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5-2019

Annual Movement Patterns and Microhabitat Use of Two Anaxyrus Species in the Southeastern Coastal Plain

Courtney Check

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Recommended Citation Check, Courtney, "Annual Movement Patterns and Microhabitat Use of Two Anaxyrus Species in the Southeastern Coastal Plain" (2019). Undergraduate Honors Theses. Paper 1289. https://scholarworks.wm.edu/honorstheses/1289

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Acknowledgements

I would like to sincerely thank my advisor, Matthias Leu, for his invaluable guidance and assistance with this project. I joined his lab as a clueless freshman who just had an interest in amphibians, and I never would have been able to develop and execute a project of this length or magnitude without his unwavering support. He is one of the most amazing mentors I have ever had, and I have developed so much as a biologist and as a person thanks to his guidance. I would also like to express gratitude to my committee members Linda Morse and Randolph Chambers for their time and input on my manuscript. I also owe a sincere thank you to the Virginia Herpetological Society, the Society for the Study of Amphibians and Reptiles, and the William & Mary Charles Center for providing the funding that made this research possible. Additionally, Olivia Windorf, Millan Khadka, and Chris Greene have all provided essential field assistance and this project would not be possible without their help. I am also grateful to Jill Ashey, Simeon Brown, Joseph Moriarty, Jesse Smyth, Trent Stafford, Matt Whalen, and Madison White for their help in the field. Lastly, a thank you to all ACER Lab members both current and alum, for providing input and advice over the last four years, and for making my research experience at William and Mary incredibly meaningful.

Abstract: Amphibian populations have declined precipitously in recent decades, and effective management plans are needed to combat this ongoing decline. However, most amphibian management plans tend to be extremely generalized, and little research has explored species- specific responses of amphibians to microhabitat or climatic variables. Using a relatively novel tracking method, I examine the annual microhabitat and movement of two Anaxyrus species (A. americanus and A. fowleri), one of which has suffered population declines, for the purpose of informing management practices regarding these species. My study found large differences in microhabitat use between the two species. A. americanus primarily utilize microhabitats associated with forest, while A. fowleri utilize a variety of microhabitats, some of which are never used by A. americanus. Additionally, A. fowleri are more mobile and cover more distance on average than A. americanus, though both species tend to move greater distances at higher temperatures, higher cumulative 3-day rainfall, and earlier in the year. Overall, my results indicate the need for management practices that are tailored to conserve species-specific habitat requirements.

Introduction

The Amphibian Crisis

Amphibian population declines have been well documented across the globe in recent decades.

As of 2004, roughly a third of all amphibians were listed under the IUCN red list, and 43.2% were experiencing some form of population decline (Stuart et al., 2004). Extinctions and local extirpations have occurred over rapid timeframes, with several species completely disappearing within ten years (Heyer et al., 1988, Lips, 2008). Numerous factors are cited as contributors to these declines, including habitat loss, poaching, climate change, invasive species, and disease

(Collins and Storfer, 2003). Notably, climate change and the fungus Batrachochytrium dendrobatidis (Bd) have been implicated in cases of sudden species extinction (De Leon et al.,

2016, Lips, 2008). The effect of these threats, however, is generally cumulative, with multiple factors playing a role in the decline of most species (Blaustein et al., 2011, Campbell Grant et al., 2016). Additionally, the impact of threats is variable across geographic regions (Campbell

Grant et al., 2016). For example, cases of sudden species extinction in the tropics have been largely linked to disease, whereas in the western United States such disappearances have usually been attributed to predation by invasive bullfrogs and fish (Bancroft et al., 2011, Adams,

1999).

In North America, amphibian populations began declining around 1960 and have continued to plummet since then (Houlahan et al., 2000). In the United States specifically, annual amphibian occupancy decreased on average 3.7% between 2002 and 2011, though for endangered species this drop is actually 11.6% (Adams et al., 2013). Bd now occurs across the entire contiguous United States, and has been linked to drastic population contractions in several native species, but disease alone does not explain the majority of amphibian declines in

North America (Carey, 1993, Gardner et al, 2007, Lanoo et al., 2011, De Leon et al., 2016).

Habitat modification and loss, on the other hand, is consistently one of the driving factors in population declines in North America (Delis et al., 1996, Harper et al., 2008, Windmiller et al.,

2008). Additionally, habitat alteration frequently facilitates or exacerbates the establishment of secondary stressors such as invasive species and/or climate change (Blaustein et al., 2011). Habitat Degradation and Amphibians

Habitat loss and degradation is the most common reason for species endangerment, and amphibians are no exception (Gardner et al., 2007, Wilcove et al., 1998). Studies on species diversity have shown that urbanization results in a decrease of both the richness and abundance of amphibians (Baldwin and DeMaynadier, 2009, Scheffers and Paszkowski, 2012).

Additionally, the IUCN estimates that 88% of threatened amphibians are affected by habitat loss or fragmentation (Baillie et al., 2004).

The biphasic nature of amphibian life cycles makes them particularly vulnerable to habitat modification, as healthy populations require both aquatic and terrestrial landscapes.

For pond-breeding species, temporary pools support the highest diversity of amphibians; when these pools are lost many species have no alternative breeding habitat (Ferreira & Beja, 2013).

However, research also indicates that even when breeding ponds remain intact, destruction of the surrounding uplands can lead to population contraction and collapse (Windmiller et al.,

2008).

The metapopulation structure of most temperate amphibian populations also makes them highly vulnerable to habitat fragmentation (Smith and Green, 2005). There is also generally a low recruitment and movement rate per generation, so consistent inter-patch connectivity is essential to maintaining populations through time (Cushman, 2006, Lehtinen et al, 1999, Rothermel, 2004). Past studies have indicated that population fluctuations and turnover in temperate amphibian species can be quite frequent, and ponds may remain empty for several years in between colonization events (Trenham et al., 2003). Furthermore, having a connected network of suitable habitat buffers populations from sporadic events that can make a single breeding site unsuitable for certain species (Petranka et al., 2004). Roads, agriculture, and forest edge can therefore pose barriers to dispersal and increase mortality in migrating adults (Fahrig et al, 1995, Titus et al., 2014, Youngquist and Boone, 2014). This, in turn, leads to a higher chance of local extinction via demographic or stochastic events (Titus et al, 2014).

An Incomplete Picture: Conservation Management and Amphibians

Despite the clear link between habitat suitability and amphibian population stability, there are still glaring gaps in the current literature. Concern over Bd has drawn focus away from habitat studies despite the substantial contribution of habitat loss to amphibian declines (Gardner et al., 2007). The cryptic nature of many amphibians and the difficulty in tracking such small organisms also means that there is a disproportionate amount of literature dealing with the aquatic larval stage of the amphibian life cycle, as this stage is restricted to specific habitats and can be raised in a lab setting (Pittman et al, 2014, Semlitsch et al., 2008). For the same reason, the research that does exist on terrestrial adults and metamorphs is usually limited to delineating general patterns of occupancy or population persistence. Habitat studies are also generally restricted to the breeding pond, as most amphibians are more easily detectable during the breeding season. Consequently, while we know that the proportion of forest cover around the breeding pond is important to many amphibians, we lack mechanistic explanations for why it is important, how they use it, or what constitutes high quality nonbreeding habitat.

This unbalanced body of literature has resulted in vague, uninformed, and often ineffective management plans for amphibians in the United States (Semlitsch and Bodie, 2003, Veysey

Powell and Babbitt, 2015). Examination of current amphibian conservation plans reveals the disconnect between managers and research. Harper et al. (2008) found that the current requirements of a 30-m buffer around aquatic resources do not support viable populations of Ambystoma maculatum and Rana sylvatica, and that no regulations exist to protect isolated wetlands of high breeding importance. Other studies indicate that the amphibian richness is lower in buffer zones than in undisturbed forest (Marczak et al, 2010). Marczak et al. (2010) hypothesized that decreased richness is due to a difference in the microclimate and habitat structure of buffer zones, but little empirical research has been conducted to investigate this hypothesis. Additionally, evidence suggests that responses to anthropogenic change are highly species-specific and targeted management is the best approach to prevent species loss, but there is very little research on the habitat use of specific species (Becker et al., 2010). As a result, managers are left with vague guidelines and must make uninformed conservation plans that are applied to all species regardless of life history.

My research seeks to address the gap in the current literature by examining the annual habitat use and movement of two native North American toads living in the Coastal Plain of

Virginia (Anaxyrus americanus and Anaxyrus fowleri) at a fine-scale. I will investigate the microhabitat use of these two species, and identify annual movement patterns using acoustic telemetry. This will provide mechanistic explanations for the importance of forest cover, and supply managers with species-specific life history data to apply in targeted management plans.

Looking at species of the Anaxyrus genus is particularly relevant to amphibian conservation, as they are highly terrestrial species that may spend as little as two weeks a year at the breeding pond, with the rest of their year being spent in upland environments. These data are also highly important to the conservation of A. fowleri, as it has suffered population declines across its range for reasons that are not fully understood. Jones and Tupper (2015) found that A. fowleri populations have decreased by approximately 53% in the mid-Atlantic over the last 15 years.

Additionally, A. fowleri is federally protected in Ontario, and listed as a species of special concern in several states in the US (Milko, 2012, Oldham, 2003). A. americanus populations, on the other hand, have remained relatively stable despite the fact that A. americanus and A. fowleri are sympatric over most of their range and share similar breeding requirements.

Therefore, my research may be able to provide insight as to the cause of A. fowleri’s decline by comparing the microhabitat and movement patterns of these two species, one of which has remained relatively stable and the other which has suffered population contractions.

Additionally, it will provide managers with a better insight into A. fowleri’s habitat requirements, allowing for more informed and effective conservation planning.

Methods

Study Sites

My study sites consisted of three public parks in the James City County and City of Williamsburg region of Virginia: College Woods (William and Mary campus – 37o16’22.4”N, 76o43’40.5”W),

Warhill Sports Complex (37o19’28.9”N, 76o45’40.2”W), and Greensprings Interpretive Trail

(37o15’06.6”N, 76o47’30.5”W). All of these sites consisted of a large central water body surrounded by a matrix of wetland, forest, grassland, and urban development. However, the

College Woods did not exhibit as extensive grassland cover as the other two sites. All three of these sites also had a network of trails, though the trails in the College Woods were composed of natural substrates and are minimally maintained, while the other sites contained well- maintained gravel trails.

Telemetry and Tracking

To follow individual toads, I used a relatively novel method known as harmonic direction finding. This method involves attaching a light (~0.4-0.7g) transponder to toads via a silicon belt. This method has been used in a few previous studies, and has been shown to not alter an individual’s movement beyond the first 24-hours following transponder attachment (Alford and

Rowley, 2007). Construction methods followed the techniques developed by Gourret et al

(2011). However, for this study, transponders and the accompanying detection device were supplied by RECCO Rescue Systems, a Swedish company that develops technology to recover avalanche victims and has recently adapted their technology for use in biological research

(http://www.recco.com/the-recco-system).

Each toad received a single transponder and belt, as well as a uniquely colored dot combination on the upper surface of the belt so that specific individuals could be identified.

During the pilot summer of tracking in 2017, I located toads every day so that general patterns could be identified. However, I reduced sampling to every other day in the fall, and once a week during the winter hibernation period. For all seasons in 2018, toads were located once every two or three days until the hibernation period, when weekly locations were resumed. Tracking occurred from June 1, 2017 until December 8, 2018, and animals were followed until they were lost, their belt detached, or environmental factors contributed to mortality. This project was approved by William & Mary’s IACUC committee prior to implementation (Protocol IACUC-

2017-02-20-11745-mleu). Microhabitat Data Collection

Every time a toad was located, I took the temperature and humidity at both ground and air level (defined as at least 30 cm above the ground) at the spot a toad was found. Soil moisture and leaf litter depth were also taken at each location, but because these elements are not well assessed by a single measurement, they were measured at four points that correspond to the corners of a 1-m2 quadrat centered on the toad. Soil moisture was measured using a FieldScout soil moisture probe with 4 in. prongs (Spectrum Technologies Inc.). Leaf litter depth was measured to the nearest centimeter using a wooden skewer with markings every 1-cm.

Habitat was assessed at two levels: substrate and microhabitat (Appendix A). Substrate is a categorical variable consisting of the various structures toads were observed in, such as tree cavities, leaf litter, and coarse woody debris. This variable allows me to quantify the use of various habitat elements by the toads. Microhabitat is broader in spatial extent, and consists of a proportional measurement of all the substrates within a 1-m2 quadrat centered on a toad (for example: 50% leaf litter, 50% coarse woody debris). This variable provides a way to quantify the broader habitat use by a toad. Both substrate and microhabitat were measured once at each location where a toad was found. If a toad was observed at a location more than once, or remained in a location for multiple days, the initial microhabitat measurement was used unless drastic changes to the microhabitat were observed (i.e. mowing or flooding).

I also recorded the diameter at breast height (DBH) of all trees in a circle at every location a toad was found. In an effort to choose a biologically meaningful measurement, the circle radius in 2017 was a running average based on the distances all toads of the same species had moved on the day the measurement was taken. In 2018, the radius remained constant, and was the average of all movement for a given species in 2017. Therefore, in 2018 the radius for

A. americanus was 6 m and the radius for A. fowleri was 12 m. If a toad was observed within one-half of the radius of a point it had previously visited, then I considered those observations to have the same circular plot (Appendix A). Due to the difference in radii, I used the density of trees rather than counts to compare differences in forest structure.

Movement

Distance measurements were also taken, including the distance between consecutive observation points, and the distance of a point from the nearest water body. In an effort to account for topographical features that could affect movement, all distances were measured in the field using a tape measure, except for cases where dense vegetation or extreme distance made this impossible. In such cases, a Geographical Information System (GIS; ArcMap 10.4) was used to calculate a distance.

Statistical Analysis

Microhabitat - Because substrate is merely intended to inform microhabitat (i.e. elucidate how toads are using their microhabitat features), it was examined solely in terms of the percent of observations per species that occurred at a given substrate.

Microhabitat importance was examined by comparing the probability of observing an

American or Fowler’s Toad (represented in the data matrix by 1 or 0, respectively) with the proportion of a given microhabitat variable in a quadrat at each observation. To do this, I used binomial generalized linear models that were then ranked using Akaike Information Criterion

(AIC). Though all variables were tested for quadratic and logarithmic relationships, these did not appear to have a higher AIC than simple linear models. All microhabitat variables were first examined as univariate models, and then the univariate models that had a higher AIC than the null model were then joined to create a series of additive models. All of the best performing models (those whose weights summed to 0.95) were then model averaged to create a single explanatory model. Variables included in microhabitat modeling included: leaf litter, vegetation

(herbaceous forbs and grasses), coarse woody debris (CWD), bare ground, natural path, gravel path, moss, tree, and water. Sites were also included as fixed effects in all models to account for geographic variation. Additionally, all observations that occurred within 24-hours of tagging or occurred after sunset (i.e. observations from the night an animal was captured) were removed.

Movement - Variables that affected the movement of each toad species were identified by modeling the distance each toad moved between observations in relation to various climatic variables. Time intervals between observations and species were also modeled against distance moved to identify potential temporal and interspecies variation. I used negative binomial generalized linear mixed models to conduct this analysis, and model selection and averaging was performed via the same method as described in the above ‘Microhabitat’ section. Variables examined in movement modeling included: ground temperature, air temperature, ground humidity, air humidity, time of year (Julian date), and rainfall. Rainfall data were obtained from the Oregon State University PRISM weather database. This database uses measurements from local weather stations to extrapolate rainfall for specific coordinates that are selected by the user. Therefore, I was able to obtain coarse rainfall data for each of my three study sites.

Because previous research has indicated that cumulative rainfall over many days can be more important to amphibians than daily rainfall, I looked at the cumulative rainfall of the days prior to each toad observation. To avoid arbitrarily picking a number of days in which to look at cumulative rainfall, I separated rainfall into different measurements corresponding to cumulative rainfall over 1-10 days and 14 days prior to each observation. However, to avoid autocorrelation of variables, I only included the rainfall variable with the lowest AIC in additive models. Likewise, if both ground and air measurements of temperature and humidity performed better than the null, I only included the measurement that had the lowest AIC. Once again, sites were also included as fixed effects, and all observations that occurred within 24- hours of tagging or occurred after sunset were removed.

Site Fidelity - Site fidelity was assessed by comparing the number of times an individual returned to a given location to the previously mentioned microhabitat variables, as well as average soil moisture and leaf litter depth. Once again, I used negative binomial generalized linear models. First, univariate models were first created for each microhabitat type, and then all variables that performed better than the null were analyzed in various combinations in additive multivariate models. To determine whether or not both species were faithful to the same kinds microhabitats, I also examined species as an interaction term with each variable. If an interactive model had a higher AIC value than a univariate model for a given variable, I used the interactive terms in the additive models. All models that performed better than the null model were then ranked and averaged using AIC to create a final predictive model.

For most variables, I determined that an individual returned to the same location if I found it within the same quadrat of a previous observation. However, when assessing site fidelity in relation to DBH, I determined that an individual returned to the same location if I found it within one-half the radius of that species’ average daily movement. I used this method for DBH because the circular plots covered a larger area than the quadrats, so observations could be lumped at coarser scales.

Results

A total of 37 A. americanus and 22 A. fowleri were followed over the two-year study period.

Between June 1, 2017 and December 8, 2018, a total of roughly 300 days was devoted to tracking. More A. americanus were followed than A. fowleri due to logistical constraints in the first year of study. Outside of the hibernation period, A. americanus were observed an average of 12 times per individual (min = 2, max = 33), and followed for an average of 28 days (min = 3, max = 77). A. fowleri, in contrast, were observed an average of 9 times per individual (min = 2, max = 37), and were followed for an average of 18 days (min = 2, max = 46).

Substrate Use

When comparing substrate use, notable differences were evident between the species. A. americanus spent the vast majority of their time under leaf litter (63.3% of observations), though tree cavities (14.3%) and coarse woody debris (11.4%) also made up large portions of observations. A. fowleri, on the other hand, used a wider variety of substrates with regularity.

The most frequently used categories by A. fowleri were vegetation (31.6%), leaf litter (24.9%), and coarse woody debris (23.2%), however burrows were also used relatively often (10.2%). A. americanus was almost never found in vegetation, and all other categories represent less than

10% of observations for a species.

Fig. 1: Substrate use as a percentage of observations for A. americanus and A. fowleri Substrate Selection in American Toads and Fowler's Toads

100%

90%

80%

70%

60%

50%

40%

Percent of Observations 30%

20%

10%

0% A. americanus A. fowleri

Leaf Litter Bare Ground Tree Cavity CWD Moss Burrow Vegetation

Microhabitat Use

Leaf litter, coarse woody debris, and vegetation proved to be the most important variables for predicting species microhabitat use. Leaf litter was strongly associated with A. americanus presence (β = 0.29), while vegetation was strongly associated with A. fowleri presence

(β = -0.94) (Table 1 and 2). Coarse woody debris was also important to predicting A. americanus presence (β = 0.16), however the proximity of this estimate to zero indicates that it may be an important factor to both species (Variables were scaled, so the best possible predictor for

American Toads would = 1 and the best possible predictor for Fowler’s Toad’s would = -1).

Table 1: Model ranking for microhabitat variables. Models highlighted in grey were used in model averaging (i.e. the sum of their weights > 0.95). Model df AIC Δ AIC AIC Weight Vegetation 4 148.04 0 0.26 Leaf Litter + CWD + Vegetation 6 148.06 0.02 0.26 Leaf Litter + Vegetation 5 148.24 0.20 0.24 CWD + Vegetation 5 148.95 0.91 0.17 Leaf Litter + CWD 5 150.83 2.79 0.06 Leaf Litter 4 154.87 6.83 0.01 CWD 4 175.59 27.55 2.7E-07 null 3 180.33 32.30 2.6E-08

Table 2: Summary of final predictive model for microhabitat. Positive effect size indicates an association with A. americanus, while negative effect size indicates an association with A. fowleri. Variable Effect Size Standard Error Weight Warhill (Intercept) 0.27 0.30 0.97 College Woods 18.73 1176.74 0.97 Greensprings -0.75 0.42 0.97 Leaf Litter 0.29 0.16 0.56 Coarse Woody Debris 0.16 0.11 0.49 Vegetation -0.94 0.30 0.93

Movement

On average, A. fowleri moved more than A. americanus (β = -0.86), even when interactions with time of year were taken into account (Appendix B). Based on all data from 2017-2018, I found that A. americanus move on average 6.4 m a day, while A. fowleri move on average 15.9 m a day. Due to the nature of my tracking method, highly mobile individuals are more likely to be lost than less mobile ones, so these averages should be considered a conservative estimate.

The greater mobility of Fowler’s Toads also likely explains why I located A. fowleri fewer times on average than A. americanus, as A. fowleri was more likely to move beyond the range of my detection.

Time interval between observations did not appear to affect distance moved (Appendix

C). For both species, time of year, ground temperature, and 3-day cumulative rain all affected daily movements. Models using ground measurements of temperature and humidity performed slightly better than air measurements, however their AIC values were extremely similar (see

Appendix D). Of all rainfall models analyzed, only cumulative 3-day rain performed better than the null. Toads moved greater distances earlier in the year (corresponding with the breeding season for both species) (β = -0.18), at higher temperatures (β = 0.10), and with greater 3-day cumulative rain (β = 0.15) (Table 3 and 4).

Table 3: Model ranking for variables that may impact daily movement. Models highlighted in grey were used in model averaging (i.e. the sum of their weights > 0.95). Model df AIC Δ AIC AIC Weight Species + Temperature + 3-day Rainfall + Date 8 3124.36 0 0.43 Species + 3-day Rainfall + Date 7 3125.18 0.82 0.28 Species + Temperature + 3-day Rainfall 7 3127.53 3.17 0.09 Species + Temperature + Date 7 3127.76 3.40 0.08 Species + Date 6 3127.97 3.60 0.07 Species + 3-day Rainfall 6 3130.14 5.78 0.02 Species + Temperature 6 3130.15 5.79 0.02 Species 5 3131.95 7.59 0.01 Temperature 5 3142.37 18.01 5.2E-05 Date 5 3143.08 18.72 3.7E-05 3-day Rainfall 5 3149.52 25.16 1.5E-06 Null 4 3150.39 26.03 9.5E-07 Time interval between observations 5 3151.08 26.72 6.7E-07 Daily Rainfall 5 3151.16 26.80 6.4E-07 Ground Humidity 5 3151.56 27.20 5.3E-07

Table 4: Summary of final predictive model for daily movement. Positive effect sizes indicate an association with long movements, while negative effect sizes indicate an association with short movements. Variable Effect Size Standard Error Weight Warhill (Intercept) 2.73 0.20 0.97 College Woods -0.08 0.24 0.97 Greensprings -0.25 0.21 0.97 Temperature 0.10 0.05 0.59 Species -0.86 0.21 0.97 3-day Rainfall 0.15 0.07 0.82 Date -0.18 0.08 0.86

Site fidelity

There was minimal difference in site fidelity between the species, indicating that both returned to locations with the same relative frequency (i.e. a model accounting for species was < 1 AIC away from the null). A. americanus site fidelity was strongly tied to an increased proportion of tree within a 1-m2 quadrat (Table 5 and 6). Site fidelity in A. fowleri, on the other hand, was linked with an increased presence of bare ground in a 1-m2 quadrat. Both species also displayed a weak site fidelity to moss. Across all DBH guilds, the DBH of trees within a relevant radius did not predict site fidelity for either species (Table 7).

Table 5: Model ranking for microhabitat variables that may impact site fidelity. Models highlighted in grey were used in model averaging (i.e. the sum of their weights > 0.95). Model df AIC Δ AIC AIC Weight Tree*Species + Bare ground*Species 9 754.72 0 0.35 Tree + Bare ground*Species 8 755.65 0.63 0.22 Tree*Species + Bare ground*Species + Moss 10 756.30 0.45 0.16 Tree + Bare ground*Species + Moss 9 757.00 0.32 0.11 Tree*Species 7 757.97 0.20 0.07 Tree + Moss 7 759.24 0.10 0.04 Tree 6 759.39 0.10 0.03 Bare ground*Species 7 762.88 0.02 0.006 Bare ground*Species + Moss 8 763.44 0.01 0.004 Moss 6 764.71 0.007 0.002 Species 5 766.03 0.003 0.001 Bare Ground 6 766.07 0.003 0.001 Moss*Species 7 766.55 0.003 0.0009 Null 4 767.01 0.002 0.0008

Table 6: Summary of final predictive model for microhabitat effects on site fidelity. For variables that do not interact with species (i.e. ‘Moss’), the positive effect size indicates an association with high site fidelity in both species. For terms with an interaction, the effect of a variable on site fidelity is equal to its term + its interaction. Therefore, because ‘Bare Ground,’ has a significant interaction with species, there is a positive effect of bare ground term on A. fowleri site fidelity (net effect size = 0.29 + (0*-0.29) = 0.29), but no effect on A. americanus site fidelity (net effect size = 0.29 + (1*-0.29) = 0). Likewise, because ‘Tree,’ has a significant interaction with species, there is almost no effect of tree on A. fowleri site fidelity (net effect size = 0.02 + (0*0.13) = 0.02), but a positive effect on A. americanus site fidelity (net effect size = 0.02 + (1*-0.13) = 0.15). Variable Effect Size Standard Error Weight Warhill (Intercept) 0.58 0.79 0.95 College Woods -0.009 0.93 0.95 Greensprings 0.17 0.85 0.95 Species 0.17 0.85 0.95 Bare Ground 0.29 0.52 0.84 Bare ground*Species -0.29 0.58 0.84 Tree 0.02 0.52 0.95 Tree*Species 0.13 0.41 0.69 Moss 0.01 0.15 0.31

Table 7: Model ranking for various DBH guilds that may impact site fidelity. No models had a higher AIC than the null, even when species was taken into account. Model df AIC Δ AIC AIC Weight Null 4 3124.36 0 0.23 DBH 6.4 - 9.1 5 3125.18 0.98 0.14 DBH >16.5 5 3127.53 1.53 0.10 No Trees 5 3127.76 1.77 0.10 DBH 5.0 - 6.4 5 3127.97 1.86 0.09 DBH 9.1 - 16.5 5 3130.14 1.88 0.09 DBH < 5 5 3130.15 1.96 0.09 No Trees*Species 7 3131.95 3.51 0.04 DBH >16.5*Species 7 3142.37 3.84 0.03 DBH 6.4 - 9.1*Species 7 3143.08 4.08 0.03 DBH 9.1 - 16.5*Species 7 3149.52 5.00 0.02 DBH 5.0 - 6.4*Species 7 3150.39 5.13 0.02 DBH < 5*Species 7 3151.08 5.31 0.02

Discussion

Microhabitat

This research is one of the first of its kind to examine fine-scale microhabitat use in specific amphibian species over the entire annual cycle. Despite the genetic similarity and sympatric range of A. americanus and A. fowleri, an analysis of their habitat use reveals vastly different strategies of accomplishing the same goal. Like many other terrestrial amphibians, both of these species seek camouflaged refuges that limit desiccation risk during the daytime hours.

However, the nature of these shelters differs greatly between the two species.

A. americanus relies primarily on leaf litter for cover, as evidenced by both substrate use and microhabitat modeling. The importance of leaf litter as a climate refuge has previously been demonstrated in Rana sylvatica and many terrestrial salamanders (Baldwin et al., 2006,

O’Donnell et al., 2014), therefore it makes sense that a forest-dwelling, terrestrial species such as the A. americanus would also utilize this cover resource. Tree cavities also seem to be important to A. americanus habitat given that they make up a relatively large portion of substrates use in this species, and that the proportion of tree in the microhabitat quadrats is associated with site fidelity in this species. Large trees (i.e. take up more space in a quadrat) are more likely to have complex basal features such as cavities and buttresses, which I frequently found toads nestled in (Piraccini, et al., 2017). These microhabitat features likely provide both shelter and climate refuges for toads, who are vulnerable to extreme temperatures and humidity due to their permeable skin. Previous studies have demonstrated the tree buttresses are associated with high abundances of temperate salamanders and neotropical leaf-litter herpetofauna, so it is not unreasonable that American Toads would also gravitate towards this resource (Piraccini et al., 2017, Whitfield and Pierce, 2005).

While A. fowleri also seek shaded daytime refuges, they use a much wider variety of shelters than their sympatric relative. Though leaf litter is used with relative frequency, vegetation, coarse woody debris, and burrows are also important microhabitats for this species.

Previous studies have found that both burrows and vegetation significantly reduce desiccation risk for amphibians, though the magnitude of this reduction can vary depending on geography and season (Lemckert and Brassil, 2000, Seebacher and Alford, 2002, Schwarzkopf and Alford,

1996). Furthermore, A. fowleri are known to associate with dry sandy soils, so their proclivity for burrowing aligns well with what is known of their natural history. Burrows have also been shown to be consistently cooler than their surrounding environments, indicating that they may act as a temperature refuge as well (Schwarzkopf and Alford, 1996). Other toad species such as

Rhinella marinus have been known to exhibit site fidelity to burrows during dry seasons for this very reason (Schwarzkopf and Alford, 1996). A fowleri’s use of burrows may also explain why bare ground is linked to high site fidelity in this species. Burrows are created in areas where exposed, sandy soil is available for digging, so it makes sense that these two elements would be correlated. However, it is also possible that these toads return to areas of bare ground due to temperature or foraging needs. Clark (1974) more frequently encountered A. fowleri on bare ground when the temperature differential of the day (i.e. the difference between the high and low temperatures) was greatest, though the correlation was weak. Some toad species are also known to select bare ground as their preferred foraging sites due to high visual contrast that increases prey visibility, and it is possible that A. fowleri return to these areas because they present optimal foraging territory (Gonzalez-Bernal et al., 2011).

Coarse woody debris appears to be a more important predictor for A. americanus presence than for A. fowleri presence, though the proximity of its coefficient to zero indicates that it is likely a relevant factor for both species. The importance of coarse woody debris as a refuge aligns with previous research in amphibians. For example, it has been shown that such debris can mitigate the effects of clear-cutting on anurans because it reduces desiccation risk (Harper et al., 2015). Similarly, Long and Prepas (2012) demonstrated that Anaxyrus boreas will select coarse woody debris as a microhabitat because it provides a humidity refuge. However, the relatively smaller importance of coarse woody debris as compared to leaf litter in this species also lines up with the behavior of other amphibians in the coastal plain. Owens et al.

(2008) found that there was no difference in amphibian abundance in Pinus taeda stands that had been experimentally manipulated to lack coarse woody debris and control stands. Likewise, in a similar study, Davis et al. (2010) found that amphibian diversity in the Coastal Plain is not tied to coarse woody debris abundance. Though neither of these studies examined A. americanus or A. fowleri abundance in their surveys, the relatively weaker response of these species to coarse woody debris indicates that they behave similarly to other amphibians in this region. Little empirical research has been conducted to determine why coastal plain species are less reliant on coarse woody debris, but some have speculated that the short fire interval and humid climate of the region may reduce the availability and need for downed woody refuges

(Davis et al., 2010). Instead, these species have adapted a reliance on more abundant landscape features such as leaf litter.

Despite some similar habitat features, however, the results of my research overwhelmingly indicate that these two species have highly different strategies for habitat use.

A. americanus utilizes mixed deciduous forest with healthy leaf litter levels and larger trees as their primary refuge. A. fowleri, on the other hand, is more drawn to habitat typical of edges and early successional forest that contains large proportions of herbaceous vegetation and exposed soils for burrowing. Consequently, this difference demonstrates that broad, generalized amphibian habitat management plans are likely to be ineffective at maintaining species diversity.

Movement

Though A. americanus and A. fowleri display variation in their microhabitat use, I found no relevant differences in these species’ movement responses to climatic variables. Both species moved greater distances on days with higher temperatures and 3-day cumulative rain.

Temperature is known to be an extremely important driver of amphibian metabolism and activity since, as ectotherms, they are incapable of regulating their own body temperature. In

American Toads specifically, higher temperatures have been linked to increased foraging success (Preest and Pough, 2015).

Similarly, rain has been linked to migratory and breeding activity in many pond-breeding amphibians, though the effects of rain on activity outside the breeding season tend to vary between species (Green et al., 2016, Hamer et al., 2008, Todd and Winne, 2006). Generally, however, rain increases landscape permeability for amphibians by reducing the threat of desiccation in areas where they would normally be at risk (Hamer et al, 2008, O’Donnell et al,

2014, Veysey et al., 2009). Therefore, it makes sense that their movements would be greater during rain events given that their overall landscape mobility has been improved. For this study, cumulative 3-day rainfall is the only variable out of the eleven rain models examined that explains some of the variation in the distance traveled by these species. This is likely because the coastal plain is an extremely humid region that already receives significant rainfall, so only severe, prolonged weather events such as multi-day rainstorms cause rain and moisture content to deviate significantly from average conditions. This tendency of the coastal plain to have humid, rainy weather may also explain why humidity was not relevant to movement in my study. Several studies have indicated that relative humidity can affect activity level and site fidelity in anurans, though plenty of others have found humidity to be unimportant to amphibian movement (Long and Prepas, 2012,

Baldwin et al., 2006). My research further reinforces that the importance of humidity to anurans likely varies across species and across geographic locations. For terrestrial toads living on the coastal plain, humidity does not seem to be a factor driving movement.

I also found substantial inter-site variation in toad movement, which highlights that regional geographic diversity is not the only factor to impact toad movement. Even across my three study sites there were large differences in toad movement. This is in line with several other multi-site studies of amphibian activity that displayed major between-site variation. For example, Popescu et al (2012) found response to clear-cutting and coarse woody debris removal was highly varied across six different study sites. In another study, Hawkes and

Gregory (2012) found that response to logging and riparian buffer retention varied wildly between sites. Overall, this demonstrates the importance of multi-site replication when studying amphibian populations, even across small geographic ranges. A relatively large portion of literature on amphibians utilizes only a single site, and this may be one reason for the prevalence of contradictory literature on amphibian movement, dispersal, and responses to climate.

Management Considerations

Amphibian management is critically underdeveloped and as a result conservation strategies are typically generalized and minimally informed. Forest buffers have previously been critiqued for being insufficient to maintain healthy amphibian populations (Semlitsch and Bodie, 2003,

Veysey Powell and Babbitt, 2015), and my study adds to this critique by demonstrating that broadly applying forest buffers as a management technique for amphibians will not be sufficient for conserving species that do not rely on forest habitat. In fact, this skewed management may provide some insight as to why A. fowleri have declined across their range while American Toads have remained relatively stable. Though buffers may protect habitat, the reliance on them means that protection is biased towards riparian corridors and forests (Dodd and Cadet, 1997) – habitat types that my research shows is not used in as large quantities by A. fowleri. Vegetation, on the other hand was an extremely important predictor of A. fowleri presence, and site fidelity in this species is tied to bare ground.

Therefore, instead of applying broad habitat protection guidelines, species-specific management must occur to ensure that threatened and declining species are actually benefitting from conservation actions. This will require significantly greater investment in targeted research that elucidates species-specific habitat requirements, particularly with regards to non-breeding habitat. These studies are typically difficult due to logistical and technological constraints, however recent advances in tracking capabilities (as shown in this research) are beginning to make these studies more feasible. In the case of the A. fowleri, my research indicates that management might be better served by expanding the types of buffers that can exist around water resources to include exposed grassland and shrub habitat, as this seems to be the habitat used more frequently by A. fowleri. Additionally, protective buffers and habitat may need to be maintained with regular disturbance regimes to prevent the eventual succession of these areas into forest. Furthermore, regional and local geography must be integrated into all amphibian management plans. The wide variation in behavior exhibited between my study sites demonstrates that amphibians are highly responsive to micro-scale climate and landscape features. Given that many amphibian species, including the ones examined in this study, have ranges that cross multiple geologic and climatic regions, it will be challenging to create broadly applicable species management strategies. Even state-wide plans can be ineffective when there are multiple landscape types within a single state (ex: Virginia, which ranges from boreal mountain forest in the west to coastal plain in the east). Instead, management should focus on developing strategies for regional ecosystems that share climatic and habitat features, such as the coastal plain or the Piedmont. Additionally, even when these plans have been developed, site-specific considerations and ground-truthing should be used extensively when developing management strategies. Goates et al. (2007) demonstrated that small seeps and wetlands can be overlooked when management plans are drafted at coarse scales, and consequently are not given the protective buffers they are supposed to have. However, they found that ground- truthing sites prior to implementation can result in much more effective protective policies that capture a greater area of amphibian habitat.

Amphibians are declining at a rapid rate globally, and the only appropriate response to this problem is the development of effective conservation strategies. However, despite the fact that amphibians are an equally or more speciose taxa than birds and mammals, they are usually managed as a group rather than with targeted conservation on the basis of habitat and/or breeding guild. If we are to adequately protect amphibian habitat, then species-specific and site-specific considerations must be taken into account. Focusing solely on landscape scale conservation and ignoring micro-scale requirements of species will result in skewed conservation that may not address the needs of declining species, such as the A. fowleri. Future research must focus on developing the body of species-specific habitat and climate requirements so as to allow for more effective, more targeted amphibian management plans to be developed.

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Appendices

Appendix A: Visual representation of categories of microhabitat

Appendix B:

Model ranking to determine if there are between-species differences in movement. Accounting for species in a model performed better than a null model. This holds true even when an interaction between time of year and species is taken into account. Note that an additive model best fits the data, hence why this model used in model comparisons rather than an interactive one. Model df AIC Δ AIC AIC Weight Species + Date 6 3127.97 0 0.66 Species*Date 7 3129.94 1.975 0.25 Species 5 3131.95 3.99 0.09 Null 4 3150.39 22.42 8.96E-06

Summary of model using ‘Species’ as sole predictive variable. Because in this model A. americanus = 1 and A. fowleri = 0, a -1 negative effect of species on distance moved, indicates that A. americanus moves less as a species overall. Variable Effect Size Standard Error Warhill (Intercept) 2.97 0.21 College Woods -0.07 0.24 Greensprings -0.30 0.22 Species -1.00 0.44

Summary of model using an interaction between species and time of year (date) as a predictive variable. Because in this model A. americanus = 1 and A. fowleri = 0, a negative effect on species on distance moved indicates that A. americanus moves less as a species overall. Time of year (date) also has a negative effect size, indicating that species move less later in the year. The interactive term is slightly positive, indicating time of year (date) mildly reduces the negative effect of species on distance moved (i.e. the two species move more similarly to each other later in the year). Variable Effect Size Standard Error Warhill (Intercept) 2.90 0.21 College Woods -0.12 0.25 Greensprings -0.28 0.22 Species -0.92 0.22 Date -0.26 0.22 Species*Date 0.05 0.24

Appendix C:

Model ranking to determine if time interval between observations affected distance moved by toads. Time interval did not have an apparent effect on distance moved. Model df AIC Δ AIC AIC Weight Null 4 3150.39 0 0.59 Interval between observations 5 3151.08 0.7 0.41

Appendix D:

Model ranking to determine whether air or ground values of temperature and humidity are better predictors of distance moved by toads. Model df AIC Δ AIC AIC Weight Ground Temperature 5 3142.37 0 0.51 Air Temperature 5 3142.55 0.91 0.46 Null 4 3150.39 8.01 0.01 Ground Humidity 5 3151.56 9.19 0.01 Air Humidity 5 3151.70 9.52 0.004