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First record of amphibian mortality associated with the fungus Batrachochytrium dendrobatidis in Catalonia (NE Spain)

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  • CRARC (Catalonian Reptiles and Amphibians Rescue Center). Masquefa. Barcelona. Spain

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We report the first cases of mortality in anurans associated with the presence of the fungus Batra-chochytrium dendrobatidis at four different localities from Catalonia (NE Iberian Peninsula, Spain). All cases were confirmed by both molecular techniques and histology. The infected individuals were two Mediterranean painted frogs (Discoglossus pictus) from Girona province found in 2018 and 2020, one Iberian waterfrog (Pelophylax perezi) from Tarragona province found in 2018, and one European common frog (Rana temporaria) from Barcelona province found in 2019. This is the first time that mortality associated with this pathogen has been confirmed in D. pictus and P. perezi. The role of the fungus as an agent possibly leading to death, in association with other external environmental factors, is discussed. These findings could suggest a recent increase of the incidence of this disease in the region. Some of these cases are particularly worrying because of their occurrence close to sites where some endemic amphibian species with extremely reduced distributions inhabit .
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Basic and Applied Herpetology 35 (2021) 000-000
DOI: https://dx.doi.org/10.11160/bah.210
CITE THIS ARTCILE AS “IN PRESS”
First record of amphibian mortality associated with the
fungus
Batrachochytrium dendrobatidis
in Catalonia
(NE Spain)
Albert Martinez-Silvestre1,*, Roser Velarde2, Rachel E. Marschang3, Iago Pérez-Novo4,
Josep F. Bisbal-Chinesta5,6, Barbora Thumsová7,8, Jaime Bosch7,9
1 Catalonian Reptiles and Amphibians Rescue Center (CRARC). 08783 Masquefa, Barcelona, Spain.
2 Wildlife Ecology & Health group (WEH) and Servei d'Ecopatologia de Fauna Salvatge (SEFaS). Departa-
ment de Medicina i Cirurgia Animals, Universitat Autònoma de Barcelona, Bellaterra, Spain.
3 LABOKLIN GmbH and Co. KG, 97688 Bad Kissingen, Germany.
4 Societat Catalana d’Herpetologia (SCH). Plaça Leonardo da Vinci, 4-5, Parc del Fòrum, 08019 Barcelona,
Spain.
5 Institut Català de Paleoecologia Humana i Evolució Social (IPHES). Edifici W3, Zona Educacional 4, Cam-
pus Sescelades, Universitat Rovira i Virgili, 43007 Tarragona, Spain.
6 Associació Herpetològica Timon (AHT). Carrer València 32, 46195, Llombai, Spain.
7 Museo Nacional de Ciencias Naturales-CSIC. José Gutiérrez Abascal 2, 28006 Madrid, Spain.
8 Asociación Herpetológica Española (AHE), José Gutiérrez Abascal 2, 28006 Madrid, Spain.
9 Research Unit of Biodiversity (CSIC, University of Oviedo-Principality of Asturias). Gonzalo Gutiérrez
Quirós s/n; Universidad de Oviedo, Edificio de Investigación. 33600 Mieres, Spain.
*Correspondence: E-mail: crarc@amasquefa.com
Received: 08 January 2021; returned for review: 25 March 2021; accepted: 29 May 2021.
We report the rst cases of mortality in anurans associated with the presence of the fungus Batra-
chochytrium dendrobatidis at four dierent localities from Catalonia (NE Iberian Peninsula, Spain).
All cases were conrmed by both molecular techniques and histology. The infected individuals
were two Mediterranean painted frogs (Discoglossus pictus) from Girona province found in 2018
and 2020, one Iberian waterfrog (Pelophylax perezi) from Tarragona province found in 2018, and
one European common frog (Rana temporaria) from Barcelona province found in 2019. This is the
rst time that mortality associated with this pathogen has been conrmed in D. pictus and P. perezi.
The role of the fungus as an agent possibly leading to death, in association with other external en-
vironmental factors, is discussed. These ndings could suggest a recent increase of the incidence of
this disease in the region. Some of these cases are particularly worrying because of their occurrence
close to sites where some endemic amphibian species with extremely reduced distributions inhab-
it.
Key words: Catalonia; chytridiomycosis; Discoglossus pictus; oubreak; Pelophylax perezi; Rana tempo-
raria.
Batrachochytrium dendrobatidis (hereafter
Bd) is currently found in all continents
where amphibians occur (Skerratt et al.,
2007) and is considered a major threat to
amphibian biodiversity worldwide (Olson
et al., 2013). However, this pathogen can be
present in the environment and on the
skin of amphibians without causing fatal
disease (Lips, 2016). In this case, infected
amphibians are considered asymptomatic
MARTINEZ-SILVESTRE ET AL.
2
carriers able to spread the pathogen to oth-
er more vulnerable species (e.g. Hansel-
mann et al., 2004). The presence of asymp-
tomatic Bd carriers is widely reported in
the literature, also within the region of
Catalonia in NE Iberian Peninsula (e.g.
BargallÓ et al., 2016; Martínez-Silvestre
et al., 2019, 2020). The rst documented Bd
infection in wild amphibian populations in
Europe dates from 1997 (Bosch et al.,
2001), when mass mortality episodes of
common midwife toads (Alytes obstetri-
cans) occurred in the Peñalara protected
area in central Spain. Additional mass
mortalities in the 2000’s were also reported
in Spain, including outbreaks in re sala-
manders (Salamandra salamandra) and
spiny toads (Bufo spinosus) from the same
location (Bosch et al., 2001; Bosch Mar-
tínez-Solano, 2006). Apart from Peñalara,
mortalities have also been detected in oth-
er montane areas of Spain, including the
Pyrenees, the Cantabrian Range (Walker
et al., 2010), and the Tramuntana Range in
Majorca island (Walker et al., 2008). Inter-
estingly, Walker et al. (2010) found an in-
creased Bd prevalence along an east-west
gradient in northern Iberia, with the east-
ern Pyrenees and the entire region of Cata-
lonia remaining free of mass mortalities
associated with Bd.
Between 2016 and 2019, following an
active health surveillance plan, numerous
routine samplings were carried out in sev-
eral areas of Catalonia. These studies led
to detection of numerous Bd infections
distributed throughout this territory, alt-
hough none of them was associated with
any mortality episode (BargallÓ et al.,
2016; Martínez-Silvestre et al., 2017, 2019,
2020; Montori et al., 2019). As a result of
this initial surveillance, an action protocol
was established, according to which, if a
dead amphibian was found during any
sampling, it was collected and sent for
analysis as quickly as possible to the Cata-
lonian Reptiles and Amphibians Rehabili-
tation Centre (CRARC).
Here we report three cases of amphibi-
ans truly aected by Bd, conrmed by
both molecular and histological methods.
Although Bd presence in Catalonia had
already been conrmed, this work pre-
sents the rst evidence of Bd-related mor-
talities detected in this region.
Materials and Methods
Field description, postmortem study and
sample collection
Case 1. Mediterranean painted frog
(Discoglossus pictus). Two male specimens
with nuptial calluses were found dead in
the Massif de Les Gavarres (Girona; Fig. 1)
during an amphibian health surveillance
study in the area (Pérez-Novo, 2018). The
rst animal (1a) was found by Environ-
ment Rangers (Cos d’Agents Rurals) of the
Catalonian government (Generalitat de Ca-
talunya) on 9 March 2018 at Riera de Rissec
(Madremanya; 41.973449°N, 2.932524°W).
The second animal (1b) was found by
members of the Catalonian Herpetological
Society (Societat Catalana d’Herpetologia,
SCH) on 12 April 2018 at the Bassa de la
Capçana (Cassà de la Selva; 41.869532°N,
2.895320°W). On 1 February 2020, another
ve Mediterranean painted frogs were
found dead, in dierent stages of decom-
position, at the same location as individual
1b (Bassa de la Capçana). Due their de-
composition status, only one animal was
AMPHIBIAN MORTALITY BECAUSE OF CHYTRIDIOMYCOSIS IN CATALONIA
3
sampled. Necropsy and skin swabbing for
PCR diagnosis were performed on the two
animals found dead in 2018, and tissue
samples (skin, liver, kidney and intestine)
were taken for histological studies from
one of them. Due to the advanced stage of
decomposition of the individuals found in
2020, only skin swabbing was possible.
Case 2. Iberian waterfrog (Pelophyax
perezi). Five carcasses were found on 20
February 2018 at the boom of a pond in
La Mussara (Tarragona; 41.257584°N,
1.026546°W; Fig. 1). During the same sam-
pling, a live adult waterfrog oating with
extended limbs was observed and imme-
diately captured for study. The animal
died on the way to CRARC and a post-
mortem study of this animal was carried
out, including skin swabs for PCR and
tissue sample collection (skin, liver, kid-
ney, stomach and intestine) for histology.
Case 3. European common frog (Rana
temporaria). A recently deceased adult ani-
mal was found on 29 April 2019 in the
Santa Fe del Montseny reservoir
(Montseny Natural Park, Barcelona;
41.768309°N, 2.470195°W; Fig. 1) by SCH
members. The animal was immediately
xed in 70% ethanol and sent to CRARC.
The animal was necropsied (despite being
xed), and skin swabs and tissue samples
for histology (skin, lung, gastrointestinal
tract, liver, spleen, gonads and kidneys)
were taken.
To prevent pathogen transmission and
cross-contamination among samples, or
even pathogen transmission among sam-
pling sites, sampling was carried out fol-
lowing strict biosecurity guidelines: all
animals were handled with nitrile gloves,
which were changed between animals in
cases of multiple mortality, and all eld
equipment, including shoes, were disin-
fected with 1% VirkonTM between sites.
Sampling and molecular methods (end
point PCR and qPCR)
Sampling was performed by swabbing
the entire skin of carcasses with sterile dry
swabs. Swab samples were stored below
Figure 1: Location of the reported cases
of Batrachohytrium dendrobatidis in am-
phibians from Catalonia. Orange areas
show the protected National (bright or-
ange) or Natural (pale orange) Parks,
including Montseny Natural Park (point
3). For a complete interpretation of the
gure, the reader is referred to the
online, coloured version of the article.
MARTINEZ-SILVESTRE ET AL.
4
4°C until they were processed in the labor-
atory, and individuals found dead were
stored in 70% ethanol. For Ranavirus
(hereafter RV) detection, liver samples
were used. End-point PCR analyses were
performed on skin swabs and liver sam-
ples to assess the presence of Bd, Batracho-
chytrium salamandrivorans (hereafter Bsal)
or RV, followed by qPCRs on positive
samples performed to quantify infection
loads. DNA was extracted using Prep-
ManTM Ultra reagent and extractions were
diluted 1:10 in DNase-free water before
PCR or qPCR amplication. PCR products
were visualized by electrophoresis on 1.5%
agarose gels using SYBRTM Save DNA gel
stain and blue light transilluminator.
qPCR analyses were performed following
Boyle et al. (2004) for Bd, a modied sin-
gleplex from Blooi et al. (2013) for Bsal,
and Leung et al. (2017) for RV. Negative
controls and standards with known con-
centrations of the corresponding pathogen
were used in each plate. Infection load of
each sample was assessed directly by the
machine software according to the refer-
ence function obtained with the standards.
A sample was assigned as positive when
the infection load was equal to or higher
than 0.1 genomic equivalents (GE) of zoo-
spores for Bd and Bsal, or 3 GEs of virions
for RV, and when the amplication curve
presented a robust sigmoidal shape. Since
qPCR was used to quantify infection loads
of previously positive samples conrmed
by end-point PCR, we did not consider
necessary to run the qPCR analyses in du-
plicate.
Figure 2: Images taken from case 1, cor-
responding to Discoglossus pictus. (a)
General view of the animal’s ventral
side. (b) Skin section (x400) stained with
haematoxylin and eosin. Arrows indicate
(from left to right) a full Bd thallus
(zoosporangium), an empty zoospore,
and a zoosporangium with discharge
tube. (c) Skin section (x1000) stained
with Gomori Methenamine Silver.
a
bc
AMPHIBIAN MORTALITY BECAUSE OF CHYTRIDIOMYCOSIS IN CATALONIA
5
Histology
Tissue samples were xed in 4% neu-
tral buered formalin before being pro-
cessed for histologic examination. One
section per tissue (liver, bone, intestine,
muscle and skin from three dierent loca-
tions, including interdigital, ventral and
dorsal integument) was examined, result-
ing in seven sections per individual. Four-
µm sections were stained with haematoxy-
lin and eosin (H/E) and Groco’s stain
(Gomori Methenamine Silver (GMS)) and
examined by light microscopy.
Results
Bd was the only considered pathogen
for which PCR analyses tested positive,
and none of the samples analysed with
molecular methods or histology showed
evidence of the presence of either Bsal or
RV.
Case 1. Discoglossus pictus. The necrop-
sy of the animals revealed a mild redness
of the ventral skin (Fig. 2a). The qPCR
analyses showed an infection load of 1099
GE for animal 1a and 855 GE for animal
1b. The individual found dead in the same
pond in 2020 presented a Bd load of 62 GE.
Histologically, and despite early autolysis,
we conrmed the presence of zoospores in
the basal layer consistent with a transmu-
ral epidermal invasion of the fungus (Fig.
2b). The GMS stain was also positive, re-
vealing numerous fungi with black-
stained wall. Active fungal reproduction
with numerous thalli and zoosporangia at
dierent stages of maturation were identi-
ed, including empty, septated zoosporan-
gia and ask-shaped discharge tubes (Fig.
2c).
Case 2. Pelophylax perezi. A reddening
of the skin was observed in the ventral
area (Fig. 3a), as in case 1. End-point PCR
analysis showed the presence of Bd, and
qPCR yielded an infection load of 1629 GE.
Histology showed extensive mild hyper-
plasia and hyperkeratosis with invasion of
the stratum corneum, and keratin layers
with numerous zoosporangia (Fig.3b) in
Figure 3: Images taken from case 2, corresponding to Pelophylax perezi. (a) General view of the
animal’s ventral side. (b) Skin section (x400) stained with haematoxylin and eosin, where multiple
Bd zoosporangia can be seen without inammatory cells.
MARTINEZ-SILVESTRE ET AL.
6
several sections of the skin (more than 2
cm2 evaluated). No inammatory cells
were seen in the dermis. Due to the clear
identication of fungal structures by H/E
stain, a GMS stain was not necessary.
Case 3. Rana temporaria. The necropsy
revealed moderate skin redness in the
groin area and proximal part of the hind
limbs. Internal haematomas were also de-
tected, corresponding to haemorrhages
aecting the coelomic cavity (Fig. 4). The
PCR was positive for Bd, although the
presence of some waste material leading to
partial inhibition of the qPCR reaction pre-
vented calculation of the infection load.
Histology was not congruent with a mor-
tal fungal cutaneous infection because on-
ly a single isolated zoospore was detected
on the stratum corneum, with secondary
bacteria detected after evaluation of sever-
al sections. In addition, an acute extensive
subcutaneous haemorrhage was the only
lesion seen in the section from the groin
area. As histological changes could not
conrm the death of this animal due to
chytridiomycosis, we concluded that
death, as well as the observed haemor-
rhages and internal bleeding, were most
likely caused by a traumatic incident.
Discussion
We describe the rst cases of mortality
related to the presence of Bd in Catalonia,
and also the rst cases of mortality due to
chytridiomycosis in D. pictus and P. perezi.
Unfortunately, we have not information
about the strains involved in these cases.
However, according to our previous re-
sults published in Byrne et al. (2019), just
the GPL strain is present in the Iberian
Peninsula. Prior to this report, Bd-related
mortality had been reported in the Iberian
Peninsula only in re salamanders, spiny
toads, and common midwife toads (Bosch
et al., 2001; Bosch Martínez-Solano,
2006; Garner et al., 2009; Walker et al.,
2010). Although mortality due to chytridi-
omycosis in the genus Discoglossus was
described only for the Tyrrhenian painted
frog (Discoglossus sardus) (Bielby et al.,
2009), most members of the family
Alytidae are highly susceptible to chytridi-
Figure 4: Dissected Rana temporaria
specimen, corresponding to case 3,
showing internal haemorrhages.
AMPHIBIAN MORTALITY BECAUSE OF CHYTRIDIOMYCOSIS IN CATALONIA
7
omycosis, including the above mentioned
A. obstetricans (Bosch et al., 2001; Walker et
al., 2010), Balearic midwife toads (Alytes
muletensis; Walker et al., 2008; Dodding-
ton et al., 2013), Betic midwife toads (A.
dickhilleni; Bosch et al., 2013; Thumsová et
al., 2021), Iberian midwife toads (Alytes
cisternasii; Bosch et al., unpublished data),
and Moroccan midwife toads (Alytes
maurus; Thumsová et al., unpublished da-
ta). Bd presence had been already de-
scribed in D. pictus from dierent localities
of Catalonia, but always with highly varia-
ble infection loads and not associated with
mortality (Saura et al., 2016; Montori et
al., 2019). Other species of the genus, in-
cluding the Moroccan painted frog (D. sco-
vazzi), have also been found to be asymp-
tomatic carriers of Bd (El Mouden et al.,
2011).
Green frogs are frequently infected
with Bd in Europe, exhibiting Bd preva-
lence values at background levels (e.g. Ita-
ly: Simoncelli et al., 2005; Adams et al.,
2008; Federici et al., 2008; Denmark: Scal-
era et al., 2008; Luxembourg: Wood et al.,
2009; Spain: Walker et al., 2010; Fernán-
dez-Beaskoetxea et al., 2016, Oficialdegui
et al., 2019; Swierland: Tobler et al., 2012;
Germany: Ohst et al., 2013; Hungary:
rÖs et al., 2018). To the best of our
knowledge, mortalities in European green
frogs associated with Bd have not been
recorded so far, and although two unwell
individuals from Poland tested positive for
Bd, eects were not associated with
chytridiomycosis (Kolenda et al., 2017). In
Catalonia, P. perezi is frequently an asymp-
tomatic carrier of Bd (e.g. BargallÓ et al.,
2016; Miras et al., 2017). Therefore, it can-
not be ruled out that the cause of death of
the individual described here as case 2 was
related to the stress of transportation to the
laboratory. Actually, sudden death of Bd-
infected individuals due to the stress of
handling is relatively common, both in the
laboratory and in the eld (J. Bosch, per-
sonal observations).
Although the cause of death of the indi-
vidual reported here as case 3 could not be
conrmed, it should be noted that this in-
fected animal was within the distribution
area of the critically endangered Montseny
brook newt (Calotriton arnoldi). A compre-
hensive study conducted between 2007
and 2011 in the whole area of the
Montseny Massif revealed an apparent
absence of Bd (ObÓn et al., 2013). However,
more recent surveys performed in 2017
and 2018 conrmed the presence of the
pathogen in this area in introduced Iberian
newts (Lissotriton boscai; Martínez-Silvestre
et al., unpublished data). Rana temporaria is
normally very resistant to the disease, be-
ing frequently infected but without exhib-
iting associated mortality (Bosch et al., un-
published data). In fact, individuals of R.
temporaria have only been found dead in
association with Bd with the concurrence
of severe episodes of mass mortalities of
highly sensitive species (Clare et al., 2016).
In those cases, only newly emerged meta-
morphs, and not adults, were found to be
aected. Even though the internal bleed-
ing detected in our case did not corre-
spond to the typical casuistry of the dis-
ease, the infection could have weakened
the animal, turning it more vulnerable to
other pathogens, or more prone to be road-
killed or predated. Changes in habits (e.g.
spending more time out of the water) or
behaviour (e.g. lethargy, loss of reexes)
MARTINEZ-SILVESTRE ET AL.
8
due to Bd infection have been described,
especially in the terminal phase of disease,
making infected animals more vulnerable
to other causes of mortality (Carey et al.,
2006; Voyles et al., 2007; Viafara et al.,
2020). In any case, the proximity of the
area where the specimen was located to
populations of C. arnoldi is worrying and
an important forewarning about the immi-
nent contact of the pathogen with this en-
demic and endangered species. Even
though the sister species Calotriton asper
has been found to be an asymptomatic
carrier of Bd (Martínez-Silvestre et al.,
2020), contact of Bd with C. arnoldi should
be prevented because specic thermal or
environmental conditions can turn basal
infections into mortalities, as described
here for D. pictus and P. perezi.
Regarding diagnosis, even when end-
point PCR tests allow a rapid detection of
the studied pathogens, a positive PCR
does not necessarily imply that the animal
is unwell. On the contrary, quantitative
PCR is generally much more sensitive than
conventional PCR and provides an estima-
tion of the infectious load. Unfortunately,
information on the burden of infection
alone is not sucient for determining the
cause of death without knowing the refer-
ence values that are lethal for the species.
In our three study cases, histology using
H/E or GMS stains from skin samples
were necessary tools to conrm that the
animals suered from the disease, ruling
out the possibility that they were merely
carriers (see Pessier, 2007). In addition, the
selection of the area where skin samples
are collected is particularly important for
histological detection. The loss of the epi-
dermis due to processing, decomposition
following death, or skin weakening caused
by the disease, can make dicult the de-
tection of characteristic lesions and, conse-
quently, the diagnosis. Furthermore, in
mild infections, hyperkeratosis can be con-
ned to foci and cause false negatives if
these are not detected and sampled during
necropsy (Borteiro et al., 2019), while in
fatal infections it is widely distributed
(Berger et al., 2005). Finally, macroscopic
symptoms such as mild skin redness were
common in almost all individuals de-
scribed here, although these symptoms are
highly nonspecic and commonly shared
between chytridiomycosis and other dis-
eases like ranavirosis (Marschang, 2019).
In mortality cases as those described
here, the following rules could be used to
conrm chytridiomycosis as the possible
cause of death and to dierentiate simple
carriers from sick individuals ruling out
other causes of mortality: (1) qPCR anal-
yses have to conrm that sick individuals
harbour much higher infection loads than
asymptomatic co-occurring conspecics;
(2) a histopathological analysis has to con-
rm that skin lesions are associated with
the presence of the fungus; and (3) the nec-
ropsy and the histological analysis of the
rest of the tissues and viscera have to sup-
port the absence of other causes of illness
or death.
The presence of Batrachochytrium in
northeast Iberian Peninsula, including Bsal
(Martínez Silvestre et al., 2019), is highly
concerning. In general, human global trav-
el and commerce is strongly associated
with the spill-over of pathogens via intro-
duced alien species (Daszak et al., 2000),
and human activities are involved as the
cause of the very recent introduction of
AMPHIBIAN MORTALITY BECAUSE OF CHYTRIDIOMYCOSIS IN CATALONIA
9
Bsal in Catalonia (Martínez-Silvestre et
al., 2019; Martel et al., 2020). In the case of
Bd, an extensive study has documented its
spatiotemporal origins, dating the emer-
gence of this pathogen to the early 20th
century (O’Hanlon et al., 2018). Since Bd
was already well distributed throughout
Europe (Garner et al., 2009) when its
emergence was reported in central Spain,
it is reasonable to suppose that the intro-
duction of Bd to the Iberian Peninsula oc-
curred a long time ago.
Despite increased surveillance of am-
phibian populations in Spain during the
last few years, no well-established proto-
cols to detect disease outbreaks have yet
been implemented in the country. As
chytridiomycosis due to Bd and Bsal are
listed by the World Organisation for Ani-
mal Health (OIE) as diseases of interna-
tional concern, it is recommended to carry
out, at least, routine controls by qualitative
PCRs in selected amphibian populations,
and histological analyses and quantitative
PCRs when mortality cases are found.
Rapid control, detection and action on am-
phibians found dead are critical action
tools for reliable diagnosis. Only the ade-
quate inclusion of these tools in eld col-
lection protocols will enable greater rates
of detection of pathogens, and the possibil-
ity of beer understanding the ecological
repercussions of these diseases.
Acknowledgement
Daniel Guinart (Parc Natural del
Montseny); Felix Amat (Museu de Gran-
ollers); Francesc Carbonell, Elena Obon,
Joan Mayné (Centre de Fauna de Torrefer-
russa), Francisco Martínez (Forestal Cata-
lana), Joaquim Soler, Zuleika Alonso and
Martina Ugrinović (CRARC and Ajunta-
ment de Masquefa), Eudald Pujol, Gerard
Carbonell, Joan Maluquer, Oriol Baena,
Fernando Loras, Joan Ferrer and Alejandro
Garcia (Societat Catalana d’Herpetologia),
Javier Burgos and Rubén Sánchez
(Associació Herpetològica Timon); Aida
Tarrago, Diego Martínez, Vanessa Ca-
denas, Ricard Casanovas, Francesc Mañas,
Albert Sanz (Generalitat de Catalunya)
and Cos d’Agents Rurals de la Generalitat
de Catalunya. Part of this study is includ-
ed in the Life Tritó del Montseny (LIFE15 /
NAT/ES/000757).
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