SEARCH

SEARCH BY CITATION

Keywords:

  • DNA polymorphism;
  • Drosophila madeirensis;
  • Drosophila subobscura;
  • rp49 gene;
  • speciation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

An ~1.6-kb fragment spanning the rp49 gene was sequenced in 16 lines of Drosophila subobscura from Madeira and in 22 lines of the endemic species D. madeirensis. Nucleotide diversity in D. subobscura from Madeira (π=0.0081) was similar to that in lines from Spain carrying the O3+4 chromosomal arrangement (π=0.0080). No significant genetic differentiation was detected between insular and continental O3+4 lines of D. subobscura. These results are compatible both with a rather recent and massive colonization, and with multiple colonization events from the continent. Nucleotide diversity in D. madeirensis (π=0.0076) was similar to that in D. subobscura, which deviates from the expectation, under strict neutrality, of a lower level of variation in an insular species with a small population size. The observed numbers of shared polymorphisms and of fixed differences between D. madeirensis and D. subobscura are compatible with the isolation model of speciation, where shared polymorphisms are due to common ancestry.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Drosophila madeirensis Monclús and D. subobscura Collins are closely related species of the obscura group that coexist in Madeira. The former species is endemic to this island, while D. subobscura has a wider distribution area that encompasses most of Europe, northern Africa, three archipelagos of the Macaronesia (Azores, Madeira and Canary Islands) and Asia Minor (Krimbas, 1992). Madeira, like the other two archipelagos, has a volcanic origin. It is a rather small island (730 km2) that arose 5–6 Myr ago (Galopin de Carvalho & Brandão, 1991). Its present fauna and flora has been therefore shaped by the set of species that have colonized the archipelago and by their subsequent evolution. Species endemism in the flora of the Madeiran archipelago is about 10% (Press & Short, 1994). This percentage is even higher for the terrestial fauna of Madeira and reaches approximately 15% for Diptera (Baez, 1993). Therefore, despite the rather close proximity of Madeira both to the continent and to the other archipelagos, it seems rather well isolated. Possible founder events during the colonization and differential selection in the island would have caused the genetic differentiation of the colonizing populations and in some cases it would have led to the origin of new species. Natural populations of D. madeirensis and D. subobscura in the islands therefore offer the opportunity of studying the colonization process and the effects of factors such as founder events and reduced migration, likely associated with insularity, on extant levels of nucleotide variation in these populations.

Populations of D. subobscura from Madeira have been characterized at the chromosomal and allozyme levels (Prevosti, 1972; Prevosti, 1974; Larruga et al., 1983; Pinto et al., 1997). Restriction map variation in the mtDNA and in the nuclear gene rp49 has also been surveyed (Afonso et al., 1990; Pinto et al., 1997; Khadem et al., 1998). At the chromosomal level, populations from Madeira are well differentiated from continental populations but are rather similar to populations from the Canary Islands (Prevosti, 1972, 1974). However, for both allozyme and mtDNA variation they are differentiated from the Canary Islands populations but not from continental populations (Larruga et al., 1983; Afonso et al., 1990; Pinto et al., 1997). In contrast, restriction fragment length polymorphism analysis of the rp49 gene region of a D. subobscura population from Madeira revealed some genetic differentiation between insular and continental populations. This study also revealed less variation in the insular than in continental populations (Rozas et al., 1995; Khadem et al., 1998).

D. madeirensis and D. subobscura diverged rather recently, about 0.6–1.0 Myr ago, according to nucleotide divergence at the rp49 gene region (Ramos-Onsins et al., 1998). They are rather similar morphologically (Monclús, 1984) and their reproductive isolation is not complete, as fertile and viable hybrids are obtained in some interspecific crosses (Khadem & Krimbas, 1991; Papaceit et al., 1991). Unlike in D. subobscura, only a few studies on intraspecific genetic variation have been conducted with D. madeirensis (González et al., 1983, 1990). Other studies have shown that the ancestors both of D. madeirensis and of extant populations of D. subobscura in Madeira are probably the result of independent colonization events from the continent (Khadem et al., 1998).

In the present study, we have sequenced the rp49 gene region in a D. subobscura population from Madeira and in a natural population of D. madeirensis. The rp49 gene (named RpL32 in Flybase) encodes the ribosomal protein 49 (or ribosomal protein L32). In D. subobscura, this gene is located in the O chromosome at band 91C, close to the proximal breakpoint of inversion 3 (Aguadé, 1988). Two alternative chromosomal arrangements for this genomic region, O3+4 and Ost, segregate in natural populations of D. subobscura. Each of these arrangements originated from the ancestral O3 arrangement by a single inversion: O3+4 by inversion 4, and Ost by inversion 3. While D. madeirensis is monomorphic for O3, this arrangement is no longer present in extant populations of D. subobscura. Unlike continental populations of D. subobscura, the population from Madeira is nearly monomorphic for the O3+4 arrangement. Present data indicate that, within this arrangement, insular and continental populations of D. subobscura are not genetically differentiated. In contrast, D. madeirensis and D. subobscura populations are highly differentiated despite their similar level of nucleotide variation. Finally, the distribution of nucleotide variation within and between species is consistent with the isolation model of speciation, where a single ancestral population splits into two descendent populations that, subsequently, remain isolated.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Fly stocks

Sixteen Drosophila subobscura and 22 D. madeirensis lines were used in the present study. Flies from both species were collected in Ribeiro Frio (Madeira, Portugal) in 1997; isofemale lines were established upon arrival in the laboratory. Highly inbred lines were subsequently obtained by 12 generations of sibmating. The gene arrangement for the O chromosome was determined for each line by observation of polytene chromosomes of salivary glands of third-instar larvae.

DNA extraction and sequencing

A modification of protocol 48 in Ashburner (1989) was used to extract genomic DNA. Despite the fact that the lines were highly inbred, single flies were used for the DNA extraction to reduce the chance of heterozygosity for the studied region. If for a particular inbred line sequencing revealed that an individual was heterozygous, additional single flies from the same inbred line were used until a homozygous individual was found; only the sequence of this individual was used in the analyses. The complete rp49 region (~1.6 kb) was amplified by the polymerase chain reaction (PCR; Saiki et al., 1988) using 20-mer oligonucleotides. The PCR products were purified with Qiaquick columns (Qiagen). Internal primers, designed at intervals of ~300 nucleotides, were used for sequencing. Both strands were cycle sequenced using the rhodamine sequencing chemistry (Perkin-Elmer) and analysed on a Perkin Elmer ABI PRISM 377 automated DNA sequencer. The newly reported sequences are deposited in the EMBL nucleotide sequence database library under accession numbers ΔJ310269–ΔJ310306.

DNA analysis

The sequences were multiply aligned using the CLUSTAL W program (Thompson et al., 1994) and edited with the MacClade program version 3.0.6 (Maddison & Maddison, 1992). The alignment was further optimized manually.

The DnaSP version 3.5 software (Rozas & Rozas, 1999) was used for the analysis of polymorphism and genetic differentiation. This program was also used to perform Tajima’s test (Tajima, 1989) and Fu and Li’s tests (Fu & Li, 1993). The significance of the corresponding test statistics was established by computer simulation using the coalescent algorithm without recombination.

The level of polymorphism was estimated as the number of polymorphic sites (S), the average number of pairwise nucleotide differences (k), nucleotide diversity (π; Nei, 1987) and expected heterozygosity per site or Watterson’s estimator (θ; Watterson, 1975). Genetic differentiation between species, populations or gene arrangements was estimated as the average number of nucleotide substitutions per site between groups (dxy). The statistical significance of genetic differentiation between groups, as estimated by Ks*, was established by the permutation test (Hudson et al., 1992a). The proportion of nucleotide diversity attributable to variation between populations, Fst, was calculated according to Hudson et al. (1992b). Fst was used to estimate the migration parameter Nm, under the island model of population structure and assuming migration-drift equilibrium (Wright, 1951; Hudson et al., 1992b).

Recombination events were identified by the four-gamete test (Hudson & Kaplan, 1985). Linkage disequilibrium was analysed between parsimony informative sites and the statistical significance of pairwise associations was obtained by the χ2 test. The Bonferroni procedure was used to correct for multiple tests (Weir, 1996).

The neighbour-joining method (Saitou & Nei, 1987), as implemented in the PAUP program (Swofford, 1998), was used for phylogenetic reconstruction using genetic distances corrected for multiple hits (Jukes & Cantor, 1969). Bootstrap values were obtained from 500 replicates.

DNA divergence models

We applied the method developed by Wakeley (1996a,b) to test whether the pattern of nucleotide variation fits to that expected under the isolation model of divergence. In this model, a single ancestral population splits into two descendent populations that remain isolated from each other for some period of time. One of the alternative models is the so-called two-population equilibrium migration model, where gene flow between populations can occur and therefore populations are not completely isolated. The isolation model (Wakeley, 1996a,b; Wakeley & Hey, 1997) can be described by four parameters: θ1, θ2, θA and τ (τ=2μt). θ1, θ2, θA are the θ values per sequence for populations 1, 2 and for the ancestral population, respectively; μ is the mutation rate per sequence per generation; and t is the time of separation measured in generations. T, the isolation time in 2N generations (where N is the effective population size), can be easily obtained from T=τ/θ1. These parameters can be estimated from four categories of sites: the number of shared polymorphic sites (SS), the number of exclusive polymorphic sites in each species (SX1 and SX2), and the number of fixed differences between species (SF). For the analysis, we refer to D. madeirensis as species 1, and to D. subobscura as species 2.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Nucleotide variation in Drosophila subobscura from Madeira

The rp49 region was sequenced in 16 lines of D. subobscura from Madeira. All lines were homozygous for the O3+4 chromosomal arrangement. The multiple alignment included 1508 sites after excluding all sites with alignment gaps. A summary of polymorphism is given in Table 1 and Fig. 1. There were 14 different haplotypes and haplotype diversity was 0.983. A total of 7 length and 46 nucleotide polymorphisms were detected. All length polymorphisms were in noncoding regions (intron and flanking regions). Two length polymorphisms could be considered complex mutational events, and the rest were microsatellites. All nucleotide polymorphisms were silent. In 17 of these polymorphisms (37%), the rarest variant was a singleton, while 29 were parsimony informative sites (28 with two variants and one with three variants). The estimated nucleotide diversity (π) was lower than the estimated heterozygosity per site (θ) (Table 1), reflecting the relatively high percentage of sites with singletons in the sample.

Table 1.   Nucleotide polymorphism in Drosophila subobscura and D. madeirensis. Thumbnail image of
image

Figure 1.  Nucleotide polymorphism at the rp49 gene region in Drosophila subobscura and D. madeirensis. Nucleotide numbering is according to Aguadé (1988). The first sequence corresponds to line J1 in Rozas & Aguadé (1994) that presents the Ost chromosomal arrangement. Lines of D. subobscura from Madeira are designated as S and those of D. madeirensis as M. Nucleotides identical to the first sequence are indicated by a dot. Length polymorphisms are not shown. A deleted nucleotide in a given polymorphic site is indicated by a gap. Polymorphic sites 27–846 correspond to the 5′ flanking region, site 944 to exon 1, sites 974–1009 to the intron, 1036–1297 to exon 2, 1388–1534 to the 3′ flanking region, and polymorphic site 1550 to the serendipity coding region.

Download figure to PowerPoint

Different tests of neutrality (Tajima, 1989; Fu & Li, 1993) were performed (Table 1). None of the tests detected a significant departure from neutral expectations in a stationary population. The test statistics were in all cases negative, which is an indication of the observed excess of low-frequency variants.

The minimum number of recombination events, as estimated by the four-gamete test (Hudson & Kaplan, 1985), was 7. In the analysis of linkage disequilibrium that included only parsimony informative sites, 54 out of 378 pairwise comparisons (14%) showed a significant association by the χ2 test (P < 0.05). Using the Bonferroni correction for multiple tests (Weir, 1996), this number dropped to 10 (3%). There was a negative relationship between the degree of linkage disequilibrium, as estimated by r 2 (Hill & Robertson, 1968), and physical distance between sites.

Genetic differentiation between D. subobscura populations from Madeira and Spain

Nucleotide variation in the D. subobscura sample from Madeira that was monomorphic for the O3+4 chromosomal arrangement was compared to variation in lines with the same gene arrangement of a population from Galicia (Spain). A summary of a previously published analysis of polymorphism in that population (Rozas & Aguadé, 1994; Rozas et al., 1999) is also given in Table 1.

The level of nucleotide diversity in the lines from Madeira (O3+4 M in Table 1) was similar to that in the O3+4 lines from Spain (O3+4 G in Table 1), but higher than in the Spanish Ost lines. Despite the similar level of nucleotide diversity in the O3+4 lines from Madeira and from Galicia, the percentage of singletons was higher in this latter sample (50%). Haplotype diversity in the sample from Madeira (0.983) was only slightly lower than in both the O3+4 (1.0) and Ost (1.0) lines from Spain.

Genetic differentiation between O3+4 lines from Madeira and Galicia was very low as measured by dxy (0.0081). In fact, there was a high proportion of shared polymorphisms, or sites segregating for the same two nucleotides, between these lines (32 out of 68 polymorphic sites in the combined sample) and no fixed differences between populations. No significant genetic differentiation between O3+4 lines from both locations was detected by the permutation test (Hudson et al., 1992a). In contrast, the sample from Madeira, like the sample of O3+4 lines from Galicia (Rozas & Aguadé, 1994; Rozas et al., 1999), was genetically differentiated from the Ost lines from Galicia (P < 0.001 for the Ks* estimator).

The lack of genetic differentiation between O3+4 lines is also reflected in the neighbour-joining tree built using the D. guanche sequence (Ramos-Onsins et al., 1998) as the outgroup (Fig. 2). In fact, O3+4 lines from both Madeira and Galicia are interspersed in the tree. On the other hand, Ost lines from Galicia form a separate cluster except for line J11, for which there is evidence of gene conversion from O3+4 (see Rozas & Aguadé, 1994).

image

Figure 2.  Neighbour-joining tree of the rp49 gene region sequences of Drosophila subobscura and D. madeirensis using D. guanche as the outgroup. Bootstrap values based on 500 replicates are given on the main nodes. The scale bar represents 0.005 nucleotide substitutions per site. O3+4D. subobscura lines from Madeira and Galicia are indicated by black and white circles, respectively.

Download figure to PowerPoint

Nucleotide variation in Drosophila madeirensis

An ~1.6-kb fragment spanning the rp49 region was sequenced in 22 lines of D. madeirensis. All lines were homozygous for the O3 chromosomal arrangement. There were 21 different haplotypes and the haplotype diversity was 0.996. A total of 4 length and 55 nucleotide polymorphisms were detected (Table 1 and Fig. 1). Like in D. subobscura, all length polymorphisms were in noncoding regions and all nucleotide polymorphisms were silent. Two length polymorphisms could be considered complex mutational events and the other two were microsatellites (Fig. 1). In 22 of the 55 polymorphic sites detected (40%), the rarest variant was a singleton, and the other 33 were parsimony informative sites (one of them with three variants).

Different neutrality tests based on polymorphism data (Tajima, 1989; Fu & Li, 1993) were performed. Like in D. subobscura, none of the tests showed a significant deviation from neutral expectations, although in all cases the test statistics were negative (Table 1).

A minimum of 13 recombination events was inferred by the four-gamete test (Hudson & Kaplan, 1985) in the history of the sample studied. In 51 out of 496 pairwise comparisons between polymorphic sites (10%), the variants were significantly associated or in linkage disequilibrium. Using the Bonferroni correction for multiple tests (Weir, 1996), this number dropped to 6 (1%).

Differentiation between D. subobscura and D. madeirensis

The level of polymorphism, measured as nucleotide diversity or π, was comparable in the samples of D. madeirensis and of D. subobscura from Madeira (Table 1). Estimated nucleotide diversity in D. madeirensis was consequently similar to that in the O3+4 lines from Galicia and higher than that in the Ost lines from that population. The percentage of singletons was rather similar in the samples of D. subobscura from Madeira and of D. madeirensis (37 and 40%, respectively), and lower than in the O3+4 and Ost lines from Galicia.

The degree of genetic differentiation, as estimated by dxy, between D. madeirensis and any of the three samples from D. subobscura (O3+4 M, O3+4 G and Ost) was rather similar (Table 2). The estimated Fst values for the three comparisons between D. madeirensis and D. subobscura were rather high. The permutation test (Hudson et al., 1992a) revealed a significant genetic differentiation between both species. The number of fixed differences was, however, rather low (Table 2). Most fixed differences between species were due to mutations in a short stretch of the 5′ flanking region (between sites 27 and 68 in Fig. 1). When this part was excluded from the analyses, the number of fixed differences dropped to 1 in all three comparisons.

Table 2.   Genetic differentiation between Drosophila subobscura and D. madeirensis. Thumbnail image of

Despite the presence of shared polymorphisms between D. madeirensis and D. subobscura (Table 2), all D. madeirensis sequences clustered in the neighbour-joining tree obtained with all the D. subobscura and D. madeirensis sequences and using D. guanche as the outgroup (Fig. 2). This cluster was supported by a much higher bootstrap value (87%) than the O3+4 and Ost clusters (51% and 53%, respectively).

Speciation models

The ψ-test statistic, proposed by Wakeley (1996b), was computed to distinguish between the isolation and migration models. Present data for D. madeirensis and D. subobscura do not allow rejection of the isolation model (ψ=0.22; P[ψ ≥ 0.22]=1.0). We applied the Wakeley & Hey (1997) formulas to obtain estimates of θ1, θ2, θA and T (Table 3). This analysis is based on the infinite-sites model. Under that assumption, shared polymorphisms represent polymorphisms present in the ancestral population. However, shared polymorphisms could also have arisen by the independent accumulation of mutations in each species (i.e. by parallel mutation). Therefore, we first tested whether the observed number of shared polymorphisms could be explained by parallel mutation. Assuming that the number of shared polymorphisms follows a hypergeometric distribution (Rozas & Aguadé, 1994), and that each nucleotide can mutate to three alternative states, we rejected the null hypothesis that shared polymorphisms were the result of parallel mutations (P[X ≥ 4]=0.0012). The similar estimates obtained for θ1, θ2 and θA (Table 3) would indicate that the ancestral population size was similar to that of the two extant descendent populations (D. madeirensis and D. subobscura, respectively). In addition, the estimated time of split indicates that these species have been isolated for a rather long time (Table 3).

Table 3.   Estimates of population parameters from the isolation model. Thumbnail image of

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Drosophila subobscura from Madeira

Populations of D. subobscura from Madeira, like those from the Canary Islands, are nearly monomorphic for the O3+4 chromosomal arrangement (Prevosti, 1971; Larruga et al., 1983). This is in contrast with the rich chromosomal polymorphism that most other populations harbour for this chromosome (Krimbas, 1992). Furthermore, the absence in the islands of arrangements present in high frequency in nearby continental populations (e.g. O3+4+8 in North-western Africa and O3+4+7 in the Atlantic coast of the Iberian peninsula) does not seem to favour the hypothesis of recent migration from the continent. Otherwise, selection against the establishment of these other arrangements in the islands should be very strong given their high frequency in those continental populations (see Khadem et al., 1998).

According to present results, the level of nucleotide variation in the rp49 gene region is similar in O3+4 lines from Madeira and the continent. Additionally, O3+4 lines from these locations are not genetically differentiated for that region. Some differentiation had been, however, detected in a previous restriction-map survey (Khadem et al., 1998). The discrepancy between the results of these studies might be partly due to the relatively low number of nucleotides sampled in the previous study and therefore to the high stochastic variance associated with nucleotide variation estimates.

Available data for chromosomal, mtDNA and rp49 variation in D. subobscura populations from Madeira and the continent do not support the hypothesis that extant populations from Madeira are the descendents of a single colonization event occurring soon after the origin of O3+4 (Khadem et al., 1998). Two different scenarios would be compatible with available data: (i) a rather recent and massive colonization of Madeira by continental D. subobscura, and (ii) multiple colonization events from the continent. In both scenarios, selection would have precluded the establishment in Madeira of chromosomal arrangements other than O3+4 (see Khadem et al., 1998). Although we cannot ascertain which scenario most likely reflects the origin of extant D. subobscura populations in Madeira, the similar level of nucleotide variation detected within O3+4 in insular and continental populations allows us to assert that the colonization of Madeira was not associated with a strong founder event.

Comparison of DNA variation between D. madeirensis and D. subobscura

The rp49 region is the first nuclear region whose variation has been analysed in a natural population from the endemic species D. madeirensis. Endemic species inhabiting rather small islands are expected to have a lower effective population size than closely related species with a worldwide distribution. Therefore, under the strict neutral model (Kimura, 1983), a lower level of nucleotide variation would be expected in endemic insular species than in mainland species. Comparison of nucleotide variation at the rp49 gene region between D. madeirensis and D. subobscura is not in agreement with that prediction. In fact, the estimated nucleotide diversity in D. madeirensis, which is monomorphic for the O3 arrangement, was similar to that estimated for O3+4 and slightly higher than for the Ost chromosomal arrangement (Table 1). The high level of nucleotide variation in D. madeirensis might be explained if ancestral populations of this species were much larger than current populations and the species had suffered a reduction in population size. This reduction could be associated with the colonization and expansion of D. subobscura in Madeira after the origin of D. madeirensis; it could also be associated with destruction of the natural habitat of D. madeirensis (laurisilva forest) occurred during the last 400 years (Doria, 1945; Frutuoso, 1979). In any case, ancestral populations of this insular species would probably be not as large as continental populations of D. subobscura. Nevertheless, the level of variation in D. madeirensis clearly indicates that the origin of this species was not associated with a strong founder event. In fact, D. madeirensis and D. subobscura exhibit a comparable level of nucleotide variation in the rp49 region and they also present a similar frequency spectrum, as measured by Tajima’s D and Fu and Li’s D and F statistics.

Present data on nucleotide variation in D. madeirensis and D. subobscura have been compared to those available for other closely related Drosophila species pairs with one insular representative. Two such pairs can be found in the triad formed by the cosmopolitan species D. simulans and the endemic species D. sechellia and D. mauritiana (Hey & Kliman, 1993; Kliman & Hey, 1993). These authors analysed nucleotide variation at the period, zeste and yolk protein 2 gene regions and found that D. simulans and D. mauritiana showed comparable levels of variation. In contrast, D. sechellia exhibited a much lower nucleotide variation than D. simulans. Our results showing an a priori unexpected high level of variation in the insular species (D. madeirensis) are quite similar to those reported for the D. simulans and D. mauritiana pair. Nevertheless, the expected positive correlation between heterozygosity and effective population size has been, and still is, a controversial issue in population genetics (Lewontin, 1974; Maynard Smith & Haigh, 1974; Gillespie, 1999, 2000). According to the strict neutral mutation model, the expected heterozygosity is a function of the effective population size (Kimura, 1983). Under several selection models, however, heterozygosity is nearly independent of the population size (Gillespie, 1999). For example, under some deleterious mutation models, heterozygosity is rather insensitive to the population size, and is thus mainly a function of the mutation rate (Gillespie, 1999). This insensitivity is also predicted by the pseudohitchhiking model (Gillespie, 2000) that considers the effect of advantageous mutations on the dynamics of neutral variation at a closely linked locus. Under this model, heterozygosity can even decrease with increasing population size.

Some shared polymorphisms between species were detected both in the sequence comparison of the period locus between D. simulans and D. mauritiana, and of the rp49 region between D. madeirensis and D. subobscura. In both cases, the data were compatible with the isolation model, where shared polymorphisms are due to common ancestry. However, shared polymorphisms between closely related species could also be due to the introgression resulting from rare hybridization between species. In fact, it has been recently proposed that, for regions not involved in reproductive isolation, introgressive hybridization might be more important than previously thought (Ting et al., 2000). If that were the case for the rp49 region, some of the observed shared variants could have originated by mutation in one of the descendent lineages after the split of the species, and they would have entered the second species gene pool by introgression. Although some hybrids between D. madeirensis and D. subobscura have been detected in recent collections (N. Khadem, unpublished results), this observation is not a proof of introgression. In addition, for a neutrally evolving gene, the relatively high number of fixed differences observed between species seems unlikely under the introgression scenario.

As in the species studied, the rp49 gene is located in a region affected by chromosomal inversions, there is an additional difficulty for explaining the presence of shared polymorphisms both if they are due to common ancestry or to introgressive hybridization. In fact, the unique origin of an inversion would a priori preclude the existence of shared polymorphisms between the ancestral and derived chromosomal arrangements. In our case, D. madeirensis is monomorphic for the ancestral O3 arrangement that went extinct in the D. subobscura lineage, while extant populations of D. subobscura segregate for the O3+4 and Ost arrangements that originated from O3. Therefore, gene transfer between arrangements, either by double crossing over or gene conversion, would be required to explain the presence of shared polymorphisms between species (Rozas & Aguadé, 1994; Rozas et al., 1999). In this case, the extent of shared polymorphism might be lower at the rp49 region than in other regions not affected by chromosomal inversions. Only analysis of multiple regions will allow establishing the effect of chromosomal polymorphism on the number of shared polymorphisms between D. madeirensis and D. subobscura. Additionally, the comparative analysis of nucleotide variation in multiple regions of D. subobscura and D. madeirensis will improve our understanding of the speciation process in Madeira.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We thank John Wakeley for sharing computer programs, Gema Blasco and David Salguero for technical support, and Serveis Científico-Tècnics, Universitat de Barcelona, for automated sequencing facilities. This work was supported by grants PB97-0918 from Comisión Interdepartamental de Ciencia y Tecnología, Spain, and 1999SGR-25 from Comissió Interdepartamental de Recerca i Innovació Tecnològica, Catalonia, Spain, to M.A., and by CITMA (Centro de Ciências e Tecnologia da Madeira) to M.K.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  • 1
    Afonso, J.M., Volz, A., Hernández, M., Ruttkay, H., González, A.M., Larruga, J.M., Cabrera, V.M., Sperlich, D. 1990. Mitochondrial DNA variation and genetic structure in Old-World populations of Drosophila subobscura. Mol. Biol. Evol. 7: 123142.
  • 2
    Aguadé, M. 1988. Nucleotide sequence comparison of the rp49 gene region between Drosophila subobscura and D. melanogaster. Mol. Biol. Evol. 5: 433441.
  • 3
    Ashburner, M. 1989. Drosophila: a Laboratory Handbook. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
  • 4
    Baez, M. 1993. Origin and affinities of the fauna of Madeira. Boletim Do Museu Municipal Do Funchal. Supplement 2: 940.
  • 5
    Doria, A.A. 1945. Estudos de história dos descobrimentos. O problema do descobrimento da Madeira. Guimarães, Portugal.
  • 6
    Frutuoso, G. 1979. Livro segundo das saudades da Terra. Ponto delgada, Portugal.
  • 7
    Fu, Y.-X. & Li, W.-H. 1993. Statistical tests of neutrality of mutations. Genetics 133: 693709.
  • 8
    Galopin De Carvalho, A. & Brandão, J. 1991. Geologia do Archipélago da Madeira. Museu Nacional de História Natural, Universidade de Lisboa.
  • 9
    Gillespie, J. H. 1999. The role of population size in molecular evolution. Theor. Popul. Biol. 55: 145156.
  • 10
    Gillespie, J. H. 2000. Genetic drift in an infinite population: the pseudohitchhicking model. Genetics 155: 909919.
  • 11
    González, A.M., Cabrera, V.M., Larruga, J.M., Gullón, A. 1983. Molecular variation in insular endemic Drosophila species of the Macaronesian archipelagos. Evolution 37: 11281140.
  • 12
    González, A.M., Hernández, M., Volz, A., Pestano, J., Larruga, J.M., Sperlich, D., Cabrera, V.M. 1990. Mitochondrial DNA evolution in the obscura species subgroup of Drosophila. J. Mol. Evol. 31: 122131.
  • 13
    Hey, J. & Kliman, R.M. 1993. Population genetics and phylogenetics of DNA sequence variation at multiple loci within the Drosophila melanogaster species complex. Mol. Biol. Evol. 10: 804822.
  • 14
    Hill, W.G. & Robertson, A. 1968. Linkage disequilibrium in finite populations. Theor. Appl. Genet. 38: 226231.
  • 15
    Hudson, R.R., Boos, D.D., Kaplan, N.L. 1992a. A statistical test for detecting geographic subdivision. Mol. Biol. Evol. 9: 138151.
  • 16
    Hudson, R.R. & Kaplan, N.L. 1985. Statistical properties of the number of recombination events in the history of a sample of DNA sequences. Genetics 111: 147164.
  • 17
    Hudson, R.R., Slatkin, M., Maddison, W.P. 1992b. Estimation of levels of gene flow from DNA sequence data. Genetics 132: 583589.
  • 18
    Jukes, T.H. & Cantor, C.R. 1969. Evolution of protein molecules. In: Mammalian Protein Metabolism (H. W. Munro, ed.), pp. 21–120. Academic Press, New York.
  • 19
    Khadem, M. & Krimbas, C.B. 1991. Studies of the genetic barrier between Drosophila subobscura and D. madeirensis. I. The genetics of male sterility. Heredity 67: 157165.
  • 20
    Khadem, M., Rozas, J., Segarra, C., Brehm, A., Aguadé, M. 1998. Tracing the colonization of Madeira and the Canary Islands by Drosophila subobscura through the study of the rp49 gene region. J. Evol. Biol. 11: 439452.
  • 21
    Kimura, M. 1983. The Neutral Theory of Molecular Evolution. Cambridge University Press, Cambridge, UK.
  • 22
    Kliman, R.M. & Hey, J. 1993. DNA sequence variation at the period locus within and among species of the Drosophila melanogaster complex. Genetics 133: 375387.
  • 23
    Krimbas, C.B. 1992. The inversion polymorphism of Drosophila subobscura. In: Drosophila Inversion Polymorphism (C. B. Krimbas & J. R. Powell, eds), pp. 127–220. CRC Press, Boca Raton.
  • 24
    Larruga, J.M., Cabrera, V.M., González, A.M., Gullón, A. 1983. Molecular and chromosomal polymorphism in continental and insular populations from the southwestern range of Drosophila subobscura. Genetica 60: 191205.
  • 25
    Lewontin, R.C. 1974. The Genetic Basis of Evolutionary Change. Columbia University Press, New York.
  • 26
    Maddison, W.P. & Maddison, D.R. 1992. Macclade: Analysis of Phylogeny and Character Evolution, Version 3.0. Sinauer, Sunderland, Mass.
  • 27
    Maynard Smith, J. & Haigh, J. 1974. The hitch-hiking effect of a favourable gene. Genet. Res. 23: 2335.
  • 28
    Monclús, M. 1984. Drosophilidae of Madeira, with the description of Drosophila madeirensis n. sp. Z. f. Zool. System. u. Evol. 22: 94103.
  • 29
    Nei, M. 1987. Molecular Evolutionary Genetics. Columbia University Press, New York.
  • 30
    Papaceit, M., San Antonio, J., Prevosti, A. 1991. Genetic analysis of extra sex combs in the hybrids between Drosophila subobscura and D. madeirensis. Genetica 84: 107114.
  • 31
    Pinto, F.M., Brehm, A., Hernández, M., Larruga, J.M., González, A.M., Cabrera, V.M. 1997. Population genetic structure and colonization sequence of Drosophila subobscura in the Canaries and Madeira Atlantic Islands as inferred by autosomal, sex-linked and mtDNA traits. J. Hered. 88: 108114.
  • 32
    Press, J.R. & Short, M.J. 1994. Flora of Madeira. HMSO, London.
  • 33
    Prevosti, A. 1971. Chromosomal polymorphism in Drosophila subobscura Coll. populations from the Canary Islands. Genét. Ibér. 23: 6984.
  • 34
    Prevosti, A. 1972. Chromosomal polymorphism in Drosophila subobscura populations from the Madeira island. Genét. Ibér. 24: 1121.
  • 35
    Prevosti, A. 1974. Chromosomal inversion polymorphism in the southwestern range of Drosophila subobscura distribution area. Genetica 45: 111124.
  • 36
    Ramos-Onsins, S., Segarra, C., Rozas, J., Aguadé, M. 1998. Molecular and chromosomal phylogeny in the obscura group of Drosophila inferred from sequences of the rp49 gene region. Mol. Phylogenet. Evol. 9: 3341.
  • 37
    Rozas, J. & Aguadé, M. 1994. Gene conversion is involved in the transfer of genetic information between naturally occurring inversions of Drosophila. Proc. Natl. Acad. Sci. USA 91: 1151711521.
  • 38
    Rozas, J. & Rozas, R. 1999. DnaSP, Version 3: an integrated program for molecular population genetics and molecular evolution analysis. Bioinformatics 15: 174175.
  • 39
    Rozas, J., Segarra, C., Ribó, G., Aguadé, M. 1999. Molecular population genetics of the rp49 gene region in different chromosomal inversions of Drosophila subobscura. Genetics 151: 189202.
  • 40
    Rozas, J., Segarra, C., Zapata, C., Alvarez, G., Aguadé, M. 1995. Nucleotide polymorphism at the rp49 region of Drosphila subobscura: Lack of geographic subdivision within chromosomal arrangements in Europe. J. Evol. Biol. 8: 355367.
  • 41
    Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B., Erlich, H.A. 1988. Primer-directed enzymatic amplification of DNA with a thermostable polymerase. Science 239: 487491.
  • 42
    Saitou, N. & Nei, M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406425.
  • 43
    Swofford, D.L. 1998. Paup: Phylogenetic Analysis Using Parsimony, Version 4.0. Sinauer Associates, Inc., Sunderland, MA.
  • 44
    Tajima, F. 1989. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123: 585595.
  • 45
    Thompson, J.D., Higgins, D.G., Gibson, T.J. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl. Acids Res. 22: 46734680.
  • 46
    Ting, C.T., Tsaur, S.C., Wu, C.-I. 2000. The phylogeny of closely related species as revealed by the genealogy of a speciation gene, Odysseus. Proc. Natl. Acad. Sci. USA 97: 53135316.
  • 47
    Wakeley, J. 1996a. The variance of pairwise nucleotide differences in two populations with migration. Theor. Popul. Biol. 49: 3957.
  • 48
    Wakeley, J. 1996b. Distinguishing migration from isolation using the variance of pairwise differences. Theor. Popul. Biol. 49: 369386.
  • 49
    Wakeley, J. & Hey, J. 1997. Estimating ancestral population parameters. Genetics 145: 847855.
  • 50
    Watterson, G.A. 1975. On the number of segregating sites in genetical models without recombination. Theor. Popul. Biol. 7: 256276.
  • 51
    Weir, B.S. 1996. Genetic Data Analysis II. Sinauer Associates, Inc., Sunderland, MA.
  • 52
    Wright, S. 1951. The genetical structure of populations. Ann. Eugen. 15: 323354.