1. Title Page
Project Title: Effectiveness of reintroductions and probiotic treatment as tools to restore the
endangered Sierra Nevada yellow-legged frog (Rana sierrae) to the Lake Tahoe Basin
Theme: Watershed, water quality, and habitat restoration
Subtheme: (2c) Increasing our understanding of special status species and communities
Principal Investigators:
Roland A. Knapp: Sierra Nevada Aquatic Research Laboratory, University of California, 1016 Mount
Morrison Road, Mammoth Lakes, CA 93546; Phone: (640) 647-0034; Fax: (877) 595-7524;
E-mail: knapp@lifesci.ucsb.edu
Vance T. Vredenburg: Department of Biology, HH 754, San Francisco State University, San
Francisco, CA 94132-1722; Phone: (415) 338-7296; Fax: (415) 338-2295; E-mail:
vancev@sfsu.edu
Collaborators:
Sarah Muskopf: U.S. Forest Service, Lake Tahoe Basin Management Unit, South Lake Tahoe
Jann Williams & Rob Grasso: U.S. Forest Service, Eldorado National Forest, Placerville
Mitch Lockhart: California Dept. of Fish and Game, High Mountain Lakes Project, Sacramento
Jeremiah Karuzas: U.S. Fish and Wildlife Service, Sacramento
Grants Contact Person:
Cara Egan-Williams, Sponsored Projects Officer, University of California, Santa Barbara, CA 931062050; Phone: (805) 893-8809; Fax: (805) 893-2611; E-mail: eganwilliams@research.ucsb.edu
Total Funding Requested: $276,292
1
2. Proposal Narrative
Abstract
The Sierra Nevada yellow-legged frog (Rana sierrae) was until recently a common inhabitant of the central
and northern Sierra Nevada, including the Lake Tahoe Basin. Because of its abundance, R. sierrae played an
important role in structuring aquatic and adjacent terrestrial ecosystems, with strong effects on nutrient cycling
and food web dynamics. Unfortunately, due primarily to the introduction of non-native fish and a novel (but
widespread) amphibian pathogen (Batrachochytrium dendrobatidis), R. sierrae is now absent from more than 90%
of its historical range and may be extirpated from the Lake Tahoe Basin. Reversing this decline will depend critically
on the removal of introduced fish from key habitats and on frog reintroductions. B. dendrobatidis infection has
limited the success of previous R. sierrae reintroduction efforts, but a recently developed probiotic treatment
against B. dendrobatidis may provide an effective method of minimizing disease impacts. In this study, we propose
to test the effectiveness of reintroductions and probiotic treatment as tools to restore R. sierrae to the Lake Tahoe
Basin. The frog treatment will involve augmenting the microbial community that inhabits the skin of R. sierrae with
Janthinobacterium lividum, a bacterium that in recent laboratory and field trials was found to strongly inhibit the
growth of B. dendrobatidis on amphibians, including R. sierrae. J. lividum is common in soil and water, and is found
naturally at low density on the skin of R. sierrae. In the first year of the project, 10 adults and 160 juveniles will be
translocated from source populations on the Eldorado National Forest to two lakes on the adjacent Lake Tahoe
Basin Management Unit. The reintroduction lakes were recently returned to their natural fishless condition and
contain high-quality habitat for R. sierrae. Prior to release at the reintroduction sites, some of the frogs will be
treated with J. lividum and the remainder will serve as untreated controls. An additional 160 juveniles will be
treated and translocated during the second year of the project. Following frog treatment and release, the effect of
J. lividum treatment on disease status and survival of frogs will be quantified over a two year period using capturerecapture methods and radio-telemetry. This research will provide critical insights into the effectiveness of J.
lividum treatment in increasing the survival of R. sierrae. If the treatment is effective, the study results could
markedly increase the success of future R. sierrae reintroduction efforts and have broad implications for the
recovery of this declining species in the Lake Tahoe Basin and throughout the Sierra Nevada.
Project justification
This proposal addresses issues in the Watershed, Water Quality, and Habitat Restoration theme, and
specifically in Subtheme 2c: Increase our understanding of special status species and communities. The Sierra
Nevada yellow-legged frog (Rana sierrae) is a special status species that was historically abundant in the central
and northern Sierra Nevada, including the Lake Tahoe Basin. As a consequence of its precipitous decline, it is
increasingly the focus of conservation and restoration efforts led by both federal and state agencies. These include
(1) an ongoing multi-agency effort led by the U.S. Fish and Wildlife Service (USFWS) to develop a Conservation
Strategy for R. sierrae, (2) the pending listing of R. sierrae under the U.S. and California Endangered Species Acts,
(3) ongoing efforts by the California Department of Fish and Game (CDFG) to develop Aquatic Biodiversity
Management Plans for watersheds throughout the Sierra Nevada (including a recently completed plan for the
Desolation Wilderness) that identify restoration opportunities for R. sierrae and other native amphibians, and
(4) efforts by the National Park Service (NPS), U.S. Forest Service (USFS), and CDFG to remove non-native fish
populations from key habitats to recover R. sierrae populations. This latter effort includes recent fish removal
projects in the Desolation Wilderness portion of the Lake Tahoe Basin Management Unit (LTBMU). The proposed
project would provide critical information and guidance on the effectiveness of frog reintroductions as a means to
reestablishing R. sierrae populations in areas from which they were previously extirpated.
2
Background and problem statement
The mountain yellow-legged frog (“MYL frog”) is a species complex composed of two closely-related taxa,
the Sierra Nevada yellow-legged frog, Rana sierrae, and the southern mountain yellow-legged frog, Rana muscosa
(Vredenburg et al. 2007). Historically, these frogs were abundant across California’s Sierra Nevada and Transverse
and Peninsular Ranges (Vredenburg et al. 2005). In the Sierra Nevada, MYL frogs occupied the majority of lake,
pond, marsh, and stream habitats, and may have been the most abundant vertebrate in these montane
ecosystems (Grinnell and Storer 1924). Because of their abundance, R. sierrae and R. muscosa played important
roles in structuring aquatic and adjacent terrestrial ecosystems, with notable effects on nutrient cycling and food
web dynamics (Finlay and Vredenburg 2007). Unfortunately, R. sierrae and R. muscosa have both declined
precipitously in recent decades and are now absent from more than 90% of their historical ranges (Vredenburg et
al. 2007). These declines have caused a variety of cascading effects, including declines in predator populations and
altered nutrient cycles (Matthews et al. 2002, Knapp 2005). In response to the steep decline of the MYL frog, R.
muscosa in southern California was listed as “endangered” under the U.S. Endangered Species Act (ESA) in 2002,
and decisions regarding the listing of R. muscosa and R. sierrae in the Sierra Nevada under both the California and
U.S. ESAs are expected by 2012 and 2013, respectively.
The decline of the MYL frog is being driven primarily by the introduction of non-native fish and the
emerging infectious disease, chytridiomycosis (Knapp et al. 2003, Vredenburg 2004, Knapp et al. 2007, Vredenburg
et al. 2010). Within the historical range of MYL frogs, most aquatic habitats were naturally fishless due to the
presence of natural barriers that prevented the upstream movement of fish from occupied downstream habitats.
Starting in the mid-1800s, several species of trout were widely introduced into fishless lakes and streams
throughout the Sierra Nevada and in montane habitats in southern California to create recreational fisheries
(Knapp et al. 2001a). Predation by trout on all MYL frog life stages resulted in marked declines of MYL frogs across
their range (Knapp and Matthews 2000, Vredenburg et al. 2005). These declines caused by introduced trout are
now being partially reversed via removal of trout populations from some sites by the NPS, CDFG, and USFS (e.g.,
Knapp et al. 2007).
Chytridiomycosis is an infectious disease of amphibians caused by the fungus Batrachochytrium
dendrobatidis (“Bd”; Longcore et al. 1999). The extraordinary virulence of Bd has caused the decline or extinction
of hundreds of amphibian species around the world during the last several decades (Skerratt et al. 2007) and
hundreds more are considered at risk as Bd spreads into new areas. The MYL frog 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 (Rachowicz et al. 2006, Vredenburg et al. 2010).
Although Bd is now virtually ubiquitous across the Sierra Nevada, some frog populations have persisted following
Bd-caused declines and have even partially recovered their previous abundance despite ongoing chytridiomycosis
(Briggs et al. 2010). Recent experiments indicate that frogs in these “persistent” populations are more resistant to
Bd than are Bd-naïve frogs (Briggs et al., in preparation), perhaps due to selection during Bd-caused die-offs for
more resistant frog genotypes. Although MYL frogs in these persistent populations are more resistant to Bd,
juvenile frogs still experience greatly increased mortality and the resulting recruitment bottlenecks cause adult
populations to be relatively small (Briggs et al. 2010, Knapp et al. 2011).
Although numerous efforts are now underway to recover MYL frog populations by removing non-native
fish, ongoing chytridiomycosis in remaining frog populations presents a major challenge for recovery efforts. This
disease typically causes recruitment failure, which has resulted in small populations with little ability to recolonize
nearby habitats. As a consequence, reintroducing frogs obtained from persistent populations into nearby suitable
habitats will often be necessary to overcome these dispersal barriers (Knapp et al. 2001b). However, because of
the recruitment failures that characterize Bd-infected MYL frog populations, the success of translocations in
reestablishing frog populations may be low unless actions are taken to reduce the impact of chytridiomycosis.
3
One of the most promising methods of mitigating the effects of chytridiomycosis is treatment of frogs
with probiotic bacteria. Amphibian skin is inhabited by a diverse microbial community, including some bacteria
that produce compounds that strongly suppress the growth of Bd (Woodhams et al. 2007). One of the best-studied
of these bacteria is Janthinobacterium lividum, a species that occurs commonly in water and soil, and is also found
in low densities on the skin of MYL frogs. In a recent laboratory experiment, augmentation of the microbial
community on the skin of MYL frogs with J. lividum decreased Bd infection intensities dramatically increased the
survival of treated frogs compared to untreated controls (Harris et al. 2009). Encouragingly, a field trial conducted
in Kings Canyon National Park in 2010 in which Bd-infected R. sierrae were treated with J. lividum also indicated
that treatment significantly increased frog survival during a one year period following J. lividum augmentation
(Vredenburg et al., unpublished data). These results have important implications for MYL frog reintroduction
efforts that attempt to use Bd-infected frogs to establish new persistent frog populations. Reintroductions, in
which frogs from persistent populations are moved to suitable but unoccupied habitats, will be an essential
component of any recovery program for MYL frogs, but recent efforts have met with limited success because of
high Bd-caused frog mortality (one of five reintroduced populations became established; Knapp et al. 2011). The
relatively low success rate that characterized these translocations could likely be significantly increased if done in
combination with treatments that reduce the impact of chytridiomycosis.
Objectives and hypotheses to be tested
The objective of the proposed study is to determine whether the success of MYL frog reintroduction
efforts can be increased by augmenting the microbial communities living on the skin of translocated frogs with
probiotic bacteria that are known to suppress the growth of Bd. Specifically, we will test the hypothesis that J.
lividum augmentation increases the survival of Bd-infected juvenile and adult R. sierrae, and improves the chances
that reintroduced frog populations will become established. If the treatments are successful, the experiment could
result in the reestablishment of R. sierrae at two locations in the Lake Tahoe Basin and provide the basis for
reestablishing additional populations on the LTBMU. More generally, the results of this experiment will provide
critically needed guidance for similar reintroductions being planned by the USFWS, NPS, USFS, CDFG, and U.S.
Geological Survey (USGS) as part of a larger effort to recover MYL frogs across their historical ranges.
Approach, methodology, and location of research
The LTBMU is an ideal location for the proposed research. Historically, R. sierrae were common across the
Lake Tahoe Basin, with known occurrences distributed from Lake Tahoe to the headwater lakes located in the
Desolation Wilderness. As is the case across its range, within the LTMBU R. sierrae has declined precipitously
during the past 50 years due primarily to widespread fish stocking and the spread of Bd, and may now be entirely
extirpated from this jurisdiction. The LTBMU contains numerous potential reintroduction sites, including several
lakes in the Desolation Wilderness from which trout populations were recently removed (Figure 1; see Relationship
to previous and current projects [page 7] for details). In addition, several relatively robust populations of R. sierrae
occur on the adjacent Eldorado National Forest (ENF) that could serve as potential sources of frogs for
reintroductions (Figure 1). As is typical for remaining R. sierrae populations across their range, these populations
are all infected with Bd and as a consequence are characterized by high juvenile mortality and resulting small
numbers of adult frogs. Despite the ongoing impacts of chytridiomycosis, several of these populations increased
markedly in recent years following the removal of non-native trout from key habitats. Unfortunately, none of these
R. sierrae populations are close enough to the fish-removal lakes in the LTBMU to allow natural recolonization
(Figure 1). Therefore, reestablishment of frogs at lakes in the Desolation Wilderness portion of the LTBMU will
require active reintroduction.
Our study is designed to accommodate the limited number of adult R. sierrae and the larger number of
juvenile R. sierrae available from frog source populations on the ENF. Specifically, we propose translocating a total
of 10 adults and 320 juveniles to two sites on the LTBMU. Prior to reintroduction, some of these frogs will be
4
treated with J. lividum and the remainder will serve as untreated controls. Post-release monitoring of adults and
juveniles will allow us to quantify the effect of J. lividum treatment on Bd infection intensity and frog survival. To
allow more intensive monitoring of adults, we will use radio-telemetry to locate all adults during each site visit.
Collectively, this research will provide important insights into the effectiveness of J. lividum treatment in increasing
the survival of R. sierrae. If the treatment is effective, this could markedly increase the success of future MYL frog
reintroduction efforts.
Juveniles: During mid-summer 2012, we will capture a total of 160 juveniles (<40 mm snout-vent length)
from the eight largest remaining R. sierrae populations in the Pyramid Creek watershed (Figure 1). We will survey
these eight sites every two days during a one week period and collect as many juveniles as possible during each
visit. Captured juveniles will be transferred to large mesh cages (2 m long x 2 m wide x 0.75 m tall) erected at each
capture site. Prior to release into the cages, each animal will be marked with an individually-numbered tag inserted
into the rear foot webbing (1 mm x 3 mm Visible Implant Alphanumeric tags: Heard et al. 2008), weighed, and
measured. We will also collect a skin swab from each juvenile using standard methods; skin swabs will be analyzed
using real-time quantitative PCR (polymerase chain reaction) to provide an estimate of Bd infection intensity (Hyatt
et al. 2007). When sufficient numbers of juveniles have been captured, we will assign them at random to one of
two groups: (1) treated with J. lividum, or (2) untreated (controls; Figure 2). To maximize the number of treated
animals available for reintroduction while maintaining high power to detect treatment effects, 70% of the animals
will be assigned to the treated group and 30% to the untreated group (Figure 2). J. lividum will be cultured in the
laboratory from skin swabs obtained from R. sierrae in the source populations. Juveniles will be exposed to J.
lividum by placing animals individually in small plastic containers that contain a concentrated solution of J. lividum.
Treatments will last for one hour. Following treatment, animals will be released back into the cages and held for an
additional 48 hours. Skin swabs collected from each frog immediately before treatment and at 24 and 48 hours
after treatment will allow us to quantify the short-term effects of J. lividum treatment on Bd infection intensity.
Juveniles in the untreated group will be handled in a manner identical to those in the treated group but the
solution to which they are exposed will be lake water.
After 48 hours, all of the juveniles in the treated group will be re-treated with J. lividum using the same
methods as described above. This second treatment will ensure that the concentration of J. lividum on the frogs’
skin is as high as possible prior to translocation. Following this second treatment, 30% of the animals in each group
(assigned at random) will be released back into the source lake (see next paragraph for details) and 70% will be
transported to the LTBMU reintroduction sites: Lake Margery and Cagwin Lake (Figure 1, Figure 2). Frogs will be
assigned to the lakes at random, with each lake receiving 50% of the frogs in each group (Figure 2). We chose these
two lakes for reintroductions because they have been fishless for several years, contain high-quality R. sierrae
habitat, and are subject to relatively low human visitation. Frogs will be transported to these sites on foot (e.g.,
Knapp et al. 2011). In summary, of the 160 juveniles initially captured, a total of 56 will be introduced into each
lake, of which 39 are treated with J. lividum and 17 are untreated controls (Figure 2).
We propose releasing 30% of treated and untreated animals back into the source populations (Figure 2)
for two reasons. First, this will allow us to separate the effect of J. lividum treatment from the effect of
translocation on frog survival. Both could have significant impacts on survival, and a failure to separate these
independent effects could confound the results. For example, if all frogs were translocated and translocation itself
had severe negative effects on the survival of juvenile frogs, this could mask a significant positive effect of J.
lividum treatment on juvenile survival. Second, if the treatment of juveniles with J. lividum significantly increases
their survival, returning 30% of the treated animals back to the source populations could at least partially
compensate for the removal of juveniles for the reintroductions.
Following release into the source populations and reintroduction lakes, we will survey all source and
reintroduced populations once per week through late-September 2012 (by October, R. sierrae become relatively
inactive and are difficult to find: Matthews and Pope 1999). Juveniles detected during surveys will be captured,
5
identified via their tag, swabbed, measured, weighed, and released. During summer 2013, we will conduct a
second round of J. lividum treatments and reintroductions using the same methods as described above (Figure 2).
Repeating the experiment during a second year will increase our statistical power to detect treatment effects and
will allow us to test for year-specific effects (e.g., if frog survival varies between years due to different weather
conditions). In addition, conducting reintroductions in both 2012 and 2013 will increase the total number of
juvenile frogs reintroduced into Lake Margery and Cagwin Lake, thereby increasing the likelihood that the
reintroductions will produce self-sustaining populations of R. sierrae. Juveniles in the source and reintroduced
populations will be monitored as in 2012. No additional treatments or reintroductions will be conducted in 2014,
but populations will continue to be monitored using the same methods as were used in 2012 and 2013, with site
visits conducted approximately once every two weeks from July to September. As in 2012 and 2013, juveniles
detected during surveys will be captured, identified via their tag, swabbed, measured, weighed, and released. Frog
recapture histories will be analyzed after years 1, 2, and 3 of the study using Program MARK, and will allow us to
quantify separately the effects of (1) J. lividum treatment, (2) frog translocation, and (3) translocation year, on
juvenile survival.
We maintain that the proposed removal of 112 juveniles per year from the source populations for
translocation to Lake Margery and Cagwin Lake (Figure 2) will have minimal effect on the source populations
because juvenile mortality in Bd-infected MYL frog populations is typically >99% (Briggs et al. 2010). Therefore, at
most 1-2 of the 112 juveniles would be expected to survive in the absence of any intervention. If the J. lividum
treatment significantly increases the survival of the 34 juveniles that are treated and released back into the source
populations (Figure 2), this would result in a net benefit to the source populations.
Adults: In mid-summer 2012, we will capture a total of 10 adult frogs (≥40 mm snout-vent length) from
the source populations (at the same time that juveniles are collected). To minimize the impact to any one
population, no more than two adults will be obtained from each population. Following capture, adults will be held
for up to two days in large mesh cages erected at each capture site (thereby providing sufficient time for all ten
frogs to be collected). Prior to release into the cages, each animal will be tagged with an 8 mm Passive Integrated
Transponder (PIT) tag inserted under the dorsal skin (Briggs et al. 2010), swabbed, weighed, and measured. Six of
the ten adults will be treated twice with J. lividum using the methods described for juveniles (i.e., animals assigned
at random as “treated” or “untreated”, second treatment conducted 48 hours after the first treatment). The four
untreated adults will be handled in the same manner as treated adults but will be exposed to lake water instead of
to J. lividum. Following the second treatment, all ten adults will be translocated to Lake Margery and Cagwin Lake
on foot. Three treated adults and two untreated (control) adults will be released into each lake. Prior to release,
we will attach a miniature radio-transmitter externally to each frog using a waist belt (Matthews and Pope 1999).
Transmitters will allow us to locate each frog on a regular basis for determination of location, Bd infection
intensity, and status (alive-healthy, alive-sick, or dead). Following release and until late-September, adult frogs at
both lakes will be located daily using radio-telemetry, and their exact position recorded using a GPS. Once per
week we will swab, measure, and weigh each live frog. For any adult frogs that die during the 2012 summer, the
cause of death will be determined whenever possible. In late-September, we will remove radio-transmitters from
all adult frogs. Because of the small number of adults available at the source populations, no additional
translocations or radio-telemetry of adults will occur in 2013 and 2014. However, in both years we will continue to
monitor these adult populations, using visual encounter surveys conducted at Lake Margery and Cagwin Lake
every two weeks from June to September. Any adults detected during surveys will be captured, identified via their
PIT tag, swabbed, measured, weighed, and released. Although our sample size of 10 reintroduced frogs is too small
to allow analysis of frog recapture histories using Program MARK, we will nonetheless be able to use the data to
provide a general description of movement patterns, Bd infection intensities, and status of adult frogs over the
three year study period.
6
Under the proposed study design, the majority of our research effort will be spent quantifying Bd
infection intensities and survival of frogs at the LTBMU reintroduction sites. However, as described above, a key
aspect of the study design calls for also conducting this frog population monitoring at the ENF source populations.
To ensure that the funds obtained from the Tahoe Research Project are spent primarily on research conducted
within the Lake Tahoe Basin, the requested funds will be used to collect frogs from the ENF source populations,
apply the J .lividum treatments, and conduct surveys of the reintroduced frog populations at Lake Margery and
Cagwin Lake (located on the LTBMU). Post-treatment surveys of the ENF source populations will be conducted
using funds provided as in-kind contributions by one of the principal investigators (R. Knapp), fisheries staff from
the ENF, and field technicians from the CDFG (see VI. Budget for details).
Relationship to previous and current projects
The proposed project is closely tied to several recently-completed or ongoing projects designed to restore
R. sierrae habitat and reverse the decline of this species. First, in 2008 the LTBMU initiated a project to restore
habitats for R. sierrae by removing non-native trout populations from eight lakes in the LTBMU portion of the
Desolation Wilderness (Figure 1). Trout removal was conducted using gill nets (Knapp and Matthews 1998).
Funding for this effort was provided by a Southern Nevada Public Land Management Act (SNPLMA) capital project
grant to the USFWS and USFS (total cost: $73,000) and by LTBMU appropriated dollars ($15,000). At the inception
of this project it was hoped that R. sierrae would naturally recolonize the target lakes following fish removal. When
this did not occur, LTBMU staff (S. Muskopf) initiated discussions with other agencies and scientists regarding the
feasibility of reintroducing R. sierrae to these lakes, and those discussions led to the development of the current
research proposal. In addition, the LTBMU fish removal project was itself part of a much larger effort being
conducted throughout California by the CDFG, NPS, and USFS to remove non-native trout populations from key
MYL frog habitats.
Second, as part of a California-wide project to recover MYL frog populations in recent years the CDFG has
conducted surveys for amphibians and non-native fish at more than 7,000 lakes and ponds across national forest
lands in the Sierra Nevada. The information obtained during these surveys is being used to develop watershedbased Aquatic Biodiversity Management Plans (ABMP) that describe (1) the current distribution of amphibians and
fish, and (2) opportunities to restore MYL frog populations and improve fisheries. An ABMP was recently
completed for the Desolation Wilderness and designated the Pyramid Creek watershed and the LTBMU fish
eradication lakes as Native Species Reserves. In these reserves, activities aimed at restoring MYL frog populations
are a high priority. To implement the actions proposed in the Desolation Wilderness ABMP, the CDFG continues to
assist the ENF and LTBMU with ongoing fish eradication efforts and amphibian surveys. Survey data from these
efforts were used to identify the R. sierrae source populations in the current proposal. The proposed R. sierrae
reintroduction project described here allows the implementation of some of the highest priority actions identified
in the Desolation Wilderness ABMP to restore R. sierrae to these Native Species Reserves.
Third, the USFWS is currently leading a multi-agency effort to develop a Conservation Strategy for R.
sierrae and R. muscosa in the Sierra Nevada. This document will identify critically important actions that must be
undertaken to reverse the decline of both species, and will focus particularly on removal of non-native fish
populations and frog reintroductions. Currently, a major limitation on the widespread use of reintroductions is the
lack of information on what methods to use to maximize the success of reintroduction efforts in Bd-positive
landscapes. The proposed project will add considerably to our understanding of the extent to which
chytridiomycosis limits reintroduction success and, if our J. lividum treatments are successful, will provide a
critically important method by which to minimize the effects of Bd. As such, results from the proposed project
could have broad implications for the design and implementation of future recovery actions undertaken on behalf
of MYL frogs across their range.
7
Strategy for engaging with managers and obtaining permits
The proposed study was developed in close coordination with staff from the LTBMU, ENF, CDFG, and
USFWS. This included discussion of alternative study designs and agency-specific permitting requirements. Key
staff in each agency also reviewed the proposal and briefed key staff (e.g., USFS Wilderness Managers on the ENF
and LTBMU) on the proposal. As a result, a broad network of people within each agency are aware of the proposal
and have provided feedback to the research collaborators on the process and timeframe for obtaining research
permits. This high level of coordination will continue throughout the planning and implementation phases of this
project. If the proposed project is funded, the principal investigators (PIs) will immediately convene a coordination
meeting with all agency collaborators to initiate the research permit process (April 2012). At this time, we will also
review the plans and timeline for all project-related research activities slated for summer 2012. To ensure that
permits are obtained in a timely manner and that all research-related questions and concerns are addressed, these
meetings will continue on a monthly basis until the start of the project in June 2012. We will also hold similar
coordination meetings in the months leading up to the 2013 and 2014 summer research seasons.
To ensure that agency staff and managers are kept informed of the results from the research, the PIs will
distribute the quarterly and annual progress reports (that are provided to the Tahoe Science Program coordinator)
to all collaborators. In addition, in January following the 2013, 2014, and 2015 field seasons, the PIs will convene a
meeting with the collaborators and other agency staff to discuss the research findings in detail. The PIs will also be
available to give presentations on the research project to any interested agency. To provide outreach to other
stakeholders and to the general public, the PIs will also work with PSW and the Tahoe Science program coordinator
to identify outreach opportunities.
Description of deliverables
Deliverables: The primary deliverables resulted from the research project will include quarterly progress
reports, annual reports, and a final report that describe the research findings to date. These reports will include
sufficient detail to ensure that relevant results can be used by collaborating agencies to guide their ongoing
development of MYL frog-related recovery actions. At the end of the project, all project-related data will be made
available to the collaborators, other interested agencies, and the general scientific community (see next paragraph
for data plan details). In addition, the study findings will be described in 1-2 manuscripts that will be submitted to
peer-reviewed journals (e.g., Conservation Biology, Ecological Applications). All collaborators will review these
manuscripts prior to submittal.
Data plan: All data will be collected on field computers (iPhones) using data entry forms developed with
Pendragon Forms software. Fields in these forms have extensive error-checking capabilities and use drop-down
menus whenever possible (e.g., VIA and PIT tag numbers) to standardize data collection and minimize data entry
errors. Data on field computers will be backed up onto external memory cards at the end of every day in the field.
Data will be transmitted via a cellular link to an Access database housed on a server at the Sierra Nevada Aquatic
Research Laboratory. This data collection system has been used extensively during the past 10 years by the PIs. At
the end of each field season, accuracy of data in the Access database will be checked using a series of Access
queries. Throughout the project, the Access database and all other project-related materials will be made available
to the collaborators. Following publication of the project-related papers in scientific journals, the Access database
and project-related data sets will be archived in the Tahoe Integrated Management System (http://www.tiims.org)
and Dryad (http://datadryad.org), where they will be publically accessible. Dryad is an international repository of
data underlying peer-reviewed articles in the basic and applied biosciences.
8
III. Project Schedule (3
Years)
9
IV. Literature Cited
Briggs, C. J., R. A. Knapp, and V. T. Vredenburg. 2010. Enzootic and epizootic dynamics of the chytrid fungal
pathogen of amphibians. Proceedings of the National Academy of Sciences, USA 107:9695-9700.
Crump, M. L., and N. J. Scott, Jr. 1994. Visual encounter surveys. Pages 84–91 in W. R. Heyer, M. A. Donnelly, R. W.
McDiarmid, L.-A. C. Hayek, and M. S. Foster, editors. Measuring and Monitoring Biological Diversity:
Standard Methods for Amphibians. Smithsonian Institution Press, Washington, D.C., USA.
Finlay, J. C., and V. T. Vredenburg. 2007. Introduced trout sever trophic connections in watersheds: consequences
for a declining amphibian. Ecology 88:2187-2198.
Grinnell, J., and T. I. Storer. 1924. Animal life in the Yosemite. University of California Press, Berkeley, California,
USA.
Harris, R. N., R. M. Brucker, J. B. Walke, M. H. Becker, C. R. Schwantes, D. C. Flaherty, B. A. Lam, D. C. Woodhams,
C. J. Briggs, V. T. Vredenburg, and K. P. C. Minbiole. 2009. Skin microbes on frogs prevent morbidity and
mortality caused by a lethal skin fungus. ISME Journal 3:818-824.
Heard, G. W., M. P. Scroggie, and B. Malone. 2008. Visible Implant Alphanumeric tags as an alternative to toeclipping for marking amphibians – a case study. Wildlife Research 35:747–759.
Hyatt, A. D., D. G. Boyle, V. Olsen, D. B. Boyle, L. Berger, D. Obendorf, A. Dalton, K. Kriger, M. Hero, H. Hines, R.
Phillott, R. Campbell, G. Marantelli, F. Gleason, and A. Colling. 2007. Diagnostic assays and sampling
protocols for the detection of Batrachochytrium dendrobatidis. Diseases of Aquatic Organisms 73:175192.
Knapp, R. A. 2005. Effects of nonnative fish and habitat characteristics on lentic herpetofauna in Yosemite National
Park, USA. Biological Conservation 121:265-279.
Knapp, R. A., D. M. Boiano, and V. T. Vredenburg. 2007. Removal of nonnative fish results in population expansion
of a declining amphibian (mountain yellow-legged frog, Rana muscosa). Biological Conservation 135:1120.
Knapp, R. A., C. J. Briggs, T. C. Smith, and J. R. Maurer. 2011. Nowhere to hide: impact of a temperature-sensitive
amphibian pathogen along an elevation gradient in the temperature zone. Ecosphere, in press.
Knapp, R. A., P. S. Corn, and D. E. Schindler. 2001a. The introduction of nonnative fish into wilderness lakes: good
intentions, conflicting mandates, and unintended consequences. Ecosystems 4:275-278.
Knapp, R. A., and K. R. Matthews. 1998. Eradication of nonnative fish by gill netting from a small mountain lake in
California. Restoration Ecology 6:207-213.
Knapp, R. A., and K. R. Matthews. 2000. Effects of nonnative fishes on wilderness lake ecosystems in the Sierra
Nevada and recommendations for reducing impacts. Pages 312-317 in Proceedings: Wilderness Science in
a Time of Change. Ogden, UT: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research
Station.
Knapp, R. A., K. R. Matthews, H. K. Preisler, and R. Jellison. 2003. Developing probabilistic models to predict
amphibian site occupancy in a patchy landscape. Ecological Applications 13:1069-1082.
Knapp, R. A., K. R. Matthews, and O. Sarnelle. 2001b. Resistance and resilience of alpine lake fauna to fish
introductions. Ecological Monographs 71:401-421.
Longcore, J. E., A. P. Pessier, and D. K. Nichols. 1999. Batrachochytrium dendrobatidis gen. et sp. nov., a chytrid
pathogenic to amphibians. Mycologia 91:219-227.
Matthews, K. R., R. A. Knapp, and K. L. Pope. 2002. Garter snake distributions in high-elevation aquatic ecosystems:
is there a link with declining amphibian populations and nonnative trout introductions? Journal of
Herpetology 36:16-22.
Matthews, K. R., and K. L. Pope. 1999. A telemetric study of the movement patterns and habitat use of Rana
muscosa, the mountain yellow-legged frog, in a high-elevation basin in Kings Canyon National Park,
California. Journal of Herpetology 33:615-624.
Rachowicz, L. J., R. A. Knapp, J. A. T. Morgan, M. J. Stice, V. T. Vredenburg, J. M. Parker, and C. J. Briggs. 2006.
Emerging infectious disease as a proximate cause of amphibian mass mortality. Ecology 87:1671–1683.
Skerratt, L. F., L. Berger, R. Speare, S. Cashins, K. R. McDonald, A. D. Phillott, H. B. Hines, and N. Kenyon. 2007.
Spread of chytridiomycosis has caused the rapid global decline and extinction of frogs. EcoHealth 4:125134.
10
Vredenburg, V. T. 2004. Reversing introduced species effects: Experimental removal of introduced fish leads to
rapid recovery of a declining frog. Proceedings of the National Academy of Sciences, USA 101:7646-7650.
Vredenburg, V. T., R. Bingham, R. Knapp, J. A. T. Morgan, C. Moritz, and D. Wake. 2007. Concordant molecular and
phenotypic data delineate new taxonomy and conservation priorities for the endangered mountain
yellow-legged frog. Journal of Zoology 271:361-374.
Vredenburg, V. T., G. M. Fellers, and C. Davidson. 2005. The mountain yellow-legged frog (Rana muscosa). Pages
563-566 in M. J. Lannoo, editor. Amphibian declines: the conservation status of United States species.
University of California Press, Berkeley, California, USA.
Vredenburg, V. T., R. A. Knapp, T. S. Tunstall, and C. J. Briggs. 2010. Dynamics of an emerging disease drive largescale amphibian population extinctions. Proceedings of the National Academy of Sciences, USA 107:96899694.
Woodhams, D. C., V. T. Vredenburg, M. A. Simon, D. Billheimer, B. Shakhtour, Y. Shyr, C. J. Briggs, L. A. RollinsSmith, and R. N. Harris. 2007. Symbiotic bacteria contribute to innate immune defenses of the threatened
mountain yellow-legged frog, Rana muscosa. Biological Conservation 138:390-398.
11
V. Figures
Figure 1. Map of the proposed 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 LTBMU lakes
from which trout were recently removed are outlined in blue and of these, the two lakes proposed for R. sierrae
reintroductions are indicated with arrows (Lake Margery, Cagwin Lake). The lakes and ponds on the ENF that are
proposed as sources of R. sierrae adults and juveniles for the J. lividum treatments and reintroductions are
outlined in black. The inset map shows the location of the project area relative to the Desolation Wilderness and
Lake Tahoe.
12
Figure 2. A schematic of the study design, showing the percentage of juvenile frogs in the treated group (white
boxes) and untreated group (gray boxes) that will be reintroduced into Lake Margery and Cagwin Lake, or released
back into the source lakes. Frogs will be treated and translocated/released in years 1 and 2 of the study, and the
numbers given in the schematic (in parentheses) describe the per-year number of frogs in each category.
13