1. Introduction
Species colonizing new environments need to adapt to novel biotic and abiotic conditions. One critical determinant of species ranges that varies with latitudes and altitudes is environmental temperature. In particular for ectotherms, such as insects, temperature plays a major role in determining species abundance and geographic distribution [
1,
2]. As a cosmopolitan species, the fruit fly
Drosophila melanogaster has adapted to a wide range of thermal environments [
3,
4,
5]. Its origin is thought to be in tropical southern-central Africa from where it spread around the world [
6,
7,
8]. After an initial expansion throughout Africa, it reached the Eurasian continent after the last glaciation around 10,000 years ago [
9,
10], and later moved on to colonize Asia and Europe [
11]. An important limiting factor while settling in Europe and at high altitudes in sub-Saharan Africa must have been temperate climates, with their low and varying temperatures.
For
Drosophila, it is known that the transcriptional output of genes can be affected by temperature [
12,
13]. The resulting expression plasticity across temperatures might have been detrimental in temperate climates if it shifts the transcriptional output away from the optimum. A high degree of expression plasticity, for instance, could have severe consequences for the intricate interactions of genes involved in development and cell differentiation. The reduction of temperature-induced expression plasticity of functionally important genes, therefore, might have been important while adapting to temperate environments, in order to buffer against fluctuations in temperature and to maintain consistent expression levels across temperatures [
14]. Indeed, temperature-induced gene expression plasticity was generally reduced in a temperate compared to a tropical population, both from Australia [
12].
The gene vestigial (
vg), well-known for its wing mutants, is a key player in the development of the
Drosophila wing. It encodes a transcription factor that plays an essential role in the development and patterning of the wing [
15]. Loss of
vg results in the failure of wings to develop [
16] and ectopic expression of
vg leads to the outgrowth of ectopic wing tissue [
17]. Known
vg mutations display a range of temperature-sensitive expression patterns [
18] indicating that DNA sequence changes at the
vg locus can cause differences in temperature-sensitivity. This makes
vg an interesting candidate for studying temperature-sensitive gene expression and the potential buffering thereof in flies from temperate climates.
We investigated temperature-sensitive expression of vg in natural populations of D. melanogaster from six different locations. These included three temperate populations from Europe, one temperate population from a high-altitude location in Africa, and two tropical populations from the ancestral species range. Temperature-induced plasticity of vg expression with higher expression at lower temperature appeared to be restricted to certain tissues and/or stages. The degree of expression plasticity differed between the populations with a higher degree in those from hot climates than in those from temperate climates. In addition, vg expression was significantly increased across temperatures in cold-temperate flies.
4. Discussion
Here we examined the expression response to temperature of the gene vg in six natural populations of D. melanogaster from different latitudes and altitudes. vg is a transcription factor that is known as a wing selector gene due to its essential role in the development and patterning of the Drosophila wing. In all four temperate populations, temperature-sensitive expression plasticity was reduced compared to the two tropical populations from the ancestral species range. Temperate populations were derived from a range of different locations including high-latitude Europe and high-altitude Africa. The consistent response to temperature across all temperate populations is consistent with positive selection acting to reduce temperature-sensitivity of vg expression in temperate climates.
We observed that reduced vg expression at 17 °C compared to the expression level in tropical flies led to a buffering of temperature-induced expression plasticity in the three population samples from warm-temperate climates. In contrast, in the cold-temperate sample from Sweden, increased vg expression at 28°C relative to the tropical and the other population samples resulted in the observed buffering effect. Given the shared demographic history of European populations, it seems likely that in addition to buffering temperature-sensitive expression in European flies, overall expression of vg is increased in the Swedish population. Other ecological constraints due to the colder climate in Sweden could be a possible explanation for the observed difference. Higher overall vg expression, for instance, might have been further beneficial in the colder climate of Sweden.
The observed direction of the expression response to temperature with higher expression at lower temperatures is typical for genes regulated by the Polycomb group (PcG) [
28,
29,
30,
31,
32]. As for many developmentally important genes, the expression of
vg is epigenetically controlled by this group of proteins. Interestingly, an earlier study found evidence for selection acting on cis-regulatory sites leading to reduced expression plasticity of another PcG-target gene in temperate flies [
33]. The selected sites were highly differentiated between African and European
D. melanogaster populations and were located in a Polycomb response element (PRE), a cis-regulatory DNA element that recruits PcG proteins to their target genes [
34].
Although
vg plays important roles in the differentiation of adult structures during development [
15,
16,
17,
35], little is known about its function in adult flies. Since the main function of
vg is in wing development [
15,
16,
17], we also looked for temperature-induced expression plasticity and its possible buffering in wing discs of wandering third instar larvae. In this tissue and at this developmental stage,
vg is in an activated state and highly expressed [
15,
27]. We chose the larval brain, in which
vg gene expression is low, as a control tissue [
27]. Temperature-sensitive expression as it is often observed for PcG-regulated genes and as we found for adults was not detected in either of the two larval tissues. Possible explanations for this include that selective pressure against such a temperature-induced expression plasticity might be much stronger in larval tissues compared to adult tissues and therefore is not observed in any of the populations. Alternatively,
vg expression is in itself not affected by temperatures in larval tissues like it is in adult tissues. At least for wing discs, the former explanation might be more likely. Mutations in
vg introns were found to cause temperature-sensitive expression of the gene in wing imaginal discs, whereas no temperature-sensitivity was observed for wild type discs [
18].
Both trans-regulatory and cis-regulatory changes might be responsible for the changes in
vg expression in adult flies. A range of temperature-sensitive expression patterns of
vg has been observed in mutant flies carrying mutations at the
vg locus [
18] indicating that DNA sequence changes at the
vg locus can cause differences in temperature-sensitivity. We assessed genetic differentiation in the
vg gene region in an attempt to identify potential candidates for cis-regulatory changes responsible for the observed expression differences. Candidate SNPs were located in the introns of
vg and upstream of
vg in a region also occupied by the insulators that demarcate the
vg gene region. As mentioned above, mutations in introns of
vg can change temperature-sensitivity of
vg expression [
18], whereas insulators are known to play important roles in ensuring PcG-mediated gene repression [
36,
37,
38,
39]. One of the Rwandan candidate SNPs was found in a region in the third intron of
vg, which, if disrupted by a mutation, was shown to result in increased temperature-sensitive expression of
vg [
18]. A common cis-regulatory mechanism between the temperate European and Rwandan populations appears to be less likely, since no shared outlier SNPs were observed. However, whether the detected candidate SNPs are actually involved in changes of
vg expression observed in this study remains unclear. Trans-regulatory changes also might play a role, as well as sites and indels that were not included in the analysis. Furthermore, elevated genetic differentiation of SNPs might also result from neutral processes such as demographic processes or due to being linked to selected sites not involved in the regulation of
vg gene expression.