Is the Italian stream frog (Rana italica Dubois, 1987) an opportunistic exploiter of cave twilight zone?
Is the Italian stream frog (Rana italica Dubois, 1987) an opportunistic exploiter of cave twilight zone?
Enrico Lunghi 0
Gentile Francesco Ficetola
0 Universität Trier Fachbereich VI Raum-und Umweltwissenschaften Biogeographie , Universitätsring 15, 54286 Trier , Germany 2 Museo di Storia Naturale dell'Università di Firenze, Sezione di Zoologia “La Specola” , Via Romana 17, 50125 Firenze , Italy 3 Natural Oasis, Via di Galceti 141, 59100 Prato , Italy 4 Vrije Universiteit Brussel, Boulevard de la Plaine 2, 1050 Ixelles, Bruxelles , Belgium 5 Department of Environmental Science and Policy, Università degli Studi di Milano , Via Celoria 26, 20133 Milano , Italy 6 Univ. Grenoble Alpes, CNRS , Laboratoire d'Écologie Alpine (LECA) , F-38000 Grenoble , France
Studies on frogs exploiting subterranean environments are extremely scarce, as these Amphibians are usually considered accidental in these environments. However, according to recent studies, some anurans actively select subterranean environments on the basis of specific environmental features, and thus are able to inhabit these environments throughout the year. We present the first study on the abundance and spatial use of the Italian stream frog, Rana italica, in subterranean environments. We monthly collected data from 66 cave sectors during a whole year (2013), recording > 120 detections of R. italica. Frogs were more frequently found close to the cave entrance, without significant differences between age classes or sexes. Adults generally were observed being higher up along cave walls compared to juveniles. Frogs abundance was higher in areas showing specific environmental features, such as warm temperature, low incident light and the presence of potential prey. Rana italica likely occupies subterranean areas characterized by a combination of microclimatic suitability and prey availability.
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Several amphibian species are known to regularly exploit subterranean environments
but, besides numerous reports concerning several salamanders species
Miller 2007, Pierce et al. 2014, Manenti et al. 2017, Soares et al. 2017, Vörös et al.
, only a few species of frogs and toads (Anura) are known to exploit these
(Fenolio et al. 2005, Köhler et al. 2010, Lunghi et al. 2014, Koller 2017)
Frogs have been usually considered as an accidental presence in subterranean
(Bressi and Dolce 1999)
, but recently many authors showed that some Anuran
species can be quite common in these sites, selecting caves with specific environmental
(Köhler et al. 2010, Rosa and Penado 2013, Biswas 2014, Lunghi et al. 2014,
Matavelli et al. 2015)
. One of these species is the Italian stream frog Rana italica
Dubois, 1987, which is endemic of Italian Apennines
(Lanza et al. 2006, Canestrelli
et al. 2008, Buono et al. 2014, Sindaco and Grieco 2014)
. According to the known
habitat requirements, R. italica is strongly bounded to freshwater environments and
usually occurs in forested areas; in some circumstances, the species is also found in
natural and artificial subterranean sites, where it seems to be able to breed
(Lanza et al.
2006, Vanni and Nistri 2006)
. Although most of the previous observations of R. italica
refer to individuals trapped in wells and vertical caves
(Bressi and Dolce 1999)
studies underlined that this frog is able to actively exploit subterranean environments
during the whole year
(Lunghi et al. 2017)
Subterranean environments are characterized by peculiar microclimatic features: in
temperate areas, they generally have a relatively constant temperature, which roughly
corresponds to the mean annual temperature at the surface. Furthermore, air
humidity is generally very high, and incoming light is only present within the first meters
after the cave entrance
(Romero 2009, Lunghi et al. 2015)
. Close to the surface (cave
entrance and twilight zone), the inner microclimate is mostly affected by external
environmental conditions, and such peculiarity can promote the abundance of troglophile
(Manenti et al. 2015, Lunghi et al. 2017)
. The microclimate characterizing the
shallow part of subterranean environments seems to be particularly suitable for Rana
(Lunghi et al. 2014)
; therefore, the use of subterranean spaces during periods
characterized by unsuitable (e.g., dry) surface conditions may be of key importance for
(Forrester et al. 2015, Lunghi et al. 2017)
. Beside a suitable
microclimate, in subterranean environments R. italica probably finds shelter from predators
(Vanni and Nistri 2006)
. Nevertheless, information on the biology of Italian stream
frogs in subterranean environments, and on the relevance of these habitats for its
conservation, is extremely limited. With this study we aim to provide the first assessment
of R. italica in subterranean environments, mostly focusing on population abundance
and habitat use.
From January 2013 to December 2013, we monthly surveyed nine subterranean
environments located in the north of Tuscan Apennines (between 43°53'17"N, 11°06'22"E
and 44°03'48"N, 10°48'09"E), where the presence of Rana italica was already assessed
by a previous study
(Lunghi et al. 2014)
. All surveyed environments did not show a
morphology hampering frogs to move freely between subterranean and outdoor areas. Three
of them were World War II refuges, one was a drainage tunnel, while the remaining six
were natural caves (Table 1). Subterranean environments were explored until the point in
which speleological equipment was necessary, and only when environmental conditions
did not pose a risk (e.g., flooding). All studied environments were divided into portions of
3-linear meters of length (hereafter sectors); this subdivision allows a good data collection
for both biotic and abiotic features characterizing subterranean environments
al. 2015, Manenti et al. 2015, Lunghi et al. 2017)
. Within each sector we recorded air
temperature and humidity (measured with a Lafayette TDP92 thermo-hygrometer;
accuracy: 0.1 °C and 0.1%) and the average incident light (obtained by averaging max and
min illuminance recorded with a Velleman DVM1300 light meter; minimum recordable
light: 0.1 lux). We recorded the abundance of Rana italica within sectors using visual
(VES; Crump and Scott 1994)
and adopting a standardized survey
method (7.5 min/sector) which limits potential effects of imperfect species detection
(Banks-Leite et al. 2014, Lunghi et al. 2017)
. Using the same procedure (VES +
standardized survey method), we assessed the presence of seven invertebrate species which
potentially represent prey items for R. italica: one dipteran (Limonia nubeculosa Meigen,
1804), three spiders (Meta menardi (Latreille, 1804), Tegenaria Latreille, 1804 sp.,
Metellina merianae (Scopoli, 1763)), one cricket (Dolichopoda laetitiae Minozzi, 1920) and
two gastropods (Chilostoma planospira (Férussac, 1832), Oxychilus draparnaudi (Beck,
1837)). All considered species have stable populations in subterranean environments,
even if they are not obligate cave species
(Lunghi et al. 2017)
. We measured the
snoutvent length (SVL) of all captured frogs (Fig. 1a); individuals with SVL ≤ 30 mm were
(Buono et al. 2014)
. Within adults, we recognized males on the
basis of secondary sexual characters (nuptial pads). For each frog, we measured the distance
from the cave entrance and the elevation from the cave ground floor.
We used Generalized Linear Mixed Models (GLMM) to assess whether abundance of
Rana italica was related to both biotic and abiotic recorded parameters. As dependent
variable we used the observed abundance of R. italica, which represents an index of
(Barke et al. 2017)
. Microclimatic features (temperature, humidity
and illuminance), biotic features (presence/absence of the seven prey species), month
of survey and sector depth were used as independent variables; sector and cave identity
were used as random factors.
We used Linear Mixed Models (LMM) to identify whether differences in spatial
use of caves occur between age classes or sexes. To assess differences between adults
and juveniles, we run two LMMs using age class and month of survey as independent
variables, while both cave and sector identity were included as random factors. The
distance of individuals from cave entrance, and the vertical position of frogs (i.e., height
above the cave floor) were the dependent variables of the two LMMs. This procedure
was repeated on adults only, replacing the independent variable age class with sex, in
order to detect differences between adult males and females.
All analyses were performed in the R environment using packages lme4, lmerTest,
nlme and unmarked
(Fiske and Chandler 2011, Douglas et al. 2015, Kuznetsova et al.
2016, Pinheiro et al. 2016, R Core Team 2016)
We performed a total of 765 surveys within 66 cave sectors (missing data due to
unfavorable climatic condition represent 4.42%), recording 122 detections of Rana italica
(60 females, 9 males, 38 juveniles and 15 unsexed individuals) (Table 1); on average
(±Error Standard; ES), we observed 1.14 ± 0.21 individuals per survey (Fig. 2).
The abundance of R. italica was strongly related to the depth of the sector (F1,98.28 =
30.40, P < 0.001), air temperature (F1,716.39 = 4.29, P = 0.038), illuminance (F1,143.82 = 8.01,
P = 0.005) and to the presence of two insect species, Dolichopoda laetitiae (F1,645.63 = 7.71,
P = 0.006) and Limonia nubeculosa (F1,730.17 = 5.50, P = 0.019). Overall, frogs were more
abundant in shallow sectors with less light and warmer temperature, and with the presence
of both D. laetitiae and L. nubeculosa. Furthermore, we detected significant differences of
frog abundance among months (F11,704.58 = 2.46, P = 0.005). March and November were
the survey months in which we recorded the highest frog abundance (Fig. 2). All other
considered variables were not significant (P > 0.17).
The distance from the cave entrance did not show significant differences between
age classes, without differences among survey months (age class: F1,67 = 0.80, P = 0.374;
month: F9,67 = 1.04, P = 0.417) (Fig. 3A), nor between sexes (sex: F1,30 = 0.56, P = 0.46;
month: F9,30 = 0.45, P = 0.894) (Fig. 3B). The use of walls was significantly different
between age classes (F1,67 = 4.92, P = 0.03) (Fig. 3C) without differences among survey
months (F9,67 = 1.86, P = 0.073); adults generally occupied higher position compared
to juveniles. Within adults, we did not detect differences between males and females
(month: F9,30 = 1.07, P = 0.413; sex: F1,30 = 0.01, P = 0.912) (Fig. 3D).
Rana italica was observed in subterranean environments throughout the year, even
though the number of observations varied between months (Fig. 2). The abundance
of Rana italica in subterranean environments was higher in areas close to the cave
entrance, but deep enough to show almost no light. These particular areas correspond
to the twilight zone
(Culver and Pipan 2014)
, where external influence is moderate
and abundance of non-strictly cave-dwelling species is the highest
(Lunghi et al. 2015,
Lunghi et al. 2017)
. The amount of incoming light represents an important factor for
R. italica, as it influences both presence and abundance of the species in subterranean
(Lunghi et al. 2017)
. Stream frogs are mostly nocturnal, and preference
for twilight areas might help them to avoid predators (Vanni and Nistri 2006);
however, we cannot exclude that the selection of sectors without light may be also related
to the avoidance of harsher climatic conditions (hot and dry), which are present close
to the surface
(Hetema et al. 2012, Everall et al. 2014)
. Temperature was an additional
parameter strongly related to abundance of R. italica. Frogs were more abundant in
relatively warm cave sectors. The temperature of cave sectors is the result of complex
interactions between external temperature, mean annual temperature, and air
circulation of karstic systems
(Badino 2004, Badino 2010, Lunghi et al. 2015)
. In winter,
temperature is highest far from the surface, thus these sectors represent ideal shelters
during hibernation. Conversely, in summer the temperature is highest close to cave
entrance where prey richness is highest
(Manenti et al. 2015, Lunghi et al. 2017)
the abundance of frogs was positively related to the presence of invertebrates such as
the cave cricket Dolichopoda laetitiae and the fly Limonia nubeculosa. Stream frogs are
generalist predators of small invertebrates, and these two insects are often abundant in
subterranean environments, therefore they have a key role in sustaining subterranean
(de Pasquale et al. 1995, Lavoie et al. 2007, Manenti et al. 2015)
Recent studies underlined that the occurrence of the cricket D. laetitiae is strongly
related to inner cave temperature, as this cricket prefers cave areas with relatively warm
(Lunghi et al. 2015, Lunghi et al. 2017)
. No particular information is
available on the seasonal occupancy of caves by Limonia nubeculosa. Therefore, the
relationship between R. italica abundance and the occurrence of these species is likely
to represent the result of trophic interactions.
As already observed for other amphibians present in subterranean environments,
individuals of R. italica probably face a trade-off between the selection of suitable
conditions, and the need of trophic resources. Thus, frogs occupy cave portions that are
deep enough to have a suitable microclimate and lack of predators, but that are not
too far from feeding areas
(Ficetola et al. 2013, Lunghi et al. 2016)
. The abundance
and the frequency of individuals found in subterranean environments suggest that
further investigations should be performed on the trophic role and the impact that R.
italica may have on the cave food web. In a study performed on the pickerel frog (Rana
palustris (LeConte, 1825)) involving analyses with both stable isotopes and stomach
contents, authors identified a significant predator activity of this species within the
(Fenolio et al. 2005)
. In our study, the correlation between R. italica and
the two insect species suggests that some trophic relationships may occur, as it is
possible that frogs are more abundant in cave sectors with the presence of potential prey.
On the other hand, it is also possible that some unrecorded features (e.g., availability of
resources) positively influence the abundance of multiple species, including both frogs
and insects. Indeed, as recently underlined for aquatic subterranean sites, amphibian
predators exploiting subterranean environments not only contribute to the top-down
control of prey, but also provide an important subsidy in terms of energy for such
(Barzaghi et al. 2017)
Frog distribution within caves was similar between age classes and sexes (Fig. 3A–B),
suggesting that there is neither spatial segregation between age classes, nor between adult
sexes. Actually, no information exists on a possible spatial segregation between
individuals of Rana italica. In our study, the abundance and diversity of potential prey observed
in subterranean environments likely promote the co-existence of different individuals.
However, the use of the subterranean space by R. italica was not limited to the cave floor,
as frogs often climbed walls (Figs 1B, 3C–D). This particular behavior, which is
generally unusual for frogs, might be compared to observations reported for other two anuran
species. In green frogs (Pelophylax synklepton hispanicus (Bonaparte, 1839)) some adults
were observed preying on high tree branches
(Mori et al. 2013)
. In artificial
subterranean environments, the Iberian frog
(Rana iberica Boulenger, 1879)
has been observed
climbing up the walls, probably looking for shelters
(Rosa and Penado 2013)
. In our
study, adults were observed more frequently being up higher along cave walls compared
to juveniles (Fig. 3C); this divergence could be related to a different climbing ability
and/or behavior between age classes.
This study represents the first analysis on the factors related to the abundance and the
spatial use of Rana italica in subterranean environments. The obtained results enable
us to hypothesize that subterranean abundance of R. italica is mostly related to
foraging activities, thus likely make this species one of the top predators of the twilight zone.
Our study underlines that the trophic role of terrestrial predators exploiting
subterranean environments should be taken into account for the ecological comprehension of
determinants which have promoted the colonization of caves. Nonetheless,
subterranean environments may also be of key importance to maintain the viability of anuran
populations in the light of the occurring global warming.
The Ministry of the Environment and Protection of Land and Sea and Provinces of
Prato and Pistoia provided authorization to perform this study (Permits n. 0046894/
PNM,15/10/2013; n. 448, 13/2/2013; n. 731, 21/02/2013). We thank L. Kaven for
checking the manuscript spelling.
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