Effect of metal stress on life history divergence and quantitative genetic architecture in a wolf...

Effect of metal stress on life history divergence and quantitativegenetic architecture in a wolf spider

F. HENDRICKX,*J.-P. MAELFAIT*� & L. LENS*

*Terrestrial Ecology Unit (TEREC), Department Biology, Ghent University, Gent, Belgium

�Institute for Nature and Forest Research (INBO), Brussels, Belgium

Introduction

Environmental stress can be defined as any environ-

mental factor that impairs fitness when first applied

(Hoffmann & Parsons, 1991; Bijlsma & Loeschcke, 2005).

Despite this negative effect on fitness, natural as well as

laboratory populations have been shown to persist under

stressful conditions, which suggests that environmental

stress may cause divergence in traits related to stress

resistance among differentially exposed populations

(Bijlsma & Loeschcke, 1997, 2005; Parsons, 2005). There

is increasing evidence for rapid micro-evolution in

populations subjected to stress, particularly for stresses

of anthropogenic origin (Parsons, 1994; Hoffmann &

Hercus, 2000; Reznick & Ghalambor, 2001; Rasanen

et al., 2003; Badyaev, 2005). Increased resistance against

ecotoxic substances such as heavy metals comprises one

of the best-documented examples of rapid micro-evolu-

tionary change in plants (Antonovics et al., 1971; Mac-

nair, 1993; Shaw, 1999; Pauwels et al., 2006), aquatic

invertebrates (Klerks & Weiss, 1987; Martinez & Levin-

ton, 1996) and terrestrial invertebrates (Posthuma & van

Straalen, 1993; Shirley & Sibly, 1999; Morgan et al.,

2007). The often very intense selection pressure caused

by metal exposure, its persistence and the fact that it is

relatively easy to measure render metal pollution to be

one of the best demonstrated classic examples of natural

selection in action. Despite the growing interest in heavy

metal contamination as driving force for physiological

Correspondence: Frederik Hendrickx, Terrestrial Ecology Unit (TEREC),

Department Biology, Ghent University, K.L. Ledeganckstraat 35, 9000

Gent, Belgium. Tel.: ++32 9 264 52 56; fax: ++32 9 264 87 94;

e-mail: [email protected]

ª 2 0 0 7 T H E A U T H O R S . J . E V O L . B I O L . 2 1 ( 2 0 0 8 ) 1 8 3 – 1 9 3

J O U R N A L C O M P I L A T I O N ª 2 0 0 7 E U R O P E A N S O C I E T Y F O R E V O L U T I O N A R Y B I O L O G Y 183

Keywords:

animal model;

Bayesian statistics;

ecological divergence;

egg size;

heavy metal;

stress resistance;

trade-off.

Abstract

Effects and consequences of stress exposure on life history strategies and

quantitative genetic variation in wild populations remain poorly understood.

We here study whether long-term exposure to heavy metal pollution may

result in alternative life history strategies and alter quantitative genetic

properties in natural populations of the wolf spider Pirata piraticus. Offspring

originating from a reference and a metal contaminated population and their

reciprocal hybrid cross were bred in a half-sib mating scheme and subse-

quently reared in cadmium contaminated vs. clean environment. Results from

this experiment provided evidence for a genetically based reduced growth rate

and increased egg size in the contaminated population. Growth rate reduction

in response to cadmium contamination was only observed for the reference

population. Animal model analysis revealed that heritability for growth rate

was large for the reference population under reference conditions, but much

lower under metal stressed conditions, caused by a strong decrease in additive

genetic variance. Heritability for growth of the metal contaminated population

was very low, even under reference conditions. Initial size of the offspring was

primarily determined by maternal effects, whereas egg size produced by the

offspring was determined by both sire and dam effects, indicating that egg size

determination is under control of the female genotype. In conclusion,

these results show that metal stress can not only affect life history variation

in natural populations, but also decreases the expression as well as the of the

amount of genetic variation for particular life history traits.

doi:10.1111/j.1420-9101.2007.01452.x

and ⁄ or life history divergence, the genetic architecture of

the traits involved and adaptive consequences remain

largely unknown (Van Straalen & Hoffmann, 2000).

A thorough understanding of the genetic (co)variance

of traits related to stress resistance is important for at least

three reasons. First, adaptation to stress not only implies

the presence of standing genetic variation for stress

resistance, but the latter can also be expected to be

related with fitness-related traits, hence resulting

in trade-offs between environments (Shirley & Sibly,

1999; Bubliy & Loeschcke, 2005). The evolution of these

‘tolerance costs’ can best be understood through resource

partitioning theory. As energy available for life history

processes such as survival, growth and reproduction is

limited, resource allocation to one trait generally means

deprivation of another (van Noordwijk & de Jong, 1986;

Stearns, 1992; Reznick et al., 2000; Roff, 2002). Physio-

logical defence mechanisms such as increased metal

excretion or the production of detoxifying enzymes

(Wilczek & Migula, 1996) of tolerant genotypes (Maroni

et al., 1987; Van Straalen et al., 1987) can therefore be

expected to reduce resource availability for growth and

reproduction (Calow, 1991). In line with this, Drosophila

melanogaster forced to evolve in a cadmium-contaminated

medium showed a significant decrease in female weight

and fecundity compared with those reared in unpolluted

environments (Shirley & Sibly, 1999). Because of the

limited number of studies addressing quantitative genet-

ics of tolerance to chemical contaminants in natural

populations (Posthuma et al., 1993; Forbes et al., 1999;

Klerks & Moreau, 2001), basic assumptions of life history

divergence because of stress tolerance remain largely

untested (Van Straalen & Hoffmann, 2000). Second,

exposure to stress may influence the expression of

heritable variation in a trait by altering the balance

between different sources of phenotypic variation, i.e.

additive genetic, dominance genetic and environmental

variation (e.g. Hoffmann & Merila, 1999; Blanckenhorn

& Heyland, 2004; Laugen et al., 2005). As a reduction in

expression of quantitative genetic variation might ham-

per the evolution of stress resistance, a better insight into

the sources of phenotypic variability in wild populations

is required to understand changes in expression of

genetic variation under ecologically relevant ecological

circumstances (Hoffmann & Merila, 1999; Pakkasmaa

et al., 2003; Charmantier & Garant, 2005; Wilson et al.,

2006). Third, adaptation to anthropogenic stresses often

results from strong directional selection for tolerance

traits. Such process might lead to alterations in the

genetic architecture of populations and, eventually to

genetic erosion, not only in traits directly related to stress

resistance, but also in fitness traits that are genetically

correlated with stress resistance (Perez & Garcıa, 2002;

Van Straalen & Timmermans, 2002; Merila et al., 2004).

This paper aims to quantify genetic variation underly-

ing life history divergence in natural populations of the

wolf spider Pirata piraticus and to assess its relationship

with metal tolerance. An earlier study demonstrated

altered life history patterns indicative for low growth

and ⁄ or low reproductive environments in populations

contaminated with Cd, Zn and Cu (Hendrickx et al.,

2003b). Although clutch mass and hence, female fecun-

dity, decreased along an increasing pollution gradient

consisting of six populations, egg size significantly

increased. In contaminated populations, females produc-

ing larger offspring tended to have lower levels of

developmental stability, as measured by fluctuating

asymmetry. This relationship was not observed for

reference populations, suggesting a fitness advantage of

producing larger offspring under environmental stress

(Hendrickx et al., 2003a). Such life history divergence

can be interpreted as adaptive under optimality models

developed by Sibly et al. (1988) and Tamate & Maekawa

(2000).

We here set up a half-sib breeding experiment where-

by F1 offspring derived from pure as well as reciprocal

hybrid crosses between individuals from the most con-

taminated and a reference population along this pollu-

tion gradient were reared under both contaminated and

reference conditions. By estimating the levels of genetic

(co)variation of life history traits size at birth, growth and

egg size, we predict that (i) reduced growth and increased

egg size have evolved as a result of tolerance cost

and adaptive life history strategies in the metal stressed

population; (ii) metal exposure decreases the additive

genetic variance proportionally more than the other

sources of phenotypic variance, resulting in decreased

realized heritabilities and (iii) long-term metal exposure

decreases the genetic variability of the investigated traits.

Materials and methods

Origin of the populations and rearing conditions

Selection of study populations was based on a previous

study where adaptive life history changes were shown to

co-vary with internal metal body burden along a pollu-

tion gradient (see Hendrickx et al., 2003b). The popula-

tions used in this study represented the two most

extreme cases along this gradient and were characterized

by profound differences in life history traits and internal

metal concentration. Reference population ‘Damvallei’

(50�58’N, 3�50’E; further referred to as ‘R’) originates

from a pristine lowland mire with a low degree of metal

contamination. Under field conditions, females of this

population had a large size, a relatively high reproductive

output and produced small eggs. A severly contaminated

population ‘Galgenschoor’ (51�18’N, 4�16’E, referred to

as ‘C’), originates from a tidal marsh along the Schelde, a

river that suffered from severe metal pollutiond during

the last four decades (Zwolsman et al., 1996). Besides the

significantly higher Cd-, Cu- and Zn-content in these

spiders, adult size and reproductive output of females

were significantly lower, but egg mass was significantly

184 F. HENDRICKX ET AL.


J O U R N A L C O M P I L A T I O N ª 2 0 0 7 E U R O P E A N S O C I E T Y F O R E V O L U T I O N A R Y B I O L O G Y

higher, than for females of population R (Hendrickx

et al., 2003b). The distance between both populations is

45 km.

Around 100 hibernating juveniles from each popula-

tion were collected during October and reared till

adulthood in a climate chamber at a constant tempera-

ture of 26 �C and a 16 : 8 h light : dark photoperiod.

They were kept individually in Petri dishes with at the

bottom a layer of plaster of Paris. As natural populations

of this species are bound to very wet and inundating

habitats (Hanggi et al., 1995), Petri dishes were kept at

100% relative humidity. Spiders were fed ad libitum with

fruit flies three times a week. To obtain optimal growth

conditions and minimal mortality, 40% wet weight

crushed dog food was added to a banana and oatmeal

based fruit fly medium (Mayntz & Toft, 2001).

Experimental design

We applied a breeding design that allowed to simulta-

neously test for within- and between-population genetic

variation in life history traits and cadmium susceptibility.

Besides pure R and C offspring (RSRD and CSCD respec-

tively; subscript S denotes Sire population and subscript

D denotes Dam population), reciprocal crosses (RSCD and

CSRD) produced hybrid offspring. To extract additive

genetic components of life history divergence, males and

females were mated according to a half-sib mating design

by allowing males of both populations to copulate with

two females of each population (four females in total).

However, this scheme could not be maintained in all

cases due to frequent injuries or killing of males by

females during copulation. Offspring originating from

copulations in which a male could not copulate with at

least one female from either population were excluded

from the experiment. The parental population consisted

of 13 R and 10 C males and 31 R and 39 C females.

Parental females produced an egg cocoon from which

spiderlings emerged after 3 weeks. F1 offspring was

obtained by randomly selecting 14 spiderlings per female,

resulting in 980 spiderlings of 16 RSRD, 23 RSCD, 15 CSRD

and 16 CSCD full sib families at the start of the

experiment. First and second instars are unable to

capture fruit flies and were therefore fed ad libitum with

collembolans (Sinella curviseta) until they reached the

third instar (after about 4 weeks). Hereafter, individuals

of each full sib family were split at random into two

equally sized groups of seven individuals, one of which

was fed with fruit flies that were transferred to a medium

containing 50 lg Cd g–1 2 days before being fed to the

spiders (rearing fruit flies on a contaminated medium for

longer time periods may affect their nutritional value and

hence confound the cadmium treatment). Spiders of

experimental and control groups were fed four flies every

third day until the age of 7 weeks when the daily portion

was augmented to six flies. Adult females were fed 12

flies per day.

Measurement of life history traits

Population differentiation and within-population vari-

ability was measured for the following life history traits:

initial weight, growth rate and egg size. As no cadmium

treatment was applied during the first 4 weeks after

emergence, its effect was only assessed on the latter two

traits. Initial weight was measured to the nearest 0.1 mg at

the age of 3 weeks (i.e. the earliest time when spiders can

be measured with high accuracy) and every third week

thereafter. None of the spiders reached adulthood before

the age of 12 weeks and this weight was taken as a proxy

of juvenile growth. However, significant mass differences

between the sexes were already present at this stage. In

order to reduce statistical complexity and increase the

number of observations within each full-sib group, we

regressed mean juvenile female weight against juvenile

male weight and transformed juvenile male weights to

juvenile female weights according to the regression

equation. Egg length (mm) and egg width (mm) were

measured to the nearest 0.01 mm. The volume of each egg

(ellipsoid) was calculated as p ⁄ 6 · egg length · (egg

width)2. The average volume of the eggs was calculated

for each spider to obtain a single observation per female.

Analysis of population differentiation

We applied a generalized linear mixed model (Proc mixed

in SASSAS v 9.1, SAS Institute, Inc., Cary, NC, USA) to test for

the significance of the main fixed effects ‘sire population’

(SPOP), ‘dam population’ (DPOP), ‘cadmium treatment’

(CD) and all two- and three-way interactions. To model

the most appropriate error structure for testing these fixed

effects, we added the random effects ‘sire’ (nested within

SPOP) and ‘dam’ (nested within Sire and DPOP) in each

cadmium treatment and also estimated the degree of

covariance between both treatments. Error terms were

allowed to differ between treatments. Fixed effects testing

was based on Type III sum of squares, with error degrees of

freedom adjusted according to Satterthwaite’s approxima-

tion as multiple error terms were included in the model

(Litell et al., 1996; Verbeke & Molenberghs, 2000).

Estimation of quantitative genetic parameters

Genetic and environmental (co)variance components

were estimated by means of the animal model (Lynch

& Walsh, 1998). Given that lS,D,t is the mean value of

offspring originating from the sire (S) and dam (D)

population combination and cadmium treatment t, the

phenotype of offspring i of the s’th sire and the d’th dam

receiving the t’th cadmium treatment (Ys,d,t,i) was mod-

elled as:

Ys;d;t;i ¼ lS;D;t þ ðas;t þ as;d;tÞ=2þ ds;d;t þ es;d;t;i

where the a’s are the breeding values, d are the maternal

and dominance effects and � are the residual errors.

Quantitative genetics and stress adaptation 185



Separating dam and sire additive genetic effects allowed

us to estimate these breeding values for each sire and

dam population separately.

The vector of a’s are modelled as random effects as

follows:

a�Cd

aþCd

� �� N

0

0

� �;

r2a;�Cd ra;�Cd;þCd

ra;�Cd;þCd r2a;þCd

� ��

where r2a,-Cd is the additive genetic variance in the

cadmium free environment, r2a,+Cd is the additive genetic

variance in the cadmium contaminated environment and

ra,-Cd,+Cd is the additive genetic covariance between

breeding values in the reference and contaminated

environment. Again, the variances of the a’s were

estimated for each population separately. The latter is

expressed as the genetic correlation across environment

(rg) rather than as a covariance and was obtained by

dividing this covariance by the square root of the product

of the variances within each environment. Estimates of

these (co)variance components were designated as VA,-Cd,

VA,+Cd and rA respectively and were obtained for both

reference and contaminated populations separately.

The applied breeding design does not allow distinguish-

ing between maternal effects and dominance deviations,

hence, both were modelled as a single random effect

represented as d and further referred to as maternal-

dominance deviations. As such, the variances in dactually represented a quarter of the variance in domi-

nance deviations and the variance in maternal effects

(Falconer & Mackay, 1996; Lynch & Walsh, 1998). They

were modelled as random vectors in an identical way as

for the additive genetic effects and estimates are referred

to as VM,D. However, as maternal-dominance deviations

can be expected to differ between cross type rather than

between populations only, estimates were initially

obtained per cross type.

Residual errors (�) are composed of additive genetic

error (�a) and dominance error and special environmen-

tal effects. The latter two errors are modelled as one

component (�w) as dominance effects cannot be esti-

mated with high precision in the current breeding design.

Hence, the error of the i’th offspring of the s’th sire and

d’th dam is modelled as:

es;d;t;i ¼ ðea;s;t;i þ ea;s;d;t;iÞ=2� �

=ffiffiffi2pþ ew;s;d;t;i

Additive genetic errors (�a) were assumed to follow the

same distribution as the breeding values. Error variances

were assumed to follow a normal distribution with mean

zero and variance r2�,,t and were allowed to differ

between the four crossing types and both cadmium

treatments. Estimates of this variance are further referred

to as VE.

Estimating these additive, maternal-dominance and

error variances allowed us to obtain separate heritabil-

ities for each population and cadmium treatment and

were calculated as:

h2ðtÞ ¼ VA=ðVA þ VM;D þ VEÞ

where t refers to the cadmium treatment.

The model was fitted using a Bayesian, MCMC approach

with Gibbs sampling in WINBUGSWINBUGS 1.4 (Spiegelhalter et al.,

2003). This permitted to estimate the full posterior

distribution of the different (co)variance components

and heritabilities directly (Merila et al., 2004). All esti-

mated parameters were given non-informative prior

distributions. For the fixed effects these were normally

distributed with mean 0 and a variance of 1000. For the

precision of the variances, these were chosen to follow a

uniform [0,1000] distribution. For the multivariate

random effects the prior distribution of the precision of

the variance–covariance matrix was chosen to follow a

Wishart distribution with R = [1 0;0 1] and 2 d.f. It

should be noted that setting a prior distribution that is

bound to zero always results in positive variance com-

ponent estimates and lower credibility intervals are

consequently always larger than zero. Hence, only

variance components with credibility intervals that are

substantially larger than zero were interpreted as being

substantial. Selection of the most parsimonious model

was based on Deviance Information Criterium (Spiegel-

halter et al., 2002). Three MCMC chains, each with

different starting values, were run simultaneously for

12 000 iterations. Convergence of the three chains was

checked by visual inspection of plots depicting the Gibbs

chains. Posterior summary statistics of the parameters

were also highly comparable for the three chains. The

first 2000 iterations were treated as burn-in and dis-

carded for the estimation of the parameters.

Results

Differentiation between populations

Week 3 weightAs the cadmium treatment was only applied from the age

of 4 weeks onwards, cadmium effects could not be

assessed. Initial weight of the spiderlings strongly

depended on female origin (DPOP: F1,771 = 10.57;

P = 0.001) whereby offspring of population R were

significantly smaller (mean = 1.87 mg, SE = 0.13) than

of population C (mean = 2.36 mg, SE = 0.10). At this age

there was no significant effect of male origin (SPOP:

F1,771 ¼ 0.76; P = 0.38) nor was there a significant

interaction between male and female origin

(F1,771 = 0.8; P = 0.37). This demonstrates that differ-

ences in offspring size in early developmental stages are

predominantly determined by female population origin.

Week 12 weightWeight at the age of 12 weeks was strongly reduced

under the cadmium treatment, although its effect

differed between the crossing types (Table 1; Fig. 1).

Differences between crossing types were more




pronounced when no cadmium treatment was applied

(Fig. 1; SPOP and DPOP effect in reference environment:

F1,19.4 = 6.45; P = 0.02 and F1,42.5 = 13.78; P = 0.0006

respectively). The fact that sire origin significantly

affected growth confirms the (partly) involvement of

genetic effects in population differentiation under refer-

ence conditions. Female origin affected weight to a

similar magnitude across cadmium treatments. Absence

of significant interactions between SPOP and DPOP

indicated that differences were largely due to additive

genetic effects. Differences in offspring weight were no

longer significant when they were exposed to cadmium

(Fig. 2; SPOP and DPOP effect in cadmium environment:

F1,21.4 = 1.98; P = 0.17 and F1,46.6 = 0.06; P = 0.81

respectively). The response to cadmium was significantly

dependent on female as well as male origin (Table 1).

Although evidence for influences of the female origin

appeared to be stronger, we found no evidence that

influences of male and female origin on cadmium

response included some dominance effects, as revealed

from a non-significant three-way interaction.

Egg sizeEgg size produced by adult F1 females was not signifi-

cantly affected by the cadmium treatment but only sire

and dam population origin (Table 2, Fig. 2). Female

offspring from parents that both originated from the

contaminated population produced significantly larger

eggs (0.34 mm3, SE = 0.007) than female offspring from

reference parents (0.28 mm3, SE = 0.007). The absence

of a significant interaction between male and female

origin confirmed the additive genetic origin of genetic

differentiation in egg size. Egg size produced by females

from the two hybrid crosses was not significantly differ-

ent (P > 0.07).

Table 1 Mixed model analysis of cadmium treatment (CD) and sire

and dam population origin (SPOP and DPOP respectively) on week-

12 weight of F1 offspring.

Effect d.f. F P-value

CD 15.6 27.1 <0.0001

SPOP 20.3 5.34 0.03

DPOP 45 4.84 0.03

SPOP · DPOP 45 0.02 0.89

CD · SPOP 15.6 4.57 0.048

CD · DPOP 43.5 19.27 <0.0001

(CD · SPOP · DPOP) 43.5 1.38 0.25

Non-significant interaction terms (between brackets) were removed

from the final model. Sire, dam and their respective interactions

with the cadmium treatment were included as random effects to

model an appropriate error structure for the fixed effects.

0 125

26

27

28

29

30

31

RsRdRsCdCsRdCsCd

Cd-treatment

Wei

ght (

mg)

(w

eek

12)

Fig. 1 Least-square mean (± SE) weight (mg) of offspring after

12 weeks as a function of cross type and cadmium treatment. Cross

type labels refer to parental origin of sires (subscript S) and dams

(subscript D) with R = reference population and C = metal con-

taminated population.

0 10.26

0.28

0.30

0.32

0.34

0.36RsRdRsCdCsRdCsCd

Cd-treatment

Egg

siz

e (m

m3 )

Fig. 2 Least-square mean (± SE) egg size (mm3) produced by

offspring of the four different cross types. Cross type labels refer to

parental origin of sires (subscript S) and dams (subscript D) with

R = reference population and C = metal contaminated population.

Table 2 Mixed model analysis of cadmium treatment (CD) and sire

and dam population origin (SPOP and DPOP respectively) on egg size

produced by F1 offspring.

Effect d.f. F P-value

CD 245 0.12 0.73

SPOP 23.5 5.88 0.02

DPOP 48.4 39.84 <0.0001

(SPOP · DPOP) 48.4 0.50 0.48

(CD · SPOP) 245 0.24 0.62

(CD · DPOP) 245 0.05 0.82

(CD · SPOP · DPOP) 245 0.00 0.99

Non-significant interaction terms (between brackets) were removed

from the final model. Sire, dam and their respective interactions

with the cadmium treatment were included as random effects to

model an appropriate error structure for the fixed effects.




Variation within populations

Week 3 weightSources of phenotypic variance for initial weight were of

similar magnitude for the four crossing types, and

obtaining estimates for each crossing type separately did

therefore not result in the most parsimonious model. A

common additive genetic, maternal-dominance and

environment specific variance was therefore estimated

over crossing types. Maternal-dominance effects contrib-

uted to the highest amount of phenotypic variance

(VM,D = 0.277; 95% CI: 0.157–0.437), followed by envi-

ronment specific residual variance (VE = 0.104; 95% CI:

0.025–0.183) and additive genetic variance (VA = 0.096;

95% CI: 0.029–0.190). Consequently, heritabilities for

initial weight were estimated to be low (h2 = 0.20), and

not substantially higher than zero (95% CI: 0.05–0.37).

Week 12 weightWe obtained additive genetic, maternal-dominance and

environmental variance estimates for each population

(cross) and cadmium treatment. The relative magnitude

of the variance components differed strongly depending

on population origin and cadmium treatment (Table 3).

For the reference population, expression of additive

genetic variance for growth was considerably larger

when spiders were reared under reference conditions

compared to the cadmium treatment. However, the

genetic correlation across environments approximated

one, indicating an absence of genotype–environment

interaction. Maternal-dominance and environment spe-

cific residual variance was in contrast highly comparable

under both cadmium treatments and hence only one

variance was estimated across environments. The stron-

ger reduction in additive genetic variance compared to

the other components consequently resulted in a signif-

icantly lower heritability for weight for the reference

population under cadmium contaminated conditions

(Fig. 3).

Estimated additive genetic variance of the contami-

nated population was in contrast much lower under

uncontaminated conditions and not statistically different

when fed with cadmium flies. As maternal-dominance

and residual variation were of comparable magnitude

under both environmental conditions, and of comparable

magnitude as for the reference populations, heritability

estimates were lower, particularly under uncontami-

nated conditions (Fig. 3).

Egg sizeVariation in egg size was mainly determined by additive

genetic and maternal-dominance effects and only for a

minor part by environment specific residual effects,

Table 3 Mean estimated additive, maternal and environmental variance components (2.5 and 97.5 percentiles between brackets) for weight

at week 12.

Source R C

VA

VA ()Cd) 47.96 [23.70; 84.6] 1.13 [0.14; 4.37]

VA (+ Cd) 4.07 [0.46; 12.40] 1.32 [0.16; 5.33]

rG 0.80 [0.51; 0.99] 0.20 [)0.76; 0.92]

RSRD RSCD CSRD CSCD

VM,D 2.92 [0.23; 9.18] 5.35 [1.86; 11.61] 3.31 [0.16; 10.80] 3.26 [0.71; 8.50]

RSRD RSCD CSRD CSCD

VE 11.19 [8.47; 14.82] 9.89 [7.31; 13.32] 8.35 [6.38;10.85] 8.37 [6.20; 11.23]

RSRD RSCD CSRD CSCD

h2 ()Cd) 0.75 [0.58; 0.86] – – 0.07 [0.05; 0.27]

h2 (+ Cd) 0.27 [0.04; 0.60] – – 0.11 [0.01; 0.37]

Cross type labels refer to parental origin of sires (subscript S) and dams (subscript D) with R = reference population and C = metal

contaminated population.

VA, genetic variance in breeding values; rG, genetic correlation across cadmium treatments; VM,D, maternal-dominance variance; VE, residual

variance.

Fig. 3 Posterior distribution (mean ± 95% credibility intervals) of

the difference in heritability for growth between the two populations

(upper panel; R = reference population, C = contaminated popula-

tion) and between the two environments (lower panel).




resulting in high estimates of narrow sense heritability

across cadmium treatments (Table 4). In parallel with

population responses, egg size variation in response to

the cadmium treatment was of small magnitude. Esti-

mates of heritability and variance components did not

differ substantially between both populations.

Discussion

Differentiation between populations

Results from this paper provide evidence for life history

divergence in two populations of wolf spiders that differ

in exposure to heavy metal contamination. The signifi-

cant effect of paternal origin on growth rate and egg size

suggests that the previously field observed population

divergence along the pollution gradient is – at least

partly – genetically based. The intermediate levels of both

life history traits in hybrid crosses, and an absence of

interactive effects between male and female origin,

further point that additive genetic components are the

main source of the observed life history variation (Lynch

& Walsh, 1998).

The weight of early instars originating from parental

reference (R) females was considerably smaller than that

of instars originating from females of the contaminated

(C) population, which is in concordance with egg size

differences recorded in both populations under natural

conditions (Hendrickx et al., 2003b). Instar size appeared

to be only affected by the population origin of the

maternal population. However, variation in egg size

produced by F1 females was additionally affected by

paternal origin. The intermediate size of eggs from both

hybrid crosses further suggests additive paternal and

maternal effects. Combining data on early instar size and

egg size demonstrate that progeny size is determined by

the mother’s genes and hence a maternal genetic effect as

has been shown for other invertebrates (Mousseau &

Dingle, 1991; Mousseau & Fox, 1998).

Apart from genetic effects, growth rates were also

affected by environmental stress levels. In absence of

cadmium stress, pure R offspring were larger than C

offspring, whereas hybrid crosses obtained intermediate

sizes. This contrasts with the larger initial size of

C offspring and indicates reduced growth of C offspring,

even in absence of environmental stress. In spiders,

exposure to heavy metals has been observed to increase

the production of detoxifying enzymes such as metallo-

thioneins (Wilczek & Migula, 1996), which is considered

to act as a physiological defence mechanism (Maro-

ni et al., 1987). Likewise, selection experiments with

D. melanogaster indicated genetically based production of

these enzymes under heavy metal contamination, even-

tually causing decreased fitness under favourable condi-

tions (Shirley & Sibly, 1999). In wolf spiders, such

mechanism is evident from the reduced growth rates

under favourable conditions in individuals locally

adapted to cadmium stress. Although in our experiment,

mean growth rates of C offspring did not exceed those of

R offspring (i.e. non-crossing reaction norms), this may

well be the case under natural conditions where cad-

mium body burdens can be expected to be even higher

then the levels reached in this experiment (50–150 lg

Cd g–1 spider, F. Hendrickx personal observation, Hend-

rickx et al., 2003c). Moreover, natural spider populations

are generally contaminated with a mixture of heavy

metals (Hendrickx et al., 2003b, 2004) that have been

shown to act synergistically (Lock & Janssen, 2002).

Egg size showed no plastic response to metal contam-

ination and differences between cross types remained

fixed across cadmium treatments. This suggests that

female wolf spiders are unable to adjust their egg size

in response to the environmental conditions experienced

during growth at population level (but see Ernsting &

Table 4 Mean estimated additive, maternal and environmental variance components (2.5 and 97.5 percentiles between brackets) for egg size.

Source R C

VA

VA ()Cd) 0.128 [0.080; 0.241] 0.108 [0.064; 0.219]

VA (+ Cd) 0.158 [0.096; 0.274] 0.162 [0.100; 0.366]

rG 0.96 [0.67; 0.99] 0.78 [0.42; 0.97]

RSRD RSCD CSRD CSCD

VM,D 0.125 [0.05; 0.350] 0.08 [0.04; 0.156] 0.117 [0.051; 0.257] 0.109 [0.049; 0.234]

RSRD RSCD CSRD CSCD

VE 0.023 [0.01; 0.05] 0.028 [0.016; 0.051] 0.051 [0.027; 0.142] 0.038 [0.017; 0.080]

RSRD RSCD CSRD CSCD

h2 ()Cd) 0.469 [0.257; 0.674] – – 0.627 [0.329; 0.923]

h2 (+ Cd) 0.411 [0.221; 0.594] – – 0.586 [0.301; 0.813]

Cross type labels refer to parental origin of sires (subscript S) and dams (subscript D) with R = reference population and C = metal

contaminated population.

VA, genetic variance in breeding values; rG, genetic correlation across cadmium treatments; VM,D, maternal-dominance variance; VE, residual

variance.




Isaaks, 1997; Fox et al., 1999; Fox & Czesak, 2000;

Guinnee et al., 2007 for examples of increased offspring

size in response to environmental variation). Given the

apparent absence of egg size plasticity in our study,

optimal egg size differences appeared to have evolved

through local adaptation, as predicted by various theo-

retical models (Smith & Fretwell, 1974; Lloyd, 1987;

McGingley et al., 1987) and demonstrated empirically in

wild populations (Einum & Fleming, 1999; Rasanen

et al., 2005).

The observed differentiation in life history traits is in

line with the pattern observed under field conditions,

where both populations are situated on the extremes of

a pollution gradient consisting of six different popula-

tions. Average metal body burdens of field captured

individuals are about 6, 5 and 1.4 times higher for Cd,

Cu and Zn respectively (Hendrickx et al., 2003b). How-

ever, as only two populations could be included in this

breeding design, other causal factors than metal pollu-

tion cannot be excluded unambiguously. First, results

from this study provide no evidence whether the

differentiation is directly caused by metal pollution or

rather because of indirect effects such as reduced prey

availability. Although the density of suitable prey items

is hard to estimate, densities of adult individuals are

highly comparable under field circumstances and aver-

age about 8–10 individuals m–2 (F. Hendrickx personal

observation). Second, contamination of the river

Schelde also includes other pollutants besides heavy

metals, which may cause, or at least reinforce, this life

history differentiation.

Within population variability

Our results show that cadmium contamination strongly

decreased the heritability for growth, but only for the

reference population. For the contaminated populations,

heritabilities for this life history trait were low, and not

affected by the applied cadmium treatment.

Although earlier studies, mainly laboratory studies on

Drosophila populations, investigating changes in herita-

bility in response to stressed conditions report an increase

in heritable variation (Hoffmann & Hercus, 2000),

decreased heritabilities under unfavourable conditions

are no exception in natural populations under more

realistic types of environmental stress (Hoffmann &

Merila, 1999, Charmantier & Garant, 2005). Most of

these studies showed that the decrease in heritability

could be attributed to an increase in environmental

variation due to stress, which results in a relative

decrease of the additive genetic variation and, conse-

quently, the heritability of that trait. We here provide

evidence that the decrease in growth heritability in the

reference populations is principally caused by a decrease

in the additive genetic variation as other sources of

phenotypic variation remained constant over cadmium

treatments.

Several patterns can explain the observed difference in

VA in this study. First, patterns of changes in additive

genetic variation were in close relationship with the

mean growth response of the different populations–

cadmium treatment combinations, indicating that the

observed differences can be attributed to scale effects

(Houle, 1992; Falconer & Mackay, 1996), i.e. a change in

the mean value of a trait following a change in its

(additive genetic) variance. However, such pattern is

unlikely to explain the change in additive genetic

variance in the present study. This can be derived from

calculating the coefficients of variation (i.e. square root of

the additive genetic variance divided by to the mean

value of the trait) which averaged 21.4% for the

reference population in the cadmium-free environment,

12.7% when cadmium treatment was applied and 5.5%

for the contaminated population across cadmium treat-

ments. Moreover, in case of scale effects, all variance

components would be expected to change consistently

whereas heritability estimates would not change in

response to changes in mean trait value.

Second, the decrease in heritable trait variation can

be understood within the framework of traits plasticity

in response to environmental variation (i.e. reaction

norms). In case of a negative additive genetic covariance

between the breeding value of growth in the reference

environment and growth rate reduction in response to

cadmium, levels of additive genetic variance will vary

along the environmental gradient and may become

close to zero at particular environmental values (Schei-

ner, 1993; Lynch & Walsh, 1998; Nussey et al., 2007).

However a similar pattern might arise if different loci

determine growth rate in both environments. For

example, the expression of growth promoting alleles

in the reference environment can be masked in part by

the expression of alleles that enhance detoxification in

the contaminated environment. If heritable variation of

the latter is low, growth heritability can be strongly

reduced under contaminated conditions. Results of the

present study can however not distinguish which of

both mechanisms prevail as the genetic correlation

across environments could not be estimated with high

accuracy.

Besides this observed decrease in heritability of growth

in the reference population, a strong decrease in growth

heritability under reference conditions was also observed

for population C. This reduction in heritability for growth,

caused by a low additive genetic variance, is likely due to

the decreased mean growth rate, suggesting a rather

permanent expression of physiological defence mecha-

nisms that decrease growth rate. This might hamper the

expression of loci that determine growth in the reference

population under reference conditions. A decrease in

additive genetic variance in response to selection on the

mean value of a trait has until now primarily been

investigated in experimental populations and by theoret-

ical models. These studies reveal that ‘antagonistic




selection’, i.e. selection towards the overall mean, being

upward in unfavourable conditions and downward in

favourable conditions, decreases environmental sensitiv-

ity, which might ultimately lead in a decreased additive

genetic variance (Falconer, 1990; Scheiner & Lyman,

1991; Scheiner, 2002). It remains however unclear if

these results can be generalized towards wild animal

populations, as the few studies that compared genetic

variability of populations subjected to long term stress

exposure gave contrasting results (e.g. Charmantier et al.,

2004; Merila et al., 2004). Other mechanisms that might

explain the observed decrease in genetic variability is a

reduction in effective population size in stressful envi-

ronments (Hoffmann & Hercus, 2000; Van Straalen &

Timmermans, 2002), which has been shown to decrease

the amount of additive genetic variance as well as

heritability (e.g. Kristensen et al., 2005). This appears

however less likely in the present population as neutral

genetic diversity, investigated on six allozymes, was

not different between both populations (F. Hendrickx,

F. Langenbick & J.-P. Maelfait unpublished data).

Egg size variation showed relatively high heritabilities

in both populations in the two environments, indicating

that there is evolutionary potential for egg size adjust-

ment to local conditions. In line with the absence of

a change in mean egg size in response to cadmium,

genetic determination appeared to be unaffected by the

cadmium treatment. The high genetic correlation dem-

onstrated that no genetic variation is present in egg size

adjustment in response to the applied cadmium treat-

ment.

Conclusion

With this experiment we give evidence that cadmium

exposure might not only reduced the growth rate of a

previously unexposed population, but moreover influ-

enced the genetic architecture of this life history trait.

Decreased realized heritabilities were observed under the

cadmium treatment, suggesting a profound decrease in

the evolutionary potential of the tested reference popu-

lation. Comparing these observations with those of a

population subject to metal pollution for several decades

revealed that growth and growth heritability were lower,

particularly under reference conditions. This demon-

strates that long-term metal pollution may cause severe

fitness costs and decrease the evolutionary potential of

particular life history traits, even when the pollution

source ceases to exist. In accordance to life history

theory, a genetically based increase in egg size is likely to

have evolved for counteracting these adverse fitness

effects.

Acknowledgments

S. Toft gave indispensable information for rearing spiders

under optimal conditions, which reduced the mortality

drastically. Many thanks to A. Alcantara and E. Bijttebier

for preparing fruit fly media and rearing collembolans

respectively. Valuable comments to this manuscript were

given by T. Backeljau, D. Eraly, S. Toft and N.M. van

Straalen. This work was supported by research funds of

Ghent University (BOF 01110498) and Fund for Scien-

tific Research Flanders (FWO-Vlaanderen) within the

framework of projects G.0202.06 and G.0247.06.

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September 2007




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