**6L]HRIDGXOW***Speleomantes*

**Figure 2.** Boxplots showing the average size (TL) of adult *Speleomantes* considered in this study: *S. a. ambrosii* (163), *S. a. ambrosii* × *S. italicus* hybrids (28), *S. a. bianchii* (46), *S. italicus* (150), *S. italicus* × *S. a. bianchii* hybrids (125). The red box indicates introduced hybrids, blue natural hybrids, while in white the parental species.

#### **4. Discussion**

Studies on hybrid populations of *Speleomantes* have been extremely limited [8,11], leaving their morphological and ecological traits largely unknown. Despite our limited sample size, especially in regards to diet, we were able to obtain interesting information to provide the basis for future research. For example, we provide the first documentation relating to the natural colonization of a new environment performed by the introduced *Speleomantes*. Forty years after their release [10] the salamanders managed to occupy at least a nearby subterranean environment with a stable population. It is therefore of great importance to regularly monitor these hybrid populations to see if they will continue to extend their distribution.

The trophic niche of *Speleomantes* is subject to temporal variations [19,26] and is therefore strongly influenced by the fluctuating availability of potential prey [27]. With this study we were able to gather only limited information on the diet of *Speleomantes* hybrids. Most of the prey consumed were Diptera, which can be highly abundant near the entrance of subterranean environments, especially during the hot season [28,29]. This is in agreement with previous studies on the *Speleomantes* diet [16,30,31]. We also observed generally higher male foraging activity, whereas natural hybrids appear to forage less. Natural hybrids are distributed in a small area of the Apuan Alps (north-western Tuscany), a territory characterized by different lithology and vegetation compared to that of other studied salamanders [1,32,33]. Diverging environmental characteristics may offer different prey availability or simply alter the peak of foraging activity in these *Speleomantes* populations [24,27]. Therefore, further and prolonged investigations on multiple hybrid populations are needed to better delineate their trophic niche [17], evaluate potential divergences [27,30], and evaluate whether they increase foraging opportunities by expanding their microclimatic niche.

A further limitation of this study is the lack of genetic information on hybrid *Speleomantes* populations. Hybrid populations can undergo complex evolutionary phenomena, for example, with selective advantages for some components of their genome. To date, the available information on the genetic characteristics of hybrid populations is limited to allozyme data, which cannot capture the complexity of genomic processes [8]. Collecting genome-wide information on salamanders is challenging because their very large genome size makes the application of different genomic tools, such as RAD sequencing, problematic. However, recent developments can allow more affordable analysis of genome-wide variation, even in species with large genomes, and in the coming years this could improve our understanding of evolutionary processes affecting these populations [31,32].

In this study we observed larger size in introduced hybrids (*S. a. ambrosii* × *S. italicus*), *S. italicus*, and the *S. a. bianchii* subspecies. We do not have data on the potential genetic effect on the size of hybrid populations. However, ecological conditions are more likely to play an important role in driving the evolution of this adaptive trait. It has been shown that *Speleomantes* are in thermal equilibrium with their surrounding environment, and that larger individuals require more time to thermoconform, and are thus potentially able to exploit a less suitable microclimate for a longer period [8,20,33], allowing them to extend their activity period and increase foraging [24]. The larger size observed in introduced hybrids may be the result of a warmer and drier local climate, whereas the smaller size of natural hybrids may have been determined by the local colder and moister climatic conditions. This remains a hypothesis that deserves further investigation. Furthermore, introduced hybrids showed a lower frequency of empty stomachs compared to other population; thus, it is possible that higher food availability allows for better feeding and growth. However, this hypothesis is not supported by the lack of differences in the body condition index. Considering the larger size of the introduced hybrids (and the associated better tolerance of harsher conditions), we recommend regular monitoring of these populations to better control their range expansion, especially in the context of global warming [34,35].

**Author Contributions:** E.L. conceived the study, analysed the data, and prepared the figures; E.L., S.M., R.M., G.F.F. and C.C. performed field surveys; E.L. and F.C. performed the recognition of stomach contents; E.L., F.C., S.M., Y.Z., R.M., G.F.F. and C.C. reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** The APC was funded by National Natural Science Foundation of China (NSFC-31972868).

**Institutional Review Board Statement:** This study was authorized by the Italian Ministry of the Environment (Ministero dell'Ambiente e della Tutela del Territorio e del Mare, PNM prot. n.56097/T-A31 of 13/11/2017 and integration, prot. n. 7513 of 17/02/2020).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data analysed in this study can be found at this link https://figshare. com/s/387b39c7de65dfc32298.

**Acknowledgments:** This study was conducted under the auspices of the Chinese Cavefish Working Group. Enrico Lunghi is supported by the Chinese Academy of Sciences President's International Fellowship Initiative for postdoctoral researchers. Caves' coordinate may be provided upon justified requests.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

