Winter habitats of bats in Texas.

Few studies have described winter microclimate selection by bats in the southern United States. This is of particular importance as the cold-adapted fungus, Pseudogymnoascus destructans, which causes the fatal bat disease white-nose syndrome (WNS), continues to spread into southern United States. To better understand the suitability of winter bat habitats for the growth of P. destructans in this region, we collected roost temperature and vapor pressure deficit from 97 hibernacula in six ecoregions in Texas during winter 2016-17 and 2017-18. We also measured skin temperature of Rafinesque's big-eared bats (Corynorhinus townsendii), Townsend's big-eared bats (C. townsendii), big-brown bats (Eptesicus fuscus), southeastern myotis (Myotis austroriparius), cave myotis (M. velifer), tri-colored bats (Perimyotis subflavus), and Mexican free-tailed bats (Tadarida brasiliensis) during hibernation to study their use of torpor in these habitats. We found that temperatures within hibernacula were strongly correlated with external air temperatures and were often within the optimal range of temperatures for P. destructans growth. Hibernacula and skin temperatures differed among species, with Rafinesque's big-eared bats, southeastern myotis, and Mexican free-tailed bats occupying warmer microclimates and having higher torpid skin temperatures. For species that were broadly distributed throughout Texas, hibernacula and skin temperatures differed within species by ecoregion; Tri-colored bats and cave myotis in colder, northern regions occupied colder microclimates within hibernacula and exhibited colder skin temperatures, than individuals of the same species in warmer, southern regions. These data illustrate the variability in microclimates used as hibernacula by bats in Texas and suggest similar variation in susceptibility to WNS in the state. Thus, monitoring microclimates at winter roosts may help predict where WNS may develop, and where management efforts would be most effective.

Introduction

The choice of overwintering habitat has numerous impacts on mammalian hibernators. Suitable hibernacula must provide an appropriate thermal environment. Defining appropriate thermal habitats has been the focus of research for decades, as the temperatures inside hibernacula profoundly influence the metabolic rates, and, therefore, the energy budgets of mammalian hibernators [1-3]. During torpor, metabolic rates decline with ambient temperatures until conditions fall below a hibernator’s thermoregulatory set point, at which point metabolic rate increases [1]. Although this relationship indicates that there are temperatures where energetic savings are greatest, hibernators often select greater temperatures for both energetic and non-energetic reasons [4-6]. Thus, habitat selection is central to the winter ecology and physiology for mammalian hibernators, and an understanding of this process can help predict how species will be affected by continued environmental change [7].

Many studies of North American subterranean bat species are focused on regions with cold winter climates (e.g., [8-9]), with less research conducted in warmer, more southern regions of the continent [10-12]. However, research outside of North America shows that seasonal torpor is not limited to cold climates or areas with severe winters. Hibernation in warmer climates appears to be typified by short bouts of torpor and relatively high torpid body temperatures [13]. Although energetic challenges of winter in these warm regions may seem less compelling than the challenges posed by colder climates, the use of seasonal torpor in subtropical regions provides testament to its importance in the life cycles of many mammals [13-17].

Unfortunately, microclimates suitable for hibernation may also place bat species in North America at risk to white-nose syndrome (WNS) [18-20]. WNS is an emerging infectious disease of North American bats caused by the cold-tolerant fungus, Pseudogymnoascus destructans, which thrives in caves, underground mines, and other features that many bats use as hibernacula [18, 21–22]. P. destructans infects dermal tissue of hibernating bats, leading to dehydration and death [23-25]. The fungus has spread rapidly from where it was first documented in North America in New York State in 2006, annually spreading to new regions [18, 26–27]. P. destructans grows optimally between 12.5° and 15.8° C, although it can persist at temperatures from 3.0–19° C [20]. Thus, P. destructans can survive in a range of habitats that are important for bats during hibernation and is likely to continue to spread to new regions and cause mass mortalities [28].

Because the effects of WNS vary among species and environmental temperatures [19, 29–30], a detailed understanding of winter habitats used by different species in different regions is needed as the causative fungus continues to spread. The need for these data is illustrated by the recent spread of P. destructans into southern United States, including Texas [31-33] where it is still uncertain if bats will develop WNS, and if so, how severe mortality might be. Although Texas bats first tested positive for P. destructans during spring 2017, no WNS mortalities or bats with histopathological symptoms of the disease have been confirmed in the state [31-33]. Understanding the unique winter ecologies of WNS-susceptible species in the southern United States is therefore imperative in understanding the spread of P. destructans and determining species and regions most at risk from WNS.

Based on existing studies, it would be reasonable to predict notably different species-specific outcomes as WNS expands to hibernacula located in warmer climates such as Texas. Webb and colleagues [8] summarized inter- and intraspecies variations in the temperatures at which various bat species were found hibernating. Although most of the data reported were collected from northern geographic ranges of North America, the authors suggested that species inhabiting cold climates can hibernate in colder conditions than species inhabiting warm climates. This prediction is notable because warmer hibernacula temperatures would likely result in higher torpid body temperatures, higher torpid metabolic rates, and greater rates of periodic arousals from torpor [34-36]. If bats in subtropical regions are adapted to frequent arousals from winter torpor, such as those seen in WNS-affected bats, impacts from the disease may be less than observed in the temperate region of North America. However, the higher hibernacula temperatures that one might expect in the subtropics may result in more favorable conditions for P. destructans to thrive compared to those farther north [11, 20], potentially resulting in more severe infections. Thus, WNS pathology in areas such as Texas is difficult to predict in the absence of data on winter habitat selection and behavior in the region. Despite this need, few researchers have studied hibernation in southern populations of bat species with broad distributional ranges [11, 37–39].

To improve our overall understanding of the suitability of bat habitats for P. destructans—and potentially WNS—in Texas, and to better inform management practices, we described the microclimates used by seven bat species as hibernacula across the state. We predicted that temperatures inside hibernacula would strongly correlate with external temperatures, and would be suitable for P. destructans growth. Based on species distribution and previous research on winter activity [40-42], we predicted that Rafinesque’s big-eared bats (Corynorhinus rafinesquii), southeastern myotis (Myotis austroriparius), and Mexican free-tailed bats (Tadarida brasiliensis) would select microclimate within hibernacula having higher ambient temperatures and lower vapor pressure deficit (VPD) than all other species, and would subsequently have warmer torpid skin temperatures. We hypothesized that two species commonly found throughout Texas, the cave myotis (Myotis velifer) and tri-colored bat (Perimyotis subflavus), would exhibit variation in their microclimate selection across the state. Because bats hibernating in cold climates would be exposed to lower ambient roost temperatures, we predicted that they would therefore have lower skin temperatures than bats in warm climates. Finally, we predicted that tri-colored bats hibernating in culverts would have higher skin temperatures than those roosting within caves due to the more exposed and variable conditions present within culverts.

Materials and methods

All methods followed ASM guidelines [43] and were approved by the Texas A&M Institutional Animal Care and Use Committee (IACUC 2015–0296).

Study area

We conducted surveys for overwintering bats throughout Texas. Texas exhibits substantial variation in landscape, environment, latitude and climate, and is characterized by 12 level III ecoregions [44-46]. Thirty-three bat species have been documented across these ecoregions, the richest diversity of bat species found in any state in the United States [47]. We conducted surveys between November and March of 2016–2017 and 2017–2018 at 97 hibernacula, including 44 caves, 45 culverts, 3 buildings, 3 bat towers, 1 tree, and 1 bat box. We obtained access to caves through information provided by the Texas Speleological Society (TSS), Texas Cave Management Association (TCMA), Texas Grottos, Texas Parks and Wildlife biologists, and private landowners, and obtained access to other sites (i.e., buildings, bat towers, tree, and bat box) from landowners and biologists. We obtained information on historic culvert bat colonies from previous literature [48-49], from the Texas Department of Transportation (TxDOT), and from biologists. We randomly selected 77 10 x 10 km grid cells across the state using the Generalized Random Tessellation Stratified (GRTS) design [50] of the North American Bat Monitoring Program (NABat). Within those grid cells we surveyed an additional 158 culverts, of which eight contained bats. All sites included in our study were located across the following six level III ecoregions: Chihuahuan Deserts, East Central Texas Plains, Edwards Plateau, South Central Plains, Southwestern Tablelands, and Texas Blackland Prairies (Fig 1).

Fig 1

Map of 97 hibernacula surveyed in Texas.

Map of the 12 level III ecoregions of Texas [46] and 30-year normal mean temperature at 4 km grid cell resolution (1981–2010) [51]. Ecoregions are numbered based on the original publication. (+) indicate the 97 hibernacula surveyed from November–March 2016–17 and 2017–18 across six of 12 level III Texas ecoregions (Chihuahuan Deserts, East Central Texas Plains, Edwards Plateau, South Central Plains, Southwestern Tablelands, Texas Blackland Prairies).

Data collection

We conducted hibernacula surveys between 08:00–17:00 hrs. To reduce disturbance, we collected all data without any animal handling. Upon entrance into the hibernacula, we identified species present based on visual characteristics and counted the number of bats belonging to each species. We collected data on all bat species observed at each site.

To assess the relationship between external and hibernacula air temperatures, and to understand how representative our single visits to hibernacula were of the entire winter season, we deployed EL-USB-2 temperature and humidity data loggers (EasyLog, Lascar Electronics, PA, USA) programmed to record internal temperature (site temperature) every hour from October–March at two caves and two culverts occupied by bats. We retrieved average external daily temperature from PRISM Climate Database [52] for those sites. These values were taken at 4 kilometer grid cell resolution.

To assess the microclimates within each site used by hibernating bats, we collected roost temperature and relative humidity (RH) within 5 cm of each hibernating bat or cluster using a temperature and humidity pen (Extech 445580). In order to take into account the relationship between temperature and RH, we calculated vapor pressure deficit (VPD) which describes the difference between the amount of moisture in the air and how much moisture the air can hold when saturated [53-54]. We converted RH to VPD by first calculating the saturation water vapor pressure (SWVP) for every roost temperature recorded and then multiplying SWVP by RH to produce the actual water vapor pressure (AWVP) [55]. VPD was calculated by subtracting AWVP from SWVP. We also measured skin temperatures of bats from a maximum distance of 0.3 m using a digital infrared thermometer (Extech; Waltham, MA) to gather baseline data on torpor behaviors in each species. We collected these skin temperature readings from the back of each bat. Although infrared thermometers are not the preferred method to measure bat skin temperature, they are a non-invasive and quick method of assessing temperature, and have been used in other research with success [56]. In order to ensure that we recorded reliable skin temperatures from torpid bats, we did not collect data from any bats that showed visible signs of arousal (e.g., vibrating, movement). To determine if bats were hibernating at the temperature within the hibernacula, we also determined substrate temperature adjacent to roosting bats.

Data analysis

To test the hypothesis that site temperature is related to external temperature, we used a Pearson product-moment correlation. To test the hypothesis that Rafinesque’s big-eared bats, southeastern myotis, and Mexican free-tailed bats would roost in areas of hibernacula with warmer roost temperatures and higher AWVP, and would therefore have higher torpid skin temperatures than all other species, we used Kruskal-Wallis H-tests (comparisons of roost temperatures and skin temperatures among species were made using separate tests). We used non-parametric statistics to analyze these data because Shapiro-Wilks tests indicated that both roost temperatures and skin temperatures for each species were not normally distributed [57]. Prior to analysis, we averaged skin temperatures, roost temperatures, substrate temperatures and VPD per site for each species to avoid pseudoreplication. We also used Pearson’s product-moment correlations to test for a linear relationship between skin temperature and substrate temperature, and test for a linear relationship between skin temperature and roost temperature.

We tested our hypothesis that skin temperatures and roost temperatures of tri-colored bats hibernating in cold climates in northern Texas (Southwestern Tablelands) would be colder than those in the warm climates of southern Texas (Chihuahuan Deserts, Edwards Plateau, South Central Plains) using Kruskal-Wallis H-test. We performed post-hoc comparisons of groups using Dunn’s tests, adjusting P-values using the Benjamini-Hochberg method to decrease the false discovery rate. Because we only found cave myotis in two ecoregions, we used a Wilcoxon rank-sum test to test our hypothesis that the skin temperatures of cave myotis in cold climates would be colder than the skin temperatures of cave myotis in warm climates. Finally, to compare roost temperatures and skin temperatures of tri-colored bats hibernating in culverts to those in caves between ecoregions, we used a Wilcoxon rank-sum test. All means are reported with standard deviations (± SD). We considered a P-value ≤ 0.05 significant for all tests. All analyses were performed in Program R v. 3.4.1 [58].

Results

We collected data from the following seven bat species: Rafinesque’s big-eared bat, Townsend’s big-eared bat (C. townsendii), big-brown bat (Eptesicus fuscus), southeastern myotis, cave myotis, tri-colored bat, and Mexican free-tailed bat. Presence of these bat species varied by ecoregion [41] (Table 1).

Table 1

Total count of bats by species for six level III ecoregions in Texas.

Ecoregion
	CD	ECTP	EP	SCP	ST	TBP
CORA	-	-	-	16	-	-
COTO	0	-	-	-	52	-
EPFU	0	0	0	2	3	0
MYAU	-	7	-	13	-	-
MYVE	0	0	46	-	112	0
PESU	10	381	191	152	30	20
TABR	0	2	20	0	0	0

Total count of bats by species (Corynorhinus rafinesquii = CORA, C. townsendii = COTO, Eptesicus fuscus = EPFU, Myotis austroriparius = MYAU, M. velifer = MYVE, Perimyotis subflavus = PESU, and Tadarida brasiliensis = TABR) for six level III ecoregions (Chihuahuan Deserts = CD, East Central Texas Plains = ECTP, Edwards Plateau = EP, South Central Plains = SCP, Southwestern Tablelands = ST, and Texas Blackland Prairies = TBP). Data were collected from 97 hibernacula in Texas between November–March 2016–2017 and 2017–2018. (0) denotes ecoregions where the species is historically present but was not documented. (-) denotes ecoregions where the species has not been previously documented.

Site temperature was highly correlated with external temperature for both occupied caves and culverts (Fig 2; P ≤ 0.05). Between October and March, the average roost temperatures for the two caves ranged from 2.29–20.90° C (Cave 1) and 12.38–21.31° C (Cave 2) (Fig 2). During the same period, the average roost temperatures for the two culverts ranged from 0.19–23.10° C (Culvert 1) and 6.21–23.00° C (Culvert 2) (Fig 2).

Fig 2

Graphs of temperature profiles for cave and culvert hibernacula.

Average daily internal (black lines) and external (gray lines) temperatures (° C) recorded at two caves and two culverts occupied by overwintering bats in Texas using an EL-USB-2 environmental data logger deployed from 1 October 2016–31 March 2017. Dashed lines indicate the dates when single-visit surveys were conducted to collect skin and microclimate temperature data from hibernating bats. Gray horizontal bars indicate the optimal growth range of P. destructans (12.5–15.8°C) [20]. r represents the correlation coefficient between internal and external temperature with corresponding degrees of freedom.

Roost temperatures differed among species (K = 30.27, d.f. = 6, P = < 0.001; Fig 3A). Rafinesque’s big-eared bats were found at hibernating in areas with significantly higher roost temperatures ( = 22.20 ± 4.21) than all other species except Mexican free-tailed bats and southeastern myotis ( = 19.67 ± 3.93; = 17.36 ± 2.49, respectively). The roost temperatures at which Mexican free-tailed bats were found did not differ significantly from the roost temperatures at which tri-colored bats, big brown bats, and cave myotis were found ( = 15.79 ± 3.69; = 11.73 ± 5.57; = 14.39 ± 4.87, respectively). Townsend’s big-eared bats were found at significantly lower roost temperatures ( = 11.18 ± 4.36) than Rafinesque’s big-eared bats, southeastern myotis, tri-colored bats, and Mexican free-tailed bats but were not significantly different to the roost temperatures at which big brown bats and cave myotis were found.

Fig 3

Box and whisker plots of average roost temperature, average skin temperature, and vapor pressure deficit for seven bat species.

Box and whisker plots displaying the median, and upper (75%) and lower (25%) quartile range of (A) average roost temperatures, (B) average skin temperatures, and (C) average vapor pressure deficit (VPD) collected between November–March 2016–2017 and 2017–2018 in Texas for the following seven bat species: Rafinesque’s big-eared bat (Corynorhinus rafinesquii = CORA), Townsend’s big-eared bat (C. townsendii = COTO), big-brown bat (Eptesicus fuscus = EPFU), southeastern myotis (Myotis austroriparius = MYAU), cave myotis (M. velifer = MYVE), tri-colored bat (Perimyotis subflavus = PESU), and Mexican free-tailed bat (Tadarida brasiliensis = TABR). Significant differences between groups (P ≤ 0.05) are distinguished by letters. Filled circles indicate outliers.

Skin temperature differed among species (K = 41.37, d.f. = 6, P = < 0.001; Fig 3B). Rafinesque’s big-eared bats and southeastern myotis had significantly higher skin temperatures ( = 20.65 ± 5.85; = 18.08 ± 2.36, respectively) than all other species except Mexican free-tailed bats ( = 18.28 ± 6.12). The skin temperatures of Mexican free-tailed bats also did not differ significantly from tri-colored bats, big brown bats, and cave myotis ( = 15.07 ± 2.87; = 10.27 ± 3.02; = 12.41 ± 4.05, respectively). Townsend’s big-eared bats had significantly lower skin temperatures ( = 9.13 ± 3.05) than Rafinesque’s big-eared bats, southeastern myotis, tri-colored bats, and Mexican free-tailed bats, but were not significantly different to skin temperatures of big brown bats and cave myotis. There was a significant positive relationship between skin temperature and substrate temperature (r113 = 0.95, P < 0.001). Skin temperature and roost temperature also had a significant linear relationship (r102 = 0.75, P < 0.001). Roost temperatures of tri-colored bats varied significantly among ecoregions (K = 11.14, d.f. = 3, P ≤ 0.01). The roost temperatures at which tri-colored bats were found was significantly warmer in the Edwards Plateau than in the Southwestern Tablelands ( = 17.88 ± 2.22; = 11.49 ± 3.92, respectively; P ≤ 0.01). The roost temperatures at which tri-colored bats were found did not differ significantly between the Chihuahuan Deserts, Edwards Plateau, South Central Plains ( = 16.28 ± 2.98; = 17.88 ± 2.22; = 15.71 ± 2.22, respectively). Roost temperatures of tri-colored bats hibernating in caves was not significantly different to those in culverts (P = 0.43; caves: n = 24, = 15.97 ± 3.77; culverts: n = 41, = 15.68 ± 3.69). VPD recorded adjacent to bats did not differ among species (K = 8.86, d.f. = 6, P ≤ 0.18; Fig 3C).

Similarly, skin temperatures of tri-colored bats varied significantly among ecoregions (K = 12.14, d.f. = 3, P ≤ 0.01). Tri-colored bats had significantly warmer skin temperatures in the Chihuahuan Deserts, Edwards Plateau, South Central Plains ( = 16.45 ± 2.19, n = 2; = 15.34 ± 2.77, n = 18; = 17.05 ± 1.52, n = 2, respectively) than in the Southwestern Tablelands ( = 8.73 ± 2.46, n = 6; P ≤ 0.05). Skin temperatures of tri-colored bats hibernating in caves did not differ from those in culverts (P = 0.19; caves: n = 27, = 14.32 ± 3.73, min = 5.35, max = 21.60; culverts: n = 41, = 15.48 ± 2.04, min = 11.00, max = 18.72). Nearby VPD did not vary significantly (K = 4.3, d.f. = 3, P > 0.05).

The roost temperatures at which cave myotis were found was significantly colder in the Southwestern Tablelands than in the Edwards Plateau (Southwestern Tablelands: n = 12, = 12.01 ± 3.94; Edwards Plateau: n = 6, = 18.31 ± 3.78; P < 0.001). Cave myotis had significantly colder skin temperatures in the Southwestern Tablelands than in the Edwards Plateau (Southwestern Tablelands: n = 10, = 9.50 ± 2.12; Edwards Plateau: n = 6, = 16.61 ± 1.80; P < 0.001). There was no significant difference in VPD between cave myotis found in the Southwestern Tablelands and in the Edwards Plateau (P > 0.05).

Discussion

We found that bats hibernate under diverse conditions across Texas, and observed broad differences in selected microclimates and torpid skin temperatures among species. Our data showed that hibernacula temperatures in Texas were suitable, and often within the optimal growth range of P. destructans, placing several bat species at risk of developing WNS. Although we did not investigate microclimate variability across the winter season at all sites, which constrains our ability to fully understand the available microclimates and species’ susceptibility to WNS, our data add significantly to our knowledge about microclimates used by bats during winter and their suitability to the growth of P. destructans.

As expected, we found a strong relationship between internal and external temperatures for both caves and culverts. However, hibernacula temperatures were not simple reflections of surface temperatures and the four hibernacula that we monitored with dataloggers for the entire winter illustrate that a diversity of thermal habitats can be found in Texas. This variation can result from several factors, including aspects of local topography and the structure of the site and its entrances [59-60]. Temperatures within each hibernacula varied throughout the winter, but minimally so during the middle of the hibernation period (December–February). Thus, although it was not feasible for us to deploy microclimate dataloggers in all hibernacula, roost temperature data taken at these locations were similar to internal site temperatures indicating that our single-visit surveys captured conditions representative of the winter season. Although these temperatures are warmer than those reported for caves and mines in temperate regions, they are similar to those reported from subtropical zones [8, 11, 55, 61].

In a review of hibernacula temperatures in North America, Perry [60] found that temperatures less than 10° C are most suitable for hibernation, and that hibernacula in areas with mean annual surface temperatures (MAST) greater than 10° C are too warm. Our data show that knowledge about hibernation ecology from the temperature region of North America may yield misleading conclusions about hibernation in the subtropical region, as we frequently found bats hibernating in temperatures greater than 15° C. Overall, we found that microclimates within hibernacula used by all species overlapped the temperature range of growth for P. destructans [20]. In fact, 22 of the 97 (22.7%) sites surveyed had microclimates within the optimal growth range (12.5–15.8° C) [20]. Four of the species found within these sites, the big brown bat, cave myotis, southeastern myotis, and tri-colored bat, are known to be susceptible to WNS [62]. While these data suggest high risk of WNS mortality, and P. destructans has been present in Texas since 2017 [31], diagnostic symptoms of WNS have yet to be documented within the state. Although WNS may yet develop in Texas, we urge researchers to investigate the possibility that hibernating bats in Texas mitigate risk to WNS through differences in winter physiology and behavior in comparison to populations farther north.

Hibernacula temperatures were not always within the optimal growth range for P. destructans, and in many cases temperatures less than 10° C were available and occupied. As we hypothesized, tri-colored bats and cave myotis in northern Texas were in deeper torpor—as indicated by lower skin temperatures—than bats of the same species in southern Texas. Tri-colored bats and cave myotis were found roosting at temperatures ranging 5.50–25.56º C and 4.67–23.67º C, respectively, and were always found torpid, as indicated by the strong correlation between skin temperature and roost temperature, and skin temperature and substrate temperature. This large range in hibernacula microclimates used by tri-colored bats is similar to values reported in previous studies (e.g., [8, 11, 48, 63–64]). The average temperature used by this species, 15.8° C, is at the upper end of the optimal growth range for P. destructans, suggesting this species is at risk in Texas. The range of microclimates used by cave myotis in our study was similar to values contained in a review by Webb and colleagues [8]. Similar to tri-colored bats, the average roost temperature for cave myotis, 14.35° C, suggesting these bats select microclimate conducive to growth of P. destructans.

Compared to tri-colored bats and cave myotis, Rafinesque’s big-eared bats, Mexican free-tailed bats and southeastern myotis were consistently found in warmer areas of the hibernacula and had warmer skin temperatures, although these values did not always differ significantly from other species. This result is consistent with previous studies of Mexican free-tailed bats, which have shown this species employs shallow torpor during winter months [41, 65–66]. Unlike the Mexican free-tailed bat, Rafinesque’s big-eared bat and the southeastern myotis have limited distributions within Texas, and are only found in regions of warmer climates. In particular, Rafinesque’s big-eared bat is only found in the South Central Plains [41]. Compared to other bats hibernating in this region, Rafinesque’s big-eared bats had the highest skin temperatures. This result is similar to that of Johnson and colleagues [42], who found this species’ winter skin temperature of 13.9° C ± 0.6 among hibernating big-eared bats in Kentucky. Johnson and colleagues [42] also found this species frequently switched among nearby hibernacula in winter. Although we did not track movements of this or any other species, the relatively high torpid skin temperatures we observed suggests this species may also be active during winter in Texas.

Contrary to predictions, there was no difference in roost VPD between species. This indicates that—when variation in temperature is taken into consideration—the bat species studied in this manuscript do not differ in their required or preferred level of roost moisture. Across species, VPD was low, suggesting that bats were found in roosts with high levels of internal atmospheric moisture. As hibernating bats are as risk of evaporative water loss (EWL), it makes sense to find these bat species in moist environments. Although not significant, the data did suggest that Mexican free-tailed bats were found in hibernacula with slightly higher VPDs than other species. As a species that tends to engage in shallow torpor during winter [41, 65–66], Mexican free-tailed bats are not as much at risk from EWL and thus can tolerate somewhat drier roosts.

Our work adds to previous studies documenting the use of culverts as hibernacula for tri-colored bats in Texas [48–49, 67]. Culverts may act similarly to artificial caves in regions where cave-forming karst is scarce, increasing roosting options. Contrary to our prediction, tri-colored bats hibernating in culverts did not have significantly different skin temperatures than those hibernating in caves. However, the range of skin temperatures of tri-colored bats roosting within caves was larger than those in culverts. Although not reported here, our observations suggest that tri-colored bats may be selecting to roost within a given temperature range within culverts as a result of their body condition, EWL, and rates of heat loss through convection and conduction [5, 9, 68]. Raesly and Gates [69] suggested that there is an interplay between external hibernacula characteristics and internal characteristics that influence roost use. This relationship, along with the lack of caves in the region, may drive occupancy of certain culverts.

The roost temperatures, skin temperatures and actual water vapor pressures we reported herein depict microclimates selected by bats within hibernacula. Further, these data speak to the vulnerability to developing P. destructans infections but cannot predict WNS mortalities, as additional factors such as length of torpor bouts, winter food availability and foraging patterns must also be considered. Nevertheless, there are some important takeaways from our study. Based on our data, and provided that P. destructans continues to spread in Texas, we hypothesize that Mexican free-tailed bats, southeastern myotis, and Rafinesque’s big-eared bats will not develop WNS in Texas. Although our data suggest that cave myotis and tri-colored bats in Texas will be affected by WNS, the lack of any WNS-affected bats in Texas to-date demands further research. Future studies should focus on gathering information on the torpor patterns (e.g., torpor bout lengths) of bats in southern US. This work will help to improve the current understanding about differences in hibernation ecology and the potential impacts of WNS on these southern occupants.

11 Jul 2019

PONE-D-19-16454

Winter habitats of bats in Texas

PLOS ONE

Dear Ms. Meierhofer,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Aug 25 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Camille Lebarbenchon

Academic Editor

PLOS ONE

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript “Winter habitats of bats in Texas” describes hibernaculum microclimate of locations selected by bat species in the southern United States. More specifically, the authors were interested in determining the suitability of winter bat habitats in regards to white-nose syndrome. The authors determined that most of the hibernacula remained in the optimal range of fungal growth, and that skin temperatures and microclimates selected varied among species and differed from other populations within different regions. This work is especially important as the fungus that causes white-nose syndrome continues to spread into areas where we are lacking information about hibernation physiology of novel species and the microclimate conditions they chose. Data such as these will be useful in predicting the impacts of the disease as it impacts novel species.

There are a few issues that I think will improve the quality of the manuscript outlined here:

• Just a personal preference, but there are a lot of abbreviations with “T” (Tr, Ts etc). Either note the abbreviation with more detail (Tskin) or spell the temperature out.

• There were inconsistences in using abbreviations versus text throughout the manuscript. Either stick with abbreviations or text.

• It may make sense to change absolute water vapor pressure to water vapor deficit (saturation pressure – absolute pressure), which would allow the authors to compare conditions across temperature.

• Lines 77- 78: The sentence is incomplete

• Lines 114-125: These are really predictions, not hypotheses

• Line 158: change “enumerated” to “counted”

• Lines 160-165: Where within the hibernacula were the loggers placed? The distance within the hibernacula would change the temperature. Additionally, how close were the measurements from PRISM to the hibernacula? And were bats located near where the dataloggers were placed?

• Line 385: EWL has not been defined yet

• Figure 2: explain what r180 represents in the Figure caption.

• Figure 3: perhaps order by group to make visual comparisons easier

• Figure 3C: switch to water vapor deficit to make comparisons easier

Reviewer #2: This study describes the temperature of bats and their roosts during hibernation in Texas. They analyze variation between bat species, ecoregions, and roost type. The results are discussed in relation to the potential infection with the fungus P. destructans, which has not affected bats in Texas yet. Based on their temperature data, the authors make conclusions about which bat species will develop or not WNS.

I enjoyed reading this manuscript. The introduction is well written and the statistical tests particularly well described. Therefore, I only have few comments, detailed below:

1) Introduction, lines 114-118: why these 3 bat species would select higher temperatures? Please explain your hypothesis.

2) Figure 1: It might be good to have a more detailed map showing the temperatures across Texas (annual mean, or for winter only). I also suggest adding information on bat species sampled in each site to give a better idea of their range. I understand that some regions were not sampled (e.g. High Plains, Southern Texas Plains): maybe not necessary to show this information in the map.

3) In the methods, the authors explain that they counted bats during their visits. Did they test if the number of bats in the hibernacula affects (increases) the roost temperature?

4) What was the target surface measured with the digital thermometer? Did the authors observe variation of temperature across the body of torpid bats? Did they point the all bat body, or only a part (back, head, ears)? I suggest to add these details for Tskin measurements.

Minor edits:

5) Line 77 : « leading to dehydration and » ? Please rephrase.

6) Line 190 : “for each species avoid pseudoreplication”. Please rephrase.

7) Line 191 : Please correct : “used Pearson’s product-moment correlations”.

8) Line 312 : Please rephrase “…temperatures, however, and the four hibernacula…”.

9) Line 315: Please correct “Temperatures within each (?) varied…”.

10) Line 345 : Please correct “the average temperature”.

11) Line 349 : Please correct “suggests”.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

23 Jul 2019

Reviewer #1: The manuscript “Winter habitats of bats in Texas” describes hibernaculum microclimate of locations selected by bat species in the southern United States. More specifically, the authors were interested in determining the suitability of winter bat habitats in regards to white-nose syndrome. The authors determined that most of the hibernacula remained in the optimal range of fungal growth, and that skin temperatures and microclimates selected varied among species and differed from other populations within different regions. This work is especially important as the fungus that causes white-nose syndrome continues to spread into areas where we are lacking information about hibernation physiology of novel species and the microclimate conditions they chose. Data such as these will be useful in predicting the impacts of the disease as it impacts novel species.

There are a few issues that I think will improve the quality of the manuscript outlined here:

• Just a personal preference, but there are a lot of abbreviations with “T” (Tr, Ts etc). Either note the abbreviation with more detail (Tskin) or spell the temperature out.

• There were inconsistences in using abbreviations versus text throughout the manuscript. Either stick with abbreviations or text.

In response to both of the above comments, we have removed abbreviations in order to improve the clarity of the manuscript.

• It may make sense to change absolute water vapor pressure to water vapor deficit (saturation pressure – absolute pressure), which would allow the authors to compare conditions across temperature.

On your suggestion, we have calculated and reanalyzed our data using vapor pressure deficit. We have made the appropriate changes in text and in Figure 3C.

• Lines 77- 78: The sentence is incomplete

We have completed this sentence to “P. destructans infects dermal tissue of hibernating bats, leading to dehydration and death”

• Lines 114-125: These are really predictions, not hypotheses

We have clarified in the text where predictions or hypotheses were stated.

• Line 158: change “enumerated” to “counted”

This has been changed as suggested.

• Lines 160-165: Where within the hibernacula were the loggers placed? The distance within the hibernacula would change the temperature. Additionally, how close were the measurements from PRISM to the hibernacula? And were bats located near where the dataloggers were placed?

Dataloggers were placed within the first third of the hibernacula as- in regions where mean ambient surface temperature (MAST) is greater than 10° C, such as in Texas, bats may roost closer to the entrance where colder external air mixes with warmer air during winter (Perry, 2013). Indeed, there is some literature for the species reported in this manuscript detailing that bats tend to roost closer to the entrance of a hibernacula (big brown bat: Tuttle, 2000; tri-colored bat: Roth, 2014, Perry, 2013; southeastern myotis: Roth, 2014; Townsend’s big-eared bat: Tennessee Bat Working Group). Bats were regularly found roosting near dataloggers. While we recognize that internal temperature will vary somewhat based upon the location of the datalogger within a hibernacula, we believe that the presentation of this data provides enough evidence to suggest that our single-visit surveys captured conditions representative of the winter season for these caves and culverts.

In regards to PRISM, measurements were taken at 4km grid cell resolution, and this has now been clarified in the text.

Perry, R. W. (2013). A review of factors affecting cave climates for hibernating bats in temperate North America. Environmental Reviews, 21, 28-39.

Roth, Z. U. (2014). A Phenological Study of Bat Communities in Southern Mississippi Caves. Master’s Thesis, 62. https://aquila.usm.edu/masters_theses/62

Tennessee Bat Working Group. http://www.tnbwg.org/TNBWG_COTO.html. Accessed 11 July 2019.

Tuttle, M. D. (200). Where the Bats Are- Part III Cave, Cliffs and Rock Crevices. BAT magazine, 18(1), http://www.batcon.org/resources/media-education/bats-magazine/bat_article/942

• Line 385: EWL has not been defined yet

We have changed this abbreviation to read “evaporative water loss”

• Figure 2: explain what r180 represents in the Figure caption.

We have clarified that ‘r represents the correlation coefficient between internal and external temperature with corresponding degrees of freedom’.

• Figure 3: perhaps order by group to make visual comparisons easier

We not quite clear on what you are suggesting but we feel that our current figure succinctly visualizes our data and allows for comparison across variables and species.

• Figure 3C: switch to water vapor deficit to make comparisons easier

We have changed this as suggested.

Reviewer #2: This study describes the temperature of bats and their roosts during hibernation in Texas. They analyze variation between bat species, ecoregions, and roost type. The results are discussed in relation to the potential infection with the fungus P. destructans, which has not affected bats in Texas yet. Based on their temperature data, the authors make conclusions about which bat species will develop or not WNS.

I enjoyed reading this manuscript. The introduction is well written and the statistical tests particularly well described. Therefore, I only have few comments, detailed below:

1) Introduction, lines 114-118: why these 3 bat species would select higher temperatures? Please explain your hypothesis.

We have clarified that these predictions were based off the known range of the species and previous research on winter activity of the species, and have provided relevant references.

2) Figure 1: It might be good to have a more detailed map showing the temperatures across Texas (annual mean, or for winter only). I also suggest adding information on bat species sampled in each site to give a better idea of their range. I understand that some regions were not sampled (e.g. High Plains, Southern Texas Plains): maybe not necessary to show this information in the map.

We have added a gradient to the figure to show the annual mean temperatures across Texas (30 year average) taken from PRISM. Although we did not sample in some ecoregions, we have chosen to show the information on the map to provide the reader with the complete information regarding the ecoregions of Texas, and allow clear comparison to the original paper from which the ecoregions were taken (Griffith et al., 2004, 2007). In regards to the bat species sampled in each site, we believe that Table 1 provides the reader with enough information regarding the range of each species without cluttering the figure.

Griffith GE, Bryce JM, Omerick JA, Comstock AC, Rogers AC, Harrison B, et al. Ecoregions of Texas. (2 sided color poster with map, descriptive text, and photographs). 2004. [cited 29 Nov 2018]. Reston: U.S. Geological Survey. Scale 1:2,500,000.

Griffith GE, Bryce S, Omernik J, Rodgers A. Ecoregions of Texas. 27 December 2007. [cited 29 Nov 2018]. Austin: Texas Commission on Environmental Quality. Available from: ftp://ftp.epa.gov/wed/ecoregions/pubs/TXeco_Jan08_v8_Cmprsd.pdf.

3) In the methods, the authors explain that they counted bats during their visits. Did they test if the number of bats in the hibernacula affects (increases) the roost temperature?

We did not investigate if the number of bats within the hibernacula affected roost temperature. As the bats were in torpor, it is extremely unlikely that they are acting as a heat source within the hibernacula. This is validated by our strong correlation between skin temperature and substrate temperature (Line 265 of original draft). Furthermore, hibernacula were large so it is unlikely that any changes in number of bats would significantly influence roost temperature.

4) What was the target surface measured with the digital thermometer? Did the authors observe variation of temperature across the body of torpid bats? Did they point the all bat body, or only a part (back, head, ears)? I suggest to add these details for Tskin measurements.

We have now clarified in the text that we measured temperature from the bats’ backs.

Minor edits:

5) Line 77 : « leading to dehydration and » ? Please rephrase.

We have completed this sentence to “P. destructans infects dermal tissue of hibernating bats, leading to dehydration and death”

6) Line 190 : “for each species avoid pseudoreplication”. Please rephrase.

We have corrected this sentence to “…for each species to avoid pseudoreplication”.

7) Line 191 : Please correct : “used Pearson’s product-moment correlations”.

Corrected as suggested.

8) Line 312 : Please rephrase “…temperatures, however, and the four hibernacula…”.

We have rephrased the sentence to improve clarity

9) Line 315: Please correct “Temperatures within each (?) varied…”.

We have corrected this sentence to “…temperatures within each hibernacula…”.

10) Line 345 : Please correct “the average temperature”.

Corrected as suggested.

11) Line 349 : Please correct “suggests”.

Corrected to ‘suggesting’.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

25 Jul 2019

Winter habitats of bats in Texas

PONE-D-19-16454R1

Dear Dr. Meierhofer,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Camille Lebarbenchon

Academic Editor

PLOS ONE

1 Aug 2019

PONE-D-19-16454R1

Winter habitats of bats in Texas

Dear Dr. Meierhofer:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Camille Lebarbenchon

Academic Editor

PLOS ONE