Departament de Genètica, Universitat de Barcelona, Barcelona, Spain; and
Department of Molecular Biology and Genetics, Cornell University
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Abstract |
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Introduction |
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The analyses of North American populations for chromosomal arrangements (Prevosti et al. 1988
), allozymes (Balanyà et al. 1994
), mtDNA restriction patterns (Latorre, Moya, and Ayala 1986
; Rozas et al. 1990
), and rp49 region restriction patterns (Rozas and Aguadé 1991
) 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 3149 individuals using different techniques (Prevosti et al. 1989
; Mestres et al. 1990
; Rozas and Aguadé 1991
). 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. 1988
; Ayala, Serra, and Prevosti 1989
).
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 2000
) 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.
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Materials and Methods |
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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 2000
); dsub05, dsub19, and dsub21 were X-linked, and the rest were autosomal. DNA was extracted from single-fly squish preps (Gloor et al. 1993
) 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 1978
), 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 1995
).
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 2000
). 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 1995
). Wright's F-statistic was estimated according to Weir and Cockerham (1984)
using the program GENEPOP, version 3.2 (Raymond and Rousset 1995
). Taking into account the difference between allele sizes, RhoST, an unbiased version of RST (Slatkin 1995
) in which allele sizes are transformed to standardize variances, was calculated using the program RST CALC, version 2.2 (Goodman 1997
). 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 1989
).
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)
, 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. 1994
) larger or smaller than that empirically observed. These simulations, using the program MULTSIM (Noor, Pascual, and Smith 2000
), 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)
and
µ2, a distance developed for the stepwise mutation model (Goldstein et al. 1995
). 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)
and the infinite-alleles model (IAM: H = 4Neµ/(1 + 4Neµ); Kimura and Crow 1964
). The dinucleotide repeat mutation rate of Drosophila melanogaster (9.3 x 10-6 per locus per generation) empirically determined by Schug et al. (1998)
was used, since it produces estimates of Ne concordant with those obtained from sequences of single nuclear genes (Pascual, Schug, and Aquadro 2000
). A similar calculation was done using the variance in repeat number (Var(nA)), for which Slatkin (1995)
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.
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Results |
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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 2000
).
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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Figure 2
shows unrooted neighbor-joining dendrograms based on chord distance (fig. 2A
) and 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
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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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 1964
) assumes that each mutation creates a novel allele, the SMM (Ohta and Kimura 1973
) assumes that new alleles arise by gain or loss of one repeat unit, and the two-phase model (TPM; Di Rienzo et al. 1994
) 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 1996
). 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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Discussion |
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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 (411 individuals) are similar to those previously obtained from data on chromosomal polymorphism (1015 individuals; Prevosti et al. 1985
) and the rp49 region (46 individuals; Rozas and Aguadé 1991
) and are close to the lower range from data on lethal genes (9149 individuals; Mestres et al. 1990
). 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. 1989
), and eight haplotypes of the rp49 region (Rozas and Aguadé 1991
) 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 1980
; Mestres et al. 1990
). 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 1986
; Prevosti et al. 1988
; Mestres et al. 1990
; Rozas and Aguadé 1991
; Balanyà et al. 1994
). 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)
and Van Oppen et al. (2000)
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. 1995
) and, to a lesser extent, within and between populations of the same species (Viard et al. 1998
; Van Oppen et al. 2000
).
When the mutation rate is high and there is a relatively large difference in average coalescence times (Rousset 1996
), FST values are biased toward greater genetic similarity (Slatkin 1995
). 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
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 1990
), while no geographic variation was found using allozymes (Buroker 1983
) and some nuclear restriction fragment length polymorphism (McDonald, Verrelli, and Geyer 1996
). In light of new genetic data on nuclear gene genealogies, Hare and Avise (1998)
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. 1999
). Similar FST estimates were found for microsatellite and allozymes in brown trout by Estoup et al. (1998)
, 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. 1988
) and mtDNA but not for allozymes (Latorre et al. 1992
) 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. 1988
). This can also be the case for some allozyme loci (e.g., the Adh locus in D. melanogaster; Berry and Kreitman 1993
). 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. 1992
) 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. 1994
). 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 1964
; Sperlich 1964
; Brncic et al. 1981
; Prevosti et al. 1984, 1988
). 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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Acknowledgements |
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Footnotes |
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1 Keywords: Drosophila subobscura,
colonization
microsatellites
founder effect
gene flow
two-phase mutation model
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
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literature cited |
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Ayala, F. J., L. Serra, and A. Prevosti. 1989. A grand experiment in evolution: the D. subobscura colonization of the Americas. Genome 31:246255.
Balanyà, J., C. Segarra, A. Prevosti, and L. Serra. 1994. Colonization of America by D. subobscura: the founder event and a rapid expansion. J. Hered. 85:427432.
Beckenbach, A. T., and A. Prevosti. 1986. Colonization of North America by the European species Drosophila subobscura and D. ambigua. Am. Midl. Nat. 115:108.
Begon, M., C. B. Krimbas, and M. Loukas. 1980. The genetics of D. subobscura populations XV. Effective size of a natural population estimated by three independent methods. Heredity 45:335350.
Berry, A., and M. Kreitman. 1993. Molecular analysis of an allozyme cline: alcohol dehydrogenase in Drosophila melanogaster on the east coast of North America. Genetics 134:869893.
Bowcock, A. M., A. Ruiz-Linares, J. Tomfohrde, E. Minch, J. R. Kidd, and L. L. Cavalli-Sforza. 1994. High resolution of human evolutionary trees with polymorphic microsatellites. Nature 368:455457.
Brncic, D., A. Prevosti, M. Budnik, M. Monclús, and J. Ocaña. 1981. Colonization of D. subobscura in Chile I. First population and cytogenetic studies. Genetica 56:39.
Buroker, N. E. 1983. Population genetics of the American oyster Crassostrea virginica along the Atlantic coast and the Gulf of Mexico. Mar. Biol. 75:99112.[ISI]
Cavalli-Sforza, L. L., and A. W. F. Edwards. 1967. Phylogenetic analysis: models and estimation procedure. Am. J. Hum. Genet. 19:233257.[ISI][Medline]
Colson, I., and D. B. Goldstein. 1999. Evidence for complex mutations at microsatellite loci in Drosophila. Genetics 152:617627.
Cornuet, J. M., and G. Luikart. 1996. Description and power analysis of two tests for detecting recent population bottlenecks from allele frequency data. Genetics 144:20012014.
Di Rienzo, A., A. C. Peterson, J. C. Garza, A. M. Valdés, M. Slatkin, and N. B. Freimer. 1994. Mutational processes of simple-sequence repeat loci in human populations. Proc. Natl. Acad. Sci. USA 91:31663170.
Estoup, A., F. Rousset, Y. Michalakis, J. M. Cornuet, M. Adriamanga, and R. Guyomard. 1998. Comparative analysis of microsatellite and allozyme markers: a case study investigating microgeographic differentiation in brown trout (Salmo trutta). Mol. Ecol. 7:339353.[ISI][Medline]
Estoup, A., C. Taillez, J. M. Cornuet, and M. Solignac. 1995. Size homoplasy and mutational processes of interrupted microsatellites in two bee species, Apis mellifera and Bombus terrestris (Apidae). Mol. Biol. Evol. 12:10741084.[Abstract]
Gloor, G. B., C. R. Preston, D. M. Johnson-Schlitz, N. A. Nassif, R. W. Phillis, W. K. Benz, H. M. Robertson, and W. R. Engels. 1993. Type I repressors of P element mobility. Genetics 135:8195.
Goldstein, D. B., A. Ruiz Linares, L. L. Cavalli-Sforza, and M. W. Feldman. 1995. Genetic absolute dating based on microsatellites and the origin of modern humans. Proc. Natl. Acad. Sci. USA 92:67236727.
Goodman, S. J. 1997. RST CALC: a collection of computer programs for calculating unbiased estimates of genetic differentiation and determining their significance for microsatellite data. Mol. Ecol. 6:881885.[ISI]
Hare, M. P., and J. C. Avise. 1998. Population structure in the American oyster as inferred by nuclear gene genealogies. Mol. Biol. Evol. 15:119128.[Abstract]
Huey, R. B., G. W. Gilchrist, M. L. Carlson, D. Berrigan, and L. Serra. 2000. Rapid evolution of a geographic cline in size in an introduced fly. Science 287:308309.
Kimura, M., and J. F. Crow. 1964. The number of alleles that can be maintained in a finite population. Genetics 49:725738.
Krimbas, C. B. 1964. The genetics of D. subobscura populations II. Inversion polymorphism in a population from Holland. Z. Vererbungsl. 95:125128.
Latorre, A., C. Hernández, D. Martinez, J. A. Castro, M. Ramón, and A. Moya. 1992. Population structure and mitochondrial DNA gene flow in Old World populations of D. subobscura. Heredity 68:1524.
Latorre, A., A. Moya, and F. J. Ayala. 1986. Evolution of mitochondrial DNA in D. subobscura. Proc. Natl. Acad. Sci. USA 83:86498653.
Leberg, P. L. 1992. Effects of population bottlenecks on genetic diversity as measured by allozyme electrophoresis. Evolution 46:477494.
Loukas, M., C. B. Krimbas, and J. Sourdis. 1980. The genetics of D. subobscura populations. XIII: a study of lethal allelism. Genetica 54:197206.
McDonald, J. H., B. C. Verrelli, and L. B. Geyer. 1996. Lack of geographic variation in anonymous nuclear polymorphisms in the American oyster, Crassostrea virginica. Mol. Biol. Evol. 13:11141118.[Abstract]
Mestres, F., G. Pegueroles, A. Prevosti, and L. Serra. 1990. Colonization of America by D. subobscura: lethal genes and the problem of the O5 inversion. Evolution 44:18231836.
Mestres, F., L. Serra, and F. J. Ayala. 1995. Colonization of the Americas by D. subobscura: lethal-gene allelism and association with chromosomal arrangements. Genetics 140:12971305.
Nei, M., T. Maruyama, and R. Chakraborty. 1975. The bottleneck effect and genetic variability in populations. Evolution 29:110.
Nei, M. 1978. Estimation of heterozygosity from a small number of individuals. Genetics 89:583590.
Noor, M. A. F., M. Pascual, and K. R. Smith. 2000. Genetic variation in the spread of Drosophila subobscura from a nonequilibrium population. Evolution 54:696703.
Noor, M. A. F., J. R. Wheatley, K. A. Wetterstrand, and H. Akashi. 1998. Western North American obscura-group. Drosophila collection data, summer 1997. Drosophila Information Service 81:136137.
Ohta, T., and M. Kimura. 1973. A model of mutation appropriate to estimate the number of electrophoretically detectable alleles in a finite population. Genet. Res. 22:201204.[ISI][Medline]
Pascual, M., F. J. Ayala, A. Prevosti, and L. Serra. 1993. Colonization of North America by D. subobscura: ecological analysis of three communities of drosophilids in California. Z. Zool. Syst. Evol. Forsch. 31:216226.
Pascual, M., M. D. Schug, and C. F. Aquadro. 2000. High density of long dinucleotide microsatellites in D. subobscura. Mol. Biol. Evol. 17:12591267.
Pemberton, J. M., J. Slate, D. R. Bancroft, and J. A. Barrett. 1995. Nonamplifying alleles at microsatellite locia caution for parentage and population studies. Mol. Ecol. 4:249252.[ISI][Medline]
Prevosti, A., R. Frutos, G. Alonso, A. Latorre, M. Monclús, and M. J. Martnez. 1984. Genetic differentiation between natural populations of D. subobscura in the Western Mediterranean area with respect to chromosomal variation. Genet. Sel. Evol. 16:143156.
Prevosti, A., G. Ribó, L. Serra, M. Aguadé, J. Balañá, M. Monclús, and F. Mestres. 1988. Colonization of America by D. subobscura: experiment in natural populations that supports the adaptive role of chromosomal inversion polymorphism. Proc. Natl. Acad. Sci. USA 85:55975600.
Prevosti, A., L. Serra, M. Aguadé, G. Ribó, F. Mestres, J. Balañá, and M. Monclús. 1989. Colonization and establishment of the Palearctic species D. subobscura in North and South America. Pp. 114129 in A. Fontdevila, ed. Evolutionary biology of transient unstable populations. Springer-Verlag, Berlin.
Prevosti, A., L. Serra, G. Ribó, M. Aguadé, E. Sagarra, M. Monclús, and M. P. Garcia. 1985. The colonization of D. subobscura in Chile: II. Clines in the chromosomal arrangements. Evolution 39:838844.
Raymond, M., and F. Rousset. 1995. GENEPOP (version 1.2): population genetics software for exact tests and ecumenicism. J. Hered. 86:248249.[ISI]
Reeb, C. A., and J. C. Avise. 1990. A genetic discontinuity in a continuously distributed species: mitochondrial DNA in the American oyster, Crassostrea virginica. Genetics 124:397406.
Rice, W. R. 1989. Analyzing tables of statistical tests. Evolution 43:223225.
Rosel, P. E., S. C. France, J. Y. Wang, and T. D. Kocher. 1999. Genetic structure of harbour porpoise Phocoena phocoena populations in the northwest Atlantic based on mitochondrial and nuclear markers. Mol. Ecol. 8:S41S54.
Rousset, F. 1996. Equilibrium values of measures of population subdivision for stepwise mutation processes. Genetics 142:13571362.
Rozas, J., and M. Aguadé. 1991. Using restriction-map analysis to characterize the colonization process of D. subobscura on the American continent. I. rp49 region. Mol. Biol. Evol. 8:447457.
Rozas, J., M. Hernández, V. M. Cabrera, and A. Prevosti. 1990. Colonization of America by D. subobscura: effect of the founder event on the mitochondrial DNA polymorphism. Mol. Biol. Evol. 7:103109.
Schug, M. D., C. H. Hutter, K. A. Wetterstrand, M. S. Gaudette, T. F. C. Mackay, and C. F. Aquadro. 1998. The mutation rates of di-, tri-, and tetranucleotide repeats in D. melanogaster. Mol. Biol. Evol. 15:17511760.
Slatkin, M. 1995. A measure of population subdivision based on microsatellite allele frequencies. Genetics 139:457462.
Sperlich, D. 1964. Chromosomale strukturanalyse und fertilitätsprüfung an einer marginalpopulation von D. subobscura. Z. Vererbungsl. 95:7381.
Van Oppen, M. J. H., C. Rico, G. F. Turner, and G. M. Hewitt. 2000. Extensive homoplasy, nonstepwise mutations, and shared ancestral polymorphism at a complex microsatellite locus in Lake Malawi cichlids. Mol. Biol. Evol. 17:489498.
Viard, F., P. Franck, M.-P. Dubois, A. Estoup, and P. Jarne. 1998. Variation of microsatellite size homoplasy across electromorphs, loci, and populations in three invertebrate species. J. Mol. Evol. 47:4251.[ISI][Medline]
Weir, B. S., and C. C. Cockerham. 1984. Estimating F-statistics for the analysis of population structure. Evolution 38:13581370.