Clinal patterns can reflect variable selection in space and are thought to be maintained by a trade-off between dispersal and local adaptation. Although clines by themselves are not sufficient to infer adaptive divergence, strong evidence of selection is obtained if those patterns can be independently replicated and understood in terms of likely fitness effects (Endler 1977). This approach has been used effectively with the invasive species Drosophila subobscura ever since its first appearance in South (Puerto Montt, Chile, 1978; Brncic and Budnik 1980) and North America (Port Townsend, WA, 1982; Beckenbach and Prevosti 1986).
An early comprehensive summary of the wealthy inversion polymorphism of D. subobscura revealed that the complex gene arrangements (with overlapping and nonoverlapping inversions) from more equatorial Palearctic populations are gradually replaced by the so-called standard gene arrangements in the five major acrocentric chromosomes as populations approach high latitudes (Krimbas and Loukas 1980). Disentangling adaptive explanations from purely historical processes that could generate this pattern (i.e., northward migration of the populations of D. subobscura after the last glacial period) was only possible when Prevosti et al. (1985) described rapid convergent evolution in the signs of the correlations between gene arrangements and latitude a few years following the American invasion. Selection must have to be strong and consistent along the latitudinal gradients to explain the rapid emergence of independent parallel clines (summarized in Balanyà et al. 2003) in such a highly dispersive species as D. subobscura (Ayala et al. 1989). Yet, not much notice was taken of the final remarks in Prevosti's et al. original work (but see Balanyà et al. 2006): “Differences in the rate of dispersal between individuals carrying different chromosomal arrangements could also have contributed to the establishment of the clines.” This point is important and recent theoretical work shows that model assumptions with random diffusive dispersal or fitness-dependent dispersal can have important implications in the structure and dynamics of clines (Armsworth and Roughgarden 2008).
The simple idea that environmental latitudinal gradients favor different inversions at different latitudes remains attractive, and several lines of evidence suggest that temperature may be the underlying selective factor. First, a long time-series experiment starting in 1976 at a northwestern Spanish population showed that the seasonal climatic cycle induces seasonal changes in inversion frequencies on chromosome O that are consistent with their latitudinal patterns (Fontdevila et al. 1983; Rodríguez-Trelles et al. 1996). Second, long-term trends were superimposed on the seasonal cycles with “southern,” low-latitude inversions increasing in frequency thus suggesting a directional response to current climate warming (Rodríguez-Trelles et al. 1996; Rodríguez-Trelles and Rodríguez 1998). More recent and comprehensive studies have shown that the genetic constitution of D. subobscura populations worldwide is indeed responding to climate change (Balanyà et al. 2006). However, it is not yet clear whether the shifts in inversion frequencies represent local selection, confound long-term trends with the presence of a shifting seasonal component (c.f. Rodríguez-Trelles and Rodríguez 2007; Balanyà et al. 2007), or simply reflect an invasion from more equatorial populations (see Santos 2007).
The putative role of temperature per se in the formation of inversion clines has been tested by letting replicated lines of D. subobscura evolve in the laboratory at different constant temperatures (Santos et al. 2005). In this experiment gene arrangements on all chromosomes generally shifted in ways inconsistent with expectations based on clinal patterns. It is nevertheless obvious that keeping flies in isolated populations under different selective regimes misses the multitude of additional factors that complicate the simplistic view in this kind of experiments: that natural selection can be embodied in its totality in laboratory population cages. Ectotherms in the wild use thermoregulatory behaviors to avoid—or at least reduce—the impact of thermal fluctuations and can maintain body temperature (Tb) within relatively narrow boundaries (e.g., by modifying daily activity patterns and selecting favorable microclimates; Stevenson 1985). Behavioral thermoregulation in D. subobscura could ameliorate effects of seasonal and latitudinal variation in air temperature, hence the actual selective pressures implicated in the rapid clinal evolution of chromosomal inversions might be weaker than originally thought (Huey et al. 2003) and could even conflict with those in the laboratory (e.g., by forcing flies that would otherwise choose colder or warmer settings to compete at fixed constant temperatures).
Seasonal variation of Tb in active D. subobscura flies from five North American populations spanning 12° latitude has been recently estimated by Huey and Pascual (2009). To evaluate if these flies can be effective thermoregulators (Hertz et al. 1993) they also measured their thermal preferences (Tp: the body temperature an organism chooses when provided with a range of potential temperatures; see Dillon et al. 2009) using a laboratory thermal gradient. The results show that mean field Tb varied from 8°C to 29°C, well outside the relatively narrow set-point range (central 50% of preferred body temperatures: 21.2°C – 25.9°C). Therefore, although D. subobscura flies can behaviorally thermoregulate, geographic shifts in ambient temperature may nevertheless be a major evolutionary force in generating the clinal patterns. A corollary of their work is to question the validity of the aforementioned laboratory thermal selection experiment because the imposed thermal regimes might not match the optimum temperatures of the various gene arrangements (Fig. 1).
Here, we explore whether the thermoregulatory behavior and thermal tolerance of D. subobscura have a genetic component. More specifically, we address whether individuals carrying different chromosomal arrangements also vary in their Tp and heat tolerance (Tko: the temperature required to knock out a fly in a water bath). Our analysis was motivated by the hypothesis that inversion clines are indeed driven by thermal selection (population cage results notwithstanding; Fig. 1), and the idea that genetic variation in thermoregulatory abilities might underlie the evolution of clines of chromosomal arrangements. Some evidence indicates that diurnal activity patterns can vary according to gene arrangements in this species (Savkovic et al. 2004), which suggests that fitness-dependent dispersal (as pointed out by Prevosti et al. 1985) due to different physiological optimal temperatures of the various inversion carriers could be at work in natural populations. Furthermore, because D. subobscura can occasionally experience injurious or lethal effects of very high Tb's—for example, larval feeding patches could become lethally hot (Feder et al. 1997), and the former maximum of 29°C for active flies in the field would certainly be considered as quite stressful by most researchers working with this species (see Krimbas 1993)—upper thermal tolerance likely imposes critical limits on fitness. Laboratory studies with D. melanogaster have associated heat-tolerance variation with genetic variation at candidate loci for thermoresistance linked to chromosomal inversions (e.g., Anderson et al. 2003; Hoffmann and Weeks 2007), and a larger than expected number of genes putatively involved in thermal adaptation in D. subobscura map inside inverted chromosomal segments (Laayouni et al. 2007). Our results show that flies carrying “cold-adapted” gene arrangements (typically “high-latitude” arrangements; see Statistical Methods below) tend to choose lower temperatures in the laboratory or have a lower heat stress tolerance, in line with what could be expected from the natural patterns. Different chromosomes are mainly responsible for the underlying genetic variation in both traits, which also explains why thermal preference and thermal tolerance were linearly independent.