Electronic supplementary material for Apfelbeck and Goymann “Ignoring the
challenge? Male black redstarts (Phoenicurus ochruros) do not increase
testosterone levels during territorial conflicts but they do so in response to GnRH”
Methodological supplement: Extraction of ethanol-plasma samples with
dichloromethane
Sample tubes containing a known amount of plasma dissolved in ethanol were
vortexed, then centrifuged (10.000 rpm) for 10 minutes and the supernatant
transferred to extraction vials. 500µl ddH2O was added to the original sample
tubes, and centrifuged again (10.000 rpm, 10 minutes). Then, the supernatant
was transferred to the extraction vials containing the plasma/ethanol mix. The
addition of water to the ethanol/plasma mixture is necessary to separate the
organic from the aqueous phase during the extraction with dichloromethane.
Then, samples were extracted with dichloromethane (DCM) after overnight
equilibration (4°C) of the plasma/ethanol/water mix with 1500 dpm of tritiated
testosterone (Perkin Elmer, Wellesley, MA, USA). The organic phase was then
separated from the aqueous phase by plunging the extraction tubes into a
methanol-dry ice bath and decanting the DCM phase into a new vial. This
extraction step was repeated to increase extraction efficiency. Then, the DCM
phase was dried under a stream of nitrogen at 40°C, samples resuspended in
phosphate buffered saline with 1 % gelatine (PBSG) and left overnight at 4°C to
equilibrate. Then we proceeded with the RIA as described in the methods
section.
Supplementary results
Morphological differences between the populations
Males of the high-altitude population were significantly heavier (F1,67=10.6,
p=0.002, Tab. S1) and had significantly longer wings (F1,68=12.3, p=0.0008, Tab.
S1) than males of the low-altitude population. Tarsus length did not differ
significantly between the two populations (F1,68=3.3, p=0.07, Tab. S1). One-yearold males weighed significantly less than adult-plumaged males (≥2 years;
F1,67=7.8, p=0.007, Tab. S1) and had significantly shorter wings (F1,68=28.4,
p<0.0001, Tab. S1). Tarsus length was not related to age (F1,68=2.5, p=0.1, Tab.
S1).
CP volume was not significantly different between adult-plumaged and
one-year-old males (F1,66=2.7, p=0.1, Tab. S1), or between males of the highand the low-altitude population (F1,66=3.2, p=0.08, Tab. S1). During the parental
phase CP volume tended to be larger than during the early breeding phase
(F1,66=3.9, p=0.05; territory establishment: 148 ± 25 mm3, parental phase:157 ±
11 mm3).
Relative increase in testosterone
As there is no general agreement on what is more biologically relevant, the
absolute increase in testosterone or the relative increase in testosterone, we
calculated general linear models with the relative increase in testosterone
(GnRH-induced / post-capture testosterone levels) as dependent variable (Tab.
S2) in addition to the absolute increase in testosterone (see manuscript).
Table S1. Morphological measurements in relation to altitude and age (means ±
95% CI).
low-altitude
high-altitude
yearlings
adults
N=52
N=30
N=37
N=45
body mass (g)
16.4g ± 0.2
17.0 ± 0.3
16.3 ± 0.3
16.9 ± 0.3
tarsus length (mm)
23.2 ± 0.2
23.4 ± 0.3
23.2 ± 0.2
23.4 ± 0.2
wing length (mm)
87 ± 1
88 ± 1
86 ± 1
88 ± 1
CP volume (mm3)
144 ± 16
166 ± 20
142 ± 18
163 ± 18
Table S2. Relative increase in testosterone (GnRH-induced testosterone/postcapture testosterone) in relation to the experimental factors and the behavioural
variables. Significant results are highlighted in bold.
Test statistic
p-value
Both populations
treatment
F1,66=0.6
0.4
breeding stage
F1,66=1.3
0.3
Location
F1,66=0.8
0.4
Age
F1,66=1.2
0.3
Low altitude
population
treatment
F1,49=0.2
0.7
Stage
F2,49=6.2
0.004
early-parental
t=-1.2 (df=49)
0.2
early-autumn
t=-2.6 (df=49)
0.01
Behaviour
approach
time within 5m
agitation
attacks (yes, no)
song (yes, no)
STI duration
attacks (yes, no)
STI
duration*attacks
F1,24=2.5
F1,24=3.2
F1,24=0.01
F1,24=0.2
F1,24=0.8
F1,26=4.4
F1,26=1.9
F1,26=8.1
0.1
0.09
0.9
0.6
0.4
0.05
0.2
0.009