Fortune favors the bold toad: urban-derived behavioral traits may provide advantages for invasive amphibian populations

Abstract

Many biological invasions occur within and between urban areas. If native species adapted to anthropogenically altered habitats are subsequently moved from an urban area in their native range to one within a novel region, then their urban-specialized phenotypes may provide them an advantage via prior adaptation. Here we examine if urban-derived behavioral traits are present within native guttural toad, Sclerophrys gutturalis, populations (Durban, South Africa) and investigate whether these localized phenotypes persisted within their invasive populations in Mauritius and Réunion. In our study, we measured boldness and exploration in populations along the toad’s invasion route and found that toads were significantly bolder in urban populations, within both native and invasive ranges. This suggests boldness increased when toads transitioned to urban living in their native range and these heightened levels of boldness were maintained within invaded urban areas. This provides evidence that a bolder phenotype was a prior adaptation that likely increased guttural toad’s invasion success. Interestingly, toad boldness returned to pre-urbanization levels within invasive populations that spread into natural areas, replicated on both islands. Exploration, on the other hand, was not increased above pre-urbanization, or pre-invasion, levels for any of the populations, and was lower in toads from Mauritius. Overall, our findings suggest that increased boldness is favored in urban habitats and that urban-derived behavioral traits may provide individuals an advantage when invading new urban landscapes.

Significance statement
Species adapting to anthropogenic landscapes have the ability to increase their invasive potential if the altered phenotypes they accrue can provide them advantages once they are transported outside their native range. Our study examined differences in behavioral traits, boldness, and exploration, along the invasion route of guttural toads, Sclerophrys gutturalis, between natural and urban sites from their native origin populations around Durban, South Africa, to their invasive populations in Mauritius and Réunion. We determined that populations were bolder in urban areas in their native range and that this increased boldness persisted in the other anthropogenic habitats within their invasive ranges, but reverted back to natural-native levels within populations that had spread into natural areas on both islands. Our findings support the growing trend that anthropogenically altered landscapes favor bolder individuals, as well as the assertion that urban-derived traits may bolster a species’ ability to establish and spread within novel landscapes.

Full text

Vol.:(0123456789)
1 3
Behavioral Ecology and Sociobiology (2021) 75:130
https://doi.org/10.1007/s00265-021-03061-w
ORIGINAL ARTICLE
Fortune favors thebold toad: urban‑derived behavioral traits may
provide advantages forinvasive amphibian populations
JamesBaxter‑Gilbert1 · JuliaL.Riley2,3· JohnMeasey1
Received: 30 March 2021 / Revised: 15 July 2021 / Accepted: 20 July 2021
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2021
Abstract
Many biological invasions occur within and between urban areas. If native species adapted to anthropogenically altered
habitats are subsequently moved from an urban area in their native range to one within a novel region, then their urban-
specialized phenotypes may provide them an advantage via prior adaptation. Here we examine if urban-derived behavioral
traits are present within native guttural toad, Sclerophrys gutturalis, populations (Durban, South Africa) and investigate
whether these localized phenotypes persisted within their invasive populations in Mauritius and Réunion. In our study, we
measured boldness and exploration in populations along the toad’s invasion route and found that toads were significantly
bolder in urban populations, within both native and invasive ranges. This suggests boldness increased when toads transitioned
to urban living in their native range and these heightened levels of boldness were maintained within invaded urban areas.
This provides evidence that a bolder phenotype was a prior adaptation that likely increased guttural toad’s invasion success.
Interestingly, toad boldness returned to pre-urbanization levels within invasive populations that spread into natural areas,
replicated on both islands. Exploration, on the other hand, was not increased above pre-urbanization, or pre-invasion, levels
for any of the populations, and was lower in toads from Mauritius. Overall, our findings suggest that increased boldness is
favored in urban habitats and that urban-derived behavioral traits may provide individuals an advantage when invading new
urban landscapes.
Signicance statement
Species adapting to anthropogenic landscapes have the ability to increase their invasive potential if the altered phenotypes
they accrue can provide them advantages once they are transported outside their native range. Our study examined differences
in behavioral traits, boldness, and exploration, along the invasion route of guttural toads, Sclerophrys gutturalis, between
natural and urban sites from their native origin populations around Durban, South Africa, to their invasive populations
in Mauritius and Réunion. We determined that populations were bolder in urban areas in their native range and that this
increased boldness persisted in the other anthropogenic habitats within their invasive ranges, but reverted back to natural-
native levels within populations that had spread into natural areas on both islands. Our findings support the growing trend
that anthropogenically altered landscapes favor bolder individuals, as well as the assertion that urban-derived traits may
bolster a species’ ability to establish and spread within novel landscapes.
Keywords AIAI hypothesis· Amphibian· Boldness· Exploration· Invasion biology· Urban ecology
Introduction
The modern era presents a host of human-related challenges
to the world’s ecosystems, with two of the most pervasive
threats stemming from the growing urban footprint and the
increasing spread of invasive species (Corlett 2015; Pelletier
and Coltman 2018; Pyšek etal. 2020). Due to the nature
of how invasive species are relocated outside their native
ranges (e.g., transportation networks, pet trade, or as a
James Baxter-Gilbert and Julia L. Riley have joint first authorship.
This article is a contribution to the Topical Collection Using
behavioral ecology to explore adaptive responses to anthropogenic
change – Guest Editors: Jan Lindström, Constantino Macias
Garcia, Caitlin Gabor
Communicated by C. R Gabor
Extended author information available on the last page of the article

Behavioral Ecology and Sociobiology (2021) 75:130
1 3
130 Page 2 of 13
biocontrol for pests), many biological invasions are innately
linked to human-dominated landscapes (Pyšek etal. 2020).
On the surface, the connection between urbanization and
biological invasion may simply reflect an increased prob-
ability (i.e., if a species lives near people, it may be more
likely to be transported, deliberately or accidentally, and
where it is moved to is more likely to be another human-
dominated landscape). Yet, there is growing evidence to
suggest that this relationship is more complex, with urban
ecosystems potentially acting as biological filters which can
promote urban-specialized phenotypes (i.e., urban evolution;
see Johnson and Munshi-South 2017) that may secondarily
increase a given species’ invasive potential (Hufbauer etal.
2012; Rey etal. 2012; González-Bernal etal. 2016; Bor-
den and Flory 2021). For example, the “anthropogenically
induced adaptation to invade” (AIAI) hypothesis posits that
species adapting to human-modified landscapes can inflate
their invasive potential by (1) increasing the likelihood of
being moved due to human proximity and (2) through the
formation of adaptive phenotypes that provide advantages
in anthropogenic habitats, which then can promote success-
ful establishment and spread after translocation (Hufbauer
etal. 2012). In part, this is due to the fact that many human-
dominated landscapes, even when geographically distinct,
share a lot of ecological and environmental characteris-
tics (e.g., the similarities between cities may be more than
between a given city and its closest natural areas). If the
AIAI hypothesis is correct, urban areas could be viewed as
“sorting grounds” for many of the world’s species, selecting
taxa flexible enough to adapt to urban landscapes and then
phenotypically increasing their invasive potential via traits
that bolster success in human and novel environments (Huf-
bauer etal. 2012). Research into how phenotypes adapted
to human landscapes can promote invasion success have
provided examples across a wide variety of taxa and bio-
logical traits, including adult plant size in weeds (Waselkov
etal. 2020), locomotory performance in lizards (Battles etal.
2019), and changes in thermal tolerance for ants and birds
(Rey etal. 2012; Jackson etal. 2015; Strubbe etal. 2015).
Behavior is an important aspect of how species overcome
challenges from novel environments and changes in animal
behavior can arise through behavioral flexibility or plasticity,
but also through fixed traits that are heritable and subject to
natural selection (Plotkin 1988; Slater and Halliday 1994;
Lapiedra etal. 2017; Thompson etal. 2018). Differences
in behavioral traits between origin and colonizing popula-
tions have been well documented in both urban ecology
(Lowry etal. 2013) and invasion biology (Hudina etal.
2014), and there are several convergent behavioral traits
associated with successful establishment and persistence of
populations within novel habitats. For example, an increase
in boldness (i.e., an individual’s propensity to take risks)
is favored in some urbanized songbirds (Evans etal. 2010;
Holtmann etal. 2017) and lizards (Pellitteri-Rosa etal. 2017;
Baxter-Gilbert etal. 2019), and so too promotes invasion
success within populations of crayfish (Pintor etal. 2008),
fish (Rehage and Sih 2004; Myles-Gonzalez etal. 2015),
lizards (Short and Petren 2008; Damas-Moreira etal. 2019),
and rodents (Malange etal. 2016). Other examples of shifts
in specific behavioral traits, like increased activity level,
aggression, exploration, and neophilia, have been similarly
observed in species living in either urban (Evans etal. 2010;
Kralj-Fišer and Schneider 2012; Thompson etal. 2018) or
invasive populations (Rehage and Sih 2004; Myles-Gonzalez
etal. 2015; Damas-Moreira etal. 2019). Of course, these
changes in behavior do not always consistently trend in the
same direction, with examples of decreased levels of bold-
ness (Putman etal. 2020) and increased neophobia (Miranda
etal. 2013) occurring in some urban populations, as well as
instances where no differences are shown for certain pheno-
types (e.g., increased boldness in urban populations, but no
differences in exploration and neophilia between urban and
rural populations; Baxter-Gilbert etal. 2019). Given that the
colonization of novel habitats can drive behavioral change
(Lowry etal. 2013; Hudina etal. 2014; Lapiedra etal. 2017),
including the formation of innate and potentially heritable
traits (Holtmann etal. 2017; Baxter-Gilbert etal. 2019) or
increased levels of behavioral flexibility (Dammhahn etal.
2020), it stands to reason that native urban populations may
promote phenotypes that could benefit individuals invad-
ing novel landscapes (i.e., prior adaptation; Hufbauer etal.
2012). This could effectively prime urbanized native taxa to
become better invaders (Borden and Flory 2021).
To test the AIAI hypothesis, we compare measures of
boldness and exploration between populations of guttural
toad, Sclerophrys gutturalis, across an urban-natural/native-
invasive gradient, following their invasion route (i.e., the
geographic pathway propagules traveled between the source
and invading populations; Estoup and Guillemaud 2010)
from their native range in Durban, South Africa, to their
invasive populations in Mauritius and Réunion (Telford
etal. 2019). Previous research from these three locations
has shown that the invasive island toad populations on both
islands have reduced body sizes and disproportionately
shorter hind limb lengths (Baxter-Gilbert etal. 2020), sug-
gesting selective pressure from these colonization events and
unique landscapes have driven physical phenotypic changes.
Given the fact that these invasive populations are known
to have undergone phenotypic divergence, as well as the
potential for urban environments to be a driver, behavioral
alterations may have also occurred and berelated to prior
adaptation through urban filters. As such, we studied toads
from natural and urban populations in Durban, Mauritius,
and Réunion to determine whether they (1) express differ-
ent levels of boldness and exploration between natural or
urban habitats within their native range, (2) maintained or

Behavioral Ecology and Sociobiology (2021) 75:130
1 3
Page 3 of 13 130
increased these phenotypic differences within the urban,
invasive habitats where they were first established, and (3)
maintained or increased these phenotypic differences once
they spread into natural habitats within the invasive range.
If urban environments are selecting for bolder and more
explorative toads, which in turn may also improve a toad’s
invasive potential (e.g., Damas-Moreira etal. 2019), then we
expect similar, higher levels of boldness and exploration to
be expressed by invasive toad populations within the urban
habitats in which they were introduced, which may also be
carried further into natural areas in their invaded ranges.
Methods
Study species andsites
Guttural toads, Sclerophrys gutturalis, are a large generalist
bufonid (maximum snout-vent length (SVL) = 140mm; du
Preez etal. 2004) with female-biased sexual size dimor-
phism (Baxter-Gilbert etal. 2020) and a broad distribution in
sub-Saharan Africa spanning from Angola in Central Africa
to Kenya in East Africa and ranging south to eastern South
Africa (see Telford etal. 2019). These toads are known
for their proclivity for thriving in human-disturbed habi-
tats (Vimercati etal. 2019) and are frequently-encountered
urban residents. For almost a century, these toads have had
invasive populations in Mauritius and Réunion, both a result
of failed biocontrol attempts (Cheke and Hume 2010), and
these alien populations express locally-specific reductions
in adult body size (i.e., insular dwarfism; Baxter-Gilbert
etal. 2020). Molecular research has confirmed that these
invasions came from the same native source population that
originated near Durban, South Africa (Telford etal. 2019).
The molecular research also supports the historic accounts
(Cheke and Hume 2010; Telford etal. 2019), which when
combined with what we know about their anthropophilic
behavior (Vimercati etal. 2019; JB-G pers obs.) suggests
the invasion route for the guttural toads likely consisted of
(1) pre-urbanization, toads existed in natural habitats in their
native range; (2) post-urbanization, toads in the immediate
vicinity of Durban started to become urbanized from 1850
onward, (3) establishment in Mauritius, in 1922 toads were
collected from the Durban area, likely from around human-
disturbed habitats, and shipped to a Mr. Regnard who was
the dock manager in Port Louis, Mauritius (Cheke and Hume
2010), who released them around human settlements and
agricultural areas to control pest insects; (4) establishment
in Réunion, in 1927, toads were collected in Mauritius, likely
from around human settlements, and shipped to a Mr. de
Villèlle (Cheke and Hume 2010), whose family estate was in
Saint-Gilles-les-Hauts, Réunion, where they were released to
control pest insects; and (5) invasive spread, on both islands
the toads numbers grew, populations spread, and the toads
invaded from human settlements and disturbed areas to natu-
ral ecosystems on both islands.
To select our sampling sites, we examined a 1 km2 area
around each prospective sampling site on Google Earth®
and used the polygon function to measure the percentage
of land cover represented by a human footprint (e.g., hard-
scape, infrastructure, impervious surfaces, and/or residential
areas). For the purposes of our study, natural areas needed
to have ≤ 1% human footprint and urban areas as those with
a human footprint of ≥ 50%, which follows similar frame-
works used by McKinney (2008), Larson etal. (2020), and
Bókony etal. (2021). Our natural-native site was a reclaimed
grassland located 110km north of Durban on a private prop-
erty which consisted of open grasslands, forest patches, and
a wetland, with the overall land covered being comprised
of ≤ 1% human footprint (Fig.1A). The urban-native site
(i.e., the Durban Botanical Gardens; established in 1849)
was within the city of Durban and was a heavily modified
greenspace which has undergone human development and
urban envelopment over the last 170years and is represented
by 81% human footprint (Fig.1B). Our sampling in the inva-
sive ranges, Mauritius and Réunion, targeted urban locations
close to where we predicted the toads were originally intro-
duced and the natural sites in these locations represent native
greenspaces which the toads have spread into. Ecologically,
both islands are similar in size, 2040 km2 (Mauritius) and
2512 km2 (Réunion) with tropical climates, and are both
considered biodiversity hotspots (Myers etal. 2000; Telford
etal. 2019). The urban-invasive site in Mauritius was located
in the village of Norte Dame, 10km from Port Louis, and
the habitat consisted of backyards, roadsides, ditches with
streams, and refuse piles, with 61% of the area being com-
prised of human footprint (Fig.1D). Our natural-invasive
site on Mauritius was in the Brise Fer forest of Black River
Gorges National Park (Fig.1C), located 40km south of Port
Louis, which is part of the last 4.4% of remaining natural
forest on the island (Hammond etal., 2015) and had < 0.1%
human footprint. Within Réunion, our urban-invasive site was
in the village of Villèlle, 2km from Saint-Gilles-les-Hauts, and
the habitat consisted of backyards, roadsides, refuse piles, and
a golf course with 58% of land cover representing human foot-
print (Fig.1F). Our Réunion natural-invasive site was located
in a natural greenspace consisting of treed and grassland habitat
outside the village of Point Payet (Fig.1E) which had < 0.1% of
human footprint and was 41km east of Saint-Gilles-les-Hauts
and adjacent to the large, protected area of Grand Étang.
Data collection
Adult toads were hand-caught during opportunistic walk-
ing surveys during time periods where toads were locally
active at each study site (Durban: February to March 2020;

Behavioral Ecology and Sociobiology (2021) 75:130
1 3
130 Page 4 of 13
Mauritius: June to July 2019; and Réunion: July 2019).
Adult size thresholds were locally specific (39mm Mau-
ritius, 36mm Reunion, and 57mm Durban; for details
see Baxter-Gilbert etal. 2020). Once captured, toads were
brought to temporary field stations at each of the loca-
tions. We housed the toads in two experimental groups
(“A” and “B”) of 16 toads each (for a total of 32 toads,
with a 50:50 sex ratio, per site), in large circular (1.83-m
diameter) plastic containers (i.e., collapsible children’s
swimming pools) outfitted with wetted sand, rocks, and
dried leaves, allowing the toads to seek shelter, encourage
normal burrowing behavior, and regulate their hydric con-
ditions. Each toad was individually marked with a unique
passive integrative transponder (PIT tag) and was given a
minimum of 24h post-capture to acclimate prior to behav-
ioral testing.
Fig. 1 Landscape images
(approximately 1 km2) show-
ing the differences in human
footprint (e.g., infrastructure,
impervious surfaces, and resi-
dential areas) between natural
and urban sites from South
Africa (native range; A natural
site with 1% footprint; B urban
site with 81% footprint), Mau-
ritius (invasive range, C natural
site with < 0.1% footprint; D
urban sites with 61% footprint),
and Réunion (invasive range,
E natural site with < 0.1%
footprint; F urban site with 58%
footprint). Images generated and
human footprint area measured
using Google Earth®

Behavioral Ecology and Sociobiology (2021) 75:130
1 3
Page 5 of 13 130
All behavioral assays occurred between 1800 and 2300h,
aligning with our observations of wild activity periods, and
room temperature (°C) was recorded for each behavioral
assay. Behavioral assays were remotely recorded using a
four-camera CCTV setup (SA Lucky ABC, IR Color CCD
Camera, model: ABC-5504H-4) with recordings being
stored in an internal DVR unit. This allowed the assays to
occur without humans being present within the room and
allowed for four toads to be independently assayed at the
same time. For this reason, recording of the behavioral
assays was done in batches (one to five in total) of four toads
each on any given night, allowing for 16 toads to be tested
per night. Additionally, the assays were conducted within
a dark room, and researchers only used dimmed red lights
within the experimental room during assay setup (detailed
below) to minimize the impact artificial white light may have
on the toad’s behavior. Furthermore, one researcher (JLR)
was responsible for carrying out the behavioral assays to
ensure consistency of the methods throughout our study. At
each of the six sites, the toads’ explorative behavior was
assayed first—taking two nights to complete, with one of
the experimental groups (i.e., group “A” or “B”) being
tested each day—followed by boldness for the following
two nights. Each toad was assayed once per behavioral trait.
Exploration assay
To begin the exploration assay, we first placed a toad under
a 114mm (D) × 81mm (H) circular acclimation chamber
within a 0.40m (L) × 0.40m (W) × 0.40m (H) arena lined
with a grid paper base. The arenas were also outfitted with
four hides (identical to the acclimation chamber) each with
one opening, to break up the blank space and allow for nor-
mal explorative behavior to occur, such as investigating
potential refuges. After 5min, the acclimation chamber was
removed, which exposed the toad to the novel environment
and the individual was left to explore for 30min. From the
video recordings, we scored the total area explored (cm2;
continuous variable) by the toad, which was calculated by
counting the number of grid squares the animal crossed
during the 30-min period including the spaces occupied
by hides. Video scoring of both assays was limited to one
researcher (JB-G) to avoid any inter-observer bias within this
study and was done so using individual ID numbers so that
the researcher was blind. We used this metric (i.e., total area
explored) as our measure of exploration (i.e., a quantitative
measure of an individual toad’s propensity to investigate its
novel surroundings).
Boldness assay
To begin our boldness assay, we exposed individuals to a
standardized “mock predation” event, wherein the toad was
flipped onto its back and allowed to right itself five consecu-
tive times within the palm of the researcher (JLR). After this,
the now “frightened” toad was placed within a single hide
facing away from the opening that was located in the center
of the same testing arena that was used in the “Exploration
assay” section. The same individual grid paper that was used
in the “Exploration assay” section lined the arena during
this assay so that the individual had familiar smells within
the testing environment. After the toad was placed in the
hide, it was filmed for 30min. From the video recordings,
we scored whether the toad exited the hide (binary vari-
able) and the time (i.e., latency) it took the toad to exit the
hide (s; continuous variable). We used these metrics as our
measures of boldness (i.e., a quantitative measure of how
quickly an individual is willing to leave the safety of a hide
after encountering a frightening situation).
Statistical analyses
All statistical tests were conducted in R version 4.0.4 (R
Core Team 2021). Before starting analyses, we explored our
data following a similar protocol as outlined in Zuur etal.
(2010). We did not find any unexplainable outliers. There
was a significant correlation between our study sites (Dur-
ban, Mauritius, and Réunion) and the room temperature doc-
umented during the behavioral assays (tested using a one-
way ANOVA, using the “lm” and “anova” function in the
R “stats” package; R Core Team 2021, for the exploration
assay: F2, 185 = 1305.30, p < 0.01; and the boldness assay:
F2, 184 = 357.99, p < 0.01). For the exploration and boldness
assays, the average room temperature varied by 6.9°C and
3.9°C between study sites, respectively (exploration assay:
Durban 30.5°C ± 0.1 SE; Mauritius 23.6°C ± 0.1 SE; Réun-
ion 21.0°C ± 0.1 SE; boldness assay: Durban 27.7°C ± 0.2
SE, Mauritius 23.9°C ± 0.1 SE, Réunion 22.0°C ± 0.1 SE).
Yet, the three response variables we selected to reflect explo-
ration and boldness were not significantly correlated to room
temperature (tested using the “lm” and “glm” function in
the R “stats” package; R Core Team 2021): the arena area
explored (cm2; β = 2.25, SE = 8.59, t1, 186 = 0.26, p = 0.79,
R2 < 0.01 as calculated using the “rsq” function from the
“rsq” R package; Zhang 2020), whether or not a toad exited
the hide (β = 0.05, SE = 0.05, z = 0.92, p = 0.36, R2 < 0.01),
and latency to exit the hide (s; β = − 40.63, SE = 20.48,
t1, 185 = − 1.98, p = 0.06, R2 < 0.02). It is important to note
that these localized temperatures reflect each popula-
tions’ regional norms. Thus, we opted not to include room
temperature in our models below, because study site and
room temperature were confounded, and room temperature
appears to have a minimal effect on these behavioral traits.
Furthermore, we did not include morphological traits in our
analyses because previous work has found these to be related
tothe study site (Baxter-Gilbert etal. 2020). For all models,

Behavioral Ecology and Sociobiology (2021) 75:130
1 3
130 Page 6 of 13
prior to interpretation, we verified the assumptions of nor-
mality and homoscedasticity of residuals. Data are presented
as predicted means ± standard error (SE) in the text, unless
otherwise specified, and α was set at 0.05 for all models.
Exploration assay
We measured the exploration of 188 adult guttural toads (93
females and 95 males). Toads were located within natural
sites in Durban, Mauritius, and Réunion (16 females and
males per study site), as well as urban sites in each of these
locations (Durban: 15 females and 16 males; Mauritius: 14
females and 15 males; Réunion: 16 males and 16 females).
Sample sizes vary slightly between behavioral assays,
because different numbers of video recordings were cor-
rupted between them (see below).
We used a linear mixed-effect model (LMM) to examine
differences in the area of the arena (cm2) explored by the
toad during the 30-min assays using the function “lmer”
in the R package “lmerTest” (Kuznetsova etal. 2017). The
LMM included the fixed effects of the study site (categori-
cal with three levels: Durban, Mauritius, or Réunion), site
type (categorical with two levels: natural or urban), and an
interaction effect between these two factors to statistically
test for the AIAI hypothesis. Additionally, the LMM also
included the fixed effect of toad sex (categorical with two
levels: female or male), as well as the random intercepts
of experimental group and within-day batch to control for
dependency among experimental groupings that occurred as
an artifact of our sampling design.
After running the LMM and verifying its assumptions, we
examined the significance of the interaction effect between
study site and type using a post hoc test for multiple com-
parisons. This was run using the function “emmeans” from
the “emmeans” R package, and the p-values generated for
these comparisons were corrected using an “mvt” adjust-
ment that uses a Monte Carlo method to produce “exact”
Tukey corrections (Lenth 2020). If the interaction was not
significant, then it was removed from the model and the
model was re-run in order to allow interpretation of the
main effects. In those cases, post hoc multiple comparisons
between all study sites were tested using the “emmeans” R
package using the same protocol as described above.
Boldness assay
We measured the boldness of 187 adult guttural toads (93
females and 94 males). These toads were located in natural
(Durban and Réunion: 16 of both sexes; Mauritius: 15 of
both sexes) and urban sites (Durban and Réunion: 16 of both
sexes; Mauritius: 14 females and 15 males).
We used a binomial generalized linear mixed-effect model
(GLMM) to examine differences whether a toad exited the
hide or not (exited = 1, stayed inside = 0), during our 30-min
boldness assay using the function “glmer” in the R package
“lmerTest” (Kuznetsova etal. 2017). This GLMM included
the fixed effects of the study site (categorical with three lev-
els: Durban, Mauritius, or Réunion), site type (categorical
with two levels: natural or urban), and an interaction effect
between study site and site type to statistically test for the
AIAI hypothesis. Additionally, the GLMM also included the
fixed effect of toad sex (categorical with two levels: female
or male), as well as the random intercepts of the experimen-
tal group and within-day experimental batch to control for
dependency among experimental groupings that occurred as
an artifact of our sampling design. We examined the signifi-
cance of interaction effects and post hoc multiple compari-
sons between study sites post hoc using the same protocol
as described above in regard to the LMM that analyzed toad
exploration tendency. Further, we analyzed the latency for
a toad to exit the hide (s) during our 30-min boldness assay
using the same LMM approach as described above.
Results
Exploration assay
The amount of area (cm2) explored by guttural toads sig-
nificantly differed between study sites (Table1) with toads
in Mauritius being significantly less explorative than their
counterparts in Durban or on Réunion (Fig.2). From our
models, and accounting for additional factors, we saw that
toads from Mauritius (692.64 ± 6.24) explored an aver-
age of 212.03 cm2 less of the arena than Durban toads
(904.67 ± 6.72) and an average of 248.58 cm2 less of the
arena than toads from Réunion (941.23 ± 6.54). Guttural
toad exploration propensity was not affected by toad sex,
whether a site was in natural or urban habitats, nor an inter-
action between study site and site type (Table1).
Boldness assay
Whether or not a toad exited the hide did not significantly
differ between study sites (Tables2 and 3, Fig.3). Toads
from urban areas were 20% more likely to exit the hide than
toads from natural areas, which represents a bolder pheno-
type (Table2, Fig.3). Toads from urban areas also tookan
average of 277s less to exit the hide (i.e., latency) than toads
from natural areas (Tables3 and 4). There was no significant
interaction between the study site and site type. In addition,
male toads were significantly more likely to exit the hide
than females acrossall locations (Table2).

Behavioral Ecology and Sociobiology (2021) 75:130
1 3
Page 7 of 13 130
Discussion
Our findings provide support for the assertion that behavio-
ral phenotypes arising from selection or being more com-
monly expressed through flexibility or plasticity, in urban
populations may provide advantages for individuals coloniz-
ing anthropogenically altered habitats outside of their native
range (i.e., AIAI hypothesis; Hufbauer etal. 2012) with
respect to increased boldness. The same trend, however, was
not observed in the toads’ tendency to explore. The bolder
phenotype of guttural toads was restricted to urban popula-
tions, with invasive toads that had expanded their range into
natural ecosystems reverting to boldness levels comparable
to that of the natural-native population (i.e., pre-urbaniza-
tion). Taken together, these findings outline three aspects of
how urban-derived behavior may contribute to the spread of
invasive populations, including that (1) the urbanization of
toads in Durban preceding their invasions likely provided
them an advantage through prior adaptation, (2) all urban
toad populations were significantly bolder than that of natu-
ral living conspecifics, and (3) that increased exploratory
behavior does not appear to be currently favored in guttural
toads above natural-native levels in any of the other five
populations we studied and appears to have decreased in the
Mauritian populations.
Across their invasion route, guttural toads were consist-
ently bolder in urban areas—either through innate, fixed
behavioral traits (Sprau and Dingemanse 2017; Baxter-
Gilbert etal. 2019), increased behavioral flexibility (Dam-
mhahn etal. 2020), or adaptive phenotypic plasticity (Yeh
and Price 2004; Partecke 2013). Furthermore, guttural toads
maintain this increased level of boldness as they established
Table 1 (a) Outcome of the linear mixed-effect model (LMM)exam-
ining differences in the arena area (cm2) explored by a toad during
our 30-min exploration assay. The interaction between study site and
site type was not significant and so it was removed and the models re-
run. Model estimates (β) of fixed effects are presented with their cor-
responding standard errors (SE), variance estimates (σ2) are supplied
for residuals and random effects, and all significant values (p < 0.05)
are bolded. Reference levels for the categorical variable are given in
brackets following the variable name. (b) We also present post hoc
multiple comparisons of arena area explored (cm2) between all study
sites, and in this case, p-values (pcorr) were corrected using an “mvt”
adjustment (Lenth 2020)
(a) Output from the linear mixed-effect model
Variable names
Fixed effects β SE t p
Intercept (Durban,
natural, female) 950.89 83.43 11.40 < 0.01
Study site (Mauritius) − 212.08 85.53 − 2.48 0.01
Study site (Réunion) 35.49 84.38 0.42 0.67
Site type (urban) − 31.90 69.45 − 0.46 0.65
Sex (male) − 61.20 69.40 − 0.88 0.38
Random effects σ2
Experimental group 4099.00
Within-day batch 0.00
Residuals 226,013.00
(b) Multiple comparisons between study sites
Study sites β SE t pcorr
Durban vs. Mauritius 212.10 85.70 2.47 0.05
Durban vs. Réunion − 35.50 84.40 − 0.42 0.92
Mauritius vs. Réunion − 247.60 85.40 − 2.90 0.02
Fig. 2 The arena area explored
(cm2) by guttural toads during
our 30-min exploration assay,
as predicted from our linear
mixed-effect model, for each
of our study sites [Durban
(native) = orange, Maur itius
(invasive) = purple, and Réunion
(invasive) = blue]. Significant
differences are denoted using a
black line with location-specific
colors at the ends located above
the boxplots. Predicted jittered
data points are shown on the left
with corresponding boxplots to
the right. In the boxplots, the
thick horizontal line represents
the median, the boxes encom-
pass the quartile ranges, and the
whiskers represent the mini-
mum and maximum of the data,
excluding outliers (points that
are 3/2 times the upper quartile)
600
700
800
900
1000
1100
Durban Mauritius Reunion
Study Site
Arena Area Explored
(
cm
2
)

Behavioral Ecology and Sociobiology (2021) 75:130
1 3
130 Page 8 of 13
their urban-invasive populations. Irrespective of the mecha-
nism that drove the expression of this phenotype, the toads
likely experienced over 70years (~ 35 generations; Vimer-
cati etal. 2017) of selection within urban/anthropogenic
areas within their native range before individuals were col-
lected and relocated to Mauritius and subsequently Réunion
(Cheke and Hume 2010). Thus, guttural toads were already
primed for living in disturbed and anthropogenically altered
habitats before they arrived (i.e., prior adaptation) which
could have provided an advantage during establishment and
localized spread in urban, invasive habitats (Hufbauer etal.
2012; Borden and Flory 2021). Our findings do not entirely
conform to the AIAI hypothesis in full(Hufbauer etal.
2012), however, because the urban-invasive populations
which spread into natural habitats, on both Mauritius and
Réunion, reverted to boldness levels that mirror that of the
natural-native population. This suggests that living within
an urban area prior to their arrival in urban, invasive habitats
may have altered their behavior in a beneficial way and that
without this step the anthropogenically altered habitats they
were released into on both islands may have proven more
challenging. Our findings also point to the adaptive value of
being able to shift behavioral traits, like boldness, higher or
lower depending on their environment—bolstering species’
persistence and invasive potential.
Boldness reflects an individual’s propensity to take risks
and, within an urban context, bolder individuals may be
more active in novel landscapes and situations due to less-
ened perceived risk, which could increase their time spent
foraging or mate searching (Réale etal. 2007; Sol etal.
2013; Sprau and Dingemanse 2017). For this reason, it is
fitting that increased boldness appears to be a convergent
and commonly noted phenotypic shift across a variety of
taxa encountering urban landscapes (Lowry etal. 2013).
An increase in boldness within urban-living individuals has
been found across numerous vertebrates (e.g., birds, Sprau
and Dingemanse 2017; fish, Rehage and Sih 2004; mam-
mals, Dammhahn etal. 2020; reptiles, Baxter-Gilbert etal.
2019) and invertebrate taxa alike (e.g., insects, Schuett etal.
2018; isopods, Houghtaling and Kight 2006; spiders, Kralj-
Fišer etal. 2017). Our findings not only support this grow-
ing trend, but further provide evidence that advantageous
behavioral traits can be maintained in populations after
being moved from one urban area to another, across entirely
different regions of the world, and that once populations
spillover into natural areas behavioral traits can shift again to
match that of native, natural norms. Interestingly, if this was
Table 2 (a) Outcome of the generalized linear mixed-effect model
(GLMM) examining differences in if a toad exited the hide during
the 30-min boldness assay. The interaction between study site and
site type was not significant, and so it was removed and the mod-
els re-run. Model estimates (β) of fixed effects are presented on the
latent (logit link) scale with their corresponding standard errors (SE),
variance estimates (σ2) are supplied for residuals and random effects,
and all significant values (p < 0.05) are bolded. Reference levels for
the categorical variable are given in brackets following the variable
name. (b) We also present post hoc multiple comparisons of the prob-
ability a toad exited the hide between all study sites, and in this case,
p-values (pcorr) were corrected using an “mvt” adjustment (Lenth
2020). These values are on the response scale (i.e., back-transformed
from logit link and the latent scale)
(a) Output from the linear mixed-effect model
Variable names
Fixed effects β SE t p
Intercept (Durban, natu-
ral, female)
− 0.52 0.41 − 1.27 0.21
Study site (Mauritius) − 0.38 0.38 − 0.99 0.32
Study site (Réunion) − 0.14 0.37 − 0.37 0.71
Site type (urban) 0.85 0.31 2.76 < 0.01
Sex (male) 0.73 0.32 2.32 0.02
Random effects σ2
Experimental group 0.08
Within-day batch 0.05
Residuals 1.00
(b) Multiple comparisons between study sites
Study sites β SE t pcorr
Durban vs. Mauritius 1.46 0.56 0.99 0.58
Durban vs. Réunion 1.15 0.43 0.37 0.93
Mauritius vs. Réunion 0.77 0.30 − 0.63 0.80
Table 3 The boldness measured during this study summarized by
study site (Durban, Mauritius, and Réunion) and site type (natural or
urban). We summarized (a) the number of toads that exited the hide
with the total number of toads we measured following and separated
using a backslash, (b) the probability of toads exiting the hides as pre-
dicted from the generalized linear mixed-effect model (see Table2a),
and (c) the latency to leave the hide (s) as predicted from a linear
mixed-effect model (see Table4a). The latter two variables are dis-
played as mean ± standard error(SE). Significant differences between
variables are shown using asterisks (*) and carets (^), respectively
showing separate comparisons, following the pertinent means and
standard errors
Boldness measure Study site Site type
Durban Mauritius Réunion Natural Urban
(a) Number of toads exited hide/total number measured 36/64 28/59 34/64 40/94 58/93
(b) Predicted probability of toads exiting hides 0.56 ± 0.02 0.47 ± 0.02 0.53 ± 0.02 0.42 ± 0.01* 0.62 ± 0.01*
(c) Predicted latency to exit the hide (s) 935.75 ± 25.04 1080.37 ± 25.78 1131.59 ± 23.39 1186.21 ± 15.89^ 909.12 ± 17.19^

Behavioral Ecology and Sociobiology (2021) 75:130
1 3
Page 9 of 13 130
to occur on a relatively short time scale, then one may pre-
sume this is a product of behavioral flexibility (Dammhahn
etal. 2020) or adaptive phenotypic plasticity (Yeh and Price
2004). However, with over 70years of urban/anthropogenic
selective forces shaping toad behavior pre-invasion, and
almost 100years of selection acting on both natural- and
urban-invasive toad populations post-invasion, the potential
for these phenotypes to be adaptive and heritable certainly
does exist. We suggest future investigations look to deter-
mine the evolutionary mechanisms driving the behavioral
differences we observed between our study populations (i.e.,
fixed vs. plastic) using appropriately robust study designs to
test for urban evolution (see Lambert etal. 2020).
Contrary to our predictions, explorative behavior did
not differ between urban and natural habitats, nor in a con-
sistent fashion along the toad’s invasion route. Although
increased levels of exploration are thought to be favored
in populations colonizing new landscapes and urban hab-
itats (Lapiedra etal. 2017; Damas-Moreira etal. 2019;
Dammhahn etal. 2020). The advantages conferred from
increased explorative behavior are most closely related
to particular stages of the invasion process—transport,
introduction, establishment, and spread (Chapple etal.
2012)—rather than within long-founded invasive popu-
lations. For example, differences between invasive and
native lizards’ explorative tendency were found in a
20-year-old invasive population of Italian Wall Lizards,
Podarcis sicula, in Portugal (Damas-Moreira etal. 2019);
however, this invasion is much more recent than the colo-
nization by guttural toads on either island. Our findings
instead seem to align with research on another invasive
amphibian, the cane toad (Rhinella marina), whereby
established island populations in Hawai’i express lower
levels of explorative behavior compared to the expanding
invasive populations in Australia, supporting the idea that
once invasive population reaches saturation in a closed
system (e.g., islands), the drive to maintain dispersive
behavioral phenotypes is relaxed (Gruber etal. 2016;
Gruber 2017). As such, it appears that, unlike boldness
within urban landscapes, if guttural toad populations had
increased their explorative behavior during their coloniza-
tion of either the urban landscape in their native range or
the ecosystems of Mauritius and Réunion, then it has not
been maintained. Rather we see a significant reduction in
explorative behavior for toads in Mauritius. Although not
following what we would expect, based on studies on the
Fig. 3 (a) The probability of a
guttural toad exiting the hide
during our 30-min boldness
assay, as predicted from our
generalized linear mixed-effect
model, for each of our study
sites by site type (urban = grey,
natural = green). (b) We also
depict the predicted probability
of exiting the hide and (c) the
predicted latency to exit the
hide for the main effect of site
type. Significant differences
are denoted using a black line
with site-type-specific colors
at the ends located above the
boxplots. Predicted jittered data
points are shown on the left
with corresponding boxplots to
the right. In the boxplots, the
thick horizontal line represents
the median, the boxes encom-
pass the quartile ranges, and the
whiskers represent the mini-
mum and maximum of the data,
excluding outliers (points that
are 3/2 times the upper quartile)
0.2
0.4
0.6
0.8
Durban Mauritiu
sR
eunion
Study Site
Probability of Exiting the Hide
(a)
0.2
0.4
0.6
0.8
NaturalUrban
Site Type
Probability of Exiting the Hide
(b)
750
1000
1250
1500
NaturalUrban
Site Type
Latency to Exit Hide (s)
(c)

Behavioral Ecology and Sociobiology (2021) 75:130
1 3
130 Page 10 of 13
explorative behavior of establishing or spreading invasive
populations (Chapple etal. 2012), this curious finding
does support the assertion that the reduced hind limb sizes
in toads from both islands may be related to a “less dis-
persive” phenotype (see Baxter-Gilbert etal. 2020). Yet,
the fact that reduced explorative behavior was only sig-
nificant in Mauritius—and not Réunion—and similarities
in boldness between urban populations of larger mainland
toads and the smaller urban conspecifics (Baxter-Gilbert
etal. 2020), this suggests that the relationship between
changes in morphological and behavioral phenotypes may
be more complex and will require further investigation.
The absence of an increase in exploration, as well as a
decrease in Mauritius, may be attributed to local factors
favoring more sedentary behavior such as increased food
availability (Lyons etal. 2017) or differences in preda-
tion levels (Huang etal. 2012); however, much more
research into the ecological differences between locations
and populations is needed (e.g., examining dietary and
trophic changes along the invasion route). Overall, we are
unable to determine whether increased toad exploration
played a role in their success as they shifted from natural
to urban, or native to invasive, habitats. All we are able to
observe now, ~ 170years after the process began, is that
a highly explorative phenotype, above the natural-native
norm, is not currently favored in any of the six populations
we studied.
Our study highlights several key aspects of the rela-
tionship between behavior, urbanization, and biological
invasions. Notably, we observed that toads from urban
habitats were significantly bolder than natural living
conspecifics, both in native and invasive ranges. If selec-
tion had favored this phenotype, as toads slowly adapted
to urban living in their native range over generations,
either through increased flexibility, adaptive plasticity, or
through rapid localized adaptation, then these acquired
phenotypes likely provided them a substantial advantage
once they were introduced to anthropogenic habitats in
both Mauritius and Réunion. This support for the AIAI
hypothesis, albeit promising, raises several new questions,
particularly regarding the evolutionary mechanisms driv-
ing these changes in behavior, but also if these behaviors
are consistent within individuals (i.e., repeatability), pre-
sent across life stages (i.e., tadpoles vs. adults), and how
this bolder phenotype specifically benefits guttural toads
in urban landscapes (i.e., urban behavioral ecology). We
recommend future research on this promising study sys-
tem to examine these questions, which should advance
our understanding of how urban habitats may be priming
native species to become better invaders and how behav-
ioral shifts can increase a given taxa’s invasive potential.
Acknowledgements We would like to thank C. Baider, M. Campbell,
A. Cheke,V. Florens, P. Kowalski, N. Mohanty,M. Mühlenhaupt, S.
Peta,S. Sauroy-Toucouère, D. Strasberg, C. Wagener, and R. Wed-
derburnfor their invaluable support and insights. We would also like
to thank Black River Gorges National Park, the Durban Botanical Gar-
dens, and the communities of Notre Dame, Villèle, and Pont Payet, and
the anonymous reviewers whose comments improved this manuscript.
Funding This work was supported by the DSI-NRF Centre of Excel-
lence for Invasion Biology (JB-G & JM), the Centre for Collabora-
tions in Africa at Stellenbosch University (JB-G), Claude Leon Foun-
dation (fellowship to JLR), and the Natural Sciences and Engineering
Research Council of Canada (fellowship to JLR).
Data availability The datasets and R code for this study are available
from Open Source Framework (OSF, DOI:https:// doi. org/ 10. 17605/
OSF. IO/ 54TAC), which can be found here:https:// osf. io/ 54tac/
Declarations
Ethics approval Ethical approval for toad captures and our experimen-
tal protocols followed animal ethics guidelines set out and approved
by the Stellenbosch University’s Research Ethics Committee (Animal
Care and Use: ACU-2020–10386). National guidelines for the use of
animals in scientific research were followed (South Africa National
Standard: The Care and Use of Animals for Scientific Purposes; SANS
Table 4 (a) Outcome of the linear mixed-effect model (LMM)exam-
ining differences in the time (s) it took a toad to exit the hide during
the 30-min boldness assay. The interaction between study site and site
type was not significant, and so it was removed and the models re-
run. Model estimates (β) of fixed effects are presented with their cor-
responding standard errors (SE), variance estimates (σ2) are supplied
for residuals and random effects, and all significant values (p < 0.05)
are bolded. Reference levels for the categorical variable are given in
brackets following the variable name. (b) We also present post hoc
multiple comparisons of arena area explored (cm2) between all study
sites, and in this case, p-values (pcorr) were corrected using an “mvt”
adjustment (Lenth 2020)
(a) Output from the linear mixed-effect model
Variable names
Fixed effects β SE t p
Intercept (Durban,
natural, female) 1181.52 160.47 7.36 < 0.01
Study site (Mauritius) 142.51 134.28 1.06 0.29
Study site (Réunion) 195.84 131.51 1.49 0.14
Site type (urban) − 276.62 108.81 − 2.54 0.01
Sex (male) − 214.92 109.26 − 1.97 0.05
Random effects σ2
Experimental group 22,381.00
Within-day batch 0.00
Residuals 55,3429.00
(b) Multiple comparisons between study sites
Study sites β SE t pcorr
Durban vs. Mauritius − 142.50 134.00 − 1.06 0.54
Durban vs. Réunion − 195.80 132.00 − 1.49 0.30
Mauritius vs. Réunion − 53.30 134.00 − 0.40 0.92

Behavioral Ecology and Sociobiology (2021) 75:130
1 3
Page 11 of 13 130
20386:2008). This work was conducted with authorization from Ezem-
velo KwaZulu-Natal Wildlife (Ordinary Permit: OP 4072/2019) and
Mauritian National Parks and Conservation Services (NP 46/3 V3).
Conflict of interest The authors declare no competing interests.
References
Battles AC, Irschick DJ, Kolbe JJ (2019) Do structural habitat modi-
fications associated with urbanization influence locomotor per-
formance and limb kinematics in Anolis lizards? Biol J Linn Soc
127:100–112. https:// doi. org/ 10. 1093/ bioli nnean/ blz020
Baxter-Gilbert J, Riley JL, Wagener C, Mohanty NP, Measey J (2020)
Shrinking before our isles: the rapid expression of insular dwarf-
ism in two invasive populations of guttural toad (Sclerophrys
gutturalis). Biol Lett 16:20200651. https:// doi. org/ 10. 1098/ rsbl.
2020. 0651
Baxter-Gilbert J, Riley JL, Whiting MJ (2019) Bold New World: urban-
ization promotes an innate behavioral trait in a lizard. Behav Ecol
Sociobiol 73:105. https:// doi. org/ 10. 1007/ s00265- 019- 2713-9
Bókony V, Ujhegyi N, Hamow KÁ, Bosch J, Thumsová B, Vörös J,
Aspbry AS, Gabor CR (2021) Stressed tadpoles mount more
efficient glucocorticoid negative feedback in anthropogenic habi-
tats due to phenotypic plasticity. Sci Total Environ 753:141896.
https:// doi. org/ 10. 1016/j. scito tenv. 2020. 141896
Borden JB, Flory S (2021) Urban evolution of invasive species. Front
Ecol Environ (published online, https:// doi. org/ 10. 1002/ fee. 2295)
Chapple DG, Simmonds SM, Wong BB (2012) Can behavioral and
personality traits influence the success of unintentional species
introductions? Trends Ecol Evol 27:57–64. https:// doi. org/ 10.
1016/j. tree. 2011. 09. 010
Corlett RT (2015) The Anthropocene concept in ecology and conserva-
tion. Trends Ecol Evol 30:36–41. https:// doi. org/ 10. 1016/j. tree.
2014. 10. 007
Damas-Moreira I, Riley JL, Harris DJ, Whiting MJ (2019) Can behav-
iour explain invasion success? A comparison between sympatric
invasive and native lizards. Anim Behav 151:195–202. https:// doi.
org/ 10. 1016/j. anbeh av. 2019. 03. 008
Dammhahn M, Mazza V, Schirmer A, Göttsche C, Eccard JA (2020)
Of city and village mice: behavioural adjustments of striped field
mice to urban environments. Sci Rep 10:13056. https:// doi. org/
10. 1038/ s41598- 020- 69998-6
du Preez LH, Weldon C, Cunningham MJ, Turner AA (2004) Bufo
gutturalis Power, 1927. In: Minter LR, Burger M, Harrison JA,
Braack HH, Bishop PJ, Kloepfer D (eds) Atlas and Red data book
of the frogs of South Africa, Lesotho and Swaziland. SI/MAB
Series #9. Smithsonian Institute, Washington, USA, pp 67–69
Estoup A, Guillemaud T (2010) Reconstructing routes of invasion
using genetic data: why, how and so what? Mol Ecol 19:4113–
4130. https:// doi. org/ 10. 1111/j. 1365- 294X. 2010. 04773.x
Evans J, Boudreau K, Hyman J (2010) Behavioural syndromes in urban
and rural populations of song sparrows. Ethology 116:588–595.
https:// doi. org/ 10. 1111/j. 1439- 0310. 2010. 01771.x
González-Bernal E, Greenlees MJ, Brown GP, Shine R (2016) Toads
in the backyard: why do invasive cane toads (Rhinella marina)
prefer buildings to bushland? Pop Ecol 58:293–302. https:// doi.
org/ 10. 1007/ s10144- 016- 0539-0
Gruber JE (2017) Geographical divergence of behavioural traits in the
invasive cane toad (Rhinella marina). PhD thesis, University of
Sydney, Sydney
Gruber J, Whiting M, Brown G, Shine R (2016) Variation in dispersal-
related behavioural traits in the cane toad (Rhinella marina). In:
Australian Society of Herpetologist Conference, Grindelwald,
Tasmania (abstract)
Hammond DS, Gond V, Baider C, Florens FBV, Persand S, Laurance
SGW (2015) Threats to environmentally sensitive areas from
peri-urban expansion in Mauritius. Environ Conserv 42:256–
267. https:// doi. org/ 10. 1017/ S0376 89291 40004 11
Holtmann B, Santos ES, Lara CE, Nakagawa S (2017) Personality-
matching habitat choice, rather than behavioural plasticity, is
a likely driver of a phenotype–environment covariance. Proc R
Soc B 284:20170943. https:// doi. org/ 10. 1098/ rspb. 2017. 0943
Houghtaling K, Kight SL (2006) Turn alternation in response to sub-
strate vibration by terrestrial isopods, Porcellio laevis (Isopoda:
Oniscidea) from rural and urban habitats in New Jersey, U.S.A.
Entomol News 117:149e154. https:// doi. org/ 10. 3157/ 0013-
872X(2006) 117[149: TAIRTS] 2.0. CO;2
Huang P, Sieving KE, Mary CMS (2012) Heterospecific informa-
tion about predation risk influences exploratory behavior. Behav
Ecol 23:463–472. https:// doi. org/ 10. 1093/ beheco/ arr212
Hudina S, Hock K, Žganec K (2014) The role of aggression in range
expansion and biological invasions. Curr Zool 60:401–409.
https:// doi. org/ 10. 1093/ czoolo/ 60.3. 401
Hufbauer RA, Facon B, Ravigne V, Turgeon J, Foucaud J, Lee CE,
Rey O, Estoup A (2012) Anthropogenically induced adaptation
to invade (AIAI): contemporary adaptation to human-altered
habitats within the native range can promote invasions. Evol
Appl 5:89–101. https:// doi. org/ 10. 1111/j. 1752- 4571. 2011.
00211.x
Jackson H, Strubbe D, Tollington S, Prys-Jones R, Matthysen E,
Groombridge JJ (2015) Ancestral origins and invasion pathways
in a globally invasive bird correlate with climate and influences
from bird trade. Mol Ecol 24:4269–4285. https:// doi. org/ 10. 1111/
mec. 13307
Johnson MT, Munshi-South J (2017) Evolution of life in urban envi-
ronments. Science 358:eaam8327. https:// doi. org/ 10. 1126/ scien
ce. aam83 27
Kralj-Fišer S, Hebets EA, Kuntner M (2017) Different patterns of
behavioral variation across and within species of spiders with
differing degrees of urbanization. Behav Ecol Sociobiol 71:125.
https:// doi. org/ 10. 1007/ s00265- 017- 2353-x
Kralj-Fišer S, Schneider JM (2012) Individual behavioural consistency
and plasticity in an urban spider. Anim Behav 84:197–204. https://
doi. org/ 10. 1016/j. anbeh av. 2012. 04. 032
Kuznetsova A, Brockhoff PB, Christensen RHB (2017) lmerTest Pack-
age: tests in linear mixed effects models. J Stat Softw 82:1–26.
https:// doi. org/ 10. 18637/ jss. v082. i13
Lambert MR, Brans KI, Des Roches S, Donihue CM, Diamond SE
(2020) Adaptive evolution in cities: progress and misconceptions.
Trends Ecol Evol 36:239–257. https:// doi. org/ 10. 1016/j. tree. 2020.
11. 002
Lapiedra O, Chejanovski Z, Kolbe JJ (2017) Urbanization and bio-
logical invasion shape animal personalities. Glob Change Biol
23:592–603. https:// doi. org/ 10. 1111/ gcb. 13395
Larson RN, Brown JL, Karels T, Riley SPD (2020) Effects of urbani-
zation on resource use and individual specialization in coyotes
(Canis latrans) in southern California. PLoS ONE 15:e0228881.
https:// doi. org/ 10. 1371/ journ al. pone. 02288 81
Lenth R (2020) emmeans: estimated marginal means, aka least-squares
means. R package version 1.4.7, https:// CRAN.R- proje ct. org/
packa ge= emmea ns
Lowry H, Lill A, Wong BB (2013) Behavioural responses of wildlife
to urban environments. Biol Rev 88:537–549. https:// doi. org/ 10.
1111/ brv. 12012
Lyons J, Mastromonaco G, Edwards DB, Schulte-Hostedde AI (2017)
Fat and happy in the city: eastern chipmunks in urban environ-
ments. Behav Ecol 28:1464–1471. https:// doi. org/ 10. 1093/ beheco/
arx109

Behavioral Ecology and Sociobiology (2021) 75:130
1 3
130 Page 12 of 13
Malange J, Izar P, Japyassú H (2016) Personality and behavioural
syndrome in Necromys lasiurus (Rodentia: Cricetidae): notes on
dispersal and invasion processes. Acta Ethol 19:189–195. https://
doi. org/ 10. 1007/ s10211- 016- 0238-z
McKinney ML (2008) Effects of urbanization on species richness: a
review of plants and animals. Urban Ecosyst 11:161–176. https://
doi. org/ 10. 1007/ s11252- 007- 0045-4
Miranda AC, Schielzeth H, Sonntag T, Partecke J (2013) Urbanization
and its effects on personality traits: a result of microevolution or
phenotypic plasticity? Glob Change Biol 19:2634–2644. https://
doi. org/ 10. 1111/ gcb. 12258
Myers N, Mittermeier RA, Mittermeier CG, da Fonseca GAB, Kent J
(2000) Biodiversity hotspots for conservation priorities. Nature
403:853–858. https:// doi. org/ 10. 1038/ 35002 501
Myles-Gonzalez E, Burness G, Yavno S, Rooke A, Fox MG (2015)
To boldly go where no goby has gone before: boldness, disper-
sal tendency, and metabolism at the invasion front. Behav Ecol
26:1083–1090. https:// doi. org/ 10. 1093/ beheco/ arv050
Partecke J (2013) Mechanisms of phenotypic responses following
colonization of urban areas: from plastic to genetic adaptation.
In: Gil D, Brumm H (eds) Avian urban ecology: behavioural and
physiological adaptations. Oxford University Press, Oxford, UK,
pp 131–142
Pelletier F, Coltman DW (2018) Will human influences on evolutionary
dynamics in the wild pervade the Anthropocene? BMC Biol 16:7.
https:// doi. org/ 10. 1186/ s12915- 017- 0476-1
Pellitteri-Rosa D, Bellati A, Cocca W, Gazzola A, Martín J, Fasola M
(2017) Urbanization affects refuge use and habituation to preda-
tors in a polymorphic lizard. Anim Behav 123:359–367. https://
doi. org/ 10. 1016/j. anbeh av. 2016. 11. 016
Pintor LM, Sih A, Bauer ML (2008) Differences in aggression, activ-
ity and boldness between native and introduced populations of an
invasive crayfish. Oikos 117:1629–1636. https:// doi. org/ 10. 1111/j.
1600- 0706. 2008. 16578.x
Plotkin HC (1988) The Role of Behavior in Evolution. MIT Press,
Cambridge, USA
Putman BJ, Pauly GB, Blumstein DT (2020) Urban invaders are not
bold risk-takers: a study of three invasive lizards in Southern Cali-
fornia. Curr Zool 66:657–665. https:// doi. org/ 10. 1093/ cz/ zoaa0 15
Pyšek P, Hulme PE, Simberloff D etal (2020) Scientists’ warning on
invasive alien species. Biol Rev 95:1511–1534. https:// doi. org/
10. 1111/ brv. 12627
R Core Team (2021) R: a language and environment for statistical com-
puting. R Foundation for Statistical Computing, Vienna, Austria,
https:// www.R- proje ct. org/
Réale D, Reader SM, Sol D, McDougall PT, Dingemanse NJ (2007)
Integrating animal temperament within ecology and evolution.
Biol Rev 82:291–318. https:// doi. org/ 10. 1111/j. 1469- 185X. 2007.
00010.x
Rehage JS, Sih A (2004) Dispersal behavior, boldness, and the link to
invasiveness: a comparison of four Gambusia species. Biol Inva-
sions 6:379–391. https:// doi. org/ 10. 1023/B: BINV. 00000 34618.
93140. a5
Rey O, Estoup A, Vonshak M etal (2012) Where do adaptive shifts
occur during invasion? A multidisciplinary approach to unrav-
elling cold adaptation in a tropical ant species invading the
Mediterranean area. Ecol Lett 15:1266–1275. https:// doi. org/ 10.
1111/j. 1461- 0248. 2012. 01849.x
Schuett W, Delfs B, Haller R, Kruber S, Roolfs S, Timm D, Willmann
M, Drees C (2018) Ground beetles in city forests: does urbaniza-
tion predict a personality trait? PeerJ 6:e4360. https:// doi. org/ 10.
7717/ peerj. 4360
Short KH, Petren K (2008) Boldness underlies foraging success of
invasive Lepidodactylus lugubris geckos in the human landscape.
Anim Behav 76:429–437. https:// doi. org/ 10. 1016/j. anbeh av. 2008.
04. 008
Slater PJB, Halliday TR (1994) Behaviour and evolution. Cambridge
University Press, Cambridge, UK
Sol D, Lapiedra O, González-Lagos C (2013) Behavioural adjustments
for a life in the city. Anim Behav 85:1101–1112. https:// doi. org/
10. 1016/j. anbeh av. 2013. 01. 023
Sprau P, Dingemanse NJ (2017) An approach to distinguish between
plasticity and non-random distributions of behavioral types along
urban gradients in a wild passerine bird. Front Ecol Evol 5:92.
https:// doi. org/ 10. 3389/ fevo. 2017. 00092
Strubbe D, Jackson H, Groombridge J, Matthysen E (2015) Invasion
success of a global avian invader is explained by within-taxon
niche structure and association with humans in the native range.
Divers Distrib 21:675–685. https:// doi. org/ 10. 1111/ ddi. 12325
Telford NS, Channing A, Measey J (2019) Origin of invasive popu-
lations of the guttural toad (Sclerophrys gutturalis) on Réunion
and Mauritius Islands and in Constantia, South Africa. Herpetol
Conserv Biol 14:380–392
Thompson MJ, Evans JC, Parsons S, Morand-Ferron J (2018) Urbani-
zation and individual differences in exploration and plasticity.
Behav Ecol 29:1415–1425. https:// doi. org/ 10. 1093/ beheco/ ary103
Vimercati G, Davies SJ, Measey J (2019) Invasive toads adopt marked
capital breeding when introduced to a cooler, more seasonal envi-
ronment. Biol J Linn Soc 128:657–671. https:// doi. org/ 10. 1093/
bioli nnean/ blz119
Vimercati G, Hui C, Davies SJ, Measey GJ (2017) Integrating age
structured and landscape resistance models to disentangle inva-
sion dynamics of a pondbreeding anuran. Ecol Model 356:104–
116. https://doi.org10.1016/j.ecolmodel.2017.03.017
Waselkov KE, Regenold ND, Lum RC, Olsen KM (2020) Agricultural
adaptation in the native North American weed waterhemp, Ama-
ranthus tuberculatus (Amaranthaceae). PLoS ONE 15:e0238861.
https:// doi. org/ 10. 1371/ journ al. pone. 02388 61
Yeh PJ, Price TD (2004) Adaptive phenotypic plasticity and the suc-
cessful colonization of a novel environment. Am Nat 164:531–
542. https:// doi. org/ 10. 1086/ 423825
Zhang D (2020) rsq: R-Squared and Related Measures. R package ver-
sion 2.0, https:// CRAN.R- proje ct. org/ packa ge= rsq
Zuur AF, Ieno EN, Elphick CS (2010) A protocol for data exploration
to avoid common statistical problems. Methods Ecol Evol 1:3–14.
https:// doi. org/ 10. 1111/j. 2041- 210X. 2009. 00001.x
Publisher's Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.

Behavioral Ecology and Sociobiology (2021) 75:130
1 3
Page 13 of 13 130
Authors and Aliations
JamesBaxter‑Gilbert1 · JuliaL.Riley2,3· JohnMeasey1
* James Baxter-Gilbert
j.h.baxtergilbert@gmail.com
* Julia L. Riley
julia.riley87@gmail.com
1 Centre forInvasion Biology, Department ofBotany
andZoology, Stellenbosch University, Stellenbosch7600,
WC, SouthAfrica
2 Department ofBotany andZoology, Stellenbosch University,
Stellenbosch7600, WC, SouthAfrica
3 Department ofBiology, Dalhousie University, Halifax,
NSB3H4R2, Canada