P092: Effectiveness of reintroductions and probiotic treatment as tools to restore the endangered yellow-legged frog (Rana sierrae) to the 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 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 in lakes. Captive rearing: The 34 R. sierrae metamorphs transported to the San Francisco Zoo were all successfully cleared of Bd, showed high survivorship, and grew rapidly. The 150 tadpoles were also cleared successfully and did well in captivity, although some mortality occurred during a brief period of unexpectedly high ammonia concentrations in the holding tanks. Many of the tadpoles were expected to have reached adult size (>40 mm snout-vent length) by summer 2014. Frog microbiome: Skin swabs collected in June and July were used to develop bacterial cultures. Approximately 60 distinct bacterial morphotypes were identified based on color, texture, and growth form, and we used molecular genetics methods to identify these morphotypes to the lowest possible level of taxonomic resolution. Samples of each morphotype were cryopreserved for use in Bd inhibition trials. Because most bacteria are not culturable, to obtain a more complete description of the frog microbiome we began designing next-generation DNA sequencing methods to identify all bacteria present in our skin swab samples.

Accomplishments and Key Findings: FY14 During FY14, we passed a major project milestone with the translocation and reintroduction of frogs into two lakes on the Lake Tahoe Basin portion of the Desolation Wilderness. On June 16, 35 adult R. sierrae that were reared at the San Francisco Zoo were transported on foot from the Echo Lakes trailhead to Lake Lucille and released. At the zoo prior to release, half of these frogs were immunized against Bd infection, and the other half served as Bd-naive controls. On June 20, we translocated 25 wild-caught adult frogs to Lake Lucille and another 25 to Jabu Lake. Half of the frogs added to each lake were collected from Pyramid Valley and the other half were collected from Rivendell Pond. On the same day, we also translocated 10 egg masses to both Lake Lucille and Jabu Lake, all of which were collected from Rivendell Pond. We also transported an additional 10 egg masses out of the backcountry and to the San Francisco Zoo for captive rearing and potential reintroduction as adults in 2015. We also continued our monthly visits to the Pyramid Valley and Rivendell Pond localities. During these 5-day visits we conducted mark-recapture surveys and collected frog skin swabs and bacterial samples. Hundreds of R. sierrae metamorphs were present at Rivendell Pond during the August trip, so 50 were collected from this site and transported to the San Francisco Zoo for captive rearing. All survived the trip and were cleared of Bd. Knapp & Vredenburg -4-

Frog donor populations: During 2014, many previously-tagged frogs were recaptured and in addition, another 201 untagged frogs were tagged in Pyramid Valley and 194 untagged frogs were tagged at Rivendell Pond. These untagged frogs included large adult frogs that had eluded capture in 2013 and smaller frogs that had recently recruited into the adult population at both sites. The total number of R. sierrae adults tagged over the two years was 433 in Pyramid Valley and 472 at Rivendell Pond. Frog movement patterns in 2014 were similar to those observed in 2013. Following breeding in the larger lakes, frogs moved into inlet and outlet streams and fringing ponds, and moved back into the larger lakes as these streams and smaller ponds began to dry up. Reasons for these seasonal movements remain unknown. Translocated/reintroduced populations: Frogs originating from the June reintroductions and/or translocations were observed repeatedly throughout the summer at both Lake Lucille and Jabu Lake. Recaptured frogs always appeared healthy and grew significantly over the summer. As is the case in the frog source populations, translocated/reintroduced frogs quickly moved from the lakes into inlet and outlet streams and nearby ponds. They subsequently moved back into the larger lakes as these adjacent habitats dried up. Frogs seen at Lake Lucille included both translocated (wild-caught) and reintroduced (zoo-reared) individuals. Captive rearing: Survival of collected egg masses and metamorphs at the San Francisco Zoo was very high. As a result, we expected to have a large number of adult R. sierrae available for reintroduction during Summer 2015. Frog microbiome: Our efforts to quantify the frog skin microbiome continued, but methodological challenges hindered our progress in achieving our objectives.

Accomplishments and Key Findings: FY15 During FY15, we conducted another round of frog translocations and reintroductions into two lakes on the Lake Tahoe Basin portion of the Desolation Wilderness. In June, 25 adult R. sierrae were translocated from a donor population (Rivendell - Eldorado NF) to Lake Lucille (LTBMU). Also in June, 35 captive-reared adult frogs were reintroduced to Lake Lucille and another 62 were reintroduced to Tamarack Lake (LTBMU). Translocated/reintroduced frog populations were monitored using capture- recapture methods and visual encounter surveys. Laboratory studies of the R. sierrae microbiome were completed, and the results provide insights into the degree to which probiotics could be used to decrease frog susceptibility to Bd. We also concluded a study that quantified the Bd-inhibiting properties of ~30 bacterial strains collected from R. sierrae in populations on the Eldorado NF and cultured in the laboratory. Translocated/reintroduced populations: Frogs from the 2014 and 2015 translocations/reintroductions were observed repeatedly throughout the summer in all three study areas (Jabu Lake, Lake Lucille and vicinity, and Tamarack Lake). Recaptured frogs always appeared healthy and grew significantly over the summer. As was observed in 2014, translocated/reintroduced frogs quickly moved from the lakes into inlet and outlet streams and nearby ponds. They subsequently moved back into the larger lakes as these adjacent habitats dried. For the zoo-reared frogs reintroduced to Lake Lucille and Tamarack Lake, Bd-exposed and control frogs were observed in approximately equal proportions, suggesting that survival of frogs in these two groups was similar over the summer. Captive rearing: The zoo-based captive rearing program was very successful at producing large numbers of healthy adult frogs for reintroduction. During the course of this study rearing methods have been fine-tuned, with important implications for the rearing of frogs from this and other populations in the future. A final cohort of captive-reared R. sierrae adults (from eggs collected at Rivendell in 2014) will be introduced to Lake Lucille and Tamarack Lake in June 2016. Knapp & Vredenburg -5-

Frog microbiome: During FY15, the analysis of all microbiome samples was completed. Results revealed that the skin microbiomes of R. sierrae tadpoles, metamorphs, juveniles, and adults were all distinct from each other, and from that of the lake environment. The tadpole microbiome was notably depauperate in bacterial species compared to later life stages. Furthermore, the skin microbiome of highly infected post-metamorphic frogs was characterized by significantly reduced species richness and evenness, and by strikingly lower variation between individuals. Over 90% of DNA sequences from the skin microbiome of highly infected frogs were derived from bacteria in a single order, Burkholderiales, compared to just 54% in frogs with lower infection levels. In a culture-dependent Bd-inhibition assay, the bacterial metabolites we evaluated all inhibited the growth of Bd. Together, these results illustrate the disruptive effects of Bd infection on host skin microbial community structure and dynamics, and suggest possible avenues for the development of anti-Bd probiotic treatments.

Conclusions This study accomplished all four of the primary objectives. Capture-mark-recapture analysis of the R. sierrae populations at the Pyramid Valley and Rivendell Pond study sites indicated that each population contained approximately 300-400 R. sierrae adults, many more than suggested by previous visual encounter surveys. Both sites contained sufficiently large populations that we were able to collect R. sierrae adults for translocation to the LTBMU study lakes and collect egg masses and metamorphs for rearing at the San Francisco Zoo. Barring significant decreases in these donor populations in future years, we anticipate that both populations could continue to serve as sources of R. sierrae for future translocation and reintroduction efforts. The R. sierrae population at Rivendell Pond, in particular, produces many egg masses and metamorphs every summer, life stages that are difficult to find at the Pyramid Valley site. During the study period covered by this grant, R. sierrae were translocated and/or reintroduced to the LTBMU study lakes (Jabu Lake, Lake Lucille, and Tamarack Lake) in 2014 and 2015. Although the grant that funded this research ended on May 31, 2016, a final reintroduction of 62 captive-reared R. sierrae adults (collected as eggs from Rivendell Pond in 2014) was conducted in June 2016. Forty of these frogs were reintroduced to Tamarack Lake and 22 were reintroduced to Lake Lucille. Results from all of the translocations/reintroductions conducted during this study include the following key points:

• Jabu Lake received a single translocation of adults and egg masses (in 2014). Following the translocation of adult frogs, numbers declined considerably in subsequent years, but frogs nonetheless were consistently observed in Jabu Lake and the small pond to the west (including in 2016). In 2015, tadpoles produced from the translocated egg masses metamorphosed, and substantial numbers of metamorphs and subadults were seen at Jabu Lake during late summer. In 2016, only a few of these subadult frogs remained but the fact that a few were observed suggests the potential for recruitment of new animals into the adult size class in 2016. In addition, the presence of second-year tadpoles at Jabu Lake in 2016 indicates successful breeding by resident adults in 2015. Successful breeding in 2016 was also confirmed by the observation of a single egg mass at Jabu Lake. Therefore, although this population has declined in size since the original translocation, it seems likely that this decline could reverse in coming years as new animals are recruited into the adult size class. Additional translocations of subadults (a size class that may be less likely to emigrate from the site than adults) may be warranted to supplement the existing population. • Lake Lucille received translocated adults (2014, 2015) and egg masses (2014), and reintroduced adults that were reared at the San Francisco Zoo (2014, 2015, 2016). Following translocation/reintroduction, frogs dispersed into the west and south-west inlet streams, Knapp & Vredenburg -6-

and both outlet streams. By 2015, tagged frogs were also detected in Lake Margery and in a pond that drains into Lake Margery from the south. Low capture probabilities of the translocated/reintroduced frogs have so far precluded a formal analysis of survival between translocated and reintroduced frogs, and for reintroduced frogs survival of immunized versus non-immunized animals. However, based on the proportion of each frog type captured, there don’t appear to be large differences in the survival of frogs in these groups. Through 2016, no tadpoles, subadults, or new (untagged) adults have been detected, suggesting that the translocated egg masses and resulting tadpoles/metamorphs had low survival and that reproduction by resident frogs has been rare or non-existent. More troubling, during the 2016 survey three dead R. sierrae adults were detected in Lake Lucille and the total number of frogs was substantially lower than in 2015. This suggests that mortality of adult R. sierrae was unusually high during the 2015-2016 winter, although the cause of this mortality remains unknown. The combination of the lack of reproduction/recruitment in this population and the recent high mortality of adults suggests that one of more barriers may exist to the establishment of this population over the long term. • Tamarack received reintroduced adults in 2015 and 2016. As with Lake Lucille, capture probabilities at this site are low. Nonetheless, reintroduced adults have been observed in Tamarack Lake itself, and in the outlet stream and ponds located on the south-east side of Tamarack Lake. Additional surveys will be needed to determine whether this population shows evidence of reproduction/recruitment in future years.

Overall, although translocated/reintroduced frogs at all three study sites appeared healthy when captured and the fact that some reproduction has occurred at Jabu Lake, it is clear that significant barriers may exist to population establishment over the long term. The microbiome findings significantly advance our understanding of the skin microbiome of R. sierrae and its interaction with Bd. However, in the near term these findings are unlikely to be useful in changing frog-Bd dynamics due to critical unanswered questions related to the degree to which the skin microbiome can be manipulated. Funding for ongoing surveys of the study populations is being pursued, and would allow key insights into whether these populations are becoming established and, if not, what barriers to establishment exist.

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Figure 1. Map of the study lakes in the Lake Tahoe Basin Management Unit (LTBMU) and adjacent Eldorado National Forest (ENF; the border between the two Forests is indicated by a black line). The two study areas on the ENF that served as frog donor populations are outlined in red: Pyramid Valley, Rivendell Pond. The LTBMU lakes from which trout were recently removed are outlined in blue and of these, the three lakes into which R. sierrae were translocated and/or reintroduced as part of the current study are indicated with black arrows (Jabu Lake, Lake Lucille, Tamarack Lake). The inset map shows the location of the project area relative to the Desolation Wilderness and Lake Tahoe.