Microsatellite Variation in Colonizing and Palearctic Populations of Drosophila subobscura

Marta Pascual, Charles F. Aquadro, Vanessa Soto and Luis Serra*

Departament de Genètica, Universitat de Barcelona, Barcelona, Spain; and
Department of Molecular Biology and Genetics, Cornell University


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
The recent colonization of North America by Drosophila subobscura has provided a great opportunity to analyze a colonization process from the beginning. A comparative study using 10 microsatellite loci was conducted for five European and two North American populations. No genetic differentiation between European populations was detected, indicating that gene flow is high among them and that the microsatellites used in the present work represent neutral markers not subject to differentiation due to selection. Extensive reduction in the number of alleles and a significant decrease in heterozygosity in colonizing populations were detected that could be explained by the founder effect and a subsequent quick but not infinite expansion. Assuming that all alleles present in the colonized area were carried by the sample of colonizers, we estimated that most probably 4–11 individuals expanded in the new area. FST and the chord distance measures reflect the colonization process more accurately, since drift has been the major force in differentiating the Old and New World populations, and thus other measures considering allele size differences, such as RhoST and {delta}µ, are less reliable for studying nonequilibrium populations. Finally, our results were consistent with the two-phase microsatellite mutational model, indicating that most alleles are generated by gain or loss of a repeat unit, while some alleles originate by more complex mutations.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Species recently and successfully invading new environments provide a great opportunity to analyze a colonization process from the beginning. Drosophila subobscura, a typical Palearctic species, colonized South and North America 2 decades ago. It was detected for the first time in North America in 1982 (Beckenbach and Prevosti 1986Citation ) and appears not to have been introduced before 1975 (Ayala, Serra, and Prevosti 1989Citation ). Its quick expansion and subsequent maintenance in natural populations indicate that it has perfectly established in the new area (Prevosti et al. 1989Citation ; Pascual et al. 1993Citation ; Noor et al. 1998Citation ). Drosophila subobscura has very rapidly adapted to the new environment and, as a consequence, has developed clines for some chromosomal arrangements (Prevosti et al. 1988Citation ) and a body size (Huey et al. 2000Citation ) similar to those found in Old World populations.

The analyses of North American populations for chromosomal arrangements (Prevosti et al. 1988Citation ), allozymes (Balanyà et al. 1994Citation ), mtDNA restriction patterns (Latorre, Moya, and Ayala 1986Citation ; Rozas et al. 1990Citation ), and rp49 region restriction patterns (Rozas and Aguadé 1991Citation ) reflect an impoverishment of genetic variation in the colonizing populations. All of these markers are polymorphic but not highly variable; a few alleles are very frequent and the rest are very rare in Palearctic populations, and only those alleles common in the Old World have been detected in North America. This indicates that the number of colonizers was not very large and has been estimated from 3–149 individuals using different techniques (Prevosti et al. 1989Citation ; Mestres et al. 1990Citation ; Rozas and Aguadé 1991Citation ). The origin of the colonizers still remains unknown; although the high degree of chromosomal polymorphism suggests a western Mediterranean origin, the presence of the O5 arrangement does not support this hypothesis (Prevosti et al. 1988Citation ; Ayala, Serra, and Prevosti 1989Citation ).

Microsatellite loci are highly polymorphic markers, distributed throughout the nuclear genome and generally neutral unless linked to loci under strong selection. The characterization of these markers in D. subobscura confirms its high variability (Pascual, Schug, and Aquadro 2000Citation ) and therefore makes them good candidates for the study of population differentiation and the colonization process. The objective of this study was to investigate, using microsatellites, the genetic variation in European and North American populations of D. subobscura. Is there genetic differentiation among Old World populations that could help us to trace the origin of the colonizers, or is gene flow sufficiently high to homogenize the gene pool in the Palearctic region in spite of the clines observed for some chromosomal arrangements? We estimated the number of colonizers and compared these results with those obtained with other genetic markers. Finally, we made inferences on the mutational process by comparison of microsatellite data between European and colonizing populations.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Population Samples
Drosophila subobscura flies were sampled in Europe and in North America in north–south transects. The five Palearctic localities sampled were Århus, Denmark (56.10°N, 10.13°E); Lille, France (50.39°N, 3.05°E); Montpellier, France (43.36°N, 3.53°E); Barcelona, Spain (41.25°N, 2.10°E); and Málaga, Spain (36.43°N, 4.25°E). The Nearctic localities from the United States were Bellingham, Wash. (48.45°N, 122.29°W), and Fort Bragg, Calif. (39.29°N, 123.43°W). The known latitudinal range occupied by D. subobscura in North America is smaller than that in the Palearctic region. The two North American populations were chosen because they were located at two points fairly distant within the North American distribution range of the species; furthermore, in these localities, the species is quite abundant, and wild individuals were available to carry out the analysis. The flies analyzed were collected in the spring of 1998 with the exception of those from Montpellier and Fort Bragg, which were collected in the autumn of 1997 and the spring of 1994, respectively. All of the specimens used in our study were wild individuals kept in ethanol or first-generation progeny of wild females; in the latter case, only one individual per isofemale line was used.

Amplification and Detection of Microsatellites
The 10 microsatellites surveyed in this study were developed from a D. subobscura subgenomic library and were the following: dsub01, dsub02, dsub04, dsub05, dsub16, dsub18, dsub19, dsub20, dsub21, and dsub27 (Pascual, Schug, and Aquadro 2000Citation ); dsub05, dsub19, and dsub21 were X-linked, and the rest were autosomal. DNA was extracted from single-fly squish preps (Gloor et al. 1993Citation ) of each line. Individuals preserved in ethanol were washed in TE overnight prior extraction. One primer of each locus was fluorescently end-labeled with 6-Fam, Hex, or Ned. We amplified dusb01, dsub18, dsub19, and dsub21; dsub02 and dsub05; and dsub04, dsub20, and dsub27 simultaneously because the results obtained were the same as those when they were amplified separately. The polymerase chain reaction (PCR) was conducted in a 20-µl reaction volume with 0.5 pmol of each primer (10 µM), 3 µl dNTP's (1 mM), 2 µl 10 x buffer, 1 U Taq polymerase (Amersham Pharmacia Biotech), and 1 µl of DNA. The total amount of primer was always the same; thus, when several loci were amplified simultaneously, the amount for each primer-locus was divided by the number of loci multiplexed. A single soak at 95°C for 5 min was followed by 30 cycles of 1 min at 95°C, 30 s at 57°C, and 30 s at 72° on a Perkin-Elmer 2400 machine. After visualization of the product in an agarose gel, the amplification reaction was diluted when necessary, mixed with 12 µl of deionized formamide and 0.5 µl of internal size marker (GeneScan-350 ROX), and loaded on an ABI PRISM 310 automated sequencer. Allele sizes were calculated using the software program GeneScan, version 3.1, and Genotyper, version 2.5 (Perkin Elmer), available from the Serveis Científico Tècnics at the University of Barcelona.

Data Analysis
Genetic diversity was quantified by the mean number of alleles per locus (nA), the observed heterozygosity (Hobs), the Hardy-Weinberg expected heterozygosity (Hexp) (Nei 1978Citation ), and the variance in repeat number (Var(nA)). Each locus in each population was tested for equilibrium and the Markov chain method was used to estimate the exact P value. For the Århus sample, gametic linkage disequilibrium between X-linked loci was assessed instead of genotypic linkage disequilibrium, since males represented 90% of the individuals analyzed. Global tests for linkage disequilibrium between all possible pairs of microsatellite loci were carried out on pooled samples for each continent. These analyses were carried out with the program GENEPOP, version 3.2 (Raymond and Rousset 1995Citation ).

For estimation of genetic variability, allele length was transformed to repeat unit number by comparison with the length of the clone in the library screen (Pascual, Schug, and Aquadro 2000Citation ). Heterogeneity of microsatellite allele frequencies was tested among all population pairs using an unbiased estimate of Fisher's exact test; a Markov chain was set with 200 batches of 1,000 iterations each to determine the exact P value using the program GENEPOP, version 3.2 (Raymond and Rousset 1995Citation ). Wright's F-statistic was estimated according to Weir and Cockerham (1984)Citation using the program GENEPOP, version 3.2 (Raymond and Rousset 1995Citation ). Taking into account the difference between allele sizes, RhoST, an unbiased version of RST (Slatkin 1995Citation ) in which allele sizes are transformed to standardize variances, was calculated using the program RST CALC, version 2.2 (Goodman 1997Citation ). Some alleles showed sizes that could not be explained by the gain or loss of whole repeat units and were probably due to insertions/deletions in the microsatellite-flanking region. Alleles differing by less than one repeat unit were pooled with the nearest integer when FST and RhoST between populations were computed. Rounding was done to include most abnormal alleles to the most frequent classes. Only autosomal loci were used to estimate genetic differentiation between populations. Statistical significance levels were determined applying a sequential Bonferroni correction (Rice 1989Citation ).

Isolation by distance among European populations was analyzed by regressing XST/(1 - XST) to the natural logarithm of their geographic distance, where XST is either FST or RhoST. The significance of the regression was determined after 5,000 permutations of a Mantel test using GENEPOP. Estimates of gene flow between population pairs were calculated according to Slatkin (1995)Citation , using his equations (15a) for RhoST and (15b) for FST. The number of chromosomes carried by the sample of colonizers was estimated by bootstrapping the probability of observing a genetic distance (1 - Psa, where Psa is the proportion of shared alleles between individuals; see Bowcock et al. 1994Citation ) larger or smaller than that empirically observed. These simulations, using the program MULTSIM (Noor, Pascual, and Smith 2000Citation ), allowed the calculation of the maximum and minimum numbers of colonists for each locus.

To assess the genetic affinities between the seven analyzed populations, two unrooted neighbor-joining trees were constructed with the autosomal loci using the chord distance of Cavalli-Sforza and Edwards (1967)Citation and {delta}µ2, a distance developed for the stepwise mutation model (Goldstein et al. 1995Citation ). Neighbor-joining trees were constructed using the (unpublished) computer package Treemake, kindly provided by S. Piry and J. M. Cornuet (CBGP-INRA, Montpellier, France) and thoroughly tested by the authors on many microsatellite data sets. Percentage bootstrap values were computed over 2,000 replications.

Effective population sizes (Ne) were estimated from all microsatellite data using heterozygosity assuming two different mutational models: the stepwise mutation model (SMM: H = 1 - [1/(1 + 8Neµ)½]; Ohta and Kimura 1973)Citation and the infinite-alleles model (IAM: H = 4Neµ/(1 + 4Neµ); Kimura and Crow 1964Citation ). The dinucleotide repeat mutation rate of Drosophila melanogaster (9.3 x 10-6 per locus per generation) empirically determined by Schug et al. (1998)Citation was used, since it produces estimates of Ne concordant with those obtained from sequences of single nuclear genes (Pascual, Schug, and Aquadro 2000Citation ). A similar calculation was done using the variance in repeat number (Var(nA)), for which Slatkin (1995)Citation showed that (Var(nA) = 4Neµ under a single-step stepwise mutation model (SSMM). By rearranging the equations, solving for Ne at each locus, and averaging across all 10 loci, we obtained estimates for all D. subobscura populations. Ne values for X-linked loci were adjusted assuming a 1:1 sex ratio of breeders so that their values corresponded to three fourths that of autosomes; similar results were obtained when only autosomal loci were used.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Microsatellite Variation
All European populations were genetically highly diverse, with many alleles at each locus (see table 1 and appendix). The number of alleles per locus ranged from 18 (dsub01) to 36 (dsub20) when pooling all data from Europe. All populations had private alleles, although always in low frequencies. The numbers of alleles per locus were much smaller in North America: the least variable locus (dsub05) had four alleles, whereas the most variable locus (dsub20) had eight. In North America, the average number of alleles for autosomal loci was 6.57, while it was 4.66 for X-linked loci.


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Table 1 Genetic Variation for 10 Microsatellite Loci in Palearctic and Colonizing North American Populations

 
Most of the alleles found in North America (either in one or in the two populations) were present in Europe as well. Some alleles found in North American populations were not detected in all European populations: one allele present in Fort Bragg and Bellingham was found only in Lille, another only in Montpellier, and a third one only in Málaga. Furthermore, four alleles were found only in North America (i.e., locus dsub18 in fig. 1 ). The proportion of private alleles was higher in the southern than in the northern European populations, probably due to the greater marginality of the northern localities. Presumably due to the founder effect, a few alleles having low frequencies in Europe had high frequencies in North America, while others abundant in all European populations were absent in the recently colonized area (fig. 1 , locus dsub20).



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Fig. 1.—Allele frequencies of European and North American populations for loci dsub18 and dsub20, pooling data for all localities within each continent

 
Expected heterozygosities (Hexp) were high in all populations and for all loci ranging from 0.535 at dsub18 in Bellingham to 0.949 at dsub05 in Málaga (see appendix). In all populations, the average observed heterozygosity (obs) was lower than unbiased average expected heterozygosity (exp) (table 1 ). Significant deviations from Hardy-Weinberg expectations were found for dsub16 in all populations and for dsub20 in all European populations with the exception of Århus. When these two loci were excluded in the comparison between Hobs and Hexp, statistically significant differences in heterozygosities were still obtained among European populations (Wilcoxon matched-pairs test; Z = 4.26, P < 0.001). When locus dsub16 was excluded while comparing the two North American populations, differences were not significant (Wilcoxon matched-pairs test; Z = 1.02, P = 0.309). Similarly, when we estimated the inbreeding coefficient (FIS) with GENEPOP, without loci dsub16 and dsub20, significant differences were found only among European populations.

No significant linkage disequilibrium was detected between pairs of loci in the European populations of D. subobscura. In North America, two associations were detected: dsub05 and dsub21, as well as dsub16 and dsub18, showed highly significant linkage disequilibrium as assessed by Fisher's exact test after Bonferroni correction. The first two loci were in the X-chromosome and the last two loci were autosomal (Pascual, Schug, and Aquadro 2000Citation ).

Population Differentiation
Genetic differentiation in allele frequencies for each population pair was highly significant between European and North American populations using Fisher's method for multiple P values and after Bonferroni correction (P < 0.001). Among European populations, heterogeneity in allele frequencies was detected between most locality pairs, with the exception of Montpellier compared with Århus, Barcelona, and Málaga, and between Barcelona and Málaga. Similar results were obtained when all loci or only autosomal loci were considered. Differences between European populations were mainly due to allele frequency differences at the autosomal locus dsub16. When this locus was excluded from the analysis, only two population pairs remained significantly heterogeneous (Málaga compared with the two northernmost localities). However, there was a significant genic differentiation for all European populations when dsub16 and dsub20 loci were excluded from the analysis using Fisher's exact test (P = 0.001). Significant heterogeneity detected between the two North American populations disappeared when locus dsub04 was excluded in the analysis: this result was due to the presence of a single long allele with a frequency of 0.183, found only in Fort Bragg.

Heterozygosity and variance in repeat number were not significantly different either between European or between North American populations as assessed by the nonparametric Kruskal-Wallis test. However, significant differences were detected when both continents were compared; European populations had higher heterozygosities (Mann-Whitney U-test; Z = 6.019, P < 0.001) and higher variances in repeat number (Mann-Whitney U-test; Z = 1.976, P < 0.05) than did North American populations.

RhoST and FST values between pairwise comparisons among European populations and between North American populations were low, with FST values being slightly lower (table 2 ). No significant correlation was observed between genetic differentiation and geographic distance between European populations; i.e., Århus was genetically more similar to Barcelona than to other geographically closer localities. As a result of the genetic similarity between localities, measurements of gene flow (Nm) between European populations were high (table 2 ). Barcelona and Málaga exhibited negative RhoST and FST values, indicating that variation within populations was much higher than that between populations and consequently that the populations were panmictic. In those cases, Nm was not estimated, and we assumed that the number of migrants was very high. When Nm was estimated between population pairs using the private-allele method (GENEPOP, option 4), the values obtained where slightly higher than those calculated using Slatkin's equation for RhoST. The estimated number of migrants among all European populations was 3.9 using RhoST, 5.8 with the private-allele method after correction for size, and 45.2 with FST.


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Table 2 Estimates of Genetic Differentiation between Drosophila subobscura Populations

 
We estimated RhoST and FST values between European and North American populations (table 2 ). The FST and RhoST values between each North American and each European population were very similar, although FST values were higher than RhoST values.

Figure 2 shows unrooted neighbor-joining dendrograms based on chord distance (fig. 2A ) and {delta}µ2 (fig. 2B ). The branching patterns of both trees are completely different; while the chord distance clearly separates the North American from the European populations (100% bootstrap values), with {delta}µ2 the two colonizing populations do not cluster together. However, most nodes are associated with bootstrap values lower than 50%, and only the Barcelona and Málaga group is preserved and yields values higher than 50% with both measures.



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Fig. 2.—Unrooted neighbor-joining trees relating the seven populations. Only the autosomal loci were used. The trees were constructed using (A) the chord distance of Cavalli-Sforza and Edwards (1967)Citation and (B) the {delta}µ2 distance of Goldstein et al. (1995). Bootstrap values are given as percentages over 2,000 replications

 
Table 3 gives estimates of N based on microsatellite heterozygosity under the IAM and SMM models and based on variance in repeat number under the SSMM. The values of N based on variance in repeat number were intermediate between the IAM and the SMM for the European populations. Southern European localities exhibited higher N values due to their higher diversity, being nonmarginal populations (table 1 ). Palearctic populations yielded higher N estimates than colonizing populations. However, values for North American populations were biased, since those models assume mutation-drift equilibrium, and colonizing populations are clearly too recent to be at equilibrium (Mestres, Serra, and Ayala 1995Citation ). N values based on variance in repeat number were equally high in Palearctic and colonizing populations.


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Table 3 Estimates of Effective Population Size (Ne)

 
Estimation of the Number of Colonizers
Drosophila subobscura populations have rapidly expanded in the colonized areas. The species reached high numbers in natural populations in as few as 1.5 years after the colonization event, as indicated by the increase in its abundance in South America (Ayala, Serra, and Prevosti 1989Citation ). In North America, a secondary founder event was detected only in the more recent expansion of D. subobscura eastward but not between coastal populations, in accordance with a quick and continuous expansion of the coastal areas (Noor, Pascual, and Smith 2000Citation ). If a population grows according to the logistic curve with an intrinsic growth rate greater than 1 (Nei, Maruyama, and Chakraborty 1975Citation ) and assuming a carrying capacity of 450,000 (Begon, Krimbas, and Loukas 1980Citation ), an average number of 7 descendants per individual is obtained. Using this estimate, we computed the maximum number of haploid genomes in the sample of colonizers with the program MULTSIM (Noor, Pascual, and Smith 2000Citation ). Since there was low genetic differentiation among European populations, assuming that the colonization of North America was the result of a single event (Ayala, Serra, and Prevosti 1989Citation ; Prevosti et al. 1989Citation ; Mestres et al. 1990Citation ; Balanyà et al. 1994Citation ), we pooled the data within each continent to estimate the number of colonizers. This number ranged from 8 to 21 haploid genomes (4–11 individuals). Although a minimal number of eight haploid genomes would explain the number of alleles found in the colonizing populations, this number is probably too small for all of them to be represented. The estimated maximum number of colonizing haploid genomes was similar even if a higher number of descendants per individual was assumed.

Implications of Our Study for Understanding the Mutational Process
The recent colonization of North America by D. subobscura offers an excellent opportunity to assess which mutational model generates allelic variation in microsatellites. The IAM (Kimura and Crow 1964Citation ) assumes that each mutation creates a novel allele, the SMM (Ohta and Kimura 1973Citation ) assumes that new alleles arise by gain or loss of one repeat unit, and the two-phase model (TPM; Di Rienzo et al. 1994Citation ) assumes that most mutations follow the SMM but allows multistep changes following a geometric distribution. We compared the observed gene diversity and the expected gene diversity computed from the observed number of alleles for all 10 loci in each population under the IAM, the SMM, and the TPM assuming 30% of multistep changes using the program BOTTLENECK (Cornuet and Luikart 1996Citation ). The results in table 4 are consistent with the TPM, since it is the only model detecting an excess of heterozygosity and thus evidence of a bottleneck in the two North American populations and no deviation in the European populations, which presumably are at mutation-drift equilibrium.


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Table 4 Probability Results of a Wilcoxon Signed-Ranks Test to Assess Bottlenecks

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Microsatellite Variation and Founder Event
The results in table 1 show a large reduction in the average number of alleles per locus and a lower average heterozygosity in the North American compared with the European populations. This is in agreement with theoretical and experimental studies of population bottlenecks, which indicate a larger impact in the number of alleles per locus than in heterozygosity between pre- and postbottleneck populations. After a bottleneck, due to the sampling process, the average heterozygosity may increase (Leberg 1992Citation ), remain the same (Balanyà et al. 1994Citation ), or decrease (Nei, Maruyama, and Chakraborty 1975Citation ; the present study), depending on the number of alleles and their frequencies. Most loci in European populations were in Hardy-Weinberg equilibrium, with the exception of loci dsub16 and dsub20. These two loci had an excess of homozygous individuals, possibly due to the presence of null alleles generated by mutations in the priming site (Pemberton et al. 1995Citation ). In North America, only the allele frequencies of locus dsub16 significantly deviated from Hardy-Weinberg expectations, suggesting the presence of a null allele in the sample of colonizers. When loci dsub16 and dsub20 were removed from the analysis, a significant reduction of observed versus expected heterozygosities in European populations was observed when applying a Wilcoxon matched-pairs test. This could indicate microspatial heterogeneity in allele frequencies contributing to a Wahlund effect in local population samples. However, the huge difference between Hexp and Hobs detected in the dsub16 and dsub20 loci (see appendix) indicates that the Wahlund effect by itself cannot explain the excess of homozygotes observed, and the existence of null alleles is probably mainly responsible for the difference.

As stated above, not all alleles frequent in Europe were found in North America, while some rare alleles were found in rather high frequencies in colonizing populations. This indicates that a rather small number of colonizers contributed to the invasion of the New World and that drift has been one of the major forces of differentiation in the colonization process. Estimates from our microsatellite data of the number of colonizers (4–11 individuals) are similar to those previously obtained from data on chromosomal polymorphism (10–15 individuals; Prevosti et al. 1985Citation ) and the rp49 region (4–6 individuals; Rozas and Aguadé 1991Citation ) and are close to the lower range from data on lethal genes (9–149 individuals; Mestres et al. 1990Citation ). Assuming that all alleles in North America derived from the Palearctic region, the minimum number of individuals contributing to the colonization of the new continent would be four, since in the colonized populations eight alleles for locus dsub20 (fig. 1 ), six arrangements of the O chromosome (Prevosti et al. 1989Citation ), and eight haplotypes of the rp49 region (Rozas and Aguadé 1991Citation ) have been found.

The comparison between endemic and colonizing populations of D. subobscura has allowed us to check the adequacy of three microsatellite mutational models: the IAM, the SMM, and the TPM. The TPM is the only one detecting a bottleneck in North American populations and no deviation in European populations. As shown by experimental data on lethal allelism, European populations have not undergone a noticeable bottleneck in the recent past (Loukas, Krimbas, and Sourdis 1980Citation ; Mestres et al. 1990Citation ). On the other hand, and due to the recent colonization, a bottleneck effect has been detected extensively in American populations with all genetic markers studied up to date (Latorre, Moya, and Ayala 1986Citation ; Prevosti et al. 1988Citation ; Mestres et al. 1990Citation ; Rozas and Aguadé 1991Citation ; Balanyà et al. 1994Citation ). These experimental facts are corroborated only by the TPM, emphasizing its adequacy. This suggests that most alleles are generated by gain or loss of a repeat unit, while some alleles originate by more complex mutations, as observed by Colson and Goldstein (1999)Citation and Van Oppen et al. (2000)Citation using a different approach.

Population Differentiation
Low population differentiation and no simple isolation-by-distance pattern were found among European populations using microsatellite loci. FST values were lower than RhoST values, although the difference was small, indicating that gene flow between populations is high and the number of migrants is large even between geographically distant populations (table 2 ). The similarity between European populations can be explained mainly by gene flow, since the Nm values estimated among them were 3.9 using RhoST, 5.8 with the private-allele method after correction for size, and 45.2 with FST. However, the similarity could also be explained, to a lesser extent, by size homoplasy. Size homoplasy has been extensively reported in interrupted and/or compound microsatellites between distantly related subspecies (Estoup et al. 1995Citation ) and, to a lesser extent, within and between populations of the same species (Viard et al. 1998Citation ; Van Oppen et al. 2000Citation ).

When the mutation rate is high and there is a relatively large difference in average coalescence times (Rousset 1996Citation ), FST values are biased toward greater genetic similarity (Slatkin 1995Citation ). This could explain why RhoST values were larger than FST values among European populations (table 2 ). On the contrary, in the comparison between North America and Europe, FST values were higher than RhoST values, since the difference in average coalescence time was relatively small and genetic drift was the dominant process. However, between North American populations, RhoST values were also larger than FST values in this case due to the presence of a long allele in locus dsub04 in only one of the two localities. The chord distance, as shown by our results (fig. 2 ), is a better estimator of divergence between populations when drift is the most important factor, while other distances considering allele size differences, such {delta}µ2, are less reliable.

Different amounts of geographic variation among populations are often estimated with different genetic markers. The American oyster exhibits large differences between populations using mtDNA (Reeb and Avise 1990Citation ), while no geographic variation was found using allozymes (Buroker 1983Citation ) and some nuclear restriction fragment length polymorphism (McDonald, Verrelli, and Geyer 1996Citation ). In light of new genetic data on nuclear gene genealogies, Hare and Avise (1998)Citation concluded that no simple explanation can account for the great variety of population genetic patterns across loci displayed by American oysters.

Population subdivision is found with mtDNA in the harbor porpoise but has not been detected with microsatellite loci (Rosel et al. 1999Citation ). Similar FST estimates were found for microsatellite and allozymes in brown trout by Estoup et al. (1998)Citation , who suggested that both categories of markers are subjected to similar selective pressures.

In D. subobscura, different amounts of geographic variation are also observed using different markers. A high degree of population structure is present between European populations for chromosomal arrangements (Prevosti et al. 1988Citation ) and mtDNA but not for allozymes (Latorre et al. 1992Citation ) or microsatellites as observed in the present study. In the case of chromosomal polymorphism, population subdivision is expected because chromosomal arrangements are under strong selection pressure (Prevosti et al. 1988Citation ). This can also be the case for some allozyme loci (e.g., the Adh locus in D. melanogaster; Berry and Kreitman 1993Citation ). On the other hand, neutral markers which are not closely linked to selected loci will reveal population structure only if migration is low and populations are small enough. The different values of gene flow among European populations of D. subobscura obtained with mtDNA (0.013) and allozymes (1.89) (Latorre et al. 1992Citation ) are lower than the values obtained with the private-allele method using microsatellites (5.8). The larger differences revealed with mtDNA may be related to the smaller population sizes of maternally inherited molecules and also to the fact that selection anywhere on the mitochondrial genome would cause differentiation due to hitchhiking, or to the fact that females are more philopatric than males.

Analysis of the Colonization with Different Genetic Markers
The recent colonization of North America by D. subobscura were studied with several markers: chromosomal arrangements, allozymes, and now microsatellites, offering the opportunity to compare the adequacy of these systems in trying to understand the colonization process. In table 5 , we summarize the mean number of alleles per locus and the mean heterozygosity in a comparison of Europe and North America. In all cases, we found a reduction in the number of alleles in the North American populations, indicative of a founder event. Allozymes are the least variable markers, and only those alleles with frequencies higher than 0.1 in European populations are present in North America (Balanyà et al. 1994Citation ). Chromosomal arrangements show intermediate variability, and some of the alleles found in North America have low frequencies in Europe (for that comparison, we considered each chromosome as a locus and each arrangement as an allele) (Krimbas 1964Citation ; Sperlich 1964Citation ; Brncic et al. 1981Citation ; Prevosti et al. 1984, 1988Citation ). Microsatellites, as found in this study, are by far the most variable markers. As in the case of chromosomal arrangements, low-frequency microsatellite alleles in Europe are present in North America. For some loci (fig. 1 , locus dsub20), alleles with frequencies higher than 0.1 in Europe were not found in North America, indicative of the small number of colonizers. As stated above, a significant reduction in heterozygosity was observed in North American populations using microsatellites as genetic markers but not using allozymes or chromosomal arrangements (table 5 ). As the intrinsic growth rates and the numbers of colonizers were the same in all three cases, differences in heterozygosity could be attributed to the higher degree of variability detected with microsatellites. In the case of chromosomal arrangements, heterozygosities, as calculated in table 5 pooling data for all localities within each continent, are overestimated due to the existence of adaptive latitudinal clines.


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Table 5 Mean Numbers of Alleles Per Locus and Heterozygosity in Europe and North America

 
The results on the colonization of North America by D. subobscura obtained with different genetic markers indicate that the number of colonizers was rather low (4–15 individuals). The chromosomal polymorphism in North America suggests that the sample of colonizers came from the western Mediterranean area, including some specimens from the north of Europe, to explain the presence of the O5 inversion in colonizing populations (Prevosti et al. 1988Citation ; Ayala, Serra, and Prevosti 1989Citation ). This could be explained either by the passive simultaneous transportation of flies from northern and southern Europe or, alternatively, by a high migration rate among European populations, as revealed by the microsatellite analysis.


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Appendix: Number of Alleles (nA), Observed (Hobs) and Expected (Hexp) Heterozygosity, Variance in Repeat Number (Var(nA)), and Number of Chromosomes Scored (n)

 

    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
We thank R. Huey, J. Balanyà, and E. Solé for providing us with flies from several localities. We also thank A. Estoup for useful comments and S. Piry and J.-M. Cornuet for providing the (unpublished) computer package Treemake. This work was supported by grant PB96-0793-C04-03 from the DGES, by grant 1998SGR-00050 from Generalitat de Catalunya, and by a Postdoctoral Fellowship to M.P. from Ministerio de Educación y Ciencia, Spain.


    Footnotes
 
Pierre Capy, Reviewing Editor

1 Keywords: Drosophila subobscura, colonization microsatellites founder effect gene flow two-phase mutation model Back

2 Address for correspondence and reprints: Marta Pascual, Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, 08028 Barcelona, Spain. mpascual{at}porthos.bio.ub.es Back


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
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Accepted for publication December 21, 2000.