ANIMAL BEHAVIOUR, 2002, 63,
doi:10.1006/anbe.2002.3046, available online at http://www.idealibrary.com on
Family, sex and testosterone effects on garter snake behaviour
RICHARD B. KING
Department of Biological Sciences, Northern Illinois University, DeKalb
(Received 7 August 2000; initial acceptance 17 October 2000;
final acceptance 7 December 2001; MS. number: A8850R)
To better understand how genes and hormones interact to affect behaviour in nature, I used a factorialdesign to test for effects of family membership, sex and testosterone level on activity and defensivebehaviour of the common garter snake, Thamnophis sirtalis
. Behaviours (latency to move, defensivestrikes, response distance) were scored prior to hormone manipulation (when snakes were 39 days of age),while sham or testosterone-containing implants were in place (190 days), following implant removal andsimulated hibernation (284 days), and when snakes were 428 days of age. Family membership hadpervasive effects on all three behaviours and on their ontogenetic trajectories, suggesting strong geneticor maternal components to behavioural variation. Sex had a significant effect on the number of defensivestrikes; females struck more frequently than males, but ontogenetic trajectories were similar between thesexes. Testosterone manipulation also had an effect on strikes: snakes in the elevated-testosteronetreatment group struck less frequently than shams while implants were in place. Sex and treatment effectson latency to move and response distance were lacking. Family*treatment interaction effects were lackingfor latency to move and number of defensive strikes but were present for response distance. Possibly,genetic or maternally induced variation in strikes is mediated through variation in circulating hormonelevels, whereas variation in response distance is mediated through receptor-level phenomena.
2002 The Association for the Study of Animal Behaviour
The interplay between genes and hormones is likely to be
ences are often investigated through hormone assays
important in shaping behavioural evolution (e.g. Crews &
and experimental manipulations (hormone implants,
Moore 1986; Crews 1987; Moore 1991; Ketterson & Nolan
ablation of endocrine glands) without regard to related-
1992). Evidence for such an interplay is accumulating
ness among individuals (Nelson 1995). Fortunately,
from twin studies and pedigree analyses of humans (e.g.
methods used to investigate genetic and hormonal influ-
Mendlewicz et al. 1999; An et al. 2000) and strain com-
ences in nature need not be mutually exclusive. By
parisons and controlled breeding designs in poultry, live-
incorporating hormonal assays and manipulations into
stock and laboratory rodents (Bates et al. 1986; Scott &
analyses of variation within and between groups of rela-
Washburn 1988). Most frequently, these studies have
tives, behavioural effects of genes and hormones can be
demonstrated high heritability for circulating levels
studied simultaneously. This paper reports the results of
of hormones and hormone-binding proteins. Linkages
such an investigation of behaviour in the common garter
between heritable variation in hormone levels and
phenotypic traits, such as morphology, life history or
High levels of genetic variation have been found in
behaviour, have been found less frequently (e.g. Bates
and allied genera for a range of behavioural
et al. 1986; Gupta & Brush 1998; Otremski et al. 2000).
traits (reviewed in Brodie & Garland 1993; Burghardt &
Especially rare are studies integrating genetic and hormo-
Schwartz 1999). Behaviours related to prey preference,
nal influences on behaviour in undomesticated species
predator avoidance and escape have been particularly
(but see Fairbairn & Roff 1999; Zera 1999; Zera & Huang
well studied, are repeatable between trials and show high
1999), perhaps due to differences in the methods of
levels of constancy over time (Brodie 1993; Brodie &
investigation typically used by behavioural geneticists
Russell 1999; Herzog & Burghardt 1988). Quantitative
and endocrinologists. Genetic effects are frequently
genetic analysis provides heritability estimates of 0.37–
assessed from patterns of variation within and between
0.42 for predator avoidance and escape behaviours
groups of relatives or from controlled breeding designs
(Arnold & Bennett 1984; Brodie 1989; Garland 1988).
(Falconer & MacKay 1996). In contrast, hormonal influ-
Studies of hormonal influences on garter snake behaviourhave focused mostly on courtship behaviour. Steroid
Correspondence: R. B. King, Department of Biological Sciences,Northern Illinois University, DeKalb, IL 60115, U.S.A. (email:
hormones appear to play an organizational role in
behavioural differentiation between males and females;
2002 The Association for the Study of Animal Behaviour
ANIMAL BEHAVIOUR, 63,
testosterone manipulations early in life (before snakes
Year and site of collection of gravid female garter snakes
first enter hibernation) modify expression of sex-typical
courtship behaviour later in life (Crews 1985; Moore &
Lindzey 1992; Whittier & Tokarz 1992).
behaviours in garter snakes (e.g. behaviours for whichgenetic data are available) is unknown. However, in somespecies of garter snakes, predator avoidance behaviour
(e.g. defensive strikes) of neonates differs between males
and females (Herzog & Burghardt 1986; Scudder &
Burghardt 1983), suggesting that gonadal hormones
might be involved. In addition, male garter snakes
emerge from hibernation earlier than females andactively search for potential mates, whereas females are
*See Figure 2 in Lawson & King (1996) for locations of study sites.
less active upon emergence. If testosterone plays anorganizational role in mate-searching behaviour (as itdoes in other aspects of courtship, Crews et al. 1985),
variation is made easier by dividing these events into two
males and females might show more general differences
groups: factors that influence the level of circulating
in activity as well (e.g. propensity to flee, Shine et al.
hormones (e.g. responsiveness to environmental stimuli,
2000). Behaviours scored in this study include latency to
responsiveness to releasing hormones, rate of secretion,
move, a measure of activity level (Herzog & Burghardt
androgen binding proteins in the plasma, hormone half-
1986) and defensive behaviour and response distance,
life), and factors that influence an organism’s response to
measures of response to an approaching threat (e.g.
a given level of hormone (receptor- and postreceptor-
Burghardt 1983; Arnold & Bennett 1984; Formanowicz
level phenomena, including receptor density, receptor
et al. 1990). Thus, this investigation serves as a test for sex
affinity, efficiency of conversion of hormone to an active
differences in nonreproductive behaviours as well as for
form and neuroendocrine interactions). Two research
possible effects of family membership and testosterone
strategies for detecting genetic variation in hormone
effects are then evident. One strategy is to test for genetic
Garter snakes have several logistical attributes that
variation in levels of circulating hormones. A second
make them well suited for the work described here.
strategy is to manipulate hormone levels and test for
Among the most important of these is large family size
genetic variation in behavioural responses.
(averaging 15.6 and ranging from 6 to 31 among 28
I used this second strategy in the research described
captive-born families; R. B. King, unpublished data),
here. Offspring born to wild-caught females were divided
which allows for an experimental design in which sex,
into three groups, a sham-manipulated group and
hormone and family effects can be tested simultaneously.
two hormone-manipulated groups. In one hormone-
Whereas previous studies have attempted to avoid the
manipulated group, testosterone levels were elevated via
potentially confounding effects of family membership on
testosterone-containing implants. In the other, testoster-
responses to hormone manipulation (Crews 1985; Crews
one levels were functionally reduced via implants con-
et al. 1985; Shine & Crews 1988), they have not explicitly
taining flutamide, which binds competitively with
tested for family effects. Another attribute of garter snakes
testosterone receptors, thus blocking testosterone’s action
is that hormonal manipulations can be carried out easily
(Liao et al. 1974; Neumann et al. 1977; Alexandre &
via hormone-containing implants (e.g. Crews 1985;
Balthazart 1987). The goal of these manipulations was
Crews et al. 1985). Finally, the social behaviour of garter
to mask individual variation in testosterone levels so
snakes consists primarily of aggregative and reproductive
that differences in receptor-level phenomena (e.g.
behaviours; intraspecific aggression appears to be lacking
receptor density, receptor affinity) might be detected.
(Burghardt 1983; Gillingham 1987; Ford & Burghardt
Comparisons between sham- and hormone-manipulated
1993). Thus, androgen levels are unlikely to be modified
siblings provide a test for an effect of testosterone
by aggressive interactions as may occur in territorial
manipulation, whereas comparisons of treatment effects
among litters provide a test for possibly heritable
The effects of hormones on behaviour typically result
variation in response to hormone manipulation.
from a cascade of events and genetic variation mightoccur at any point in this cascade. The effect of testoster-one starts with an internal or external environmental
stimulus triggering the production of releasing hormones
by the hypothalamus, the releasing hormones triggerrelease of gonadotropins by the anterior pituitary
I obtained study animals by collecting gravid females in
and these subsequently trigger secretion of testosterone.
the wild (Ottawa Co., Ohio, U.S.A.) and maintaining
Testosterone travels to receptors in the central nervous
them in captivity until parturition. Females were col-
system where it is involved in regulation of gene tran-
lected in late May and early June 1994, 1995 and 1996
scription which ultimately affects behaviour and mor-
(Table 1) and housed individually until parturition.
phology (Hadley 1984). The task of detecting genetic
Following parturition in late July–early August, I classified
GARTER SNAKE BEHAVIOUR
Timing of behavioural tests, hormone manipulation, simu-
ing testosterone levels and the time course of hormone
lated hibernation and collection of blood samples from neonatal
manipulation, I collected blood samples from neonates at
195 days of age (while implants were in place), 318 days
of age and 437 days of age. I also scored neonates for a setof morphological characters that were analysed separately(unpublished data). Upon completion of the study, sur-
viving animals were returned to the wild or maintained incaptivity for use in captive breeding.
Hormone Manipulation and Assay
Hormone levels were manipulated using subcutaneous
implants. Implants consisted of 7-mm lengths of empty
diameter, Baxter T5715-3), tubing filled with 4–5 mm of
crystalline testosterone (Sigma T-1500), or tubing filledwith 4–5 mm of flutamide (Sigma F-9397). Implants were
*Ages shown refer to the start of behavioural testing.
sealed with silicon adhesive and inserted at mid-bodythrough a small lateral incision 2–3 scale rows above theventer (following Crews 1985; Crews et al. 1985). Prior to
neonates by sex by everting the hemipenes of males, then
implantation, snakes were immobilized via hypothermia.
measured them and placed them in individual cages for
Ethanol was used to clean the skin prior to implantation.
captive maintenance. Fresh water was available continu-
ously and food (large earthworms for gravid females,
Laboratories Abbott Park, Illinois, U.S.A.) and cloth first-
small earthworms or earthworm pieces for neonates) was
aid tape was used to seal incisions.
provided three times a week. The room in which snakes
Blood samples (100–300 l) were collected from caudal
were housed was maintained at about 26 C and 50% RH
with a 12:12 h light:dark photic cycle. A heating cable
heparinized syringe (Bush & Smeller 1978). Blood
running under one end of the cages provided gravid
samples were centrifuged and the plasma fraction was
females with a thermal gradient ranging from room
frozen for hormone analysis following completion of
bleed collection from all snakes. Testosterone levels were
I divided litters containing at least four males and four
determined by radioimmunoassay as described by King
females into sham- and hormone-manipulation treat-
et al. (2000). Steroids were ether-extracted from plasma
ments. Most families were divided into sham- and
(50–200 l diluted to 1 ml with distilled water) and stan-
elevated-testosterone treatment groups, or sham- and
dards (1.95–500 pg testosterone in 100 l methanol)
flutamide-treatment groups, but a few large families were
added to a radioimmunoassay containing 3H testosterone
divided into sham-, elevated-testosterone and flutamide-
(ca. 5000 cpm; New England Nuclear NET-370) and
treatment groups. Within families, at least two males and
testosterone antibody (1:60 000 dilution). Samples and
two females were assigned to each treatment. I scored
standards were incubated overnight at 4 C, after which
neonates for three behaviours at 39 days of age (Table 2).
bound and unbound steroid was separated using a
Implants were inserted when snakes were 109 days of age
charcoal-dextran suspension. After centrifugation, the
and behaviours were scored again when snakes were 190
supernatant from each sample was added to vials contain-
days of age, after implants had been in place for 81 days.
ing scintillation cocktail (Bio-Safe II, Research Products
Implants were then removed and snakes were placed in
International, Mt Prospect, Illinois, U.S.A.), and counted
simulated hibernation (7 C, 0:24 h LD photic cycle) for
in a Beckman liquid scintillation counter. Testosterone
70 days. Behaviours were scored again following removal
levels were determined using a logit–log curve-fitting
from hibernation when snakes were 284 days of age. A
programme. Testosterone levels at 195, 318 and 437 days
final set of behavioural scores was obtained when snakes
were determined in separate assays. Intra-assay variability
were 428 days of age. The schedule of hormone manipu-
was 10%, interassay variabitity was 13%, and assay
lation and hibernation used here approximately parallels
sensitivity ranged from 3 to 3327 pg per sample at 195
that used by Crews (1985) in his analysis of garter snake
days, 5–1275 pg per sample at 318 days, and 6–2065 pg
courtship behaviour. The schedule of behavioural tests
was designed to detect (1) family and sex effects on
body used here (provided by G. Niswender, Colorado
behaviour soon after birth (39 days), (2) activational
State University), had high cross-reactivity with 5-
effects of testosterone on behaviour (190 days), and (3)
organizational effects of testosterone on behaviour fol-
reactivity with androstenedione (2%). However, because
lowing emergence from hibernation (284 days) or later in
dihydrotestosterone accounts for a relatively small pro-
life prior to adulthood (428 days) (in nature, garter snakes
portion of total androgens in garter snakes (Crews
reach adulthood at 2–3 years of age, Rossman et al. 1996).
et al. 1985; Mason & Crews, 1985), the assay provides a
To document the effectiveness of implants in manipulat-
reasonably accurate estimate of testosterone levels.
ANIMAL BEHAVIOUR, 63,
age and repeated measures multivariate analysis of vari-ance (MANOVA) of behaviour over the entire course of
I scored three different measures of garter snake behav-
the experiment (O’Brien & Kaiser 1985; Potvin et al.
iour, latency to move, defensive behaviour and response
1990; von Ende 1993). In these analyses, scores for a
distance. Each behaviour was scored on two consecutive
given behaviour at different ages were dependent vari-
days and averaged across days for analysis. Behavioural
ables; sex, family and treatment were between-subjects
tests were conducted in an environmental room main-
factors; and time (MANOVA only) was a within-subjects
tained at 22 C. Latency to move and defensive behaviour
factor. MANOVA identifies two general sources of vari-
(total number of strikes at a stationary and a moving
ation: ‘between-subjects’ and ‘within-subjects’ effects.
stimulus) were scored sequentially in a carpeted 75-cm
Between-subjects effects (main effects of and interactions
diameter arena. I measured latency to move by placing a
among the between-subjects factors) reflect differences in
snake in the centre of the arena under an 8-cm diameter
a given response variable among factor levels over the
opaque cover for 2 min, raising the cover from behind
entire course of an experiment and can arise during an
one-way glass, and recording the time elapsed until the
experiment or from pre-existing differences present at the
snake moved its head outside an 11-cm diameter circle
start of an experiment. In the present study, between-
marked on the carpet. After 30 s, I recorded the number of
subjects effects were useful in identifying overall effects of
strikes at a stationary stimulus. I held the stimulus (my
sex and family. In contrast, within-subjects effects (main
finger) about 2 cm in front of the snake’s head and
effects of within-subjects factors and interactions among
recorded the number of strikes in a 1-min interval. After
within-subjects and between-subjects factors) reflect dif-
another 30 s, I recorded the number of strikes at a moving
ferences in how the score of a given response variable
stimulus. In this test I wiggled my finger rapidly from side
changes over time. Within-subjects effects, if significant,
to side, and again, recorded the number of strikes in
provide unambiguous evidence for a treatment effect on
1 min (strikes at a stationary stimulus and strikes at a
moving stimulus follow Herzog & Burghardt 1986; see
I conducted analyses using SPSS 10.0 statistical soft-
also King & Turmo 1997). I summed the number of strikes
ware. All ANOVA and MANOVA made use of type III
at the stationary stimulus and at the moving stimulus for
sums of squares. Pillai’s trace was used in MANOVA
significance testing. The assumption of equality of vari-
I recorded response distance beginning on the day
ance was tested using Levene’s test. Where this assump-
following completion of the other behavioural measures.
For this test, I placed a snake under an 8-cm diameter
examined to ensure that there was no systematic relation-
ship between group means and variances. Examination of
the frequency distribution of residuals revealed no
paper silhouette of a bird’s head measuring 4 cm
marked departures from normality. Each behaviour was
8 cm high with beak and eyes marked in black)
tested separately (see Results for evidence of the absence
was positioned at the other end of the arena. Plexiglas
of any strong correlation among behaviours). Because
and a removable partition separated the snake from the
some mortality occurred over the course of the investiga-
rest of the arena. After 2 min, I lifted the cover and
tion, sample size was maximized over each time interval
removed the opaque partition and then moved the pred-
by conducting separate analyses of behaviour at 39–190
ator from side to side and towards the snake in 10-cm
days, 39–284 days and 39–428 days. For analyses of
increments using a long rod from behind one-way glass. I
behaviour at 39–284 days and 39–428 days, sample size
recorded the distance at which the snake first responded
criteria were relaxed from two to one offspring per sex per
(e.g. by orienting towards the threat or fleeing rearward).
treatment. This precluded testing the highest-order inter-
Snakes that showed no change in behaviour were given a
action in these analyses but this interaction was consist-
10. Thus, snakes that responded to a distant
ently nonsignificant at 39 days of age (see Results) and
stimulus had high response distance scores and snakes
over 39–190 days. Comparison of analyses using the
that failed to respond or responded only to a nearby
original versus relaxed sample size criteria revealed no
qualitative difference in the conclusions reached (analy-
Behavioural tests were conducted blind to sex and
ses not shown). For analyses of behaviour at 39–284 days
treatment of snakes and to previous behavioural scores.
and 39–428 days, reverse Helmert contrasts were used to
To meet assumptions of normality and equality of vari-
identify over which time intervals significant changes in
ance more closely in the analyses described below, I
behaviour occurred. Except as noted below, results were
transformed latency to move using natural logarithms,
qualitatively similar for analyses of behaviour at 39–190
the number of strikes by adding one and computing the
days, 39–284 days and 39–428 days and so for simplicity
square root, and the response distance by adding 10 and
only analyses of the entire 39–428 day interval are pre-
dividing by 110 (converting response distance to a pro-
sented here. Observed effect size ( 2, computed by SPSS as
portion) and then computing the arcsine of the square
of committing a type II error given the observed effectsize) were computed for all analyses. Observed power is
necessarily low when observed effect size is small. For this
I tested for sex, family and treatment effects using
reason, minimum detectable effect size ( 2
analysis of variance (ANOVA) of behaviour at 39 days of
GARTER SNAKE BEHAVIOUR
Table 3. P
values from repeated measures multivariate analysis of variance of behaviour in sham- versus
flutamide-treated garter snakes
Because the primary interest of this analysis is in treatment effects on changes in behaviour over time (i.e. thetime*treatment interaction), only within-subjects sources of variation are shown. P
values less than 0.05 are shown
to hypothesis and error degrees of freedom and F
successive days. Repeatability was highest when snakes
were tested at 39 days of age (r
=0.62, 0.78 and 0.30 for
page 177). Effect sizes 2=0.01, 0.06 and 0.14 correspond
latency to move, strikes and response distance (N
to small, medium and large effect sizes (Cohen 1988;
animals from 26 families). Repeatability at 190, 284 and
428 days of age ranged from 0.43 to 0.56 for latency to
I initially compared sham- and flutamide-treated
move, 0.65–0.73 for strikes and 0.09–0.19 for response
snakes to determine whether these two groups might be
=213, 176 and 165 sham-treated animals from
pooled in order to increase sample size in subsequent
24 families at 190, 284 and 428 days, respectively).
analyses. The general absence of time*treatment interac-tion effects while implants were in place (see Results),together with the observation that testosterone levelswere uniformly low in all but the elevated-testosterone
treatment snakes, suggested that the use of flutamide tofunctionally reduce testosterone levels was unnecessary.
Therefore, I pooled sham- and flutamide-treated snakes
Ninety-three offspring (43 males, 50 females) belonging
and conducted two subsequent sets of analyses. The first
to seven families were divided into sham- and flutamide-
involved tests for family and sex effects on behaviour in
treatment groups (N
=50 and 43, respectively). MANOVA
the pooled sham- and flutamide-treatment groups and
revealed no significant within-subjects effects of treat-
the second involved tests for treatment effects between
ment (i.e. no time*treatment, time*family*treatment,
the elevated-testosterone treatment group and the pooled
time*sex*treatment, time*family*sex*treatment effects)
sham- and flutamide-treatment groups. Comparison of
on latency to move or response distance over any time
analyses in which sham- and flutamide-treatment snakes
interval (Table 3). MANOVA revealed a significant
were not pooled with those in which these treatments
time*treatment effect on strikes over the entire experi-
were pooled revealed no qualitative difference in the
ment (Table 3, 39–428 days; P
=0.047). However, this
conclusions reached (analyses not shown).
source of variation was far from significant over 39–190days (P
=0.745) or 39–284 days (P
time*treatment effect arose only during the 284–428 day
Repeatability was computed as the intraclass corre-
lation between measures of each behaviour across
ANIMAL BEHAVIOUR, 63,
power of tests for within-subjects effects involving treat-
(Table 4, Fig. 1) (power was sufficient to detect medium
ment was sometimes low and minimum detectable effect
size was large ( 2>0.14) over the entire experiment (Table3). However, analysis over 39–190 days had power suffi-
cient to detect a medium time*treatment effect size( 2=0.058). Analysis of covariance, with family and treat-
Two hundred and sixty-nine offspring (132 males, 137
ment as factors and ln(snout–vent length) as a covariate,
females) belonging to 17 families were used to test for
revealed that ln(testosterone) did not differ between
differences in behaviour between testosterone-treatment
males belonging to sham- and flutamide-treatment
group and the pooled sham- and flutamide-treatment
groups while implants were in place (195 days:
=113 and 156, respectively). Crystalline testos-
terone was still present in implants upon their removal.
minimum 2=0.098) or following implant removal (318
Radioimmunoassay of blood samples collected when
snakes were 195 days old (after completion of the second
set of behavioural tests and before implants were
removed) revealed that implants had a marked effect on
2 =0.115). Given these results, sham- and
circulating testosterone levels. Testosterone levels aver-
flutamide-treatment groups were pooled for the analyses
aged 87.3 pg/ml (95% confidence interval=65.5, 116.4)
among 66 sham- and flutamide-treatment males com-pared
testosterone-treatment males and 8636.0 pg/ml (6840.4,10 903.1) in 49 testosterone-treatment females (means
Family and Sex Effects
and confidence intervals backtransformed from naturallogarithms). Testosterone levels in elevated-testosterone
Three hundred and seventy-seven offspring (188 males,
treatment animals approached those reported in young
187 females) belonging to 26 families were used to test for
male garter snakes during a pulse of high testosterone
sex and family effects on behaviour prior to hormone
that occurs shortly after birth (24 540–122 490 pg/ml,
manipulation. ANOVA revealed highly significant family
Table 1 in Crews et al. 1985). However, this pulse was not
effects on all three behaviours (Table 4). In addition,
evident in another natricine snake (King et al. 2000).
there was a highly significant effect of sex on strikes;
Testosterone levels in elevated-testosterone treatment
females struck more frequently than males. No significant
animals were similar to those seen in adult male
family*sex interactions were present. Power was sufficient
garter snakes (1400–72 000 pg/ml, Table 1 in Weil 1985).
to detect small sex effects and medium to large family*sex
Radioimmunoassay of blood samples collected when
effects (Table 4). Consistent with ANOVA results,
snakes where 318 days and 436 days of age revealed
MANOVA revealed significant between-subjects effects of
hormone manipulations had only temporary effects
family on all three behaviours and of sex on strikes over
on testosterone levels. At 318 days of age, testosterone
the entire experiment (Table 4). There were significant
levels averaged 78.1 pg/ml (95% confidence inter-
within-subjects effects of time and of family (i.e. a
val=62.0, 98.4) among 76 sham- and flutamide-treatment
time*family interaction) on all three behaviours, indicat-
males compared with 82.1 pg/ml (59.8, 112.8) in 53
ing that behavioural scores changed over time and that
testosterone-treatment males. At 436 days of age, testos-
the pattern of change differed among families (Table 4,
terone levels averaged 1037.4 pg/ml (95% confidence
Fig. 1). Reverse-Helmert contrasts indicated that signifi-
interval=691.1, 1557.3) among 69 sham- and flutamide-
cant time effects were present over all intervals except
treatment males compared with 1192.1 pg/ml (730.8,
when contrasting latency at 428 days with previous times
1944.8) in 50 testosterone-treatment males. Analysis of
and when contrasting strikes at 190 days with 39 days
covariance, with family and treatment as factors and
(Table 4). Latency decreased slightly between 39 and 190
ln(snout–vent length) as a covariate, revealed no effect of
days and increased following emergence from simulated
treatment on ln(testosterone) at 318 days (F
hibernation (Fig. 1). Strikes remained constant from 39 to
190 days, decreased following emergence from simulated
hibernation (284 days) and then increased by 428 days
observed power=0.353, minimum 2=0.035).
(Fig. 1). Response distance decreased from 39 to 190 to
MANOVA over the full 428-day experiment revealed
284 days and then increased by 428 days (Fig. 1). Reverse-
Helmert contrasts revealed that, with one exception,
treatment (time*treatment, time*family*treatment, time*
time*family effects were significant for all three behav-
sex*treatment) were consistently nonsignificant (Table
iours over all time intervals (Table 4). The one exception
5). For strikes, within-subjects effects involving treat-
was the contrast of response distance at 428 days with
ment were also nonsignificant over the full 428-day
previous times. Time*family effects are evident in profile
experiment. However, the time*treatment interaction
plots (Fig. 1): some families showed increases while other
approached significance over the full experiment (Table
families showed decreases in a given behaviour over a
=0.076) and was significant in analyses of strikes at
given time interval. The time*sex interaction was consist-
ently nonsignificant, indicating that changes in the
behaviour of males and females occurred in parallel
reverse-Helmert contrasts indicated that a significant
GARTER SNAKE BEHAVIOUR
ANIMAL BEHAVIOUR, 63,
Profile plots showing variation in estimated marginal means for latency to move (ln transformed seconds), number of strikes
(square-root transformed) and response distance (arcsine square-root transformed proportions) at 39 days, 190 days, 284 days and 428 days.
(a) Variation among families, with families represented by separate lines. (b) Variation between male (—"—) and female (– –C– –) garter
snakes (bars represent ±1 standard error of estimated mean). The between-subjects effect of family and the within-subjects time-by-family
interaction were significant for all three behaviours; the between-subjects effect of sex was significant for strikes. Snakes underwent simulated
hibernation between 213 and 283 days ( ).
GARTER SNAKE BEHAVIOUR
Table 5. P
values from repeated measures multivariate analysis of variance of behaviour in testosterone-treated versus pooled sham- and
flutamide-treated garter snakes
3,125 11.57 <0.001
0.218 0.060 0.999
0.283 0.124 1.000
3,125 86.09 <0.001
0.674 0.060 1.000
0.484 0.124 1.000
3,125 43.81 <0.001
0.513 0.060 1.000
0.177 0.124 1.000
0.122 0.124 0.991
0.124 0.124 0.992
Because the primary interest of this analysis is in treatment effects on changes in behaviour over time (i.e. the time*treatment interaction), only
within-subjects sources of variation are shown. P
values less than 0.05 are shown in bold.
treatment effect occurred while testosterone-containingimplants were in place: the contrast between 190 days
and 39 days was significant but other contrasts were
nonsignificant (Table 5). The direction of the treatmenteffect paralleled the difference seen between males and
females in that males (Fig. 1) and testosterone-treatmentanimals (Fig. 2) showed lower frequencies of strikes than
did females and sham-treatment animals, respectively.
For response distance, the time*family*treatment interac-
tion was significant over the full 428 day experiment but
(time*treatment, time*sex*treatment) were nonsignifi-cant (Table 5). Again, reverse-Helmert contrasts indicated
testosterone-containing implants were in place: only the
contrast between 190 days and 39 days was significant(Table 5). However, the time*family*treatment effect was
nonsignificant in analyses of response distance measured
and at 39, 190 and 284 days (MANOVA: F
Profile plot showing variation in estimated marginal means
=0.091). Other within-subjects effects on response dis-
testosterone-treated (—"—) and pooled sham- and flutamide-
tance involving treatment (time*treatment, time*sex*
treated garter snakes (– –x– –) (bars represent ±1 standard error of
treatment) were consistently nonsignificant. Power was
estimated mean). The within-subjects time-by-treatment interaction
was significant over the 39–190-day interval. Implants were in place
between 109 and 196 days ( ) and snakes underwent simulated
time*family*treatement effects (Table 5).
hibernation between 213 and 283 days ( ).
ANIMAL BEHAVIOUR, 63,
Correlations among measures of the same behaviour over time and among different behaviours
Entries represent Pearson product-moment correlations among residuals from MANOVA analysis of behaviour over the full 428-dayexperiment. N
=167. Statistically significant correlations (following adjustment for multiple tests by use of α=0.05/48 for correlations amongdifferent behaviours and α=0.05/6 for correlations among measures of the same behaviour over time) are shown in bold. Correlationsobtained from analyses of behaviour at 39 days, 39–190 days and 39–284 days were similar in magnitude.
Correlations among Behaviours
analysis (Brodie & Garland 1993) to behaviours measuredat 39 days of age yields heritability estimates of 0.65
Pearson correlations among residuals of the three
(approximate 95% confidence interval: 0.37, 0.95) for
behavioural scores at 39 days were nonsignificant
latency, 0.56 (0.30, 0.86) for strikes, and 0.33 (0.13, 0.56)
for response distance (heritability was computed as
=0.090). Significant positive correlations
from a two-factor ANOVA with family and sex included
were typically present between measures of the same
as factors; confidence intervals were computed as in
behaviour over time (Table 6). These correlations were
Becker 1992, following modification for the inclusion of
strongest for strikes (range 0.457–0.590), intermediate for
sex as a factor). These estimates assume that litters consist
latency to move (0.197–0.385), and weakest (and some-
of full siblings and that maternal effects are negligible.
times nonsignificant) for response distance (
Evidence is mounting that in some natricines, including
0.286). Small but significant negative correlations were
, multiple paternity within litters is common-
present between strikes at 190 days and response distance
place (Barry et al. 1992; Garner 1998; Gibson & Falls
at 190 days and between strikes at 284 days and response
1975; McCracken et al. 1999; Prosser 1999; Schwartz et al.
distance at 190 days (Table 6). Use of residuals removes
1989). By itself, multiple paternity should lead to under-
the effects of sex, family membership and treatment on
estimates of h
2 using full sibling analysis. King et al.
(2001) have taken advantage of the occurrence ofmultiple paternity within litters to explore the possibility
that maternal effects also contribute to between-familyvariation. Based on an analysis of four litters each sired by
Perhaps the clearest result of this investigation is the
two males (eight sireships total), it appears that maternal
pervasive effect family membership had on garter snake
effects may markedly inflate estimates of h
2 in natricines
behaviour. Such family effects have been found consist-
obtained using full sibling analysis. The nature of these
ently in studies of the behaviour of natricine snakes
maternal effects remains unexplored and may include
(garter snakes and their allies) (reviewed by Brodie &
both maternal environmental effects (effects of the com-
Garland 1993; Burghardt & Schwartz 1999; see also
mon uterine environment shared by littermates) and
Burghardt et al. 2000). However, this study goes further in
maternal genetic effects (effects of maternal genotype on
documenting the presence of significant time*family
interactions, indicating that ontogenetic trajectories also
In the present study, variation between families may
vary among families (Fig. 1). Patterns of variation within
have been inflated by year and site effects: gravid females
and between families have been used previously to esti-
were collected in 3 years and from five sites (Table 1).
mate the heritability of defensive behaviour in natricine
Efforts were made to minimize year effects by using
snakes (Brodie & Garland 1993; Burghardt & Schwartz
uniform rearing and testing conditions. Furthermore, site
1999; Burghardt et al. 2000). Applying a full sibling
effects are likely to be small because sites were separated
GARTER SNAKE BEHAVIOUR
by less than 27 km and molecular genetic analyses sug-
in testosterone level between treatment groups were non-
gest that gene flow among them is common (Bittner
significant. This suggests that testosterone has an activa-
1999; Lawson & King 1996). Examination of family
tional effect on strikes. In contrast, testosterone has an
means revealed no consistent year or site effects on the
organizational effect on garter snake courtship behaviour:
results presented here. However, small but significant
elevating testosterone levels early in life elicits courtship
differences in behaviour have been observed in wild-
behaviour later in life, well after effects on circulating
caught adult garter snakes from these sites (R. B. King & T.
hormone levels have passed (Crews 1985).
Manipulation of testosterone level had a similar effect
A second clear result of this investigation is the pres-
on males and females (time*sex*treatment effects were
ence of sex differences in strikes but not in latency or
consistently nonsignificant). However, it is unknown
response distance. For all three behaviours, my analyses
whether this effect was a direct result of testosterone
were of sufficient power to detect even small differences
manipulation or an effect mediated through the conver-
between males and females using Cohen’s (1988) effect
sion of testosterone to dihydrotestosterone (DHT) or
size criteria (Table 4). Thus, the absence of sex effects on
oestrogen. Significantly, cells that concentrate testoster-
latency and response distance appears to be biologically
one and oestrogen are equally common in many areas of
meaningful and not simply a result of type II error. The
the brains of male and female garter snakes, including
effect of sex on strikes differs from the family effect
areas such as the amygdala, which is associated with
described above in that it is smaller in magnitude and
limbic responses like defensive behaviour (Halpern et al.
does not include an ontogenetic component: ontogenetic
1982). Further experiments that directly manipulate DHT
changes in strikes among males parallel those among
or oestrogen levels or that manipulate testosterone levels
females (Fig. 1). Sex differences in behaviour have been
in the presence of inhibitors such as 5- -reductase (the
demonstrated in laboratory studies of some neonatal
enzyme responsible for conversion of testosterone to
natricines but not others. Male Butler’s garter snakes, T.
DHT) or aromatase (the enzyme responsible for conver-
, and southern water snakes, Nerodia fasciata
sion of testosterone to oestrogen) would be useful (Moore
more frequently than do females (Scudder & Burghardt
1983; Herzog & Burghardt 1986). In contrast, among the
A fourth result of this investigation is the presence of a
common garter snakes tested here, females struck more
time*family*treatment interaction effect on response dis-
frequently than males. Sex differences are apparently
tance but not on latency to move and strikes: families
lacking in other natricine snakes (e.g. T. melanogaster
responded differently to hormone manipulation for
T. ordinoides, T. radix
, N. cyclopion
, N. rhombifera
, N. sipe-
response distance. This result suggests that variation in
; Scudder & Burghardt 1983; Herzog & Burghardt
response distance among families may be mediated
1986; E. D. Brodie III, unpublished data; A. Queral-Regil &
through receptor- or postreceptor-level phenomena. In
R. B. King, unpublished data; R. B. King & D. Anderson,
contrast, families responded similarly (or not at all) to
unpublished data). However, detection of sex differences
hormone manipulation for latency to move and strikes
may require moderately large sample sizes. In contrast to
although admittedly, my analyses were only of sufficient
the results presented here, an earlier study of 90 common
power to detect large effects using Cohen’s (1988) effect
garter snakes born to seven females failed to reveal any
size criteria (Table 5). Taken at face value, the lack of
difference between males and females (Herzog &
time*family*treatment interaction effects on latency and
Burghardt 1986). Sex differences in diet, foraging behav-
strikes suggests that variation between families in these
iour, spatial patterns and movement have been observed
in snakes in the wild (Gregory et al. 1987; Shine 1991;
Reinert 1993). Whether the sex difference in strikes docu-
among males, families differ in circulating testosterone
mented here and in the studies cited above is related to
levels (R. B. King, J. H. Cline & C. J. Hubbard, unpub-
behavioural differences in nature is unknown.
lished data; see also King et al. 2001), it is possible that
A third result of this investigation is an apparent effect
genetic or maternally induced variation in some garter
of testosterone manipulation on strikes. Although this
snake behaviour (e.g. strikes) is mediated through pro-
effect was relatively small, the difference between treat-
cesses that influence circulating hormone levels (e.g.
ment groups parallels the difference seen between males
responsiveness to environmental stimuli, responsive-
and females. Males (the sex with intrinsically higher
ness to releasing hormones, rate of secretion, androgen
testosterone levels) struck less frequently than did
binding proteins in the plasma, hormone half-life).
females and snakes that received implants containing
Detecting an effect of individual variation in circulat-
testosterone showed a decrease in strike frequency
ing testosterone level on behaviour using the data
relative to sham-manipulated animals. Sexually mono-
gathered in this study is complicated by the fact that
morphic behaviours (latency, response distance) were
testosterone level changes over time, precluding its use as
a covariate in the repeated measures analyses used here to
although, admittedly, small treatment effects may have
test for family, sex and treatment effects. In addition,
gone undetected because my analyses were only of suffi-
testosterone was not assayed in all individuals (e.g. pilot
cient power to detect medium effects using Cohen’s
assays indicated only marginally detectable levels in
(1988) effect size criteria (Table 5). The effect of treatment
sham- and flutamide-treatment females, and insufficient
on strikes was only evident while implants were in place.
blood volume was obtained from some individuals). To
Following implant removal, differences in behaviour and
avoid these problems, I used analysis of covariance
ANIMAL BEHAVIOUR, 63,
(ANOVA) to test for a possible effect of testosterone level,
the course of this investigation (Fig. 1, Table 4). One
sex and family on behaviour among 91 testosterone-
interpretation is that larger (older) snakes are less vulner-
treatment animals from 15 families at 190 days of age
able to predators and thus are less responsive to an
(while implants were in place). I also used ANCOVA to
approaching threat. Temporal changes in latency to move
test for an effect of testosterone level and family on
and strikes may be associated with emergence from simu-
behaviour using data on 73 males from 10 families at 284
lated hibernation. Snakes were slower to move and struck
and 428 days (after implants had been removed and
less frequency at 284 days (1–2 days after emergence)
testosterone had returned to baseline). Covariation
than on earlier test dates (Fig. 1, Table 4). Changes in
between testosterone level and strikes was consistently
behaviour associated with recovery of physiological func-
nonsignificant despite the fact that strikes was the one
tions following emergence from hibernation have been
behaviour that most clearly showed a testosterone treat-
reported in natural populations of garter snakes as well
ment effect. Covariation between testosterone level and
behaviour did achieve statistical significance for response
North American natricine snakes have become a model
distance among testosterone-treatment animals at 190
system in evolutionary quantitative genetics (Brodie &
days of age (P
=0.049) and for latency to move among
Garland 1993; King & Lawson 1995; Arnold & Phillips
males at 284 days (P
=0.041) but not at other times. Thus,
1999). Much previous work with these snakes has focused
evidence that individual variation in testosterone levels
on estimating heritability, genetic correlation and selec-
has detectable effects on behaviour is equivocal at best.
tion on quantitative characters in an effort to assess
However, the results of thus study suggest that future
potential for evolutionary change. These studies have
experiments designed specifically to test for such effects
been groundbreaking in demonstrating how genetic cor-
relations among traits may constrain evolutionary
Garter snakes grew markedly over the course of this
change (Arnold 1988), how combinations of traits (e.g.
investigation, from a mean of 1.6 g at birth to a mean of
behaviour and morphology) can be the target of correla-
19.7 g at 438 days of age. Furthermore, females exceed
tional selection (Brodie 1989), and how gene flow can
males in body size as adults, a difference thought to be
slow adaptive evolution (King & Lawson 1995). In con-
mediated by an inhibitory effect of testosterone on
trast, the work reported here and other recent studies of
growth of males (Crews et al. 1985). Thus, it is possible
natricines have focused on how proximate mechanisms
that the differences in garter snake behaviour among
(e.g. hormonal pathways, maternal effects, experience)
families, sexes and treatments reported here are attribu-
mediate the expression of quantitative traits (Burghardt
table to differences in body size. Several lines of evidence
et al. 2000; King et al. 2001). This emphasis on proximate
suggest that this is not the case (a more detailed analysis
mechanisms is complimentary to an evolutionary quan-
of family, sex and testosterone effects on garter snake
titative genetic approach. One interest in evolutionary
morphology will be presented elsewhere). (1) Neither
quantitative genetics is the relative constancy of genetic
mass nor snout–vent length (SVL) covaried significantly
with behaviour at 39, 190 or 284 days of age. SVL did
covariance matrix, G
) (Arnold & Phillips 1999). A con-
covary significantly with latency to move at 428 days of
simplifies prediction of long-term evolutionary
age (ANOVA with sex, family and treatment as factors,
change but implies that such change is constrained by the
=0.016). (2) Size residuals and behaviour residuals gen-
specific form of G
. Tests for constancy have typically
erated from mutivariate analyses that included latency,
involved comparisons of G
among populations or closely
strikes, response distance and mass or SVL as dependent
related taxa (e.g. Arnold & Phillips 1999). Alternatively,
variables, and sex, family and treatment as factors were
insight into the constancy of G
can come from investiga-
uncorrelated at 39, 190 and 284 days of age. Residual SVL
tions of proximate mechanisms. Studies such as this one
was positively correlated with residual latency at 428 days
can reveal the degree to which suites of traits are influ-
enced by common pathways (e.g. a single hormonal
by this age, larger snakes were slower to move than were
control mechanism) and the ease with which expression
smaller snakes. (3) Differences in body size between males
of such traits might become uncoupled. The observation
and females and between treatment groups, although
that number of strikes is influenced by testosterone levels,
statistically significant, were small. At 438 days, males
whereas latency to move and response distance are
exceeded females in SVL by just 1.4% (358 versus
not, suggests that these behaviours may evolve relatively
353 mm) and females exceeded males in mass by 2.6%
(20.1 versus 19.6 g). At 195 days (immediately following
More generally, this study demonstrates the utility of
implant removal), testosterone-treated animals exceeded
combining quantitative genetic and endocrinological
sham- and flutamide-treated animals in SVL by less than
approaches to the study of behaviour. Evidence is
1% (276 versus 278 mm) and sham- and flutamide-
accumulating for heritable variation in circulating levels
treated animals exceeded testosterone-treated animals in
of a range of hormones and hormone-binding proteins
(e.g. Jaquish et al. 0000; Meikle et al. 1986, 1988a, b;
Although variation in body size does not appear to
Zarazaga et al. 1998). Furthermore, a number of studies
explain the family, sex or treatment effects on behaviour
provide evidence for correlations between hormonal vari-
reported here, increasing body size may have contributed
ation and variation in other phenotypic traits, including
to temporal changes in behaviour. This is especially true
behaviours related to activity, avoidance and aggression
for response distance, which decreased consistently over
(e.g. Glowa et al. 1992; Compaan et al. 1993; Gupta &
GARTER SNAKE BEHAVIOUR
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