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https://doi.org/10.1007/s11692-024-09635-6
Introduction
Cities are highly transformed environments characterized
by a set of conditions with important, but not yet fully
explored, consequences for biodiversity (Grimm et al.,
2008; Seto et al., 2012). Urbanization leads to increased lev-
els of habitat fragmentation, pollution (i.e., air, water, soil,
acoustic, and light pollution), and changes in biotic (spe-
cies community richness and structure) and abiotic (chemi-
cal and physical parameters) conditions, which results in
novel environments (Cadenasso et al., 2007; Grimm et al.,
2008; Oke, 1973). While some species are unable to persist
in cities, others have managed to develop stable and well
adapted urban populations (e.g., the brown rats and feral
pigeons) (McKinney, 2008). Populations inhabiting urban
areas usually exhibit phenotypic, physiological, or ecologi-
cal modications that result from novel selective pressures
associated to these environments, providing interesting
study systems for exploring contemporary evolution in
Lucía Alarcón-Ríos
alarconrioslucia@gmail.com
Guillermo Velo-Antón
guillermo.velo@uvigo.es
1 Departamento de Biología de Organismos y Sistemas,
Universidad de Oviedo, C/ Valentín Andrés Álvarez s/n,
Oviedo 33071, Spain
2 Universidade de Vigo, Facultade de Bioloxía, Grupo GEA,
Edicio de Ciencias Experimentais, Vigo E-36310, Spain
3 Centro de Investigação em Biodiversidade e Recursos
Genéticos, CIBIO, InBIO Laboratório Associado,
Universidade do Porto, Campus de Vairão, Vairão
4485-661, Portugal
4 BIOPOLIS Program in Genomics, Biodiversity and Land
Planning, CIBIO, Campus de Vairão, Vairão
4485-661, Portugal
5 Departament de Biologia Evolutiva, Ecologia i Ciències
Ambientals (BEECA) IRBio, Institut de Recerca de la
Biodiversitat, Universitat de Barcelona, Barcelona, Spain
Abstract
The environmental transformations associated with cities are expected to aect organisms at the demographic, phenotypic,
and evolutionary level, often negatively. The prompt detection of stressed populations before their viability is compro-
mised is essential to understand species’ responses to novel conditions and to integrate urbanization with biodiversity
preservation. The presumably stressful conditions of urban environments are expected to aect organisms’ developmental
pathways, resulting in a reduction of the ecacy of developmental stability and canalization processes, which can be
observed as increased Fluctuating Asymmetry (FA) and Phenotypic Variance (PV), respectively. Here, we investigated
whether patterns of phenotypic variation of urban populations of a fully terrestrial salamander, Salamandra salamandra
bernardezi, are aected by urban settings compared to surrounding native forest populations. We sampled populations
within and around the city of Oviedo (northern Spain) and used geometric morphometrics to compare morphological
dierentiation, head shape deviance from the allometric slope, PV, and FA. We also compared morphological patterns
with neutral genetic and structure patterns. We observed increased levels of dierentiation among urban populations and
in PV within certain of them, yet no dierences in allometric deviance and FA were detected between habitats, and no
morphological measures were found to be correlated with genetic traits. Our results do not support a clear negative impact
of urban conditions over salamander populations, but rather suggest that other ecological and evolutionary local processes
inuence morphological variation in this urban system.
Keywords Amphibians · Canalization · Fluctuating Asymmetry · Geometric Morphometrics · Stressors
Received: 17 May 2023 / Accepted: 2 April 2024
© The Author(s) 2024
Urban Life Aects Dierentiation and Phenotypic Variation but not
Asymmetry in a Fully Terrestrial Salamander
LucíaAlarcón-Ríos1,2,3,4 · AntigoniKaliontzopoulou5· DavidÁlvarez1· GuillermoVelo-Antón2,3,4
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Evolutionary Biology
anthropic environments (Diamond & Martin, 2021; Johnson
& Munshi-South, 2017; Santangelo et al., 2022). Indeed, the
modication of eco-evolutionary dynamics in cities results
in particular patterns of variability within urban areas,
such as an increase of intraspecic phenotypic variation
(Thompson et al., 2022). Understanding how organisms
vary in response to urbanization, as well as how they are
aected by the novel selective pressures, is crucial to pre-
dict the responses of biodiversity to human-dominated areas
and foster conservation strategies that may conciliate urban
development with biodiversity preservation (Collins et al.,
2021; McDonnell & Hahs, 2015).
Many of the biotic and abiotic factors related to urbaniza-
tion can alter organismal life histories, and with them the
amount of energy invested in reproduction and develop-
ment in urban-dwelling populations (Diamond & Martin,
2021; Kolonin et al., 2022; Sepp et al., 2018; Snell-Rood et
al., 2015). When those factors negatively aect individual
homeostasis and tness, ultimately compromising popu-
lation viability, they are considered as stressors (Parsons,
2005). Among the many outputs of stressful conditions,
development is one of the rst levels to be disturbed. It is a
highly regulated process that includes stability mechanisms
to buer deviations from the predetermined developmental
pathway caused by genetic or environmental disturbances
(i.e., canalization and developmental stability) (Debat &
David, 2001; Waddington, 1942; Willmore et al., 2007).
But when conditions (e.g., environmental) exceed certain
thresholds, the ecacy of such regulatory mechanisms is
reduced, producing suboptimal phenotypes, which may ulti-
mately cause a reduction in tness (Clarke, 1995; Møller,
1997). Deviance from the predetermined phenotype can
have direct eects on tness, aecting for example indi-
vidual’s survival (Martín & López, 2001; Tocts et al., 2016)
or mating success (Møller & Thornhill, 1998). At the same
time, levels of variability around an optimal phenotype
can be considered an indicator of the ecacy of regulatory
mechanisms underlying development, as phenotypic varia-
tion resulting from disturbed development usually arises
before any other trait that signicantly compromises popu-
lation survival is aected (e.g., reproductive output). Thus,
it can be used as a biomarker to precociously detect stressed
populations (“early warning system” Clarke, 1995).
The impact of stressors over developmental accuracy
may arise at two dierent organismal scales: at the popu-
lation level, as an outcome of the disturbance of develop-
mental canalization processes, which results in an increased
phenotypic variance (PV) across individuals; and at the
individual level, resulting from the disturbance of devel-
opmental stability within individuals, usually measured as
an increase in uctuating asymmetry (FA) or the random
deviation from bilateral symmetry (Beasley et al., 2013;
Debat & David, 2001; Palmer & Strobeck, 1986; Willmore
et al., 2007). The assessment of developmental disturbance
using the aforementioned phenotypic variation measures
(i.e., PV and FA) is a widely accessible, inexpensive, and
non-invasive methodology, which allows increasing sample
sizes and the spectrum of study systems. Considering the
number of potential stressors that converge in urban envi-
ronments, the assessment of developmental precision using
phenotypic traits can help to evaluate the health of urban
populations and constitute a preliminary approach to inves-
tigate the consequences of urban environments on a wide
range of urban-dwelling organisms (e.g., plants: Shadrina
et al., 2020; invertebrates: Weller & Ganzhorn, 2004; birds:
Vangestel & Lens, 2011; amphibians: Zhelev et al., 2019;
reptiles: Lazić et al., 2015; sh: Allenbach, 2011; and mam-
mals: Puckett et al., 2020).
The potential to persist in urban environments and the
magnitude and direction of the eects of urbanization are
highly dependent upon species’ specic attributes and life-
history traits (Becker et al., 2007; Evans et al., 2011; Pyron,
2018). For instance, generalist species with high mobility
(e.g., birds and ying insects) and short generation times are
more prone to successfully persist, and even adapt (Salmón
et al., 2021), in highly modied habitats than specialists
with reduced dispersal abilities, long generation times, and
with traits that increase their sensitivity to certain stressors,
such as ectothermic physiology or ground dwelling life
(McDonnell & Hahs, 2015; Møller, 2009; Vergnes et al.,
2014). Despite the fact that the development of ectotherms
is more sensitive to environmental factors than that of endo-
therms (Eyck et al., 2019; Noble et al., 2018) studies on
urban ecology among vertebrates are biased towards birds
and mammals, while reptiles and amphibians remain largely
understudied (Collins et al., 2021). Specically, amphibian
species are expected to be highly aected by urbanization
because of their relatively reduced mobility, high sensitivity
to environmental cues (i.e., pollutants, through their perme-
able skin), and the common biphasic life-cycle that usually
includes egg-lying and an aquatic larval phase (Hamer &
McDonnell, 2008; Pyron, 2018). Nonetheless, amphib-
ians present a wide variety of reproductive modes, ranging
from fully aquatic life-cycles and external development,
either direct or biphasic, to fully terrestrial species in which
development is completed within the parents’ body (and all
combinations in between) (Nunes-de-Almeida et al., 2021).
Thus, species with external development, both aquatic or
terrestrial, have been reported to be especially sensitive to
hydroperiod, habitat availability and connectivity, environ-
mental-pollutants, and the presence of predators (Becker et
al., 2007; Hamer & McDonnell, 2008; Suazo-Ortuño et al.,
2008; Trimble & Van Aarde, 2014). Conversely, those repro-
ductive strategies in which development occurs internally,
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Evolutionary Biology
including metamorphosis when it exists, are expected to be
aected dierently by novel environmental factors and may
present dierent conservation requirements.
Here, we evaluated developmental disturbance in mor-
phological variation (i.e., head shape) from urban popu-
lations of a fully terrestrial salamander with internal
development (pueriparity sensu Greven, 2003) comparing
phenotypic variation measures between urban and forest
populations of the pueriparous Salamandra salamandra
bernardezi (Alarcón-Ríos et al., 2020; Buckley et al., 2007;
Mulder et al., 2022) in the city of Oviedo (northern Spain)
and surrounding areas (Fig. 1a). This system is particularly
well-suited for urban research in fully terrestrial amphibians
due to the following reasons: (i) patterns of neutral genetic
diversity and structure of urban and surrounding popula-
tions from Oviedo have already been studied and linked to
historical and demographic processes (Álvarez et al., 2015;
Lourenço et al., 2017). This allows for morphological com-
parisons between and within habitats under a well-known
evolutionary history. (ii) A recent study found a higher pro-
portion of morphological deformities within urban popu-
lations compared to forest ones (Velo-Antón et al., 2021)
indicating the presence of factors that inuence the mor-
phology of some urban populations. (iii) The study of mor-
phological variation within this species has been previously
optimized using geometric morphometrics to study dorsal
head shape (Alarcón-Ríos et al., 2017), which provides
high-resolution tools for studying the developmental eects
of urban environments in this organism. The head is a com-
plex and functionally relevant structure (Hanken & Hall,
1993) and, thus, a likely target of selection. It is also highly
variable within S. salamandra (Alarcón-Ríos et al., 2020a;
Bas & Gasser, 1994), even at local scales (Alarcón-Ríos et
al., 2017). Indeed, some of the aforementioned deformities
in Oviedo populations appeared in the head (Velo-Antón et
al., 2021), supporting head shape as a suitable structure to
assess morphological variation in response to urban stress-
ors. Finally, (iv) while most studies investigating amphib-
ians´ phenotypic responses to anthropic alterations have
focused on species with aquatic stages or terrestrial eggs
(e.g., Rubbo & Kiesecker, 2005; Parris, 2006; Wilk et al.,
2020; but see Iglesias-Carrasco et al., 2017), to our knowl-
edge, the phenotypic changes of amphibians with a fully
internal embryonic development have never been exam-
ined, making this a particularly interesting system for future
comparisons with other reproductive strategies.
We aim to answer four fundamental questions: (1) are
urban populations of S. s. bernardezi morphologically dif-
ferentiated from neighbouring forest populations? Due to
the high functionality of the trait under study (head mor-
phology), the ecological disparities between habitats,
and the historical isolation of urban populations from
surrounding ones (see Lourenço et al., 2017) we expect
some degree of dierentiation between urban and forest sal-
amanders. (2) Do urban populations exhibit higher levels of
developmental disturbance than forest populations? Based
on the high sensitivity of amphibians to environmental dis-
turbance (Hamer & McDonnell, 2008; Pyron, 2018), and
the observed higher incidence of deformities within urban
populations (Velo-Antón et al., 2021), we hypothesize a
lower performance of the mechanisms involved in devel-
opmental stability and morphological canalization in urban
populations, resulting in higher levels of uctuating asym-
metry (FA) and phenotypic variance (PV). (3) Are levels of
developmental instability (i.e., FA) and phenotypic variance
(i.e., PV) associated with genetic variation (heterozygosity
and relatedness) and eective population size (Ne) in these
populations? Although Oviedo populations present levels of
genetic variation comparable to forest ones, they generally
show higher levels of relatedness and smaller Ne, which
together with their higher isolation (Lourenço et al., 2017)
might be reected in increased FA and PV levels (e.g.,
Garrido & Pérez-Mellado, 2014; Eterovick et al., 2016).
Finally, (4) are patterns of phenotypic and genetic dier-
entiation among urban populations generally concordant?
Considering that genetic dierentiation among Oviedo
city populations result from drift processes and bottleneck
events (Lourenço et al., 2017), a concordance between both
patterns would point to similar processes of random accu-
mulation of variance acting on morphology, while a devi-
ance would point to local sources of variation within the
urban environment.
Materials and methods
Sampling and Study site
We sampled adult individuals during rainy nights between
October-November 2020 in 10 urban populations from
Oviedo, and nine neighbouring native forest populations
(Table 1). Due to species-specic activity patterns, whereby
males are more active during the sampling period, and the
diculty of accessing certain urban populations, which are
located within historic buildings, enclosed convents, and
private estates, together with their small size, obtaining
either no females or a suciently large number of them to
yield consistent results was not feasible in many of them.
Thus, to avoid unbalanced sampling between sexes and
populations that may add noise or provide unreliable results,
only adult males were studied. Urban sampling points were
considered as dierent populations as they are genetically
isolated from each other (Lourenço et al., 2017). All popu-
lations from native forest (hereafter ‘forest’) were located
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Evolutionary Biology
Shape data Acquisition: Geometric Morphometrics
For the study and comparison of head morphological varia-
tion and asymmetry between urban and forest populations
we applied landmark-based geometric morphometrics
(GM), adapting the methodology described in Alarcón-Ríos
et al. (2017), which has been shown to accurately identify
shape dierences at a very ne biological scale (popula-
tion) while maintaining a low, non-systematic measure-
ment error. We digitized 13 xed landmarks and 32 sliding
semilandmarks using tpsDig2 v 2.21 (Rohlf, 2015), which
capture main morphological features of the dorsal view
of salamanders’ head: snout, upper jaw, eyes and parotid
glands (Fig. 1d). One of us (L.A-R) digitized all individuals
twice to take measurement error into account. In the total
sample size (N = 377), we did not consider any individual in
which image quality or the existence of any morphological
condition, such as missing eyes (Velo-Antón et al., 2021),
prevented the accurate digitization of all landmarks.
To explore patterns of head morphology between (urban
vs. forest sites) and within habitats, and examine develop-
mental stability we rst performed a Procrustes ANOVA
using the bilat.symmetry function from the R-package geo-
morph (Adams et al., 2021; Baken et al., 2021). This func-
tion implements a Procrustes ANOVA with individual, side,
and their interaction as main factors to test for the presence
of Directional Asymmetry (DA, corresponding to the side
within forested patches of variable sizes (Fig. 1a) primar-
ily composed of oaks (Quercus robur), chestnuts (Casta-
nea sativa), and birches (Betula spp.), with some riparian
species, and a well-developed understory. Forest sampling
points were located more than two kilometres apart from
each other, the maximum distance of dispersion in this
species (Hendrix et al., 2010). All forest populations were
within a radius of nine kilometres from the city to keep geo-
graphic environmental variation between habitat groups to a
minimum (Fig. 1). We examined 377 individuals (188 from
urban and 189 from forest sites) (Table 1).
After collection, animals were anesthetized (benzocaine;
Ethyl 4-aminobenzoate; Sigma-Aldrich, Darmstadt, Ger-
many. Product number: E1501. Ref.: 12,909) to facilitate
animal handling and data collection. We obtained high-res-
olution pictures of the dorsal view of the head following the
methodology for image acquisition described in Alarcón-
Ríos et al. (2017), and released all animals, after recovery
from anaesthesia, at the place of capture within the follow-
ing 24 h. Salamanders were captured and processed under
the collection permits provided by the regional government
of Asturias, Spain (Nº Expte: AUTO/2020/671). All appli-
cable national and institutional guidelines for the care and
use of animals were followed.
Table 1 Summary table of all studied populations from urban and forest habitats, sample sizes, shape variation (mean FA index and PV values
considering size eects by population) and genetic traits (Ho, R and Ne). Genetic and demographic data was obtained from Lourenço et al., 2017
Population Code Habitat N mean
FA index
PV Ho R Ne
Facultad Biología FB Urban 26 1.870 1.10 × 10− 3 0.678 0.094 46
Plaza de Toros PT Urban 20 1.862 9.25 × 10− 4 0.68 0.099 126
Club de Tenis TEN Urban 18 1.901 1.08 × 10− 3 0.737 0.125 30
San Pedro de los Arcos ARC Urban 19 1.864 1.24 × 10− 3 0.658 0.136 70
Jardines Seminario SEM Urban 18 1.876 8.50 × 10− 4 0.641 0.129 348
Calle Muérdago-Otero OTE Urban 21 1.871 9.21 × 10− 4 0.721 0.137 21
Campus del Milán MIL Urban 17 1.866 9.65 × 10− 4 0.658 0.265 15
Patio Catedral CAT Urban 33 1.862 1.07 × 10− 3 0.663 0.116 22
Patio Peregrinos PER Urban 8 1.868 4.79 × 10− 4 - - -
Monasterio MON Urban 8 1.863 1.05 × 10− 3 0.619 0.288 18
Total Urban 188
San Miguel de Lillo LIL Forest 18 1.867 9.41 × 10− 4 - - -
Villamar VMA Forest 14 1.843 7.56 × 10− 4 - - -
Bendones BEN Forest 19 1.850 1.4 × 10− 3 - - -
Pozoval POZ Forest 32 1.862 8.30 × 10− 4 - - -
Latores LAT Forest 17 1.879 7.66 × 10− 4 - - -
Brañes BRA Forest 26 1.851 8.52 × 10− 4 - - -
Soto del Rey REY Forest 22 1.866 9.12 × 10− 4 - - -
Laviada LAV Forest 15 1.860 6.87 × 10− 4 - - -
La Folguera FOL Forest 26 1.863 8.49 × 10− 4 - - -
Total Forest 189
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Evolutionary Biology
components of developmental (in)stability (see further on
for details on statistical procedures).
We implemented all GM procedures and statistical analy-
ses using packages geomorph v. 4.0 (Adams et al., 2021;
Baken et al., 2021) and RRPP v. 1.0 (Collyer & Adams,
2018, 2019) in the R language for statistical programming R
(R Development Core Team, 2022). We assessed statistical
signicance based on 10,000 random permutations, using
Residual Randomization (Collyer et al., 2015).
Morphological Dierentiation between and within
Habitats
We rst explored global patterns of morphological variation
between and within habitats. For this purpose, we compared
the symmetric component of head shape between habitats
using distance-based Procrustes ANOVAs and including
sampling population as a factor nested within habitat. As
eect) and/or Fluctuating Asymmetry (FA, corresponding
to the individual × side interaction) while accounting for
measurement error (following Klingenberg et al., 2002). We
performed these tests in all populations separately and on
the complete data set to identify coherent shape components
across the entire sample.
After testing for asymmetry patterns, we aligned the
average of head shape coordinates from both replicates of
each individual using a generalized least-squares Procrustes
superimposition (GPA; Rohlf & Slice, 1990; Rohlf, 1999)
to standardize in size, location and orientation. Then, we
repeated the procedures of bilat.symmetry function to obtain
averaged coordinates of mirrored individual congurations,
thus isolating only the symmetric shape component for
downstream analyses of shape variation across populations;
while we used the total (averaged across individual repli-
cates and superimposed) shape variation to examine some
Fig. 1 Map displaying the distri-
bution of studied populations in
urban (blue) and forest (orange)
habitats. The inset shows the
location of Oviedo city in the
Iberian Peninsula (Spain and
Portugal) (a). Picture of Pozoval
(POZ) as an example of a forest
population (b). Picture of Pere-
grinos (PER) as an example of an
urban population (c). Landmarks
(red circles) and semilandmarks
(white circles) recorded on the
dorsal view of salamanders’ head
for geometric morphometrics
analysis (d)
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Evolutionary Biology
(Adams et al., 2021; Baken et al., 2021). In both analyses
we accounted for size eects on shape including CS as a
covariate in the model. Populations PV from dierent habi-
tats were then compared using a permutational ANOVA.
As a second measure of developmental canalization,
we calculated the deviation from the size-shape allometric
relationship following Lazic et al. (2015), also considering
only the symmetric part of shape variation. As both habitats,
and populations within them, presented the same allome-
tric slopes (see Results), we used a common allometric line
for all individuals. We then tested for dierences in these
deviations from the allometric line through a permutational
ANOVA with habitat and population nested within habitat
as factors.
Fluctuating Asymmetry PatternsFluctuating Asymmetry
Patterns
To evaluate dierences in the level of developmental insta-
bility between habitats and among populations we rst
extracted an individual unsigned asymmetry index (FA
index) for head shape. This index was calculated as the
square root of sum of squared dierences between original
and reected copies of bilateral landmark coordinates fol-
lowing Lazić et al. (2015). Then, we tested whether levels of
FA diered between habitats and across populations within
each habitat through a permutational ANOVA with habitat
and population, nested within habitat, as factors. Then, to
evaluate whether levels of FA varied with size, and if there
were dierences in allometric relationship between FA and
size in each habitat and among populations we repeated the
permutational ANOVA including size as a covariate.
Association between head Morphology and Genetic
Background in Urban Populations
We evaluated the correlation between patterns of morpho-
logical variation in urban populations (population PV and
mean FA of each population) and population mean hetero-
zygosity (Ho), relatedness (R) and eective population size
(Ne) using permutation tests with 1,000 resampling cycles
without replacement. Genetic and demographic data were
obtained from a previous study in which sampling was con-
ducted four years earlier (Lourenço et al., 2017), and which
includes all urban populations analyzed in this study except
for population PER (Table 1).
To evaluate the role of drift as driver of morphological
dierentiation among urban populations we examined the
correlation between the matrix of pairwise genetic distances
(FST) (extracted from Lourenço et al., 2017) and the matrix
of pairwise morphological distances (Euclidean distances
of population mean shapes after correcting for size eects)
a measure of head size we used the logarithm of Centroid
Size (logCS), calculated as the square root of the summed
squared distances of each landmark from the centroid of the
landmark conguration (Dryden & Mardia, 2016), which is
uncorrelated with shape in the absence of allometry. Then,
we analysed the allometric relationship between head shape
and size by repeating the Procrustes ANOVA including head
size as a covariate and its interaction with habitat and popu-
lation (nested within habitat). This allowed us to investigate
the covariation between head shape and size, test for com-
mon allometric slopes between habitats and among popu-
lations within habitats and evaluate habitat and population
dierentiation in shape while accounting for size eects on
shape.
To further explore levels of dierentiation among pop-
ulations within each habitat, and determine which habitat
presented higher levels of dierentiation among the popu-
lations within it we used the function morphol.disparity in
geomorph package (Adams et al., 2021; Baken et al., 2021).
We compared Procrustes variances across all populations
within each habitat, calculated from the residuals of a model
that considered population size-corrected shape.
To visually explore patterns of dierentiation in morpho-
space of studied populations we produced a two rst prin-
cipal components plot that included least-squares means for
each analysed population after accounting for allometric
shape variation and 95% nonparametric condence ellipses
for each group as a measure of the precision of group mean
estimation.
Morphological Signs of Developmental Disturbance
To investigate whether the urban environment aected
salamander head development we compared three dierent
components of shape variation between habitats: total head
shape variance (PV), deviations from the shape-size allo-
metric slope as a measure of canalization, and uctuating
asymmetry (FA) as a measure of developmental instability
(Lazić et al., 2015).
Head Shape Variance and Deviations from Allometric
SlopeHead Shape Variance and Deviations from Allometric
Slope
We rst tested whether urban populations exhibited higher
levels of head shape variance (PV) than forest ones as a
proxy of the levels of developmental canalization. Using the
symmetric component of head shape variation (see above),
we estimated: (1) overall PV within each habitat consider-
ing habitat mean; (2) PV within each population, consid-
ering population mean in the model, using the function
morphol.disparity as implemented in the package geomorph
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Evolutionary Biology
comparing habitats considering population PV (Table 1), we
found no signicant dierences between them (Table 3a).
Signicant dierences in the degree of deviation from
the allometric slope existed among populations within each
habitat but not between habitats (Table 3b).
Fluctuating Asymmetry Patterns
Procrustes ANOVAs performed for head shape revealed the
existence of DA and FA (i.e., signicant side and individual
× side terms respectively), both when populations are con-
sidered separately (Table S1) or when they are all analysed
together (Table 4). However, our results did not nd dier-
ences in FA levels between habitats or among populations.
Moreover, FA did not vary with head size, and the lack of
signicant dierences between habitats and among popula-
tions persisted when accounting for head size (Table 3c).
Associations between Morphology and Genetic
Background in Urban Populations
We nd a marginally signicant positive relationship
between Ho and FA (r = 0.653; P = 0.046). However, this
relationship was no longer signicant after removing an
extreme outlier value (TEN) (r = 0.179; P = 0.315). We
therefore did not have strong evidence that either of het-
erozygosity (Ho), relatedness (R), or eective population
size (Ne) were correlated with levels of developmental
instability (mean FA index) (R: r = -0.223, P = 0.699; Ne:
r = 0.069, P = 0.265) or levels of PV (phenotypic disparity
within each population) (Ho: r = -0.009, P = 0.495; R: r =
-0.043, P = 0.519; Ne: r = -0.528, P = 0.949) among urban
populations.
using the Mantel test as implemented in the R package eco-
dist (Goslee & Urban, 2007).
Results
Morphological Dierentiation between and within
Habitats
Symmetric shape comparisons between habitats did not
show signicant dierences between individuals from
urban and forest populations (Table 2a). Conversely, indi-
viduals from both habitats diered in head size (Table 2b),
and an allometric relationship between shape and size
existed (Table 2c). However, when size eects were con-
sidered, urban and forest populations still did not dier in
shape (Table 2c).
Procrustes ANOVAs also showed a signicant varia-
tion across populations within each habitat, irrespective of
size eects on shape (Table 2c). Furthermore, the degree
of dierentiation among populations was greater among
urban than among forest populations when considering
size eects on shape (dierentiation among urban popula-
tions:10.04 × 10− 4; dierentiation among forest popula-
tions: 8.91 × 10− 4; P = 0.045) (Fig. 2).
Morphological Signs of Developmental Disturbance
Head Shape Variance and Deviation from Allometric Slope
Analysis comparing levels of overall PV between habitats
resulted in signicantly higher levels of morphological dis-
parity among urban individuals (Urban PV: 12.03 × 10− 4;
Forest PV: 10.26 × 10− 4; P = 0.007) (Fig. 2). However, when
Table 2 Results of the permutational ANOVAs used to test dierences in head shape (a) and size (b) between habitats and among populations
within habitats, shape-size covariation, head shape dierentiation between habitats and among populations after accounting for allometric varia-
tion and common allometric slopes (c). Signicant values are in bold
df SS F ZP
a) Head Shape
Habitat 1 0.008 2.063 1.573 0.061
Population 17 0.066 3.832 10.656 < 0.001
Residuals 358 0.364
b) Head Size
Habitat 1 1.074 18.515 2.830 0.001
Population 17 0.986 4.765 5.717 < 0.001
Residuals 358 4.358
c) Head shape including size as covariate
Size 1 0.010 9.828 5.086 < 0.001
Habitat 1 0.008 2.222 1.698 0.046
Population 17 0.063 3.743 10.193 < 0.001
Size × Habitat 1 0.001 0.730 -0.379 0.644
Size × Population 17 0.020 1.164 1.206 0.117
Residuals 339 0.337
1 3
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Table 3 Results of the permutational ANOVAs used to test for dierences between habitats and across populations within each habitat in pheno-
typic variance (PV) (a), the deviation from the allometric slope (b) and in uctuating asymmetry (FA) index (c), without and while considering
variation in size. Signicant values are in bold
Df SS F ZP
a) Phenotypic Variance Habitat 1 2.96 × 10− 8 0.689 0.231 0.420
Residuals 17 7.29 × 10− 7
b) Deviation from allometric slope Habitat 1 0.007 1.868 1.374 0.088
Population 17 0.063 3.671 10.202 < 0.001
Residuals 358 0.359
c) Fluctuating Asymmetry Index i) No considering size
Habitat 1 0.009 3.815 1.447 0.070
Population 17 0.039 0.724 -0.765 0.774
Residuals 358 1.124
ii) Including size
Size 1 0.002 0.606 0.206 0.438
Habitat 1 0.007 2.982 1.233 0.110
Population 17 0.039 0.709 -0.818 0.789
Size x Habitat 1 0.001 0.254 -0.270 0.607
Size x Population 17 0.038 0.698 -0.844 0.802
Residuals 339 1.085
Fig. 2 Means and 95% condence ellipses of the rst and second prin-
cipal components of head shape variation across individuals in each
studied population from both habitats. Deformation grids depict shape
change at the extremes of the rst axis in comparison to the overall
mean. Shape change has been magnied by a factor of three to facili-
tate visualization
1 3
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Evolutionary Biology
(Thompson et al., 2022). The city of Oviedo was founded
during the late 8th century and urban salamander popula-
tions became progressively isolated following the expan-
sion and urbanization of the city, suering bottlenecks and
drift processes that shaped the levels and structure of neutral
genetic diversity (Lourenço et al., 2017). Drift and founder
eects associated to reduced population size and high levels
of isolation are expected to decrease intrapopulation genetic
and phenotypic variation and increase interpopulation dif-
ferentiation (Johnson & Munshi-South, 2017; Thompson
et al., 2022). Accordingly, urban salamander populations
present higher interpopulation dierentiation than forest
ones in head shape. Specically, PER and MON popula-
tions, which have the lowest sample size (N = 8) and occur
in small courtyards within the Cathedral (0.023 and 0.101
Ha respectively) (Fig. 1c), are two of the most dierenti-
ated populations along the rst axis of variance (Fig. 2).
Salamanders from the Cathedral (CAT, PER and MON) are
considered to be isolated since the construction of Oviedo’s
rst wall 12 centuries ago (Lourenço et al., 2017). Remark-
ably, other urban populations exhibiting considerable dier-
entiation such as TEN and SEM (Fig. 2), are those in which
isolation occurred longer ago, not considering the Cathedral
ones (Lourenço et al., 2017). Thus, it could be plausible that
time since isolation, and thus drift, may explain to some
extent phenotypic dierentiation among urban populations.
At the same time, cities are known to present high spa-
tial and temporal heterogeneity (Alberti et al., 2020), and
thus, observed pattern of dierentiation might also result
from local adaptive processes operating dierently across
the city. The mosaic of habitats within the city, which ranges
from city parks to stony courtyards (Fig. 1c, Lourenço et
al., 2017), may lead to ecological dierences across sites
(e.g., dierences in type and availability of preys, shelter, or
predators), which together with the restriction of gene ow
among urban populations might favour local adaptation pro-
cesses with the subsequent dierentiation among popula-
tions (Kozak et al., 2005; Littleford-Colquhoun et al., 2017;
Marques et al., 2022). In the present study, the lack of cor-
relation between phenotypic and neutral genetic dierentia-
tion patterns, suggests that local adaptive processes within
the city may be contributing to head shape dierentiation,
in a ‘city-archipelago’ fashion (Littleford‐Colquhoun et al.,
2017). However, in addition to local adaptation, we cannot
Similarly, we nd no signicant correlation between
genetic (FST) and phenotypic distances among populations
(Mantel r = 0.339; P = 0.271).
Discussion
Cities represent an extreme transformation of the original
habitats that are expected to aect organisms inhabiting
them at dierent levels (e.g., phenotypic, demographic, evo-
lutionary). Stress-related changes in phenotypic and life-
history traits are rarely detected until population viability
is severely compromised, but the indirect evaluation of the
ecacy of developmental processes can allow the timely
detection of urban populations under stress (Clarke, 1995).
Urban populations’ developmental pathways are expected
to be aected due to the exposition to urban stressors result-
ing in an increased developmental instability (i.e., FA) and
a higher frequency of phenodeviants (i.e., PV). Our results
in the fully terrestrial salamander S. s. bernardezi partially
support these predictions, as we found inconsistent results
between dierent measurements of developmental distur-
bance: we observed a higher PV in almost all urban popu-
lations, but no dierences in PV, allometric deviance and
FA levels between urban and forest habitats, which prevents
us from conrming a clear negative impact of urban con-
ditions over salamander populations, as quantied through
the study of head-shape developmental processes. On the
other hand, we found higher morphological dierentiation
among urban populations than among forest ones, suggest-
ing the existence of some mechanism driving phenotypic
divergence within Oviedo city.
Increased Urban Phenotypic Dierentiation but
Inconsistent Patterns in PV
Increased phenotypic variability is a general trend in
anthropic areas worldwide, and it can arise from develop-
mental, ecological and evolutionary processes (Alberti et
al., 2017; Thompson et al., 2022). On the one hand, high
phenotypic dierentiation among populations within a city
might result from eco-evolutionary processes associated to
the urban environment, such as isolation time, environmen-
tal heterogeneity, and the modication of selective pressures
Table 4 Results of the Procrustes ANOVA used to test for the presence of Directional (DA, side eect), and Fluctuating Asymmetry (FA, individual
× size eect) on the entire sample. Signicant values are in bold
df SS F ZP
Individual 376 1.753 4.238 -2.516 0.994
Side 1 0.160 145.271 7.969 < 0.001
Individual × Side 376 0.414 6.522 58.042 < 0.001
Individual × Side × Replicate 754 0.127
1 3
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Evolutionary Biology
higher phenotypic diversity observed in some populations
from the city.
No Dierences in Fluctuating Asymmetry or
Allometric Deviance
Contrary to our predictions, we did not nd evidence of a
disruption of developmental processes in FA or allometric
deviance. First, uniformity of allometric slopes across popu-
lations and habitats points to similar mechanisms controlling
head shape variation with respect to size. Additionally, the
lack of signicant dierences in the deviance from the allo-
metric slope between habitats reveals that allometric mech-
anisms are not altered in urban environments. Secondly,
urban populations do not exhibit higher FA than forest ones.
Here, we should emphasize that developmental stability is
trait-specic (Lazić et al., 2013; Karvonen et al., 2003), and
it depends upon the trait functionality, which determines the
strength of the control mechanisms underlying development
(Leamy & Klingenberg, 2005; Palmer & Strobeck, 1986). In
this sense, the head is a highly functional and relevant struc-
ture that is expected to be strongly buered developmen-
tally. As such, disturbances in developmental stability may
occur in a very ne scale, if at all, and not be easy to identify
in this structure. In addition, the degree of asymmetry can
vary along ontogeny. Our sampling design only includes
adult males, so the results can be biased by two dierent
mechanisms: selection against asymmetric individuals and
lower tness (Tocts et al., 2016), although the frequency of
head deformities in adult salamanders within urban popula-
tions (Velo-Antón et al., 2021) suggest no drastic eects of
asymmetry on survival; and the existence of buering devel-
opmental mechanisms that compensate asymmetry through-
out development (Lazić et al., 2016), although trends and
underlying mechanisms seem to be highly variable across
species (Lazić et al., 2017). Thus, investigating FA levels at
earlier stages of development might uncover dierent pat-
terns and the existence of stressors in urban environments.
Furthermore, our study did not observe a clear relation-
ship between levels of genetic diversity, Ne, relatedness,
and levels of head shape asymmetry or phenotypic variance
across urban populations. Similarly, those relationships
did not appear between genetic diversity and deformity
frequencies neither in a previous study (Velo-Antón et al.,
2021). Other studies exploring the relationship between
genetic diversity and developmental stability in other organ-
isms have provided highly variable results (Eterovick et al.,
2016; Garrido & Pérez-Mellado, 2014; Gilligan et al., 2000;
Graham et al., 2010; Pertoldi et al., 2006; Vøllestad et al.,
1999). However, it is important to note that environmental
and genetic factors may aect tness in a combined way,
and high genetic diversity might constitute a protection
completely rule out drift processes as an underlying driver
of morphological dierentiation among urban populations
(Clegg et al., 2002). Additionally, cities might modify natu-
ral and sexual selection pressures (i.e., relaxed selection)
(Lahti et al., 2009; Rodewald & Arcese, 2017; Santangelo et
al., 2022). For instance, the high morphological dierentia-
tion observed among urban salamander populations might
result from dierences in selection pressures over mor-
phological specialization across populations, allowing for
higher, and potentially adaptively successful diversication
in urban morphologies (e.g., Falvey et al., 2020). However,
further research on the consequences of head shape changes
in relationship with habitat structure and heterogeneity
would be necessary to evaluate more accurately the factors
underlying functional dierentiation between urban sites.
On the other hand, all those eco-evolutionary processes
that concur within cities can also help to understand observed
levels of variability (i.e., PV) in head shape within urban
populations. Despite a reduction in intrapopulation pheno-
typic diversity is expected in small and isolated urban popu-
lations (Johnson & Munshi-South, 2017; Thompson et al.,
2022), we observed that they generally present higher PV
values (but not signicantly dierent from forest ones), with
the exception of PER, which is the less variable studied pop-
ulation (Table 1). This trend could be suggesting a reduction
in the ecacy of the mechanisms buering developmental
canalization processes, understood as an organismal prop-
erty that promotes the production of consistent phenotypes
from a common genetic basis (Waddington, 1942; Willmore
et al., 2007). The disruption of canalization-related mecha-
nisms can have a genetic basis (e.g., expression of cryptic
genetic variation, recessive alleles, or mutations), or may
result from external factors that modify the accurate func-
tioning of developmental buering (see Takahashi, 2019
and references therein). Accordingly, phenotypic variability
has been observed to increase in inbred populations (Réaale
& Ro, 2003), or under stressful environmental conditions
such as nutritional or thermal stress in some organisms
(Gonzalez et al., 2014; Homann & Hercus, 2000; Ima-
sheva et al., 1999). Although with the data at hand we cannot
determine the exact mechanisms underlying the observed
generalized increase in morphological variability of urban
populations, we did not nd any correlation between genetic
diversity or inbreeding measures and phenotypic variance.
Indeed, Oviedo urban populations do not show signicantly
lower levels of genetic diversity than populations outside
the city (Lourenço et al., 2017), and thus, similar levels of
phenotypic variation could be expected. Therefore, it seems
more plausible that, in this system, the performance of
developmental canalization mechanisms could be impaired
in some populations due to environmental factors associated
to specic urban areas, which would in turn result in the
1 3
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Evolutionary Biology
although processes such as drift cannot be completely ruled
out, remarkable dierences in head morphology among
urban populations and the lack of correlation of such dif-
ferentiation with neutral genetic patterns indicate the pos-
sibility of distinct eco-evolutionary processes inuencing
phenotypic disparity within cities.
As a concluding remark, in their recent review Thompson
and collaborators (2022) drew attention to two key aspects of
urban evolutionary studies. First, the importance of consid-
ering phenotypic disparity, more than means, when assess-
ing phenotypic consequences of urbanization. Second, the
relevance of sampling design (i.e., population denition),
and the implications of making groups by contrasting habi-
tats not considering heterogeneity and structure within each
of them. Our results are in line with both ideas, as important
patterns of dierentiation between habitats only arise when
considering morphological variation, but not when compar-
ing morphological means. Indeed, the higher morphological
dierentiation that exists among urban populations high-
lights the need of considering multiple populations to cover
the heterogeneity that could exists within each habitat to
accurately understand the patterns that arise and pinpoint
the possible underlying mechanisms.
Supplementary Information The online version contains
supplementary material available at https://doi.org/10.1007/s11692-
024-09635-6.
Acknowledgements We thank R. Álvarez for help during eld
work and two anonymous reviewers and the editor for their insight-
ful comments and reviews. This work was supported by National
Funds through FCT—Foundation for Science and Technology to
G.V.-A. (SALOMICS: grant number PTDC/BIA-EVL/28475/2017;
ANTHROPOPHIBIAN: grant number PTDC/BIA-CBI/2278/2020).
L.A.-R. was supported by a post-doctoral research contract in SALO-
MICS project and a ‘Margarita Salas contract’ funded by the Euro-
pean Union - NextGenerationEU, Recuperation, Transformation and
Resilience Plan by Spanish Ministry of Universities, on the basis of
the University of Oviedo (Spain) call, Ref: MU-21-UP2021-030;
GVA was supported by the FCT (CEECIND/00937/2018), and by a
Ramón y Cajal research grant (Ref. RYC-2019-026959-I/AEI/https://
doi.org/10.13039/501100011033); A.K. is supported by a Ramón y
Cajal research grant co-funded by the Spanish State Research Agency
and the European Social Fund (RYC2019-026688-I/AEI/https://doi.
org/10.13039/501100011033).
Author Contributions L. A.-R. and G. V.-A. designed the study. L. A.-
R., G. V.-A. and D.A. carried out sampling and data collection. L.A.-R.
and A.K. carried out analysis, L.A.-R. led the writing to which all au-
thors contributed. All authors read and approved the nal manuscript.
Funding This work was supported by National Funds through FCT—
Foundation for Science and Technology to G.V.-A. (SALOMICS: grant
number PTDC/BIA-EVL/28475/2017; ANTHROPOPHIBIAN: grant
number PTDC/BIA-CBI/2278/2020). L.A.-R. was supported by a
post-doctoral research contract in SALOMICS project and a ‘Margari-
ta Salas contract’ funded by the European Union - NextGenerationEU,
Recuperation, Transformation and Resilience Plan by Spanish Ministry
of Universities, on the basis of the University of Oviedo (Spain) call,
against other environmental stressors (Joubert & Bijlsma,
2010; Kristensen et al., 2006). Thus, the lack of dierences
in FA between urban and forest populations may result from
the existence of moderate to high levels of genetic diversity
in Oviedo (Lourenço et al., 2017), which are very similar, or
even higher, to other distant populations occurring in larger
forest areas throughout several S. salamandra subspecies
(Antunes et al., 2018, 2021; Lourenço et al., 2019; Velo-
Antón et al., 2012). Indeed, a high Ne/N ratio was found in
a urban salamander population in Oviedo (Álvarez et al.,
2015), suggesting putative mechanisms of genetic compen-
sation (e.g., high levels of multiple paternity; Alarcón-Ríos
et al., 2020) to prevent inbreeding depression, which could
also buer against developmental disturbances.
Finally, specic life-history traits of the study system
should be considered when interpreting the impacts of
urban environments. For S. salamandra, in particular, the
pueriparous reproductive mode of the examined popula-
tions reduces their direct exposition to stressors (e.g., pollut-
ants) during embryogenesis, since the developing embryos
remain protected within the mother’s body until birth after
metamorphosis (Buckley et al., 2007). Given that this is the
most sensitive stage of development (Møller, 1996; Pineda
et al., 2012), this might explain the absence of clear mor-
phological signs of developmental disturbance within the
urban habitat reported here. Pueriparity is a key trait for
the persistence of viable salamander populations across
Oviedo, in patches where water bodies for reproduction are
not available (Álvarez et al., 2015; Lourenço et al., 2017).
Furthermore, genetic Álvarez et al., 2015; Lourenço et al.,
2017) and morphological (this study) results suggest that
despite inhabiting a highly transformed environment, these
pueriparous salamanders have managed to persist and main-
tain stable urban populations. However, deciphering the role
that this evolutionarily derived reproductive mode may play
in the capacity of amphibian populations to cope with envi-
ronmental stressors requires comparative studies including
larviparous forms.
Conclusions and implications for urban evolutionary
studies
The lack of unambiguous evidence to conrm stressed
urban salamander populations, via developmental distur-
bance measures (i.e., developmental stability and canaliza-
tion processes), together with the reasonably larger levels of
genetic diversity observed across Oviedo urban populations
(Lourenço et al., 2017), suggest that the viability of Oviedo
urban salamanders is not apparently compromised. How-
ever, the increased levels of phenotypic variation observed
in most urban populations point to several potential scenar-
ios that would need further investigation. On the other hand,
1 3
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Ref: MU-21-UP2021-030; GVA was supported by the FCT (CEEC-
IND/00937/2018), and by a Ramón y Cajal research grant (Ref. RYC-
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Open Access funding provided thanks to the CRUE-CSIC agreement
with Springer Nature.
Data Availability Datasets used in the present study can be found in
Figshare https://doi.org/10.6084/m9.gshare.19771855.v2
Declarations
Competing Interests The authors declare no competing interests.
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