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.

biospeleology, Anuran, amphibian, cave biology, prey, microclimate, spatial use, food web

Several amphibian species are known to regularly exploit subterranean environments but, besides numerous reports concerning several salamanders species (Niemiller and Miller 2007, Pierce et al. 2014, Manenti et al. 2017, Soares et al. 2017, Vörös et al. 2017), only a few species of frogs and toads (Anura) are known to exploit these environments (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 environments (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 features (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), recent 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 species (Manenti et al. 2015, Lunghi et al. 2017). The microclimate characterizing the shallow part of subterranean environments seems to be particularly suitable for Rana italica (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 population survival (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.

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).

Figure 2. 

Average monthly observation (±ES) of Rana italica within studied caves.

The abundance of R. italica was strongly related to the depth of the sector (F_1,98.28 = 30.40, P < 0.001), air temperature (F_1,716.39 = 4.29, P = 0.038), illuminance (F_1,143.82 = 8.01, P = 0.005) and to the presence of two insect species, Dolichopoda laetitiae (F_1,645.63 = 7.71, P = 0.006) and Limonia nubeculosa (F_1,730.17 = 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 (F_11,704.58 = 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: F_1,67 = 0.80, P = 0.374; month: F_9,67 = 1.04, P = 0.417) (Fig. 3A), nor between sexes (sex: F_1,30 = 0.56, P = 0.46; month: F_9,30 = 0.45, P = 0.894) (Fig. 3B). The use of walls was significantly different between age classes (F_1,67 = 4.92, P = 0.03) (Fig. 3C) without differences among survey months (F_9,67 = 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: F_9,30 = 1.07, P = 0.413; sex: F_1,30 = 0.01, P = 0.912) (Fig. 3D).

Figure 3. 

Boxplots indicating differences in the use of cave spaces. Difference between A age classes (Adults/ Juveniles) and B adult sexes (Females/Males) in the use of the subterranean surface area; differences between C age classes and D adult sexes in the use of cave walls. Diagonal bar inside the box represents the median.

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 environments (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). Indeed, 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 trophic networks (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 temperature (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 cave habitat (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 environments (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.