Yosemite Valley (the Valley) is a glacially-carved valley on the western slope of the Sierra Nevada within Yosemite National Park (Fig. 1). The Valley elevation ranges from 900 to 1280 m, spans across ~ 1500 ha, and contains the Merced Wild and Scenic River. The north and south sides of the Valley are bounded by steep granitic cliffs and the east and west sides of the valley are bounded by steep waterfalls or cascades. Yosemite Valley is a particularly good place for bullfrog eradication as bullfrogs do not inhabit the high elevation (> 6000 ft.) aquatic tributaries that feed into the Merced River in the Valley. As a result, the only possible way for the non-assisted colonization of bullfrogs to the Valley would be from downstream source populations. The dramatic geological features of the Merced River canyon downstream from the Valley likely impede any natural migration of bullfrogs from neighboring populations.
The National Park Service (NPS) collected data on bullfrog removal efforts sporadically. The history of bullfrog removal in the Valley is a patchwork of raw data as well as anecdotal accounts (history of eradication effort in the Valley is summarized in Table 1). Bullfrog removal started opportunistically in the mid-1990s by a single NPS employee who sporadically removed bullfrogs at breeding locations for less than a month a year until 2004. Bullfrog efforts began in earnest in the Valley in 2005 when the NPS hired 2 full time restoration technicians specifically for bullfrog removal. From 2005 to 2015, a 1 or 2-person crew worked 1–4 mo./year surveying and removing bullfrogs throughout the Valley. During this time, the NPS collected data on the number of bullfrogs removed and did not record negative bullfrog sightings, the sex of bullfrogs removed, survey covariates, or the presence or densities of native anurans. After 2015, the NPS continued surveying for bullfrogs using traditional visual surveys as well as environmental DNA (eDNA) techniques. All bullfrog surveys and removal efforts occurred during the onset of breeding (mid-May) until the end of the summer (late August).
To identify breeding bullfrog populations, the NPS conducted visual surveys for egg masses and larvae (tadpoles) around all available breeding habitat: slack water, ponds, or stagnant streams (Fig. 1). Bullfrog egg masses, which are formed as large gelatinous mats, are unlike native anuran egg masses of other species in Yosemite Valley, and therefore distinguishing species was possible via visual detection. Crews conducted breeding surveys in late spring and early summer (mid May to early June) during daylight hours (0900–1600 h; see Table 1 for yearly survey effort). During surveys, crews walked slowly through the water bodies removing any egg masses and larvae observed using dip nets, paint strainers, or 5-gallon buckets. Occasionally, crews would use a backpack electrofisher (Smith-Root, Vancouver, WA) or seine nets to remove larvae.
The NPS surveyed for and removed adult and sub-adult bullfrogs during night visual surveys from the onset of bullfrog breeding (May/June) until the end of summer (August/September; see Table 1 for yearly survey effort). Crews used 200+ lumen flashlights to locate and stupefy bullfrogs via eyeshine. Crews captured bullfrogs using a variety of methods: hand grabbing, spearing (trident pole spears outfitted with rubber sling), dip netting, seine netting, or shooting with pellet rifles. On occasion, crews caught frogs using hook and line methods. Crews also attempted to trap individuals using funnel traps with “Alive Lure™”, a mechanical lure that looks and vibrates like an insect placed inside the traps as an attractant. (The funnel trap method was not successful in the Valley as no frogs were captured.)
The Ahwahnee Hotel reflection pond was an artificial concrete structure that sustained a breeding population of bullfrogs. Along with eradication activities stated above, in spring 2006, NPS drained the pond to remove all life stages of bullfrogs. This is the only water body that could be drained in the Valley. All other water bodies were naturally occurring.
We humanely euthanized all bullfrogs depending on life stage and capture method. Crews placed all collected egg masses on shorelines to dry. For adults and sub-adults captured via pole spear, crews would immediately euthanize the frog using skull blunt force trauma and pithing (Underwood et al. 2013). Crews euthanized all other adult and sub-adult bullfrogs using a buffered solution of MS 2-22 at a concentration of 2–3 g/l solution at pH 7.0–7.5 and/or skull blunt force and pithing protocol.
Environmental DNA surveys and long term monitoring
In 2015, Yosemite NPS began surveying for bullfrogs using aquatic eDNA sampling, an alternative survey technique sensitive to species at low densities. Species detection using eDNA methods is accomplished by collection and identification of trace DNA particles originating from shed skin cells, feces, etc., that are extracted from water samples (Taberlet et al. 2012). Environmental DNA methods have been used to detect aquatic amphibian species at low densities (Rees et al. 2014) and have been found to be more effective at detecting bullfrogs than traditional survey methods (Dejean et al. 2012; Goldberg et al. 2018).
For eDNA surveys, crews collected filtered water samples from suitable bullfrog habitats where breeding or presence was previously known (Fig. 1). The amount of water filtered as well as samples collected varied over the years as the NPS refined eDNA collection techniques. In 2015 and 2016, we collected a 50 ml samples every 40 m around the perimeter or length of each site as this sampling strategy was proven effective for trout species (Kamoroff and Goldberg 2018; see also Dunker et al. 2016; Table 2). In 2018, we collected 250–500 ml water samples every 40 m around the perimeter or length of each site to attempt to collected 1–2 l sample from each site (a standard eDNA sampling amount; Rees et al. 2014). However filter clogging and time limitation limited volume collected at some sites (Table 2). To detect any contamination from field equipment, we collected a 250–500 ml field blank using distilled or deionized water per site or per sampling day. We filtered all water samples using a 0.45 µm cellulose nitrate filter membrane with a 47 mm diameter filter funnel (Thermo Fisher Scientific). We used the same collection, filtration, storage, and DNA extraction method described in Kamoroff and Goldberg (2018). To determine if bullfrog DNA was present in the samples collected, we analyzed the extracted DNA from the filtered water samples in triplicate using a quantitative polymerase chain reaction (qPCR) and a previously published American bullfrog qPCR assay (Strickler et al. 2015). We included an exogenous internal positive control to ensure no PCR inhibition had occurred (IPC; Applied Biosystems) and that DNA would indeed amplify if it were present in a sample. We ran inhibited samples though OneSteptm PCR Inhibitor Removal spin columns (Zymo Research). If inhibition was still present, we diluted the samples 1:10 and re-analyzed. We created and analyzed negative extraction and qPCR controls with every batch and plate.
We considered the species to have been detected in a sample if all 3 qPCR reaction replicates tested positive. If 1 or 2 of the technical replicates tested positive, we reanalyzed the samples in triplicate. We confirmed the presence of DNA if any reanalyzed replicates tested positive during the 2nd round. We stored samples at 4 °C between qPCR runs to minimize DNA degradation caused by heat or multiple freeze–thaw events. We considered a technical replicate to be positive if an exponential increase occurred at any point of the 50 cycles (as described by Goldberg et al. 2013, see also Ellison et al. 2006).
Additional survey methods included the installation of song meters at known bullfrog sites in the Valley. From 2016 to 2018 we deployed 1 song meter at 1–2 locations where we had previously detected bullfrogs via eDNA surveys. The NPS set the song meters in early spring (April–June) during bullfrog breeding and retrieved them late summer (August–September).
The focus of the removal efforts was to directly manage for non-native species. The project was not set up as a research experiment, rather effort and time was spent when resources were available. Over the past ~ 15 years, the project has been managed by different personnel. All project managers had the same goal for removal/eradication of non-native bullfrogs, however methods, efforts, and protocols fluctuated. As a result, we did not conduct visual surveys or eDNA surveys in a systematic approach that would allow for the determination of detection probability of either method.