Amphibians are facing population declines and extinctions, and protecting and supplementing refuges can help species survive [22]. However, the microhabitat requirements of most species are unknown, and artificial refuges have not been well tested for amphibians [5, 22]. In this study, we first evaluated the post-reproductive dormancy site selection of R. dybowskii in experimental enclosures to determine its dormancy site preferences. We then investigated the effects of shelter and shade on R. dybowskii behaviour and survival following post-reproductive dormancy.
Effects of temperature on habitat selection
Among the conditions that influence the activity or distribution of frogs, the most important may be temperature and water [25, 26]; in particular, low temperature may be a major inducer of post-reproductive dormancy in R. dybowskii [21]. In the present study, R. dybowskii live mainly under leaves, soil, and stones, and different dormant sites were significantly different. According to wood frog post-breeding habitat utilization, frogs prefer locations with deeper leaf litter layers, warmer air, and less humidity and light [27]. Post-breeding adult wood frogs rarely migrate long distances and seek areas near water to avoid desiccation and predators [16]. The habitat of post-breeding salamanders was positively associated with slash cover and negatively associated with grass, tree basal area, and humidity, and salamander habitats had leaves that were deeper and more humid [28]. Despite the similarity of the microhabitats chosen by these animals, there are differences. It is possible that other animals choose these habitats for feeding and living, whereas brown frogs select habitats for dormancy. In this study, in the early stage of post-reproductive dormancy, the average temperature over a 20-d period was lower than 10 °C (Fig. S3), while R. dybowskii chose a cool and hidden microhabitat for dormancy instead of an exposed habitat with a faster temperature rise [11, 29]. These places are not exposed to sunlight, the temperature is lower than that of unobstructed places, the temperature fluctuations are relatively small, and the humidity is greater.
Many species, including R. dybowskii, require both aquatic and terrestrial habitats, yet there is insufficient information on patterns of fine selection in these habitats [30]. Moisture is a necessary requirement for nearly all amphibians [26, 31], which select dormant habitats based on both humidity and temperature, especially humidity [22]. In the present study, after spawning, R. dybowskii was mostly dormant in terrestrial habitats near the spawning pools. Most R. dybowskii hibernate in water; young R. dybowskii that have not reproduced still hibernate in water [32]; however, in this study, R. dybowskii rarely chose to hibernate in water after breeding in water. In spring, variable water temperatures do not provide a cool resting environment for R. dybowskii [33], and R. dybowskii continuously enters and departs the water, which may not be conducive to dormancy. Away from water, amphibians are at risk of desiccation, and the nearby spawning pond provides many suitable humidity gradients, which may provide a suitable humidity environment after breeding; thus, sites that they choose to occupy are likely to contain elements that reduce this risk [34].
The effect of temperature (shade) on frog behaviour
Temperature impacts animals, especially ectotherms, in all areas of their existence and survival [9]. Latitude, altitude, weather, and habitat composition affect organism temperatures [22, 35]. Our research has shown that there is no difference in the survival rate of frogs under different shade and time conditions. Some studies have shown that shade affects amphibian populations, and it may be that shade affects amphibians differently at different developmental levels. Captive-bred and reintroduced dusky gopher frogs prefer open canopies and rich ground plants and have a high survival rate [2]. Comparing the reactions of six species of tropical amphibian tadpoles inhabiting thermally opposed open and shade habitats revealed that open habitat species frequently adapt more rapidly than shade habitat species [9]. Tree removal from historically open-canopy ponds promotes the recovery of open-habitat species [6]. In contrast, shade can impact the growth rate of gopher frogs, prolong the larval phase, lower survival rates, and diminish the quality of metamorphosis [36]. The amount of heat-related mortality at some propagation locations can be decreased by increasing the canopy cover [6]. There are differing views on the effects of shade on amphibian behaviour and populations, especially as climate change is causing an accelerated increase in average temperatures and extreme heat events [9].
During dormancy (between 25 April and 10 May), there was no significant effect of shading on the activity. Shade has no discernible impact on R. dybowskii activity at this time since it has previously entered dormancy to find a suitable inactive spot and goes dormant [22]. Rana dybowskii may be mainly affected by temperature (they hibernate mainly in shelters); low temperature inhibits frog activity [25]. Our studies have shown that frogs have different activity frequencies under different shade and time conditions, with a particularly pronounced performance after 4 May. Captive-bred and reintroduced dusky gopher frogs prefer open canopies and rich ground plants [2]. Terrestrial ectotherms adjust their body temperatures behaviourally to maintain ideal body temperatures by varying their daily or seasonal use of shady or sunny microhabitats [37, 38]. To maintain optimal body temperatures, R. dybowskii behaviourally regulates body temperature by altering the daily or seasonal use of shaded or sunny microhabitats [37, 39]
Refuges and burrow
Amphibians use refuges or shelters for a variety of reasons, including ambush feeding, buffering, reducing osmoregulatory and thermal stresses, and predator avoidance [40]. Often to lessen the threat of predation or to improve adaptability and access to resources, animals must consider the physical characteristics of the shelter (such as size and construction materials), as well as the surrounding environmental conditions (vegetation, entrance orientation, and distance to resources) [5]. In the present study, the role of refuges may be different during dormant and active periods.
Almost all R. dybowskii in our study used shelters (rocks and loose soil holes) during the dormant period; some frogs even dug holes to make shelters during dormancy (Fig. 4). The typical harsh spring conditions of rain, wind and cold do not provide favourable conditions for R. dybowskii to end their dormancy (Green et al. 2016). Thus, the fate of R. dybowskii exposed to harsh springtime conditions will depend on whether they can find locally abundant refuges that buffer against life-threatening conditions [11, 12]. Although we have not examined the microclimate within the shelter, previous research indicates that tree hollows create a more stable microclimate than their surroundings, sustaining lower temperatures and greater humidity during the day and higher temperatures and lower humidity at night [41]. Previous research has demonstrated that common frog microhabitats (e.g., soil, tree holes) can reduce exposure to temperature extremes by 14–31 times, hence reducing population vulnerability by a factor of 108 and potentially reducing death during extreme weather events [11]. By providing a proper microclimate, refuges or shelters may have thermoregulatory and osmoregulatory effects; they may also minimize physiological stress in terrestrial anurans induced by heat, cold, and drought [42,43,44]. During the early stages of dormancy, the shelter may also function as concealment in areas where R. dybowskii repose, allowing them to evade predators and providing a safe environment for the frogs during dormancy [40].
In this study, the majority of frogs found refuge or shelter at the experimental site. Frogs in soil shelters were more active than those in stone shelters, but there was no difference in their survival rates. Previous research has demonstrated that individuals without shelter spend more time digging and that all frogs spend a considerable amount of time beneath shelter [40]. Significant impacts of shelter (plant provision) on captive red-eyed tree frog body size and growth rates suggest a fitness benefit [18]. Frogs hiding in underground shelters had a 22% higher survival rate than frogs not hiding underground compared to frogs released into burrows; frogs released at ponds had a 33% lower survival rate, spent less time underground, and moved farther and more often in search of shelter [2]. Several studies have found increased movement of amphibians on ground shelters without litter or on bare ground [45]. The soil shelters in this study were more likely to be damaged with wind and rain than the stone shelters, and the frog's cover was damaged and the frequency of activity increased. Our results are consistent with these studies, suggesting that bare ground and limited shelter availability may be important drivers of increased movement and decreased settlement in poor habitats, possibly in response to higher predation and desiccation risks [4].
The two strategies used by frogs to dig burrows to avoid desiccation appear to be different, and the type of soil has an effect on both strategies [8, 46]. In sandy or crumbly soils, anurans can easily burrow, and when the earth is dry, these frogs tunnel even deeper to remain below the dry front [46]. To prevent dehydration, frogs prefer to stay in moist soil, and several species can often dive to a depth of 90 cm [8]. Anurans that inhabit dense, clay-rich soils use a different technique. Anurans tunnel 10–30 cm below the soil's surface in this soil [47]. As the earth around them dries, the frogs construct a cocoon from exfoliated skin and mucus [46]. In this study, the soil cut-off was deeper than the rock-medium cave. It may be that loose soil is easier to burrow into. Amphibians may rehydrate by absorbing water from the soil around them; therefore, it was thought that when burrowing anurans were exposed to dry conditions, they would dig deeper into the shelter to find moisture and rehydrate [8, 46]. However, irregularly wet rocks tend to aid in water conservation, resulting in shallower borrows.
Conservation implications
There are currently few meaningful management actions that will have a tangible effect on amphibians' vulnerability to climate change [6]. Numerous potentially beneficial but untested actions could be implemented into local or regional amphibian management plans, programs, and activities [6]. Examples include retaining or supplementing artificial and natural shelters to mitigate desiccation and thermal stress and adjusting the shade fabric covering habitats to maintain an ambient temperature [6]. The complexity of the environment, specifically shelter or the provision of cover, may be a crucial factor to consider when constructing enclosures for captive amphibian rearing [18]. Captive red-eyed tree frogs exhibit a satisfactory growth rate under covered conditions, indicating that providing shelters is preferable for this type of frog [18, 48]. For Xenopus, shelter cover appears to confer behavioural benefits but has no impact on the growth or body conditions of this type of frog, whereas for nonmodel taxa, shelter cover might exert positive effects on growth as well as behaviour [18, 40].
In this study, stones were used as a shelter during the dormant period for R. dybowskii in terrestrial habitats. It is necessary to consider a type of shelter conducive to a particular frog's dormancy. Previous research suggested that PVC pipes and cover boards could provide suitable shelter for some amphibians [6, 13]. The temperature change in PVC pipes is usually larger than that under natural shelters [6]. We recommend the use of a thin cystosepiment as a shelter or refuge, as cystosepiment has many advantages, such as it does not press down on R. dybowskii, does not irritate their skin, is easy to clean and disinfect, and is slow to conduct heat in direct sunlight. However, in the dormant state, many R. dybowskii individuals are crowded together, pressing against each other; if some individuals move, the remainder may be easily disturbed [49]. Therefore, more research is needed to better understand frog microhabitat needs and to evaluate different shelter designs [6].