Effectiveness of Reintroductions and Probiotic Treatment As Tools to Restore the Endangered Sierra Nevada Yellow-Legged Frog (Rana Sierrae) to the Lake Tahoe Basin

P092: Effectiveness of reintroductions and probiotic treatment as tools to restore the endangered Sierra Nevada yellow-legged frog (Rana sierrae) to the Lake Tahoe Basin Project period: July 1, 2012 to May 31, 2016 Final Report: August 4, 2016 Principal Investigator: Roland Knapp; Co-principal Investigator: Vance Vredenburg Background A century ago the Sierra Nevada yellow-legged frog (Rana sierrae) was common in lakes, ponds, and streams in the mid and upper elevations of the central and northern Sierra Nevada. Unfortunately, R. sierrae has declined precipitously in recent decades and is now absent from more than 90% of its historical range, including all of the Lake Tahoe Basin. As a consequence, R. sierrae was listed as “endangered” under the U.S. Endangered Species Act in 2014. The decline of R. sierrae is being driven primarily by the extensive introduction of non-native trout into naturally fishless habitats and the emerging infectious disease, chytridiomycosis. Nearly all lakes and streams at higher elevations in the Sierra Nevada were naturally fishless, but starting in the late 1800s several species of trout were widely introduced into these habitats. Predation by trout on R. sierrae has caused widespread declines and extirpations of R. sierrae populations. Chytridiomycosis is an infectious disease of amphibians caused by the fungus Batrachochytrium dendrobatidis (“Bd”). The extraordinary virulence of Bd has caused the decline or extinction of hundreds of amphibian species around the world during the last several decades and hundreds more are considered at risk as Bd spreads into new areas. R. sierrae is particularly susceptible to Bd, and the spread of this pathogen across California during the past 30 years has caused the loss of hundreds of frog populations from remaining fishless habitats in the Sierra Nevada. Fortunately, some R. sierrae populations have persisted following Bd-caused declines, and recent experiments indicate that frogs in these “persistent” populations are more resistant to Bd than are Bd-naïve frogs. Although numerous efforts are now underway to recover R. sierrae populations by removing non-native trout, ongoing chytridiomycosis in the remaining frog populations presents a major challenge for recovery efforts. Even in R. sierrae populations that are persisting with this disease, chytridiomycosis often causes recruitment failure that may decrease the chances of successful population reestablishment. This may be particularly the case when frogs from persistent populations are translocated to nearby habitats or are used in captive rearing efforts because of the typically small number of frogs involved in such efforts. Therefore, the success of translocations and reintroductions in reestablishing frog populations may be low unless actions are taken to maximize the number of translocated/reintroduced frogs, and/or reduce the impact of chytridiomycosis. Two possible methods to reduce frog susceptibility to chytridiomyocosis are (1) augmenting the frog skin microbiome of translocated frogs with probiotic bacteria that reduce Bd infection intensities, and (2) exposure of zoo- reared (Bd-naïve) frogs to Bd to prompt an immune response that could serve to protect frogs from future Bd infection. Objectives The overall objective of the current study was to re-establish self-sustaining populations of R. sierrae in the Desolation Wilderness portion of the Lake Tahoe Basin (managed by the Lake Tahoe Basin Management Unit – LTBMU). We hoped to accomplish this by obtaining R. sierrae from populations on the adjacent Eldorado National Forest that are persisting despite ongoing chytridiomycosis, and Knapp & Vredenburg -2- translocating these animals to adjacent suitable habitats in the Lake Tahoe Basin. In addition, early life- stage R. sierrae collected from the frog source populations were to be reared to adulthood at the San Francisco Zoo, exposed to Bd and then cleared to stimulate an immune response against Bd, and then reintroduced into the lakes on the LTBMU. Some of the reintroduction sites are the same sites used for frog translocations, and some would only receive reintroduced (captive-reared frogs). The specific study objectives were as follows: 1. Using mark-recapture methods, estimate the number of R. sierrae adults present in the Eldorado National Forest populations proposed as sources of frogs for the translocations. These population size estimates are critical for determining how many frogs can be collected from these populations. 2. Given the initial uncertainty as to whether the source populations were sufficiently large to allow collection of any adult frogs, develop a frog rearing facility at the San Francisco Zoo and use this facility to rear R. sierrae tadpoles and metamorphs to adulthood for possible use in the frog translocations. 3. Identify candidate probiotic bacteria for use in the bacterial augmentation treatments. Ideal bacteria would be present naturally on R. sierrae in the proposed source populations, strongly reduce the growth of Bd, and be able to colonize frogs and persist on frogs at high densities for months or years. 4. After Objectives 1-3 are achieved, translocate frogs from the source populations to the reintroduction sites. Before releasing translocated frogs all animals will be treated with probiotic bacteria to reduce their susceptibility to Bd. Survival of translocated frogs will be quantified using mark-recapture methods. Identification of probiotic bacteria was more complicated and took longer than originally thought. As a result, Objective 3 was not met until the final year of the project. Therefore, translocated and/or reintroduced R. sierrae were not subject to probiotic augmentation as part of this project. Accomplishments and Key Findings: FY13 During FY13, we focused our research efforts on Objectives 1-3. Specifically, this included (1) an intensive R. sierrae mark-recapture effort in the Pyramid Valley area to quantify frog population size, habitat use, and movement patterns, (2) collection of samples to characterize the microbial communities on frogs, and (3) collection of samples to describe Bd infection intensities on frogs. In mid- June we initiated a mark-recapture study on the two proposed frog source populations: (1) Pyramid Valley, and (2) an isolated pond located on the northwest side of Lake Aloha that we refer to as “Rivendell Pond” (Figure 1). Pyramid Valley contains a large complex of lakes (Pyramid Lake, Waca Lake) and unnamed ponds connected by ephemeral streams. Breeding occurs primarily in the lakes, but frogs disperse to adjacent ponds and streams during the summer. The Rivendell Pond site contains a pond and an adjacent perennial stream. As in Pyramid Valley, breeding occurs in the pond but many adults occupy the stream habitat during the summer months. During our visit all habitats at both sites were searched for adult frogs on three consecutive days. Adults were captured using hand nets, and were PIT tagged, swabbed, weighed, measured, and released. Separate swabs were collected for quantifying Bd loads, culturing bacteria, and determining bacterial community composition. To initiate the rearing of R. sierrae at the San Francisco Zoo, we also collected 34 R. sierrae metamorphs and 150 first-year tadpoles from Rivendell Pond and transported them out of the backcountry and to the zoo. All animals survived the transport and at the zoo were cleared of Bd using the anti-fungal drug itraconazole. Frog donor populations: During FY13, a total of 510 adults were tagged. Similar numbers of frogs were tagged at Pyramid Valley and Rivendell Pond: 232 versus 278, respectively. Given that substantial Knapp & Vredenburg -3- numbers of frogs captured during the September trip remained untagged, both populations are likely considerably larger than the number of tagged frogs alone would indicate. This large number of adults at both sites was unexpected because previous visual surveys suggested the presence of no more than 50 adults at either location. This finding that both proposed source populations are much larger than previously believed was very important because it suggested that moderate numbers of adults could be collected from both sites for use in a frog translocation effort. Given the relatively low susceptibility of adults to Bd, use of adults (instead of metamorphs or tadpoles) would greatly increase the chances that the translocations would be successful in allowing the establishment of reproducing frog populations. At both sites, following breeding in the deeper lake habitats nearly all adults moved to stream habitats. Similar habitat preferences have been documented for R. sierrae populations elsewhere in the Sierra Nevada. In Pyramid Valley, >90% of adults were observed in the Pyramid Lake outlet and west inlet. Small numbers of frogs were observed in ponds scattered throughout Desolation Valley, but very few frogs were seen at Waca Lake. In September, when flows in the Pyramid outlet were reduced to a trickle, frogs left this habitat. Some entered Pyramid Lake but surprisingly some moved all the way into the west inlet, a considerable distance. At Rivendell Pond, >90% of adults were found in the stream that flows to the north of the pond. Frogs appeared to be moving back into the pond in September, at which time flows in the stream were greatly reduced. The reason for the strong preference by adult frogs for stream habitats is unclear, but may be related to the higher amounts of cover available for frogs in streams compared to

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