TY - JOUR
AU1 - B, Kubiak, Bruno
AU2 - Renan, Maestri,
AU3 - de Almeida, Thamara S,
AU4 - R, Borges, Leandro
AU5 - Daniel, Galiano,
AU6 - Rodrigo, Fornel,
AU7 - de Freitas, Thales R O,
AB - Abstract A major interest of evolutionary biologists is to understand which environmental features are associated with morphological 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 provide 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. digging adaptations, divergent selection, geometric morphometrics, mammalian skull, natural selection, phenotypic plasticity, scratch-digging, tooth-digging INTRODUCTION Environmental features are associated with morphological and behavioural modifications in many species (Herrel et al., 2008; Losos & Mahler, 2010). One of the major interests of evolutionary biologists is to understand the factors that guide these changes over microevolutionary time scales. In this context, one goal of evolutionary studies is to identify the factors that influence the different morphologies of the structures involved in locomotion and other physical activities of animals. Research on modifications related to digging is particularly useful for this purpose, especially for animals that occupy excavated burrows. Several rodent lineages have evolved this lifestyle independently in different parts of the world, leading to parallel morphological specializations, such as having a cylindrical body and reduced eyes, ears and tail (Nevo, 1979; Lacey et al., 2000). It is also known that habitat differences (e.g. soil hardness, type of vegetation cover) may influence subterranean rodent excavation activities, resulting in different evolutionary responses in the mode of excavation, including scratch-digging, tooth-digging or a combination of the two (Lehmann, 1963; Dubost, 1968; Hildebrand, 1985; Lessa & Thaeler, 1989; 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 modifications 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 strategies 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 macroevolutionary patterns associated with selection for differences in craniodental and postcranial modifications. 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 complementary 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 postcranial 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 secondarily 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 characteristics 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 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., 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 occupation 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 characteristics that influence patterns of morphological variation related to excavation strategies in subterranean rodents. Figure 1. View largeDownload slide 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). Figure 1. View largeDownload slide 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). 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 differences 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 individuals 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. MATERIAL AND METHODS Soil hardness We used the data generated by Galiano et al. (2014, 2016) and Kubiak et al. (2015) to investigate differences 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 characteristics are not fully developed in both size and shape, which could bias the analyses. All humeri, skulls and mandibles were deposited with the specimen collection 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 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 specimens. 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 investigate the relationship between bite force and shape in the dorsal, ventral and lateral views of the skull and the mandible. 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 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 (F1,7 = 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 (F7,72 = 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 (F7,72 = 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 (89.94 ± 18.64°) and sand dunes (89.28 ± 5.30°) (T = 0.90, P = 0.184). Figure 2. View largeDownload slide 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. Figure 2. View largeDownload slide 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. Table 1. Mean (± SD) estimated bite force and skull length in Ctenomys minutus from sand dune and sand field habitats Sand dunes (13 males and 17 females) Sand fields (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 Sand dunes (13 males and 17 females) Sand fields (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 View Large Table 1. Mean (± SD) estimated bite force and skull length in Ctenomys minutus from sand dune and sand field habitats Sand dunes (13 males and 17 females) Sand fields (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 Sand dunes (13 males and 17 females) Sand fields (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 View Large PLS analyses indicated that the shapes of all skull and mandible views are strongly correlated with estimated bite force (dorsal, r = 0.81; ventral, r = 0.74; 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 the closer position of landmarks 1 and 7 on the mandible (Fig. 3D). Figure 3. View largeDownload slide 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). Figure 3. View largeDownload slide 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). 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 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 addition to the facet of the ulna and the articulation of the facet of the ulna. The ventral view also showed differences 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 articulation. In ventral view, individuals from the sandy dunes had a larger humerus (Fig. 4). Additionally, in 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). 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 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 Dorsal view Ventral view Distal/proximal view 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. View Large 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 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 Dorsal view Ventral view Distal/proximal view 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. View Large Table 3. Number of correctly classified specimens based on shape of the humerus of Ctenomys minutus between habitat types, and the percentage of correct classification for the three views Sand fields Sand dunes Percentage Dorsal view  Sand fields 17 7 74  Sand dunes 5 11 69 Ventral view  Sand fields 20 03 87  Sand dunes 5 11 69 Distal/proximal view  Sand fields 14 10 61  Sand dunes 8 8 50 Sand fields Sand dunes Percentage Dorsal view  Sand fields 17 7 74  Sand dunes 5 11 69 Ventral view  Sand fields 20 03 87  Sand dunes 5 11 69 Distal/proximal view  Sand fields 14 10 61  Sand dunes 8 8 50 A jackknife, leave-one-out, cross-validation procedure was used to classify the specimens. View Large Table 3. Number of correctly classified specimens based on shape of the humerus of Ctenomys minutus between habitat types, and the percentage of correct classification for the three views Sand fields Sand dunes Percentage Dorsal view  Sand fields 17 7 74  Sand dunes 5 11 69 Ventral view  Sand fields 20 03 87  Sand dunes 5 11 69 Distal/proximal view  Sand fields 14 10 61  Sand dunes 8 8 50 Sand fields Sand dunes Percentage Dorsal view  Sand fields 17 7 74  Sand dunes 5 11 69 Ventral view  Sand fields 20 03 87  Sand dunes 5 11 69 Distal/proximal view  Sand fields 14 10 61  Sand dunes 8 8 50 A jackknife, leave-one-out, cross-validation procedure was used to classify the specimens. View Large Figure 4. View largeDownload slide 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. Figure 4. View largeDownload slide 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. DISCUSSION Our results provide strong evidence that parapatric 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, demonstrating that the digging strategies are not evolutionarily exclusive and corroborating findings by Morgan (2009) and Marcy et al. (2016). However, modifications 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 variation 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 secondarily, 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 corroborate this pattern, allowing us to infer that the excavation 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 effectively 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 individuals 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 changes in individual skull shape. Higher estimated bite forces are associated with a shorter jaw, implying a shortening of the out-lever arm of mandibular muscles, which provides a mechanical advantage; this phenomenon has already been described for other species of Ctenomys in relation to the shortening of the skull (Versi, 2002; Lessa et al., 2008; Barčiová et al., 2009; Becerra et al., 2014). Even though individuals occupying the sandy fields have a larger angle of incisor procumbency, these differences are very small and not significant when compared with data from animals in sand dunes. The relationship between incisor procumbency angle and improvements in excavation remains controversial, and some authors have provided evidence that there is an association between rostrum–incisor procumbency and soil hardness (Reig & Quintana, 1992; Mora et al., 2003; Lessa et al., 2008; Marcy et al., 2016), Echeverría et al. (2017) suggested that incisor procumbency angle might not be related to biomechanical advantages in excavation. Our results demonstrate that intraspecific modifications are not very subtle when comparing animals in habitats with markedly different soil hardness; thus, further evaluation is required to confirm whether changes in procumbency of the upper incisor are related to different strategies of excavation. The modifications in humerus morphology we found are similar to those described in other studies comparing subterranean and non-subterranean rodents (Hildebrand, 1985; Lessa & Stein, 1992; Fernández et al., 2000; Morgan & Verzi, 2006; Lessa et al., 2008; Morgan & Álvarez, 2013) and in studies making comparisons between strictly subterranean species (Vassallo, 1998; Steiner-Souza et al., 2010; Morgan et al., 2017). These modifications are related to areas that function as extensor muscles and can generate better mechanical advantages for forearm movements and fixation of the scapulohumeral joint (Steiner-Souza et al., 2010). The distal displacement of landmarks 1 and 10 (same 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 pectoral 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 excavation 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 excavation strategies between populations and that soil characteristics are closely related to the vital activities 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 estimated bite force. In addition to interhabitat differentiation, males exhibit greater estimated bite force than females in both habitats. Similar results were described 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 Ctenomys, in which males engage in aggressive interactions 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 morphology 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, representing 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 evidence that different habitats influence the characteristics 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, measurements 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 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 addition, 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. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher's web-site: 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. 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). REFERENCES Adams DC , Otárola-Castillo E . 2013 . geomorph: anr package for the collection and analysis of geometric morphometric shape data . Methods in Ecology and Evolution 4 : 393 – 399 . Google Scholar Crossref Search ADS Álvarez A , Vieytes EC , Becerra F , Olivares AI , Echeverría A , Verzi DH , Vassallo AI . 2015 . Diversity of craniomandibular morphology in caviomorph rodents: an overview of macroevolutionary and functional patterns . In: Vassallo AI , Antenucci D , eds. Biology of caviomorph rodents: diversity and evolution . Buenos Aires : Sociedad Argentina para el Estudio de los Mamíferos , 199 – 228 . Barčiová L , Šumbera R , Burda H . 2009 . 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TI - Evolution in action: soil hardness influences morphology in a subterranean rodent (Rodentia: Ctenomyidae)
JF - Biological Journal of the Linnean Society
DO - 10.1093/biolinnean/bly144
DA - 2018-11-08
UR - https://www.deepdyve.com/lp/oxford-university-press/evolution-in-action-soil-hardness-influences-morphology-in-a-mbOIsAmdep
SP - 1
VL - Advance Article
IS - 
DP - DeepDyve
ER -