Methodological supplement




Дата канвертавання25.04.2016
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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-year-old 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 high- and 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

N=52


high-altitude

N=30


yearlings

N=37


adults

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/post-capture 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

F1,24=2.5

0.1

time within 5m

F1,24=3.2

0.09

agitation

F1,24=0.01

0.9

attacks (yes, no)

F1,24=0.2

0.6

song (yes, no)

F1,24=0.8

0.4

STI duration

F1,26=4.4

0.05

attacks (yes, no)

F1,26=1.9

0.2

STI duration*attacks

F1,26=8.1

0.009


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