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Evolution in action: soil hardness influences morphology in a subterranean

rodent (Rodentia: Ctenomyidae)

Article  in  Biological Journal of the Linnean Society · January 2018

DOI: 10.1093/biolinnean/bly144/5165651

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Biological Journal of the Linnean Society, 2018, XX, 1–11. With 4 figures.

Evolution in action: soil hardness influences morphology

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in a subterranean rodent (Rodentia: Ctenomyidae)
BRUNO B. KUBIAK1,2*, , RENAN MAESTRI1, , THAMARA S. DE ALMEIDA1,
LEANDRO R. BORGES1, DANIEL GALIANO3, RODRIGO FORNEL4 and
THALES R. O. DE FREITAS1
1
Instituto de Biociências, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9500, 91501-
970 Porto Alegre – RS, Brazil
2
Departamento de Ciências Biológicas da Universidade Regional Integrada do Alto Uruguai e das Missões
– Campus de Frederico Westphalen, Av. Assis Brasil 709, 98400-000 Frederico Westphalen – RS, Brazil
3
Universidade Federal da Fronteira Sul, Campus Realeza, Rua Edmundo Gaievisk, 1000, 85770-000,
Realeza, PR, Brazil
4
Programa de Pós-graduação em Ecologia, Universidade Regional do Alto Uruguai e das Missões –
Campus de Erechim, Av. Sete de Setembro 1621, 99709-910, Erechim – RS, Brazil

Received 9 July 2018; revised 27 August 2018; accepted for publication 28 August 2018

A major interest of evolutionary biologists is to understand which environmental features are associated with mor-
phological and behavioural characteristics of species. Intraspecific studies addressing this question provide the best
evidence for ecology-driven evolution over short time scales. Here, we evaluated whether two adjacent habitats differ
in soil hardness and whether skull and forelimb morphology and estimated bite force differ between populations
of a single species from sand fields and sand dunes. We used a total of 39 humeri and 88 skulls and mandibles of
Ctenomys minutus from both habitats to estimate the bite force and generate morphometric data. Our results pro-
vide strong evidence that parapatric populations, occupying adjacent habitats, can respond differently in particular
circumstances. This indicates that C. minutus uses different strategies (i.e. scratch-digging and tooth-digging) in the
excavation of tunnels, and both of them respond to changes in soil hardness, establishing that the strategies are not
evolutionarily exclusive. This difference is probably a consequence of the harder soils found in the sand fields, which
are more difficult to excavate. Our results suggest the presence of divergent selection or strong phenotypic plasticity
in the excavation-related morphology of populations occupying different habitats.

ADDITIONAL KEYWORDS:  digging adaptations – divergent selection – geometric morphometrics – mammalian

skull – natural selection – phenotypic plasticity – scratch-digging – tooth-digging.

INTRODUCTION modifications related to digging is particularly

useful for this purpose, especially for animals that
Environmental features are associated with mor-
occupy excavated burrows. Several rodent lineages
phological and behavioural modifications in many
have evolved this lifestyle independently in different
species (Herrel et al., 2008; Losos & Mahler, 2010).
parts of the world, leading to parallel morphological
One of the major interests of evolutionary biolo-
specializations, such as having a cylindrical body and
gists is to understand the factors that guide these
reduced eyes, ears and tail (Nevo, 1979; Lacey et al.,
changes over microevolutionary time scales. In this
2000). It is also known that habitat differences (e.g.
context, one goal of evolutionary studies is to identify
soil hardness, type of vegetation cover) may influence
the factors that influence the different morpholo-
subterranean rodent excavation activities, resulting
gies of the structures involved in locomotion and
in different evolutionary responses in the mode of
other physical activities of animals. Research on
excavation, including scratch-digging, tooth-digging
or a combination of the two (Lehmann, 1963; Dubost,
*Corresponding author. E-mail: busnelo@hotmail.com 1968; Hildebrand, 1985; Lessa & Thaeler, 1989;

© 2018 The Linnean Society of London, Biological Journal of the Linnean Society, 2018, XX, 1–11 1
2  B. B. KUBIAK ET AL.
Lessa, 1990; Lessa et al., 2008; Giannoni et al., 1996;
Stein, 2000; Mora et al., 2003; Barčiová et al., 2009).
These two evolutionary responses involve different
strategies and structures during excavation. Scratch-
digging involves the use of the claws and forelimbs for
the construction of tunnels (Dubost, 1968; Hildebrand,
1985; Reichman & Smith, 1990; Nevo, 1999), whereas
tooth-digging involves the use of incisor teeth and the
skull to remove soil (Lessa & Thaeler, 1989; Vassallo,
1998). The use of these two strategies can lead to the
specialization of different skeletal characteristics,
with tooth-digging resulting in craniodental modifica-
tions and scratch-digging in postcranial modifications
(Vassallo, 1998; Lagaria & Youlatos, 2006; Morgan &
Verzi, 2006; Verzi & Olivares, 2006; Barčiová et al.,
2009; Hopkins & Davis, 2009; Morgan, 2009, 2015;
Steiner-Souza et al., 2010; Becerra et al., 2012; Morgan
& Álvarez, 2013; Echeverría et al., 2014, 2017; Álvarez
et al., 2015; Marcy et al., 2016; Borges et al., 2017).
However, most studies have addressed these strat-
egies separately, with a few exceptions that have
studied them in combination (Lessa & Thaeler, 1989;
Lessa & Stein, 1992; Lessa et al., 2008; Morgan et al.,
2017). Furthermore, these studies generate data that
allow inferences to be made regarding the macro-
evolutionary patterns associated with selection for
differences in craniodental and postcranial modifica-
tions. Nevertheless, to our knowledge no studies have
addressed the differentiation of the two strategies in
a single species or analysed the microevolutionary
results in distinct populations. Such single-species
studies are important for the generation of com-
plementary information regarding the factors that
influence selection for craniodental and postcranial
structure.
Among subterranean rodents, species in the genus
Ctenomys possess characteristics that make them an
ideal experimental model for testing the factors that
influence the differentiation of craniodental and post-
cranial structures related to excavation activities.
These animals perform most vital activities below
the soil surface, constructing and inhabiting tunnel
systems that they excavate predominantly using the
scratch-digging strategy (Lehmann, 1963) and second-
arily using the incisors for burrowing (tooth-digging)
(Dubost, 1968; Ubilla & Altuna, 1990; Vassallo, 1998;
Stein, 2000). Among subterranean rodents, Ctenomys
has the highest richness, with ~70 species (Freitas,
2016), although the soil conditions and predominant
digging behaviour for most of them have not been
well described (Ubilla & Altuna, 1990; Vassallo, 1998;
Stein, 2000; Lessa et al., 2008). These species produce
a series of variations according to the different char-
acteristics of the habitats that they inhabit, many of
them related to excavation (e.g. variation in bite force,
changes in the shape and size of structures such as
© 2018 The Linnean Society of London, Biological Journal of the Linnean Society, 2018, XX, 1–11
the skull and humerus, and differences in mode of
digging; Nevo, 1979; Vassallo, 1998; Lacey et al., 2000;
Morgan & Verzi, 2006; Morgan, 2009, 2015; Steiner-
Souza et al., 2010; Becerra et al., 2012; Morgan &
Álvarez, 2013; Echeverría et al., 2014; Álvarez et al.,
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2015; Borges et al., 2017). Ctenomyids are herbivorous
rodents, feeding on both aerial and underground plants
collected. They cut vegetable aerial parts directly from
the surface by exiting burrow openings, moving through
small areas on the surface (Vassallo, 1998; Lacey et al.,
2000; Lopes et al., 2015). Ctenomyids are distributed
among an extensive variety of habitat types, but are
mainly found in open vegetation (savannas, deserts
and dunes) and, in some cases, in forest habitats
(Lacey et al., 2000; Stolz et al., 2013; Gardner et al.,
2014; Ojeda et al., 2015); consequently, these species
can occupy sites with various soil characteristics. This
diversity in habitat types might be related to the use of
two forms of excavation, allowing the successful occu-
pation of diverse environments, especially those that
might be considered challenging (e.g. hard soils, soils
with a high proportion of roots; Morgan et al., 2017).
However, each species typically occurs in only a single
habitat type (Lacey et al., 2000). In contrast to the
general distribution patterns for the genus, Ctenomys
minutus occupies two types of habitats: sand fields and
sand dunes (Freitas, 1995; Lopes et al., 2013; Galiano
et al., 2014, 2016) (Fig. 1). These habitats have marked
differences in plant biomass, with sand fields having
significantly higher above- and below-ground values
(Galiano et al., 2014; Kubiak et al., 2015). Moreover,
the home range size of this species varies with habitat,
with individuals living in sand dunes tending to have
larger home ranges (Kubiak et al., 2017). The selection
of food items also differs between the two habitat types
(Lopes et al., 2015). Evaluating this scenario can help
to elucidate the relevant environmental character-
istics that influence patterns of morphological vari-
ation related to excavation strategies in subterranean
rodents.
In this study, we evaluate whether postcranial
(humerus morphology) and craniodental structure
(skull morphology) and estimated bite force of
C. minutus differ in populations from different habitats
(sand dunes vs. sand fields) and whether these differ-
ences are related to soil hardness. We hypothesize that
soil hardness affects excavation-related morphology,
with the specific prediction that individuals inhabiting
sand fields should have a stronger bite force than indi-
viduals inhabiting sand dunes owing to differences in
habitat features with potential to influence selection
on bite force (i.e. plant biomass and soil hardness).
In addition, we also predict differences in excavation
strategies between individuals that might generate
changes in skull and humeral morphological features
related to digging.
 HABITAT-DRIVEN MORPHOLOGICAL DIFFERENCE  3

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Figure 1.  Geographical distribution of Ctenomys minutus in the coastal plain of southern Brazil in sand dunes and sand
fields. Squares represent sampling sites for soil hardness in sand fields (white squares) and sand dunes (grey squares), and
circles represent sample locations for skulls in sand fields (white circles) and sand dunes (grey circles).

MATERIAL AND METHODS
Soil hardness
We used the data generated by Galiano et al. (2014,
2016) and Kubiak et al. (2015) to investigate differ-
ences in soil hardness at nine different locations: six
areas in sand fields and three areas in sand dunes
(see Fig. 1). A minimum of ten measurements of soil
hardness were made in each area (all were occupied
by C. minutus) using a soil penetrometer (see details
provided by Galiano et al., 2014, 2016; Kubiak et al.,
2015). We used soil hardness at depths of 10 and
20 cm in the analyses because these correspond to the
soil depth that this species inhabits (Galiano et al.,
2014).
Sampling
We evaluated 88 skulls and mandibles of adult
C. minutus (juveniles were not used in this study)
to estimate bite force and to generate morphometric
data. We used 38 skulls and mandibles from specimens
collected at six locations within the sand dune habitat
(13 males, 17 females and eight not identified). We
also evaluated 50 skulls and mandibles collected from
eight locations in the sand field habitat (28 males and
22 females) (Fig. 1). Likewise, we used the humeri of
39 individuals, 16 from the sand dunes (six males and
ten females) and 23 from the sand fields (12 males,
nine females and two undetermined). We excluded
juveniles from the sample based on the small size of
the skull and humeri, because morphological charac-
teristics are not fully developed in both size and shape,
which could bias the analyses. All humeri, skulls and
mandibles were deposited with the specimen collec-
tion at the Laboratório de Citogenética e Evolução at
the Departamento de Genética of the Universidade
Federal do Rio Grande do Sul. Collection numbers
and locations of each are presented in the Supporting
Information (Table S1).
Bite force estimations and
upper incisor procumbency
Bite force was estimated for each individual using
the method proposed by Freeman & Lemen (2008).
Two measurements were taken of the lower incisor:
(1) length (anterior–posterior length); and (2) width
(medial–lateral width). The following formula was
subsequently applied: Zi = [(anterior–posterior length)
× 2(medial–lateral width)]/6, where Zi is the index of
incisor strength. Freeman & Lemen (2008) found that
this index correlates closely with individual bite force
measurements in vivo, with a correlation coefficient
of 0.96. After determining Zi, we used the regression
equation provided by the same authors to transform

© 2018 The Linnean Society of London, Biological Journal of the Linnean Society, 2018, XX, 1–11
4  B. B. KUBIAK ET AL.
values to newtons. See the Supporting Information
(Table S1) for individual bite force values.
Procumbency of the upper incisor was measured
as the ‘angle of Thomas’ (Reig et al., 1965). In the
lateral view of the skull, this angle is delimited by the
grinding plane of the molariforms and the straight line
going through the tip of the incisor and the posterior
ridge of its alveolus (see Lessa et al., 2008; Echeverría
et al., 2017). Differences in procumbency of the upper
incisor between habitats were verified by a t-test.
Geometric morphometrics approach
We used the same humeri, skulls and mandibles to
obtain the shape variables. We used a digital camera
(Nikon Coolpix P100, 13.1 megapixels, 3648 × 2736
resolution) to produce images of the mandible and
the dorsal, ventral and lateral view of the skull of
each specimen. The position and distance between the
camera and the subjects were the same for all speci-
mens. We chose 29 landmarks that were digitized in
the dorsal view of the skull, 30 in the ventral view and
21 in the lateral view (Fernandes et al., 2009), and
13 in the mandible (Fornel et al., 2010). We chose 19
landmarks in the dorsal view of each humerus, 19 in
the ventral view and seven in the distal/proximal view
(Steiner-Souza et al., 2010; for landmark positions, see
Supporting Information, Figs S1, S2). The anatomical
landmarks were digitized using TPSDig2 v.2.17 (Rohlf,
2015). The resulting matrices of coordinates were
superimposed through generalized Procrustes analysis
(GPA), which removes the effects of scale, orientation
and position. Geometric morphometric procedures
were performed with the geomorph package (Adams &
Otárola-Castillo, 2013).
Statistical analyses
To evaluate differences in soil hardness and vegetation
cover between the two habitat types, we performed
ANOVAs. We used the average hardness values for
comparison among areas to avoid pseudo-replication.
Plots of the residuals were checked for normality and
homoscedasticity.
We used an ANCOVA to test for relationships
between estimated bite force and habitat type after
controlling for skull length and sex. Estimated bite
force correlates significantly with body size (Freeman
& Lemen, 2008) and may exhibit sexual dimorphism,
so we used size (skull length) and sex as covariates
to control for this. We used a series of partial least-
squares (PLS) analyses (Rohlf & Corti, 2000) to inves-
tigate the relationship between bite force and shape in
the dorsal, ventral and lateral views of the skull and
the mandible.
© 2018 The Linnean Society of London, Biological Journal of the Linnean Society, 2018, XX, 1–11
We used a MANCOVA to verify differences in
humerus shape in the different habitats, also using
size and sex as covariables. An ANOVA was used to
analyse differences in humerus size between habitats.
We also used a linear discriminant analysis with a
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leave-one-out cross-validation procedure to evaluate
the percentages of correct assignation of humerus
between the sand dunes and sand fields. All analyses
were performed using the R program (R Core Team,
2016) with the vegan (Oksanen et al., 2012) and
geomorph packages (Adams & Otárola-Castillo, 2013).
RESULTS
Comparisons of soil hardness showed that the sand
fields have harder soils than the sand dunes at depths
of both 10 and 20 cm (F 1,7 = 10.68, P = 0.013 and
F1,7 = 18.59, P = 0.003, respectively; Fig. 2). Individuals
from sand fields have a stronger estimated bite force
than individuals from sand dunes (F 7,72  = 15.35,
P < 0.001), and males have a stronger estimated bite
force than females (F7,72 = 12.47, P < 0.001). We also
found a positive correlation between skull length
and estimated bite force (F 7,72 = 140.64, P < 0.001).
However, there was no interaction between sex and
habitat, because males have a stronger estimated bite
force in both habitat types. The estimated bite force
and average skull lengths are summarized in Table 1.
No difference was found between procumbency of the
upper incisor of individuals occupying sand fields
Figure 2.  Soil hardness expressed as the pressure (kg/
cm2) needed to penetrate the soil at two different depths
(10 and 20 cm) from sampling sites in sand dunes (white
boxes) and sand fields (grey boxes). The left-hand y-axis
indicates hardness values at 10 cm depth, and the right-
hand y-axis indicates hardness values at 20 cm depth.
Asterisks indicate a significant difference between habitat
types.
 HABITAT-DRIVEN MORPHOLOGICAL DIFFERENCE  5

(89.94 ± 18.64°) and sand dunes (89.28 ± 5.30°)
(T = 0.90, P = 0.184).
PLS analyses indicated that the shapes of all skull
and mandible views are strongly correlated with esti-
mated bite force (dorsal, r = 0.81; ventral, r = 0.74;
the closer position of landmarks 1 and 7 on the man-
dible (Fig. 3D).
In addition, differences in the shape and size of the
ventral view of the humerus were found for animals
in the different habitat types (Table 2). However, the

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lateral, r = 0.74; and mandible, r = 0.60; Fig. 3).
Visualization of the changes in shape described
by the PLS shape vector (derived from skull data)
showed that the highest values of estimated bite
force were associated with rostral enlargement and
retraction of the skull base in the dorsal and ventral
views. At the opposite end of the same shape vector,
lower estimated bite force values were associated
with shortening of the rostrum and zygomatic arch
and an increase in the skull base (Fig. 3A, B). In the
lateral view of the skull, higher estimated bite force
values were associated with an increase in skull
height (Fig. 3C), and the jaw was relatively shortened
in association with higher bite force, as indicated by
dorsal view of the humerus showed a difference only in
shape and not in size, and the distal/proximal view did
not demonstrate statistically significant differences in
the shape and size of the humerus between the habitat
types (Table 2). The differences in shape in both the
ventral and the dorsal view were more noticeable in
the humeral head, specifically in the condyle, in add-
ition to the facet of the ulna and the articulation of the
facet of the ulna. The ventral view also showed differ-
ences in the deltopectoral crest, and the dorsal view
presented a greater tuberosity in the humeral head
and a difference in the radial facet and radial articu-
lation. In ventral view, individuals from the sandy
dunes had a larger humerus (Fig. 4). Additionally, in

Table 1.  Mean (± SD) estimated bite force and skull length in Ctenomys minutus from sand dune and sand field habitats

Sand dunes Sand fields

(13 males and 17 females) (28 males and 22 females)

Bite force (N) Skull length (mm) Bite force (N) Skull length (mm)

Males 50.94 ± 10.19 44.27 ± 2.57 59.31 ± 10.41 44.72 ± 1.94

Females 44.53 ± 7.72 43.08 ± 2.87 45.08 ± 6.83 41.78 ± 1.79
Total 46.93 ± 9.43 43.48 ± 2.68 53.04 ± 11.43 43.22 ± 2.47

Figure 3.  Representation of conformational changes associated with negative (grey lines; lower bite force) and positive
(black lines; higher bite forces) partial least-squares vectors in the cranium in dorsal (A), ventral (B) and lateral (C) views
and in the mandible in lateral view (D).

© 2018 The Linnean Society of London, Biological Journal of the Linnean Society, 2018, XX, 1–11
6  B. B. KUBIAK ET AL.

Table 2.  MANOVA of humerus shape (R2, F- and P-values) by size, sex and habitat type and ANOVA of centroid size (R2,
F- and P-values) between sexes and habitats in Ctenomys minutus

Dorsal view Ventral view Distal/proximal view

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R2 F P R2 F P R2 F P

Shàpe
Size 0.154 6.810 0.001** 0.0428 1.688 0.092 0.050 2.019 0.047*
Sex 0.020 0.453 0.980 0.0557 1.100 0.326 0.053 1.055 0.355
Habitat 0.057 2.504 0.017* 0.065 2.582 0.002** 0.043 1.695 0.070
Size
Sex 0.129 2.673 0.076 0.217 5.444 0.031* 0.138 1.887 0.076
Habitat 0.020 0.516 0.344 0.104 5.239 0.021* 0.0175 0.428 0.433

*P ≤ 0.05; *P ≤ 0.001.

both the dorsal and the ventral view of the humerus,
landmarks 1 (dorsal) and 10 (ventral) were located
more distally in individuals from the sandy fields
(Fig. 4). Linear discriminant analysis of each humerus
view indicated that individuals in the sandy fields had
slightly higher percentages of correct classification
than those in sand dunes (Table 3). In addition, the
ventral and dorsal views showed similar and overall
higher values (> 70%) of correct classification, whereas
the distal/proximal view showed a low level of correct
classification (Table 3).
DISCUSSION
Our results provide strong evidence that parapa-
tric populations, occupying adjacent habitats, can
respond differently in particular circumstances.
We demonstrate large differences in soil hardness
between the two different habitats occupied by
C. minutus; sandy fields have harder soils at depths
of 20 and 10 cm, which are the depths used by this
species for construction of tunnels (Gastal, 1994). We
also found significant differences in the structures
related to different excavation strategies between
habitats: estimated bite force and skull morphology
(related to tooth-digging) and humerus morphology
(related to scratch-digging). These differences might
suggest the use of both strategies by C. minutus in
the excavation of tunnels and that the animals use
them depending on changes in soil hardness, demon-
strating that the digging strategies are not evolution-
arily exclusive and corroborating findings by Morgan
(2009) and Marcy et al. (2016). However, modifica-
tions in humerus morphology, even if significant,
were less expressive than the skull modifications,
and estimated bite force, which is closely related to
skull morphology, showed the most pronounced vari-
ation between habitats.
Scratch-digging is considered to be the main
strategy within Ctenomys, which requires vigorous
scraping movements involving postcranial elements.
These species use the tooth-digging strategy second-
arily, with the incisors being used to loosen soil and
remove obstacles, such as roots, rocks and harder soils
(Dubost, 1968; Ubilla & Altuna, 1990; Giannoni et al.,
1996; Vassallo, 1998; Stein, 2000). Our data corrob-
orate this pattern, allowing us to infer that the exca-
vation strategy and the modifications involved are
guided by habitat characteristics (e.g. soil hardness).
However, to confirm this pattern it would be necessary
to analyse the digging behaviour of individuals in
both habitats. Vassallo (1998) found similar results
for Ctenomys australis and Ctenomys talarum; both
species use scratch-digging in areas with soft soils and
frequently use incisors only to cut roots. However, in
areas with more compact soils, only C. talarum effect-
ively uses the incisors for excavation, which generates
advantages in terms of excavation efficiency. Our data
cannot confirm which habitat type was first occupied
by C. minutus, but we find an association between
soil hardness and morphology based on the observed
differences in bite force in the different habitat types.
This might be because the use of tooth-digging as a
strategy depends on this habitat characteristic (i.e. soil
hardness). Because these habitats vary not only in soil
hardness but also in the availability of plant biomass,
the diet of the species might also be affected. Lopes
et al. (2015) found that individuals inhabiting sand
fields consume mainly plants of the families Poaceae
(68.69%), Fabaceae (17.88%) and Araliaceae (9.77%),
whereas individuals inhabiting sand dunes consume
Asteraceae (30.02%), Poaceae (29.80%) and Araliaceae
(18.31%). Therefore, differences in the diet of the indi-
viduals in different habitats might also be influencing
the observed pattern of bite force.
Differences in the estimated bite force of animals
occupying different habitats are closely related to

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 HABITAT-DRIVEN MORPHOLOGICAL DIFFERENCE  7

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Figure 4.  Representation of conformational changes associated with negative (black lines; sandy dunes) and positive (grey
lines; sandy fields) partial least-squares vectors in dorsal (A) and ventral (B) views of the humerus. Landmarks 2 and 7
represent the proximal part of the humerus, in each view.

Table 3.  Number of correctly classified specimens based small and not significant when compared with data
on shape of the humerus of Ctenomys minutus between from animals in sand dunes. The relationship between
habitat types, and the percentage of correct classification incisor procumbency angle and improvements in exca-
for the three views vation remains controversial, and some authors have
provided evidence that there is an association between
Sand fields Sand dunes Percentage rostrum–incisor procumbency and soil hardness (Reig
& Quintana, 1992; Mora et al., 2003; Lessa et al., 2008;
Dorsal view
Marcy et al., 2016), Echeverría et al. (2017) suggested
  Sand fields 17 7 74
that incisor procumbency angle might not be related to
  Sand dunes 5 11 69
biomechanical advantages in excavation. Our results
Ventral view
demonstrate that intraspecific modifications are
  Sand fields 20 03 87
not very subtle when comparing animals in habitats
  Sand dunes 5 11 69
Distal/proximal view
with markedly different soil hardness; thus, further
  Sand fields 14 10 61
evaluation is required to confirm whether changes
  Sand dunes 8 8 50 in procumbency of the upper incisor are related to
different strategies of excavation.
A jackknife, leave-one-out, cross-validation procedure was used to
The modifications in humerus morphology we
classify the specimens. found are similar to those described in other studies
comparing subterranean and non-subterranean rodents
changes in individual skull shape. Higher estimated (Hildebrand, 1985; Lessa & Stein, 1992; Fernández
bite forces are associated with a shorter jaw, implying et al., 2000; Morgan & Verzi, 2006; Lessa et al., 2008;
a shortening of the out-lever arm of mandibular Morgan & Álvarez, 2013) and in studies making compar-
muscles, which provides a mechanical advantage; this isons between strictly subterranean species (Vassallo,
phenomenon has already been described for other 1998; Steiner-Souza et al., 2010; Morgan et al., 2017).
species of Ctenomys in relation to the shortening of These modifications are related to areas that function
the skull (Versi, 2002; Lessa et al., 2008; Barčiová as extensor muscles and can generate better mech-
et al., 2009; Becerra et al., 2014). Even though indi- anical advantages for forearm movements and fixation
viduals occupying the sandy fields have a larger angle of the scapulohumeral joint (Steiner-Souza et al., 2010).
of incisor procumbency, these differences are very The distal displacement of landmarks 1 and 10 (same

© 2018 The Linnean Society of London, Biological Journal of the Linnean Society, 2018, XX, 1–11
8  B. B. KUBIAK ET AL.
point viewed in dorsal and ventral views) indicates the
site of greatest expansion of the deltoid process with
respect to the shaft of the humerus. This suggests a
greater mechanical advantage of the deltoid and pec-
toral muscles, by increasing the in-lever arm of these
muscles for use in harder soils (Echeverría et al., 2014;
Elissamburu & Vizcaíno, 2004). These modifications
are less pronounced than interspecific modifications
(Vassallo, 1998; Lessa et al., 2008; Steiner-Souza et al.,
2010; Morgan & Álvarez, 2013; Morgan et al., 2017), but
they are nonetheless important because they represent
the first demonstration of such intraspecific changes.
Similar trends have been observed in phylogenetically
closely related species occupying similar habitats, such
as C. minutus and Ctenomys lami, which show fewer
differences from one another than phylogenetically
distantly related species or species occupying different
habitats. Furthermore, the percentage reclassification
based on linear discriminant analysis was low for
these two species (Steiner-Souza et al., 2010), as in the
present study, owing to the similarities between traits.
Differences between the size of the humerus were
also similar to those described by Steiner-Souza et al.
(2010), in which the authors compared four species
of Ctenomys and showed that species occupying soils
with extreme differences in hardness have a smaller
humerus in harder soils and a larger humerus in softer
soils. The authors attributed this to the restriction of
size imposed by harder soils, which is related to exca-
vation activities and the selection of the optimal size
for digging (i.e. smaller animals might be selected
for in habitats with harder soil).
Soil hardness and plant cover are closely linked to
the ecology of subterranean rodents and can influence
their distribution (Miller, 1964; Reichman & Jarvis,
1989), excavation strategies (Hildebrand, 1985; Stein,
2000), changes in skull morphology (Barčiová et al.,
2009) and bite force on a macroevolutionary scale
(Borges et al., 2017). Furthermore, the occupation of
different habitat types influences behavioural aspects
of C. minutus, such as home range size (Kubiak et al.,
2017), in that animals occupying sand dunes have
larger home ranges than animals occupying sand
fields. Combining this with our results, we infer that
soil hardness also influences differentiation in exca-
vation strategies between populations and that soil
characteristics are closely related to the vital activi-
ties of subterranean animals and are thus key factors
for selection on species or population characteristics,
such as size, shape, home range area (Heth, 1989; Lövy
et al., 2015) and distribution (Miller, 1964; Reichman &
Jarvis, 1989), possibly influencing species divergence.
Ctenomys minutus showed sexual dimorphism in esti-
mated bite force. In addition to interhabitat differenti-
ation, males exhibit greater estimated bite force than
females in both habitats. Similar results were described
© 2018 The Linnean Society of London, Biological Journal of the Linnean Society, 2018, XX, 1–11
for C. talarum (Becerra et al., 2011), corroborating the
idea that differentiation in bite force between sexes
should be a result of sexual selection. This selection is
likely to be associated primarily with male dominance,
owing to the polygynous mating system of the genus
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Ctenomys, in which males engage in aggressive interac-
tions with other males (Zenuto et al., 1999a, b). Genetic
data corroborate these results, and although C. minutus
individuals have the same karyotype (2n = 46a) in both
sand fields and sand dunes (Freitas, 1997), molecular
markers such as mitochondrial DNA show differences
in haplotypes between habitats. This was also described
using microsatellite DNA, which suggests a lack of gene
flow between the habitats (Lopes et al., 2013). Ctenomys
minutus did not show sexual dimorphism in morph-
ology of the humerus, similar to what has already been
described in a previous study of this species (Steiner-
Souza et al., 2010).
A potential drawback of our analyses is the indirect
estimates of bite force from linear measurements of the
lower incisor using the formula derived by Freeman &
Lemen (2008). The formula was derived for a sample
including specimens of 13 species of rodents, repre-
senting a large range of mandible shapes and sizes
and also encompassing a wide range of lifestyles. Such
estimates are well suited to the interspecific level, but
show mixed support in the intraspecific level (Freeman
& Lemen, 2008), and this and other estimators of bite
force can show imprecise results at the intraspecific
level (Ginot et al., 2018). However, we have strong evi-
dence that different habitats influence the character-
istics of individuals from adjacent populations. Our
results show that there is variation in the size and
shape of the humerus and skull of C. minutus, related
to the habitat occupied, and these modifications are
related to excavation activities. However, measure-
ments of in vivo bite force are necessary to confirm the
results presented.
In summary, our data point to the possibility of
divergent selection in excavation strategies in C. minutus
populations occupying sand dunes and sand fields. In
addition, our results, together with those described by
Kubiak et al. (2017), indicate that this species shows
differentiation in two characteristics (home range size
and modifications in structures involved in excavation
activities) that is directly associated with habitat.
Therefore, we infer that habitats with differences in soil
hardness and vegetation cover can directly influence
behavioural characteristics in subterranean rodents, as
has been proposed for other species (Heth, 1989; Rosi
et al., 2000; Spinks et al., 2000; Sumbera et al., 2003;
Romañach et al., 2005). Consequently, this divergent
selection could lead to sympatric speciation, as reported
in recent studies (Polyakov et al., 2004; Hadid et al.,
2013; Li et al., 2015; Lövy et al., 2015; Šklíba et al.,
2016). Unfortunately, the results generated here do not
 HABITAT-DRIVEN MORPHOLOGICAL DIFFERENCE  9
allow us to discriminate accurately between adaptation
and phenotypic plasticity, the two probable outcomes
of selection driven by habitat. Future studies should
address whether differences in the estimated bite force
are a result of adaptation or simply an expression of
phenotypic plasticity (e.g. by evaluating bite force in
newborns and juveniles from both habitats). In add-
ition, studies evaluating other postcranial structures
and muscles involved in excavation activities might
help us to gain a better understanding of the influence
of soil hardness on the selection of digging strategies for
subterranean rodents.
ACKNOWLEDGEMENTS
We are grateful to our colleagues from the Laboratório
de Citogenética e Evolução of the Departamento de
Genética at Universidade Federal do Rio Grande do Sul
for support during various stages of this research. We
thank Aldo I. Vassallo and the other anonymous reviewer
for their helpful comments. This study was supported by
the Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq), the Coordenação de Apoio de Pessoal
de Nível Superior (CAPES) and the Fundação de Amparo
a Pesquisa do Rio Grande do Sul (FAPERGS).
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SUPPORTING INFORMATION
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Table S1. Full list, with identification numbers, for the collection in Laboratório de Citogenética e Evolução da
Universidade Federal do Rio Grande do Sul, of the sex, habitat, locality and estimated bite force (in newtons) of
all skulls of Ctenomys minutus used in the study.
Figure S1. Landmarks used to capture shape from the dorsal (A), ventral (B) and lateral (C) view of the skull and
the left side view of the mandible (D) in Ctenomys minutus.
Figure S2. Landmarks used to capture shape from the dorsal (A), ventral (B) and distal (C) view of the humerus
in Ctenomys minutus.

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