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Altitudinal variation in lifespan of Drosophila melanogaster populations from the Firtina Valley, northeastern Turkey
Journal of Thermal Biology 61 (2016) 91–97
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Journal of Thermal Biology journal homepage: www.elsevier.com/locate/jtherbio
Altitudinal variation in lifespan of Drosophila melanogaster populations from the Firtina Valley, northeastern Turkey Nazli Ayhan a,b, Pinar Güler a,c, Banu Sebnem Onder a,n a
Hacettepe University, Faculty of Science, Department of Biology, 06800 Beytepe, Ankara, Turkey UMR Emergence des Pathologies Virales (EPV: Aix-Marseille University – IRD 190 – Inserm 1207 – EHESP), Marseille, France c Institut für Zoologie, Universität Regensburg, Regensburg 93040, Germany b
art ic l e i nf o
a b s t r a c t
Article history: Received 30 May 2016 Received in revised form 31 August 2016 Accepted 2 September 2016 Available online 4 September 2016
Studies of altitudinal changes in phenotype and genotype can complement studies of latitudinal patterns and provide evidence of natural selection in response to climatic factors. In Drosophila melanogaster, latitudinal variation in phenotype and genotype has been well studied, but altitudinal patterns have rarely been investigated. We studied populations from six different altitudes varying between 35 m and 2173 m in the Firtina Valley in northeastern part of Turkey to evaluate clinal trends in lifespan under experimental conditions. Lifespan in the D. melanogaster populations was examined in relation to altitude, sex, temperature (25 °C and 29 °C), and dietary yeast concentration (5 g/L and 25 g/L). As expected high temperature decrease lifespan in all populations. However, it was shown that lifespan was slightly affected by dietary stress. We found that lifespan decreases significantly under thermal stress conditions with increasing altitude. Moreover, there was a slightly negative relationship between altitude and lifespan, which was closely associated with climatic factors such as temperature and precipitation, may suggest local adaptation to climate. & 2016 Elsevier Ltd. All rights reserved.
Keywords: Altitudinal gradient Clinal variation Food stress Lifespan Local adaptation Temperature stress
1. Introduction Life history traits represent a focal point for natural selection to produce the best lifetime strategies that increase fitness in a particular environment (Baur and Raboud, 1988). Species that occur in different environments should present different patterns of life history traits in their different populations due to different local selection pressures. This variation may be the result of either adaptation or phenotypic plasticity (Stearns, 1989). Clinal variation is one of the most common types of geographical variation that occurs along latitude and altitude gradients in diverse groups of animals and plants (Futuyma, 1998). Geographical variation in traits related to fitness is determined by genetic differentiation in many species (Adrion et al., 2015; Karl et al., 2009; Takahashi et al., 2011; van Delden and Kamping, 1997). Analyzing these clinal patterns in fitness-related traits is one of the main objectives of studies of adaptive evolution (Sambucetti et al., 2006). Temperature is one of the significant selective factors acting on geographical clines in traits of adaptive importance (Norry et al., 2001; Reeve et al., 2000). Geographical and clinal patterns in fitness-related traits, such as morphometric, physiological, and n
Corresponding author. E-mail address: [email protected] (B.S. Onder).
http://dx.doi.org/10.1016/j.jtherbio.2016.09.002 0306-4565/& 2016 Elsevier Ltd. All rights reserved.
life-history traits, have been found in Drosophila populations (Adrion et al., 2015; Arthur et al., 2008; Bubliy and Loeschcke, 2005; Hoffmann and Shirriffs, 2002; Pitchers et al., 2013; Robinson and Partridge, 2001; Sambucetti et al., 2006; Schmidt and Paaby, 2008). Depending on the Drosophila species studied, latitudinal clines have also been found in allele frequencies and chromosomal inversion frequencies (de Jong and Bochdanovits, 2003; Fabian et al., 2012; Kapun et al., 2016; Knibb, 1982; Paaby et al., 2010; Simões et al., 2012). Parallel geographical clines in molecular markers and/or quantitative traits within and among species are an important source of information for studies of local adaptation (Adrion et al., 2015; Reinhardt et al., 2014; Schrider et al., 2016). Many life-history traits, such as lifespan, are known to correlate with external factors. Lifespan is highly variable among populations and may vary with latitude, altitude, and temperature (Duyck et al., 2010; Tatar et al., 1997). Latitudinal or altitudinal patterns in lifespan have been documented in a number of species (Munch and Salinas, 2009) including Melanoplus sanguinipes / devastator (Tatar et al., 1997), Margaritifera margaritifera (Ziuganov et al., 2000), some sub-tropical anuran species (Morrison et al., 2004), Lycaena tityrus (Karl and Fischer, 2009), Ceratitis rosa (Duyck et al., 2010), Homo sapiens (Burtscher, 2014), D. buzzatii (Norry et al., 2006), and D. melanogaster (Schmidt and Paaby, 2008; Sgrò et al., 2013). These studies suggest that lifespan may contribute to adaptive differentiation across habitats. However,
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there is relatively little information regarding to how lifespan varies with geography and with different environmental conditions (Hoffmann et al., 2003; Mitrowski and Hoffmann, 2001; Schmidt and Paaby, 2008; Trotta et al., 2006). The strong relationship between climatic factors and life-history traits is well known, and these results come mostly from investigation of organisms collected from different geographical locations, particularly from latitudinal gradients. It is possible, however, that climatic differences do not directly contribute to differences in fitness-related traits owing to the limited gene flow between distant populations (Bubliy and Loeschcke, 2005; Pitchers et al., 2013). We studied D. melanogaster populations from six different localities between 35 m and 2173 m in the Firtina Valley in northeastern Turkey. The valley is covered with warm deciduous forests extending without interruption in lowlands. Stands of dense trees, reaching over 30 m in height, surrounded the valley in the lowlands (0–1000 m), whereas meadows and grasslands are dominant in the subalpine and alpine zones (above 1000 m). The dense tree coverage isolates the valley from wind, and wind activity within the valley is therefore highly reduced. The valley receives abundant rainfall all year long with mean precipitation values reaching 1296.5 mm. Temperatures are usually low with a yearly mean of 13.5 °C. The highest temperatures are recorded in July and August with mean values of approximately 21.7 °C, although intra-day temperatures can vary depending on sunlight and rainfall. Relative humidity is high in the lowlands and constant throughout the year with mean values of approximately 73–82% (Saglam and Caglar, 2007). The most drastic environmental changes occur in the subalpine and alpine zones, where trees are limited and open landscapes are more common. Higher altitudes possess harsher and unstable environments with lower air temperatures and oxygen
levels, and increased wind and radiation levels. High altitudes are thus typically unsuitable for insects like fruit flies that mostly feed and breed on rotting fruits. Limited vegetation leads to limited food sources for fruit flies and prevents them from occupying the highlands. These decreases in fruit supplies along altitudinal gradients may limit high-altitude colonization by fruit flies that have ripe fruit requirements. Because of all these heterogeneous environmental conditions along altitudinal transects in the valley, the Firtina Valley is well suited for studies of adaptation to different environments at narrow geographical scales. The aim of this study was to examine the variation in lifespan in relation to local climatic variables in populations collected along a 40-km altitudinal transect in the Firtina Valley in Turkey, with an average mean annual temperature difference of 10 °C between the lowest and highest elevation points. By analyzing lifespan, we expected to identify the selectively important climatic factors under natural conditions, and by rearing the populations under different temperatures and diets, we also aimed to investigate plastic responses to temperature and diet. Phenotypic plasticity is thought to play an important role in the adaptation of populations to changing local environments (Schlichting and Pigliucci, 1998).
2. Materials and methods 2.1. Origin of the Flies Used Fly collection was conducted in August 2012 at six different elevations along an altitudinal transect in the Firtina Valley, Rize, Turkey (Fig. 1), using banana-baited plastic bottle traps. Geographical coordinates were determined with a GPS (Table 1). The
Fig. 1. Map showing the six sampling localities along the altitudinal gradient from Firtina Valley, Turkey.
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Table 1 Climatic and geographical parameters for the D. melanogaster collection localities. Population ID
Collection Locality
Latitude
Longitude
Altitude (m)
Taverage (°C)
Tmax (°C)
Tmin (°C)
MAP (mm)
PWM (mm)
PDM (mm)
YD CH SY CT EY KK
YAMAÇDERE ÇAMLIHEMŞİN ŞENYUVA ÇAT ELEVİT YAYLASI KALE KÖY
41.16N 41.05N 41.01N 40.86N 40.85N 40.79N
40.99E 41.00E 40.99E 40.94E 41.01E 40.96E
35 309 407 1320 1908 2173
13.6 12.5 12.5 8.9 5.7 3.8
25.2 25.8 26.0 26.9 24.0 22.2
2.6 0.3 0.0 6.5 10.0 12.0
1857 1440 1393 652 620 644
239 177 170 73 82 90
80 77 76 40 37 36
Taverage ¼ mean annual temperature; Tmax ¼ maximum temperature; Tmin ¼ minimum temperature; MAP ¼ mean annual precipitation; PWM ¼ precipitation wettest month; PDM ¼ precipitation driest month.
Table 2 Three-way ANOVAs for the effects of altitude, sex, and yeast on lifespan of D. melanogaster populations at two different rearing temperatures: (a) 25 °C, and (b) 29 °C. (a) Source of variance (25 °C)
df
Sum of squares Mean square F value P value
Altitude Yeast Sex Altitude Yeast Altitude Sex Yeast Sex Altitude Yeast Sex Error (b) Source of variance (29 °C)
5 1 1 5 5 1 5
13,342.126 2310.438 766.843 2161.538 562.782 116.128 1488.889
Altitude Yeast Sex Altitude Yeast Altitude Sex Yeast Sex Altitude Yeast Sex Error
2668.425 2310.438 766.843 432.308 112.556 116.128 297.778
11.291 9.776 3.245 1.829 0.476 0.491 1.260
o0.0001 0.002 0.072 0.104 0.794 0.483 0.279
1022 241,528.938 236.330 df Sum of squares Mean square F value P value
5 1 1 5 5 1 5
32,821.151 0.025 770.947 1182.252 2638.282 1588.935 648.190
1037 79,528.403
6564.230 0.025 770.947 236.450 527.656 1588.935 129.638
85.593 0.000 10.053 3.083 6.880 20.719 1.690
o0.0001 0.986 0.002 0.009 o0.0001 o0.0001 0.134
76.691
climatic characteristics of the sites vary from low to high elevation with decreasing temperature and precipitation (Table 1). Climatic data on the collection localities were obtained from the WorldClim database (Hijmans et al., 2005). 2.2. Experimental design Flies from different localities were used to set up six mass populations of D. melanogaster. All flies were reared on a standard cornmeal-sugar-yeast (CSY) Drosophila medium and kept at 25 °C on a 12L/12D schedule. Experiments were conducted after all wild-caught flies reached the second laboratory generation. To estimate lifespan, unmated 3–5-day-old flies were collected from the third laboratory generation from each population and sex, and allowed to mate for fixing the mortality cost of mating and reproduction for both sexes. After 24 h, flies were sorted by sex and transferred to vials according to population and sex for each experimental temperature and dietary combination. Each vial consisted of 10 flies of one sex, with five replicates for all experimental groups. Three times a week the flies were transferred to new vials containing fresh medium until the last fly was dead. Lifespan assays were carried out in standard (25 g/L yeast) and restricted (5 g/L yeast) CSY food medium and under standard (25 °C) and high temperature (29 °C) conditions. Each temperature diet population sex trial was conducted with 50 flies. In total, 2107 individual flies were observed for the lifespan assays.
2.3. Data analyses Each population was represented by five vials containing 10 individuals of each sex in each experiment. After verification of normality, we analyzed the temperature datasets separately because of the large effect of experimental temperature. In order to test for any relevant interactions among yeast, sex, and altitude (population), an ANOVA was performed for the two different rearing temperatures using these variables as fixed factors. In order to reveal local adaptation to environmental conditions, Pearson's correlation coefficient (r) values between lifespan and geo-climatic variables were calculated using whole dataset. To minimize the risk of Type I error from multiple tests, we applied a sequential Bonferroni correction (Rice, 1989). For α ¼0.05, the critical P value was 0.00089. As the data were found to follow normal distribution after Kolmogorov-Smirnov test, we used parametric statistics. All statistical analyses were performed using IBM SPSS Statistics software 20.0.
3. Results We measured lifespan in both sexes from six populations across two temperatures and two different diets. To avoid measuring laboratory adaptation, we preserved the genetic variation available by starting the experiments soon after capture from the field. Statistical summaries of lifespan data at different temperatures and diets are shown in Table 2 and Fig. 2. As expected, temperature stress had a significant effect on mean lifespan in all populations (Fig. 2 c, d), with flies of both sexes exhibiting a strong decrease in mean lifespan with increasing temperature. However, dietary stress had little effect on mean lifespan (Fig. 2), with the mean lifespan of populations and sexes varying among rich and poor diets in different ways. Moreover, there was heterogeneity in lifespan and stress responses among populations. In Table 2, lifespan values are compared according to population/altitude, sex, and dietary regime using multiple comparison tests following three-way ANOVAs. As can be seen from Table 2, at the 25 °C temperature, the analyses of variance showed that lifespan was significantly affected by altitude and diet, but not by sex, and with no significant interaction between these factors. At the 29 °C temperature, lifespan data revealed a significant effect of both altitude and sex, and significant interactions of altitude-bysex, altitude-by-yeast, and sex-by-yeast, even though there was no effect of yeast on lifespan (Table 2 b). Mean lifespan varied between altitudes under all environmental conditions, and the ANOVA revealed a significant effect of altitude on lifespan. In other words, results showed a significant lifespan differences among populations. Pearson correlation coefficients were estimated to measure the strength of the association between lifespan and the geographic and climatic variables (Table 3). We found similarities and differences between sexes in correlation analyses of lifespan, altitude,
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Fig. 2. Means ( 7 SE) for lifespan of D. melanogaster females (filled symbols) and males (open symbols) from six different altitudes: (a) flies reared at 25 °C and 5 g/L yeast amount; (b) flies reared at 25 °C and 25 g/L yeast amount; (c) flies reared at 29 °C and 5 g/L yeast amount; (d) flies reared at 29 °C and 25 g/L yeast amount.
and climatic variables. There was a negative correlation between mean lifespan and altitude at both temperatures, both yeast levels, and in both sexes (Table 3). After employing the Bonferroni correction for controlling Type 1 error, significance of these correlations was evaluated at the 0.05/number-of-pairwise comparison level. There was a significant negative correlation between lifespan and altitude at 29 °C for females (Table 3, restricted food r¼ 0.385, p o188 0.0001; standard food r ¼ 0.312, p o0.0001). However, lifespan and altitude tended to have a slightly negative correlation for the other groups. We found a significant correlation between altitude and lifespan for only two of the eight groups after the Bonferroni
correction (Table 3). The positive correlation of lifespan with mean annual precipitation, wettest month precipitation, and driest month precipitation is remarkable. Statistically significant r-values were obtained between precipitation and lifespan in six groups of flies kept at 29 °C, whereas no significant correlations were determined between precipitation and lifespan for the flies reared at 25 °C (Table 3). Precipitation thus significantly influenced lifespan under temperature stress, especially in females. Correlations among mean annual temperature, maximum and minimum temperature, and lifespan showed differences by sex. Specifically, lifespan in females, but not in males, at 29 °C, showed a positive correlation
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Table 3 Pearson correlation coefficients for the correlations between lifespan and geographic and climatic variables. Taverage ¼ mean annual temperature; Tmax ¼maximum temperature; Tmin ¼ minimum temperature; MAP ¼mean annual precipitation; PWM ¼ precipitation wettest month; PDM ¼precipitation driest month. 25°C Temperature 5 g/L Yeast
Geography Altitude Temperature Taverage Tmax Tmin Precipitation MAP PWM PDM
29°C Temperature 25 g/L Yeast
5 g/L Yeast
25 g/L Yeast
Females
Males
Females
Males
Females
Males
Females
Males
0.053
0.147n
0.097
0.009
0.385nnn
0.133n
0.312nnn
0.155nn
0.047 0.015 0.053
0.128n 0.056 0.152n
0.090 0.017 0.102n
0.004 0.097 0.012
0.362nnn 0.164nn 0.377nnn
0.095 0.095 0.127n
0.283nnn 0.127n 0.297nnn
0.131n 0.017 0.147nn
0.055 0.052 0.100
0.193nn 0.201nn 0.207nn
0.125n 0.128n 0.133n
0.047 0.054 0.055
0.381nnn 0.367nnn 0.408nnn
0.195nn 0.208nn 0.169nn
0.303nnn 0.288nnn 0.322nnn
0.171nn 0.168nn 0.172nn
Values in bold were significant following sequential Bonferroni correction. n
p o0.05. p o 0.001. p o0.01.
nnn nn
with varying temperature conditions (Table 3). Four of six positive correlations were significant after Bonferroni correction.
4. Discussion Lifespan showed variation among populations along the altitudinal gradient in the Firtina Valley. In standard laboratory conditions, without food or temperature stress, lifespan showed no significant correlation with climatic and geographic variables. The results from the temperature-stressed flies are quite different. Females at 29 °C under both dietary conditions exhibited a significant correlation between geographic and climatic variables and lifespan. Mean lifespans of populations at high temperature were significantly determined by the altitude and climatic variables of the point of origin of the population, particularly in female individuals. Lifespan in temperature-stressed environments is related to the environmental conditions at a population's point of origin, which suggests an adaptive response to thermal stress. Rigid diet stress or malnutrition decreases lifespan (Fadamiro et al., 2005; Karl and Fischer, 2009; Tatar et al., 2014). In our results, there was virtually no overall difference in lifespan between the feeding treatments (5 g/L and 25 g/L yeast), and this pattern cannot be readily explained. Unsuitable or poor food resources, as in our study the 5 g/L diet group, functions as an environmental stress factor. These results might suggest that genetic variance under feeding stress in natural D. melanogaster populations does not contribute to the ageing response. It is known that dietary stress influences ageing in laboratory-adapted Drosophila lines (Burger et al., 2010; Doroszuk et al., 2012; Mair and Dillin, 2008). Environmental adaptation may shape the stress response to artificial environmental changes. Populations that are newly collected from nature are probably adapted to poor dietary conditions. This could be the reason why these populations have the same ageing pattern under both dietary conditions. On the other hand, as expected, high temperature was found to be a very powerful stress for ageing in all populations, and it decreased lifespan in a significant way. The relationship between altitude and lifespan was significant under high-temperature stress, and a negative correlation between altitude and lifespan might suggest adaptation to the generally cooler conditions at high elevation. Decreases in lifespan with increasing altitude suggest lower heat-stress resistance in high-altitude populations. Cold tolerance has been suggested to be a common feature in high altitude populations of
Drosophila (Sørensen et al., 2005) and populations from lower latitudes exhibit higher tolerance to heat (Hoffmann et al., 2005). It is known that longevity and stress tolerance are positively correlated (Vermeulen and Loeschcke, 2007). We found that sex differences interacted with yeast levels and altitude at 29 °C. When we compared the data for all the populations from different altitudes, female flies generally lived longer than the males. Under stressful combination of very high temperature and very low food level, males from high altitudes (1320, 1908, 2173 m) and females from low altitudes (35, 309, 407 m) were more stress tolerant and lived much longer. As expected for ectothermic organisms, lifespan is strongly dependent on environmental temperature (Norry and Loeschcke, 2002; Norry et al., 2006), with shorter lifespans and increased metabolic activity at higher temperatures (Speakman, 2005; Terblanche et al., 2005). In our study, interactions between temperature and altitude reflected some marginal variation in lifespan in response to temperature. The population EY, in particular, which was collected at 1908 m above sea level, showed a very short lifespan for both sexes and yeast concentrations. Mean lifespan of the EY population decreased two fold when exposed to high temperature in contrast to standard temperature. The EY population showed a significant response to temperature, probably because of a higher sensitivity of the population to environmental stress. High-altitude populations had shorter lifespans than those of low-altitude populations. This pattern is in agreement with observations of reduced longevity in high-altitude populations of D. buzzatti (Norry et al., 2006), D. melanogaster (Fabian et al., 2015), and Melanoplus sanguinipes and M. devastator (Tatar et al., 1997). Regardless of environmental conditions, high altitudes are more hazardous (Lencioni, 2004). In addition to altitudinal differentiation, we found in general an increase in lifespan in low-altitude populations kept under food stress at standard temperatures. This is in agreement with the increase in lifespan observed in the copper butterfly Lycaena tityrus (Karl and Fischer, 2009) under food stress and the increased starvation-survival of low-altitude populations of D. takahashii and D. nepalensis (Parkash et al., 2005). In addition to stress response differentiation, we found that the low-altitude populations YD and CH were more tolerant of food stress. Thus, there was a clear difference in lifespan between food-stressed populations and those under standard dietary conditions. An indirect approach of correlating trait variability with local climatic conditions at each population's point of origin can help to
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explain a trait's adaptive significance. Temperature generally decreases with increasing altitude (Table 1). Similarly, precipitation also decreases with increasing altitude. Temperature and precipitation parameters at the points of origin are negatively correlated with altitude as well as with lifespan. Thus, these climatic parameters may influence life history trait or stress tolerance, but their significance remains unclear. MAP, PWM, and PDM are positively correlated with lifespan (Table 3) and some of the r-values are significant. Significant correlation observed between lifespan and precipitation for the females aged at 29 °C could be a result of a relationship between precipitation and stress tolerance. Very few studies are available on the effect of precipitation on life history traits, although Fabian et al. (2015) showed that fecundity in D. melanogaster populations was negatively correlated with the amount of rainfall, and in other Drosophila species it has been observed that humidity is positively correlated with desiccation and negatively correlated with starvation tolerance (Parkash and Munjal, 2000; Parkash et al., 2005).
5. Conclusions Altitudinal variation and differences among populations give rise that lifespan could be under selection for local adaptation. Geographical and climatic variables are related to lifespan and we may infer that temperature and precipitation variables at the site of origin of the populations are the potential selective factors for the observed changes in lifespan and stress response, thus supporting the importance of climatic adaptation.
Acknowledgements This paper is dedicated to the memory of Prof. Dr. Ali Nihat Bozcuk (1941–2015), whose mentorship and inspiration was crucial to this research. We thank Ibrahim Aslan for assistance with collections, and we are grateful to Cagasan Karacaoglu for the climatic data and Utku Perktas for graphical aid. We thank also two anonymous reviewers for their suggestions to improve the manuscript. This work was supported by the Hacettepe University Scientific Research Coordination Unit (Grant number 012 D06 601 012).
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Temperature and clinal variation in larval growth efficiency in Drosophila melanogaster. J. Evol. Biol. 14, 14–21. Saglam, I.K., Caglar, S.S., 2007. Local population size and dynamics of the color polymorphic bush cricket Isophya rizeensis Sevgili, 2003 (Orthoptera: Tettigoniidae) within the Fırtına Valley. Turk. J. Zool. 31, 1–8. Sambucetti, P., Loeschcke, V., Norry, F.M., 2006. Developmental time and size-related traits in Drosophila buzzatii along an altitudinal gradient from Argentina. Hereditas 143, 77–83. Schlichting, C.D., Pigliucci, M., 1998. Phenotypic Evolution: A Reaction Norm Perspective. Sinauer Associates, Sunderland, MA. Schmidt, P.S., Paaby, A.B., 2008. Reproductive diapause and life-history clines in North American populations of Drosophila melanogaster. Evolution 62, 1204–1215. Schrider, D.R., Hahn, M.W., Begun, D.J., 2016. Parallel evolution of copy-number variation across continents in Drosophila melanogaster. Mol. Biol. Evol. 33, 1308–1316. 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Journal of Thermal Biology journal homepage: www.elsevier.com/locate/jtherbio
Altitudinal variation in lifespan of Drosophila melanogaster populations from the Firtina Valley, northeastern Turkey Nazli Ayhan a,b, Pinar Güler a,c, Banu Sebnem Onder a,n a
Hacettepe University, Faculty of Science, Department of Biology, 06800 Beytepe, Ankara, Turkey UMR Emergence des Pathologies Virales (EPV: Aix-Marseille University – IRD 190 – Inserm 1207 – EHESP), Marseille, France c Institut für Zoologie, Universität Regensburg, Regensburg 93040, Germany b
art ic l e i nf o
a b s t r a c t
Article history: Received 30 May 2016 Received in revised form 31 August 2016 Accepted 2 September 2016 Available online 4 September 2016
Studies of altitudinal changes in phenotype and genotype can complement studies of latitudinal patterns and provide evidence of natural selection in response to climatic factors. In Drosophila melanogaster, latitudinal variation in phenotype and genotype has been well studied, but altitudinal patterns have rarely been investigated. We studied populations from six different altitudes varying between 35 m and 2173 m in the Firtina Valley in northeastern part of Turkey to evaluate clinal trends in lifespan under experimental conditions. Lifespan in the D. melanogaster populations was examined in relation to altitude, sex, temperature (25 °C and 29 °C), and dietary yeast concentration (5 g/L and 25 g/L). As expected high temperature decrease lifespan in all populations. However, it was shown that lifespan was slightly affected by dietary stress. We found that lifespan decreases significantly under thermal stress conditions with increasing altitude. Moreover, there was a slightly negative relationship between altitude and lifespan, which was closely associated with climatic factors such as temperature and precipitation, may suggest local adaptation to climate. & 2016 Elsevier Ltd. All rights reserved.
Keywords: Altitudinal gradient Clinal variation Food stress Lifespan Local adaptation Temperature stress
1. Introduction Life history traits represent a focal point for natural selection to produce the best lifetime strategies that increase fitness in a particular environment (Baur and Raboud, 1988). Species that occur in different environments should present different patterns of life history traits in their different populations due to different local selection pressures. This variation may be the result of either adaptation or phenotypic plasticity (Stearns, 1989). Clinal variation is one of the most common types of geographical variation that occurs along latitude and altitude gradients in diverse groups of animals and plants (Futuyma, 1998). Geographical variation in traits related to fitness is determined by genetic differentiation in many species (Adrion et al., 2015; Karl et al., 2009; Takahashi et al., 2011; van Delden and Kamping, 1997). Analyzing these clinal patterns in fitness-related traits is one of the main objectives of studies of adaptive evolution (Sambucetti et al., 2006). Temperature is one of the significant selective factors acting on geographical clines in traits of adaptive importance (Norry et al., 2001; Reeve et al., 2000). Geographical and clinal patterns in fitness-related traits, such as morphometric, physiological, and n
Corresponding author. E-mail address: [email protected] (B.S. Onder).
http://dx.doi.org/10.1016/j.jtherbio.2016.09.002 0306-4565/& 2016 Elsevier Ltd. All rights reserved.
life-history traits, have been found in Drosophila populations (Adrion et al., 2015; Arthur et al., 2008; Bubliy and Loeschcke, 2005; Hoffmann and Shirriffs, 2002; Pitchers et al., 2013; Robinson and Partridge, 2001; Sambucetti et al., 2006; Schmidt and Paaby, 2008). Depending on the Drosophila species studied, latitudinal clines have also been found in allele frequencies and chromosomal inversion frequencies (de Jong and Bochdanovits, 2003; Fabian et al., 2012; Kapun et al., 2016; Knibb, 1982; Paaby et al., 2010; Simões et al., 2012). Parallel geographical clines in molecular markers and/or quantitative traits within and among species are an important source of information for studies of local adaptation (Adrion et al., 2015; Reinhardt et al., 2014; Schrider et al., 2016). Many life-history traits, such as lifespan, are known to correlate with external factors. Lifespan is highly variable among populations and may vary with latitude, altitude, and temperature (Duyck et al., 2010; Tatar et al., 1997). Latitudinal or altitudinal patterns in lifespan have been documented in a number of species (Munch and Salinas, 2009) including Melanoplus sanguinipes / devastator (Tatar et al., 1997), Margaritifera margaritifera (Ziuganov et al., 2000), some sub-tropical anuran species (Morrison et al., 2004), Lycaena tityrus (Karl and Fischer, 2009), Ceratitis rosa (Duyck et al., 2010), Homo sapiens (Burtscher, 2014), D. buzzatii (Norry et al., 2006), and D. melanogaster (Schmidt and Paaby, 2008; Sgrò et al., 2013). These studies suggest that lifespan may contribute to adaptive differentiation across habitats. However,
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there is relatively little information regarding to how lifespan varies with geography and with different environmental conditions (Hoffmann et al., 2003; Mitrowski and Hoffmann, 2001; Schmidt and Paaby, 2008; Trotta et al., 2006). The strong relationship between climatic factors and life-history traits is well known, and these results come mostly from investigation of organisms collected from different geographical locations, particularly from latitudinal gradients. It is possible, however, that climatic differences do not directly contribute to differences in fitness-related traits owing to the limited gene flow between distant populations (Bubliy and Loeschcke, 2005; Pitchers et al., 2013). We studied D. melanogaster populations from six different localities between 35 m and 2173 m in the Firtina Valley in northeastern Turkey. The valley is covered with warm deciduous forests extending without interruption in lowlands. Stands of dense trees, reaching over 30 m in height, surrounded the valley in the lowlands (0–1000 m), whereas meadows and grasslands are dominant in the subalpine and alpine zones (above 1000 m). The dense tree coverage isolates the valley from wind, and wind activity within the valley is therefore highly reduced. The valley receives abundant rainfall all year long with mean precipitation values reaching 1296.5 mm. Temperatures are usually low with a yearly mean of 13.5 °C. The highest temperatures are recorded in July and August with mean values of approximately 21.7 °C, although intra-day temperatures can vary depending on sunlight and rainfall. Relative humidity is high in the lowlands and constant throughout the year with mean values of approximately 73–82% (Saglam and Caglar, 2007). The most drastic environmental changes occur in the subalpine and alpine zones, where trees are limited and open landscapes are more common. Higher altitudes possess harsher and unstable environments with lower air temperatures and oxygen
levels, and increased wind and radiation levels. High altitudes are thus typically unsuitable for insects like fruit flies that mostly feed and breed on rotting fruits. Limited vegetation leads to limited food sources for fruit flies and prevents them from occupying the highlands. These decreases in fruit supplies along altitudinal gradients may limit high-altitude colonization by fruit flies that have ripe fruit requirements. Because of all these heterogeneous environmental conditions along altitudinal transects in the valley, the Firtina Valley is well suited for studies of adaptation to different environments at narrow geographical scales. The aim of this study was to examine the variation in lifespan in relation to local climatic variables in populations collected along a 40-km altitudinal transect in the Firtina Valley in Turkey, with an average mean annual temperature difference of 10 °C between the lowest and highest elevation points. By analyzing lifespan, we expected to identify the selectively important climatic factors under natural conditions, and by rearing the populations under different temperatures and diets, we also aimed to investigate plastic responses to temperature and diet. Phenotypic plasticity is thought to play an important role in the adaptation of populations to changing local environments (Schlichting and Pigliucci, 1998).
2. Materials and methods 2.1. Origin of the Flies Used Fly collection was conducted in August 2012 at six different elevations along an altitudinal transect in the Firtina Valley, Rize, Turkey (Fig. 1), using banana-baited plastic bottle traps. Geographical coordinates were determined with a GPS (Table 1). The
Fig. 1. Map showing the six sampling localities along the altitudinal gradient from Firtina Valley, Turkey.
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Table 1 Climatic and geographical parameters for the D. melanogaster collection localities. Population ID
Collection Locality
Latitude
Longitude
Altitude (m)
Taverage (°C)
Tmax (°C)
Tmin (°C)
MAP (mm)
PWM (mm)
PDM (mm)
YD CH SY CT EY KK
YAMAÇDERE ÇAMLIHEMŞİN ŞENYUVA ÇAT ELEVİT YAYLASI KALE KÖY
41.16N 41.05N 41.01N 40.86N 40.85N 40.79N
40.99E 41.00E 40.99E 40.94E 41.01E 40.96E
35 309 407 1320 1908 2173
13.6 12.5 12.5 8.9 5.7 3.8
25.2 25.8 26.0 26.9 24.0 22.2
2.6 0.3 0.0 6.5 10.0 12.0
1857 1440 1393 652 620 644
239 177 170 73 82 90
80 77 76 40 37 36
Taverage ¼ mean annual temperature; Tmax ¼ maximum temperature; Tmin ¼ minimum temperature; MAP ¼ mean annual precipitation; PWM ¼ precipitation wettest month; PDM ¼ precipitation driest month.
Table 2 Three-way ANOVAs for the effects of altitude, sex, and yeast on lifespan of D. melanogaster populations at two different rearing temperatures: (a) 25 °C, and (b) 29 °C. (a) Source of variance (25 °C)
df
Sum of squares Mean square F value P value
Altitude Yeast Sex Altitude Yeast Altitude Sex Yeast Sex Altitude Yeast Sex Error (b) Source of variance (29 °C)
5 1 1 5 5 1 5
13,342.126 2310.438 766.843 2161.538 562.782 116.128 1488.889
Altitude Yeast Sex Altitude Yeast Altitude Sex Yeast Sex Altitude Yeast Sex Error
2668.425 2310.438 766.843 432.308 112.556 116.128 297.778
11.291 9.776 3.245 1.829 0.476 0.491 1.260
o0.0001 0.002 0.072 0.104 0.794 0.483 0.279
1022 241,528.938 236.330 df Sum of squares Mean square F value P value
5 1 1 5 5 1 5
32,821.151 0.025 770.947 1182.252 2638.282 1588.935 648.190
1037 79,528.403
6564.230 0.025 770.947 236.450 527.656 1588.935 129.638
85.593 0.000 10.053 3.083 6.880 20.719 1.690
o0.0001 0.986 0.002 0.009 o0.0001 o0.0001 0.134
76.691
climatic characteristics of the sites vary from low to high elevation with decreasing temperature and precipitation (Table 1). Climatic data on the collection localities were obtained from the WorldClim database (Hijmans et al., 2005). 2.2. Experimental design Flies from different localities were used to set up six mass populations of D. melanogaster. All flies were reared on a standard cornmeal-sugar-yeast (CSY) Drosophila medium and kept at 25 °C on a 12L/12D schedule. Experiments were conducted after all wild-caught flies reached the second laboratory generation. To estimate lifespan, unmated 3–5-day-old flies were collected from the third laboratory generation from each population and sex, and allowed to mate for fixing the mortality cost of mating and reproduction for both sexes. After 24 h, flies were sorted by sex and transferred to vials according to population and sex for each experimental temperature and dietary combination. Each vial consisted of 10 flies of one sex, with five replicates for all experimental groups. Three times a week the flies were transferred to new vials containing fresh medium until the last fly was dead. Lifespan assays were carried out in standard (25 g/L yeast) and restricted (5 g/L yeast) CSY food medium and under standard (25 °C) and high temperature (29 °C) conditions. Each temperature diet population sex trial was conducted with 50 flies. In total, 2107 individual flies were observed for the lifespan assays.
2.3. Data analyses Each population was represented by five vials containing 10 individuals of each sex in each experiment. After verification of normality, we analyzed the temperature datasets separately because of the large effect of experimental temperature. In order to test for any relevant interactions among yeast, sex, and altitude (population), an ANOVA was performed for the two different rearing temperatures using these variables as fixed factors. In order to reveal local adaptation to environmental conditions, Pearson's correlation coefficient (r) values between lifespan and geo-climatic variables were calculated using whole dataset. To minimize the risk of Type I error from multiple tests, we applied a sequential Bonferroni correction (Rice, 1989). For α ¼0.05, the critical P value was 0.00089. As the data were found to follow normal distribution after Kolmogorov-Smirnov test, we used parametric statistics. All statistical analyses were performed using IBM SPSS Statistics software 20.0.
3. Results We measured lifespan in both sexes from six populations across two temperatures and two different diets. To avoid measuring laboratory adaptation, we preserved the genetic variation available by starting the experiments soon after capture from the field. Statistical summaries of lifespan data at different temperatures and diets are shown in Table 2 and Fig. 2. As expected, temperature stress had a significant effect on mean lifespan in all populations (Fig. 2 c, d), with flies of both sexes exhibiting a strong decrease in mean lifespan with increasing temperature. However, dietary stress had little effect on mean lifespan (Fig. 2), with the mean lifespan of populations and sexes varying among rich and poor diets in different ways. Moreover, there was heterogeneity in lifespan and stress responses among populations. In Table 2, lifespan values are compared according to population/altitude, sex, and dietary regime using multiple comparison tests following three-way ANOVAs. As can be seen from Table 2, at the 25 °C temperature, the analyses of variance showed that lifespan was significantly affected by altitude and diet, but not by sex, and with no significant interaction between these factors. At the 29 °C temperature, lifespan data revealed a significant effect of both altitude and sex, and significant interactions of altitude-bysex, altitude-by-yeast, and sex-by-yeast, even though there was no effect of yeast on lifespan (Table 2 b). Mean lifespan varied between altitudes under all environmental conditions, and the ANOVA revealed a significant effect of altitude on lifespan. In other words, results showed a significant lifespan differences among populations. Pearson correlation coefficients were estimated to measure the strength of the association between lifespan and the geographic and climatic variables (Table 3). We found similarities and differences between sexes in correlation analyses of lifespan, altitude,
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Fig. 2. Means ( 7 SE) for lifespan of D. melanogaster females (filled symbols) and males (open symbols) from six different altitudes: (a) flies reared at 25 °C and 5 g/L yeast amount; (b) flies reared at 25 °C and 25 g/L yeast amount; (c) flies reared at 29 °C and 5 g/L yeast amount; (d) flies reared at 29 °C and 25 g/L yeast amount.
and climatic variables. There was a negative correlation between mean lifespan and altitude at both temperatures, both yeast levels, and in both sexes (Table 3). After employing the Bonferroni correction for controlling Type 1 error, significance of these correlations was evaluated at the 0.05/number-of-pairwise comparison level. There was a significant negative correlation between lifespan and altitude at 29 °C for females (Table 3, restricted food r¼ 0.385, p o188 0.0001; standard food r ¼ 0.312, p o0.0001). However, lifespan and altitude tended to have a slightly negative correlation for the other groups. We found a significant correlation between altitude and lifespan for only two of the eight groups after the Bonferroni
correction (Table 3). The positive correlation of lifespan with mean annual precipitation, wettest month precipitation, and driest month precipitation is remarkable. Statistically significant r-values were obtained between precipitation and lifespan in six groups of flies kept at 29 °C, whereas no significant correlations were determined between precipitation and lifespan for the flies reared at 25 °C (Table 3). Precipitation thus significantly influenced lifespan under temperature stress, especially in females. Correlations among mean annual temperature, maximum and minimum temperature, and lifespan showed differences by sex. Specifically, lifespan in females, but not in males, at 29 °C, showed a positive correlation
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Table 3 Pearson correlation coefficients for the correlations between lifespan and geographic and climatic variables. Taverage ¼ mean annual temperature; Tmax ¼maximum temperature; Tmin ¼ minimum temperature; MAP ¼mean annual precipitation; PWM ¼ precipitation wettest month; PDM ¼precipitation driest month. 25°C Temperature 5 g/L Yeast
Geography Altitude Temperature Taverage Tmax Tmin Precipitation MAP PWM PDM
29°C Temperature 25 g/L Yeast
5 g/L Yeast
25 g/L Yeast
Females
Males
Females
Males
Females
Males
Females
Males
0.053
0.147n
0.097
0.009
0.385nnn
0.133n
0.312nnn
0.155nn
0.047 0.015 0.053
0.128n 0.056 0.152n
0.090 0.017 0.102n
0.004 0.097 0.012
0.362nnn 0.164nn 0.377nnn
0.095 0.095 0.127n
0.283nnn 0.127n 0.297nnn
0.131n 0.017 0.147nn
0.055 0.052 0.100
0.193nn 0.201nn 0.207nn
0.125n 0.128n 0.133n
0.047 0.054 0.055
0.381nnn 0.367nnn 0.408nnn
0.195nn 0.208nn 0.169nn
0.303nnn 0.288nnn 0.322nnn
0.171nn 0.168nn 0.172nn
Values in bold were significant following sequential Bonferroni correction. n
p o0.05. p o 0.001. p o0.01.
nnn nn
with varying temperature conditions (Table 3). Four of six positive correlations were significant after Bonferroni correction.
4. Discussion Lifespan showed variation among populations along the altitudinal gradient in the Firtina Valley. In standard laboratory conditions, without food or temperature stress, lifespan showed no significant correlation with climatic and geographic variables. The results from the temperature-stressed flies are quite different. Females at 29 °C under both dietary conditions exhibited a significant correlation between geographic and climatic variables and lifespan. Mean lifespans of populations at high temperature were significantly determined by the altitude and climatic variables of the point of origin of the population, particularly in female individuals. Lifespan in temperature-stressed environments is related to the environmental conditions at a population's point of origin, which suggests an adaptive response to thermal stress. Rigid diet stress or malnutrition decreases lifespan (Fadamiro et al., 2005; Karl and Fischer, 2009; Tatar et al., 2014). In our results, there was virtually no overall difference in lifespan between the feeding treatments (5 g/L and 25 g/L yeast), and this pattern cannot be readily explained. Unsuitable or poor food resources, as in our study the 5 g/L diet group, functions as an environmental stress factor. These results might suggest that genetic variance under feeding stress in natural D. melanogaster populations does not contribute to the ageing response. It is known that dietary stress influences ageing in laboratory-adapted Drosophila lines (Burger et al., 2010; Doroszuk et al., 2012; Mair and Dillin, 2008). Environmental adaptation may shape the stress response to artificial environmental changes. Populations that are newly collected from nature are probably adapted to poor dietary conditions. This could be the reason why these populations have the same ageing pattern under both dietary conditions. On the other hand, as expected, high temperature was found to be a very powerful stress for ageing in all populations, and it decreased lifespan in a significant way. The relationship between altitude and lifespan was significant under high-temperature stress, and a negative correlation between altitude and lifespan might suggest adaptation to the generally cooler conditions at high elevation. Decreases in lifespan with increasing altitude suggest lower heat-stress resistance in high-altitude populations. Cold tolerance has been suggested to be a common feature in high altitude populations of
Drosophila (Sørensen et al., 2005) and populations from lower latitudes exhibit higher tolerance to heat (Hoffmann et al., 2005). It is known that longevity and stress tolerance are positively correlated (Vermeulen and Loeschcke, 2007). We found that sex differences interacted with yeast levels and altitude at 29 °C. When we compared the data for all the populations from different altitudes, female flies generally lived longer than the males. Under stressful combination of very high temperature and very low food level, males from high altitudes (1320, 1908, 2173 m) and females from low altitudes (35, 309, 407 m) were more stress tolerant and lived much longer. As expected for ectothermic organisms, lifespan is strongly dependent on environmental temperature (Norry and Loeschcke, 2002; Norry et al., 2006), with shorter lifespans and increased metabolic activity at higher temperatures (Speakman, 2005; Terblanche et al., 2005). In our study, interactions between temperature and altitude reflected some marginal variation in lifespan in response to temperature. The population EY, in particular, which was collected at 1908 m above sea level, showed a very short lifespan for both sexes and yeast concentrations. Mean lifespan of the EY population decreased two fold when exposed to high temperature in contrast to standard temperature. The EY population showed a significant response to temperature, probably because of a higher sensitivity of the population to environmental stress. High-altitude populations had shorter lifespans than those of low-altitude populations. This pattern is in agreement with observations of reduced longevity in high-altitude populations of D. buzzatti (Norry et al., 2006), D. melanogaster (Fabian et al., 2015), and Melanoplus sanguinipes and M. devastator (Tatar et al., 1997). Regardless of environmental conditions, high altitudes are more hazardous (Lencioni, 2004). In addition to altitudinal differentiation, we found in general an increase in lifespan in low-altitude populations kept under food stress at standard temperatures. This is in agreement with the increase in lifespan observed in the copper butterfly Lycaena tityrus (Karl and Fischer, 2009) under food stress and the increased starvation-survival of low-altitude populations of D. takahashii and D. nepalensis (Parkash et al., 2005). In addition to stress response differentiation, we found that the low-altitude populations YD and CH were more tolerant of food stress. Thus, there was a clear difference in lifespan between food-stressed populations and those under standard dietary conditions. An indirect approach of correlating trait variability with local climatic conditions at each population's point of origin can help to
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explain a trait's adaptive significance. Temperature generally decreases with increasing altitude (Table 1). Similarly, precipitation also decreases with increasing altitude. Temperature and precipitation parameters at the points of origin are negatively correlated with altitude as well as with lifespan. Thus, these climatic parameters may influence life history trait or stress tolerance, but their significance remains unclear. MAP, PWM, and PDM are positively correlated with lifespan (Table 3) and some of the r-values are significant. Significant correlation observed between lifespan and precipitation for the females aged at 29 °C could be a result of a relationship between precipitation and stress tolerance. Very few studies are available on the effect of precipitation on life history traits, although Fabian et al. (2015) showed that fecundity in D. melanogaster populations was negatively correlated with the amount of rainfall, and in other Drosophila species it has been observed that humidity is positively correlated with desiccation and negatively correlated with starvation tolerance (Parkash and Munjal, 2000; Parkash et al., 2005).
5. Conclusions Altitudinal variation and differences among populations give rise that lifespan could be under selection for local adaptation. Geographical and climatic variables are related to lifespan and we may infer that temperature and precipitation variables at the site of origin of the populations are the potential selective factors for the observed changes in lifespan and stress response, thus supporting the importance of climatic adaptation.
Acknowledgements This paper is dedicated to the memory of Prof. Dr. Ali Nihat Bozcuk (1941–2015), whose mentorship and inspiration was crucial to this research. We thank Ibrahim Aslan for assistance with collections, and we are grateful to Cagasan Karacaoglu for the climatic data and Utku Perktas for graphical aid. We thank also two anonymous reviewers for their suggestions to improve the manuscript. This work was supported by the Hacettepe University Scientific Research Coordination Unit (Grant number 012 D06 601 012).
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