Mitigating Reptile Road Mortality: Fence Failures Compromise Ecopassage Effectiveness

Abstract

Roadways pose serious threats to animal populations. The installation of roadway mitigation measures is becoming increasingly common, yet studies that rigorously evaluate the effectiveness of these conservation tools remain rare. A highway expansion project in Ontario, Canada included exclusion fencing and ecopassages as mitigation measures designed to offset detrimental effects to one of the most imperial groups of vertebrates, reptiles. Taking a multispecies approach, we used a Before-After-Control-Impact study design to compare reptile abundance on the highway before and after mitigation at an Impact site and a Control site from 1 May to 31 August in 2012 and 2013. During this time, radio telemetry, wildlife cameras, and an automated PIT-tag reading system were used to monitor reptile movements and use of ecopassages. Additionally, a willingness to utilize experiment was conducted to quantify turtle behavioral responses to ecopassages. We found no difference in abundance of turtles on the road between the un-mitigated and mitigated highways, and an increase in the percentage of both snakes and turtles detected dead on the road post-mitigation, suggesting that the fencing was not effective. Although ecopassages were used by reptiles, the number of crossings through ecopassages was lower than road-surface crossings. Furthermore, turtle willingness to use ecopassages was lower than that reported in previous arena studies, suggesting that effectiveness of ecopassages may be compromised when alternative crossing options are available (e.g., through holes in exclusion structures). Our rigorous evaluation of reptile roadway mitigation demonstrated that when exclusion structures fail, the effectiveness of population connectivity structures is compromised. Our project emphasizes the need to design mitigation measures with the biology and behavior of the target species in mind, to implement mitigation designs in a rigorous fashion, and quantitatively evaluate road mitigation to ensure allow for adaptive management and optimization of these increasingly important conservation tools.

Full text

RESEARCH ARTICLE
Mitigating Reptile Road Mortality: Fence
Failures Compromise Ecopassage
Effectiveness
James H. Baxter-Gilbert
1¤
, Julia L. Riley
2¤
, David Lesbarrères
1
, Jacqueline D. Litzgus
1
*
1Department of Biology, Laurentian University, Sudbury, Ontario, P3E 2C6, Canada, 2Magnetawan First
Nation, Britt, Ontario, P0G 1A0, Canada
¤Current address: Department of Biological Sciences, Macquarie University, Marsfield, New South Wales,
2122, Australia
*jlitzgus@laurentian.ca
Abstract
Roadways pose serious threats to animal populations. The installation of roadway mitiga-
tion measures is becoming increasingly common, yet studies that rigorously evaluate the ef-
fectiveness of these conservation tools remain rare. A highway expansion project in
Ontario, Canada included exclusion fencing and ecopassages as mitigation measures de-
signed to offset detrimental effects to one of the most imperial groups of vertebrates, rep-
tiles. Taking a multispecies approach, we used a Before-After-Control-Impact study design
to compare reptile abundance on the highway before and after mitigation at an Impact site
and a Control site from 1 May to 31 August in 2012 and 2013. During this time, radio teleme-
try, wildlife cameras, and an automated PIT-tag reading system were used to monitor reptile
movements and use of ecopassages. Additionally, a willingness to utilize experiment was
conducted to quantify turtle behavioral responses to ecopassages. We found no difference
in abundance of turtles on the road between the un-mitigated and mitigated highways, and
an increase in the percentage of both snakes and turtles detected dead on the road post-
mitigation, suggesting that the fencing was not effective. Although ecopassages were used
by reptiles, the number of crossings through ecopassages was lower than road-surface
crossings. Furthermore, turtle willingness to use ecopassages was lower than that reported
in previous arena studies, suggesting that effectiveness of ecopassages may be compro-
mised when alternative crossing options are available (e.g., through holes in exclusion
structures). Our rigorous evaluation of reptile roadway mitigation demonstrated that when
exclusion structures fail, the effectiveness of population connectivity structures is compro-
mised. Our project emphasizes the need to design mitigation measures with the biology
and behavior of the target species in mind, to implement mitigation designs in a rigorous
fashion, and quantitatively evaluate road mitigation to ensure allow for adaptive manage-
ment and optimization of these increasingly important conservation tools.
PLOS ONE | DOI:10.1371/journal.pone.0120537 March 25, 2015 1/15
OPEN ACCESS
Citation: Baxter-Gilbert JH, Riley JL, Lesbarrères D,
Litzgus JD (2015) Mitigating Reptile Road Mortality:
Fence Failures Compromise Ecopassage
Effectiveness. PLoS ONE 10(3): e0120537.
doi:10.1371/journal.pone.0120537
Academic Editor: Gregorio Moreno-Rueda,
Universidad de Granada, SPAIN
Received: September 17, 2014
Accepted: January 23, 2015
Published: March 25, 2015
Copyright: © 2015 Baxter-Gilbert et al. This is an
open access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: Due to ethical and
legal restrictions, data are available upon request.
Interested researchers may contact the
corresponding author, Jacqueline Litzgus, to request
the data.
Funding: Financial support for this research was
provided by Magnetawan First Nation, Laurentian
University, Ontario Ministry of Natural Resources,
Ontario Ministry of Transportation (MTO) HIIFP
Grant, and NSERC Discovery Grant. Opinions
expressed in this paper are those of the authors and
may not necessarily reflect the views and policies of
the MTO. The funders had no role in study design,

Introduction
Increasing rates of urbanization, with associated habitat destruction and fragmentation, have
led to the imperilment of much the world’s biodiversity [1,2]. Roads and traffic present some
of the longest lasting effects from both point-source mortality and enduring habitat and popu-
lation fragmentation [1,3]. The threats posed by roads extend from individual mortality to
population-wide effects, as barriers within populations can lead to loss of genetic diversity and
isolation [4]. Over the last two decades, the field of road ecology has grown to include examina-
tion of numerous taxa and incorporates a wide variety of disciplines, all with the common goal
to better understand the interaction between roads and wildlife [1,5,6,7]. Closely tied to this
research effort is the development of mitigation strategies aimed at protecting wildlife from the
negative effects of roads [8], with particular focus on large-bodied species [9,10]. Far fewer
studies have examined effectiveness of road mitigation for small-bodied species [11,12]. Rep-
tiles, considered one of the most imperilled groups of animals globally [13], have been substan-
tially affected by the proliferation of roads [14,15,16], and conservation strategies to minimize
threats posed by roads are being implemented [17,18]. Yet, few studies have evaluated the ef-
fectiveness of these conservation efforts [7].
Threats to reptiles from roads are multifaceted and often relate to the species’specific eco-
logical and life-history traits, behaviors, and movement patterns [19,20,21]. Turtles regularly
encounter roads during long-distance seasonal movements, and road mortality of adults leads
to population declines because of the “bet-hedging”life history of turtles that requires high
adult survivorship for population persistence [14,22]. Turtles are particularly susceptible be-
cause up to 98–100% of individuals can be killed during their first road-surface crossing at-
tempt [18]. Similar to turtles, seasonal movements of snakes also require road crossings, and
road mortality has been identified as a population-level threat to several species [23,24,25].
Snakes bask on road surfaces to absorb radiant heat; this behavior prolongs exposure to traffic
and increases the likelihood of collisions [26]. Further increasing the threat to reptiles, 2.7% of
drivers will intentionally run over snakes [27].
Mitigation measures have been designed to reduce road mortality by installing exclusion
structures (e.g., fences, gravity walls), and to reduce fragmentation by installing population con-
nectivity structures (e.g., ecopassages, bridges) [17,18,27]. The integration of such structures
into highway designs is becoming increasingly common for a wide variety of affected wildlife,
yet the effectiveness of these mitigation measures is rarely quantified [7,19,28,29]. This lack of
assessment is a concern because functional and cost-effective conservation measures are critical
to the recovery of imperiled populations, especially given the limited funds available for conser-
vation projects [7]. Evaluation is needed to provide a framework for effective and logistically fea-
sible mitigation that can be regularly implemented into roadways [6,30,31].
Effective roadway mitigation measures must meet the following criteria [19,28,32]: 1) re-
duction in abundance of wildlife on roads, 2) maintenance of habitat connectivity and dispersal
routes, and 3) prevention of prey-trap formation. To this end, we quantitatively assessed these
mitigation criteria for an assemblage of reptiles along a major roadway in Ontario, Canada.
Our study used these criteria to rigorously assess the effectiveness of the mitigation via four
methods: i) Before-After-Control-Impact (BACI) study to examine change in reptile abun-
dance on roads, ii) radio telemetry to examine reptile movements around roads, iii) a willing-
ness to utilize (WTU) experiment to assess likelihood of ecopassage use, and iv) monitoring
ecopassages using wildlife cameras and an automated passive integrated transponder (PIT-tag)
reading system to quantify reptile and predator presence. If the exclusion structures are effec-
tive at preventing reptiles from accessing the highway, we expect a significant decrease in the
abundance of reptiles on the highway post-mitigation (criterion 1; method i). Concurrently, if
Mitigating Reptile Road Mortality
PLOS ONE | DOI:10.1371/journal.pone.0120537 March 25, 2015 2/15
data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared
that no competing interests exist.

the connectivity structures are effective at promoting population and habitat connectivity, then
we expect that individuals should use the ecopassages to gain access to resources on either side
of the highway without exposing them to an increased risk of mortality from collisions with ve-
hicles or potential predators within ecopassages (criteria 2–3; methods ii-iv).
Methods
Study area and mitigation measures
Mitigation measures were constructed to reduce reptile road mortality and provide safe cross-
ing options along a newly expanded section of Highway 69/400 in central Ontario, Canada.
This major thoroughfare runs north-south and bisects the Georgian Bay coastline of Lake
Huron, one of Canada’s richest areas of reptile biodiversity [33]. In addition, the area has a
high number of reptiles designated as species at risk (SAR [34]). The highway expansion and
associated increase in traffic present long-lasting and significant threats to 6 species of turtles
(5 SAR) and 12 species of snakes (5 SAR) in the region [34].
Our study was conducted over two years at two sites located 50 km apart: (1) an Impact site
near Burwash, Ontario, Canada and (2) a Control site at Magnetawan First Nation. The survey
areas at both sites consisted of a 13-km section of Highway 69/400 with comparable reptile di-
versity [34], habitats (i.e., wetland mosaics interspersed with upland rocky outcrops and mixed
forests), and traffic volumes [35]. The Impact site was a 2-lane un-mitigated highway during
2012 and a 4-lane mitigated highway in 2013. The Control site, a 2-lane highway, remained
un-mitigated during both study years.
The mitigation measures at the Impact site consisted of an exclusion structure (reptile fenc-
ing) and three population connectivity structures (ecopassages). The reptile fencing consisted
of heavy-gauge plastic textile extending 0.8 m above- and 0.2 m below-ground with a 0.1 m
wide lip running perpendicular underground. The fence was affixed to the base of a 2.3 m tall
chain-link fence intended to exclude large mammals from the highway (Fig. 1A and 1B). Sec-
tions of reptile fencing were installed along the highway at the Impact site in areas that were
identified as important habitat and to be potential hotspots for reptile road crossings. The rep-
tile fence connected the three ecopassages (spaced 450–600 m apart), and extended beyond the
north ecopassage by 600 m and beyond the south ecopassage by 150 m. Each ecopassage con-
sists of two 3.4 m x 2.4 m x 24.1 m concrete box culverts that cross the north-, and south-
bound lanes of the highway (Fig. 1C). A fenced 15.3 m gap connects each culvert through the
median between the lanes (Fig. 1C), allowing ample light to enter the ecopassages [36].
All summary data are reported as means, followed by 1 SE. The significance level of α=0.05
was used for all statistical tests, and all statistical analyses were conducted in R (version 2.15.0, R
Development Core Team, 2012).
Effectiveness of the exclusion structures
BACI study (criterion 1). The BACI study examined the differences in abundance of rep-
tiles on the highway between the Before (2012) and After (2013) periods. Simultaneous surveys
were conducted by car at the Impact and Control sites three times daily (09:00, 18:00, 22:00)
from 1 May to 31 August each year. Daily roadside walking transects (RWT; at approximately
10:00) were conducted on foot and covered 2 km of highway. At the Impact site, the RWT was
located in an area with continuous mitigation (in the After period, 2013), while the RWT at the
Control site spanned an area with habitats similar to those at the Impact site. Over the two-
year study period, a total of 1,934 surveys were conducted. Location and mortality status were
recorded for any reptile found on the highway during both the driving and RWT surveys. To
determine if individuals were being recaptured, each live reptile captured on the highway was
Mitigating Reptile Road Mortality
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individually marked and released at a safe distance off the highway in the direction it was head-
ing. If the reptile was deceased, the individual was removed from the road to avoid being re-
counted.
Differences between reptile abundances on the highway between the Before and After peri-
ods were examined using a Poisson generalized linear model (GLM) for non-parametric count
data, including the fixed effects of period (Before, After), site (Control, Impact), and the inter-
action between these two effects. To ensure consistency in findings, differences in relative
abundances of snakes and turtles at the two sites were also compared between Before and After
periods by a paired two-sample t-test. The percentage of dead reptiles detected out of the total
reptiles recorded on the road during surveys was also calculated at the Control and Impact
sites during both the Before and After periods.
Effectiveness of the connectivity structures
Radio telemetry (criterion 2). The spatial ecologies of two SAR turtles at the Impact site
were studied using radio telemetry to examine animal movements, road crossings, and to
Fig 1. Mitigation measures completed during the fall of 2012 along Highway 69/400 in central Ontario, Canada. These measures include reptile
fencing consisting of a heavy gauge plastic geotextile extending 0.8 m above- and 0.2 m below-ground with a 0.1 m wide lip running perpendicular
underground (A). The fence was affixed to a 2.3 m tall large mammal, wire fence and was installed in areas believedto pose a risk to reptiles (B). Three
ecopassages were built within the fenced area and each consists of two 3.4 m x 2.4 m x 24.1 m concrete box culverts (C), separated by a 15.3 m gap for
increased light (D).
doi:10.1371/journal.pone.0120537.g001
Mitigating Reptile Road Mortality
PLOS ONE | DOI:10.1371/journal.pone.0120537 March 25, 2015 4/15

determine average dispersal distance (the linear distance an individual would travel between
crossing structures or to circumvent exclusion structures during a movement event, calculated
as the square root of home range size [37]). During both the 2012 and 2013 active seasons,
adult Blanding’s turtles (Emydoidea blandingii,n= 10, threatened [34]) and snapping turtles
(Chelydra serpentina,n= 12, special concern [34]) captured within 1 km of the highway were
outfitted with radio transmitters (R1920, Advanced Telemetry Systems). Individuals were
tracked every 2 to 3 days and locations were recorded using a handheld GPS unit (eTrex Vista,
Garmin).
Home range sizes (95% minimum convex polygons [38]), and the number of highway cross-
ings per individual were calculated and tallied (ArcGIS 10.0, ESRI). Home range sizes did not
differ between species (F
1,27
= 0.15, p= 0.70), sexes (F
1,27
= 0. 01, p= 0.93), or the Before and
After periods (F
1,27
= 0.01, p= 0.92) allowing the data to be pooled to create an average home
range size for all turtles. Average home range size was then used to calculate a local SAR turtle-
specific dispersal distance [37]. Individual home ranges overlapping with the highway post-
mitigation and road-crossing locations were counted for all radio-tracked turtles.
Willingness to utilize experiment (criterion 2). Between 1 May to 31 August 2013, adult
painted turtles (Chrysemys picta; males n= 24; females n= 30) were collected from a wetland
2.5 km west of the highway at the Impact site. Individuals were transported to a testing site at
the east entrance of an ecopassage. Painted turtles were used for the willingness to utilize
(WTU) experiment because of their ability to navigate using the sun [39,40], and their ability
to return to their home range [41]. During the experiment, the ecopassage was located between
the individual (east of the highway) and its home wetland (west of the highway), creating a sce-
nario in which a turtle was motivated to move in a specific direction similar to seasonal move-
ments to critical habitat (e.g., overwintering or nesting sites). Also, painted turtles were used
for behavioral trails because we could capture them in high numbers, they are frequently found
on roads [42], and because previous studies have examined their willingness to use ecopassages
in laboratory and arena settings [36,43].
Prior to the experiment, individuals were temporarily outfitted with a radio transmitter
(R1680, Advanced Telemetry Systems) and placed in an acclimation box 5 m from the entrance
of the ecopassage. The turtle was left to acclimate to the sun’s position (to provide a cue for
navigation), the substrate, and the noise and smell of the highway for 10 min. After the accli-
mation period, the box was remotely opened by a researcher situated behind a blind [20]. Tur-
tle movements were monitored from behind the blind to assess the individual’s interactions
with the ecopassage. Each individual’s behavior was ranked using a measure of crossing success
on a scale from 0–2: 0) not willing to use, walked away; 1) made no choice, remained at en-
trance; 2) willing to use, crossed into, or through, the culvert [44]. After 20 min, or if an indi-
vidual moved greater than 10 m out of the testing area, the turtle was collected and its location
was recorded. All WTU tests occurred within less than 8 h of capture. After the WTU test, the
turtle was marked to prevent reuse, and returned to the original site of capture within 12 hours.
Due to the importance of connectivity within populations for both sexes [45], and because
no difference was detected between the sexes in WTU scores (χ
22
= 0.79, p= 0.67), the data
were pooled for analyses. A Pearson chi-squared test was used to compare WTU scores from
our experiments to scores for turtles tested in an arena study (n= 190 [43]). The proportion of
turtles in our study that refused to use the ecopassage (scored 0), that made no decision regard-
ing the ecopassage (scored 1), and that were willing to use the ecopassage (scored 2) were com-
pared to similar data obtained during the first 30 min of testing conducted in the arena study
(91/190 (48%) refused, 10/190 (5%) no decision, 89/190 (47%) used ecopassages [43]).
Ecopassage monitoring (criteria 2 & 3). Wildlife cameras (TrophyMAX, Bushnell) were
installed in the entrances of the ecopassages to monitor use by reptiles and potential reptile
Mitigating Reptile Road Mortality
PLOS ONE | DOI:10.1371/journal.pone.0120537 March 25, 2015 5/15

predators [17,11]. Cameras were mounted to the ceiling of the culvert, and aimed directly at
the ground to maximize frame coverage. During the day, the motion-sensors set at the highest
sensitivity, triggered photo capture. Because slow-moving terrestrial ecotherms are difficult to
detect with infrared motion sensors at night, cameras were programmed to automatically take
a picture every minute between the hours of 1830 to 0630 [11].
Additionally, automated passive integrated transponders (PIT-tag) readers (HPR, Biomark)
with loop antennas (BIO 10 Antenna, Biomark) were installed in the central ecopassage at the
Impact site prior to the 2013 field season. The antennas spanned both entrances of the ecopas-
sage, providing information on the number of individuals entering the ecopassage, the number
successfully exiting the other side, and duration of the crossing event. The readers constantly
scanned and logged the PIT-tag number of any animal that passed through the loop antenna,
and the date and time of crossing. During the 2013 active season, adult turtles (n= 38: 6
painted turtles; 15 Blanding’s turtles; 17 snapping turtles) and snakes (n= 20: 8 eastern garters-
nakes, Thamnophis sirtalis; 12 northern watersnakes, Nerodia sipedon) found within 1 km of
the highway were captured and individually marked with a subcutaneously-injected PIT-tag
(HPT12, Biomark).
All research was conducted under approved Laurentian University Animal Care Committee
protocols (AUPs 2008–12–02 and 2013–03–01, and was authorized by Magnetawan First Na-
tion’s Chief and Council and the Ontario Ministry of Natural Resources.
Results
Effectiveness of the exclusion structures
BACI study (criterion 1). A total of 960 road surveys were conducted in 2012, and 974
were conducted in 2013. We recorded 618 snakes and 378 turtles on the highway at both sites
combined, and the percentage of dead snakes detected on the road was 83% and for turtles was
84%. In all cases, 2–9 times more animals were found dead on the road than alive, depending
on site and time period (Table 1). These levels of mortality are far higher than would be consid-
ered sustainable for many reptile species—especially snapping turtles based on their life history
[46] and known population densities at this latitude [47]. Alarmingly, the percentage of dead
reptiles detected at the Impact site increased by 20% for turtles and 25% for snakes between the
Before and After periods, while the Control site had an increase of only 2% for turtles and 11%
for snakes.
The Poisson GLM demonstrated no significant interaction between period (Before and
After) and site (Control and Impact) for turtles (z
488 =
-0.05, p= 0.57) but there was a signifi-
cant interaction for snakes (z
488 =
3.60, p<0.01); however, this interaction was due to an in-
crease in snakes recorded at the Control site rather than a decrease in snake presence at the
Impact site. The paired two-sample t-test corroborated our findings. Relative turtle abundance
Table 1. Number (proportion) of reptiles, alive (AOR) and dead (DOR), observed on the road between samples periods (Before, After) and sites
(Control, Impact).
Taxa Before After
Control Impact Control Impact
AOR DOR AOR DOR AOR DOR AOR DOR
Turtle 20 (14%) 121 (86%) 18 (32%) 39 (68%) 15 (12%) 108 (88%) 8 (14%) 49 (86%)
Snake 41 (24%) 131 (76%) 26 (32%) 55 (68%) 34 (12%) 261 (88%) 7 (10%) 63 (90%)
Total 61 152 44 94 49 369 15 112
doi:10.1371/journal.pone.0120537.t001
Mitigating Reptile Road Mortality
PLOS ONE | DOI:10.1371/journal.pone.0120537 March 25, 2015 6/15

(Control relative to Impact) on the highway did not differ between the Before and After periods
(t
121
= 0.81, p= 0.42) (Fig. 2A). In contrast, relative snake abundance on the highway differed
between the Before and After periods (t
121
= -3.78, p<0.01); however, snake abundance on
the road was not substantially reduced at the Impact site post-mitigation and the statistical dif-
ference between the Before and After periods is attributed to the increase in snake abundance
on the road at the Control site (Fig. 2B).
Effectiveness of the connectivity structures
Radio telemetry (criterion 2). The average home range size for all turtles combined was
42.5 ha (SE 12.4), and the calculated dispersal distance was 652 m. Post-mitigation, there were
11 road crossings by 3 of the turtles (2 snapping turtles, and 1 Blanding’s turtle) and 1 snapping
turtle passed through the exclusion fence but did not cross the highway. Based on telemetry
Fig 2. Daily abundance of reptiles on the highway for each survey period (Before and After) did not
differ for turtles (A), but did differ for snakes (B) when considering survey sites (Impact (●) and
Control ()). The parallelism between the solid and dashed lines visually represents no significant interaction
between site and period for turtles (A; GLM z
488 =
-0.05, p= 0.57), while this interaction was significant for
snakes (B; GML z
488 =
3.60, p<0.01). Yet, a strong reduction in snake abundance at the Impact site was still
not seen between periods, thus the interaction is due to the large increase in snake abundance observed at
the Control site during the After period.
doi:10.1371/journal.pone.0120537.g002
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locations, the successful road crossings were not likely through an ecopassage, but rather
through a drainage culvert that was incorporated into the mitigation fence.
Willingness to utilize (WTU) test (criterion 2). Most turtles did not make a decision re-
garding use of the ecopassages within the allotted time (n= 37/54; 69%). Of the individuals
that did make a decision, more than twice as many turtles refused to use the ecopassage (n=
12/54; 22%) than were willing to enter (n= 5/54; 9%). When our results were compared to
those from a previously reported arena study [43], we found that far fewer individuals were
willing to use an ecopassage below an active highway than in a testing arena (χ
22
= 863.52, p<
0.001).
Ecopassage monitoring (criteria 2 & 3). A total of 485 individual animals were photo-
graphed in the ecopassages, consisting of at least 23 non-reptile and 3 reptile species (S1 Table).
Ducks and geese (Family Anatidae) were present in 40.2% of photographs and were the most
common taxa recorded using the ecopassages. In contrast, reptiles were one of the least photo-
graphed taxa (in 2.0% of photos). Painted turtles were photographed in the ecopassages on 6 oc-
casions (4 adults and 2 hatchlings; 1.2% of photos). An adult snapping turtle (0.2% of photos),
and 3 northern watersnakes were also photographed in the ecopassages (0.6% of photos). Addi-
tionally, during regular camera maintenance, snapping turtle tracks were observed that were not
associated with a photograph, and a live juvenile red-bellied snake (Storeria occipitomaculata)
was also observed within an ecopassage. A number of potential reptile predators were also seen
using the ecopassages: Ardea herodias (8.9% of photos), Procyon lotor (8.2%), Neovison vison
(2.3%) and Canis latrans (1.7%).
Although frequent tests of the automated PIT-tag reader occurred, only two PIT-tagged ani-
mals were recorded in the ecopassage: a watersnake and a painted turtle. In both cases, the
readers did not detect a complete crossing (i.e., PIT-tags were not logged at both entrances). In-
stead, it appears that the individuals either retreated from the entrance of the ecopassage after
approaching the reader, or circumvented the exclusion fencing within the highway median be-
tween the ecopassages.
Discussion
To determine the effectiveness of exclusion and connectivity structures, we examined whether
the mitigation resulted in a reduction in abundance of reptiles on roads (criterion 1), while
maintaining habitat connectivity and dispersal routes (criterion 2) without forming a prey-trap
(criterion 3). By taking a broad, multispecies approach we were able to examine effectiveness of
the mitigation to determine if it had the desired wide-ranging ecological impact (i.e., across
multiple species and demographic groups). We found that the success of the connectivity struc-
tures was reliant upon the success of the exclusion structures, and that the entire mitigation
system was compromised at our site because of failures in the materials, implementation, and
design of the exclusion structures. Our findings demonstrate the importance of designing ro-
bust, biologically-relevant exclusion structures to mitigate road mortality of a variety of small-
bodied terrestrial and semi-aquatic animal taxa.
Lack of reduction in abundance of reptiles on the road (criterion 1)
The first criterion of our evaluation was not satisfied because the reptile fence did not prevent
turtles from gaining access to the road surface, and the percentage of dead turtles detected on
the road increased by 20% during the post-mitigation period. In contrast, surveys post-mitiga-
tion did record a difference in relative abundance of snakes on the road; however, this differ-
ence was not due to a decrease in the abundance of snakes on the highway at the Impact site,
but rather due to the prevention of a parallel proportional increase in snake abundance as
Mitigating Reptile Road Mortality
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observed at the Control site in 2013. Furthermore, there was a 25% increase in percentage of
dead snakes detected on the road at the Impact site in the post-mitigation period, indicating
that although the fence may have prevented a proportional increase in abundance on the road,
as seen at the Control site, many more individuals were killed on the highway post-mitigation.
The increase in the percentage of dead turtles and snakes detected on the road post-mitiga-
tion may be attributed to a corralling effect of the exclusion fence on individuals who gained
access to the highway via fence-gaps. Reptiles may then be forced to spend an increased
amount of time on, or adjacent, to the highway in search of a gap in the fence through which to
pass on the other side of the road [48]. Another potential cause of the increase in the percent-
age of dead reptiles on the road may have been the increase in road surface area from 2-lanes
pre-mitigation to 4-lanes post-mitigation. Although increases in road width may increase rep-
tile exposure to traffic, one of the goals of mitigation is to reduce road mortality and if an over-
all reduction is not seen, then mitigation cannot be deemed effective regardless of the changes
in road width. In fact, in many cases, highway improvement projects involve the widening of
highways, thus our results are representative of what wildlife is truly experiencing on the land-
scape. Considering that there was no relative reduction in turtle abundance, and an increase in
both snake and turtle mortality were observed, we conclude that a non-continuous, flexible-
plastic fence is not capable of reducing reptile abundance on a highway. The inability of the
fencing tested in our study should not be generalized to all exclusion structures; several studies
have documented high success rates with various styles of exclusion structures [17,18]. This
begs the question, why was the fence in our study not effective?
Close examination of the fence revealed a suite of issues that rendered this style of exclusion
structure incapable of preventing reptiles from gaining access to the highway, including failures
in both the material and installation. One of the main issues was over 115 gaps located along
the 3 km of sectionally-fenced highway resulting from rips, holes, and washouts. Furthermore,
during the spring melt, up to 30% of the fence was semi-submerged, which allowed reptiles to
easily swim or climb over the fence. Between areas that were deliberately left unfenced and the
many unintentional gaps, approximately 2/3 of the sectionally-fenced area was permeable. The
distance between locations of reptiles found on the highway in the fenced area (n= 91) to the
closest known gap in the fence averaged only 38.3 m (SE 4.2). This is a relatively small distance
for reptiles to cover given their spatial ecologies [25,49], clearly indicating that animals were
easily gaining access to the road through the gaps.
The need to increase the effectiveness of exclusion structures is evident, particularly in light
of both the high levels of road mortality documented and the permeability of the type of fence
used in our study. We suggest the use of more durable materials in the design of exclusion
structures. Flexible-plastic fencing is prone to rips and tears, quickly degrades over the short-
term, and requires regular maintenance [18]. Also, both plastic and metal mesh fences are easi-
ly climbed by many reptile species [18,50]. High water levels and drainage must be taken into
consideration so that the threats of washouts and flooding are minimized, as exposure to water
will degrade or destroy exclusion structures. Roads are built to be long-lasting structures, and
mitigation measures should be equally long-lasting. An alternative exclusion structure to fenc-
ing would be a concrete or steel gravity wall fitted into the sloped gravel between the shoulder
and ditch, which would provide a solid long-lasting barrier known to be effective [17]. Al-
though incurring a higher initial cost, this more durable exclusion structure may be far more
cost-effective compared to intense maintenance (e.g., annual maintenance costs can equal in-
stallation cost of geotextile fencing [51]) that is necessary to maintain this type of fencing over
the long term [18]. Furthermore, as previous evaluation indicates [17], concrete or steel gravity
walls are more biologically effective (i.e., they successfully reduce road mortality of animals),
which is a crucial consideration in order to effectively protect imperiled, ecologically-important
Mitigating Reptile Road Mortality
PLOS ONE | DOI:10.1371/journal.pone.0120537 March 25, 2015 9/15

species. When planning mitigation measures it is also important to take a pragmatic approach,
such as using a cost-benefit analysis [52] including consideration of factors such as: initial cost,
maintenance costs, human-wildlife collision costs, effectiveness, benefits to target species, over-
all ecological benefit, human benefit, and the lifespan of the structure. The relative weighing of
the factors used to determine what style of mitigation is warranted and effective for ungulates
can be distinctly different than those for reptiles, particularly because mitigation measures for
ungulates are often implemented to off-set the costs of human-wildlife collisions [52], rather
than as a conservation tool. When the goal of mitigation measures is conservation, then long-
lasting exclusion structures are required to ensure that conservation efforts are successful at re-
ducing the abundance of wildlife on roadways to a level that is biologically significant for
population viability.
Maintenance of habitat connectivity and dispersal routes (criterion 2)
The ecopassages demonstrated low use, and likely would have had more crossings if the exclu-
sion fencing was functioning properly. The wildlife cameras and haphazard encounters showed
that 4 reptile species used ecopassages. However, the number of observations documented
within the ecopassages (n= 12) was much lower than the number of individuals found on the
highway during the same timeframe (n= 127). Additionally, data from the two automated
PIT-tag readers detected only 2 of the 54 individual reptiles implanted with PIT-tags [42]. This
further indicates that although reptiles were observed using the ecopassages to facilitate move-
ments between or within habitats, the number of reptiles using the ecopassage to cross was
only 9% of the number using the road surface to cross.
Our two-year radio telemetry study of Blanding’s and snapping turtles at the Impact site es-
tablished that home range sizes did not change in response to the changes in the highway and
that 27% of the radio-tagged individuals had home ranges that overlapped with the highway
(n= 6/22). These individuals had critical habitats (i.e., nesting and overwintering sites) and sea-
sonal habitats (i.e., basking and foraging sites) on both sides of the highway. The locations of
road crossings, used to move between critical and seasonal habitats, were all within the calcu-
lated dispersal distance of the turtles in our study (652 m) to an ecopassage, suggesting that al-
though the turtle had access to ecopassages, they choose not to use them. In fact, alternative
methods of crossing were used that involved circumventing the fence (n= 1/4 turtles), or cross-
ing via a drainage culvert (1.0 m diameter; n= 3/4 turtles) incorporated into the fence. The use
of the drainage culvert is believed to be a result of its placement in an open channel in the cen-
ter of the wetland, an observed movement corridor, rather than a preference by individuals to-
wards small, flooded, poorly-lit drainage culverts. The use of the drainage culvert rather than
the large well-lit ecopassages illustrates the importance of carefully selecting ecopassage loca-
tions. If ecopassages are constructed for only a limited number of species, then specific habitats
can be targeted; however, if the goal is to mitigate for a wide variety of species, or if habitat se-
lection by a target species varies due to sex and age-class, then diversifying ecopassage loca-
tions (e.g., movement corridors in wet and dry, lowland and upland locations) may assist in
maximizing likelihood of use. Overall, the turtles did not use the ecopassages as travel routes,
which together with the low use by reptiles in general, translates to a failure in this criterion of
effectiveness.
The WTU test provided valuable insight regarding the lack of ecopassage use observed dur-
ing our study as turtles were twice as likely to refuse using an ecopassage as they were to use it.
This was significantly different from results obtained in an arena study [43] and we suggest
that such differences may be because we tested ecopassage use beneath a live highway with the
sights, sounds, and smells of traffic. Our findings thus provide a more realistic understanding
Mitigating Reptile Road Mortality
PLOS ONE | DOI:10.1371/journal.pone.0120537 March 25, 2015 10 / 15

of a turtle’s response to an ecopassage. Although the previously reported arena study also tested
variable culvert apertures [43], all of which were smaller than that in our study, the fact that
turtles were less likely to use a large, well lit ecopassage with ample natural substrate was a sur-
prising result. The relatively higher usage observed in arena studies may have been driven by
the experimental design in which the turtle was only provided a single option—moving
through the ecopassage (simulating a crossing) with no opportunity to escape [36,43]. If con-
servation biologists are to achieve rates of ecopassage usage as high as those reported for arena
studies, the associated exclusion structure needs to remove any other crossing option (e.g.,
crossing over, under, through gaps in the fence, or circumnavigation around the fence). To
achieve this goal, exclusion structures guiding individuals toward ecopassages should remain
continuous beyond the target species’dispersal distance [37], rather than merely the length of
suitable habitat (e.g., the distance along which suitable habitat abuts a road). Without other
crossing options, wildlife should be far more likely to use an ecopassage. In our study, as a re-
sult of the numerous failures in the exclusion structure at the Impact site, turtles that refused to
use the ecopassage were provided with a multitude of other crossing options. We urge future
studies to examine how other reptile species, particularly squamates, and small to medium-
sized mammals, respond to ecopassages as these areas of research are sorely lacking.
Due to the high cost associated with the installation of ecopassages, it is important that their
likelihood of use is ensured. Through better understanding of an animal’s willingness to use
ecopassages and species-specific dispersal distances between and around ecopassages, conser-
vation biologists and wildlife managers can more effectively design mitigation measures that
will work properly, in turn optimizing the structure’s ecological value and offsetting its initial
monetary costs.
Prevention of prey-trap formation (criterion 3)
A common concern regarding crossing structures is their potential to be prey-traps [32]. We
observed no predation of reptiles within the ecopassages. Yet, of the observed wildlife in the
ecopassages (n= 485 observations), 22.3% were known reptile predators [25,49]. In contrast,
reptiles only accounted for 2.8% of observed wildlife within the ecopassages. Thus, at the cur-
rent level of reptile use, the ecopassages in our study would be highly ineffective for a predator
to use as a hunting location. Most likely, predator presence in the ecopassage is simply related
to road crossings. Similar findings refuting the prey-trap hypothesis have been noted for both
small and large mammals [32,53]. However, if reptile abundance was to be increased within
the ecopassages, then further examination of the potential for prey-trap formation would
be required.
Conclusion
The use of road mortality mitigation measures is crucial for the conservation of biodiversity
[1,7,8]. However, the specific mitigation measures we tested were deemed ineffective because
of their inability to reduce the abundance of reptiles on the road, and the limited use of the eco-
passages. Our study further demonstrates that conservation decisions need to be supported by
solid science, and it is critical that highway designers and wildlife managers rigorously and
thoroughly test the effectiveness of mitigation measures [7,29]. Our study is important because
it examined the effectiveness of the mitigation measures currently being implemented into
road projects, and did so with a taxa-wide approach. Unlike previous studies that focused on
key species and specific aspects of demographic differences in crossing rates and locations, our
study takes the approach that if mitigation measures are to be deemed effective, they should be
able to reduce overall percentage of dead reptiles on the road, maintain connectivity, and
Mitigating Reptile Road Mortality
PLOS ONE | DOI:10.1371/journal.pone.0120537 March 25, 2015 11 / 15

prevent prey-trap formation regardless of changes in road width, speed limits, and inter-popu-
lation variability in crossing behavior. If a road is to be considered mitigated for a group, or bet-
ter yet, multiple groups of species, then the mitigation measures used should result in an
overall functional level of protection for the complement of populations, including all
demographic subgroups.
As global biodiversity decreases [54] and threats to animal populations are identified and
become more wide-spread, we must strive to increase our level of protection for rare and im-
periled species beyond current norms. Furthermore, mitigation methods should also be de-
signed to offer protection to common species. Mitigation is more than a political mandate
satisfying a piece of legislation, it is a means to create infrastructure that directly reduces the
negative impacts of development on wildlife [17]. Mitigation measures should be designed to
last over the long-term (i.e., the lifespan of the road). Materials used for exclusion structures
need to be enduring (e.g., concrete gravity walls, solid steel barriers), and should be incorporat-
ed into highway engineering and provided the same level of scrutiny given to road construc-
tion. The effectiveness of population connectivity structures relies on the effectiveness of
exclusion structures. Although we observed some use of ecopassages, it occurred at low rates,
particularly because other, albeit more risky, crossing options existed. Ecopassages are effective
and worth their monetary cost when the associated exclusion structures are working. If care is
taken to properly assess the effectiveness of mitigation, and steps are taken to adaptively man-
age the negative impacts of roads, we can further our efforts to stem the tide of species loss, and
better conserve and protect the natural world.
Supporting Information
S1 Table. Animals observed crossing through ecopassages under the highway. Vertebrate
fauna passing through three ecopassages under Highway 69, Burwash, Ontario, Canada, were
recorded on wildlife cameras from 1 May to 31 August 2013. Crossings are indicated by num-
ber of photos taken and percentage of total photos taken.
(DOCX)
Acknowledgments
We thank J. Baxter-Gilbert, S. Boyle, G. Hughes, R. Maleau, K. Tabobondung, D. Jones, L.
Monck-Whipp, and C. Neufeld for assistance in the field, and the community of Magnetawan
First Nation for their support. We would also like to thank the three reviewers for their insights
and suggestions.
Author Contributions
Conceived and designed the experiments: JBG JLR DL JDL. Performed the experiments: JBG
JLR. Analyzed the data: JBG JLR. Contributed reagents/materials/analysis tools: DL JDL.
Wrote the paper: JBG JLR DL JDL.
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