A non-invasive genetic survey of the Pine marten ( Martes martes ) in the western river Po plain (italy): preliminary results
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ABSTRACT: An hair trapping protocol, with camera trapping surveillance, was carried out on the south-western side of the Etna, inhabited by an abundant population of the European wildcat. We aimed to collect hair for genetic analysis on the base of a field study conducted in Switzerland, where valerian tincture had been used to attract wildcats to rub again wooden sticks and therefore leaving hairs. We placed 18 hair trapping stations, plus one camera trap per scented wooden stick, 1 km away from each other for 60 days (October 29 2010 to December 28 2010). The rate of "capture" success (1 capture / 24.5 trap-days) by camera trapping was substantially the same as those obtained during previous surveys performed in the same study area without the use of any attractants. No wildcats were photographed while rubbing against the wooden sticks, neither any wildcat was interested in the scent lure. We discuss limitations of the hair trapping, providing possible explanations on the failure of valerian tincture, while suggesting some field advices for future monitorings.Hystrix 07/2012; · 0.35 Impact Factor
- Acta theriologica 07/2011; 56(3):199-207. · 0.95 Impact Factor
Hystrix It. J. Mamm. (n.s.) 19 (1) (2008): 77-80
A NON-INVASIVE GENETIC SURVEY OF THE PINE
MARTEN (MARTES MARTES) IN THE WESTERN RIVER PO
PLAIN (ITALY): PRELIMINARY RESULTS
ALESSANDRO BALESTRIERI1, ARITZ RUIZ-GONZÁLEZ2, LUIGI RE-
MONTI1, BENJAMÍN J. GÓMEZ-MOLINER2, SARA GENOVESE1,
LAURA GOLA3, CLAUDIO PRIGIONI1*
1Dipartimento di Biologia Animale, Università di Pavia, Piazza Botta, 9 27100 Pavia;
*Corresponding author, e-mail: firstname.lastname@example.org
2Department of Zoology and Animal Cell Biology, Zoology Laboratory, Facultad de
Farmacia, Universidad del País Vasco (UPV-EHU), C/ Paseo de la Universidad 7
01006 Vitoria, Spain
3Parco Fluviale del Po e dell'Orba, tratto Vercellese-Alessandrino, Piazza
Giovanni XXIII 6, 15048 Valenza (AL)
Received 3 March 2008; accepted 16 June 2008
RIASSUNTO - Monitoraggio genetico non invasivo della martora (Martes martes) nella
pianura padana occidentale: risultati preliminari. Tra il 2003 e il 2004, due esemplari di-
martora (Martes martes) sono stati investiti da automezzi sulla sponda sinistra del fiume Po
(R. N. Garzaia di Valenza, Piemonte). Per accertare la presenza stabile della specie nella
Riserva e lungo il tratto vercellese-alessandrino del Parco fluviale del Po, è stato applicato
un metodo PCR-RFLP per l’analisi di mtDNA di origine fecale. A partire da gennaio 2007,
sono state raccolte 212 feci attribuibili al genere Martes in base a forma e dimensioni.
L’analisi di un campione di 117 feci (55%) ha permesso di identificare 10 feci di martora e
15 di faina (M. foina), confermando la presenza della martora nella Riserva e accertandola
per la prima volta in un’area collinare in sponda destra del Po (Camino, AL), circa 32 km a
monte della Riserva. Resta da valutare se la scarsa resa dell’amplificazione del DNA
(21,4%) sia dovuta alla degradazione del materiale genetico o ad un numero elevato di feci
di volpe (Vulpes vulpes) erroneamente attribuite a Martes durante la fase di raccolta.
Parole chiave: PCR-RFLP, Martes foina, species identification, faecal DNA
The Italian range of the pine marten
(Martes martes) includes the Alps, the
Apennines, Sardinia, Sicily and the Isle of
Elba, but little is known about numbers and
trend of its populations (Genovesi and De
The pine marten is believed to occur pri-
marily in well structured forest habitats
(Buskirk, 1992), but in southern Europe it
has shown great ecological plasticity, hav-
ing been reported in coppices, the Mediter-
ranean maquis and cultivated land with
woodland fragments (De Marinis and Mas-
seti, 1993; Pittiglio, 1996). In northern Ita-
ly, the pine marten is mainly associated
with deciduous and coniferous forests be-
tween 1000 and 2000 m a.s.l. (Martinoli,
2001), but in the last ten years it has been
reported also for several localities of the
western River Po plain (NW Italy), where
road kills have highlighted the presence of
this mustelid in both arable land and urban
Balestrieri et al.
surroundings. The penetration of the pine
marten into agricultural areas probably fol-
lows the main watercourses, which still of-
fer patches of semi-natural woodland
(Balestrieri et al., 2008).
Road kills are unpredictable events which,
if regularly filed over long periods, can
help to draw a large-scale picture of species
distribution, but are ineffective when trying
to examine their actual range and habitat
preferences at a local scale. On the con-
trary, the systematic survey of field signs,
mainly faeces, can yield reliable results
about the distribution, abundance and habi-
tat requirements of carnivores (Sadlier et
The pine marten lives sympatrically across
a large part of Europe with the closely re-
lated stone marten (Martes foina), which is
quite similar in terms of morphology and
feeding habits (Marchesi et al., 1989).
The monitoring of European pine marten
populations is hindered by our inability to
distinguish the faeces from the two species,
making indirect survey methods unreliable.
Moreover martens’ faeces can be confused
with those of other carnivores, such as, for
northern Italy, the red fox Vulpes vulpes
and the polecat Mustela putorius (Davison
et al., 2002).
Nonetheless, recent progress in molecular
techniques has supplied several non-
invasive genetic methods for species identi-
fication (review in Ruiz-González et al.,
2008) which can be applied to field moni-
In February 2003 and March 2004, two
pine martens were killed by cars inside the
Natural Reserve "Garzaia di Valenza", a
12.3 km2 wide protected area on the left
bank of the River Po, included in the River
Po Park (SE Piedmont, NW Italy). The
whole territory is flat, mainly cultivated.
Small wood patches border an abandoned
river meander and three naturalized artifi-
To determine the stable presence of the spe-
cies in the Reserve and investigate its dis-
tribution along the western River Po plain
we applied a polymerase chain reaction -
restriction fragment length polymorphism
(PCR-RFLP) method specifically designed
for distinguishing between M. martes and
M. foina by the analysis of faecal mito-
chondrial deoxyribonucleic acid (mtDNA;
Ruiz-González et al., 2008).
Since January 2007, a total of 212 “marten-
like” faeces has been collected through
weekly surveys along fixed transects. Dur-
ing the first three months, eight areas were
surveyed (Fig. 1); successively, the moni-
toring was limited to the two areas regu-
larly providing some faecal samples, the
above cited Reserve and a hilly area on the
right bank of the river, some 32 km up-
stream (included between the towns of
Camino and Pontestura, Alessandria prov-
Figure 1 - Study area (River Po Park) with
the eight sampling stations (grey dots: posi-
Faecal samples were initially assigned to
the genus Martes if less than 10 mm large
and to the red fox when larger than 15 mm.
Samples with intermediate width were at-
tributed on the basis of their overall ap-
A portion (about 30%) of each “marten-
like” faecal sample was picked up with
Martes genetic survey
sticks and preserved in 96% ethanol and by
freezing until DNA extraction, the rest was
retained for dietary analysis. The UTM co-
ordinates of each sample were filed and
projected onto a GIS (Arcview 3.1 ESRI).
The faecal mtDNA extraction procedure
was based on the protocol described by
Gómez-Moliner et al. (2004). Two specifi-
cally designed primers were used, which
generate 276 bp long amplicons. These
primers amplify the DNA from the two
Martes species and from the other four
Mustela species, whilst the DNA from
foxes or martens’ prey render no amplicons
(except for 900 bp long amplicons from
Sorex spp.). The simultaneous use of the
restriction enzymes RsaI and HaeIII differ-
entiated M. martes from M. foina and them
both from the other carnivore species
whose DNA is amplified by the selected
primers (see Ruiz-González et al., 2008 for
The genetic analysis of a sub-sample of
117 faeces (55%) has yielded 25 identifica-
tions, corresponding to 10 M. martes scats
and 15 M. foina scats. The presence of the
pine marten has been confirmed in the Re-
serve and ascertained for the first time in
the Camino-Pontestura area.
The rate of success of DNA amplification
was 21.4%, a much lower rate than that ob-
tained in a previous survey carried out in
the northern Iberian peninsula by the same
PCR-RFLP method (88%; Ruiz-González
et al., 2008).
Further analyses are needed to understand
if these results depend on overlong interval
between consecutive surveys - causing
DNA degradation in the older faeces -, an
overlong storage period of the samples be-
fore analysis - which could increase the
probability of DNA degradation -, a high
rate of faeces misidentification - as re-
ported for low density pine marten popula-
tions in Great Britain (Davison et al., 2002)
-, or a combination of these factors.
In man-made habitats, the residual patches
of semi-natural vegetation provide cover,
suitable den-sites and food to many spe-
cies, particularly elusive carnivores. Until a
few years ago, in the study area four me-
dium-size carnivores had been reported, the
red fox, the stone marten, the polecat and
the badger (Meles meles). The latter mainly
rely on earthworms and maize (Balestrieri
et al., 2004), whilst little is known about
the polecat, the presence of which has not
been confirmed by our analyses; the diet of
the red fox and Martes species widely
overlap, probably as a consequence of the
low trophic diversity of the agricultural ar-
eas in the plain (authors’ unpublished data).
The two Martes species are considered to
compete for the same resources (Delibes,
1983), even if they can co-exist in many
areas (Genovesi, 1993; Ruiz-González et
al., 2008), exploiting the same microhabi-
tats (Posluszny et al., 2007). In the study
area, according to their body weight ratio,
the fox is likely to play the role of the
dominant species (Prigioni et al., 2008),
whilst the stone marten should be the least
competitive (Hermann, 1994). We argue
that, in conditions of food shortage, the en-
try of the pine marten could alter the exist-
ing ecological relationship within the car-
nivore guild to the detriment of the stone
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