The genetic structure of drosophila subobscura populations from the islands of majorca and minorca (balearic islands, spain) based on allozymes and mitochondrial dna

ABSTRACT The genetic structure of seven populations of _Drosophila subobscura_ from different locations on Majorca and Minorca (Balearic Islands, Spain) was studied using two types of

markers: allozyme and mitochondrial DNA restriction analyses. Both markers showed congruent results. In the allozyme data, when the _Acph-1_ locus was excluded from the joint _F_ST

statistics, only three out of 21 comparisons were statistically significant, lending support to the hypothesis of low genetic differentiation. The mtDNA restriction analyses showed two

haplotypes at a high frequency (more than 40% each), irrespective of the location considered, and a number of endemic haplotypes at very low frequencies (not higher than 2% each). The

analyses of the genetic structure yielded a pattern similar to the allozymes. The cytonuclear disequilibrium analyses showed the difficulty of detecting cytonuclear associations in natural

populations because they are mainly transient. SIMILAR CONTENT BEING VIEWED BY OTHERS POPULATION STRUCTURE AND GENETIC DIVERSITY OF NON-NATIVE AOUDAD POPULATIONS Article Open access 10 June

2021 DIVERGENCE TIME ESTIMATION USING DDRAD DATA AND AN ISOLATION-WITH-MIGRATION MODEL APPLIED TO WATER VOLE POPULATIONS OF _ARVICOLA_ Article Open access 08 March 2022 EVOLUTIONARY HISTORY

AND COLONIZATION PATTERNS OF THE WING DIMORPHIC GRASSHOPPER _DICHROPLUS VITTATUS_ IN TWO ARGENTINEAN BIOMES Article Open access 21 February 2022 INTRODUCTION _Drosophila subobscura_ is a

Palaearctic species of the _obscura_ subgroup of _Drosophila_, which is endemic throughout Europe (except central and northern Scandinavia) as well as the Middle East, northern Africa and on

the Atlantic islands of the Azores, Madeira and the Canaries. Natural populations have been extensively studied during the latter half of this century. Among the genetic traits studied,

allozymes (Pinsker et al., 1978; Cabrera et al., 1980; Latorre et al., 1992), which give a uniform allele distribution irrespective of which locus or geographical area is considered, and

inversion polymorphisms (Menozzi & Krimbas, 1992; Orengo & Prevosti, 1996), which exhibit clear clinal frequency distributions, are particularly relevant. In recent years, mtDNA

studies have increased our knowledge of this species. Previous studies on the distribution of mtDNA haplotypes in the Old World populations of _D. subobscura_ have shown the presence of two

widespread and equally frequent haplotypes, as well as a set of less common haplotypes generally present in never more than a single locality (Afonso et al., 1990; Latorre et al., 1992; Moya

et al., 1993; González et al., 1994; García-Martínez et al., 1998), with the exception of the Canary Islands, where an endemic haplotype is the most frequent. The same distribution pattern

has been detected in the New World colonizing populations of _D. subobscura_ (Rozas et al., 1990). It is still an unresolved question which evolutionary processes account for this

distribution. Studies concerning the genetic dynamics of the mtDNA and experiments in different species have shown inconclusive results with respect to selection or neutrality, such as for

example in _D. melanogaster_ (Kilpatrick & Rand, 1995), _D. pseudoobscura_ (MacRae & Anderson, 1988), _D. subobscura_ (Fos et al., 1990), _D. pseudoobscura_ and _D. persimilis_

(Hutter & Rand, 1995). A shared aspect in most of these experiments is the possible cytonuclear interactions between the mtDNA and the nuclear markers which may affect fitness. The

detection of such interactions is difficult, because we do not know which kind of relationship exists or between which nuclear markers it is established. The cytonuclear disequilibria method

(Asmussen & Basten, 1994) could be used to detect such fitness interactions. The purpose of this work was to determine the genetic structure of natural populations of _D. subobscura_

from the islands of Majorca and Minorca (Balearic Islands) in relation to allozymic and mtDNA markers. At the same time, we have tried to detect cytonuclear interactions between the mtDNA

and some nuclear markers in an attempt to understand the population dynamics of mtDNA haplotypes of this species. MATERIALS AND METHODS We studied approximately 500 isofemale lines of _D.

subobscura_ in seven samples from the Balearic Islands: three on the island of Majorca (MA.1, MA.2 and MA.3) and four on Minorca (MI.1, MI.2, MI.3 and MI.4). The three samples from Majorca

were collected at the same location, a pine forest near Esporles, in spring and autumn 1992 and in spring 1993. Three of the Minorcan samples were collected in a pine forest from the north

of the island in autumn 1994, spring 1995, and spring 1996, and the last one in another pine forest from the south of the island in spring 1996, 20 km away from the first location. The flies

were collected with conventional traps of fermented bananas. Once in the laboratory, females were placed individually into a tube with food and kept in an incubator at 19°C. When the _F_1

larvae appeared, females were used for allozyme analyses and the offspring were used to determine maternal mitochondrial haplotypes. ALLOZYME ANALYSIS The allozyme analyses were carried out

using starch gel electrophoresis as described by Ayala et al. (1972) and Brewer (1970). The following 12 enzymes (13 loci) were analysed in each fly: alcohol dehydrogenase (_Adh_), octanol

dehydrogenase (_Odh_), superoxide dismutase (_Sod_), malate dehydrogenase (_Mdh-1_), isocitrate dehydrogenase (_Idh_), xanthine dehydrogenase (_Xdh_), fumarase (_Fum_), catalase (_Cat_),

acid phosphatase (_Acph-1_, _Acph-2_), phosphoglucomutase (_Pgm-1_), alkaline phosphatase (_Aph-4_) and esterase (_Est-5_). The APH-4 and EST-5 enzymes were only studied in the last two

populations from Minorca in an attempt to find allozymes with alleles of intermediate frequencies. Some of these enzymes have a close functional relationship with mitochondrial metabolism

(i.e. FUM, MDH-1, IDH). All the enzymes from a single fly were assayed simultaneously, by cutting slices from the starch gel after electrophoresis to reveal the different allozymes in each

one. In a few cases slices were lost, which caused slightly variable sample sizes. EXTRACTION AND DIGESTION OF MTDNA An enriched fraction of mtDNA was obtained by the method described by

Latorre et al. (1986). This fraction was digested with five restriction enzymes. Three of the enzymes (_Eco_RI, _Eco_RV and _Hin_dIII) recognize sequences of 6 bp, whereas the other two

(_Hpa_II and _Hae_III) sequences of 4 bp. These enzymes were selected for their capability of detecting mtDNA polymorphisms (Afonso et al., 1990; Latorre et al., 1992). The fragments

obtained by digestion were separated on horizontal 0.8–2.0% agarose gels. To determine fragment size, lambda DNA digested with _Hin_dIII and lambda DNA double digested with _Hin_dIII–_Eco_RI

were used as size standards. After electrophoresis, gels were stained with 0.1 μg ethidium bromide mL−1 and visualized with a 260-nm UV light transilluminator. A mtDNA restriction map was

obtained by means of all possible single and double digestions of the mtDNA. The different restriction patterns obtained, using a given enzyme and the haplotypes, were named according to the

notation of (Latorre et al. 1986, 1992). STATISTICAL ANALYSIS Allele and genotype frequencies were estimated for each system and population from the allozymic electrophoretic data.

Hardy–Weinberg equilibria were tested using the BIOSYS-1 program (Swofford & Selander, 1989). The Bonferroni procedure was used to correct the deviations in the χ2-test that originated

from the number of tests performed (Weir, 1990). Heterogeneity was analysed by the _F_-statistics (_F_IS, _F_IT, _F_ST) (Wright, 1965). The degree of mtDNA differentiation within and between

populations (_V_w and _V_b), as well as the degree of population subdivision (_N_ST) were estimated following Lynch & Crease (1990), with a computer program kindly supplied by the

authors. We used Tajima’s _D_-test (Tajima, 1989) to test for any departure from neutrality for the mtDNA haplotype distribution in the populations. The cytonuclear interactions were

performed following the methodology developed by Asmussen & Basten (1994), by calculating the cytonuclear disequilibria and considering a diallelic nuclear locus and two cytoplasmic

haplotypes. RESULTS Table 1 shows the allele frequencies of the 13 isozyme loci studied in the seven samples. _Aph-4_ and _Est-5_ were studied in two of the Minorcan populations. _Cat_ and

_Acph-2_ were only analysed in the Majorcan populations. Most of the enzymes were polymorphic, although some populations had a single fixed genotype; however, interestingly, _Xdh_ was

monomorphic in all Majorcan population, but not in the Minorcan ones. Most allozymes were in Hardy–Weinberg equilibrium (those not in equilibrium were mainly in the Minorcan samples). The

global _F_ST values were significant in all cases, indicating a genetic structure among the samples. _Acph-1_ was the enzyme that made the greatest contribution to this differentiation,

although this was mainly because of sample 2 from Minorca, which clearly exhibited a different proportion of alleles. The total _F_ST was 0.075, but when the analyses were made without

_Acph-1_, this value decreased to 0.030, which was also significant. For the _F_ST between paired populations (Table 2), 10 out of 21 comparisons were significant when _Acph-1_ was included

(above the diagonal). This proportion decreased to three out of 21 when _Acph-1_ was not included (below the diagonal). Without _Acph-1_, the seven samples did not show any clear genetic

differentiation. Figure 1 (bottom) shows the mtDNA cleavage map of the five restriction enzymes, based on the physical mtDNA map of _D. yakuba_ (Clary & Wolstenholme, 1985). We found 21

polymorphic sites and eight conserved regions. The NADH complex genes were concentrated in nine variable (43%) and three conserved (37%) sites. Latorre et al. (1992) found 69% and 17%,

respectively. Table 3 shows the different polymorphic sites which defined every haplotype found in the populations. As in other populations, haplotypes I and II were the most frequent

(92.5%), with haplotype II being commoner than I. The less common haplotypes accounted for only 7.5% and, in general, they were unique for each population. Similar results were obtained by

González et al. (1994). The relationship between haplotypes I and II and the rare ones is also indicated in Fig. 1, which represents an unrooted phylogenetic tree of the 26 haplotypes of _D.

subobscura_ mtDNA. Ten haplotypes were derived from haplotype I and 14 from haplotype II, caused by unique mutations in the mtDNA, with the exception of haplotypes XXI, XXII and XXV, which

had passed through two mutational steps. Table 4 shows the mitochondrial DNA differentiation within and between populations, after Lynch & Crease (1990). Most of the observed variation

was concentrated within populations. The total amount of mtDNA polymorphism may be estimated by the average number (_V_w) of substitutions per nucleotide site for random pairs of haplotypes

from the same population, plus the average number (_V_b) between populations. In our data, _V_w=0.00498 ± 0.00415 and _V_b=−0.00002 ± 0.00006. The fraction of the nucleotide variation

between populations, _N_ST, was −0.004 ± 0.063, which was not significantly different from zero. Tajima’s _D_-test (Tajima, 1989) was used to test departures of the mtDNA haplotypic

distribution from the neutrality hypothesis. The rationale of the test is that in a panmictic population, under the neutral mutation model, no difference would be expected between the

average number of nucleotide differences (i.e. nucleotide heterozygosity) and the number of segregating sites. Table 5 gives the estimates of _D_ for each of the seven samples; for the three

samples from Majorca, for the samples from Minorca and for the pooled population. No significant departures from neutrality were found in the different samples, nor in the Majorcan

population, at the 5% level, although the Minorcan and the total populations did show a significant negative departure from neutrality, thus indicating that there were excessive numbers of

rare haplotypes. To test the relationship between the mtDNA haplotypes and nuclear markers, a set of cytonuclear disequilibria analyses was made with haplotypes I and II. The analyses with

the samples separated did not show any cytonuclear disequilibria. All the samples were analysed as a unique population, because of the genetic similarity of the seven samples studied at the

enzymatic level (with the exception of _Acph-1_). Only a slightly significant cytonuclear disequilibrium was detected (χ21=4.85) in the cytonuclear estimator of nuclear heterozygotes versus

mtDNA haplotypes of _Acph-2_. Apart from this, no other cytonuclear disequilibrium estimator was significant in any enzyme. DISCUSSION We have carried out an extensive study of seven samples

of _D. subobscura_ from the islands of Majorca and Minorca at the allozymic and mtDNA level. At the allozymic level we did not observe any clear genetic differentiation among the samples.

Although the _F_ST values were statistically significant, the tests of paired genetic differentiation did not show great differences between the samples. The only exception was when _Acph-1_

was included in the analyses, but this was caused by the different allozymic frequency of sample 2 from Minorca. Therefore, we consider that the populations of Majorca and Minorca have no

clear genetic differences between them. When the mtDNA was studied, the pattern agreed with that found in other populations, that is, I and II are present in all populations at high

frequencies, whereas certain rare ones occur at low frequencies. The test we used to determine the average number of substitutions per nucleotide per pair of random haplotypes within and

between populations (Lynch & Crease, 1990) was not significant, which indicates that there is no between-population heterogeneity. This result is similar to that reported by González et

al. (1994) in _D. subobscura_ or by Baba-Aissa et al. (1988) and Nigro (1988) for _D. simulans_. The mtDNA polymorphism observed in natural populations can be interpreted in several ways,

such as interactions with nuclear polymorphisms, random genetic drift, or direct selection on the mtDNA haplotypes (Latorre et al., 1992; Moya et al., 1993; García-Martínez et al., 1998).

The _D_-values of the Tajima test for mtDNA non-neutral evolution were not significant in the seven samples. Nevertheless, we detected significant negative _D_-values for the entire Minorcan

population and the total population. According to Tajima (1989), a significant negative value might be the result of purifying selection or a population bottleneck. Although purifying

selection might generate a negative _D_-value, it would be expected to be observed more significantly in all the populations analysed and this was not the case in our study. Therefore, we

think that the explanation based on a population bottleneck is more acceptable. It is possible that the Minorcan population as a whole may not yet have reached equilibrium, probably as a

consequence of seasonal periodic bottlenecks, followed by an expansion. The result would be an excess of rare haplotype polymorphism while the population was expanding. On the contrary, the

Majorcan population seems to be more stable. The significance of the total population would be the result of the influence of the Minorcan population. Cytonuclear associations between

nuclear markers and mtDNA haplotypes or some kind of genetic hitchhiking are difficult to detect by means of cytonuclear disequilibria parameters. Unless fitness interactions are extremely

strong, the disequilibrium parameters are quickly going to be negligible (Babcock & Asmussen, 1996). Only some transient disequilibria could be expected as a consequence of genetic

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the moment the samples were taken. Because of the adaptive value of the inversions, experiments in population cages (i.e. a new environment different from that of nature) might generate

strong genetic hitchhiking on different and neutral competing haplotypes. The detection of these associations could help us to understand the dynamics of mtDNA polymorphism in natural

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of mating. _Evolution_, 19: 395–420. Article  Google Scholar  Download references ACKNOWLEDGEMENTS This work has been supported by grants PB93-0690 and PB96-0793 from DGES (Spain). AUTHOR

INFORMATION AUTHORS AND AFFILIATIONS * Laboratori de Genètica, Departament de Biologia, Facultat de Ciències, Universitat de les Illes Balears, Palma de Mallorca (Balears), Spain José A

Castro, Misericòrdia Ramon & Antònia Picornell * Institut Cavaniltes de Biodiversitat i Biologia Evolutiva i Departament de Genètica, Universitat de València, Burjassot (València), Spain

Andrés Moya Authors * José A Castro View author publications You can also search for this author inPubMed Google Scholar * Misericòrdia Ramon View author publications You can also search

for this author inPubMed Google Scholar * Antònia Picornell View author publications You can also search for this author inPubMed Google Scholar * Andrés Moya View author publications You

can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to José A Castro. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS

ARTICLE Castro, J., Ramon, M., Picornell, A. _et al._ The genetic structure of _Drosophila subobscura_ populations from the islands of Majorca and Minorca (Balearic Islands, Spain) based on

allozymes and mitochondrial DNA. _Heredity_ 83, 271–279 (1999). https://doi.org/10.1038/sj.hdy.6885500 Download citation * Received: 15 April 1998 * Accepted: 30 March 1999 * Published: 01

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allozymes * _Drosophila subobscura_ * Majorca * Minorca * mtDNA haplotypes