Mitochondrial DNA Haplotype Frequencies in Natural and Experimental Populations of Drosophila subobscura

Corresponding author: Andrés Moya, Departament de Genètica, Facultat de Biología, Universitat de València, Dr. Moliner, 50, 46100 Burjassot, Valencia, Spain, andres.moya{at}uv.es (E-mail).

Communicating editor: C.-I WU

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The evolution of Drosophila subobscura mitochondrial DNA has been studied in experimental populations, founded with flies from a natural population from Esporles (Majorca, Balearic Islands, Spain). This population, like other European ones, is characterized by the presence of two very common (>96%) mitochondrial haplotypes (called I and II) and rare and endemic haplotypes that appear at very low frequencies. There is no statistical evidence of positive Darwinian selection acting on the mitochondrial DNA variants according to Tajima's neutrality test. Two experimental populations, with one replicate each, were established with flies having a heterogeneous nuclear genetic background, which was representative of the composition of the natural population. Both populations were started with the two most frequent mitochondrial haplotypes, but at different initial frequencies. After 13 to 16 generations, haplotype II reached fixation in three cages and its frequency was 0.89 by generation 25 in the fourth cage. Random drift can be rejected as the force responsible for the observed changes in haplotype frequencies. There is not only statistical evidence of a linear trend favoring a mtDNA (haploid) fitness effect, but also of a significant nonlinear deviation that could be due to a nuclear component.

A recurrent observation in studies of mitochondrial DNA evolution in Old and New World populations of Drosophila subobscura during more than 10 yr is the high prevalence of two haplotypes and the sporadic appearance of low-frequency endemic, i.e., rare, ones (LATORRE et al. 1986 , LATORRE et al. 1992 ; AFONSO et al. 1990 ; ROZAS et al. 1990 ; MOYA et al. 1993 ; GONZALEZ et al. 1994 ). It is yet an unresolved question which evolutionary forces account for this distribution. Recent studies based on mtDNA nucleotide sequences of D. melanogaster and related species (KANEKO et al. 1993 ; BALLARD and KREITMAN 1994 ; Rand et al. 1994 ), of Mus species (NACHMAN et al. 1994 ) and of humans and chimpanzees (NACHMAN et al. 1996 ) have shown deviations from a neutral model, which have been interpreted as caused by a large class of mildly deleterious mutations. On the contrary, fitness components studies in the evolution of natural and experimental populations, carried out with Drosophila, have shown a diversity of results with very different interpretations. On the basis of changes in the mtDNA haplotype frequencies in experimental populations and fitness estimates, MACRAE and ANDERSON 1988 , MACRAE and ANDERSON 1990 and JENKINS et al. 1996 have suggested that mtDNA variants of Drosophila pseudoobscura evolve in a nonneutral fashion. SINGH and HALE 1990 argued that these results can be interpreted as caused by an incompatibility system, similar to the one promoted by Wolbachia endosymbionts in Drosophila simulans (NIGRO and PROUT 1990 ). FOS et al. 1990 showed that natural selection acts on mitochondrial DNA haplotypes in D. subobscura, but they also attributed an important effect to the nuclear genetic background. Their results and conclusions have been criticized by both HUTTER and RAND 1995 and KILPATRICK and RAND 1995 who argued that population cages were not replicated. KILPATRICK and RAND 1995 did not obtain evidence of selection in competition experiments between mitochondrial haplotypes in Drosophila melanogaster, and their results were interpreted as caused by a temporary hitchhiking of nuclear background on mtDNA (BABCOCK and ASMUSSEN 1996 ). On the contrary, similar experiments between D. pseudoobscura and Drosophila persimilis (HUTTER and RAND 1995 ) identified mtDNA as a source of fitness variation and also found evidence of cyto-nuclear interactions affecting fitness.

The main goal of the present work is to test the hypothesis of neutral evolution in the mtDNA of D. subobscura. For this purpose, we take advantage of previous studies on experimental evolution and temporal and geographic mtDNA haplotype distribution of D. subobscura (see above), to study haplotype frequency changes in two replicated experimental populations. These populations were derived from a representative set of isofemale lines from a natural population whose mtDNA haplotype frequencies have also been studied.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The natural population:
D. subobscura flies were captured in a pine forest near Esporles (Majorca Island, Spain) with conventional traps of fermented banana. Once in the lab, 204 females were placed individually in a vial with food and kept in an incubator at 19°. When the F₁ larvae appeared, females were used to determine their maternal mitochondrial haplotypes (see below).

Extraction and digestion of mtDNA isofemale lines:
An enriched fraction of mtDNA was obtained as previously described (LATORRE et al. 1986 ). Routinely, enough DNA could be extracted from 15 individuals for digestion with at least five restriction enzymes. The following restriction enzymes were used: EcoRI, EcoRV and HindIII (recognition sequence of 6 bp), and HpaII and HaeIII (recognition sequence of 4 bp). Previous studies have shown that these restriction enzymes reveal a rich mtDNA polymorphism in D. subobscura (AFONSO et al. 1990 ; LATORRE et al. 1992 ).

Digested DNAs were separated on horizontal 0.6–1.2% agarose gels in 0.5x TBE containing 0.1 µg/ml EtBr. Lambda phage DNA digested with HindIII and double digested with HindIII-EcoRI was used as a size standard.

A mtDNA restriction map was obtained by means of all possible single and double digestions. The different restriction patterns and haplotypes are named according to the notation given by LATORRE et al. 1986 , LATORRE et al. 1992 .

Presence of Wolbachia:
To exclude an incompatibility system in D. subobscura promoted by the presence of Wolbachia, a PCR assay using 16S rDNA Wolbachia-specific primers was carried out. Primers were designed according to O'NEILL et al. 1992 . These primers amplify a fragment of 869 bp. Two different strains of Sitophilus oryzae containing Wolbachia were used as positive controls. In addition, two D. subobscura regions were used as positive control for DNA amplification: the V4-18S rDNA (the V4 variable region of the 18S rDNA) and the ND5 mtDNA (the subunit 5 of the NADH dehydrogenase). The sequences of the primers are as follows: 18SV4up-5'-ACCTGCAGCAGCCGCGGTAATTCCAGC-3' and 18SV4down-5'-GCTCTAGACGTACTTGGCAAATGCTTTCGC-3' (that amplify a fragment of 427 bp) and ND5up-5'-TGACCAGCTAGCTATTCTGATC-3' and ND5down-5'-GCTATAGCTAGCCCCTACAC-3' (that amplify a fragment of 984 bp). PCR conditions were as follows: an initial denaturation for 5 min at 94° and 30 cycles of denaturation at 94° for 1 min, annealing at 55° for 1 min and extension at 72° for 2 min. Total DNA was extracted as in MARTINEZ et al. 1992 but excluding the alkaline treatment.

Figure 1 shows a sample PCR obtained with the three sets of primers from DNA belonging to D. subobscura individuals from Mallorca (Esporles, this study), Menorca and Valencia populations, as well as DNA from the two strains of S. oryzae. As it can be observed there was no amplification in D. subobscura with the Wolbachia-specific primers.

View larger version (90K): In this window In a new window Download PPT slide   Figure 1. PCR amplifications. Lanes A, B, and C correspond to total D. subobscura DNA from one isoline from Mallorca (Esporles, this study), Menorca and Valencia (Spain), respectively, amplified with Wolbachia 16S rDNA-specific primers. Lanes G, H, and I correspond to the same DNAs amplified with V4-18S rDNA primers, and lanes K, L, and N with ND5 primers. Lanes D, J, and O correspond to negative controls from each set of primers. Lanes E and F correspond to Wolbachia DNA from two strains of S. oryzae amplified with 16S rDNA-specific primers. M is the 100-bp ladder molecular marker (Pharmacia, Piscataway, NJ) and M' is the 1-kb ladder molecular marker (GIBCO BRL, Gaithersburg, MD).

Discrete generation experiments:
Population cages were initiated with flies derived from 51 D. subobscura isofemale strains from Esporles (Majorca, Spain). A total of 20 isofemale strains bore mitochondrial haplotype I and the other 31 haplotype II.

Two experimental populations, both replicated (denoted as A1 and A2, and B1 and B2, respectively) were initiated at the same time with 1000 individuals (500 males and 500 females) representative of the genetic composition of the natural population from Esporles. The starting frequencies in cages A1 and A2 were 30% for haplotype I (300 individuals, 15 individuals from each isofemale strain) and 70% for haplotype II (700 individuals, 22 or 23 individuals from each isofemale strain). The starting frequencies of mitochondrial haplotypes in cages B1 and B2 were 70% for haplotype I (700 individuals, 35 individuals from each isofemale strain) and 30% for haplotype II (300 individuals, 9 or 10 individuals from each isofemale strain).

Cages were started with 13 food cups and kept at 19°. Egg laying lasted for 6 days, after which the food cups with eggs and larvae were moved to a new cage. When the F₁ appeared, 13 more food cups were added and left 6 days for egg laying; this set of food cups was then moved to a new population cage. All cages followed a similar cycle of discrete generations.

Estimation of mtDNA haplotypes frequencies in experimental populations:
About 100 individuals (see RESULTS) were sampled each generation and used for extraction of mtDNA and estimation of haplotype frequencies. mtDNA was again extracted according to LATORRE et al. 1986 with the following modifications. Two flies were homogenized in an Eppendorf tube with 50 µl of buffer I (10 mM Tris-HCl, 60 mM NaCl, 5% sucrose, 10 mM EDTA, pH 7.8). After homogenization, 62.5 µl of buffer II (300 mM Tris-HCl, 1.25 % SDS, 5 % sucrose, 10 mM EDTA, pH 9.0) were added and tubes were incubated at 65° for 30 min. After incubation, 18 µl of 3 M potassium acetate (pH 4.8) were added and tubes were kept at -20° for 15 min. Tubes were centrifuged in a microfuge at maximum speed for 15 min. The supernatant was transferred to a fresh tube, and an equal volume of isopropanol was added, mixed and incubated at room temperature for 10 min. DNA was precipitated by centrifugation at maximum speed for 15 min, washed with 70% ethanol, vacuum dried, and resuspended in 11.5 µl of TE buffer.

DNA was digested with restriction endonuclease HaeIII in a final volume of 15 µl. This enzyme cuts genomic DNA frequently but has only a few recognition sites in the mitochondrial genome of D. subobscura (a single restriction site in haplotype II and two sites in haplotype I). In agarose gels, mtDNA bands are, therefore, easily distinguishable against a background of genomic DNA by visual inspection (one 15.8-kb band for haplotype II and two, 9.0- and 6.8-kb, bands for haplotype I). Because DNA was extracted from two flies simultaneously, three different restriction fragment patterns could be distinguished: a single 15.8-kb band (indicating that the two flies carried haplotype II); two bands of 9.0 and 6.8 kb (indicating that the two flies carried haplotype I) and three bands of 15.8, 9.0 and 6.8 kb (indicating that each fly had a different mtDNA haplotype). Partial digests can be easily distinguished because of both the intensity of the bands and the presence of undigested molecular forms, i.e., supercoiled and relaxed.

Statistical analyses:
The intensity of selection acting on mtDNA haplotypes was analyzed following two methods. The first method (FISHER and FORD 1947 ; SCHAFFER et al. 1977 ) tests the null hypothesis that changes observed in haplotype frequencies can be explained as the result of random drift. FISHER and FORD 1947 showed that the effective size of a population in each generation and the sample size taken from it can be used to generate a matrix of covariances between the haplotype frequencies observed in the various generations. By means of a ² statistic this covariance matrix can be used to test the hypothesis that random drift alone accounts for the observed changes in haplotype frequency. This method is sensitive only to fairly large differences in selective values, i.e., of the magnitude s = 0.05 (SCHAFFER et al. 1977 ). The second method is based on a model of linear change in frequency due to selection, which may be used to detect selection coefficients as small as 0.01 (SCHAFFER et al. 1977 ). The magnitude of the selection differential per generation is measured by a linear parameter and the significance for such a linear trend as well as nonlinear deviations thereof are evaluated by ² tests with 1 and g - 2 degrees of freedom respectively, where g is the number of generations elapsed between the samples. Both tests use haplotype frequencies which are arcsin squared transformed and given in radians. It should be noted that curvilinearity is substantially decreased by this transformation, which has the effect of expanding the scale at the ends of the haplotype frequency range. Additionally, the test can respond to the linear component in a trend, even if the underlying phenomenon is nonlinear.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

mtDNA restriction enzyme analysis carried out on 204 flies from the natural population of Esporles yielded a total of 10 haplotypes (Table 1). Haplotype I (39.2%) and haplotype II (56.9%) accounted for more than 95% of all haplotypes. These two haplotypes differ by a single HaeIII site. Haplotypes IV, V, VII, VIII, IX and X differ by a single site from haplotype II, whereas haplotype III and VI differ by a single site from haplotype I. TAJIMA's (1989) D statistic was used to test the deviation from neutrality and yielded a D value of -1.29, which is not statistically significant, at the 0.05 level, for a sample size of 204 individuals.

Flies belonging to the natural population of Esporles that had a heterogeneous, representative nuclear background (see MATERIALS AND METHODS) and bore either mtDNA haplotype I or II were placed in experimental population cages and allowed to evolve for a number of generations (Table 2). Two replicates for each initial starting frequency of both haplotypes were used. The experimental protocol was designed to minimize the potential effects of random drift by starting each generation with about a thousand eggs, larvae and pupae competing for resources in food cups. Monitoring the frequency change of haplotype II versus I showed that replicates gave similar results. When haplotype II was started at a higher frequency (70%) it reached fixation at generations 14–15. The same tendency toward fixation of haplotype II was observed when its starting frequency was 0.3. A decrease of haplotype II in population cage B2 was observed in the first three generations, which may be attributed to some uncontrolled factor. By generations 4–5 it regained the starting frequency and, from that moment, it increased until the last checked generation (25), where its frequency was 88.7%. The B1 population cage reached fixation at generation 16.

Results from ² tests for the presence of a selective pressure favoring haplotype II are given in Table 3. The tests have been carried out assuming two different effective numbers of females, i.e., N_f = 250 or 1500. Irrespective of these numbers, the ² values for testing random genetic drift were always highly significant in all cages (P < 0.01), clearly disallowing the null hypothesis of random genetic drift. In all cases, linear selection was statistically significant (P < 0.01). Additionally, in spite of the linearizing effects of the arcsin squared transformation (see MATERIALS AND METHODS), a nonlinear deviation was also statistically significant (P < 0.01).

View this table: In this window In a new window   Table 3. 2 values under either random drift (selective neutrality) or linear directional change for the four D. subobscura experimental populations

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In the survey of mtDNA haplotype frequencies in the Esporles population (Table 1) haplotype II was found at higher frequency (56.9%) than haplotype I (39.2%). A similar frequency distribution was observed in a study of mtDNA from a different D. subobscura population in which extensive and recurrent seasonal sampling was carried out (GONZALEZ et al. 1994 ). In fact, the higher frequency of haplotype II than that of haplotype I seems to be the pattern in this species, at least during the last 12 years. From 1985 to 1997, the mtDNA restriction analysis of 1684 flies, thoroughly sampled over the distribution range of the species (LATORRE et al. 1986 ; AFONSO et al. 1990 ; MOYA et al. 1993 ; GONZALEZ et al. 1994 ; and other unpublished results from our laboratory), show the following overall distribution: 673 with haplotype I (40%), 860 with haplotype II (51%), and 151 with rare haplotypes (9%). A possible explanation for the stable frequencies of the two haplotypes is balancing selection (CLARK 1984 ). In that case, however, Tajima's D would be positive, rather than negative. A negative value is compatible with either a purifying selection or a population bottleneck. However, it is too risky to conclude that mtDNA of D. subobscura is evolving neutrally based only on a test that has been performed with eight polymorphic sites. To perform appropriate neutrality tests at the nucleotide level, we are currently sequencing 984 bp of the ND5 gene from more than 45 mtDNA haplotypes of D. subobscura from different locations, i.e., haplotypes I and II as well as some rare ones, and Drosophila guanche, which is closely related to D. subobscura. Tajima's D, and FU and LI's (1993) D and F do not show significant deviation from neutrality or, when significant, they are negative, in agreement with the results obtained here. Moreover, MCDONALD and KREITMAN's (1991) test, does not show departure from neutrality (C. MARTÍNEZ, R. BUENO, A. GONZÁLEZ, E. BARRIO, A. MOYA, unpublished results). Taking all these results together, we have not found evidence of positive selection in the evolution of mtDNA of D. subobscura. On the contrary, the stability of the frequencies of haplotypes I and II over more than twelve years may be accounted for by their drifting in a population of very large effective size.

If haplotypes I and II are neutral in nature, the fixation of haplotype II in the population cage experiments deserves an explanation. A point to be mentioned is that population cages do not constitute a natural environment. Consequently, factors promoting positive selection of haplotype II in population cages could be counterbalanced by other factors under natural conditions. Regarding experimental populations, balancing selection, frequency-dependent selection, some sort of habitat-specific selection or genetic hitchhiking of mtDNA by a nuclear target are ad hoc explanations because we have no clear physiological/ecological evidence for a mechanism. FOS et al. 1990 showed that when two mtDNA haplotypes of D. subobscura compete in experimental populations one or the other approaches fixation depending on the nuclear background with which they are associated. The two populations used in that report were from very different geographic origins (one from Canary Islands, Spain, bearing the endemic mtDNA haplotype VIII, and the other from Helsinki, Finland, with mtDNA haplotype I), and different lines, homozygous for all chromosomal inversions, were synthesized. The nuclear background effect was manifested because the mtDNA haplotype that reached fixation in each case was the one placed within its own nuclear background.

The population cage experiments were set up with different lines of wild flies belonging to the Esporles population evolving under relatively high effective population sizes (see MATERIALS AND METHODS). Thus, in addition to the segregating mtDNA variation, there is considerable nuclear variation segregating as well. For example, the rich and adaptive inversion polymorphism of D. subobscura in Mediterranean populations is well known (KRIMBAS and LOUKAS 1980 ; ORENGO and PREVOSTI 1996 ). This raises the possibility that the shift in mtDNA haplotype frequencies could be caused by not only to selection acting on mtDNA variants, but also by temporary hitchhiking of mtDNA haplotype II by the selected nuclear genetic variation (KILPATRICK and RAND 1995 ; BABCOCK and ASMUSSEN 1996 ) or a functional coadaptation between nuclear and mitochondrial genomes (CLARK and LICKEGAARD 1988; MACRAE and ANDERSON 1988 ; FOS et al. 1990 ; HUTTER and RAND 1995 ). It must be remarked (see Table 3) that there is statistical evidence not only of a linear trend favoring a mtDNA (haploid) fitness effect, but also of a significant nonlinear deviation that could be due to a fitness effect of a nuclear component on the haplotype trajectories.

	FOOTNOTES

¹ These authors contributed equally to this work.

	ACKNOWLEDGMENTS

We greatly appreciate the suggestions made by the associate editor and one anonymous reviewer. We also acknowledge E. BARRIO, S.F. ELENA, F. GONZÁLEZ and R. VAN HAM for their helpful criticisms and the Servicio de Bioinformática de la Universitat de València for computing facilities. We are indebted to A. HEDDI who kindly supplied us with two strains of S. oryzae, harboring Wolbachia, and to B. SABATER for preparing the figure. This work has been supported by grants PB93-0690 and PR95-228 from The Direccíon General de Investigacíon Cienfica y Técnica (Spain), and by grant PB96-0793 from the Dirección General de Enseanza Superior (Spain). J.G.M. and J.A.C. have been recipients of a fellowship from the Ministerio de Educación y Ciencia (Spain).

Manuscript received March 13, 1997; Accepted for publication March 9, 1998.

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