SALAMANDRA 54(2) 101–108 15 MayReproduction 2018 ISSNin European 0036–3375 cave salamanders Comparative reproductive biology of European cave salamanders (genus Hydromantes): nesting selection and multiple annual breeding Enrico Lunghi1,2,3, Claudia Corti2, Raoul Manenti4, Benedetta Barzaghi4, Salvatore Buschettu5, Claudia Canedoli6, Roberto Cogoni7, Giovanni De Falco5, Francesca Fais5, Alessandra Manca5, Valentina Mirimin5, Manuela Mulargia8, Cinzia Mulas8, Martina Muraro4, Rosalba Murgia5,10, Michael Veith1 & Gentile Francesco Ficetola4,11 1) Department of Biogeography, University of Trier, Trier, Germany 2) Natural History Museum of University of Florence, Section Zoology “La Specola”, Firenze, Italy 3) Natural Oasis, Prato, Italy 4) Department of Environmental Science and Policy, University of Milan, Milano, Italy 5) Speleo Club Oristanese, Oristano, Italy 6) Department of Environment and Territor Science, University of Milan-Bicocca, Milano, Italy 7) Unione Speleologica Cagliaritana, Quartu Sant’Elena, Italy 8) Speleo Club Nuoro, Nuoro, Italy 9) Gruppo Grotte Ogliastra, Perdasdefogu, Italy 10) Fondazione IMC – International Marine Centre – Onlus, Oristano, Italy 11) Univ. Grenoble Alpes, CNRS, Laboratoire d’Écologie Alpine (LECA), Grenoble, France Corresponding author: Enrico Lunghi, e-mail: [email protected], Biogeography Department, Trier University, Universitätsring 15, 54286 Trier, Germany Manuscript received: 30 June 2017 Accepted: 24 December 2017 by Alexander Kupfer Abstract. Information on the life history of European cave salamanders remains limited. Despite a handful of studies carried out both under natural and controlled conditions, one of the least known aspects concerns the reproductive and breeding behaviour. Here we present information on the breeding behaviour of all the eight European Hydromantes spe- cies collected during four years of intensive monitoring (2014–2017). We provide information on the breeding phenology, suggesting that the breeding seasons are likely linked to environmental variables. Nesting sites were used repeatedly in different years by different females. Our data indicated a seasonality of mating activity and the possibility of sperm stor- age. Furthermore, we report on the presence of a single large-sized oviductal egg in H. imperialis. Finally, we documented oviparity in Hydromantes sarrabusensis, the only species that was suggested to be viviparous. Key words. Speleomantes, Plethodontidae, cave, Amphibia, Urodela, mating behaviour, Mediterranean, biospeleology, sea- sonality. Introduction tal conditions, i.e., high moisture and cool temperature (Spotila 1972). Underground habitats (e.g., caves, mines, European cave salamanders (genus Hydromantes; see cracks and other epikarst-environments) are therefore op- Wake 2013 for discussion on nomencalture) include eight timal refuges from adverse outside environmental condi- species distributed in Italy and in a small part of South- tions (Ficetola et al. 2012, Culver & Pipan 2014, Lunghi Eastern France (Lanza et al. 2006a, Sillero et al. 2014). et al. 2014a). Even though underground detection of sala- The two main characteristics of these amphibians are the manders is higher in summer, these habitats are exploit- lack of lungs and direct development (Lanza et al. 2006b). ed throughout the year, suggesting that they are extremely Lack of lungs forces these animals to rely on cutaneous and suitable for these species (Lunghi et al. 2015a); moreover, bucco-pharyngeal respiration only (Lanza et al. 2006b), besides the suitable microclimate, they mostly lack preda- which occurs efficiently only in specific environmen- tors (Pastorelli & Laghi 2006, Manenti et al. 2016, Sal- © 2018 Deutsche Gesellschaft für Herpetologie und Terrarienkunde e.V. (DGHT), Mannheim, Germany Available at http://www.salamandra-journal.com 101 Enrico Lunghi et al. vidio et al. 2017a), and, therefore, represent an optimal only one breeding season occurs during the warmer pe- choice to carry out a delicate life-phase such as reproduc- riods of the year (Papinuto 2005, Lunghi et al. 2014b, tion. Lunghi et al. 2015b, Murgia et al. 2016). b) Do cave sala- Giving the elusive habits of Hydromantes, many of their manders show nest site selection? Observations performed life-history traits are still poorly known, and reproduction in nature described the location chosen by females to nest, is among the less known traits. During the last years, sev- but it is not known whether and how females select the eral studies on the biology and physiology of these sala- breeding site, nor if nest-site fidelity occurs. The present manders have been performed to better understand their long-term study attempts to answer the above mentioned reproduction (all related papers are gathered in the latest questions, adding unpublished insights to the behaviour of review written by Lanza et al. 2006b). Sexual maturity in these salamanders. Furthermore, for the first time ovipar- Hydromantes is generally reached at the third or fourth ity in Hydromantes sarrabusensis is reported, a species that year, while in the big-sized Sardinian species is likely was thought to be viviparous (Lanza et al. 2006b). reached a bit later (Salvidio 1993, Salvidio et al. 2017b). Courtship has been observed throughout the year, while the deposition of spermatophores was observed only in Materials and methods autumn (for further details on courtship see also Bruce et Data collection al. 2000, Lanza et al. 2006b). However, given that in these species the pachytene spermatocytes do not degenerate During a period of four years (2014–2017) we performed during the cold season, it is likely that European cave sal- extensive field activities with the aim to cover as much amanders can mate throughout the year (Mertens 1923, as possible the distribution range of the eight European Lanza 1959). Hydro mantes species (Lanza et al. 2006a). Overall, we Besides some physiological analyses, only a few studies surveyed > 150 underground sites (caves and mines) where performed under controlled conditions were focused on the salamanders were present. Twenty-two of them (sev- Hydromantes’ breeding behaviour (Durand 1967, Lanza en for H. flavus, three for H. supramontis, three for H. im­ et al. 2006b, Oneto et al. 2010, Oneto et al. 2014). Re- perialis, three for H. genei, three for H. sarrabusensis and cently some authors monitored clutches in nature, im- three for H. ambrosii) were repeatedly surveyed during the proving the knowledge on this topic and allowing com- entire study period. Surveys on H. flavus were performed parison with findings obtained in controlled conditions during all seasons, while for the other species they were (Papinuto 2005, Lunghi et al. 2014b, Lunghi et al. 2015b, mainly concentrated in spring/early summer and autumn. Murgia et al. 2016). The first field report on the breeding During each survey, we first checked if salamanders were behaviour was provided by Papinuto (2005), who moni- performing courtship behaviour. These salamanders adopt tored one clutch of H. genei in an abandoned mine. The the so called “vaccination”: after mounting on the female, female was observed curled up on her eggs for nearly five males use their pre-maxillary teeth to produce scratches months until hatching occurred. This behaviour not only on the females skin in order to transfer the hedonic secre- allows the transfer of her skin secretion to the eggs provid- tions produced by their mental gland directly in the female ing protection against bacteria and fungi, but also protects circulatory system; all these actions are performed with the nest from intruders (Lanza et al. 2006b, Oneto et al. the purpose to increase the female interest and make her 2014). Monitoring activities on clutches was also carried willing to collect the spermatophora (Lanza et al. 2006b). out on H. italicus, H. flavus and H. imperialis (Lunghi et We then measured snout–vent length (SVL, using a plastic al. 2014b, Lunghi et al. 2015b, Murgia et al. 2016). Find- transparent rule) to the nearest mm in females salaman- ings of these studies were generally consistent with those ders, considering gravid those showing fully developed observed for H. strinatii in controlled conditions (Oneto eggs (~3–5 mm in diameter) visible in the abdominal cav- et al. 2010, Oneto et al. 2014). Embryos require a long ity; when possible, we also recorded salamanders’ distance time to develop before being ready to hatch, and during from the cave entrance. Using Linear Mixed Models, we this span of time the mother rarely leaves the clutch unat- tested whether gravid females occupy different cave are- tended (Lanza et al. 2006b, Lunghi et al. 2014b, Oneto et as when compared to the non-gravid ones. For this anal- al. 2014). After hatching, the mother provides parental care ysis we used the square-root transformed distance from to the hatchlings for some weeks before they leave the nest the cave entrance as dependent variable, female condi- (Oneto et al. 2010, Lunghi et al. 2015b). Murgia et al. tion (gravid/non-gravid) and species identity as depend- (2016) also observed that the highest density of nest sites ent variables, and cave identity as random factor. We then was found in a section of a cave showing the most stable used Linear Models to test whether gravid females showed microclimate, characterized by high humidity levels (close larger size. To avoid pseudo-replication, for this analysis to saturation) and a yearly fluctuation of temperature of we only used the females