we can ask whether the departures from neutrality can be accounted for by demographic events such as a population expansion. Demography is expected to influence all sites in the nuclear genome in the same way, whereas selection is predicted to have more localized effects (Andolfatto and Przeworski 2000; Galtier, Depaulis, and Barton 2000; Hahn, Rausher, and Cunningham 2002). Thus, to address this issue, we need information on nucleotide variation in other gene regions. Although data on nucleotide variation are relatively scarce in D. buzzatii, recent surveys have revealed a significant excess of rare variants in other nuclear genes such as the Est-A locus (Gómez and Hasson 2003) and in three anonymous noncoding regions (Laayouni et al. 2003). Interestingly, data from this last survey are from a single population, thus avoiding the possible effect of pooling lines from different geographic origin. The same is true for a subset of 13 sequences in the Est-A data set (Gómez and Hasson 2003). Moreover, negative results of Tajima's D statistic have been reported in surveys of nucleotide variation in the mtDNA (Rossi et al. 1996; De Brito, Manfrin, and Sene 2002).

In addition, the significant R2 test, one of the most powerful in the detection of population expansions (Ramos-Onsins and Rozas 2002) and robust to the presence of some recombination (Ramos-Onsins, personal communication), gives additional support to the hypothesis that D. buzzatii has passed through a recent population expansion.

Thus, a population size change seems to be the most suitable explanation for the patterns of nucleotide variation observed in the Xdh region in D. buzzatii.

Population Structure and Cactus Hosts
Despite the wealth of population genetic studies in Drosophila (reviewed in Powell [1997]), very few have addressed the relationship between ecological variables and genetic population structure. The scarcity of such studies is probably a result of the lack of appropriate ecological and population biological data for most Drosophila species (Pfeiler and Markow 2001). However, cactus breeders as D. buzzatii and D. koepferae, may provide suitable model systems to test the hypothesis of a relationship between habitat continuity and genetic population structure (Shoemaker and Jaenike 1997; Pfeiler and Markow 2001). The prediction under such model is that species restricted to patchy habitats experience greater restriction in gene flow among populations than those utilizing continuously distributed habitats. Our present study seems to be in line with this prediction. On one hand, populations of D. koepferae, a species associated with long lasting, predictable habitats for the larvae such as columnar cacti (Heed and Mangan 1986; Etges 1993; Hasson, Naveira, and Fontdevila 1992; Fanara, Fontdevila, and Hasson 1999; Fanara and Hasson 2001; authors' field observations) are genetically structured. On the other hand, D. buzzatii, associated to a more continuously distributed resource, such as prickly pears, exhibits a lower level of population subdivision. However, further studies based on larger samples per population and more loci will help us evaluate whether the patterns outlined in the present study are particular for the gene studied or constitute distinctive features of these species adapted to live in South American deserts.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
We thank P. Cohen for help in adjusting the experimental conditions and for training in the molecular techniques in the first stage of this project. We also thank J. S. F. Barker for sharing some of his collected flies (Catamarca and Tilcara lines) and the Antp/{Delta}5 balanced stock, J. Rozas for yielding the new version of DnaSP, S. Ramos-Onsins for many helpful suggestions and for sharing his program CalcTest for the resampling methods, and Serveis Científico-Tècnics at Universitat de Barcelona for automated sequencing facilities. We acknowledge A. Navarro, J. Wakeley, and two anonymous reviewers for many useful comments on an earlier version of this manuscript. R.P. is a doctoral fellow of CONICET, and E.H. is member of Carrera del Investigador Científico (CONICET). This work was supported by grants from Universidad de Buenos Aires, CONICET, and Fundación Antorchas, Argentina to E.H., and by grants PB97-0918 and BMC2001-2906 from Comisión Interdepartamental de Ciencia y Tecnología, Spain to M.A.


    Footnotes
 
Wolfgang Stephan, Associate Editor Back


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 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
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Accepted for publication August 19, 2003.





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