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Journal of Herpetology, Vol. 54, No. 2, 235–244, 2020
Copyright 2020 Society for the Study of Amphibians and Reptiles
A Long-Term Study on Massasaugas (Sistrurus catenatus) Inhabiting a Partially Mined
Peatland: A Standardized Method to Characterize Snake Overwintering Habitat
ANNE R. YAGI,
1,2,3
R. JON PLANCK,
4
KATHARINE T. YAGI,
2
AND GLENN J. TATTERSALL
2
1
8Trees Inc., 11 Berkwood Place, Fonthill, Ontario, L0S 1E2, Canada
2
Department of Biological Sciences, Brock University, 500 Glenridge Avenue, Saint Catharines, Ontario L2S 3A1, Canada
4
Deceased.
ABSTRACT.—Temperate snakes occupy overwintering sites for most of their annual life cycle. Microhabitat characteristics of the
hibernaculum are largely undescribed, yet are paramount in ensuring snake overwintering survival. We hypothesized that snakes
survive hibernation within a vertical subterranean space that we termed a ‘‘life zone’’ (LZ), that is aerobic and flood and frost free
throughout winter. We studied an isolated, endangered population of Massasaugas (Sistrurus caten atus) inhabiting an anthropogenically
altered peatland and monitored the subterranean habitat during a period of environmental stochasticity. Initial radio telemetry confirmed
that snakes moved between altered and natural habitats during the active season and showed hibernation-site fidelity to either habitat.
We used a grid of groundwater wells and frost tubes installed in each hibernation area to measure LZ characteristics over 11 consecutive
winters. The LZ within the impacted area was periodically reduced to zero during a flood–freeze cycle, but the LZ in the natural area was
maintained. Model selection analysis revealed that soil depth and flood status best predicted LZ size. Thermal buffering and
groundwater dissolved oxygen increased with LZ size, and annual Massasauga encounters were significantly correlated with LZ size.
This analysis suggests a population decline occurred when LZ size was reduced by flooding. Our data give support to the importance and
maintenance of an LZ for successful snake hibernation. Our methods apply to subterranean hibernation habitats that are at risk of
environmental stochasticity, causing flooding, freezing, or hypoxia.
Snakes inhabit a range of climates, and those living in
temperate ecosystems must adapt behaviorally and physiolog-
ically to the seasonal changes in their environment (Gregory,
1982; Huey, 1982; Ultsch, 1989). Temperate snakes have adapted
to their climate by employing a biphasic life-history strategy
(Huey, 1982). During the above-ground or the active season,
surface temperatures are conducive for movement, foraging,
growth, and reproduction (Harvey and Weatherhead, 2006a).
When surface temperatures are suboptimal, snakes retreat
below ground during the overwintering or hibernation phase
(Cowles, 1941; Gregory, 1982; Ultsch, 1989; Costanzo and Lee,
2013). In some latitudes or altitudes, the overwintering phase
may account for more than half of the animal’s annual life cycle
(Gregory, 1982). A typical snake overwintering site consists of a
subterranean space with access to the surface that includes
crayfish and mammal burrows, rotting tree roots, fractures in
bedrock, caves, or holes weathered by wind, or water (Weath-
erhead and Prior, 1992; Johnson, 1995; Kingsbury and Coppola,
2000; Harvey and Weatherhead, 2006b). The depth, width, or
ecological characteristics of the subsurface wintering site are not
well understood (Prior, 1997), however, likely vary with soil
type, geology, hydrology, vegetation, climate, and topography.
Radiotelemetry has provided insights into hibernation ther-
mal ecology, the locations of hibernacula, site fidelity (Mac-
artney et al., 1989; Johnson, 1995; Sage, 2005; Harvey and
Weatherhead, 2006b), and the timing of ingress and egress
(Nordberg and Cobb, 2016). Surgically implanted temperature
data loggers or hygrochron data loggers fused to rattles and
doubly labeled water have also been used to study the
ecophysiology of hibernation (Agugliaro, 2011; Nordberg and
Cobb, 2016). Because small numbers of relatively old and large
snakes are monitored during radiotelemetry studies (Johnson,
1995; Harvey and Weatherhead, 2006b; Refsnider et al., 2012;
Zappalorti et al., 2014), survival rates are difficult to determine
(Jones et al., 2012). Radiotelemetry studies cannot locate all
hibernation sites used by solitary hibernators. Therefore,
estimating population level impacts using radiotelemetry alone
may be inaccurate (Jones et al., 2012). Furthermore, the invasive
surgical procedures associated with radiotelemetry itself may
contribute to snake mortality (Lentini et al., 2011). Locating
hibernacula using more invasive methods (Cowles, 1941; Sage,
2005; Burger et al., 2012) can directly change the behaviors of
hibernating snakes or alter the physical conditions of the site.
During hibernation, snakes survive by freeze avoidance or
supercooling and exhibit variable tolerances to water inunda-
tion and hypoxia. Snakes generally avoid freezing by retreating
into a thermally buffered subterranean space in the autumn and
remaining there until spring (Costanzo and Claussen, 1988;
Storey and Storey, 1992; Gienger and Beck, 2011). Snakes are
freeze-intolerant (Costanzo and Claussen, 1988; Storey and
Storey, 1996; Costanzo and Lee, 2013), even species with very
northerly distributions (Churchill and Storey, 1992; Lee and
Constanzo, 1998; Andersson and Johansson, 2001; Costanzo and
Lee, 2013). Supercooling (Storey and Storey, 1996) is only a
short-term strategy for snakes at high latitude (Storey and
Storey, 1992; Andersson and Johansson, 2001). There is some
indirect evidence that hibernating snakes may drown if the
hibernaculum floods (Viitanen, 1967; Prestt, 1971; Shine and
Mason, 2004; Harvey et al., 2014), although access to water
during overwintering can be beneficial to overall survival
(Costanzo, 1989a,b; Prior, 1997; Costanzo et al., 2001). Snakes
may survive flooding events in the field (May et al., 1996; Seigel
et al., 1998) and in the laboratory (Constanzo, 1989a; Todd et al.,
2009). However, the ability to remain completely submerged for
the entire duration of hibernation seems unlikely and may vary
among species and within species depending on hibernacula
site quality (Gregory, 1982; Ultsch, 1989; Rollinson et al., 2008).
The survival of overwintering ectothermic vertebrates de-
pends on the reduction in metabolic rates when exposed to cold
environmental temperatures (reduced O
2
demand) and the
ability to obtain oxygen via cutaneous respiration (Costanzo,
3
Corresponding author. E-mail: anne.yagi@8trees.ca
DOI: 10.1670/18-143
1989a; Tattersall and Boutilier, 1997; Schulte, 2015). Snakes in
hibernacula may experience poor air quality when flooding,
snow, or ice block atmospheric oxygen exchange (Boutilier,
1990), particularly when the surrounding organic soils produce
toxic biogases (Moore and Knowles, 1989; Charman et al., 1994).
Measurement of hibernaculum air quality is challenging,
although assessment of groundwater dissolved oxygen
(GWDO) could provide an indirect measure of aerobic quality
changes within a hibernaculum.
We studied Massasauga (Sistrurus catenatus) hibernation
habitat using a subterranean grid of wells and frost tubes at
two sample areas (Mined and Not Mined), a system that allowed
us to avoid disturbing the ecological functions of the hibernation
site or the hibernating snakes themselves. A 4-yr radiotelemetry
study (i.e., 2000-2004) averaging 5.2 Massasaugas per year with 5
individuals followed over multiple years (Yagi and Tervo, 2005)
demonstrated that (1) individuals moved between Mined and
Not-Mined habitats during the active season, (2) individuals
exhibited site fidelity to either Mined or Not-Mined hibernation
sites across years, and (3) not all hibernacula could be identified.
To survive, we assumed that snakes would move within a
vertical subterranean space that is aerobic and flood and frost free
(Fig. 1), and we named this space the ‘‘life zone’’ (LZ). We
measured groundwater level (GWL), frost depth (FD), ground-
water temperature (T
GW
), and dissolved oxygen (GWDO) at each
site and hypothesized that (1) if an LZ requires a frost- and flood-
free subterranean space, then we expect the LZ size to be affected
by environmental variables, such as precipitation and tempera-
ture that cause flooding and freezing; (2) if the thermal and
aerobic quality of the subterranean space is affected by the size of
the LZ, then we predict an increasing quality with LZ size; and
(3) if successful snake hibernation requires the presence of an LZ,
then we expect to find increased snake overwintering survival
with larger LZ size and higher quality. Additionally, we propose
a standardized method for measuring, delineating, and commu-
nicating the characteristics of the LZ within terrestrial and semi
terrestrial habitats using a long-term case study of an endangered
Massasauga (Sistrurus catenatus) population.
MATERIALS AND METHODS
Study Site.—The study site is a partially drained and strip-
mined peatland wetland ecosystem within the Great Lakes
lowland region near Port Colborne Ontario, Canada (428520500’N,
798150000’W, datum WGS 84). The wetland is approximately
1,700 ha containing naturalized and remnant swamp, marsh, and
bog vegetation communities within an organic soil basin
underlain by thick clay soils (Browning, 2015). Constructed
watercourses (i.e., drains) intersect both the organic and
underlying clay layer; thus, shortening the hydroperiod (Brown-
ing, 2015; Fig. 2). When peat mining ceased in the 1990s, increases
in internal drain water levels occurred because of intrinsic and
extrinsic factors including vegetation growth, sediment accumu-
lations, woody debris, constructed peat dams, Beaver (Castor
canadensis) dams (Yagi and Litzgus, 2012; Browning, 2015), and
stochastic wet weather events (Environment and Climate Change
Canada, 2016). The wetland is isolated by surrounding agricul-
tural, industrial, and rural land use and is more than 200 km from
other Massasauga populations.
Measurement of the LZ.—We initiated our LZ study near the end
of the initial radiotelemetry study (Yagi and Tervo, 2005)
sampling between the winter of 2003–2004 through the winter
of 2013–2014. Mined and Not-Mined LZ study areas were chosen
based upon hibernation site fidelity data. More than 1 km of
forested peatland separates the LZ study areas. The study areas
are also within different hydrologic subcatchments (Browning,
2015) but otherwise have a similar vegetation community (Betula
pendula,Aronia melanocarpa,Vaccinium sp., Rubus sp.) with holes
at the surface. The Mined site is low-lying with exposed organic
soil surface flattened and compacted from past peat mining
activities. The Not-Mined site is higher in elevation with more
microtopography.
Groundwater level (GWL [cm]), FD (cm), groundwater
temperature (T
GW
[8C]), GWDO (mg/L), and snow height
(cm) were measured manually in a well-field grid within the
Mined and Not-Mined hibernation study areas for 11 winters.
Winters were defined annually from 1 November to 30 April.
Detailed methods for constructing and sampling the LZ well-
field grid are in Appendix 1. The LZ (cm) calculation equals the
difference between the top-of-well-pipe water-level (T) and the
height of well above ground (H) plus FD (LZ =T-[H +FD]);
or simply LZ =GWL -FD (Fig. 3). Observations of surface
flooding or ice formation in hibernation areas constituted a zero
LZ. We calculated the LZ groundwater thermal buffering
function (T
BUFF
8C) for each study area as the difference
between T
GW
and daily mean air temperature (T
AIR
), or T
BUFF
=T
GW
-T
AIR
(Environment and Climate Change Canada,
2016).
An important hydrological event occurred 3 yr into the LZ
study when the Mined study area (0.8 ha) and approximately
317 ha within the central peat barrens flooded for the first time
(Fig. 2; Yagi and Litzgus, 2012). The first flood event occurred
during a stochastic storm from 10 October 2006 to 13 October
2006 that included mixtures of snow, rain, and freezing
temperatures (Environment and Climate Change Canada,
2016). The flooded area accounted for over 30% of the central
peat barrens, and the Mined study area continued to experience
FIG. 1. Life-zone (LZ) model hypothesis for terrestrial and
semiterrestrial hibernating Massasaugas. Life zone is the vertical
subterranean space that remains aerobic, not frozen, and flood free
throughout hibernation.
236 A. R. YAGI ET AL.
flood events over the next five winters (i.e., winter of 2006–2007
to winter of 2010–2011). The flood events were followed by a
dry weather cycle with a severe drought and central peatland
wildfires in 2012 (Fig. 2).
Because we were unable to maintain a strict weekly sampling
schedule over the 11-yr study period, and because field
equipment sometimes failed, temporal gaps in our data set
exist. For missing GWL, we used the average between
subsequent measurements and assumed the levels did not
change drastically between visits. We used the average daily FD
value for the study area to fill weekly gaps to complete the LZ
calculation. Although our approach had a smoothing effect on
the data, we were able to retain the spatial context. The data
correction occurred 146 times out of 3170 (4.6%). We omitted
blank GWDO and T
GW
data from further analysis.
Massasauga Population.—We used annual (April–November)
mark–recapture (M-R) data from 1998 to 2016 to monitor
population trends (Ontario Ministry of Natural Resources and
Forestry, unpubl. data). All captured adults were tagged
subcutaneously with a passive integrated transponder (PIT;
Parent, 1997). We identified neonatal snakes by their head and
mottle pattern. We estimated age from several biological
parameters such as snout–vent length (SVL), weight, reproduc-
tive maturity, and the number of rattles within a complete rattle
sequence. We assumed that two rattles form per year for this
population, which is consistent with local data and other
rattlesnake populations at similar latitudes (Fitch, 1985; Mac-
artney et al., 1990; Aldridge and Brown, 1995). All encountered
snakes were aged in this manner to confirm the presence in the
population and allow us to discern a preflood group from
postflood recruits. The number of snakes considered to be adults
(N
adults
) was aged 3 yr and older.
We did not use traditional calculations for population
estimates because of the cryptic nature of Massasaugas, our
low annual recapture rates, and uncertainty in annual survival
because of the presence of impaired habitat and environmental
stochasticity. We instead built a time series of known individ-
uals (N
indiv
) for the overall sampling area and LZ study period.
All encountered snakes per year plus those presumed present
because of their age estimates from future encounters (N
indiv
=
new captures [C] +recaptures [R] +known undetected [U]). We
calculated the yearly rate of detection (D) as (C +R)/N
indiv
.
Snakes not recaptured up to 10 yr after the last encounter were
hence removed from the sample population. We calculated the
proportion of adults in the population (P
adults
)asN
adults
/N
indiv
.
Statistical Analyses.—We used the program R to complete all
statistical analyses and graphing (version 3.5.0; R Core Team,
2018). We tested parameters and model residuals for normality
using a Shapiro-Wilk test and visually using qq-plots. We used
nonparametric tests (i.e., Kruskal-Wallis and Dunn’s test) when
transformations failed to correct for violations of parametric
statistical assumptions.
Weather Data Analysis.—We downloaded 16 winters (1998–
2014) of historical weather data from the nearest weather station
with vetted data (Port Colborne or Hamilton, Ontario). This
period encompassed the broader 16-yr Massasauga population
study, including the 11-yr LZ study. Thus, it allowed us to
contextualize the LZ study within recent winter climate trends
FIG. 3. Hibernation habitat study methods developed for a partially
mined peatland ecosystem inhabited by a Massasauga population
located in southern Ontario, Canada. Life-zone methods include a grid
of groundwater wells paired with a frost tube in each study area (Mined
and Not Mined). Life zone (LZ) equals the groundwater level (GWL)
minus frost depth (FD). All measurements are relative to the ground
surface.
FIG. 2. The Massasauga population study area is a partially strip-
mined peatland located in southern Ontario, Canada. (A) shows the
amount of surface water present during dry weather cycles (1998–2005;
2011–2014). (B) shows the amount of surface water present during a wet
weather cycle (2006–2011). The first known flood event occurred from 10
October 2006 to 13 October 2006. Hibernation study areas are data
sensitive.
MASSASAUGA OVERWINTERING HABITAT AND SURVIVAL 237
concerning precipitation and temperature. We partitioned the
monthly weather data to match the LZ study period and
extracted four weather variables for analysis: precipitation (snow,
rain, total) and the number of freezing days (i.e., mean daily
temperature <08C; Environment and Climate Change Canada,
2016). We conducted a principal component analysis (PCA) to
reduce the winter weather records into useful categories for
analysis. We employed a K-means cluster analysis with the K-
means function to assess the sums of squares based on the
different number of clusters. By plotting the total within-groups
sums of squares against the number of clusters, the sudden
change in slope depicted an optimal value of three winter types
(Everitt et al., 2011). We partitioned the three winter types into
groups defined by temperature and precipitation trends: cold–
dry (winter type 1), mild–wet (winter type 2), and cold–wet
(winter type 3). We achieved a data reduction of the same
weather data using PCA with the FactoMineR package (L ˆe et al.,
2008). All variables were centered and scaled to unit variance
before the PCA. The predominant axis (PC1) described a measure
of rainfall (from dry to wet), and the second axis (PC2) combined
thenumberofdaysbelow08C (from mild to cold) and total
precipitation as snow.
Life Zone Analysis.—The LZ is the space between the FD and
GWL. Therefore, according to our hypothesis, when GWL
exceeds the ground surface or frost reaches the GWL, LZ size
equals zero. However, we used negative LZ values to avoid zero
truncation issues during the model analysis (Zuur et al., 2009).
Fixed and random effects (Table 1) were analyzed with respect to
the response variables LZ size and natural log-transformed
GWDO and T
BUFF
using linear mixed effect models on the full
data set with the lmer function from the lmerTest package.
Adjusted degrees of freedom were estimated using the
Satterthwaite method. A model selection analysis using the AIC
c
function from the AICcmodavg package was used to assess the
fixed effects that best predict LZ size, ln GWDO, and T
BUFF
(Akaike, 1987). A Type III ANOVA was used to analyze the
significance between fixed effects within the Akaike top model.
Massasauga Survival Analysis.—We used a linear model to
assess differences in detection by flood groupings. An annual
flood survival estimate was calculated for the preflood group
accounting for known deaths. We used a generalized linear
model with a Poisson distribution to compare the previous
winter’s fixed effects (LZ size, T
BUFF
,GWDO,T
GW
,snowheight)
against the following active season N
indiv
(Table 1). Akaike model
selection was used to determine the top model. AType II analysis
of deviance was used to assess the significance of the fixed-effects
within the Akaike top model, using the ANOVA function from
the R car package.
RESULTS
Winter Weather Trends.—The PCA combined local winter
environmental conditions into PC1 (rainfall) and PC2 (snow
and number of freezing days), which clustered into three winter
types (Fig. 4; Table 2). Winter type 1 (cold–dry) occurred four
times but only once during the LZ study. Winter type 2 (mild–
wet) occurred four times during the LZ study. Winter type 3
(cold-wet) occurred six times (Fig. 4).
LZ Size Dynamics.—The Not-Mined area has significantly
deeper organic soils (mean =167.9 cm, SD =53.3) vs. Mined
(mean =71.3 cm, SD =20.7; t
(30)
=8.49, P<0.001). Maximum
recorded frost depth (29.8 cm) was in Not-Mined habitat during
winter 2007 after the first flood event. The Mined LZ size was
significantly larger before than after flooding (P<0.001; Table 3).
Mined LZ size was reduced to zero periodically (312/658
measurements =47% zeros) during flood events (Fig. 5). Not-
Mined LZ size was not significantly different throughout the 11-
yr study (P=0.49; Table 3; Fig. 5). The model that best explained
the variance in LZ size included the fixed effects of site soil depth
(cm) and flood condition (Table 4). Type III ANOVA indicated
TABLE 1. Summary of parameters used in the model selection analysis for each habitat and encounter response variable from an 11-yr hibernation
habitat study on a Massasauga population located in southern Ontario, Canada. Linear mixed-effects models are (1) Life-zone size (LZ [cm]), (2) LZ
thermal buffering function (T
BUFF
), (3) groundwater dissolved oxygen (GWDO [mg/L]), (4) a generalized linear model compares the previous winter’s
fixed effects against the following active-season Massasauga encounters (N
indiv)
.
Response variable Fixed effects Random effects Data
LZ (cm) Weather cycle (dry or wet)
Flood (before and after)
Site (Mined and Not Mined)
Site soil depth (cm)
PC1, PC2, winter type (1–3)
Year
Well ID
Date
Full data set n=3,170
T
BUFF
(D8C) and ln (GWDO) (mg/L) LZ (cm)
Snow height (cm)
Flood (before and after)
Site (Mined and Not Mined)
Site soil depth (cm)
PC1, PC2, winter type (1–3)
Year
Well ID
Date
Full data set n=2,281
Massasauga encounters (N
indiv
) Mean LZ (cm)
Mean snow-height (cm)
Mean GWDO (mg/L)
Mean T
GW
(8C)
Mean T
BUFF
(D8C)
Generalized linear models Annual means n=11
TABLE 2. Summary of PCA eigenvalues for winter environmental
data from a Massasauga population in southern Ontario, Canada. We
downloaded historical weather data for the study area from the
Environment and Climate Change Canada weather data website
(1998–2014). Variables of interest include total rain, total snow, total
precipitation, and the number of days when the mean temperature was
below zero.
Importance of components PC1 PC2 PC3
Variance 1.93 1.6 0.47
% of variance 48.26 39.95 11.79
Cumulative % of variance 48.26 88.21 100
238 A. R. YAGI ET AL.
that site soil depth and flood condition were significant effects on
LZ size (Table 5).
Groundwater Temperature.—Mined T
GW
declined significantly
following flood events (P<0.001), whereas Not-Mined T
GW
did
not (P=0.08; Table 3). T
GW
was not measurable when a well was
flooded or frozen (361 out of 3,170 sampling events, 11.4%),
causing data gaps and imbalances in the Mined area data set
during the flood maxima, winter 2008. There were also 75 out of
3,170 (2.3%) sampling events that were too dry to measure
groundwater attributes. Mined T
BUFF
was not significantly
different due to flooding (P=0.11; Table 3); and Not-Mined
T
BUFF
was significantly greater after flooding (P<0.001; Table 3).
The top model for T
BUFF
included the significant fixed effects of
LZ size, snow height, site soil-depth, flood-condition, and PC1
(Tables 4 and 5).
Groundwater Dissolved Oxygen.—Mined GWDO (mg/L) was
significantly reduced when flooded (P<0.001) and Not-Mined
GWDO significantly increased (P<0.001; Table 3). Mined
ln GWDO was significantly and positively correlated with mean
LZ size before flooding (F
1,34
=41.25, R
2
=0.55, P<0.001) but
not after (F
1,41
=0.48, R
2
=0.01, P=0.49; Fig. 6). Although the
Not-Mined area did not flood, it did undergo the same wet and
dry weather cycles. The relationship between Not-Mined mean
GWDO and mean LZ size was significant before (F
1,32
=2.6, R
2
=
0.14, P=0.03) but not after (F
1,51
<0.001, R
2
<0.001, P=0.99)
the first flood event (Fig. 6). The top model that best explained
the variance in ln GWDO included the fixed effects LZ size, PC1,
flood condition, and snow height (Table 4). Type III ANOVA for
the top model showed that LZ and PC1 were significant factors,
whereas snow height and flood condition were not (Table 5).
Massasauga Population Dynamics.—Mean snake detection (D)
increased in the study area from before (mean =0.30, SD =0.11)
to during (mean =0.52, SD =0.37) and decreased after (mean =
0.38, SD =0.19) the flood events. There was no significant
difference in D among these flood groupings (F
2,8
=0.68, P=
0.53). Therefore, we used before and after the first flood event
groups for subsequent analyses. Total search effort during the 11-
yr study averaged 735 person hours per year, SD =354. The
lowest N
indiv
occurred in 2008, and the preflood group low
occurred in 2010 (Fig. 7). The preflood group encounters declined
by 33% after the first flooded winter, followed by 76% after the
second, and 90% by the third winter (Fig. 7). The mean age of
N
indiv
in 2004 was 2.5 yr 60.50 SE (P
adults
=0.45), increased to
4.1 yr 60.46 S.E. in 2007 (P
adults
=0.79), and declined to 1 yr 6
0.6 S.E. by 2010 (P
adults
=0.11; Appendix 2). The Akaike top
model that predicted N
indiv
included LZ size over the fixed
effects T
BUFF
,snowheight,T
GW,
and GWDO (Table 6). The N
indiv
and previous winter’s mean LZ was a significant positive
FIG. 4. PCA plot depicting the winter-type categories developed for winter environmental data for a Massasauga population located in southern
Ontario, Canada. We analyzed local annual winter (November–April) environmental data: total precipitation, rainfall, snow, and the number of days
mean temperature is below zero (Environment and Climate Change Canada, 2016).
TABLE 3. Life-zone summary measurements mean 6SD from Mined and Not-Mined study areas before and after first flood event which initiated a
5-yr stochastic flooding period followed by receding floodwaters (asterisk denotes significant Pvalue).
Life zone measures
Mined Not mined
Before After Test statistic Before After Test statistic
LZ size (cm) 40.4 612.6,
n=834 9.2 612.5,
n=658
v
2
=898.6, df 1*,
P<0.001 51.3 615.9,
n=771 50.4 616.7,
n=907
v
2
=0.48, df 1,
P=0.49
T
GW
(8C) 5.62 61.66,
n=579 4.23 62.03,
n=290
v
2
=113.6, df 1*,
P<0.001 5.95 61.48,
n=641 6.10 61.54,
n=718
v
2
=3.1, df 1,
P=0.08
T
BUFF
=T
GW
-T
AIR
6.17 63.83,
n=579 6.03 64.56,
n=290
v
2
=2.55, df 1,
P=0.11 6.96 64.89,
n=641 8.44 64.76,
n=718
v
2
=35.7, df 1*,
P<0.001
GWDO (mg/L) 2.42 61.82,
n=579 1.45 61.50,
n=290
v
2
=64.0, df 1*,
P<0.001 1.8 61.40,
nn=641 2.71 62.10,
n=718
v
2
=61.42, df 1*,
P<0.001
MASSASAUGA OVERWINTERING HABITAT AND SURVIVAL 239
relationship with an incidence response ratio of 0.06 (v
2
=28.8, df
=1, r
2
=0.34, P<0.001; Fig. 8).
DISCUSSION
In our study, anthropogenic habitats and natural habitats
show important differences in life-zone functions during times
of environmental stochasticity. Important factors affecting LZ
size in our model analysis were site, soil depth, snow height,
flood condition, and winter type. The significant relationship
between LZ size and flood condition supported our first
hypothesis that environmental factors, such as precipitation
and temperature, affect the size of the LZ. Thermal buffering
and GWDO quality attributes significantly improved in natural
areas that maintained an LZ compared to peat-mined habitats
that did not. The significant relationship between LZ size, T
BUFF
,
and GWDO supports our second hypothesis that LZ size affects
thermal and aerobic quality. Finally, the significant relationship
between LZ size and Massasauga encounters indirectly sup-
ports our third hypothesis that Massasauga survival increases
with LZ size. Because natural habitats only maintained an LZ,
they likely provided areas of refugia for the Massasauga
population.
In our LZ model, we considered that snakes might die from
either drowning, freezing, or asphyxiation. However, only the
top model showed a positive correlation between N
indiv
and LZ
size. In altered ecosystems, a large LZ may provide the capacity
needed to maintain survival during times of environmental
stochasticity. In natural bog ecosystems, a shallow LZ may also
provide thermal stability because there is an elevated and stable
groundwater table (Smolarz et al., 2018). At our site, we have a
degraded ombrotrophic bog ecosystem without stable hydrol-
ogy or stable Massasauga population. Therefore, relationships
between population trends and LZ functions may be confound-
ed by these complexities. In the case of GWDO, we did not
directly measure the air oxygen content in occupied hibernac-
ula. Yet, over half the measurements of GWDO were hypoxic,
which indicates that cutaneous respiration, as a flood survival
strategy, may not be possible here (Costanzo and Lee, 1995;
Tattersall and Boutilier, 1997; Jackson, 2007). Therefore, main-
tenance of an aerobic space within the hibernation site for
oxidative metabolism is an important consideration in over-
wintering survival. Because subterranean air spaces naturally
have low atmospheric oxygen exchange, winter types vary, and
flooding reduces soil oxygen content, additional monitoring is
needed to establish the relationship between LZ size, survival,
GWDO, snow height, and air space O
2
(Boutilier, 1990;
FIG. 5. Massasauga hibernation habitat weekly life-zone size (LZ cm)
by study site (Mined and Not Mined), over time (November 2003–April
2014). The study site is a partially strip-mined peatland located in
southern Ontario, Canada. Life-zone size was zero for at least 1 wk
during winters (2006–2011) because of surface flooding and freezing
during a wet weather cycle. Error shown shaded in gray is 695%
confidence interval (CI).
TABLE 4. Akaike top five linear mixed effect models and the null model results for an 11-yr Massasauga hibernation habitat study located in
southern Ontario, Canada. The models are (A) Life-zone size (LZ [cm]), (B): thermal buffering function (T
BUFF
), and (C) groundwater dissolved oxygen
(ln GWDO). Fixed effects include winter type (1–3) or PC1, or PC2, site (Mined or Not Mined) or site soil depth (cm), flood (before and after) or
weather cycles (dry and wet), and LZ size (cm). For all analyses, random effects were Well ID, date, and year (null model). The top models are marked
by an asterisk.
Model AICc DAIC
c
df x
(A) LZ
Site soil depth +flood 22,785.1 0 7 0.72*
Site soil depth +flood +PC1 22,787.0 1.9 8 0.28
Winter type +site soil depth 22,805.8 20.7 8 <0.001
Flood 22,812.3 27.2 6 <0.001
Weather cycle 22,825.4 40.4 6 <0.001
Null 22,830.6 45.5 5 <0.001
(B) T
BUFF
LZ +snow height +flood +site soil depth +PC1 6,267.5 0 10 0.8395*
LZ +snow height +flood +PC1 6,271.5 4.1 9 0.1101
LZ +snow height 6,275.2 7.7 7 0.0179
LZ +snow height +flood 6,276.1 8.7 8 0.011
LZ +snow height +flood +PC2 6,277.1 9.7 9 0.0067
Null 6,323.1 55.7 5 <0.001
(C) ln GWDO
LZ +snow height +flood +site soil depth +PC1 4,994.5 0 9 0.7874*
LZ +snow height +site soil depth 4,997.3 2.8 8 0.1948
Site soil depth +snow height 5,004.2 9.7 7 0.0062
PC1 +PC2 5,004.4 9.9 7 0.0056
PC1 5,005.2 10.7 6 0.0038
Null 5,006.9 12.4 5 0.0016
240 A. R. YAGI ET AL.
Cavallaro and Hoback, 2014). Survival experiments that control
snake overwintering locations, coupled with LZ measures,
would provide direct support of our hypothesis that the
presence of an LZ supports overwinter survival.
Our population trends are based upon Massasauga encoun-
ters that are backcast using age and refined annually with new
M-R and age estimate data. Therefore, N
indiv
is more reliable in
the past and underestimated in the most recent years. Our M-R
data are affected by the challenges of finding a cryptic species.
Yet our snake detection rate doubled during the first three flood
years, then dropped to zero in the fourth flood year (i.e., 2010).
Zero captures in 2010 are an important finding, considering
there was a spatial search effort bias toward the central peat
barrens as we were trying to recapture flood survivors. After
2010 we evened out the search effort and captured Massasaugas
in the Not-Mined habitats that were previously undetected.
Declining encounters and the significant correlation with LZ
size provide indirect evidence of reduced survival in the
wetland during this period of environmental stochasticity. We
also presented evidence that the mean age of N
indiv
increased by
a factor of 1.6 from 2004 to 2007, then declined by a factor of 4.1
by 2010. The increase in the mean age estimate suggests (1) low
recruitment or reproductive output, (2) that adults had a higher
initial survival rate than their offspring, or (3) a combination of
factors. The subsequent decline in mean age by 2010 is likely
related to successful overwintering in the younger age classes.
Because postflood encounters were almost exclusively in Not-
Mined habitats that maintained an LZ, females giving birth in
higher elevations may have aided neonatal dispersal and
survival. Adults likely moved to higher-elevation areas and
overwintered successfully, emigrated beyond the survey areas,
or did not survive hibernation in low-lying areas because of site
fidelity. This study presents indirect evidence of the latter. With
recent flooding events and the challenges in monitoring a
recovering cryptic species, the relationship between LZ func-
tions and survival requires further investigation. We should
avoid overinterpreting these results because our summarized
TABLE 5. The top-ranked linear mixed-effects model ANOVA results for the 11-yr Massasauga hibernation habitat study located in southern
Ontario, Canada. The hibernation habitat study examined the following environmental effects: (A) life-zone size (LZ cm), (B) LZ thermal buffering
function (T
BUFF
), and (C) LZ aerobic quality function (GWDO). Random effects were well ID, date, and year (* denotes significant Pvalue).
Fixed effects SS MS Num df Den df Fvalue Pvalue
(A) LZ size ~site soil depth +flood condition
Site soil depth 2,373.9 2,373.9 1 46.311 40.879 <0.001*
Flood 4,078.3 4,078.3 1 8.611 70.228 <0.001*
(B) T
BUFF
~LZ size +snow height +flood condition +PC1 +site soil depth
LZ cm 19.31 19.31 1 918.7 35.34 <0.001*
Snow height 5.49 5.49 1 2186.4 10.05 <0.01*
Flood 4.20 4.20 1 171.2 7.69 <0.01*
PC1 6.04 6.04 1 170.5 11.05 <0.01*
Site soil depth 3.47 3.47 1 50.0 6.35 0.01*
(C) ln GWDO ~LZ size +snow height +flood condition +PC1
LZ cm 6.22 6.221 1 396.56 14.56 <0.001*
PC1 6.66 6.6563 1 3.51 15.58 0.02*
flood 2.36 2.3623 1 3.30 5.53 0.09
Snow height 0.10 0.0968 1 375.79 0.23 0.63
FIG. 6. Massasauga hibernation habitat weekly groundwater
dissolved oxygen (mean GWDO mg/L) by weekly life-zone size
(mean LZ cm). The study area is a partially mined peatland located in
southern Ontario, Canada. Mined and Not-Mined areas are grouped by
before (2003–2005) and after (2006–2014) first flood event. Error shown
shaded in gray is 695% CI.
FIG. 7. Massasauga encounters (N
indiv
) from 2004–2014 are
calculated from the long-term (1998–2016) encounter data set for an
isolated population located in southern Ontario, Canada. N
indiv
includes
all encountered adult and juvenile Massasaugas plus those presumed
present due to their age estimates from future encounters. The preflood
group encounter trend is indicated to discern postflood recruitment.
MASSASAUGA OVERWINTERING HABITAT AND SURVIVAL 241
data may mask the true relationship between encounters and
hibernation habitat quality.
Across the Massasauga range, hibernation sites are often, but
not always, found in habitats with an elevated groundwater
table (Johnson, 1995; Parent, 1997; Harvey and Weatherhead,
2006b). Habitats that maintain an LZ may offer increased
thermal stability and population viability (Pomara et al., 2014;
Smolarz et al., 2018). The presence of a consistent snow layer
may also provide important thermal buffering, especially when
considering the presence of a thermally stable groundwater
table and the thickness of the LZ. In addition to the increased
frequency of stochastic events that reduce LZ size, the presence
of a snow layer at our study site in southern Ontario is
inconsistent between years. Increasingly episodic polar vortices
(Charlton and Polvani, 2007) may also increase freeze risk in
areas, especially when there is a lack of snow cover and a small
LZ. Modeling LZ size and quality functions within various
climate-change scenarios under ecologically relevant conditions
are important next steps in this research (Pomara et al., 2014).
Expanding LZ monitoring into other habitats and soil types are
warranted where there is a flood or freeze concern.
Furthermore, demonstrating causality by multiple potential
factors is challenging, especially when subsurface mortality and
unknown hibernacula locations prevent the confirmation of
survival. The continual presence of potentially poor habitat that
is periodically used by Massasaugas during dry weather cycles
may indicate the presence of an ecological trap (Battin, 2004).
From a conservation perspective, mortality events caused by an
ecological trap may increase the extinction risk of a population
that may already be compromised by small size, lack of rescue
by isolation, genetic drift, and uncertainty surrounding adap-
tation to environmental stochasticity (Battin, 2004; Bradke et al.,
2018). Future studies on this topic should provide opportunities
to study the effects of climate change, habitat quality, winter
severity, and the relevance to reptile hibernation success in
northern climates.
Acknowledgments.—All procedures involving animals fol-
lowed the Ontario Ministry of Natural Resources and Forestry
(OMNRF) regulations, and Animal Care permit 98–55 to 16–55.
We have withheld locations of hibernation study areas because
of data sensitivity. Environment Canada Habitat Stewardship
Fund, OMNRF, and the World Wildlife Fund provided funding
support. We thank our partners, Land Care Niagara, Niagara
Peninsula Conservation Authority, National Massasauga Re-
covery Team, Toronto Zoo, B. Johnson, J. Litzgus, Vittie family,
Yagi family, J. Durst, M. Browning, K. Frohlich, D. Mills, A.
Parks, D. Denyes, M. Karam, and C. Blott. This article is
dedicated to the memory of R. Tervo (d. 8 November 2008) and
R. J. Planck (d. 29 October 2015).
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Accepted: 19 January 2020.
Published online: 15 May 2020.
APPENDIX 1
Detailed Methods for Measuring the Life Zone (LZ).—Groundwater
wells paired with frost tubes were installed, forming a grid pattern,
across both Mined and Not-Mined hibernation study areas. Thirty-
meter and 50-m spacings were used for Mined and Not-Mined well
fields, respectively. Weekly measurements averaged 12 wells for
Mined (0.8 ha) and 24 wells in the Not-Mined (3.75 ha) areas. We
placed wells and frost tubes approximately 1 m away from any
surface hole, to avoid impacting existing snake burrows. We made
groundwater wells from 1.83-m length of black 5-cm diameter ABS
pipe and drilled several 0.5-cm-diameter holes perpendicular
through the sides of the bottom one-third portion, and then placed
a cap on top (Fig. 3). To install a well, we bored a vertical hole into
the ground surface up to 1.2-m depth using a hand-operated 5-cm-
diameter soil auger. We placed the well into the hole and tamped
down the soil around the pipe at the ground surface interface. We
also measured the depth to clay (i.e., organic soil depth [cm]) at
installation. Soil-depth measurement was limited to the maximum
length of the auger ~1.2 m. Fibric soil and subterranean spaces were
also noted when they occurred.
We constructed a frost tube in two parts, following a design
developed by the U.S. Forestry Service (Patric and Fridley, 1969). For
the first part, we made an outer casing from 1.3-cm-diameter PVC
conduit, and cut it into 60–100-cm lengths depending on soil depth.
We covered one end with 3Meduct tape and pushed the casing
vertically into the ground, flush to the surface. We located the casing
approximately 1 m from the well. For tighter soils, we recommend
the use of metal rebar to make the hole. We constructed the inner
second part from 0.95-cm outer-diameter clear, flexible tubing sealed
at the bottom with a cured waterproof sealant and a 2-cm piece of
wooden doweling to form a plug. The flexible tubing fits tightly
inside the outer casing. We delineated the corresponding ground
surface by marking the clear tube with an indelible felt-tipped pen.
We filled the clear tube with red-food-dye colored water up to this
mark. We used waterproof tape to secure the top of the frost tube to
prevent water leakage and attached a guidewire through the tape to
a nearby post or shrub to help locate the frost tube under the snow.
We measured frost depth (FD [cm]) as ice formation within the red
water-filled tube which changes color from red to clear, giving a
distinct visual demarcation of ice. We measured GWL (cm) in the
well from the top of the pipe using an electronic measuring tape
(Heron Instruments Little Dippere22 m), which beeps when it
encounters water. LZ size (cm) equals the space between FD and
GWL (Fig. 3). Manual data collection occurred weekly between 1
November and 31 April, from 2003 to 2014.
We also measured temperature (T
GW
[8C]) and dissolved oxygen
(GWDO [mg/L]) within the top 10-cm groundwater layer inside the
well, using a DO meter (YSI 55 or 550 models) calibrated to zero
salinity, and surface elevation (183 m above sea level). We
calculated the LZ groundwater thermal buffering function (T
BUFF
[8C]) for each study area as the difference between T
GW
and daily
mean air temperature (T
AIR
; Environment and Climate Change
Canada, 2016). We also measured total snow height (cm) including
any ice accumulations, using a measuring tape. The flooded zone
was mapped from field observations with the use of a handheld
Garmin GPS (model eTrex 20x) and digitized into orthogonal
corrected aerial imagery with ÓArcGIS mapping software. We
recorded surface flooding and ice formation at a well site, and
where this occurred, we scored LZ equal to zero centimeters.
APPENDIX 2. A summary of Massasauga population data collected during 11 active seasons following each winter’s hibernation study (2004–2014)
located in southern Ontario, Canada. Encounters (N
indiv
), mean age (with standard error), detection rate (C+R/N
indiv
), and flood state from 2004 to
2014 are included. N
indiv
is the number of new captures, recaptures, and known-undetected snakes, calculated by backcasting age in the population
time series. Flooding events started in the winter of 2006–2007 and continued through to winter of 2010–11.
Year N
indiv
Proportion adults Mean age Age SE Detection rates Description
2004 29 0.45 2.5 0.4 0.36 Preflood
2005 23 0.48 3.1 0.5 0.17 Preflood
2006 21 0.76 3.9 0.6 0.38 Preflood
2007 14 0.79 4.1 0.5 0.64 Flood
2008 7 0.57 3.4 1.2 0.57 Flood
2009 8 0.25 1.5 0.8 0.63 Flood
2010 9 0.11 1.0 0.5 0.00 Flood
2011 10 0.10 1.8 0.5 0.30 Receding flood with flood events
2012 10 0.40 2.3 0.6 0.10 Postflood with wildfires
2013 10 0.60 2.9 0.7 0.40 Postflood
2014 8 0.62 3.6 1.0 0.50 Postflood
244 A. R. YAGI ET AL.
