Dynamics of an emerging disease drive large-scale
amphibian population extinctions
Vance T. Vredenburga,1, Roland A. Knappb, Tate S. Tunstallc,d, and Cheryl J. Briggsd
aDepartment of Biology, San Francisco State University, San Francisco, CA 94132-1722;bSierra Nevada Aquatic Research Laboratory, University of California,
Mammoth Lakes, CA 93546;cDepartment of Integrative Biology, University of California, Berkeley, CA 94720-3140; anddDepartment of Ecology, Evolution,
and Marine Biology, University of California, Santa Barbara, CA 93106-9610
Edited* by David B. Wake, University of California, Berkeley, CA, and approved March 25, 2010 (received for review December 6, 2009)
Epidemiological theory generally suggests that pathogens will not
cause host extinctions because the pathogen should fade out when
the host population is driven below some threshold density. An
emerging infectious disease, chytridiomycosis, caused by the fungal
pathogen Batrachochytrium dendrobatidis (Bd) is directly linked to
the recent extinction or serious decline of hundreds of amphibian
species. Despite continued spread of this pathogen into uninfected
areas, the dynamics of the host–pathogen interaction remain un-
known. We use fine-scale spatiotemporal data to describe (i) the
invasion and spread of Bd through three lake basins, each contain-
ing multiple populations of the mountain yellow-legged frog, and
(ii) the accompanying host–pathogen dynamics. Despite intensive
sampling, Bd was not detected on frogs in study basins until just
before epidemics began. Following Bd arrival in a basin, the disease
spread to neighboring populations at ≈700 m/yr in a wave-like pat-
tern until all populations were infected. Within a population, infec-
tion prevalence rapidly reached 100% and infection intensity on
individual frogs increased in parallel. Frog mass mortality began
only when infection intensity reached a critical threshold and re-
peatedly led to extinction of populations. Our results indicate
that the high growth rate and virulence of Bd allow the near-
simultaneous infection and buildup of high infection intensities
in all host individuals; subsequent host population crashes there-
fore occur before Bd is limited by density-dependent factors. Pre-
venting infection intensities in host populations from reaching
this threshold could provide an effective strategy to avoid the
extinction of susceptible amphibian species in the wild.
amphibian declines|Batrachochytrium dendrobatidis|chytridiomycosis|
emerging infectious disease|Rana muscosa
The majority of contemporary extinctions are typically at-
tributed to anthropogenic changes such as habitat destruction,
overexploitation, and species introductions. Disease is generally
not considered a major driving force in extinctions, in part be-
cause simple epidemiological theory suggests that a pathogen
will fade out when its host population is driven below some
threshold density (1, 2). Class Amphibia provides one of the
best-documented examples of contemporary biodiversity loss,
with ≈43% of the more than 6,600 described species currently
threatened with extinction (3). Remarkably, an emerging in-
fectious disease, chytridiomycosis, is directly linked to the recent
extinction or serious decline of hundreds of amphibian species
(4). The effect of chytridiomycosis on amphibians has been de-
scribed as the greatest loss of vertebrate biodiversity attributable
to disease in recorded history (4), and although doubts about the
importance of disease in driving global amphibian declines have
been expressed (5), these have largely been overcome by weight
of evidence (4, 6–8).
Chytridiomycosis is caused by the fungal pathogen Batracho-
chytrium dendrobatidis (Bd), whose only known host is larval and
adult amphibians. This pathogen was described in the late 1990s
(6, 9) and is now known from six continents (4). The infective
stage is a free-living flagellated zoospore that encysts in the skin
arth’s biodiversity is increasingly threatened with extinction.
of an amphibian and develops into a zoosporangium. Zoospor-
angia produce zoospores via asexual reproduction [it remains
unclear whether sexual reproduction also occurs (10, 11)], and
the zoospores are released into the environment through a dis-
charge tube. Tadpoles are typically little affected by chy-
tridiomycosis, but sublethal and lethal effects are known (12, 13).
Effects of chytridiomycosis on frogs are highly variable, with
frogs of some species dying from the disease within weeks and
others experiencing few negative effects (4). Chytridiomycosis
likely causes frog mortality by severely disrupting epidermal
functions and causing osmotic imbalance (14, 15). However, it
remains unknown how chytridiomycosis is able to cause the ex-
tinction of its amphibian hosts, an outcome that would require
that Bd not be severely limited by density-dependent factors. The
objective of our study was to describe frog–Bd dynamics by
measuring both Bd prevalence in populations and infection in-
tensity in individual frogs during chytridiomycosis epizootics
(epidemics in nonhuman species) in naive frog metapopulations
(we use the term “metapopulation” to mean a collection of
populations connected by dispersal) (16, 17). In doing so, we
reveal the heretofore unknown importance of infection intensity
as a factor allowing Bd to drive amphibian populations to ex-
tinction. We also sought to describe the rate of spread by Bd
through these metapopulations, which is information critical to
understanding the potential vectors of this pathogen.
The rapid decline of California’s mountain yellow-legged frog
(a species complex consisting of Rana muscosa and Rana sierrae)
(18) is emblematic of global amphibian declines (3). Historically,
these two species inhabited thousands of lakes and ponds in
California’s Sierra Nevada (where this study took place) (19).
Both of these closely related species are highly aquatic and have
a multiyear tadpole stage that allows them to breed successfully
in the cold water bodies typical of the high elevation portions of
this mountain range. Despite the fact that the majority of their
habitat is fully protected, these frogs have disappeared from
>93% of their historic range during the past several decades
(18). As a consequence of this decline, the mountain yellow-
legged frog has gone from being one of the most common ver-
tebrates in the Sierra Nevada to one classified as “critically en-
dangered” (3). One of the earliest recorded cases of Bd infecting
amphibians in western North America (1975) was in R. muscosa
specimens from the Sierra Nevada (20); these specimens were
originally identified as Rana boylii, but subsequent inspection by
one of the authors (V.T.V.) indicated that they are actually
R. muscosa. Since then, Bd has spread across this mountain
Author contributions: V.T.V., R.A.K., and C.J.B. designed research; V.T.V., R.A.K., T.S.T.,
and C.J.B. performed research; V.T.V., R.A.K., and C.J.B. analyzed data; and V.T.V., R.A.K.,
and C.J.B. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
Freely available online through the PNAS open access option.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
| May 25, 2010
| vol. 107
| no. 21
range, causing the extinction of hundreds of mountain yellow-
legged frog populations (21, 22).
Our study area comprised three lake basins: Milestone, Sixty
Lake,andBarrett LakesinSequoia–KingsCanyonNational Park,
CA (Fig. S1). The three basins were separated from each other by
20–50 km. At the beginning of our study, we found no evidence of
chytridiomycosis in the frog populations in these lake basins, but
all three basins were immediately adjacent to basins in which
chytridiomycosis epizootics and subsequent frog population
extinctions had recently occurred. At the inception of our study
(1996–2000), the three study basins, Milestone, Sixty Lake, and
Barrett Lakes, contained 13, 33, and 42 frog populations, re-
spectively, and represented the most intact remaining meta-
populationsofthesespecies.To quantify trendsinpopulation size
before and after Bd-caused epizootics, we used repeat surveys of
all 88 frog populations over a 9–13-year period. Frog surveys were
conducted 1–5 times per year at each population in Milestone
Basin (R. muscosa: 2000, 2003–2008), 1–12 times per year in Sixty
LakeBasin (R.muscosa: 1996–2008),andonce peryear in Barrett
Lakes Basin (R. sierrae: 1997, 2002–2008), for a total of 1,995
surveys (yearly average = 1.8 surveys × population−1). We mea-
sured Bd prevalence and infection intensity (expressed as zoo-
spore equivalents × swab−1) using a real-time quantitative PCR
assay (23) conducted on skin swabs (24) collected from frogs in
2004–2008 (n = 4,591). Before the availability of the PCR assay,
tadpole mouthpart inspections (25) were used for assessments of
Bd prevalence (2002–2005, n = 1,389).
We detected Bd in Milestone Basin in June 2004, in Sixty Lake
Basin in August 2004, and in Barrett Lakes Basin in July 2005
(Fig. 1). In the relatively small Milestone Basin, Bd spread to
virtually all populations within a single year (Fig. 1 A and B). In
the larger Sixty Lake and Barrett Lakes Basins, it took 3–5 years
for Bd to spread to all frog populations (Fig. 1 F–O). Our most
detailed within-season Bd occurrence data were collected in
Sixty Lake Basin, and these data allowed us to quantify the
pattern and rate of Bd spread. In Sixty Lake Basin, the distance
from the original Bd outbreak site (Fig. 1F) to subsequently
infected populations increased linearly with time (linear re-
gression through the origin: R2= 0.85, P < 0.001), consistent
with a wave-like pattern (Fig. 1 F–J). The slope of the regression
line indicated an average rate of Bd spread (±1 SE) of 688 ±
64 m·yr−1. The pattern and rate of spread in Barrett Lakes Basin
(where we collected skin swabs only once per year; Fig. 1 K–O)
were qualitatively similar to those measured in Sixty Lake Basin.
In 48 of the 88 frog populations, Bd assays (n = 1,341 swabs,
909 mouthpart inspections) were conducted before the beginning
of Bd-caused epizootics. We used results from these assays to
calculate the probability that Bd was present on frogs at these
Frogs present, Bd-negative
Frogs present, Bd-positive
Frogs present, Bd-status unknown
initial detection of Bd. Depicted are Milestone Basin (A–E), Sixty Lake Basin (F–J), and Barrett Lakes Basin (K–O). Lake color (green, yellow, and black) shows
the Bd infection and frog population status, and the light gray shaded region surrounds the area in which frog populations were Bd-positive in each year.
Lakes shown with a thick black outline are fishless, and a thin gray outline indicates that nonnative fish were present (details on the historic fish distribution
are presented in SI Text). The infection status of frog populations depicted in A and K is based on mouthpart surveys of 459 tadpoles. The infection status of
frog populations in B–J and L–O is based on 4,591 skin swabs analyzed using a real-time PCR assay.
Maps of the three study metapopulations showing the spread of Bd and frog population status (adults only) during a 4-year period following the
| www.pnas.org/cgi/doi/10.1073/pnas.0914111107Vredenburg et al.
sites during the early part of our study but not detected (i.e.,
false-negative result). These calculations were based on the as-
sumption that the true prevalence of Bd was 5%. For 33 (69%)
of the 48 populations, the probability of false-negative results
was less than 0.05 (median = 0.02; Table S1). For the best-
sampled populations (22 populations for which >30 swabs or
mouthpart inspections were collected before detection of Bd),
the median probability of false-negative results was 1.6 × 10−3
(Table S1). These results strongly suggest that Bd was not
present in frog populations in Milestone, Sixty Lake, and Barrett
Lakes Basins in the early years of our study.
Soon after the detection of Bd, major declines in frog pop-
ulations were observed in all three study basins (Fig. 2) and were
coincident with observations of hundreds of dead and dying frogs
(Fig. S2). By 2008, the number of adult frogs in Milestone Basin
had declined from 1,680 (frog counts averaged over all surveys
conducted before Bd arrival) to 22 (Fig. 2A), from 2,193 to 47 in
Sixty Lake Basin (Fig. 2B), and from 5,588 to 436 in Barrett Lakes
Basin (Fig. 2C). Similarly, by 2008, adult frogs were extinct from 9
of13populationsin Milestone Basin,27of33populations in Sixty
Lake Basin, and 33 of 42 populations in Barrett Lakes Basin (Fig.
1 E, J, and O). Based on high rates of population extinctions in
nearby basins in the 10 years following Bd arrival, we expect that
most, if not all, of the still-extant populations will also go extinct
during the next 3 years as the remaining tadpoles metamorphose
and succumb to chytridiomycosis (21).
To quantify the effect of Bd arrival on frog population growth
rates, we compared population growth rates in (i) the years before
Bd arrival, (ii) the year of Bd arrival, and (iii) the year after Bd
arrival. There was a significant decrease in the frog population
growth rate in the year of Bd arrival compared with the growth
rate in the same populations before Bd arrival [Fig. 3; mean dif-
ference ingrowthrate ([before Bd arrival] −[year ofBd arrival]) =
1.8, paired t test: t = 2.9, df = 42, P < 0.01] and an even larger
decrease in the year following Bd arrival [Fig. 3; mean difference
in growth rate ([before Bd arrival] − [year after Bd arrival]) = 3.2,
paired t test, t = 7.5, df = 36, P << 0.01]. Therefore, the decrease
in the frog population growth rate began with the arrival of Bd
and was clearly evident within 1 year after the detection of Bd.
We used detailed within-season data from the eight most in-
tensively sampled populations in Milestone and Sixty Lake Basins
to describe the frog–Bd dynamics during epizootics. Following
the detection of Bd in these populations, adult frog populations
invariably crashed to extinction (n = 7) or near-extinction (n = 1;
Fig. 4A). On the date when Bd was detected, both prevalence
and infection intensity were relatively low (prevalence: median =
0.42, range = 0.05–1; infection intensity: median = 13.4, range =
0.2–3,843.0). In all populations, Bd prevalence increased rapidly,
and in all but one case, it reached 100% (97% in the remaining
case), often in less than 50 days (Fig. 4B). Infection intensity in-
creased exponentially; the within-year rate of increase (? x ± 1 SE)
was 0.15 ± 0.02 × day−1(Fig. 4C). Declines in frog numbers
were generally not evident until an average infection intensity
of ≈10,000 zoospore equivalents per swab was reached [maxi-
mum infection intensity at time of population crash (? x ± 1 SE) =
11,775 ± 5,851 zoospore equivalents × swab−1; Fig. 4 A and C].
Exceeding this threshold consistently resulted in mass mortality
and rapid population decline (Fig. 4A). Bd prevalence and in-
fection intensity remained high even in the last surviving frogs
following population crashes (Fig. 4 B and C). Frogs swabbed
during the second summer after the outbreak (>300 days post-
outbreak; Fig. 4 B and C) were all newly metamorphosed sub-
adults (which had survived the winter as tadpoles). The fact that
subadults have much higher infection intensities than do adults
populations during 1996–2008 before and after the detection of Bd: Mile-
stone Basin (A), Sixty Lake Basin (B), and Barrett Lakes Basin (C).
Total number of adult and subadult frogs in the three study meta-
growth rate (rt) of three categories of frog populations: (i) populations
before detection of Bd (rtfor each lake averaged over all years before Bd
arrival), (ii) populations during the year in which Bd was detected, and (iii)
populations 1 year after Bd was detected. In each case, rt= ln(Nt) − ln(Nt−1),
where Ntis the number of adult frogs in the lake in year t. Box plots display
the median yearly frog population growth rate (horizontal line), 25th and
75th percentiles (gray boxes), 10th and 90th percentiles (whiskers), and all
points that lie outside of the 10th and 90th percentiles (•). Data are from 88
frog populations located in all three study basins (1996–2008).
Box plots showing the effect of Bd arrival on the yearly population
Vredenburg et al.PNAS
| May 25, 2010
| vol. 107
| no. 21
likely explains the high infection intensities even at the end of
the epizootic when very few frogs remained (Fig. 4 A and C).
Most of the frog study populations were sampled for Bd for at
least 1 year before epizootics began. In all 48 of these frog pop-
ulations, we found no evidence of Bd until just before the ob-
served frog die-offs. Therefore, we suggest that Bd was absent
from the three study metapopulations before 2004. Two studies in
Central America (6, 7) also reported the absence of Bd until just
before frog die-offs were observed. The apparent absence of Bd
before frog die-offs is critically important in resolving the con-
tinued debate about whether Bd is a novel pathogen sweeping
through naivehost populations(7,8,26)ora widespreadendemic
pathogen that has emerged as a result of changing environmental
conditions such as those caused by climate warming (Bd thermal
optimum hypothesis) (27). Implicit in the Bd thermal optimum
hypothesis is the presence of Bd in amphibian populations before
chytridiomycosis epizootics (28). Our results indicate that Bd was
likely not present on amphibians in our study populations until
just before epizootics began. Therefore, our data do not support
the Bd thermal optimum hypothesis but are consistent with Bd
as a novel pathogen spreading through naive host populations.
Data from the intensively sampled Sixty Lake Basin meta-
population indicated that Bd spread as a distinct wave at a rate
of 688 m·yr−1, and rates of spread in Milestone and Barrett
Lakes Basin were qualitatively similar. This rate of spread is
much lower than rates reported for Bd in Central and South
America and Australia (17–282 km·yr−1) (8, 29), but it is unclear
if these differences in rate of spread are real or are the result of
different spatial scales of sampling used in our study compared
with previous studies. In our study system, the observed pattern
of Bd spread within a metapopulation is consistent with frog
movement patterns, suggesting that frogs may be an important
agent of dispersal at this scale (these frogs are known to move
only several hundred meters between lakes in a single summer)
(17, 30). However, the continuing between-basin spread of Bd
and the lack of evidence for interbasin frog movement (17, 18)
suggest the involvement of unknown additional vectors. Other
possible between-basin dispersal agents include more vagile
sympatric organisms, including amphibians (e.g., Pseudacris
regilla), insects, or birds.
Before our study, the only data available on frog–Bd dynamics
during disease outbreaks showed a temporal correlation between
increases in Bd prevalence and amphibian population decline
(7), but that study did not include any measurement of infection
intensity. As a consequence, the dynamics of this disease were
only partially described until now. Our quantification of infection
intensity provided a key insight into how Bd causes host extinc-
tions. Temporally intensive sampling at multiple frog pop-
ulations showed that the very high growth rate and virulence of
Bd in mountain yellow-legged frogs allowed the near-simulta-
neous infection and buildup of high infection intensities in all
host individuals. Subsequent host population crashes therefore
occurred before Bd could be limited by density dependence, host
immune response, or other factors.
Chytridiomycosis is a major driver of an ongoing global mass
extinction event (31) in amphibians, but field interventions
designed to reduce disease impacts by altering Bd–host dynamics
have only just begun. Our results show a primary role for in-
fection intensity in driving the population extinctions that typi-
cally follow these epizootics. This suggests that interventions
designed to prevent Bd infection intensity on frogs from reaching
the critical lethal threshold could reduce the probability of
population extinction. Interventions could include capturing
frogs immediately in front of the Bd wave and releasing them
back into the same habitat after the Bd wave has passed and
pathogen pressure has declined following die-offs of resident
frog populations or reducing the density of infective Bd zoo-
spores by treating a large proportion of frogs during epizootics
with antifungal drugs (32, 33) and releasing them back into the
same habitat. In both cases, the goal of interventions would not
be to eradicate the pathogen from the targeted habitats, because
this would not be feasible, but, instead, to reduce pathogen
transmission rates and thus increase host survivorship (34).
Given a known rate of Bd spread in our study system and the
resulting knowledge of exactly where the Bd front is within
remaining frog metapopulations, the results of the current study
create a unique opportunity to test these approaches, the results
of which will be of critical importance to the global conservation
Study Area Description. The three study watersheds are in Sequoia–Kings
Canyon National Park (milestone: 36°38′57″ N, 118°27′28″ W; Sixty Lake: 36°
49′03″ N, 118°25′24″ W; Barrett Lakes: 37°04′52″ N, 118°31′35″ W; Fig. S1).
Milestone and Sixty Lake Basins contain the southern mountain yellow-
legged frog (R. muscosa), and Barrett Lakes Basin contains the closely related
Sierra Nevada yellow-legged frog (R. sierrae (18). These basins are located in
the subalpine and alpine zones and contain 13–42 oligotrophic lakes and
ponds (elevation range: 3,030–3,790 m), all of which are naturally fishless.
Milestone and Sixty Lake Basins before and after detection of Bd: frog
counts (adults + subadults) from visual encounter surveys (A); infection
prevalence, defined as the fraction of skin swabs collected from each pop-
ulation on each date positive for Bd (B); and infection intensity, defined as
the average zoospore equivalents on swabs collected from each population
on each date (C). Data are from frog populations that were sampled more
than once per year, experienced >80% declines by the end of 2006, and for
which the decline in the number of frogs was >10. This last criterion ex-
cluded populations that were very small before Bd arrival. Populations were
aligned along the x axis such that “0” represents the date on which each
frog population began to decline. This was calculated for each population by
determining the date at which the number of postmetamorphic frogs
dropped below 20% of the average population count before that point.
Frog–Bd dynamics in eight intensively sampled populations in
| www.pnas.org/cgi/doi/10.1073/pnas.0914111107 Vredenburg et al.
Nonnative trout (primarily Oncorhynchus mykiss and Salvelinus fontinalis)
have been introduced into many Sierra Nevada lakes to provide recreational
fishing opportunities, and their negative impacts on mountain-yellow leg-
ged frogs are well known (35–37). The active season for frogs in the study
basins is from early June to mid-October; the basins are typically covered by
several meters of snow during the winter.
Frog Surveys. We used diurnal visual encounter surveys (38) of entire water
body perimeters to describe the abundance of adult (≥40 mm snout–vent
length) and subadult (<40 mm snout–vent length) mountain yellow-legged
frogs at all water bodies in the study basins (36, 39). In these species, counts
from surveys are highly correlated with estimates of population size
obtained using mark-recapture techniques. These frogs’ high detectability
during visual surveys is a consequence of a diurnal habit, spending the
majority of the active season at the water–land interface (30, 40), and not in
terrestrial habitats (41), and occupying structurally simple habitats (e.g.,
subalpine lakes, alpine lakes) in which the lack of submerged logs or aquatic
vegetation provides few places for frogs to hide.
Disease Prevalence and Infection Intensity. Weused frog skinswabs and areal-
time quantitative PCR assay to quantify Bd prevalence and infection intensity
(23, 24). Swabs were stroked across a frog’s skin in a standardized way: five
strokes on each side of the abdominal midline, five strokes on the inner thighs
30strokes × frog−1). Swabs were air-dried in the field and stored individuallyin
labeled microcentrifuge tubes before PCR analysis. We used standard Bd DNA
extraction and real-time PCR methods (23, 24), except that swab extracts were
analyzed singly instead of in triplicate (42). We defined infection intensity as
the number of “zoospore equivalents” per swab. Zoospore equivalents were
calculated by multiplying the genomic equivalent values generated during the
real-time PCR assay by 80; this multiplication accounts for the fact that DNA
extracts from swabs were diluted 80-fold during extraction and PCR. For
calculations of Bd prevalence, swabs were categorized as Bd-positive when
zoospore equivalents were ≥1 and as Bd-negative when zoospore equivalents
were <1. Before the availability of the PCR assay, we determined the infection
status (infected/uninfected) of frog populations using inspections of tadpole
mouthparts (upper jaw sheaths). Tadpole mouthpart anomalies can have nu-
merous causes, but in R. muscosa and R. sierrae, mouthpart anomalies are an
accurate indicator of chytridiomycosis (25).
Bd Disinfection Procedures. To ensure that Bd was not spread between frog
populations by field sampling activities, we disinfected all field gear by
immersion in 1% sodium hypochlorite or 0.01% quaternary ammonia for 5
min (43). In Milestone and Barrett Lakes Basins, disinfection was performed
whenever moving between frog populations. In Sixty Lake Basin, where the
distribution of Bd was very well known during each summer, we divided the
area into discrete units based on geography and Bd infection status (infec-
ted/uninfected) and disinfected gear when moving between units.
Rate of Bd Spread. Calculations of Bd spread rate in Sixty Lake Basin were
based on the date of earliest Bd detection: August 22, 2004. For each newly
infected frog population in this basin, we calculated (i) the minimum
straight-line distance from the original outbreak sites (Fig. 1F) and (ii) the
number of days between August 22, 2004 and the date on which Bd was
detected. The slope from a linear regression model of distance as a function
of time provided the rate of spread. The regression included only pop-
ulations that became infected by the autumn of 2006. The intercept of the
regression (±1 SE) was not significantly different from zero (189 ± 223 m);
thus, the regression line was forced through the origin. Lakes that had not
become infected by the autumn of 2006 were situated significantly further
from the site of initial Bd detection than lakes that became infected (logistic
regression: P < 0.01, df = 28).
ACKNOWLEDGMENTS. Research permits were provided by Sequoia–Kings
State University; and University of California, Santa Barbara Institutional
Animal Care and Use Committees. We thank the staff at Sequoia–Kings Can-
yon National Park for logistical support and many technicians for their help
in collecting field data and running PCR assays. This work was funded by
National Institutes of Health Grant R01ES12067 and National Science Foun-
dation Grant EF-0723563 as part of the joint National Science Foundation–
National Institutes of Health Ecology of Infectious Disease program.
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