First Data on Nesting Ecology and Behaviour in the Imperial Cave Salamander Hydromantes Imperialis

Home , Imperial cave salamander

NORTH-WESTERN JOURNAL OF ZOOLOGY 11 (2): 324-330 ©NwjZ, Oradea, Romania, 2015 Article No.: 151505 http://biozoojournals.ro/nwjz/index.html

First data on nesting ecology and behaviour in the Imperial cave salamander Hydromantes imperialis

Enrico LUNGHI1,2,3*, Rosalba MURGIA4,5, Gianni DE FALCO4, Salvatore BUSCHETTU4, Cinzia MULAS6, Manuela MULARGIA7, Claudia CANEDOLI8, Raoul MANENTI9 and Gentile Francesco FICETOLA8,10,11

1. Natural Oasis, Via di Galceti 141, 59100 Prato, Italy. 2. Universität Trier Fachbereich VI Raum- und Umweltwissenschaften Biogeographie, Campus I, Gebäude N Universitätsring 15, 54286 Trier, Germany. 3. Museo di Storia Naturale dell’Università di Firenze, Sezione di Zoologia “La Specola”, Via Romana 17, 50125 Firenze, Italy. 4. Speleo Club Oristanese, Via Canepa 8, 09170 Oristano, Italy. 5. IMC-International Marine Centre, Torregrande, 09170 Oristano, Italy. 6. Gruppo Grotte Ogliastra, Via Pascoli snc, 08046 Perdasdefogu, Italy. 7. Speleo Club Nuoro, Via Tunisi 3, 08100 Nuoro, Italy. 8. Dipartimento di Scienze dell’Ambiente e del Territorio e di Scienze della Terra, Università degli Studi di Milano-Bicocca, Piazza della Scienza 1, 20126 Milano, Italy. 9. Dipartimento di Bioscienze, Università degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy. 10. Laboratoire d’Ecologie Alpine (LECA), Université Grenoble-Alpes, F-38000 Grenoble, France. 11. CNRS, LECA, F-38000 Grenoble, France. *Corresponding author, E. Lunghi, E-mail: [email protected]

Received: 24. November 2014 / Accepted: 03. May 2015 / Available online: 11. November 2015 / Printed: December 2015

Abstract. During the dry season, the European Plethodontid salamanders (genus Hydromantes) usually occupy underground environments (i.e. caves), where they can find cold temperatures and high moisture. Hydromantes breed in hypogean environments, where they usually lay eggs in hidden shelters. Mothers perform a long-lasting parental care of the eggs, which also continues after hatching. Due to the cryptic habitat and behaviour, their breeding biology is poorly known. Most of the available data refer to observations in captivity, while data from wild populations are scarce and deal with the findings of single nests. Here we report the first study on the Imperial cave salamander H. imperialis nesting ecology and behaviour, by performing quantitative observations on multiple nests. We found four nests in a cave located in Central Sardinia. We monitored them through five months, recording environmental features. Nests were associated with cold, humid and dark sectors of the cave, but sectors with nests did not show greater climatic stability than the superficial ones. Nests were continuously attended by females; temporary desertion became more frequent when temperatures were high and it was later in the season. Newborns were attended by their mothers for up to 52 days after hatching. The comparison of breeding biology across multiple Hydromantes species suggests earlier hatch in population/species living in warmer areas, with similar post-hatch brood attendance among species.

Keywords: Microclimate, temperature, humidity, parental cares, brood attendance, cave fauna, amphibians.

Introduction environments are avoided during the hottest and driest seasons (Cimmaruta et al. 1999, Lanza et al. Cave salamanders of the genus Hydromantes Gis- 2006). On the other hand, hypogean environments tel, 1848 are fully terrestrial and lay eggs in sub- represent a shelter in which Hydromantes can find aerial environments (Lanza et al. 2006). Such fea- suitable microclimatic conditions even during un- tures force these salamanders to live and repro- favourable periods (e.g. hot and dry summers) duce in environments with specific microclimate, (Salvidio et al. 1994, Vignoli et al. 2008, Ficetola et such as cold temperature and high moisture (Fice- al. 2012, Lunghi et al. 2014a). In these habitats, Hy- tola et al. 2012, Lunghi et al. 2014a). In Europe dromantes select the most appropriate zone to there are eight species of Hydromantes, five of breed and to perform their long-lasting egg care which are endemic to Sardinia (Sillero et al. 2014). (Stefani & Serra 1966, Papinuto 2005, Lunghi et al. Seven of these species belong to the sub-genus 2014b). Until few years ago the knowledge on the Speleomantes, while one species belongs to the sub- reproduction of these salamanders was quite genus Atylodes (Wake 2013). In Mediterranean ar- scarce (Lanza et al. 2006). Recent studies per- eas, cave salamanders can exploit both epigean formed both in natural and semi-natural condi- and hypogean environments; however outdoor tions improved our understanding of their repro- Multiple nesting of Hydromantes imperialis 325 ductive biology, and showed the complexity of eggs are characterized by high humidity, low light parental cares in these salamanders (Stefani & and temperature (as observed for the adults of Serra 1966, Papinuto 2005, Lanza et al. 2006, Oneto other Hydromantes species (Ficetola et al. 2013)); ii) et al. 2010, Oneto et al. 2013, Lunghi et al. 2014b, sectors with eggs have particularly stable micro- Oneto et al. 2014). Observations in both natural climatic conditions (Lunghi et al. 2014b). Second, and controlled environments can be useful to un- we quantitatively recorded the brooding behav- derstand the breeding behaviour of these sala- iour of females, and evaluated whether it was re- manders. Oneto et al. (2010, 2014) recently studied lated to specific microhabitat features (Oneto et al. the parental cares of H. strinatii, by performing ob- 2010). In particular we assessed whether the peri- servations on individuals under semi-natural con- ods in which females leave the nest unattended ditions. H. strinatii females constantly attended the (Oneto et al. 2010) were related to specific envi- nests, keeping their body in touch with the eggs ronmental conditions. (Oneto et al. 2010). During egg care, the mother rarely moved from the nest; however, temporary desertion of the nest became more frequent after Material and methods all eggs had hatched (Oneto et al. 2010). Post- Surveys were conducted during a herpetological field ac- hatching parental care lasted six weeks, during tivity performed in a cave located in Oristano District in which newborns tried to keep their body in touch Central Sardinia (39° 53'N, 8° 58' E). The presence of H. with the mother as much as they could. During imperialis was assessed using visual encounter surveys in this time, the mothers moved away from the nest June 2014, following the same procedure described by only occasionally, and they actively defended the Ficetola et al. (2013). After the first observation of broods against conspecific intruders (Oneto et al. clutches, the site was repeatedly surveyed until the com- plete abandonment of all nests by mothers and newborns: 2014). However, observations performed in wild from 15 June to 25 October 2014 the cave was surveyed 20 conditions, and on a wider range of species, are times. Eggs or brooding females were never touched or needed to assess the generality of results obtained directly disturbed. Using a metric wheel we measured the under controlled conditions. Until now, informa- maximum depth of the cave (from the entrance to the tion on the breeding behaviour of Hydromantes in deepest point) and determined the position of each nest nature was extremely limited and available for (distance from the entrance and height from the ground). three species only. Papinuto (2005) observed a We also measured the height and width of each nest’s entrance. Starting from the entrance, we divided the cave clutch of H. genei in an abandoned mine, and no- environment in 3-meter sectors. This size approximately ticed that the mother stayed constantly close to the corresponds to the size of Hydromantes home-ranges dur- eggs, rarely moving away from the nest and only ing their underground activity (Salvidio et al. 1994). In for a short time. Newborns left the nest after two each sector we recorded microclimatic parameters [air weeks. Lunghi et al. (2014b) reported two observa- temperature, relative humidity and max/min incident tions: a clutch of H. flavus in a deep zone of a cave light (illuminance)] using an EM882 multi-function Envi- system and a clutch of H. italicus in an abandoned ronmental Meter (PCE Instruments; minimum illuminance 0.01 lux). Data on temperature and relative humid- mine. In these species, the mothers were never ob- ity was recorded 17 times through the whole period. As served far from the nest, and post-hatch parental previous studies suggest that Hydromantes select cold, cares lasted from one week to one month, in H. humid and dark sectors of caves (Ficetola et al. 2012, flavus and H. italicus, respectively. However, due Lunghi et al. 2014a), we used one-tailed t-test to evaluate to the difficulty of accessibility and finding cave whether nest sites were associated with these microhabi- salamander nests, all the studies from natural en- tat conditions. We used Levene’s test to assess whether vironments reported observations on single nests, eggs were associated with areas with particularly stable microclimate (as hypothesized by Lunghi et al. 2014b), by without possibilities to compare the nesting be- testing whether sectors in which nest were present varied haviour of different females of the same popula- significantly in respect to temperature and humidity, tion, and only recorded few quantitative data. compared to the first sector of the cave (parameters re- Here we report the first study on the Imperial corded within the cave, 3 m from the cave entrance). cave salamander H. imperialis nesting ecology and Moreover, we used Generalized linear mixed models behaviour, by performing quantitative observa- (GLMM) assuming binomial error distribution to test if tions on multiple nests. First, we assessed whether mothers move away from their nest under specific microclimatic features or in particular periods. To analyse the selection of nesting areas was related to cave mother’s choice to move away from nest, we considered environmental features. Specifically, we tested two as dependent variable female attendance to the nest dur- non-exclusive hypotheses: i) cave sectors with ing each survey. As independent variables, we considered

326 E. Lunghi et al. microclimatic conditions (temperature, relative humidity), day of survey, and presence of hatched eggs. Nest identity was considered as random factor. We used the Aikake’s Information Criterion corrected for small sample size (AICc) to identify the combination of variables that explains attendance/nonattendance of females to the nest (Stephens et al. 2007). If a model showed AICc higher than a simpler nested model, it was not included among the candidate models (Richards et al. 2011). We tested significance of each variable included into the best model (i.e. the model with lowest AICc) using a likelihood ratio test. Prior to performing analyses, humidity was transformed using square-root arcsine, while illuminance was log-transformed to better meet the assumptions of para-

metric analyses. All statistical analysis were performed in Figure 1. Seven eggs provided with peduncle that draws R 3.1.1 (R Development Core Team 2014). them close (third nest; 15 June).

Results

We found four clutches of Hydromantes imperialis during the first survey performed on 15 June 2014. The maximum cave depth was 30 m. Nests were located at different points through the development of the cave, into small holes present on cave walls (Table 1). Sectors with nests showed significantly higher humidity (t7.03 = -2.26, P =

0.029) and lower illuminance (t7 = 2.05, P = 0.04) compared to sectors without nests. Furthermore, sectors with nests tended to have lower temperature, although this test was marginally non- significant (t8.68 = 1.66; P = 0.067). Considering temperature, not a single nest was located in cave sec-

tors with lower variability than the first sector of Figure 2. First nest during a survey on 15 June: eggs show the cave (Levene’s test: all P ≥ 0.29). Similarly, sec- high transparency and embryos and yolk are clearly tors with nests did not show lower variability than recognisable. the first sector in respect to humidity (all P ≥ 0.10). Clutches were composed of 7-11 eggs each, all provided with a peduncle (Fig. 1). During the first Table 2. Candidate AIC models created using the following dependent factors: Day (day of survey); Eggs hatch surveys (June) eggs were transparent and the em- (presence of hatched eggs); Temp (air temperature); bryos inside were perfectly observable (Fig. 2 and Hum (relative humidity); Day*T (interaction between Fig. 3a). In early July almost half of the yolk was day of survey and air temperature). absorbed and embryos started to have a defined Eggs Day Temp Hum Day*T df AICc delta weight shape, allowing us to recognize various morphol- hatch ogy features of the animals. In late July-early Au- -1.612 -4.615 0.08958 5 31.0 0.00 0.71 gust embryos had filled up all the available space -0.04962 1.0960 4 33.8 2.77 0.18 inside the eggs, making their aspect more dark 2 34.7 3.70 0.11 and opaque (Fig. 3b-c). The first newborns were observed on 16 August in the nest furthest from of their broods, rarely moving away from them the entrance, while in the other nests the first (Table 1). The best-AIC model suggested that nests newborns appeared on 25 and 28 August (Table 1; were more frequently unattended by mothers Fig. 3d). Eggs from the same nest did not hatch when temperature was higher (nearly significant simultaneously, but required up to 10 days for effect; χ21 = 3.404, P = 0.06) and in the last part of hatch completion (Table 1). breeding season (χ21 = 4.52, P = 0.03) (Table 2). A During surveys mothers constantly took care significant positive interaction between

Multiple nesting of Hydromantes imperialis 327

Table 1. Position and morphology of nests and data on their occupancy: Distance (distance from entrance); Height (height from the ground); Size of the nest-hole (height and width of the entrance of nests); First born (date of first eggs hatched); All born (date when all eggs were hatched); Empty (date when the nest was completely uninhabited); Nest unattended (date when mother left unattended the nest). * The fourth nest was not checked between 14 and 26 September due to the high risk of stepping on salamanders that were covering the ground floor.

Distance from Size of the Nest Height First born All Born Empty entrance nest-hole unattended 1st nest 14.8 m 100 cm 2 x 2 cm 28/08 3/09 25/10 17/08 2nd nest 17.6 m 50 cm 5 x 2 cm 25/08 3/09 20/09 7/09 3rd nest 20 m 20 cm 3 x 2 cm 28/08 7/09 13/09 7/09 4th nest 21.7 m 150 cm 4 x 3 cm 16/08 25/08 27/09* 9/08

Figure 3. Third nest observed at different times. A) On 23 June eggs were transparent and embryos in early developmental stage are detectable; B) On 9 August embryos were grown up and showed an advanced body-shape; C) On 17 August yolks were continuously less visible due to embryos development; D) First salamander hatched on 28 Au- gust.

Table 3. Mean hatch date and length of post-hatch parental cares, in different studies on Hydroman- tes nests, from natural and semi-natural environments. Mean annual temperature is the mean annual temperature of the outdoor environment, obtained from the world climate (Hijmans et al. 2005).

Mean Post-hatch Mean Number Species Study outdoor parental cares hatch date of clutches temperature (days) H. genei (Papinuto 2005) 31 August 1 13.7 14 H. imperialis present study 24 August 4 15.0 6-52 H. italicus (Lunghi et al. 2014b) 11 September 1 12.0 30 H. flavus (Lunghi et al. 2014b) 26 August 1 15.9 7 H. strinatii (Oneto et al. 2010, 2014) 6 October 3 10.2 42

328 E. Lunghi et al.

temperture and day of survey indicated that the lected for nesting did not show particularly high effect of temperature was particularly important microclimatic stability, compared to the areas of later in the season (χ21 = 5.10, P = 0.024). the cave closest to the surface, i.e. where the high- Before leaving the nests unattended, females est microclimatic variability is expected (Romero changed eggs position, by moving them to the 2009). This suggests that the microclimatic stabil- deepest and most hidden part of the nest-holes. ity, that is characteristic of the whole cave envi- After hatching, newborns followed their mothers ronment (Romero 2009), is sufficient to maintain into and outside the nests for 6-52 days (mean: 27). suitable microhabitat conditions during the sev- Sometimes some newborns explored areas just eral months required for egg development (Lanza outside the nest, while the mother remained inside et al. 2006). To lay their eggs, females probably se- with the rest of her brood. This behaviour was ob- lect the first suitable area of the cave (cold and served 6 times, most frequently for the deepest humid enough) which is at the same time not too nest: we found newborn salamanders outside of far from food resources (Ficetola et al. 2013). This the fourth nest 4 times and only one time outside choice might help females to obtain food without of the first and the second. By the 25 October all distancing too much from their clutches. Female nests were abandoned. choice might therefore result from the trade-off between environmental features, clutch security and food availability. Discussion Clutch size was similar to the one previously reported for other species (Lanza et al. 2006). For This study describes the first finding of multiple all the nests, hatching was relatively synchronous, nests for the genus Hydromantes (Speleomantes) un- toward the end of the summer, similar to what der natural conditions, by analysing the first col- was observed in all the other Hydromantes species lection of quantitative parameters on environ- (Papinuto 2005, Lanza et al. 2006, Lunghi et al. mental features potentially related to nest site 2014b) (Table 3). The end of the summer may be choice and parental care behaviour for the genus. an extremely suitable period for juveniles, as it Before this study, Hydromantes clutches in natural generally corresponds to the end of the dry sea- environments have been observed only few times son, and thus juveniles may move outside of the (Stefani & Serra 1966, Papinuto 2005, Lunghi et al. caves, where food availability is higher. Neverthe- 2014b), while clutches of H. imperialis were only less, across the different Hydromantes species, av- observed in captivity (Lanza et al. 2006). Clutches erage hatch date was significantly related to the of H. imperialis were inside small holes located on mean annual temperature: eggs hatched signifi- cave walls, as observed by studies on the other cantly earlier in populations living in the warmest species (Stefani & Serra 1966, Papinuto 2005, areas (Table 3; linear regression, weighted for the Lunghi et al. 2014b). Females look for hidden number of clutches, between mean hatch date and places in order to protect their eggs as much as mean annual temperature: F1,3 = 52.2, P = 0.005, R2 possible (Lanza et al. 2006, Pastorelli & Laghi 2006, = 0.93). Cave temperature is strongly related to the Oneto et al. 2010). Normally, observed females mean annual outdoor temperature (Romero 2009), laid their eggs not too close to the entrance, in ar- and in amphibians high temperature increases me- eas that present suitable microclimatic features tabolism, accelerating embryo development (Mor- (cool temperature, high moisture and darkness), rison & Hero 2003). Overall, the good synchrony with a pattern of microhabitat selection similar to of hatches, with earlier hatch in the warmer locali- the one observed by individuals of other Hydro- ties, suggests that the whole breeding cycle may mantes species (Ficetola et al. 2013, Lunghi et al. follow a similar seasonal pattern across the whole 2014a). During previous observations, females Hydromantes genus. with eggs were always relatively far from the en- Within a nest, eggs hatched over a relatively trance, and previous work suggested that these long period (Table 1). As was observed for H. stri- areas might be characterized by high microcli- natii (Oneto et al. 2010), eggs from all 4 nests of H. matic stability (Lunghi et al. 2014b). Females of H. imperialis never hatched in the same day: a single imperialis also selected nest-holes relatively far clutch required up to 10 days for all newborns to from the entrance, and the recording of microcli- come out of their eggs (Fig. 3d). matic parameters in multiple occasions allowed to Mothers look carefully after their broods, at- test this hypotheses. Actually, the cave sectors se- tending and protecting them against predators

Multiple nesting of Hydromantes imperialis 329 and infections (Lanza et al. 2006, Oneto et al. Papinuto, Francesco Papinuto, Denise Atzei, Luca 2014). However, it is already known that some- Buschettu, Giancarlo Cadeddu, Rita Cadeddu, Alfredo times mothers leave their nest unattended (Oneto Camedda, Franca Contu, Gabriele Espis, Salvatore Manca, Carla Meli, Gianfranco Muzzetto, Simona et al. 2010). From our observations we could see Putzolu, Cristina Santona, Loredana Tegas. This study that the mothers’ choice to distance for a while was allowed by the Italian Ministry of the Environment, from their nest was related to some environmental out withstanding of the DPR 357/97 , Prot. 0040002. features. Mothers left nests mostly when air temperature was relatively high and during later periods. However, before moving away, mothers References moved the eggs in a concealed part of the nest. During our observations we always saw mothers Cimmaruta, R., Forti, G., Nascetti, G. Bullini, L. (1999): Spatial curled on their eggs inside holes; however, in two distribution and competition in two parapatric sibling species of European plethodontid salamanders. Ethology Ecology & instances, before leaving the nest, mothers hid the Evolution 11: 383-398. eggs in the backside of nest walls, making their Ficetola, G.F., Pennati, R. Manenti, R. (2012): Do cave salamanders detection extremely difficult (or impossible). For occur randomly in cavities? An analysis with Hydromantes strinatii. Amphibia-Reptilia 33: 251-259. all species, after hatching, mothers continued to Ficetola, G.F., Pennati, R. Manenti, R. (2013): Spatial segregation occupy the nest with newborns in average for one among age classes in cave salamanders: habitat selection or month, with great differences among females (min social interactions? Population Ecology 55: 217-226. Hijmans, R.J., Cameron, S.E., Parra, J.L., Jones, P.G. Jarvis, A. (2005): 6 days, max 52 days). The post-hatch nest atten- Very high resolution interpolated climate surfaces for global dance overlaps with what has been observed in land areas. International Journal of Climatology 25: 1965-1978. other species (Table 3), suggesting a lack of inter- Lanza, B., Pastorelli, C., Laghi, P. Cimmaruta, R. (2006): A review of systematics, taxonomy, genetics, biogeography and natural specific differences in post-hatching behaviour, history of the genus Speleomantes Dubois, 1984 (Amphibia but rather some variation among females, which Caudata Plethodontidae). Atti del Museo Civico di Storia could depend on both environmental features and Naturale di Trieste 52. Lunghi, E., Manenti, R. Ficetola, G.F. (2014a): Do cave features physical status. As observed by Oneto et al. (2010) affect underground habitat exploitation by non-troglobite newborns never feed before leaving definitively species? Acta Oecologica 55: 29-35. the nest. Time of permanence inside the nest after Lunghi, E., Manenti, R., Manca, S., Mulargia, M., Pennati, R. Ficetola, G.F. (2014b): Nesting of cave salamanders (Hydromantes hatching might thus also depend on how long flavus and H. italicus) in natural environments. Salamandra 50: would last the yolk absorption by newborns 105-109. (Lunghi et al. 2014b). However, in contrast to the Morrison, C. Hero, J.M. (2003): Geographic variation in life-history characteristics of amphibians. Journal of Animal Ecology 72: observations performed on H. strinatii (Oneto et al. 270-279. 2010), in our study newborns were more inclined Oneto, F., Ottonello, D., Pastorino, M.V. Salvidio, S. (2010): to explore areas around the nest, even without Posthatching Parental Care in Salamanders Revealed by Infrared Video Surveillance. Journal of Herpetology 44: 649-653. their mother, as also reported by Papinuto (2005). Oneto, F., Ottonello, D., Pastorino, M.V. Salvidio, S. (2013): Nuovi This difference in behaviour might be due to inter- dati sul comportamento di cura parentale nei caudati: specific behavioural differences or to the different Hydromantes (Speleomantes) strinatii (Amphibia, Plethodontidae). Atti IX Congresso Nazionale della Societas Herpetologica environmental conditions (natural vs. semi- Italica, Bari-Conversano 26-30 settembre 2012. pp. 204-207. In: natural) which may influence the behaviour of Scillitani, G., Liuzzi, C., Lorusso, L., Mastropasqua, F. Ventrella, both mother and newborns. P. (eds), Atti IX Congresso Nazionale della Societas Herpetologica Italica, Bari-Conversano 26-30 settembre 2012. Our study adds new insights on the brooding Eds. Pineta, Conversano (Bari). behaviour of European cave salamanders; how- Oneto, F., Ottonello, D., Pastorino, M.V. Salvidio, S. (2014): ever information remains limited to a handful of Maternal care and defense of young by the plethodontid salamander Speleomantes strinatii (Aellen, 1951). Scripta species (Table 3), and for each species no more Herpetologica: Studies on Amphibians and reptiles in honour of than one population has been investigated. Fur- Benedetto Lanza. pp. 129-136. In Capula, M. Corti, C. (eds), ther researches, comparing the results of multiple Scripta Herpetologica: Studies on Amphibians and reptiles in honour of Benedetto Lanza. Edizioni Belvedere, Latina. nests and multiple populations will be necessary Papinuto, S. (2005): Sul ritrovamento ed il monitoraggio di una to better understand the complex breeding behav- nidiata di Speleomantes genei (Temminck & Schlegel, 1838) iour of these cryptic salamanders. (Amphibia Urodela Plethodontidae) in una galleria mineraria dell'Inglesiente (Sardegna sud-occidentale). Sardegna

Speleologica 22: 3-6. Pastorelli, C. Laghi, P. (2006): Predazione su Speleomantes italicus (Amphibia, Caudata, Plethodontidae) da parte di Meta menardi Acknowledgements. We are grateful to many friends that (Arachnida, Araneane, Metidae). Riassunti 6° Congresso took part in this long survey: Valentina Mirimin, Sandro Nazionale della Societas Herpetologica Italica. In Riassunti 6° Corona, Francesca Fais, Sandro Murru, Silvestro

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