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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.
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