Ecologia Mediterranea

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