AMPHIBIAN AND REPTILE DISEASES

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1 AMPHIBIAN AND REPTILE DISEASES 37 AMPHIBIAN AND REPTILE DISEASES Herpetological Review, 2018, 49(1), by Society for the Study of Amphibians and Reptiles Expanding Knowledge of Batrachochytrium dendrobatidis in Wyoming, USA The disease caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd; Longcore et al. 1999) has been linked to amphibian declines worldwide (Berger et al. 1998), and has been associated with declines of native amphibians in Wyoming, USA (Muths et al. 2008; Murphy et al. 2009; Pilliod et al. 2010). However, the Wyoming state distribution of Bd has not been examined. Studies of Bd occurrence and prevalence in Wyoming, as in other parts of the Rocky Mountain region, have focused on certain mountain ranges (Patla et al. 2016; Bd-maps 2017), and particularly on the Western Toad (Anaxyrus boreas; e.g., Muths et al. 2008; Murphy et al. 2009; Pilliod et al. 2010). Other mountain ranges and much of the plains and intermountain basins in the region contain species and habitats for which Bd has either not been sampled or results have not been formally published. Due in part to concerns over potential negative impacts of Bd on amphibians, our goal was to consolidate Bd sampling results from organizations and agencies that regularly survey for amphibian in Wyoming in order to assess the spatial distribution of Bd across Wyoming and its prevalence in the state s native amphibian species. The Wyoming Game and Fish Department, Wyoming Natural Diversity Database, and the United States Forest Service sampled post-metamorphic amphibians for Bd as part of standardized survey efforts to inventory or monitor amphibians from 2002 to Survey sites varied in size and structure, consisting of discrete natural and manmade aquatic features or collections of aquatic features within a drainage (e.g., oxbows, stock ponds, streams, and permanent and temporary ponds) across much of the state of Wyoming. Sampling occurred from April through JAMES A. ERDMANN Department of Biological Sciences, Southeastern Louisiana University, SLU 10736, Hammond, Louisiana 70402, USA WENDY A. ESTES-ZUMPF* Wyoming Game and Fish Department, 528 S. Adams St., Laramie, Wyoming 82070, USA CHARLOTTE SNOBERGER Wyoming Game and Fish Department, 3030 Energy Lane, Casper, Wyoming 82609, USA ZACHARY J. WALKER Wyoming Game and Fish Department, 260 Buena Vista, Lander, Wyoming 82520, USA AMY POCEWICZ The Nature Conservancy, 258 Main St., Lander, Wyoming 82520, USA *Corresponding author; Wendy.Estes-Zumpf@wyo.gov September, with the majority of samples collected between May and August. We combined results into a single Bd database to facilitate statewide surveillance of the pathogen. Sampling methods followed techniques outlined in Livo (2004a) and Hyatt et al. (2007). Using a sterile cotton swab, we rubbed the ventral surface and each inner thigh and foot of an individual amphibian 3 5 times. We placed swabs in screw-cap vials containing 70 95% ethanol and refrigerated the vials until they were shipped to the laboratory for testing. The majority of samples were analyzed at Pisces Molecular, LLC (Boulder, Colorado, USA) using a Bd DNA PCR assay modified from Annis et al. (2004). A portion of samples from Wyoming Toads Table 1. The number of Batrachochytrium dendrobatidis (Bd) positive samples (+) and the total number of samples collected (N) across all 23 counties in Wyoming, USA from County + / N % Bd-positive Albany 119 / % Big Horn 0 / 18 0% Campbell 9 / 41 22% Carbon 80 / % Converse 5 / 25 20% Crook 0 / 4 0% Fremont 6 / 21 29% Goshen 6 / 27 22% Hot Springs 0 / 5 0% Johnson 20 / % Laramie 0 / 7 0% Lincoln 35 / % Natrona 6 / 29 21% Niobrara 2 / 12 17% Park 10 / 26 38% Platte 0 / 8 0% Sheridan 24 / % Sublette 194 / % Sweetwater 0 / 2 0% Teton 133 / % Uinta 2 / 2 100% Washakie 0 / 4 0% Weston 17 / 38 45%

2 38 AMPHIBIAN AND REPTILE DISEASES Fig. 1. Major mountain ranges and ecoregions in Wyoming, USA, and distribution of Batrachochytrium dendrobatidis (Bd) in Wyoming, USA based on 1611 samples collected from 11 amphibian species during surveys from (Anaxyrus baxteri) was tested for Bd at the San Diego Zoo s Amphibian Disease Lab (Escondido, California, USA) using RealTime Taqman PCR (Boyle et al. 2004). Thus, naïve prevalence for Wyoming Toads could be biased high relative to other species due to the increased sensitivity of qpcr in detecting Bd. We tested samples individually, but some samples were pooled within drainages to reduce costs (N = 88). Samples pooled across multiple species within the same drainage (N = 23) were excluded from species-level analyses. Because we pooled some samples within drainages, we mapped results with respect to watersheds (10-digit hydrologic unit codes; HUCs; Simley and Carswell 2009). We collected Bd samples from 1611 individual amphibians at 543 individual wetland sites in 65 of 107 HUCs sampled across Wyoming and from 11 of the state s 12 amphibian species (Fig. 1). Sample sites varied in elevation from 1062 m to 3176 m. We documented Bd in 16 counties (Table 1) and every major ecoregion in Wyoming (Fig. 1; Omernik 1987; Omernik and Griffith 2014). We detected Bd at 283 of 543 (52%) sites sampled and in 9 of 11 species (Table 2). We did not detect Bd in either

3 AMPHIBIAN AND REPTILE DISEASES 39 Table 2. Detection of Batrachochytrium dendrobatidis (Bd) in 11 amphibian species in Wyoming, USA sampled in The number of Bd-positive samples (+) and the total number of samples collected (N) are summarized for each species across all sites, individuals, and single-species pooled samples. *Samples pooled from 3 8 individuals within a species and analyzed as a single sample; excludes results from samples pooled across multiple species. Sites Individuals Pooled samples* Species + / N % Bd-positive + / N % Bd-positive + / N %-Bd positive Ambystoma mavortium 5 / 25 20% 1 / 39 3% 4 / 4 100% Anaxyrus baxteri 1 / 1 100% 76 / % - Anaxyrus boreas 58 / 94 62% 68 / % 5 / 5 100% Anaxyrus cognatus 1 / 3 33% 1 / 2 50% - Anaxyrus woodhousii 14 / 65 22% 1 / 55 2% 1 / 5 20% Lithobates pipiens 31 / 84 37% 28 / % 2 / 2 100% Lithobates sylvaticus 24 / 54 44% 26 / % 6 / 6 100% Pseudacris maculata 155 / % 30 / % 37 / % Rana luteiventris 43 / 73 59% 51 / % 6 / 6 100% Spea bombifrons 0 / 5 0% 0 / 14 0% - Spea intermontana 0 / 1 0% 0 / 1 0% - Table 3. Seasonal detection of Batrachochytrium dendrobatidis (Bd) in 11 amphibian species in Wyoming, USA, sampled in Earlyseason samples were collected April June. Late-season samples were collected July September. The number of Bd-positive samples (+) and the total number of samples collected (N) are summarized across seasons for each species. *Data presented for individual samples and do not include pooled samples. Early-season* Late-season* Species + / N % Bd-positive + / N % Bd-positive Ambystoma mavortium 0 / 15 0% 0 / 19 0% Anaxyrus baxteri 26 / 45 58% 50 / 83 60% Anaxyrus boreas 28 / 87 32% 40 / % Anaxyrus cognatus 1 / 2 50% 0 / 0 na Anaxyrus woodhousii 1 / 32 3% 0 / 23 0% Lithobates pipiens 22 / 76 29% 6 / 83 7% Lithobates sylvaticus 5 / 27 19% 21 / % Pseudacris maculata 18 / % 12 / 70 17% Rana luteiventris 7 / 35 20% 41 / % Spea bombifrons 0 / 12 0% 0 / 2 0% Spea intermontana 0 / 1 0% 0 / 0 na Total 108 / % 170 / % spadefoot species (Spea intermontana, S. bombifrons); however, sample sizes for both these species were low (N = 1 and N = 15, respectively); a minimum sample size of 60 is recommended for assessing Bd occurrence when it is at low prevalence (Skerratt et al. 2008). Bd was most prevalent (naïve prevalence = proportion of samples testing positive for Bd) in Boreal Chorus Frogs (Pseudacris maculata), Columbia Spotted Frogs (Rana luteiventris), and Western Toads, with Bd detected at over 50% of sites where these species were sampled (Table 2). We documented Bd in federally endangered Wyoming Toads (and in Boreal Chorus Frogs) as early as 2007 at the one Wyoming Toad reintroduction site sampled for this analysis. Captive-reared Wyoming Toads have been released at this site since Bd is also known to have occurred since at least 2000 at the location where this species was first rediscovered (USFWS 2015). Our results indicate that Bd is widespread across Wyoming, occurring in at least 9 of the state s 12 amphibian species and in 16 of 23 counties and all major ecoregions in Wyoming. By consolidating Bd sampling results from organizations working throughout the state, our study is the first to assess the statewide distribution of Bd in Wyoming. Results identify areas of high Bd prevalence, such as the Wind River Range in west-central Wyoming (Fig. 1). This rugged and remote mountain range, most of which is designated wilderness, had one of the highest Bd rates in the state with 97 of 142 (68%) samples testing positive. Our results also identify regions and species for which minimal sampling data exists. For example, although over 50% of sites sampled tested positive for Bd, the majority of sampling occurred in montane regions. In general, lower elevations, such as the Wyoming Basin in central Wyoming, had low Bd prevalence but also had few samples as of 2014 (Fig. 1). These results allow resource managers to target information gaps in their region when conducting future amphibian inventory or monitoring surveys. We also documented an expansion in the spatial extent of Bd in the Bighorn Mountains in north-central Wyoming, with amphibians testing positive for Bd from localities where samples from previous surveys tested negative (Ross et al. 2014). This mountain range, isolated from the rest of the Rocky Mountains,

4 40 AMPHIBIAN AND REPTILE DISEASES supports a disjunct population of Columbia Spotted Frogs, a glacial relict population of Wood Frogs (Lithobates sylvaticus), and one of the few remaining high-elevation populations of Northern Leopard Frogs (L. pipiens) in Wyoming. Since first detected in Wood Frogs in the Bighorn Mountains in 2010 (Ross et al. 2014), Bd appears to have spread north to one of the largest breeding populations of the Bighorn Columbia Spotted Frog, with 5 of 8 samples testing positive for Bd in 2014 (Anicka Kratina- Hathaway, University of Wyoming, pers. comm.). Continued research is needed to determine how the apparent spread of Bd in this mountain range will affect these unique amphibian communities. Failure to detect Bd in the Plains Spadefoot and Great Basin Spadefoot could be a reflection of the small sample sizes, although previous studies have not detected Bd in metamorphosed individuals from either species (Livo 2004b; Carey and Livo 2009; Richardson et al. 2014). Spadefoots spend the majority of time away from water and typically breed in ephemeral playas and ponds in arid plains and basins and have notably short larval periods (Altig and McDiarmid 2015), which may limit their exposure to Bd. The ephemeral nature and often very warm water temperatures of these breeding ponds might further limit the ability of Bd to persist (Johnson et al. 2003). However, other North American spadefoot species have tested positive for Bd (e.g., Scaphiopus couchii, Scaphiopus holbrookii, Spea multiplicata; Tupper et al. 2011; Sigafus et al. 2014). All other amphibian species that tested positive for Bd in this study have previously tested positive for the fungus (Bd-maps 2017). Species-specific Bd prevalences reported in this study do not necessarily reflect prevalence across Wyoming because samples were not collected randomly from across each species range. Temporal biases in sampling (annual and seasonal; Kriger and Hero 2007a) and whether swabbed individuals were in water at the time of capture (Hossack et al. 2013) could further influence results. Samples collected in the spring or fall in temperate regions often exhibit higher presence and pathogenicity of Bd than those taken in the hotter summer months (Berger et al. 2004). Overall Bd prevalence in this study was similar between samples collected early in the season (25%; April June) versus later in the summer (24%; July September; Table 3); however, our study was not designed to test for seasonal differences in Bd prevalence and samples sizes were not evenly distributed across early and late season periods (Table 3). Results of this study will be used to identify gaps in our knowledge of the distribution of Bd in Wyoming, as well as amphibian populations of potential concern, thus allowing resource managers to focus future sampling efforts. Current knowledge of Bd environmental persistence based on factors such as temperature, aridity, and permanence of water features can also be used to make predictions to inform future sampling and surveillance efforts in Wyoming and abroad (Kriger and Hero 2007b; Bustamante et al. 2010; Forrest and Schlaepfer 2011; Xie et al. 2016). Although Bd is negatively impacting some amphibian populations in Wyoming, specifically Western (Boreal) Toads (USFWS 2017) and the endangered Wyoming Toad (USFWS 2015), the influence of Bd on other amphibians in Wyoming is largely unstudied and likely varies among species and populations (Lips 2016; Gervasi et al. 2017). Continued Bd sampling efforts combined with ongoing amphibian inventory and monitoring studies in Wyoming will aid our ability to assess impacts of Bd on other amphibian populations and better understand its relationship to climate and habitat. Acknowledgments. We thank the United States Forest Service and the Bureau of Land Management for supporting amphibian Bd monitoring on lands managed by those agencies in Wyoming. The Wyoming Game and Fish Department herpetology program and the University of Wyoming s Biodiversity Institute provided additional funding for Bd analyses. We thank the Wyoming Natural Diversity Database for help developing our statewide Bd database. We sincerely thank the many biologists, biological technicians, and citizen scientists who helped collect Bd samples from Wyoming s amphibians over the past 13 years. Lastly, we thank D. H. Olson, E. L. Muths, and B. R. Hossack for their thoughtful reviews and for significantly improving the quality of this manuscript. 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