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- Materials and methods
For ectotherm species, such as insects, temperature has long been recognized as a major environmental factor responsible for species abundance and geographic distribution (Andrewartha & Birch 1954; Precht, Christophersen & Hensel 1955; Cossins & Bowler 1987; Leather, Walters & Bale 1993). Ambient temperature varies according to daytime and season, so that natural populations are often exposed to heat or cold stress (Gibbs, Perkins & Markow 2003). The capacity to adapt to and tolerate such stresses is crucial for the persistence of populations (Hoffmann & Parsons 1991, 1997; Addo-Bediako, Chown & Gaston 2000; Chown, Addo-Bediako & Gaston 2002; Hoffmann, Sorensen & Loeschcke 2003; Klok & Chown 2003). In temperate countries, species must tolerate cold conditions during winter and have developed a diversity of adaptive mechanisms to do so, including the occurrence of diapause and the production of antifreeze compounds (Leather et al. 1993; Graham, Walker & Davies 2000).
Drosophilid species form convenient models with which to investigate ectotherm responses to stress because they are found across a full range of habitats from the Arctic to the equator. Drosophila melanogaster is a cosmopolitan species, and its broad geographical range is accompanied by genetic variation in a diversity of traits, which, in most cases, vary progressively with latitude (David & Capy 1988): this pattern suggests that local climate and especially temperature is the selective factor. Latitudinal clines have been demonstrated for several morphological traits (e.g. Capy, Pla & David 1993) and also for traits directly related to fitness, such as viability, rate of development, larval competitive ability, egg production and tolerance to stresses (heat, cold, desiccation, starvation) (Stanley & Parsons 1981; Boulétreau-Merle et al. 1982; Davidson 1990; James & Partridge 1995, 1998; Guerra et al. 1997; Karan et al. 1998; Hoffmann et al. 2003).
A specific problem in thermal adaptation investigations arises from the fact that most morphological and physiological traits exhibit a large amount of phenotypic plasticity related to growth temperature. For several morphological traits, the response curves are now well described and adaptive variations in the shape of the reaction norms are known across species or geographical populations (see David, Gibert & Moreteau 2004). However, plasticity in fitness traits is less commonly investigated because measurements at the adult stage include variation, which is a consequence of both growth and experimental temperature (Huey et al. 1999).
In the present work, we analyse cold tolerance in D. melanogaster with a recently developed assay (David et al. 1998), that measures the time needed for recovering from chill coma after cold treatment. Recovery from chill coma induced at 0 °C provided a clear-cut discrimination between temperate and tropical species (Gibert et al. 2001). Comparison of geographical populations in nine cosmopolitan species also showed that temperate strains recovered faster than tropical ones, suggesting a genetic adaptation (Gibert et al. 2001). Such adaptive genetic variations were confirmed and latitudinal clines were found in Australian populations of two species, D. melanogaster (Hoffmann, Anderson & Hallas 2002) and D. serrata (Hallas, Schiffer & Hoffmann 2002). Here we investigate a global set of populations of D. melanogaster and demonstrate a latitudinal cline which might exist on all continents. We also analyse the role of growth temperature and show that most adaptive variability arises from phenotypic plasticity. By changing the developmental temperature the adult phenotype varies from cold tolerance (as found in temperate species) to cold sensitivity (as found in purely tropical ones).
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- Materials and methods
Recovery time after a cold shock at 0 °C is a laboratory assay aimed at measuring the cold sensitivity of a given Drosophila strain. Like most laboratory assays, it does not correspond to a situation occurring in nature. So the question is: are our results relevant to natural selection and adaptation? We believe that our assay provides a positive answer for four main reasons.
First it has been shown (Gibert et al. 2001) that recovery from chill coma induced at 0 °C permits an unambiguous classification of species as either tropical or temperate. Second, a brief survey (Gibert et al. 2001) revealed that this distinction between tropical and temperate species is also observed in other insect orders, suggesting a need for more extensive comparisons. Third, we have shown here that genetic variations among world populations of D. melanogaster vary according to a latitudinal cline. Populations living at high latitudes under temperate climates recover more quickly (i.e. are more cold tolerant) than tropical populations. Fourth, and still more interestingly, we show here that recovery time is a plastic trait, depending on developmental temperature. The results are in the expected direction for an adaptive plasticity: a development at low (12 °C) temperature considerably increases the cold tolerance of adults so that D. melanogaster (an Afrotropical species) approaches the values typical of purely temperate species (Gibert et al. 2001).
To our knowledge, this is the first time that a linear reaction norm based on development according to growth temperature has been observed in Drosophila. Other fitness traits, such as viability, offspring production, male fertility or adult longevity, exhibit a generally sharp decrease at both ends of the thermal range (Cohet & David 1978; David et al. 1983; Pétavy et al. 2001; Chakir et al. 2002). In such cases, phenotypes may be interpreted as pathological, reflecting the deleterious effects of extreme developmental temperatures. However, in the case of cold tolerance plasticity clearly has a beneficial effect. In this respect, chill coma recovery appears to support the beneficial acclimation hypothesis (Leroi, Bennett & Lenski 1994) but it could also be interpreted as ‘colder is better’ (Huey et al. 1999). Analogous trade-offs between cold tolerance and fertility were also observed after acclimation of D. melanogaster adults at 11 °C (Bubliy et al. 2002). The fact that a given environment may result in pathological effects on some fitness traits but in beneficial effects on others illustrates the complexity and diversity of plastic responses, and the difficulty of generalized interpretations.
In a temperate country such as France, autumn generations do develop at temperatures close to 12 °C. This is shown by examining morphometric traits and knowing their reaction norms. The two most informative traits are female abdomen pigmentation and wing/thorax ratio (David et al. 1994; Pétavy et al. 2002) which both exhibit monotonically decreasing reaction norms. A low developmental temperature in adult flies collected in October and November is inferred by the observation of a very dark pigmentation and high wing/thorax ratio (J. R. David, unpublished observations). During winter, adults developed at a low temperature probably overwinter in sheltered places, although this is not well understood (Izquierdo 1991). They are probably submitted to repeated cold stresses and the capacity to recover rapidly is an advantage, which is also correlated to a better survival (J. R. David, unpublished observations).
From an evolutionary point of view, a consensus exists that D. melanogaster is native to tropical Africa (David & Capy 1988) and that it could have reached Europe several millennia ago, independently of modern human transportation (Lachaise et al. 1988). The cold adaptation seen in temperate populations may be the consequence of a long-lasting directional selection. The observation of a similar adaptive plasticity in the ancestral tropical populations is, however, unexpected since temperatures approaching 0 °C and able to induce a chill coma are not encountered in nature. Two opposite kinds of explanation may be proposed. First, we may assume that plasticity, which presumably implies some specific changes in the nervous system (see David et al. 1998), is a by-product of some more general functional changes related to growth temperature. For example, a lowering of temperature in ectotherms increases the level of unsaturated fatty acids, so that the fluidity of cell membranes is kept stable (Cossins & Bowler 1987; Hazel 1995). In Drosophila, it has been shown in several species that acclimation to low temperature resulted in a qualitative change in the proportion of monoenes and diens, and also in a decrease in the length of fatty acids (Ohtsu, Kimura & Katagiri 1998). In this respect, chill coma plasticity might be considered as an exaptation, according to Gould & Vrba (1982). Second, it is also possible that D. melanogaster, in spite of its Afrotropical origin, was already adapted to cold. Cold temperatures are encountered in the mountains of tropical countries and the occurrence of mountain populations in that species is known. Indeed, the collection of a wild living male in a natural habitat at an altitude of 3000 m was already mentioned (David & Tsacas 1981). In other words, D. melanogaster could have been somehow preadapted to the colonization of temperate countries by first colonizing afrotropical mountains. To examine this hypothesis, it would be interesting to investigate colonizing species which, like D. ananassae (Morin et al. 1997) or Zaprionus indianus (Karan, Moreteau & David 1999), are readily transported by humans, but are restricted to tropical places and apparently unable to tolerate cold winters.