1. Introduction
The expansion of a species into new territories provides the opportunity for adaptation to novel environmental conditions. Although such adaptation is of fundamental interest to evolutionary biologists, the identification of locally adaptive traits, along with their underlying molecular and genetic bases, has proven to be challenging. Given its well-understood genetics and experimental tractability,
Drosophila melanogaster has become a leading model organism for studying the molecular basis of adaptation. These studies typically involve the comparison of DNA sequence and/or gene expression variation between populations from the ancestral (sub-Saharan African) and derived (cosmopolitan) species ranges [
1,
2,
3,
4,
5,
6,
7], or among multiple populations spanning an environmental gradient, such as a latitudinal cline [
8,
9,
10]. Clinal variation has been detected for several types of genetic polymorphism in
D. melanogaster, including chromosomal inversions [
9], allozymes [
11,
12], and single nucleotide polymorphisms (SNPs) [
13,
14]. Moreover, some genetic variants show repeated oscillations in frequency across seasons in temperate populations [
15]. Collectively, these studies suggest that a considerable amount of genetic and expression variation may be maintained within
D. melanogaster due to local or seasonal adaptation, although detailed functional studies of the underlying causal variants and how they exert their effects on a relevant organismal phenotype remain rare [
16,
17,
18,
19,
20,
21].
Global populations of
D. melanogaster are polymorphic for a 49-bp deletion in the 3′ untranslated region (UTR) of the
Metallothionein A (
MtnA) gene, as shown in
Figure 1 [
18,
22,
23]. This deletion is present at high frequency (34–100%) in cosmopolitan populations but low frequency (0–8%) in sub-Saharan Africa and is absent in other
Drosophila species [
18,
23,
24], suggesting that it is a derived mutation that has spread to high frequency following the species’ out-of-Africa expansion. Furthermore, the frequency of the deletion increases with distance from the equator in Europe, North America, and Australia, which is consistent with adaptation to temperate environments [
18]. The deletion is associated with increased
MtnA expression and reporter gene experiments have demonstrated that, in a common genetic background, the deletion causes an expression increase in the range of 1.4 to 2.3-fold [
18]. The deletion also is associated with increased tolerance to oxidative stress in isofemale lines derived from Europe and Asia [
18]. Taken together, these results suggest a scenario in which the
MtnA 3′ UTR deletion is selectively favored in temperate environments due to the increased oxidative stress tolerance that it confers, which is mediated by higher
MtnA expression levels. The importance of oxidative stress as a selective factor in temperate populations is further supported by studies of the
Bari-Juvenile hormone epoxy hydrolase (Bari-Jheh) transposable element insertion, which is present at high frequency in temperate regions and upregulates expression of the
Jheh genes, leading to increased oxidative stress tolerance [
25,
26]. In the case of
MtnA, the 3′ UTR deletion is presumably deleterious under some environmental conditions, which would explain why it remains polymorphic within temperate populations, as well as its rarity in sub-Saharan Africa and other tropical and sub-tropical regions.
In the current study, we determine the frequency of the MtnA 3′ UTR insertion/deletion (indel) polymorphism in a large sample of wild-caught flies of both sexes from a temperate European population (Munich, Germany) across seasons and years. This allows us to test for potential forms of balancing selection, such as seasonally fluctuating selection, overdominant selection, or sexual antagonism, which could be involved in the maintenance of this polymorphism. We further examine the effect of the deletion on MtnA expression in nearly-isogenic lines derived from this population. We also use publicly available data to test for associations between the deletion, MtnA expression, and oxidative stress tolerance in a large North American population sample. Finally, by re-analyzing genome sequence data from a sub-Saharan African population, we determine the frequency of the deletion in the ancestral species range and test whether it is consistent with ancient standing variation or more recent admixture with non-African flies.
4. Discussion
By genotyping wild-caught
D. melanogaster from a European population and re-analyzing publicly available genomic data from an African and a North American population, we were able to gain a better functional and population genetic understanding of the
MtnA 3’ UTR indel polymorphism. Consistent with a previous study [
18], we find that the deletion allele is present at high frequency in cosmopolitan populations, but low frequency within sub-Saharan Africa. In the few sub-Saharan African lines that had the deletion, it was embedded within a genomic region sharing high sequence similarity with European lines, as shown in
Figure 4. Thus, the deletion is unlikely to represent standing genetic variation in the ancestral population, but instead a new mutation that increased in frequency following the species’ out-of-Africa migration. In a German population, the deletion appears to have remained at a remarkably stable frequency of approximately 90% for over a decade. However, despite its stable frequency and clinal distribution across continents, we found no evidence for balancing selection acting on the indel polymorphism in this population. The deletion did not vary significantly in frequency across years or seasons, or between the sexes, as shown in
Table 1. Thus, there was no evidence for seasonally fluctuating or sexually antagonistic selection acting on this polymorphism. Similarly, we did not detect an excess of heterozygotes, as would be expected for overdominant selection, as shown in
Table 2.
The above findings suggest that the
MtnA indel polymorphism has been subjected to a more complex form of selection. One possibility is that the polymorphism affects a fitness component other than viability, which could not be detected by measuring allele frequencies in adult flies, even with large sample sizes. Another possibility is that the deletion is consistently favored by positive selection in the German population but remains polymorphic in this population due to repeated migration of flies from more tropical populations where the deletion is at intermediate frequency (45–65%) [
18] and may be under balancing selection. A third possibility is that
MtnA is just one of many loci involved in polygenic adaptation. In a scenario where multiple genetic variants of small effect influence a selected trait, alleles may rise in frequency rapidly, but then level off at an intermediate frequency without going to fixation in the population [
49]. Our functional analyses are consistent with this interpretation: the two traits we investigated, gene expression and oxidative stress tolerance, both showed evidence of being influenced by variation at multiple loci. In terms of
MtnA gene expression, the use of nearly-isogenic lines allowed us to determine the effect of the indel polymorphism while minimizing the contribution of other variants across the genome. This indicated that the magnitude of the effect of the indel polymorphism differed depending on the genomic background, as shown in
Figure 2. Furthermore, among the DGRP lines, the indel explained only 8–23% of the observed
MtnA expression variation. Similarly, oxidative stress tolerance is influenced by many loci: 154 SNP loci showed a significant association with survival on MSB in a previous study of the DGRP lines [
45]. The
MtnA gene was not among the candidates influencing MSB tolerance in the previous study. However, that study focused only on SNP markers and, thus, did not consider the
MtnA indel polymorphism, which does not show strong linkage to SNPs in this region of the genome [
18]. The previous study also found susceptibility to oxidative stress to be sexually dimorphic, with many variants displaying sexually antagonistic effects [
45]. While we found evidence that the magnitude of the effect of the indel polymorphism on both
MtnA expression and oxidative stress tolerance was sex-dependent, as shown in
Figure 3, the response was in the same direction in males and females, suggesting that the polymorphism has a concordant effect on fitness in both sexes. This is in agreement with there being no evidence for sexual antagonism influencing the frequency of the polymorphism in adult flies, as shown in
Table 1.
Catalán et al. [
18] found that increased
MtnA expression, as well as the presence of the
MtnA 3’ UTR deletion, was associated with increased survival in the presence of hydrogen peroxide, a reactive oxygen species (ROS). While ROS are natural products of metabolism and serve important functions in the cell, high levels, which can be introduced by environmental factors such as UV light or toxins, lead to oxidative stress. Organisms cope with oxidative stress through the induction of antioxidant or free radical scavenger genes, as well as other mechanisms such as apoptosis and cell cycle arrest [
50,
51]. Thus, it is unsurprising that previous studies found that the transcriptional response to oxidative stress depends on the stress-inducing agent [
50,
52,
53] and that genes associated with oxidative stress tolerance are typically specific to the agent of induction [
45]. This is thought to be a product of the different mechanisms of action these agents employ that result in toxicity [
45]. Our results provide insight into the mechanism through which
MtnA expression improves oxidative stress tolerance. We found that increased
MtnA expression, as well as the
MtnA deletion allele, is associated with increased tolerance to MSB, but not paraquat, as shown in
Figure 3. While paraquat toxicity is primarily driven by redox cycling, MSB toxicity primarily occurs through other reactions with biomolecules unrelated to superoxide formation [
54]. It has been suggested that metallothioneins may play a role as scavengers of free radicals in the oxidative stress response [
55], which is in line with our findings.