RESEARCH ARTICLE
2015 | VOLUME 32 | PAGES 23-29
The increase of an amphibian population:
11 years of Rana temporaria egg-mass monitoring
in 30 mountain ponds
Rocco Tiberti1,2*
2.
1.
Alpine Wildlife Research Centre, Gran Paradiso National Park, Degioz 11, 11010 Valsavarenche, Aosta, Italy
University of Pavia, DSTA-Dipartimento di Scienze della Terra e dell’Ambiente, Via Adolfo Ferrata 9, 27100 Pavia, Italy
Mount Guglielmo in the Italian Alps is an ancient transhumance area where man-made ponds for watering
cattle are an ancestral component of the landscape and represent the most important breeding sites
for common frogs (Rana temporaria) in the study area. Egg-masses in 30 mountain ponds from the
Mount Guglielmo were counted from 2005-2015 to monitor the population trends of R. temporaria,
after a putative population decline due to lethal bacterial infections which affected the tadpoles from
2002 to 2006. Egg-mass counts were also used to understand how the recovery of ponds from the
natural process of drying-out and the replacement of traditional ponds with pools waterproofed with
polymers influence the population dynamics of R. temporaria and the suitability of the ponds as
breeding sites. This R. temporaria population increased over the study period, suggesting that the
population was able to withstand long lasting larval mortality. Population increase was also sustained
by the recovery of some dried ponds, but the modern pools were less suitable than traditional ponds
for R. temporaria reproduction. These results suggest that the persistence of the traditional practices
related to transhumance (e.g. maintenance of man-made ponds) in the study area is probably of pivotal
importance for the conservation of the local herpetofauna.
INTRODUCTION
Amphibians diseases are a major cause of the global amphibian decline crisis (Daszak et al., 1999), and
are a primary factor determining the natural dynamics of wild populations (Anderson & May, 1986). To understand
the real extent of the global amphibian decline crisis, biologists need to distinguish between natural population
fluctuations and abnormal declines that may be directly or indirectly due to human actions (Pechmann et al., 1991;
Gardner, 2001). Long-term studies, encompassing population fluctuations or cycles, can help biologists learn more
about this problem (Meyer et al., 1998; Loman & Andersson, 2007). In any case, mortality events attributable to
infectious diseases should be carefully monitored to assess its impact on amphibian populations.
In the present 11-year study, egg-mass counts from 30 mountain ponds were used to assess the population
dynamics of Rana temporaria Linnaeus, 1758 on Mount Guglielmo massif (Italian Alps). The present study was
initiated to quantify the demographic effects of a series of widespread mass die-offs affecting R. temporaria
tadpoles at many ponds in the study area (Tiberti, 2011). Around 2002, die-offs of R. temporaria tadpoles were
reported for the first time, often resulting in mass mortality of the entire tadpole population within a pond and
affecting most ponds in the entire area (Tiberti, 2011). Other common amphibian species (Bufo bufo and Triturus
carnifex; Tiberti in press) were apparently unaffected by the epidemics. Die-offs have been ascribed to bacterial
infections caused by Aeromonas sp. (Tiberti, 2011), often associated to epidemics of red-leg disease (Rigney et al.,
1978). Aeromonas sp. is an ubiquitous bacterial genus living in free waters, on the skin and in the digestive tract of
Received 12 Mach 2015
*Corresponding author
Accepted 04 October 2015
Published Online 14 October 2015
© ISSCA and authors 2015
[email protected]
ROCCO TIBERTI
amphibians without causing infections, and is considered an opportunistic pathogen, infecting immunodepressed
hosts (Carey, 1993). Therefore, the spread of the epidemic could have been facilitated by other immunosuppressant
density dependent (e.g. overcrowding) or environmental factors (see Meyer et al., 1998), or by the presence of
other, not detected, pathogens (Tiberti, 2011). Despite not being able to determine the root causes, such die-offs
have not been reported in the study area (at least with the same severity and spatial extent) since 2007.
Mount Guglielmo is an ancient area of alpine transhumance (the seasonal transfer of livestock to high
altitude pastures). Due to its geology (dominated by calcareous rocks with many karst landforms), surface water
is largely absent above 1000 m a.s.l. and water for cattle was traditionally obtained by constructing ponds. These
anthropogenic habitats are an ancestral part of the cultural landscape (alpine transhumance dates back to Neolithic;
Festi et al., 2014) and represent virtually the only surface aquatic habitat in high altitude pasturelands in the
study area. In this context traditional land use practices (e.g. pond construction and maintenance) sustain valuable
ecosystems for amphibians. However modernization in the water supply for cattle has meant that some traditional
ponds (dug into the ground, with a clay bottom) were replaced with large artificial pools (waterproofed with
polymers sheets). Thus, a second objective of this study was to assess R. temporaria use of modern artificial ponds
compared to traditional transhumance ponds
MATERIALS AND METHODS
Study area
Mount Guglielmo (1957 m a.s.l.) is an isolated massif between the Trompia and Camonica valleys
(Brescia Prealps, Italy) (fig. 1). In the study area there are 30 mountain man-made ponds for watering cattle, which
sustain Bufo bufo and Triturus carnifex populations and are the most important breeding sites for R. temporaria in
the study area. The mean altitude of the ponds was 1535 m a.s.l. (range: 1166-1863 m a.s.l. ), mean surface area
was 706 m2 (range: 140-3400 m2), and mean depth was 0.81 m (range: 0.2-2.0 m; values based on the maximum
seasonal depths measured in May-July 2006 ). Nearby ponds (situated less than 1 km apart) were clustered into
four groups (A-D; fig. 1), thus R. temporaria could probably disperse among ponds in the same group. Most of
the ponds had a clay bottom, but ponds A2, C2, C3, D3, D6, and D8 (fig. 1) had an artificial substrate made of
waterproof polymers.
Egg-mass counts
Obtaining accurate counts of R. temporaria egg-masses is possible because their clutch size is large (up to
4500 eggs; Nöllert & Nöllert, 1992), they are easily visible in shallow water, and laid simultaneously by numerous
frogs in a confined area (Bernini & Razzetti, 2006). For 11 years (2005-2015), one to three surveys per pond were
performed annually to count the egg-masses deposited in all mountain ponds in the study area (N = 30, fig. 1).
Surveys were conducted just after the putative breeding season by walking along the entire edge of each pond.
The date of the first survey was decided based on the weather conditions (e.g. persistence of the snow cover) and
on the pond features (e.g. altitude and exposure) during the usual breeding period in the study area, which was
highly predictable (between the 21 April and 10 May). Assuming that the time of reproduction should be the same
in the ponds closer together, repeat surveys were needed when the egg-masses had not yet been laid in a certain
area/group of ponds or when amplexing pairs were still observable in the ponds. The position and developmental
stage (freshly laid or not) of the egg-masses was recorded at each survey to not underestimate counts when repeat
surveys were conducted. Of 330 potential egg mass counts (30 sites for 11 years) over the study, only 15 were
not conducted, when i) the ponds were still ice-covered and a second survey was not performed, or ii) when the
survey was done after the eggs were already hatched, which rarely occurred. During the egg-mass surveys, the
number of adult Triturus carnifex along the shorelines was counted because their presence can potentially affect
the suitability of the ponds as a breeding site of R. temporaria (Tiberti, 2011). When surveys were repeated, the
maximum number of Triturus carnifex was used as an index of abundance.
Statistical analysis
A linear mixed effects model (LME) was used to test if the observed trend in the time series of egg-mass
counts was significant, including the effects of some potentially important covariates, and accounting for the
repeated counts in the same ponds and groups of ponds. Transformed of annual egg-mass counts (log+1) were
added to the model as a dependent variable, the group of ponds as a random effect and each pond as a nested
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Figure 1. Mount Guglielmo summit (triangle) bordered by the 1000 m a.s.l. contour line. Dotted lines delimit a 500 m buffer area around the
numerated ponds belonging to the groups A-D.
random effect. The year of the survey (from year 1 to 11), the pond altitude (centered at 1500 m a.s.l.), the pond
area (m2), the number of adult newts along the shoreline, the substrate of the pond (artificial vs. natural), and the
mean temperature and the precipitation during i) the breeding season (from 21 April to 10 May), ii) the previous
hibernating season (November-April), and iii) the previous activity season (May-October), were added to the
model as covariates. In particular, the counts of Triturus carnifex were added to the model as their presence can
affect the suitability of the ponds as a breeding site for R. temporaria (Tiberti, 2011), while the climatic variables
were added as they could affect the survival and the energy allocated to reproduction of overwintering frogs, the
survival and fat reserves of the frogs at their feeding grounds, and the mildness of the climatic conditions during the
breeding period (Meyer et al., 1998). Meteorological covariates were measured at the weather station of Pisogne,
Brescia (45°49’02”N, 10°09’00”E, 842 m a.s.l.), 6.8 km from the peak of Mount Guglielmo. An “autoregressive
moving average” class (corARMA) was used to model the temporal autocorrelation structure of the residuals,
previously calculated with the function ACF (Autocorrelation Function) (Crawley, 2012). The LME was fitted by
Maximum Likelihood following Zuur et al. (2009) using the nlme package of the statistical environment R version
3.1.1 (R Development Core Team, 2010). The MuMIn package (Bartoń, 2011) was used following Grueber et
al. (2011) to select the best fitting models (ΔAICc < 4) among the models including all possible combinations of
the fixed covariates. We report the 95% confidence intervals of the averaged parameter estimates and the relative
importance of the covariates provided by the function model.avg of MuMIn. Linear regression was used to check
if the used climatic covariates showed a significant trend during the study period.
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ROCCO TIBERTI
RESULTS
The number of egg-masses oviposited in the study area increased significantly from 2005 to 2015 (fig. 2,
tab. 1). The number of egg-masses oviposited in natural ponds (38.2 ± 58.5; mean ± sd) was significantly higher
than artificial ponds (3.5 ± 11.2; table. 1). In 2014, a decline of the egg-mass counts was observed (fig. 2), although
this could be partially influenced by missing data (4 missing counts from ponds B7, D5, D6, and D7 due to icecovered conditions during the last survey, which, due to the impossibility of performing a second survey, made it
impossible to account for the probable later ovipositions).
There was no evidence that climatic variables (mean temperature or precipitation) had a significant effect
on oviposition (tab. 1) and there was not a relationship between climatic variables and egg-mass counts. Olenly
precipitation during the hibernating season showed a significant increasing trend over the study period (tab. 2), but
LME results exclude a causal relationship with the increase in the number of egg-masses in the study area (tab. 1).
Figure 2. Time series of the egg-mass counting in the whole Mount Guglielmo area and divided by groups of ponds (A-D).
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Table 1. Fixed effect results from a generalized mixed effect model testing the effect of several covariates on the log+1 transformed of the abundance of R. temporaria egg-masses in pastureland ponds of Mount Guglielmo. All the observations (N = 285) have been collected over 11 years
(2005-2015) in 30 ponds. Legend: Beta = Averaged parameter estimate; Lower 95% CI and Upper 95% CI = lower and upper 95% confidence
intervals; RVI = Relative Variable Importance (from 0 to 1). Fixed terms whose 95% confidence intervals around parameter estimates do not
include 0 are considered significant and have been marked in bold. Temp.: mean temperature; Prec.: precipitation amount.
Fixed term
Beta
Lower 95% CI
Upper 95% CI
Intercept
0.838
-2.779
4.455
RVI
-
Year
0.148
0.078
0.218
1.00
Altitude
0.000
-0.002
0.002
0.10
Area
0.000
0.000
0.001
0.19
Newts
0.003
-0.003
0.017
0.38
Substrate (artificial vs. natural)
-1.592
-2.560
-0.624
1.00
Temp. breeding season
0.003
-0.201
0.232
0.19
Prec. breeding season
0.001
-0.001
0.004
0.35
Temp. previous Winter
-0.074
-0.230
0.015
0.69
Prec. previous Winter
0.000
-0.001
0.000
0.48
Temp. previous Summer
0.068
-0.059
0.375
0.43
Prec. previous Summer
0.000
-0.001
0.001
0.14
DISCUSSION
Because the breeding biology of R. temporaria (e.g. aggregate breeding and single clutch per female)
suggest that egg-mass counts provide an accurate estimate of the number of reproductive females (Crouch &
Paton, 2000), the observed increase in the number of egg-masses likely indicates an increase of the number of
breeding females, and probably of the effective population size over this 11-year study.
The climatic variables (mean temperatures and precipitations, tab. 1) were chosen as potentially important
factors affecting the survival of hibernating R. temporaria, their survival/resource allocation during the activity
period (Lardner & Loman, 2003) and the suitability of the breeding season. Climatic factors may affect effective
population sizes of several amphibian species (McCaffery & Maxell, 2010), but the present results are consistent
with those from other studies, where R. temporaria populations were relatively unaffected by a number of climatic
variables (Elmberg, 1990; Meyer et al., 1998; Tattersall & Ultsch, 2008). Probably the effects of climatic factors
are masked by the importance of density dependent regulation within the population. For example, an amphibian
population experiencing a rapid expansion can be relatively unaffected by climatic factors, of course in the absence
of catastrophic climatic events.
It is likely that the observed population increase was the results of the end of the epidemic (Tiberti, 2011).
These results suggest that some populations of R. temporaria have the capacity to withstand high larval mortality.
Unfortunately historical data on the abundance of R. temporaria before the observed larval mortality events are not
available and it is impossible to determine if the observed increases represent post-epidemic recovery. Therefore, it
is not possible to know how the observed population increases compares to population sizes before the epidemic.
Female R. temporaria take 2-5 years to reach sexual maturity (Guarino et al., 2008). Therefore, the effects
of the epidemic on the population size might not become evident for years. Such process may explain the pattern
of ongoing decline observed in pond group D in 2005-2008 (only 9 egg-masses were counted in ponds group D in
2008) after the end of epidemic in 2006. It is possible that in group D the monitoring period included part of the
putative decline.
The persistence of the studied population after high tadpole mortality could be explained in several ways.
First R. temporaria has a high reproductive potential, which allows populations to recover from a small number of
surviving individuals. Moreover this species is distributed widely (Kuzmin et al., 2009) and could re-colonize the
extirpated habitats. Also R. temporaria longevity (6-15 years; Guarino et al., 2008) could explain the persistence
of the studied population: adults can breed for multiple years and, if recruitment failure only occurs for a limited
number of years, it is possible that adult persistence could buffer populations against tadpole mortality. Indeed,
compared to the mortality of post-metamorphic stages, increased mortality in anuran larvae is less likely to affect
the overall population growth rate and the population viability (Biek et al., 2002). However, recurrent epidemic
events can sometimes cause persistent declines and local extinctions in R. temporaria populations (Teacher et al.,
2010), suggesting not to rely too much on their capacity to withstand epidemics.
There are other factors potentially contributing to the observed trend in egg-mass counts. For example,
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ROCCO TIBERTI
Table 2. Linear regression of climatic variables against time (Year). Temp.: mean temperature; Prec.: precipitation amount. Significant P-values
(<0.05) have been marked in bold.
Model
Beta
t-test
P
Temp. previous Summer ~ Year
-0.102
-1.50
0.17
0.11
Prec. previous Summer ~ Year
28.48
1.72
0.12
0.16
Temp. previous Winter ~ Year
0.03
0.26
0.80
-0.10
Prec. previous Winter ~ Year
40.61
2.27
<0.05
0.29
Temp. breeding season ~ Year
-0.08
-0.76
0.47
-0.04
Prec. breeding season ~ Year
6.29
1.13
0.29
0.03
Adjusted R2
the sudden onset of R. temporaria egg-masses in group C is the result of the increased hydroperiod of a dry pond
(C4, fig. 1) in an area where R. temporaria was usually absent, probably due to the presence of a particularly large
population of Triturus carnifex, which could prevent the reproduction of R. temporaria (Tiberti, 2011). This new
pond was quickly colonized by R. temporaria, which established a small population.
Without a regular maintenance (e.g. removal of vegetation and sediments and re-waterproofing of the
bottom), most of the mountain ponds in the study area will probably disappear within a few years to decades, due
to their progressive burial or to the infiltration of water at the clay bottom. Pond recovery is a common practice
among the local farmers (nine pond restorations were detected in ten years) in the study area, where transhumance
still produce an actual water demand for cattle. The survival of alpine transhumance is probably favorable for
amphibians and their breeding habitats, preventing the progressive abandonment of the rural landscape, which is
often viewed with concern for the likely negative consequences for biodiversity due to land use change (e.g. Hartel
& von Wehrden, 2013). However, sometimes the modernization of agricultural practices has meant that traditional
ponds - much more suitable as breeding sites for R. temporaria (tab. 1) - were replaced by artificial large pools
with bottoms made of waterproofing polymers. These pools, due to their steep shore, may also become a lethal trap
both for amphibians and other animals, such as small mammals, marmots, and reptiles which can be often found
therein drowned (personal observation). This is somehow in contrast with the results from other study areas, where
the widespread construction of artificial ponds - less prone to drying out than natural ones - produced a population
increase in R. temporaria (Cooke, 1972). Probably artificial ponds can become more attracting for R. temporaria
in a context of general loss of natural ones, but in the area of the Mount Guglielmo frogs can find both the habitats
within a short distance, and their choice falls on natural ponds.
ACKNOWLEDGEMENTS
I thank Fiorenzo and Paola for their help and company during the field work.
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