Ecologia Mediterranea
Ecologia Mediterranea
Ecologia Mediterranea
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subsequently 12 hours later. During the cold<br />
season, from November to March, the traps<br />
were disarmed at night.<br />
Captured animals were transferred from the<br />
trap to a sealed transparent plastic box, used<br />
as a pre-anaesthetisation chamber, in which<br />
cotton imbibed with ethyl ether was introduced.<br />
Each animal was weighed to evaluate<br />
the exact dose of anaesthetic and intramuscularly<br />
injected with ketamine. Anaesthesia<br />
lasted an average of 10 minutes per animal.<br />
During this period some samples of hair bulb<br />
were taken and immediately kept frozen at<br />
– 80 o C and each captured animal was marked<br />
with an individually numbered transponder<br />
tag (Trovan, UK), inserted subcutaneously<br />
between the shoulder blades.<br />
Genetic data<br />
We used 11 polymorphic microsatellites loci,<br />
isolated from other mustelid species (Table 1)<br />
and tested in weasel by Pertoldi et al. (2006)<br />
and Wang et al. (2002).<br />
DNA was extracted from hair samples using<br />
the ChargeSwitch® Forensic DNA Purification<br />
Kit (Invitrogen, Inc.), validated for DNA<br />
extraction from human hair bulb. DNA amplification<br />
was performed in the following steps:<br />
at 94 o C for 2 min, then 40 cycles of denaturation<br />
at 94 o C for 15 s, annealing at 52.5 o C<br />
for 15 s, polymerization at 72 o C for 15 s, and<br />
final polymerization at 72 o C for 10 min.<br />
Annealing temperature for MVI57 locus was<br />
57.5 o C. The forward (F) primer was fluorescently<br />
labelled with different markers (HEX,<br />
TAMRA, 6-FAM); then PCR products were<br />
analysed by electrophoresis by a DNA AB310<br />
automated sequencer (Applied Biosystems,<br />
Inc.).<br />
ecologia mediterranea – Vol. 38 (2) – 2012<br />
Pilot study of genetic relatedness in a solitary small carnivore, the weasel: implications for kinship and dispersal<br />
Table 1 – Used microsatellite loci and their features.<br />
Allele number and heterozygosity (H) were<br />
calculated using the program GIMLET<br />
v.1.3.3. (Valiere 2002; http://pbil.univlyon1.fr/software/gimlet).<br />
Deviations from Hardy-Weinberg equilibrium<br />
were tested using the program GENEPOP v.4.<br />
(Raymond and Rousset 1995;<br />
http://kimura.univmontp2.fr/~rousset/genepop).<br />
Bonferroni corrections for multiple comparisons<br />
were used to find critical significance<br />
levels for both locus MLUT20 and MER022<br />
(equivalent to p ≤ 0.05). Finally, genetic kinship<br />
among animals was analyzed using the<br />
program KINGROUP v.2.0.8.+ (Konovalov et<br />
al. 2004; http://code.google.com/p/kingroup).<br />
We tested two kinds of first order of relatedness<br />
(primary hypothesis): parent/offspring<br />
and full siblings versus the null hypothesis of<br />
unrelatedness, using the likelihood formulas<br />
of Goodnight and Queller (1999).<br />
Results<br />
During the study, 14 weasels were captured<br />
(11 males and 3 females).<br />
All the 11 analyzed microsatellite loci were<br />
found polymorphic, with a variable number<br />
of alleles (Table 1). The average number of<br />
alleles per locus was 5.36.<br />
Mean observed (Ho) and expected (He) heterozygosity<br />
for the 11 loci within the studied<br />
population (N=14), were 0.46 and 0.62<br />
respectively (Table 1), ranging from 0.07 to<br />
0.93 for Ho and from 0.07 to 0.85 for He.<br />
After Bonferroni corrections, comparison<br />
between Ho and He for both locus MLUT20<br />
and MER022 showed p values ≥ 0.05. So no<br />
significant deviations from Hardy-Weinberg<br />
Source species Locus primer name Reference N alleles P value Ho He<br />
Mustela lutreola MLUT20 Cabria et al. 2007 9 0,02* 0,64 0,83<br />
MLUT25 Cabria et al. 2007 2 - 0,07 0,07<br />
MLUT27 Cabria et al. 2007 6 0,83 0,71 0,63<br />
MLUT35 Cabria et al. 2007 10 0,07 0,93 0,85<br />
Meles meles MEL1 Bijlsma et al. 2000 8 0,83 0,71 0,75<br />
Mustela erminea MER009 Fleming et al. 1999 5 0,11 0,50 0,66<br />
MER022 Fleming et al. 1999 4 0,01* 0,57 0,70<br />
MER041 Fleming et al. 1999 4 0,17 0,43 0,46<br />
Gulo gulo GGU216 Duffy et al. 1998 4 0,29 0,14 0,64<br />
GGU234 Duffy et al. 1998 5 0,34 0,19 0,76<br />
Mustela vison MVI057 O’Connell et al.1996 2 1,00 0,19 0,50<br />
* Means that Bonferroni correction was applied.<br />
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