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Ó Springer 2006 Genetica (2006) 128:81–93 DOI 10.1007/s10709-005-5537-7 Altitudinal patterns for longevity, fecundity and senescence in Drosophila buzzatii F.M. Norry1, P. Sambucetti1, A.C. Scannapieco1 & V. Loeschcke2 1 Departamento de Ecologı´a y Evolución, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, (C-1428-EHA) Buenos Aires, Argentina (Phone: (++54-11)-45763300-219/(++54-11)-43624548; Fax: (++54-11)-45763384; E-mail: [email protected]); 2Department of Ecology and Genetics, University of Aarhus, Ny Munkegade, Bldg. 540, DK-8000, Aarhus C, Denmark Received 10 August 2005 Accepted 28 November 2005 Key words: Drosophila, early fecundity, elevational gradient, population-by-temperature interaction, senescence rate, sex-specific variation Abbreviations: CBM – cactus-based medium; CPHR – Cox’s Proportional Hazards Regression; GLIM – generalized linear model; IDM – instant Drosophila medium Abstract We tested for variation in longevity, senescence rate and early fecundity of Drosophila buzzatii along an elevational transect in Argentina, using laboratory-reared flies in laboratory tests performed to avoid extrinsic mortality. At 25 °C, females from lowland populations lived longer and had a lower demographic rate of senescence than females from highland populations. Minimal instead of maximal temperature at the sites of origin of population best predicted this cline. A very different pattern was found at higher test temperature. At 29.5 °C, longevity of males increased with altitude of origin of population. No clinal trend was apparent for longevity of females at 29.5 °C. There was evidence for a trade-off between early fecundity and longevity at non-stressful temperature (25 °C) along the altitudinal gradient. This trait association is consistent with evolutionary theories of aging. Population-by-temperature and sex-by-temperature interactions indicate that senescence patterns are expressed in environment specific ways. Introduction Senescence is the progressive decline in survival and reproduction with age. This degenerative process is unlikely to strongly contribute to mortality when harsh environments persist. In addition, mortality in the wild is mainly due to extrinsic causes such as cold, starvation, predation, desiccation and/or infection. Therefore, wild animals do not often live long enough to become very aged, so that the intensity of natural selection will decline with age (Rose, 1984; Finch, 1990; Partridge & Mangel, 1999; Kirkwood & Austad, 2000). Evolutionary theories of aging postulate that the nearly absent or decreased selection in late ages will result in (i) an accumulation of late-acting deleterious mutations (the mutation accumulation theory, Medawar, 1952) and/or (ii) an increase in frequency of alleles with late deleterious but early beneficial effects (the antagonistic pleiotropy theory, Williams, 1957). In model organisms like Drosophila, laboratory populations have successfully been used to test hypotheses on the evolution of longevity and senescence. For instance, different studies in Drosophila have examined the experimental evolution of life span by using artificial selection on age at reproduction (Rose, 1984, 1991; Finch, 82 1990; Partridge & Fowler, 1992; Roper, Pignatelly & Partridge, 1993; Zwaan, Bijlsma & Hoekstra, 1995; Partridge & Mangel, 1999). A general result of these studies of experimental evolution was that longevity consistently diverged between the ageselected lines and their controls, and a general correlate of this selection response has been reduced early fecundity in the long-lived flies (Rose, 1999 ; Partridge & Mangel, 1999). These general findings from experimental evolution in Drosophila are consistent with evolutionary theories of aging (Kirkwood & Austad, 2000), though cultural artifacts could have influenced associations between longevity and fecundity in some of the studies of experimental evolution (Promislow & Tatar, 1998; Linnen, Tatar & Promislow, 2001). An approach that can complement work with experimental evolution populations is to use laboratory stocks recently derived from the wild to test for clinal trends of variation in both longevity and early fecundity of Drosophila at controlled temperature. This approach can be used under experimental conditions of reduced or absent extrinsic mortality in order to test for geographical and/or environmental variation in senescence patterns (e.g., Reznick, 1993; Tatar, Grey & Carey, 1997; Reznick et al., 2004). If a longevity cline occurs along a thermal gradient, the cline can best be studied at different temperatures since genotype-by-temperature interactions can be evolutionary important for this trait (e.g., Vieira et al., 2000; Norry & Loeschcke, 2002a, b). At high temperature, many genes that might be involved in genotype-by-environment interactions strongly change in their expression level (Sørensen et al., 2005a), including the gene coding for the 70 KD heat-shock protein, which clinally varies in its heat-induced expression (Sørensen et al., 2005b). Clinal patterns of senescence might potentially shift with high temperature and/or other factors because of genotype-by-environment interactions. In studies of geographic variation in longevity, an important prediction from evolutionary theories of senescence is that when a harsh environment persists for some populations it should lead to increased intrinsic mortality and higher reproductive effort early in life relative to other populations which experience more benign environmental conditions. However, if the probability of extrinsic mortality depends on some individual conditions, negative correlations can be found between the extrinsic force of mortality and the rate of senescence (Reznick et al., 2004; Bronikowski & Promislow, 2005). Geographical clines of many fitness-related traits are often influenced by thermal adaptation because climate partially depends on geography. For instance, geographical patterns of climatic adaptation have been inferred on the basis of traits that vary clinally with latitude in model organisms including Drosophila and other ectotherms (e.g., David & Bocquet, 1975; James & Partridge, 1995; Capy, Plá & David, 1993; van’t Land et al., 1999; Huey et al., 2000; Wilson, 2001; Weeks, McKechnie & Hoffmann, 2002; Santos et al., 2004; Lindgren & Laurila, 2005). Mitrovski and Hoffmann (2001 ) have found evidence for latitudinal variation in both reproductive pattern and adult survival over winter in field-caged D. melanogaster originating from a latitudinal gradient along the East coast of Australia. Further, a candidate gene for aging (Methuselah) was found to clinally vary with latitude in Drosophila (Schmidt, Duvernell & Eanes, 2000). Thus, latitudinal clines have been found for many fitness-related traits and some of their putative candidate genes have been identified including genes that influence thermal adaptation and/or longevity (Schmidt et al., 2000 ; Weeks, McKechnie & Hoffmann, 2002; Frydenberg, Hoffmann & Loeschcke, 2003). Regions at high altitude and latitude share some similar extreme environmental conditions (Lencioni, 2004), but altitudinal gradients have been much less explored than latitudinal gradients in studies of clinal variation in Drosophila. However, altitudinal clines can often be detected on narrower geographical scales than latitudinal gradients (Etges, 1989; Tatar, Grey & Carey, 1997; Bubliy & Loeschcke, 2004; Berner, Körner & Blanckenhom, 2005; Sørensen et al., 2005b). Here we report an empirical analysis of both longevity and early fecundity in populations of the cactophilic D. buzzatii derived from an elevational gradient of north-western Argentina where significant thermal differences persist (Table 1). This species occurs in several phytogeographical areas from lowlands to highlands of Argentina (Hasson et al., 1995). Predictions from evolutionary theories of senescence might apply for geographical variation in D. buzzatii. For instance, longevity and early fecundity are expected to be negatively correlated along elevational clines, if there is a trade-off 83 Table 1. Sites of D. buzzatii populations sampled in Northwest Argentina. Estimated minimal and maximal mean temperatures (Tmin and Tmax, see Materials and methods for details) are given for each altitude (m above sea level) and cactus species are shown for each collection site Population altitude (m) Latitude (°) Longitude (°) Tmin (°C) Tmax (°C) Cactus species 202 27.48 64.18 6 34 O.q. 401 28.52 66.15 4 35 O.q.; T.t.; S.c.; C.v. 709 739 33.25 24.38 66.25 65.03 4 3 33 33 O.p.;.T.c. O.q.; S.c.; C.v. 879 26.08 65.11 3 33 O.q.; T.t.; C.v. 1654 26.06 65.58 1 26 O.s.; T.t. 1855 26.27 66.02 0 25 O.s.; T.t. 2263 26.35 65.51 )2 24 O.s. Key to cactus species: O.p., Opuntia panpeana; O.q., O. quimilo; O.s., O. sulphurea; T.t., Trichocereus terschekii; T.c., T. candicans; C.v., Cereus validus. between these traits. Additionally, longevity could be expected to be lower in highland than in lowland populations because of harsh environmental conditions in high elevations (Mani, 1962; Lencioni, 2004). For instance, winter cold stress might increase mortality in highland populations, as Mitrovski and Hoffmann (2002) have shown that adult mortality increases when temperatures are particularly low for Drosophila in the wild. Moreover, wild D. buzzatii feed and breed mainly on rotting tissues (hereafter rots) of different species of cacti of the genus Opuntia, but the host cactus species vary with altitude (Table 1). Rots of the host species O. sulfurea are much more ephemeral and patchily distributed than rots of the bigger host species such as O. quimilo and O. panpeana (Hasson, Naviera & Fontdevila, 1992), which are nearly absent at high elevations (Table 1). In spite of harsh thermal conditions in winter, D. buzzatii actually overwinters at least as larvae even at the highest elevations sampled in the present study (Table 1; rots support alive larvae during winter at high elevations, F.N.’s personal observation), where neutral markers in sharp contrast to inversion polymorphisms show no association with geography (Rodriguez et al., 2000). Therefore, altitudinal adaptive variation for fitness-related traits can be tested in this species (Dahlgaard, Hasson & Loeschcke, 2001; Sørensen et al., 2005b). In the present study we test whether or not mean longevity decreases and senescence rate increases as the altitude of origin of populations increases. Second, we test for population-by-temperature interactions (attributable to genotype-by-environment interactions) as a source of altitudinal variation in longevity. Finally, we compare early fecundity between the extremes of the elevational gradient studied, as well as across five populations, testing whether the among-population differences are consistent with a trade-off between early fecundity and life span at non-stressful temperature. Materials and methods Strains Wild flies were collected using banana baits at eight localities in Argentina along an elevational transect in mid-April, 2003 (Table 1). Mass cultures were set up using 40–50 wild flies per locality, with a 50:50 sex ratio approximately. One hundred inseminated females of the laboratory G2 generation were transferred from each mass culture to individual vials to set up isofemale lines. D. koepferae, a sibling species of D. buzzatii, was present at some collection sites and they were identified by examining the male genitalia (Vilela, 1983). After 4 laboratory generations (the laboratory G4 generation, when all lines were checked for species), 22 isofemale lines of D. buzzatii were inter-se crossed within each population, and a mass culture was established for each population of origin. All strains were maintained at 25 °C, with three standard bottles per population and 70–100 flies per bottle, with the prevailing (approximately 84 50:50) sex ratio. Standard bottles were 90 55-mm shell bottles containing 40 ml of instant Drosophila medium (hereafter IDM, Carolina Biological Supply, Burlington, NC, USA). Measurement and analyses of longevity and mortality First instar larvae from the laboratory G7 generation were collected from each mass population by using a small spoon with agar and yeast paste. Larvae were placed at a density of 35 in 95 20mm shell vials containing 6 ml of IDM. All cultures were kept in a temperature-controlled room at 25 °C. The eclosing adults were collected, sexed under light CO2 anesthesia, and used for measurement of longevity. For each population, either four or five vials each containing 10 males plus 10 females were set up at each of two experimental temperatures, 25 °C (5 vials) and 29.5 °C (4 vials). The flies were transferred to fresh vials every 1 d (29.5 °C experiment) or 2 d (25 °C experiment) and at each transfer all vials were examined for dead flies. The number of vials was gradually reduced as deaths occurred, with surviving adults being kept at a density as close to 20 per vial as possible. Longevity data (in days) were first subjected to a generalized linear model (GLIM) with Gamma error and log-link function for each test temperature, using sex as fixed factor (d.f. = 1) and altitude of origin of population as a continuous variable effect (d.f. = 1). Additionally, to test for altitudinal clines separately for each sex, longevity data (in days) were loge-transformed to remove dependence of variances on means in linear regression analyses of mean longevity on altitude of origin of population in each sex. In order to test for any relevant interactions among temperature, sex, and altitude (population), an ANOVA was performed using these three variables as fixed factors. Age-specific mortality rate (lx) was estimated as the continuous form of age-specific mortality, where lx = )ln(1 ) qx), qx = dx/Nx, dx is the number of flies dying in the interval x to x + 1 and Nx is the number alive at day x (Elandt-Johnson & Johnson 1980 ; Tatar, Grey & Carey, 1997). Very late ages (with less than 4 survivors) were excluded from the reported analysis because of small sample size although reported estimates were non-significantly different from estimates based on the whole set of data. Maximum likelihood theory provides straightforward significance tests for determining the best model for the observed data, by comparing the Gompertz, Gompertz-Makeham, logistic, and logistic-Makeham models (Pletcher, 1999, http:// www.hcoa.org/scott/softw-winmodest.asp). For all populations, excepting 29.5 °C-males from 709 m above sea level, the Gompertz model provided the best fit although other mortality models yielded similar patterns. Estimates of the rate parameter (b in Gompertz function) were obtained via maximum likelihood using WinModest (Pletcher, 1999). Sample sizes larger than ours are necessary to estimate the intercept parameter (Pletcher, 1999). Estimates of b were used to test for an effect of altitude on senescence rate by regressing them on the altitude of origin of population. Clinal variation in mortality rate was further tested with the semiparametric Cox’s Proportional Hazards Regression (CPHR) model (Cox, 1972), using the STATISTICA package (StatSoft, 1999). In CPHR, a baseline survival curve is systematically flexed up or down by each independent variable, and the method computes a coefficient (B) for each of them as well as a total chi-square statistic. A positive B-value indicates a significant association with higher mortality. Independent variables in CPHR were altitude as well as latitude in order to adjust for any possible latitudinal variation. To control for any possible size effects (e.g., Norry & Loeschcke, 2002a), the size of experimental individuals was measured as thorax length by using a micrometer ocular. Then altitudinal variation in mortality was also tested by using body size as a covariate in CPHR. Thermal data were used as predictor variables in some analyses. Minimal mean temperatures were average daily minimal temperatures for the coldest month and maximal mean temperatures were average daily maximal temperatures for the hottest month, using data from the Argentine Meteorological Service web site www.meteofa.mil.ar (temperatures for each particular altitude were obtained by interpolation using records for La Rioja, Salta, Santiago del Estero and La Quiaca). Assay for adult longevity over standard versus cactus-based media Elevational populations differ in host cactus species (Table 1). Pilot assays were performed at 85 25 °C in the laboratory G8 generation to compare longevity using instant Drosophila medium (IDM) versus cactus-based media (CBM) from two Opuntia species. Field-collected rots of O. quimilo and O. sulphurea were frozen at )20 °C until the time of assay with CBM. This assay was performed for flies derived from a lowland (401 m above sea level) and a highland (1855 m above sea level) population that differ in more than one cactus species as shown in Table 1. Small but nonautoclaved pieces of frozen rots (approximately 6 ml in total) of either O. quimilo or O. sulphurea were placed in a 95 20-mm shell vial as the only source of food at 25 °C. Longevity was simultaneously scored using these CBM vials as well as IDM vials, following otherwise the same procedure described above for IDM, with 10 flies of each sex per vial and five vials per population and food medium. Flies were transferred to vials with fresh medium every 2 d. Early fecundity Fecundity was studied at 25 °C in the laboratory G7 generation under a 12:12 L:D cycle for a subset of five populations including the two extremes of the elevational gradient studied (202, 709, 1654, 1855, 2263 m above sea level, Table 3). Three other populations from the same elevational transect (401, 739, 879 m above sea level, Table 1) were not analyzed for fecundity because of constraints of time to simultaneously score this trait in all populations. Shell vials containing a small spoon with agar plus yeast were set up for our stocks derived from the corresponding altitudes. One 1 d-old female plus two 1 d-old males per vial and 15 vials per population were set up to score fecundity. Number of eggs was scored from spoons every 2 days (when flies were transferred to vials with fresh spoons) until the death of each female. Absolute early fecundity was first estimated for each female (our observation unit) as the number of eggs laid within about the first one-third (more precisely, the 1/3.5 first fraction) of its whole adult life. Early fecundity was further tested as a measure relative to overall fecundity (Promislow, 1995). Specifically, relative early fecundity was estimated for each female (our observation unit) as the ratio of the number of eggs laid within the first 1/3.5 fraction of its whole adult life to the total number of eggs laid during lifetime (Promislow, 1995). Results Survival curves are presented in Figure 1 for males and females at 25 and 29.5 °C. GLIM results indicated that longevity both differed between the sexes and varied with altitude of origin of population [GLIM with (1) sex as a fixed categorical factor and (2) altitude of origin as a continuous variable effect as specified in Materials and methods: Wald1 = 18.08*** for (1) and 11.88*** for (2) at 25 °C; Wald1 = 27.27*** for (1) and 7.97*** for (2) at 29.5 °C. ***p < 0.005]. Consistent with GLIM results, mean longevity showed significant interactions between test temperature and altitude of origin of population as well as between sex and altitude in an ANOVA on loge-transformed longevity in days (Table 2). Further, mean longevity at 25 °C declined with the altitude of origin of population in females but not in males (Figure 2; linear regression statistics for females are: R2 = 0.66, beta = )0.81*, *p < 0.05). A very different pattern was found at 29.5 °C (Figure 2b), with mean longevity showing an increase with altitude of origin of population in males but not in females (Figure 2; linear regression statistics for males are: R2 = 0.59, betaaltitude = 0.97*, betalatitude = 0.94*, p < 0.05). Age-specific mortality rate is shown in Figure 3 for males and females at temperatures where clinal altitudinal variation was found. The b-parameter, which can be interpreted as the demographic rate of senescence (Finch et al., 1990 ), significantly increases with the altitude of origin of population in females at 25 °C and decreases but not significantly in males at 29.5 °C (Figure 4; linear regression statistics are: R2 = 0.51, beta = 0.71* for females at 25 °C; R2 = 0.11, beta = )0.32 for males at 29.5 °C *p < 0.05). No clinal pattern was found for b-estimates in both males at 25 °C and females at 29.5 °C (results not shown). Estimates of b were generally higher at 29.5 °C than at 25 °C (partially shown in Figure 3), which is consistent with the fact that flies live longer at 25 °C than at 29.5 °C (Figure 2). CPHR analysis indicated that altitude had an effect on mortality rate at 25 °C, with a significant association of female mortality with altitude of origin but no clinal variation in male mortality (Table 3). Further, CPHR confirmed that male mortality significantly decreased with altitude at 86 females at 25°C females at 29.5°C (b) 1 0.8 proportion alive proportion alive (a) 0.6 0.4 0.2 0 0.8 0.6 0.4 0.2 0 0 10 20 (c) 30 40 age (days) 50 60 70 0 5 (d) males at 25°C 1 0.8 0.8 0.6 0.4 0.2 0 10 15 20 age (days) 25 30 25 30 males at 29.5°C 1 proportion alive proportion alive 202 m 401 m 709 m 739 m 879 m 1654 m 1855 m 2263 m 1 0.6 0.4 0.2 0 0 10 20 30 40 50 60 70 age (days) 0 5 10 15 20 age (days) Figure 1. Survival curves of males and females of laboratory-reared D. buzzatii are shown for two adult test temperatures, 25 and 29.5 °C, for populations from an altitudinal gradient in north-western Argentina. Altitude in this and subsequent figures is given in m above sea level. Table 2. ANOVA on ln (longevity in days) performed to test for effects of (1) adult test temperature, (2) sex, (3) altitude of origin of population and the respective interactions for laboratory-reared D. buzzatii along an altitudinal gradient from Argentina. As a result of escaped flies, d.f. of error fell below the number from the initial cohort size Source of variation d.f. MS F 369.60*** (1) Temperature 1 76.491 (2) Sex 1 6.412 30.985*** (3) Altitude 7 0.467 2.255* (1) (2) 1 0.384 1.854 (1) (3) 7 1.050 5.0719*** (2) (3) 1 0.447 2.1623* 1 1217 0.182 0.207 0.878 Interactions (1) (2) (3) Error *p < 0.05; ***p < 0.005. 29.5 °C (Table 3). Identical conclusions were achieved when variation in body size was held constant. Altitudinal effects were still significant for both females at 25 °C and males at 29.5 °C when using body size as a covariate of mortality (CPHR, Baltitude = 0.23, v21 = 14.1*** for females at 25°C; Baltitude = )0.18, v21 = 11.21*** for males at 29.5 °C; no CPHR statistics was significant for males at 25 °C and females at 29.5 °C ***p < 0.005). We also tested for associations between thermal variables and mortality. CPHR coefficients were significant for Tmin at 25 °C (Table 3). In contrast, neither minimal nor maximal mean temperature of the origin site of population predicted mortality at 29.5°C (Table 3). In a two-population assay for longevity at 25 °C, no significant variation was found for mean longevity when estimated using instant Drosophila medium (as in the whole experiment) versus two cactus-based media [Table 4; ANOVA with (1) type of medium, (2) population and (3) sex as fixed factors: (1) F(1, 566) = 8.83, (2) F(2, 566) = 1.05, (3) F(1, 566) = 18.62, (1) (2) F(2, 566) = 0.28, (1) (3) F(1,566) = 0.91, (2) (3) F(2, 566) = 1.32, (1) (2) (3) F(2, 566) = 0.41]. In addition, age-specific 87 flies at 25°C ln (longevity) (a) 3.5 3.4 3.3 3.2 3.1 3 2.9 2.8 2.7 0 500 1000 1500 altitude 2000 2500 flies at 29.5°C ln (longevity) (b) 2.8 2.75 2.7 2.65 2.6 2.55 2.5 2.45 2.4 tude (Table 5, Figure 5), the difference being highly significant (t-test, t29 = 41.50*** and 4.64*** for absolute and relative early fecundity, respectively ***p < 0.005). Additionally, early fecundity was analyzed for all five populations that were measured for this trait (Table 5). Per capita profiles for fecundity are given in Sambucetti et al. (2005) for several of the here studied populations. Early fecundity significantly increased with the altitude of origin of population (linear regression statistics are: R2 = 0.85, beta = 0.92* for absolute early fecundity; R2 = 0.87, beta = 0.93* for relative early fecundity, *p < 0.05). Further, we tested for an among-population correlation between mean longevity and early fecundity over trait means from all five populations (Figure 5). Both traits, early fecundity and mean longevity, were negatively correlated across populations (Figure 5, Pearson correlation, r = )0.98** and )0.96** for absolute and relative fecundity respectively, **p < 0.01). Discussion 0 500 1000 1500 altitude 2000 2500 Figure 2. Mean longevity (in ln-days) of males and females at two test temperatures, 25 °C (a) and 29.5 °C (b), are shown for laboratory-reared D. buzzatii from an altitudinal gradient in Argentina. Filled circles and solid lines are estimates for females; empty circles and dashed lines are estimates for males. All lines were estimated from linear regression analysis indicated in the text. Error bars correspond to SE of the mean. mortality rate was not significantly different across these culture media (results not shown). Early fecundity at 25 °C was first compared for the two populations originating from the ends of the cline. These two divergent populations, originating from 202 and 2263 m above sea level, strongly differ not only in mean longevity but also in the demographic rate of aging in females at 25 °C as well as in males at 29.5 °C (tests for the null-hypothesis of identical b-parameters are: b202 m = 0.1288 versus b2263 m = 0.3220, v21 = 12*** for 25 °C-females, and b202 m = 0.6105 versus b2263 m = 0.3007, v12 = 4.07* for 29.5 °C-males. *p < 0.05; ***p < 0.005). As expected from the antagonistic pleiotropy theory, relative early fecundity was consistently higher for the shortest-lived population from high altitude (2263 m above sea level) than for the longest-lived population from the lowest alti- Longevity of D. buzzatii females at 25 °C decreased with the altitude of origin of population, and minimal instead of maximal mean temperature of origin site best predicts this clinal pattern. Further, estimates of age-specific mortality rate and CPHR analysis showed similar results, with mortality rate increasing with altitude. In addition, the shortestlived population from 2263 m above sea level showed high early fecundity when compared to the longest-lived population of the other extreme of the elevational gradient studied. Early fecundity at 25 °C was positively correlated with altitude, and this trait was negatively correlated with 25 °Clongevity across populations. The longevity and age-specific mortality trends presumably reflect genetic variation in intrinsic mortality because extrinsic causes of mortality were experimentally removed, and because all experimental individuals were reared in a common environment. The negative correlation between 25 °C-longevity and altitude disappeared in females at 29.5 °C, and males from high elevations and high latitude at 29.5 °C lived longer than males from lowlands and low latitude. CPHR analysis confirmed such a temperature dependent variation (Table 3). Thus, geographical variation in longevity is both sexspecific and temperature-dependent in D. buzzatii. 88 females at 25°C 202 m 401 m 709 m 739 m 879 m 1654 m 1855 m 2263 m (a) 1 ln (mortality rate) 0 -1 -2 -3 -4 -5 0 10 20 30 40 50 age (days) males at 29.5°C (b) 0 ln (mortality rate) -1 -2 -3 -4 -5 5 10 15 age (days) 20 25 Figure 3. Age-specific mortality rate for females at 25 °C (a) and males at 29.5 °C (b) from altitudinal clines for mean longevity in laboratory-reared D. buzzatii north-western Argentina. Previous studies have shown that both body size and resistance to thermal stress, two traits that are often correlated with longevity (e.g., Partridge & Farquhar, 1983; Norry & Loeschcke, 2002b, 2003), clinally vary with latitude in D. melanogaster (e.g., Coyne & Beecham, 1987; James & Partridge, 1995; van’t Land et al., 1999; see Hoffmann, Anderson & Hallas, 2002, for thermal resistance traits). Other studies have shown that highland populations are less resistant to heat stress than lowland populations of D. buzzatii in the same geographic region studied here (Sørensen, Dahlgaard & Loeschcke, 2001; Sørensen et al., 2005b). We found that the altitudinal trend of longevity in this species does not appear to be a result of altitudinal variation in body size, which is consistent with studies of latitudinal variation in other species of Drosophila (Mitrovski & Hoffmann, 2001). Specifically, mortality was still associated with altitude of the population of origin when controlling for variation in body size. Overall, these results parallel findings by Tatar, Grey and Carey (1997) in other ectotherms, grasshoppers. Genotype-by-environment interaction is a longknown mechanism of evolutionary importance for fitness traits. For senescence traits, interpopulation or interspecific phenotypic variation on which selection could act is often greater at benign than at high temperature because of genotype-byenvironment interactions (e.g., Dudycha, 2003; Sambucetti et al., 2005). In Drosophila, genotypeby-environment interaction appears to be a general property of the genetic basis that controls variation in longevity (Vieira et al., 2000). We did not find 89 Table 3. Cox Proportional Hazards Regression statistics estimating clinal associations between laboratory-measured mortality of D. buzzatii at two test temperatures and geographical as well as thermal variables. Analyses were performed separately for geographical and thermal variables as well as for each sex. Tmin and Tmax are given in Table 1. p-Values were corrected for multiple comparisons by using the sequential Bonferroni method (Rice, 1989) Test temp. Predictor variables Females B (±SE) 25 °C 29.5 °C 25 °C v21 B (±SE) v12 Geography Altitude 0.23 (0.07)** Latitude 0.07 (0.06) 12.36** 0.09 (0.06) 2.57 0.08 (0.07) Geography Altitude 0.05 (0.07) Latitude 0.07 (0.07) )0.17 (0.07)* 1.43 9.98* )0.19 (0.07)* Temperature )0.35 (0.07)** 0.06 (0.06) 15.23** )0.19 (0.07)* 0.04 (0.05) 9.15* Tmin )0.06 (0.06) 1.31 )0.07 (0.06) 1.29 Tmax 0.04 (0.06) Tmin Tmax 29.5 °C Males Temperature 0.08 (0.05) *p < 0.05; **p < 0.01. evidence for interactions between population and standard versus cactus-based culture media in longevity. However, we found an interaction between temperature and the altitude of origin of population to be a significant source of variation in longevity of D. buzzatii. Previous studies have shown that genetic variation in adult survival is partly influenced by the second chromosome polymorphism in wild populations of this species (Norry et al., 1995; reviewed in Hoffmann, Sgrò & Weeks, 2004), and this polymorphism varies clinally with altitude (Hasson et al., 1995). One candidate gene for genotype-by-environment interactions that maps within polymorphic inver- sions is hsp70 (Ranz, Segarra & Ruiz, 1997). Previous work showed that Hsp70-expression decreases by selection for extended life in D. melanogaster at 25 °C (Norry & Loeschcke, 2003). In addition, the heat-induced expression of this heatshock protein (Hsp70) is higher in highland than in lowland populations of D. buzzatii (Sørensen, Dahlgaard & Loeschcke, 2001; Sørensen et al., 2005b), and hsp70 gene maps at the same region as a QTL for knockdown resistance to heat stress in D. melanogaster (Norry, Dahlgaard & Loeschcke, 2004). These previous observations are consistent with the present finding that longevity at 25 °C, as opposed to longevity under warmer conditions Table 4. Mean longevity (in ln-days ± SD) of laboratory-reared males and females at 25 °C is shown for two populations of different altitudinal origin which were tested on three different food substrates: IDM is instant Drosophila medium; CBM-O. q. is a cactus-based medium of O. quimilo; and CBM-O. s. is a cactus-based medium of O. sulphurea. Information for both populations from different altitude (m above sea level) is given in Table 1 Food substrate Population altitude (m) Males Female IDM 401 3.26±0.27 3.06±0.51 CBM O .q. 401 3.25±0.30 3.22±0.29 3.04±0.53 CBM O. s. 401 3.27±0.22 IDM 1654 3.18±0.47 2.88±0.58 CBM O. q. CBM O. s. 1654 1654 3.17±0.51 3.18±0.52 3.00±0.65 2.96±0.58 90 females at 25°C 0.7 males at 29.5°C b parameter 0.6 0.5 0.4 0.3 0.2 0.1 0 0 500 1000 1500 altitude 2000 2500 Figure 4. Estimates of the b-parameter of the Gompertz model are plotted against altitude of origin of population for females at 25°C and males at 29.5 °C in laboratory-reared D. buzzatii. All estimates were obtained with Winmodest (Pletcher, 1999). (29.5 °C), decreases with the altitude of origin of wild populations. A pattern of sex-specificity was found for altitudinal variation in longevity, which could be more evident because of genotype-by-temperature interactions. Specifically, longevity at 25 °C shows a negative elevational cline in females but not in males whilst mean longevity at high temperature (29.5 °C) shows a positive elevational cline in males but not in females. Similar trends were observed for age-specific mortality rates (though non-significantly in males at 29.5 °C), and were further confirmed by Cox-regression analysis (Table 3). The cause(s) for this sex-specificity is still unknown but other studies suggest that sexspecific interactions with temperature can be a plausible mechanism maintaining genetic variation in longevity. For instance, about 17 QTL were Table 5. Mean values (±SD) for both absolute and relative estimates of early fecundity at 25 °C for laboratory-reared females from five D. buzzatii populations sampled along an elevational gradient in Argentina. Information for these populations from different altitude (m above sea level) in given in Table 1 Population altitude (m) Early fecundity Absolute Relative 202 36.89±2.04 0.31±0.07 709 41.12±1.65 0.32±0.03 1654 55.36±1.72 0.39±0.04 1855 49.40±1.81 0.37±0.04 2263 67.58±2.01 0.45±0.08 detected for life span in D. melanogaster (Nuzhdin et al., 1997 ), all of which were sex- or environment-specific, and more than 50% of them had sexually antagonistic or antagonistic pleiotropic effects across environments including temperature experienced by adults (Vieira et al., 2000). Sexby-temperature interactions were also detected for longevity in both cold-selected lines and their control in D. melanogaster (Norry & Loeschcke, 2002b). In D. buzzatii, second chromosome inversions appear to exhibit sex-specific and sexually antagonistic effects on longevity in the wild (Norry et al., 1995; Rodriguez et al., 1999 ). The present results suggest that temperature is a factor causing sex-specific interactions in a longevity cline in D. buzzatii. Neutral markers, including allozyme loci that are not linked to polymorphic inversions, consistently show no association with altitude and latitude in D. buzzatii originating from the same geographic region as sampled in the present study (Rodriguez et al., 2000). In this context, the action of natural selection rather than drift and migration could best explain the clinal pattern in senescencerelated traits found along the altitudinal gradient studied. High altitude should be associated with hazardprone environments due to a combination of severity, seasonality, unpredictability and variability (Lencioni, 2004). There is an altitudinal limit of distribution for insects as the high degree of harshness of high elevations includes reduced quantity and quality of food, high risk of desiccation, low precipitation, strong wind, and very low 91 202 m 709 m 1654 m 1855 m 2263 m absolute early fecundity (a) relative early fecundity (b) 100 90 80 70 60 50 40 30 20 10 0 2.7 2.8 2.9 3 3.1 ln (longevity) 3.2 3.3 2.7 2.8 2.9 3 3.1 ln (longevity) 3.2 3.3 0.6 0.5 0.4 0.3 0.2 0.1 0 Figure 5. Early fecundity (±SE) is plotted against mean gevity for five populations originating from an altitudinal dient in north-western Argentina. Both the absolute (a) relative (b) estimates of early fecundity are shown (see for details). longraand text temperature (Mani, 1962; Lencioni, 2004). For instance, mortality was found to increase when temperature is particularly low for adult Drosophila in the wild (Mitroski & Hoffmann, 2002). Evolutionary theories of senescence predict that increased probability of extrinsic mortality due to hazard-prone environments will impact on life span by increasing both the intrinsic mortality rate and early fecundity at normal temperatures (Finch, 1990; Rose, 1991; Partridge & Mangel, 1999; Kirkwood & Austad, 2000). If high elevations are associated with environmental harshness, both reduced longevity and increased early fecundity in highland populations might be expected as discussed above. The present study provides the first test for an elevational cline in longevity, senescence rate and early fecundity in Drosophila. Fecundity also shows latitudinal patterns in D. melanogaster (Hoffmann et al., 2003). Our results with D. buzzatii are consistent with results by Tatar, Grey and Carey (1997) in grasshoppers, indicating that accelerated senescence at nonstressful temperature can evolve in insects in high elevations. Low temperature at high elevation can lead to a very limited reproduction at late age, since both pre-freezing and freezing temperatures should increase the risk of mortality (Mitroski & Hoffmann, 2002), especially of aged insects. Coldstress survival is known to decrease with age, and this trait is highly correlated with knock-down temperature (Hori & Kimura, 1998) so that aged flies may be more vulnerable to predation and less active in late reproduction at cooler climates. Furthermore, both the breeding and feeding resources are dramatically more abundant for D. buzzatii in the lowland populations than in the highland populations where the substrates used by this species are very patchily distributed (Hasson, Naviera & Fontdevila, 1992). However, we emphasized that although all these observations are consistent with a pattern of accelerated senescence in highland populations, future work is required to test for the cause(s) of elevational or altitudinal clines of aging. For instance, a hypothesis alternative to the hypothesis of reduced longevity in highland populations is that extended longevity could have evolved by countergradient selection to compensate for the environmentally short growing season in high elevations (Conover & Schultz, 1995 ). However, such a pattern was apparent for males at 29.5 °C but the opposite pattern was found in females at 25 °C. Countergradient selection is not expected to impact on longevity if mortality in the wild is mainly due to extrinsic rather than intrinsic causes. Accelerated aging at high elevation could evolve by pleiotropy (if there is a trade-off between early reproduction and longevity or late reproduction) and/or by an accumulation of late-acting deleterious mutations. The pattern of differences in early fecundity observed both between the extremes of the elevational cline and across populations is consistent with a trade-off between longevity and early fecundity. Future work is required to test for this and other possible causes of elevational clines in senescence of insects. Acknowledgements We are indebted to Doth Andersen and Trine Gammelgaard for assistance with field work and Stuart Barker as well as two anonymous review- 92 ers for helpful comments on the manuscript. This research was supported by a frame and a center grant from the Danish Natural Sciences Research Council to V.L., as well as by grants from the University of Buenos Aires (X139), ANPCYT and CONICET (PIP5805) to F.N. References Berner, D., C. Körner & W.U. Blanckenhom, 2005. Grasshopper populations across 2000 m of altitude: is there life history adaptation? Ecography 27: 733–740. Bronikowski, A.M. & D.E.L. Promislow, 2005. Testing evolutionary theories of aging in wild populations. Trends Ecol. Evol. 20: 271–273. Bubliy, O.A. & V. Loeschcke, 2004. Variation of life-history and morphometrical traits in Drosophila buzzatii and Drosophila simulans collected along an altitudinal gradient from a Canary island. Biol. J. Linn. Soc. 84: 119–136. Capy, P., E. Plá & J.R. David, 1993. Phenotypic and genetic variability of morphometric traits in natural populations of Drosophila melanogaster and Drosophila simulans. I. Geographic variations. Genet. Select. Evol. 25: 517–526. Conover, D.O. & E.T. Schultz, 1995. Phenotypic similarity and the evolutionary significance of countergradient variation. Trends Ecol. Evol. 10: 248–252. Cox, D.R., 1972. Regression models and life-tables. J. R. Stat. Soc. B 34: 187–220. Coyne, J.A. & E. Beecham, 1987. Heritability of two morphological characters within and among natural populations of Drosophila melanogaster. Genetics 117: 727–737. Dahlgaard, J., E. Hasson & V. Loeschcke, 2001. Behavioral differentiation in oviposition activity in Drosophila buzzatii from highland and lowland populations in Argentina: plasticity or thermal adaptation? Evolution 55: 738–747. David, J.R. & C. Bocquet, 1975. Similarities and differences in latitudinal adaptation of two Drosophila sibling species. Nature 257: 588–590. Dudycha, J.L., 2003. A multi-environment comparison of senescence between sister species of Daphnia. Oecologia 135: 555–563. Elandt-Johnson, R. & N.L. Johnson, 1980. Survival Models and Data Analysis. Wiley, New York. Etges, W.J., 1989. Chromosomal influences on variable life histories along an altitudinal transect in Drosophila robusta. Am. Nat. 133: 83–110. Finch, C.E., 1990. Longevity, Senescence and the Genome. University of Chicago Press, Chicago. Frydenberg, J., A.A. Hoffmann & V. Loeschcke, 2003. DNA sequence variation and latitudinal associations in hsp23, hsp26 and hsp27 from natural populations of Drosophila melanogaster. Mol. Ecol. 12: 2025–2032. Hasson, E., H. Naviera & A. Fontdevila, 1992. The breeding sites of the Argentinean species of the drosophila mulleiri complex (subgenus Drosophila repleta group) Revista Chilena Hist. Nature 65: 319–326. Hasson, E., C. Rodrı́guez, J.J. Fanara, H. Naveira, O.A. Reig & A. Fontdevila, 1995. The evolutionary history of Drosophila buzzatti. XXVI. Macrogeographic patterns of inversion polymorphism in New World populations. J. Evol. Biol. 8: 369–384. Huey, R.B., G.W. Gilchrist, M.L. Carlson, D. Berrigan & I. Serra, 2000. Rapid evolution of a geographic cline in size in an introduced fly. Science 287: 308–309. Hoffmann, A.A., A. Anderson & R. Hallas, 2002. Opposing clines for high and low temperature resistance in Drosophila melanogaster. Ecol. Lett. 5: 614–618. Hoffmann, A.A., M. Scott, L. Partridge & R. Hallas, 2003. Overwintering in Drosophila melanogaster populations: outdoor field cage experiments on clinal and laboratory selected populations help to elucidate traits under selection. J. Evol. Biol. 16: 614–623. Hoffmann, A.A., C.M. Sgrò & A.R. Weeks, 2004. Chromosomal inversion polymorphism and adaptation. Trends Ecol. Evol. 9: 482–488. Hori, Y. & M.T. Kimura, 1998. Relationship between cold stupor and cold tolerance in Drosophila (Diptera: Drosophilidae). Environ. Entomol. 27: 1297–1302. James, A.C. & L. Partridge, 1995. Thermal evolution of rate of larval development in Drosophila melanogaster in laboratory and field populations. J. Evol. Biol. 8: 315–330. Kirkwood, T.B. & S.N. Austad, 2000. Why do we age? Nature 408: 233–238. Lencioni, V., 2004. Survival strategies of freshwater insects in cold environments. J. Limnol. 63(Suppl. 1): 45–55. Lindgren, B. & A. Laurila, 2005. Proximate causes of adaptive growth rates: growth efficiency variation among latitudinal populations of Rana temporaria. J. Evol. Biol. 18: 820–828. Linnen, C., M. Tatar & D. Promislow, 2001. Cultural artifacts: a comparison of senescence in natural, laboratory-adapted and artificially selected lines of Drosophila melanogaster. Evol. Ecol. Res. 3: 877–888. Mani, M.S., 1962. Introduction to High Altitude Entomology – Insect Life Above the Timber-Line in the North-West Himalaya. Methuen & Co., Ltd, London. Medawar, P.B., 1952. An Unsolved Problem in Biology. Lewis, London. Mitrovski, P. & A.A. Hoffmann, 2001. Postponed reproduction as an adaptation to winter conditions in Drosophila melanogaster: evidence for clinal variation under seminatural conditions. Proc. R. Soc. Lond. B. Biol. Sci. 268: 2163–2168. Norry, F.M., J. Dahlgaard & V. Loeschcke, 2004. Quantitative trait loci affecting knockdown resistance to high temperture in Drosophila melanogaster. Mol. Ecol. 13: 3585–3594. Norry, F.M. & V. Loeschcke, 2002a. Temperature-induced shifts in associations of longevity with body size in Drosophila melanogaster. Evolution 56: 299–306. Norry, F.M. & V. Loeschcke, 2002b. Longevity and resistance to cold stress in cold-stress selected lines and their controls in Drosophila melanogaster. J. Evol. Biol. 15: 775–783. Norry, F.M. & V. Loeschcke, 2003. Heat-induced expression of a molecular chaperone decreases by selecting for long-lived individuals. Exp. Geront. 38: 673–681. Norry, F.M., J.C. Vilardi, J.J. Fanara, E. Hasson & C. Rodriguez, 1995. An adaptive chromosomal polymorphism affecting size-related traits, and longevity selection in a 93 natural population of Drosophila buzzatii. Genetica 96: 285–291. Nuzhdin, S.V., E.G. Pasyukova, C.L. Dilda, Z-B. Zeng & T.F.C. Mackay, 1997. Sex-specific quantitative trait loci affecting longevity in Drosophila melanogaster. Proc. Natl. Acad. Sci. (USA) 94: 9734–9739. Partridge, L. & M. Farquhar, 1983. Lifetime mating success of male fruitflies (Drosophila melanogaster) is related to their size. Anim. Behav. 31: 871–877. Partridge, L. & K. Fowler, 1992. Direct and correlated responses to selection on age at reproduction in Drosophila melanogaster. Evolution 46: 76–91. Partridge, L. & M. Mangel, 1999. Messages from mortality: the evolution of death rates in the old. Trends Ecol. Evol. 14: 438–442. Pletcher, S.D., 1999. Model fitting and hypothesis testing or age-specific mortality data. J. Evol. Biol. 12: 430–439. Promislow, D.E.L., 1995. New perspectives on comparative tests of antagonistic pleiotropy using Drosophila. Evolution 49: 394–397. Promislow, D.E.L. & M. Tatar, 1998. Mutation and senescence: where genetics and demography meet. Genetica 102/ 103: 299–314. Ranz, J.M., C. Segarra & A. Ruiz, 1997. Chromosomal homology and molecular organization of Mulleŕs elements D and E in the Drosophila repleta species group. Genetics 145: 281–295. Reznick, D., 1993. New model system for studying the evolutionary biology of aging: Crustacea. Genetica 91: 79–88. Reznick, D.N., M.J. Briant, D. Roff, C.K. Ghalambor & D.E. Ghalambor, 2004. Effect of extrinsic mortality on the evolution of senescence in guppies. Nature 431: 1095–1099. Rice, W.R., 1989. Analyzing tables of statistical tests. Evolution 43: 223–225. Rodriguez, C., J.J. Fanara & E. Hasson, 1999. Inversion polymorphism, longevity, and body size in a natural population of Drosophila buzzatii. Evolution 53: 612–620. Rodriguez, C., R. Piccinali, E. Levy & E. Hasson, 2000. Contrasting population genetic structures using allozymes and the inversion polymorphism in Drosophila buzzatii . J. Evol. Biol. 13: 976–984. Roper, C., P. Pignatelly & L. Partridge, 1993. Evolutionary effects of selection on age at reproduction in larval and adult Drosophila melanogaster. Evolution 47: 445–455. Rose, M., 1984. Laboratory evolution of postponed senescence in Drosophila melanogaster. Evolution 38: 1004–1010. Rose, M., 1991. Evolutionary Biology of Aging. Oxford University Press, Oxford. Rose, M.R, 1999. Genetics of aging in Drosophila. Exp. Gerontol. 34: 577–585. Sambucetti, P., J.G. Sørensen, V. Loeschcke & F.M. Norry, 2005. Variation in senescence and associated traits between sympatric cactophilic sibling species of Drosophila. Evol. Ecol. Res. 7: 915–930. Santos, M., P.F. Iriarte, W. Céspedes, J. Balanyà, A. Fontdevila & L. Serra, 2004. Swift laboratory thermal evolution of wing shape (but not size) in Drosophila subobscura and its relationship with chromosomal inversion polymorphism. J. Evol. Biol. 17: 841–855. Schmidt, P.S., D.D. Duvernell & W.F. Eanes, 2000. Adaptive evolution of a candidate gene for aging in Drosophila. Proc. Natl. Acad. Sci. (USA) 97: 10861–10865. Sørensen, J.G., J. Dahlgaard & V. Loeschcke, 2001. Genetic variation in natural populations of Drosophila buzzatii: down-regulation of Hsp70 expression and variation in heat stress traits. Funct. Ecol. 15: 289–296. Sørensen, J.G., F.M. Norry, A.C. Scannapieco & V. Loeschcke, 2005b. Altitudinal variation for stress resistance traits and thermal adaptation in adult Drosophila buzzatii from the New World. J. Evol. Biol. 18: 829–837. Sørensen, J.G., M.M. Nielsen, M. Kruheffer, J. Justesen & V. Loeschcke, 2005a. Full genome gene expression analysis of the heat stress response in Drosophila melanogaster. Cell Stress Chaperon. 10: 312–329. StatSoft, 1999. STATISTICA for Windows (Computer Program Manual). StatSoft Inc., Tulsa. Tatar, M., D.W. Grey & J.R. Carey, 1997. Altitudinal variation for senescence in Melanophus grasshoppers. Oecologia 111: 357–364. van’t Land, J.P., P. van Putten, B. Zwaan, A. Kamping & W. van Delden, 1999. Latitudinal variation in wild populations of Drosophila melanogaster: heritabilities and reaction norms. J. Evol. Biol. 12: 222–232. Vieira, C., E.G. Pasyukova, Z. Zeng, J.B. Hackett, R.F. Lyman & T.F.C. Mackay, 2000. Genotype–environment interaction for quantitative trait loci affecting lifespan in Drosophila melanogaster. Genetics 154: 213–227. Vilela, C.R., 1983. A revision of the Drosophila repleta species (Diptera-Drosophilidae). Revista Basileira Entomol. 27: 1– 114. Weeks, A.R., S.W. McKechnie & A.A. Hoffmann, 2002. Dissecting adaptive clinal variation: markers, inversions and size/stress associations in Drosophila melanogaster from a central field population. Ecol. Lett. 5: 756–763. Wilson, R.S., 2001. Geographic variation in thermal sensitivity of jumping performance in the frog Limnodynastes peronii. J. Exp. Biol. 204: 4227–4236. Williams, G.C., 1957. Pleiotropy, natural selection and the evolution of senescence. Evolution 11: 398–411. Zwaan, B.J., R. Bijlsma & R.F. Hoekstra, 1995. Direct selection on life span in Drosophila melanogaster. Evolution 49: 649–659.