Reichard, D. G., O'Neal, D. M., and Ketterson, E. D.

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Indiana University Bloomington 
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Ketterson / Nolan Research Group Collection 
This document is part of a collection that serves two purposes. First it is a public archive for data and 
documents resulting from evolutionary, ecological, and behavioral research conducted by the Ketterson- 
Nolan research group. The focus of the research is an abundant North American songbird, the dark-eyed 
junco, Junco hyemalis, and the primary sources of support have been the National Science Foundation and 
Indiana University. The research was conducted in collaboration with numerous colleagues and students, 
and the objective of this site is to preserve not only the published products of the research, but also to 
document the organization and people that led to the published findings. Second it is a repository for the 
works of Val Nolan Jr., who studied songbirds in addition to the junco: in particular the prairie warbler, 
Dendroica discolor. This site was originally compiled and organized by Eric Snajdr, Nicole Gerlach, and Ellen 
Ketterson. 
Context Statement  
This document was generated as part of a long-term biological research project on a 
songbird, the dark-eyed junco, conducted by the Ketterson/Nolan research group at Indiana University. For 
more information, please see IUScholarWorks (https://scholarworks.iu.edu/dspace/handle/2022/7911). 
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you agree to the license terms outlined at https://scholarworks.iu.edu/dspace/
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For additional information, visit the Ketterson/Nolan Lab community on the IUScholarWorks repository
Behavioral Consequences of Elevated Testosterone in 
Female Dark-Eyed Juncos (Junco hyemalis) 
Dustin G. Reichard1, Dawn M. O’Neal2, and Ellen D. Ketterson2 
1St. Mary’s College of Maryland, St. Mary’s City, MD 
2Indiana University, Bloomington, IN 
Through experimental elevation of plasma testosterone (T), novel phenotypes can be created and tested for relative 
fecundity and viability and thus for likelihood of being favored by natural selection. Previous studies have shown that 
elevated T in male dark-eyed juncos (Junco hyemalis) results in greater fitness due to increased mating success, 
despite reductions in parental care. The constraint hypothesis predicts that this fitness gain might be offset by 
deleterious effects of elevated T in females, which may explain why high T males have not evolved. In the female dark-
eyed junco, previous studies have suggested that females are behaviorally insensitive to T during the incubation stage, 
which argues against the constraint hypothesis. In this study, in an effort to determine potential fitness consequences 
associated with elevated T, we asked whether experimentally elevated T affects maternal aggression towards 
conspecifics during incubation or brooding of nestlings. Incubating females implanted with T-filled or empty implants 
were presented with a conspecific male at the nest, and attack intensity was measured for fifteen minutes. Brooding 
behavior of females and male provisioning were observed for three hours when nestlings were three days old. 
Increased T had no effect on maternal aggression, providing additional support for female insensitivity to T during 
incubation. Conversely, high T females spent less time on the nest and spent less time brooding than control females, 
suggesting that aspects of female parental behavior are sensitive to T during the nestling stage. Further study will 
show whether these aspects might prove detrimental and conceivably constrain the evolution of T in females or males.  
ABSTRACT 
INTRODUCTION RESULTS 
-Experimentally elevated T in 
male dark-eyed juncos  
reduces parental care, but  
increases overall fitness due to 
extra pair fertilizations.1   
-Why are we not seeing 
selection for higher male T?   
-If elevated T results in a fitness 
loss in females, then females 
could constrain high T levels in 
males. 2   
-Alternatively, female traits 
ordinarily sensitive to 
androgens could be insensitive 
if the fitness costs associated 
with sensitivity are high.3 
-What effect might high T have 
on maternal aggression and 
nestling brooding? 
-No significant effect of high T on 
maternal aggression during 
conspecific nest intrusions  
-High T females spent 
significantly less time on the nest 
than control females (p<0.05) 
(Mean Diff. – 10.00 minutes) 
-High T females spent 
significantly less time brooding 
than control females (p<0.05) 
(Mean Diff. – 8.49 minutes) 
-No significant difference in 
female provisioning rate between 
treatments (p>0.05) 
-Males mated to high T females 
provisioned young significantly 
more often than males mated to 
control females (p<0.05) (Mean 
Diff. – 0.90 provisions/hr) 
 
METHODS 
-89 females received 5 mm 
subcutaneous silastic implant of 
crystalline T or an empty implant 
-After clutch complete, simulated 
nest intrusion with caged 
conspecific male for 15 minutes, 
recorded male and female 
proximity to the cage (1m and 
5m) and aggressive behaviors 
(dives and hits)  
-Day 3 after hatching, nest 
videotaped for 3 hours and 
brooding and provisioning rates 
scored 
S
Q
R
T 
(T
im
e 
(m
in
))
 C-females 
T-females 
0.00 
1.00 
2.00 
3.00 
4.00 
5.00 
6.00 
7.00 
1m 5m On Nest 
Proximity to Intruder (m)  
T-females=15; C-Females=19 
Female Response to Male Intruder  
DISCUSSION 
-This study provides the first 
evidence of female sensitivity to 
increased plasma testosterone in 
relation to parental behavior.  
-Previous research done during 
the egg incubation stage 
supported female insensitivity to 
high T.4,5   
-Results from the nest intrusions 
suggest that maternal aggression 
is not mediated by testosterone. 
-Male’s increased provisioning 
rate may counteract negative 
effects of reduced female 
brooding.   
-Results are consistent with the 
constraint hypothesis. More data 
needed on nestling growth rates, 
survivorship, and fitness of 
offspring as adults.   
LITERATURE CITED 
1Reed, W.L. et al. 2006. American Naturalist. 167. 
2,4Clotfelter, E.D. et al. 2004. Hormones and Behavior. 
46:171-17 
3,5Lynn, S.E.. et al. 2004. Hormones and Behavior. 47: 
170-177. 
Female Time On Nest 
1.90 
2.00 
2.10 
2.20 
2.30 
2.40 
2.50 
2.60 
2.70 
Treatment 
To
ta
l T
im
e 
O
n 
N
es
t (
hr
s)
 T-Females 
C-Females 
* 
n=10 n=15 1.80 
1.90 
2.00 
2.10 
2.20 
2.30 
2.40 
2.50 
Ti
m
e 
S
pe
nt
 B
ro
od
in
g 
(h
rs
) 
Female Brooding 
Treatment 
T-Females 
C-Females 
* 
n=10 n=15 
Female Provisioning (Day 3 Young) 
0.00 
0.50 
1.00 
1.50 
2.00 
2.50 
3.00 
Treatment 
N
es
tli
ng
 fe
ed
in
gs
/h
ou
r 
T-Females 
C-Females 
n=10 n=15 
0.00 
0.50 
1.00 
1.50 
2.00 
2.50 
3.00 
3.50 
4.00 
N
es
tli
ng
 fe
ed
in
gs
/ h
ou
r 
Male Provisioning (Day 3 Young) 
Female Treatment 
T-Mates 
C-Mates * 
n=10 n=15 
Proximity to Intruder (m) 
T-females=15; C-females=19 
T-Mates 
C-Mates 
0.00 
0.20 
0.40 
0.60 
0.80 
1.00 
1.20 
1.40 
1.60 
1.80 
1m 5m 
S
Q
R
T 
(T
im
e 
(m
in
))
 
Male Response to Male Intruder 
HYPOTHESES 
-T-females will exhibit increased 
aggression towards 
conspecifics at the nest to 
correlate with increased 
aggression in captive studies 
-As the primary caregiver, 
females will remain insensitive 
to testosterone during the 
nestling stage. 
Behavioral Consequences of Elevated Testosterone in 
Female Dark-Eyed Juncos (Junco hyemalis) 
Dustin G. Reichard1, Dawn M. O’Neal2, and Ellen D. Ketterson2 
1St. Mary’s College of Maryland, St. Mary’s City, MD 
2Indiana University, Bloomington, IN 
Through experimental elevation of plasma testosterone (T), novel phenotypes can be created and tested for relative 
fecundity and viability and thus for likelihood of being favored by natural selection. Previous studies have shown that 
elevated T in male dark-eyed juncos (Junco hyemalis) results in greater fitness due to increased mating success, 
despite reductions in parental care. The constraint hypothesis predicts that this fitness gain might be offset by 
deleterious effects of elevated T in females, which may explain why high T males have not evolved. In the female dark-
eyed junco, previous studies have suggested that females are behaviorally insensitive to T during the incubation stage, 
which argues against the constraint hypothesis. In this study, in an effort to determine potential fitness consequences 
associated with elevated T, we asked whether experimentally elevated T affects maternal aggression towards 
conspecifics during incubation or brooding of nestlings. Incubating females implanted with T-filled or empty implants 
were presented with a conspecific male at the nest, and attack intensity was measured for fifteen minutes. Brooding 
behavior of females and male provisioning were observed for three hours when nestlings were three days old. 
Increased T had no effect on maternal aggression, providing additional support for female insensitivity to T during 
incubation. Conversely, high T females spent less time on the nest and spent less time brooding than control females, 
suggesting that aspects of female parental behavior are sensitive to T during the nestling stage. Further study will 
show whether these aspects might prove detrimental and conceivably constrain the evolution of T in females or males.  
ABSTRACT 
INTRODUCTION RESULTS 
-Experimentally elevated T in 
male dark-eyed juncos  
reduces parental care, but  
increases overall fitness due to 
extra pair fertilizations.1   
-Why are we not seeing 
selection for higher male T?   
-If elevated T results in a fitness 
loss in females, then females 
could constrain high T levels in 
males. 2   
-Alternatively, female traits 
ordinarily sensitive to 
androgens could be insensitive 
if the fitness costs associated 
with sensitivity are high.3 
-What effect might high T have 
on maternal aggression and 
nestling brooding? 
-No significant effect of high T on 
maternal aggression during 
conspecific nest intrusions  
-High T females spent 
significantly less time on the nest 
than control females (p<0.05) 
(Mean Diff. – 10.00 minutes) 
-High T females spent 
significantly less time brooding 
than control females (p<0.05) 
(Mean Diff. – 8.49 minutes) 
-No significant difference in 
female provisioning rate between 
treatments (p>0.05) 
-Males mated to high T females 
provisioned young significantly 
more often than males mated to 
control females (p<0.05) (Mean 
Diff. – 0.90 provisions/hr) 
 
METHODS 
-89 females received 5 mm 
subcutaneous silastic implant of 
crystalline T or an empty implant 
-After clutch complete, simulated 
nest intrusion with caged 
conspecific male for 15 minutes, 
recorded male and female 
proximity to the cage (1m and 
5m) and aggressive behaviors 
(dives and hits)  
-Day 3 after hatching, nest 
videotaped for 3 hours and 
brooding and provisioning rates 
scored 
S
Q
R
T 
(T
im
e 
(m
in
))
 C-females 
T-females 
0.00 
1.00 
2.00 
3.00 
4.00 
5.00 
6.00 
7.00 
1m 5m On Nest 
Proximity to Intruder (m)  
T-females=15; C-Females=19 
Female Response to Male Intruder  
DISCUSSION 
-This study provides the first 
evidence of female sensitivity to 
increased plasma testosterone in 
relation to parental behavior.  
-Previous research done during 
the egg incubation stage 
supported female insensitivity to 
high T.4,5   
-Results from the nest intrusions 
suggest that maternal aggression 
is not mediated by testosterone. 
-Male’s increased provisioning 
rate may counteract negative 
effects of reduced female 
brooding.   
-Results are consistent with the 
constraint hypothesis. More data 
needed on nestling growth rates, 
survivorship, and fitness of 
offspring as adults.   
LITERATURE CITED 
1Reed, W.L. et al. 2006. American Naturalist. 167. 
2,4Clotfelter, E.D. et al. 2004. Hormones and Behavior. 
46:171-17 
3,5Lynn, S.E.. et al. 2004. Hormones and Behavior. 47: 
170-177. 
Female Time On Nest 
1.90 
2.00 
2.10 
2.20 
2.30 
2.40 
2.50 
2.60 
2.70 
Treatment 
To
ta
l T
im
e 
O
n 
N
es
t (
hr
s)
 T-Females 
C-Females 
* 
n=10 n=15 1.80 
1.90 
2.00 
2.10 
2.20 
2.30 
2.40 
2.50 
Ti
m
e 
S
pe
nt
 B
ro
od
in
g 
(h
rs
) 
Female Brooding 
Treatment 
T-Females 
C-Females 
* 
n=10 n=15 
Female Provisioning (Day 3 Young) 
0.00 
0.50 
1.00 
1.50 
2.00 
2.50 
3.00 
Treatment 
N
es
tli
ng
 fe
ed
in
gs
/h
ou
r 
T-Females 
C-Females 
n=10 n=15 
0.00 
0.50 
1.00 
1.50 
2.00 
2.50 
3.00 
3.50 
4.00 
N
es
tli
ng
 fe
ed
in
gs
/ h
ou
r 
Male Provisioning (Day 3 Young) 
Female Treatment 
T-Mates 
C-Mates * 
n=10 n=15 
Proximity to Intruder (m) 
T-females=15; C-females=19 
T-Mates 
C-Mates 
0.00 
0.20 
0.40 
0.60 
0.80 
1.00 
1.20 
1.40 
1.60 
1.80 
1m 5m 
S
Q
R
T 
(T
im
e 
(m
in
))
 
Male Response to Male Intruder 
HYPOTHESES 
-T-females will exhibit increased 
aggression towards 
conspecifics at the nest to 
correlate with increased 
aggression in captive studies 
-As the primary caregiver, 
females will remain insensitive 
to testosterone during the 
nestling stage. 


Behavioral consequences of elevated testosterone in female dark-eyed juncos (Junco hyemalis)

https://scholarworks.iu.edu/dspace/bitstream/2022/15379/1/17%20Reichard2007SICB.pdf

Behavioral consequences of elevated testosterone in female dark-eyed juncos (Junco hyemalis)

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