Evolution of Molar Shape in Didelphid Marsupials (Marsupialia: Didelphidae): Analysis of the Inﬂuence of Ecological Factors and Phylogenetic Legacy

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Zoological Journal of the Linnean Society, 2014. With 9 ﬁgures

Evolution of molar shape in didelphid marsupials (Marsupialia: Didelphidae): analysis of the inﬂuence of ecological factors and phylogenetic legacy

MARIA AMELIA CHEMISQUY1,4*, FRANCISCO J. PREVOSTI1,2,4, GABRIEL MARTIN3,4 and DAVID A. FLORES1,4

1División Mastozoología, Museo Argentino de Ciencias Naturales ‘Bernardino Rivadavia’, Ciudad Autónoma de Buenos Aires, Argentina 2Departamento de Ciencias Básicas, Universidad Nacional de Luján, Luján, Buenos Aires, Argentina 3Laboratorio de Investigaciones en Evolución y Biodiversidad, Facultad de Ciencias Naturales Sede Esquel, Universidad Nacional de la Patagonia ‘San Juan Bosco’, Esquel, Chubut, Argentina 4Consejo Nacional de Investigaciones Cientíﬁcas y Técnicas (CONICET), Ciudad Autónoma de Buenos Aires, Argentina

Received 29 January 2014; revised 25 August 2014; accepted for publication 26 August 2014

The diversity of items consumed by modern didelphids, varying from mostly fruits in Caluromys Allen to mostly small vertebrates in Lutreolina O. Thomas, may cause changes in molar size and shape. We evaluated the morphometric variation of the ﬁrst and third upper and lower molars of 16 genera of didelphid marsupials, with the aim of assessing the relationship between molar shape change, diet and phylogeny. We used a geometric morphometric approach to analyse how shape changes with diet. We mapped shape onto the phylogeny of the group to reconstruct ancestral states and analyse the evolution of molar shape. Finally, we statistically estimated the effect of size, diet and phylogeny on molar shape. All the analyses indicated little correlation between diet and molar shape and a strong correlation between the position of each genus on the phylogeny and molar shape. We believe that the wide ecological niche used by most of the groups (at least regarding diet) makes the evolutionary changes not strong enough to override pre-existing differences that occur among clades, and the absence of highly diet- specialized species (e.g. hypercarnivory or obligate folivory) causes the need for retaining a molar shape that can be useful to process different kinds of food items.

ADDITIONAL KEYWORDS: canonical phylogenetic ordination – evolutionary constraints – geometric morphometrics – shape optimization.

INTRODUCTION Velázquez & Scaglia, 1992; Dumont, Strait & Friscia, 2000; Abello, Ortiz Jaureguizar & Candela, 2012), and Molars are complex structures that possess great vari- also as characters for phylogenetic analyses (e.g. Reig, ation among species, from simple conical structures in Kirsch & Marshall, 1987; Goin & Pardiñas, 1996; dolphins to complex molars with folds and trans- O’Leary & Geisler, 1999; Voss & Jansa, 2009; Prevosti, verse plates in capybaras (Ungar, 2010). As well as the 2010; Archibald & Averianov, 2012; O’Leary et al., 2013). other teeth, they play an important role in taxonom- The basic tribosphenic molar is composed of an upper ic analyses, and have been widely used to infer the element with three pointed principal cusps (paracone, diet of extinct species (e.g. Goin & Montalvo, 1988; Goin, metacone and protocone) connected with cutting crests, and a lower element comprising the trigonid, or shearing end (also with three principal cusps and crests), *Corresponding author. E-mail: [email protected] and the talonid, or crushing heel (Luo, Cifelli &

Kielan–Jaworowska, 2001). In occlusal view, tribosphenic metatherians, such as a ‘V-shaped’ centrocrista (Reig molars constitute a system able to perform different et al., 1987; but see Case, Goin & Woodburne, 2005). functions during mastication (Crompton & Hiiemae, Although within modern didelphids there are no major 1969, 1970). The upper row forms a continuous series changes in molar morphology (Voss & Jansa, 2009), of triangles with their apices directed toward the middle the diversity of items consumed by their members, from line of the oral cavity. The triangular spaces formed mostly frugivorous in Caluromys Allen to highly car- by these structures are coupled by a series of invert- nivorous in Lestodelphys Tate (Vieira & Astúa de ed triangles formed by the trigonid of the inferior el- Moraes, 2003), could be reflected in changes in molar ements. In this way, the talonids of the inferior row size and shape, such as the relative position of crests contact the lingual (or inner) half of the superior el- and cusps (see, for example, Goin et al., 1992). ements (i.e. the protocone) (Crompton & Hiiemae, 1969, In this study, we evaluated the morphometric vari- 1970). ation of the first and third upper and lower molars The relative position of molar cusps and crests is of 13 extant and three extinct genera of didelphid the main characteristic to analyse when trying to infer marsupials, with the main aim being to assess the re- diets, and many authors have developed different ap- lationship between molar shape change, diet and phy- proaches that relate molar shape to diet. For example, logeny. We used a traditional geometric morphometric Goin et al. (1992) analysed the orientation of the approach to analyse how shape changes with diet. We metacrista and the paracristid using the angle that also mapped shape onto the phylogeny of the group those crests form with the mesiodistal axis of the molar to reconstruct ancestral states and analyse the evo- series in Didelphoidea to differentiate between carni- lution of molar shape in Didelphidae. Finally, using vores, insectivores and omnivores. Kay (1975) used the an approach similar to canonical phylogenetic ordina- length of the shearing blades of the second lower molar tion (Giannini, 2003), we statistically estimated the effect to separate fruit-eating primates from leaf-eating pri- of size, diet and phylogeny on the shape of the molars. mates. The same approach was followed by Strait (1993) to discriminate between insectivores feeding on hard MATERIAL AND METHODS and soft objects. New and more complex analyses use three-dimensional molar modelling to relate crest ori- SAMPLING entation and cusp size and shape with diet, taking into We digitized the first and third upper and lower molars account the effects of occlusion and wear in molars of 103 specimens corresponding to 13 extant genera (Evans, 2005; Evans & Sanson, 2006; Evans et al., 2007). of Didelphidae and three extinct taxa, analysing as All these contributions (and many others not men- many representatives of each genus as we could (Table 1, tioned for the sake of brevity) show that mammal molars see Supporting Information, Table S1). For the highly can be good indicators of differences in diet habits, some- speciose genera Marmosa Gray, Monodelphis Burnett, thing that can be explained by their function in food Philander Brisson and Thylamys Gray we included rep- processing. resentatives of more than one species. This was done Despite this relationship between molar shape and to account for as much variation as possible within each diet, there is a general agreement that the phylogenetic genus, although molar morphology has been found history of a group also influences molar morphology mostly to be uniform for each (Voss & Jansa, 2009). (e.g. Kay & Hylander, 1978; Strait, 1993; Caumul & Therefore, samples were pooled to show one general Polly, 2005; Ungar, 2010; Klukkert, Teaford & Ungar, pattern per genus. 2012). This explains why tooth morphology has Goin et al. (1992) showed that there are no differ- been used for more than a century to classify and iden- ences in tooth morphology between males and females tify mammals, and to establish their phylogenetic in Monodelphis dimidiata (J.A. Wagner), one of the most relationships. dimorphic species within New World marsupials (Pine, Didelphid marsupials have primitive tribosphenic Dalby & Matson, 1985). Moreover, Martin (2005) men- molars, the upper ones with a well-developed buccal tions the lack of sexual dimorphism for teeth in stylar shelf, and five stylar cusps (Meléndez, 1990; Lestodelphys halli (O. Thomas), derived from a very Hillson, 2005). Lower molars have a well-developed large dataset of upper and lower molars, while the same trigonid and talonid, and twinned entoconid and pattern is described for the genus Thylamys by Martin hypoconulid, which are well separated from the (2008). Therefore, we assumed that there is no sexual hypoconid (Meléndez, 1990; Ungar, 2010). The dimorphism in didelphid molars and included both sexes. ‘didelphoid’ molar model is an ancient morphology Goin et al. (1992) also showed bilateral asymmetry already established in the Cretaceous (Fox, 1987). between left and right molars. Consequently, we only However, living didelphids and some Neogene extinct digitized right molars. As teeth acquire full adult size marsupials exhibit a relatively derived molar mor- from the start (Hillson, 2005), both juveniles and adults phology with respect to most Cretaceous and Paleogene were included, selecting only molars with little wear.

Table 1. The number of specimens of each molar includ- Dental terminology (Fig. 1B, D) partially follows Voss ed per genus, and diet classification assigned to each genus & Jansa (2009). For upper molars, 26 landmarks and (following Vieira & Astúa de Moraes, 2003) semi-landmarks were digitized on the following positions: 1, stylar cusp E (StE); 2–14, semi-landmarks de- Genus M1 M3 m1 m3 Diet scribing the labial margin of the tooth; 15, apex of stylar cusp A (StA); 16, apex of stylar cusp B (StB); 17, apex Caluromys J.A.Allen 3 2 4 4 I of stylar cusp D (StD); 18, apex of the metacone; 19– Chironectes Illiger 4 2 3 3 V 24, semi-landmarks describing the centrocrista; 25, apex Cryptonanus Voss, 42 6 4II of the paracone; 26, apex of the protocone (Fig. 1A). Lunde & Jansa For lower molars, 13 landmarks and semi-landmarks Didelphis Linnaeus 11 9 11 7 III were digitized on the following positions: 1, apex of Gracilinanus Gardner & 22 2 1II the hypoconid; 2, apex of the hypoconulid; 3, apex of Creighton Hyperdidelphys Ameghino 2 2 2 1 V the entoconid; 4, apex of the metaconid; 5, apex of the Lestodelphys Tate 3 2 3 3 V paraconid; 6, anterior border of the anterior cingu- Lutreolina O. Thomas 9 4 7 6 V lum; 7–11, semi-landmarks describing the anterobasal Marmosa Gray 4 3 3 4 II cingulum; 12, posterior border of the anterobasal cin- Marmosops Matschie 2 2 2 2 II gulum; 13, apex of the protoconid (Fig. 1C). The ap- Metachirus Burmeister 4 3 4 4 IV plication MakeFan6 (Sheets, 2003) was used to ensure Monodelphis Burnett 10 6 8 9 IV consistent placement of the semi-landmark coordi- Philander Brisson 5 5 4 4 IV nates using an equiangular and equidistant criterion Thylamys Gray 7 5 6 5 IV to place them, depending on the structure. In the case Thylatheridium Reig 3 * 3 8 V of upper molars, a 20-line fan was placed in the labial Thylophorops Reig 5 2 2 10 V margin radiating from the most concave point of the centrocrista, and a 12-line comb was placed on the *This taxon was not included in the analyses of third upper centrocrista from the paracone to the metacone. For molars due to excessive wear of the available materials. lower molars, a 10-line fan was placed on the anteri- or cingulum radiating from the notch between paraconid Diet categories used for all analyses (Table 1) were and protoconid. Both landmarks and semi-landmarks based on Vieira & Astúa de Moraes (2003), who clas- were then digitized using the software tpsDIG 2.12 sified the genera of Neotropical marsupials in five ordered (Rohlf, 2008a). Unaligned landmark coordinates are categories from mostly frugivorous (I) to mainly car- available as Supporting information, Data S1. nivorous species (V). Extinct genera were placed under the category V, as they are all described as highly carnivorous (Reig et al., 1987; Goin & Montalvo, 1988; Goin MULTIVARIATE ANALYSES OF SHAPE & Pardiñas, 1996). Diet classification of fossil taxa is Landmark configurations were aligned by perform- based mostly on dental anatomy, although sometimes ing a Procrustes superimposition, using the program considering different aspects of dentition from those TPSrelw 1.46 (Rohlf, 2008b). For the alignment, semi- analysed here (e.g. Goin et al., 1992). Consequently, we landmarks were slid using the minimum bending energy might possibly fall in a circular argument when we method (Bookstein, 1997). The distribution of speci- include the diet information of extinct taxa in some of mens on shape space was analysed by performing a the analyses, inflating the relationship between diet principal component analysis (PCA) of the Procrustes and dental morphology. However, because we found that coordinates using the software MorphoJ (Klingenberg, that was a weak relationship (see Results), we think 2011). that including extinct taxa is not necessarily prob- The relationship between tooth morphology and diet lematic for our work and allows us to widen the taxo- was explored using a between-group PCA, which proj- nomic sample and the scope of our study. ects the data onto the principal components of the group means (Mitteroecker & Bookstein, 2011). Between- IMAGES AND LANDMARKS group PCA leads to a better group separation than the Molars in occlusal view were digitally imaged using a ordinary PCA, but avoids the overfitting achieved by stereomicroscope with image capture capability. For both the canonical variate analysis and the linear discri- upper and lower molars, teeth were orientated placing minant analysis, and preserves the original Procrustes the molar neck parallel to the lens plane. Images were distances in the shape space produced by the super- carefully checked to avoid errors due to misorienta- imposition (Mitteroecker & Bookstein, 2011; Seetah, tion, and when necessary they were repeated until the Cardini & Miracle, 2012). Between-group PCA was per- right orientation was achieved. All images contained formed using the package ade4 (Dray & Dufour, 2007) a scale. for the software R (R Development Core Team, 2012).

Figure 1. Occlusal views of the third upper (A, B) and lower (C, D) didelphid molars. A and C, molars of Didelphis albiventris showing the landmarks and semilandmarks used. B and D, didelphid molars illustrating features of crown morphology discussed in the text. Squares, landmarks; circles, semilandmarks. See text for a detailed description of landmarks. Abbreviations: ac, anterior cingulum (light grey shading); cc, centrocrista; co, cristid obliqua; ect, ectoﬂexus; Ent, entoconid; ento, entocristid; Hyp, hypoconid; Hypd, hypoconulid; Me, metacone; Med, metaconid; meta, metastylar corner (grey shading); Pa, paracone; Pacr, paracristid; Pad, paraconid; para, parastylar corner (dark grey shading); postcd, postcristid; prePa, preparacrista; Pr, protocone; Prcr, protocristid; Prd, protoconid; prePr, preprotocrista; posMe, metacrista; posPr, postprotocrista; StA, stylar cusp A; StB, stylar cusp B; StC, stylar cusp C; StD, stylar cusp D; StE, stylar cusp E; Ta: talonid; Tri: trigonid.

To analyse the shape transformation associated with et al., 2010). We used the tree resulting from the analy- the distribution in the between-group PCA, shape was sis of five concatenated genes published by Voss & Jansa regressed against the coordinates of each specimen on (2009: fig. 33). Extinct taxa (indicated by †) were placed the first and second principal components (PC1 and following the final cladogram published by Reig PC2). et al. (1987: fig. 68) for Thylatheridium† Reig and Thylophorops† Reig, and the cladogram obtained by Goin & Pardiñas (1996) for Hyperdidelphys† Ameghino. PHYLOGENETIC RECONSTRUCTION OF The optimization was performed using the soft- ANCESTRAL SHAPE ware TNT (Goloboff, Farris & Nixon, 2008), using a Landmark configurations were optimized on the phy- grid of ten cells, a window of two cells and a nesting logeny, through spatial optimization using maximum level of the grids of four. Although specimens were pre- parsimony, as described by Catalano, Giannini & aligned using a Procrustes superimposition, we also Goloboff (2010) and Goloboff & Catalano (2011). The improved the alignments using a dynamic superim- algorithm optimizes the landmark configurations on position approach, which simultaneously superim- phylogenetic trees using a modification of the Sankoff poses and maps landmark configurations (Catalano & optimization (see Goloboff & Catalano, 2011). This Goloboff, 2012). method allows the reconstruction of the displacement of each landmark – in two dimensions in this case – as if they were independent ‘characters’. The CORRELATION BETWEEN SHAPE, SIZE, summation of individual landmark optimizations (i.e. DIET AND PHYLOGENY the reconstruction of their ancestral position in each To evaluate the effect of phylogeny, size and diet on node) gives the ancestral landmark configuration of the shape, we used a method inspired by the canoni- every node of the tree (for more details see Catalano cal phylogenetic ordination proposed by Giannini (2003),

© 2014 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014 EVOLUTION OF MOLAR SHAPE IN DIDELPHIDAE 5 as it is the only comparative method that allows the PC2 showed specimens with longer m1s and the op- partitioning of how much of the shape (a multidimen- posite trend in the remaining characters (Fig. 2). The sional variable) is explained by the factors men- graph showed a broad superposition of taxa. However, tioned above. The method consists of performing a it is important to note the placement of Caluromys redundancy analysis (RDA; Legendre & Legendre, 1998) on the upper left quadrant, Lestodelphys (with other using shape (i.e. the landmark configurations of the specimens of Thylamyini) on the lower left quadrant, aligned specimens) as the community matrix and and Metachirus Burmeister and Hyperdidelphys†on centroid size, diet and phylogeny as constraints. the positive end of PC1 (Fig. 2). The other taxa were Phylogenetic information was taken from the matrix placed in intermediate positions of the graph, al- representation using parsimony of the tree (MRP), which though in general terms, the Didelphini–Metachirini consists of a binary matrix where the columns repre- group tended to be separated on PC1 from the sent the membership of each taxon to a clade (see Thylamyini and Marmosini. No clear pattern of dis- Giannini, 2003). We analysed the effect of the three tribution was found between specimens and the dif- factors together as constraints, of each factor sepa- ferent diet categories, except for category I, which was rately as a constraint and the effect of each factor sepa- placed on the upper left quadrant by itself and sepa- rately using the remaining factors as a conditioning rated from the others (Fig. S1). matrix. This last analysis allowed us to evaluate the PC1 and PC2 from the analysis of the first upper individual effect of each factor, excluding the interac- molar (M1) explained 28.41 and 16.54% of the total tion with the other variables (Legendre & Legendre, variance, respectively. Specimens placed on the nega- 1998). To discard collinear variables from the model tive end of PC1 showed molars with a shorter and we proceeded as follows: first, we evaluated the per- transversally orientated metacrista, mesially dis- centage of shape variation explained by each vari- placed StD that is closer to StB, and a less pro- able independently using an RDA (i.e. size, diet and nounced ectoflexus (Fig. 3), while specimens placed on each node of the phylogeny); second, we entered in the the positive end of the PC1 showed the opposite trend. model the variable that explained the most variation The negative end of PC2 showed specimens with more and estimated the Akaike information criterion (AIC; symmetrical molars, with a shorter metacrista and Godínez-Domínguez & Freire, 2003) value for that paracrista, a more convex labial margin, a less in- model. Then, we entered in the model each of the reflected centrocrista and a distally displaced protocone maining variables in order of explanation percentage (Fig. 3), while the opposite trend was observed on the until the AIC became constant. The variables that did positive end of PC2. In contrast to what was ob- not reduce the AIC were excluded from the final model. served in the first lower molar, the PCA of the M1 To account for individual variation on the entire showed less superposition of taxa. Caluromys was placed sample, but to avoid the effect of over-representing in the negative end of PC1, clearly separated from the certain genera (as they had more specimens ana- remaining taxa. Marmosa, Marmosops Matschie and lysed), each of the RDAs was performed 10 000 times Cryptonanus Voss, Lunde & Jansa were placed on the using a resampled matrix that included only one speci- positive end of PC2, while Chironectes Illiger was at men randomly chosen per genus each time. Resampling the opposite end of the plot (Fig. 3). Lestodephys, of the data, RDAs with their significance and AIC es- Hyperdidelphys† and Thylatheridium† were placed on timations were performed using the R package vegan the lower right quadrant of the plot. The remaining (Oksanen et al., 2013). taxa were placed in intermediate positions between these extremes (Fig. 3). Diet categories showed a broad superposition except for category I, placed on the negative end of PC1. There is almost no overlap between RESULTS categories II and III, which are placed on the posi- SHAPE ANALYSES tive and negative ends of PC2, respectively. However, PC1 and PC2 of the analysis of the first lower molar both categories are broadly overlapped by categories (m1) explained 23.65 and 18.28% of the total vari- IV and V (Fig. S2). ance, respectively. Specimens placed on the negative In the analysis of the third lower molar (m3), PC1 end of PC1 showed molars with a larger trigonid and and PC2 explained 34 and 16.83% of the total vari- a smaller talonid, a distal displacement of the protoconid ance, respectively. The shape change observed along and the metaconid, and a narrower anterior cingu- each axis was very similar to the change observed along lum (Fig. 2), while the opposite trend was observed PC1 and PC2 for m1 (Fig. 4). However, in this analy- on the positive end of PC1. PC2 showed specimens sis, the distribution of the taxa changed compared with with shorter molars on its negative end, with rela- the analysis of m1, with Lutreolina Thomas, tively longer trigonids and shorter talonids, and a more Hyperdidelphys†, Thylatheridium† and species of robust anterior cingulum (Fig. 2). On its positive end, Thylamyini placed mostly on the negative side of PC1,

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Figure 2. First lower molar (m1) shape variation along the first two principal components (PC) from the PCA of the Procrustes coordinates, showing the distribution of taxonomic groups. Deformation grids show the extreme shape of each PC. other species of Didelphini on the positive end of PC1, a deeper ectoflexus and a more inflected centrocrista Caluromys placed on the positive end of PC2 and (Fig. 5). Specimens placed on the positive end of PC1 Metachirus on the negative end (Fig. 4). Regarding diet showed longer M3s, with the opposite pattern on the categories, there was no clear ordination pattern, except remaining characters (Fig. 5). On the negative end of for the placement of taxa within category I on the upper PC2 specimens showed a distally displaced StA, mesially left quadrant. Note, however, a trend for taxa within displaced StD, distolabially displaced StE, distolingually category II to be placed on the negative end of PC1, displaced metacone and a less inflected centrocrista, and taxa of category III on the positive end of PC1. while specimens placed on the positive end of PC2 Nonetheless, there was a broad superposition among showed the opposite trend. Didelphis Linnaeus was the five categories (Fig. S3). placed on the positive end of PC1, near Caluromys, In the analysis of the third upper molar (M3), PC1 which was also placed on the positive end of PC2. Speci- and PC2 explained 35.47 and 21.29% of the total vari- mens of Lutreolina, Hyperdidelphys† and Thylophorops† ance, respectively. Specimens placed on the negative were found on the negative end of PC2, while end of PC1 showed a shorter M3, with a longer Lestodelphys was placed on the negative end of PC1 metacrista and paracrista, a labially displaced StB and (Fig. 5). The remaining taxa were placed in intermedi- StD, an expanded stylar shelf caused mainly by the ate positions throughout the different axes, with a trend lingual displacement of the paracone and the metacone, towards the negative end of PC1 (Fig. 5). Regarding

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Figure 3. First upper molar (M1) shape variation along the first two principal components (PC) from the PCA of the Procrustes coordinates, showing the distribution of taxonomic groups. Deformation grids show the extreme shape of each PC. diet, categories I and III are separated from the re- bution of specimens on the plots, its effect is not strong maining categories, on the positive end of PC2 and PC1, and is not concentrated in only one of the principal respectively. The remaining categories form a contin- components but is distributed in, at least, PC1 and uum, displaced to the negative end of PC1, from the PC2. negative end of PC2 for category V to the positive end of PC2 for categories II and IV (Fig. S4). The effect of size on the PCAs was evaluated by re- RELATIONSHIP BETWEEN TOOTH gressing, for each data set, PC1 and PC2 with size (i.e. MORPHOLOGY AND DIET log centroid size). Except for PC2 of the m3 analysis, On the between-group PCA (B-G PCA) of m1 we did there was a significant correlation between shape and not find an improvement of the results obtained by the size in all the cases. However, the percentage of shape PCA, showing a broad overlap among diet categories, explained by size was low in most cases, and reached with the exception of category I. This category was only 50% for PC1 of M3 (m1 42.97 and 12.87%; m3 located on the negative end of PC1, apart from the re- 19.82 and 1.13%; M1 7.96 and 25.59%; M3 53.12 and maining categories, with a morphological pattern that 23.64% for PC1 and PC2, respectively). Consequent- showed a smaller anterior cingulum and a broader ly, although allometry is playing a role in the distri- trigonid (Fig. S5).

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Figure 4. Third lower molar (m3) shape variation along the ﬁrst two principal components (PC) from the PCA of the Procrustes coordinates, showing the distribution of taxonomic groups. Deformation grids show the extreme shape of each PC.

Between-group PCA of M1 showed a better separa- egory II, both being separated from categories V and tion of diet categories (Fig. S6). Diet category I was IV along the first axis, while category III was orien- clearly separated on the positive end of both axes tated towards the positive end of the second axis (Fig. 6). (i.e. upper right quadrant of Fig. S6). The remaining Shape changes towards the positive end of PC1 in- categories form a continuum, with diet categories II cluded a narrower molar, smaller talonid, distally dis- and IV separated along PC1, and categories V and placed paraconid and larger anterior cingulum (Fig. 6). III forming a cluster separated from category IV along Towards the positive end of PC2, m3 became narrow- PC2 (Fig. S6). Variation towards the positive end of er, the entoconid and hypoconulid became distolabially PC1 can be associated with a longer M1, a more in- displaced, while the hypoconid and protoconid were flected centrocrista, a shallower ectoflexus, distally dis- mesiolingually displaced (Fig. 6). In the plot of M3, the placed metacone, a narrower stylar shelf at the five diet categories were clearly better separated (Fig. 7). metastylar corner and a mesially displaced StD (Fig. S6). Categories II, IV and V were separated along PC2, while Variation towards the positive end of PC2 was categories I and III were separated from each other also associated with a wider molar (labiolingually), three along PC2 and from the other three categories with a shallower ectoflexus and a less inflected along PC1 (Fig. 7). Towards the positive end of PC1, centrocrista, a shorter metacrista, and twinned StB M3 became shorter and broader, molars showed a and StD (Fig. S6). sharper centrocrista and deeper ectoflexus, a larger The separation of diet categories obtained using the stylar shelf, and labially displaced StB and StD (Fig. 7). between-group PCA was better for both upper and lower Shape changes towards the positive end of PC2 in- third molars (i.e. m3 and M3). The plot of m3 showed cluded the labial displacement of StA, mesial displace- a continuum among the five diet categories, with the ment of StE, labial displacement of StB and StD, along centroid of category I located inside the ellipse of cat- with a separation of both cusps from each other, lingual

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Figure 5. Third upper molar (M3) shape variation along the first two principal components (PC) from the PCA of the Procrustes coordinates, showing the distribution of taxonomic groups. Deformation grids show the extreme shape of each PC. displacement of the paracone, metacone and protocone, on most of the landmarks from the root pattern (i.e. and a more inflected ectoflexus and centrocrista (Fig. 7). Caluromys), the main changes being the shortening of the molar, the lengthening of the metacrista caused by a mesial displacement of the metacone and a labial SHAPE CHANGES ALONG PHYLOGENY displacement of StE, a reduction of the anterior The optimization of M3 showed changes both on the cingulum evidenced by the posterior displacement of terminal taxa and on the internal nodes, being more landmark 15, and mesial displacement of the protocone. important on the former (Fig. S7). The node that groups The terminal node of Monodelphis showed a larger all Didelphinae (node 1, Fig. 8) showed displacement stylar shelf, a less inflected centrocrista, labial

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-3.0

-4.0 -5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 PC1 Labial

Mesial

Figure 6. Scatter plots resulting from the between-group PCA of the third lower molar (m3), summarizing differences between the five diet categories. White squares with the Roman numeral of each diet category represent the centroid of the distribution for that category. Deformation grids show the extreme shape of each PC. displacement of the protocone, distal displacement of ing of the metacrista, a mesial displacement of StA the metacone and StD, and mesial displacement of StB and distal displacement of the protocone (Fig. 8). Stylar (Fig. 8). The node that groups tribe Thylamyini showed cusp E was distally displaced, and the protocone was a longer metacrista and a deeper ectoflexus (node 4, labially displaced on the node that groups the remain- Fig. 8). The next node that showed important changes ing genera of Didelphini (node 10, Fig. 8). Didelphis was the one that groups Thylamys and Lestodelphys, showed a longer molar, with a shorter metacrista, a with a shortening of the molar, a distolabial displace- smaller stylar shelf and a less inflected centrocrista ment of StB and distolingual displacement of StD, and (Fig. 8). A less inflected ectoflexus and mesial displace- a deeper ectoflexus (node 6, Fig. 8). Lestodelphys showed ment of StD were observed on the node that groups a shortening of the molar, an expansion of the stylar Thylophorops†, Lutreolina and Hyperdidelphys† (node shelf (mainly at the metastylar corner), a lingually dis- 11, Fig. 8). Thylophorops† showed a longer and more placed protocone and a less inflected centrocrista; in distally displaced metacrista, and a mesially dis- Thylamys StB and StD were lingually displaced (Fig. 8). placed protocone (Fig. 8). Hyperdidelphys† showed The lingual displacement of StB and StD was also ob- a similar pattern, but with a narrower stylar shelf, a served in the node that groups Metachirini and distally displaced StA and anterior cingulum, and a Didelphini, and in Metachirus (node 8, Figs 8 and S7). lingually displaced protocone (Fig. 8). Lutreolina showed Metachirus also showed a longer metacrista, and a la- a shorter molar, with a less inflected centrocrista, a bially inflected centrocrista. The node of Didelphini poorly developed ectoflexus, a distolabially displaced showed a distolabial displacement of the protocone StD and a labially displaced protocone (Fig. 8). The op- and a less inflected centrocrista (node 10, Fig. S7). timization of M1 shows a similar pattern, but changes Chironectes showed a shallower ectoflexus, a shorten- are more subtle in most specimens analysed (Fig. S8).

9.00

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Labial

Mesial

Figure 7. Scatter plots resulting from the between-group PCA of the third upper molar (M3), summarizing differences between the ﬁve diet categories. White squares with the Roman numeral of each diet category represent the centroid of the distribution for that category. Deformation grids show the extreme shape of each PC.

The optimization of m3 also showed more changes tribe Marmosini, the node that groups Thylatheridium† on the terminal taxa than on the internal nodes and Monodelphis showed a lingual displacement of the (Fig. S9). The Didelphinae node showed a labial dis- hypoconid and a distolabial displacement of the placement of the entoconid, a mesial displacement of metaconid (node 3, Fig. 9), while in Thylatheridium† the hypoconulid, a lingual displacement of the protoconid there was an enlargement of the trigonid, and in and a larger anterior cingulum (node 1, Fig. 9). Inside Monodelphis the entoconid and the hypoconulid were

Node 1 Monodelphis Caluromys Monodelphis 2 Marmosa Marmosops

1 4 Thylamys 6 Node 4 Node 6 Lestodelphys 5 Gracilinanus 3 7 Cryptonanus Metachirus

8 Chironectes Thylamys Lestodelphys Philander 9 12 Didelphis 10 Thylophorops 11 Lutreolina Labial Hyperdidelphys Mesial

Metachirus Chironectes Node 10 Node 11

Didelphis Thylophorops Lutreolina Hyperdidelphys

Figure 8. Phylogeny showing a summary of the optimization for the third upper molar (M3). Numbers on the branches indicate node number. Taxon names and nodes in bold indicate the optimizations being shown. Deformation grids show the changes with respect to the previous node.

Caluromys Node 1 Node 3 Marmosa 2 3 Thylatheridium Monodelphis Marmosops 1 5 7 Thylamys Thylatheridium Monodelphis Lestodelphys 6 Gracilinanus 4 8 Cryptonanus Metachirus 9 Chironectes Marmosops Thylamys 10 13 Philander Didelphis 11 Thylophorops Labial 12 Lutreolina Mesial Hyperdidelphys

Lestodelphys Metachirus Node 12

Thylophorops Lutreolina Hyperdidelphys

Figure 9. Phylogeny showing a summary of the optimization for the third lower molar (m3). Numbers on the branches indicate node number. Taxon names and nodes in bold indicate the optimizations being shown. Deformation grids show the changes with respect to the previous node. twinned, the paraconid was distolingually displaced and placed paraconid, metaconid and entoconid and a the anterior cingulum was larger (Fig. 9). Within tribe mesially displaced hypoconulid (Fig. 9). Thylamys showed Thylamyini, the main changes are on the terminal taxa, a smaller trigonid with a distally displaced paraconid while the basal node showed only a distolabial dis- and protoconid, and a larger anterior cingulum (Fig. 9). placement of the protoconid, and a labial displace- Lestodelphys showed a distally displaced entoconid ment of the metaconid (node 5, Fig. S9). Marmosops (Fig. 9), while Gracilinanus Gardner & Creighton showed a distally displaced hypoconid, a labially dis- showed a mesiolingual displacement of the protoconid

Table 2. Results of the redundancy analyses of molar morphology, diet size and phylogeny

m1 M1 m3 M3

% P % P % P % P

Total 72.71 0.2272 81.75 0.0041 85.71 0.0004 85.53 0.0002 Diet 15.09 0.0176 21.51 0.0001 9.68 0.0705 20.3 0.0005 Diet partial var. 6.02 0.9331 5.97 0.1593 3.5 0.4608 4.77 0.3154 Size 10.73 0.0037 10.83 0.0713 13.35 0.0347 25.01 0.0003 Size partial var. 3.67 0.5115 4.51 0.3299 4.9 0.3622 4.78 0.3757 Phylogeny 62.09 0.0261 72.42 0.006 76.19 0.0001 74.25 0.0001 Phylogeny partial var. 48.19 0.5758 47.58 0.0764 64.68 0.0022 40.15 0.0034

%, percentage of variance explained by each analysis; P, probability for each analysis (bold numbers are significant at P < 0.05); partial var, partial variance explained by one factor (e.g. diet) but not by the others (i.e. size, phylogeny). and metaconid, a mesial displacement of the paraconid, node 13 in M1, node 12 in lower molars and M3, nodes a lingual displacement of the entoconid, a distal dis- 7 and 3 in M3, and nodes 8 and 4 in lower molars placement of the hypoconulid, a labial displacement and M1 (see Figs 8, 9, S8–S10 for node numbers). The of the hypoconid and a smaller anterior cingulum model using three factors as constraints (diet, size and (Fig. S9). The anterior cingulum became larger in phylogeny) explained between 72.71 and 85.71% of the Metachirus, the metaconid and hypoconulid were total shape variation (Table 2). Diet explained between mesially displaced, the hypoconid was distolingually 9.68 and 21.51% of the variance in all molars, while displaced and the paraconid was distally displaced the proportion explained exclusively by this variable (Fig. 9). The basal node of Didelphini showed only a was much lower and statistically not significant mesial displacement of the hypoconid (node 10, Fig. S9). (Table 2). Centroid size accounted for 3.67–4.9% of the Chironectes showed a longer talonid with a labially dis- total variation when using the remaining factors as placed hypoconid, and a shorter trigonid with a lingually conditioning matrix, and 10.73–25.01%, when using size displaced protoconid, paraconid and metaconid, the dis- as a constraint (Table 2). Finally, phylogeny ex- placement being much larger on the protoconid, and plained between 62.09 and 76.19% of the variation, al- a larger anterior cingulum (Fig. S9). The node that though it explained only between 40.15 and 64.68% groups Philander and Didelphis showed a mesiolingual of the shape variation by itself (Table 2). displacement of the protoconid (node 13, Fig. S9). The node that groups Lutreolina, Thylophorops† and Hyperdidelphys† showed a labial displacement of the DISCUSSION metaconid, a mesiolabial displacement of the paraconid, a distolabial displacement of the protoconid, a Because teeth are the principal structure involved in distolingual displacement of the hypoconid and a smaller the mechanical breakdown of food in mammals, the anterior cingulum (node 12, Fig. 9). In Thylophorops† morphology of cusps and crests in occlusal view should the protoconid was lingually displaced, the paraconid be expected to show different levels of correlation with distolabially displaced, the hypoconid distolingually dis- diet (e.g. Kay & Sheine, 1979; Strait, 1993; Evans, 2005; placed and the anterior cinugulum was larger (Fig. 9). Evans & Sanson, 2006; Evans et al., 2007; Hogue & Lutreolina showed a labially displaced hypoconid, a ZiaShakeri, 2010). Surprisingly, our results seem to mesiolingually displaced hypoconulid and a distally dis- contradict this premise, as we found low correlation placed metaconid (Fig. 9). Hyperdidelphys† showed a between molar shape and diet. The PCA of the labial displacement of the metaconid and protoconid, Procrustes coordinates of the four molars showed a broad a mesiolabial displacement of the paraconid, a superposition of diet categories, except category I (rep- distolingual displacement of the hypoconulid, and a resented only by the genus Caluromys), which showed distolabial displacement of the entoconid (Fig. 9). The a trend separating it from the remaining diet catego- optimization of m1 showed similar changes (Fig. S10). ries (Figs 2–5). By using a BG-PCA, which maxim- izes the separation between the centroids (or means) of the different groups without distorting the shape CORRELATION BETWEEN MORPHOLOGY, PHYLOGENY, distances (Mitteroecker & Bookstein, 2011; Seetah et al., SIZE AND ECOLOGY 2012), we obtained a better separation of the diet cat- For all molars, the final model chosen by AIC omitted egories in third molars; however, there was overlap four nodes of the phylogeny: node 2 in all four molars, of all categories in m3, and categories II, IV and V in

M3 (Figs 6, 7). Even some specimens of category IV parallel to that axis (Goin et al., 1992). The plot of M3 were placed near the centroid of the distribution of cat- showed a clear example of the constraint caused by egory V and vice versa in M3 (Fig. 7). In the between- the most demanding food item in Lestodelphys (de- group analysis of first lower and upper molars, we did manding in the sense of the functional requirements not observe a better separation in the distribution of needed to process the food item), as this species is placed diet categories. Most of them overlapped and even the on the negative end of PC1, having a long metacrista centroids of some categories were placed inside the con- transverse to the mesiodistal axis of the tooth row fidence ellipses of the other categories (Figs S5, S6). (Fig. 5). By contrast, taxa that consume (or are thought Such differences in fitting tooth shape variation to any to consume) a much smaller proportion of inver- diet category are expected, because in adult marsu- tebrates, such as Lutreolina, Hyperdidelphys† and pials with permanent dentition, the last molars (M3, Thylophorops†, were placed on the negative end of PC2, m3–m4) are placed in an optimal place from a with the metacrista more parallel to the mesiodistal biomechanical view (see Werdelin, 1987). Consequent- axis of the tooth row (Fig. 5). Hogue & ZiaShakeri (2010) ly, we expect a better correlation between these molars also found inconsistencies between the length of molar and diet than between anterior molars and diet. crests and the proportion of insects or leaves eaten by There are many factors that could be obscuring the a broader group of marsupials. Similarly to what relationship between molar shape and diet. Didelphid happens with Lestodelphys, they found that carnivo- marsupials have primitive tribosphenic molars, which rous species that consume even a small proportion of due to their generalized shape are flexible and allow insects have long shearing crests in m3, characteris- the consumption of different kinds of food (Goin et al., tic of insectivorous species. Thus, the absence of a better 1992). This is reflected in their dietary flexibility, with correlation between diet and molar shape could be the main items consumed by this group of marsu- related to the absence of highly specialized species, and pials being a mix of small vertebrates, arthropods, fruits the need for retaining a shape that can be useful to and nectar in different proportions (Vieira & Astúa de process different kinds of food items. In this context, Moraes, 2003; Ungar, 2010). In fact, the diet classifi- the basic pattern of a tribosphenic molar appears to cation used in this contribution showed a continuum be the best morphology when coping with mixed diets. from mostly frugivorous to mainly carnivorous species, This does not mean that New World marsupials are where none of the neotropical marsupials fed exclu- not subject to adaptive pressures (with a subsequent sively on plant material, invertebrates or vertebrates diversion from the generalized pattern), but implies (Vieira & Astúa de Moraes, 2003). For example, one that the generalized tribosphenic molar morphology oc- of the most carnivorous species of the group, curring in these species represents the best solution Lestodelphys halli, does not feed exclusively on ver- to different diets, working effectively for most species tebrates, but also consumes insects (Martin, 2008), which with only minor deviations to reflect changes in the can be an important item in their diet in some local- main diet preferences. Therefore, small changes in mor- ities (Zapata et al., 2013). As this is a small species phology would be represented by minor differences in, (total length less than 250 mm, mass 60–90 g; Pearson, for example, the orientation and length of the 2007), it is possible that diet would shift seasonally metacrista, the relationship between protocone/ depending on prey availability and/or with age, with talonid size, trigonid–talonid proportions and length younger individuals consuming more arthropods and of the cristid obliqua. Our placing of the studied genera older ones being active rodent hunters (Martin & in the feeding categories we used allowed us to assess Udrizar Sauthier, 2011). Arthropods, as well as leaves, those differences, despite them being relatively small. have a high proportion of structural carbohydrates in Another possible explanation for the weak relation- their composition, which are usually difficult to digest ship between molar shape and diet could be a recent and need to be ground into smaller pieces for easier radiation of the group, where the ecological speciali- digestion (Kay & Sheine, 1979). Consequently, the con- zation occurred faster than the morphological changes. sumption of arthropods might create a structural con- However, Meredith et al. (2008) estimated a date of ap- straint that the shape of the molars is more in proximately 40 Mya for the splitting of Didelphinae from accordance with an insect-based diet than with a meat Caluromys, and 30 Mya for the splitting of Monodelphis based one, even when the first item is not an impor- from the remaining Didelphinae. Thus, we believe tant item in the diet of this marsupial. Species spe- that the morphological uniformity of the molars is cialized in eating insects tend to have longer shearing unlikely to be attributable to a recent radiation of the crests than other species (Kay & Hylander, 1978; Kay group. & Sheine, 1979; Hogue & ZiaShakeri, 2010), and these One important factor to take into account when ana- crests tend to be more transverse than longitudinal lysing our results is the impact of the phylogenetic with respect to the mesiodistal axis of the molar series, legacy. The canonical phylogenetic analyses showed that while in carnivorous species the crests tend to be more in all analysed molars, phylogeny alone (without taking

© 2014 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014 16 M. A. CHEMISQUY ET AL. into account the interaction with other variables) ex- phylogeny (Figs 8 and S7). The clade formed by plained between 40 and 65% of the variance, while diet Lutreolina, Thylophorops† and Hyperdidelphys† (all car- never explained more than 6% (the model was never nivores) show long metacristae, more longitudinally ori- statistically significant, compared with phylogeny which entated to the mesiodistal axis of the tooth row, while was significant for the third molars). Without exclud- Lestodelphys, which is placed in a distant node with ing the interaction between variables, the percentage the insectivorous genus Thylamys, shows the metacrista of variance explained by diet rises to 10–20%, but the orientated more transverse to the mesiodistal axis of percentage explained by the phylogeny also in- the tooth row, a change of shape also present in the creases, rising to 62–72%. Thus, at least half of the optimization of the node of that clade (node 6, Fig. 8). shape variance explained by diet is related to Something similar can be observed in the optimiza- phylogenetic relationships, meaning that only a small tion of m3, where the length and orientation of the part of the molar shape was influenced by diet during paracristid follows the same pattern as the metacrista. evolution of the group. An example of this effect of In the analysis of m3 we also included representa- phylogenetic legacy can be found in the similar molar tives of the carnivorous genus Thylatheridium†, which shape described for Lestodelphys and Thylamys despite is the sister group to the insectivorous/carnivorous genus some differences in diet. The same trend was ob- Monodelphis. The optimization of Thylatheridium† served with allometry, which could be interpreted as showed a long transversely orientated paracristid, a a structural constraint (Hall, 1999). The largest pro- feature more related to insectivory than to carnivory portion of the molar shape is exclusively related to the (Fig. 9). Consequently, the dental morphology exhib- position of each genus on the phylogenetic tree, which ited by the ancestor of each taxon clearly influences is difficult to explain given that phylogeny is not dental shape, causing species with similar diets to have an evolutionary constraint but rather the pattern of different molar shapes. evolutionary change over time, or the correlation of In some cases, we also observed convergent shapes cladogenetic events and morphological patterns (Losos, as a solution to similar diet constraints. For example, 2011). What is usually termed phylogenetic con- the PCA of M1 showed several carnivorous species from straint or phylogenetic inertia is related to restric- different phylogenetic lineages grouped in the lower tions on the evolutionary trajectories imposed by right quadrant of the graph (Figs 3 and S2), conse- previous adaptations (Johnson, McKinney & Sorenson, quently exhibiting similar molar shapes. This conver- 1999), and includes several genetic, developmental and gence was also observed in the optimization of M1 fitness constraints on evolution, which is ultimately and M3 in the phylogeny, where a larger stylar related to natural selection (Shanahan, 2011). In our shelf, mainly at the metastylar corner, was observed results, we found a strong phylogenetic effect (meaning independently in several carnivorous/faunivorous a slow rate of character evolution related to the rate genera: Lestodelphys, Monodelphis and the clade of cladogenesis; Losos, 2011), but the data available Thylophorops†+Lutreolina + Hyperdidelphys† (Fig. S7). make the real constraints behind this historical pattern What remains to be tested is the shape optimization difficult to distinguish. We believe that the broad eco- of the molars of Caluromys, a group placed on the logical niche used by most of the groups/genera studied other end of the diet variation, being mostly frugivorous. herein (at least regarding diet) did not generate enough Future analyses including taxa placed outside selective pressure on molar morphology to override pre- Didelphidae will help to describe the evolutionary existing differences that occur among clades, subse- history of the molar shape of a group that shows quently confounding the relationship between diet and such a distinctive morphology (at least as seen in our tooth shape. Additionally, because a large proportion results). of shape variation is related to phylogeny and not to The constraints posed to the molar shape by the size or diet, it is possible that this part of the vari- phylogenetic legacy have been described previously by ation is related to genetic drift, which could be trans- many authors in other mammal groups (e.g. Kay & lated on a macroevolutionary scale to an evolutionary Hylander, 1978; Strait, 1993) and even in marsupials model similar to a random walk or Brownian motion (Hogue & ZiaShakeri, 2010). However, this is the first or other models where the change occurred at specia- study to clearly show the bias caused by the phylogenetic tion (Felsenstein, 1985). legacy using new methodologies to reconstruct the The molar shape optimization analysis on phylog- ancestral shape of the molars throughout the phylogeny reinforces the ideas mentioned above. When ana- eny of the group. The pattern observed in the lysing the optimization of M3 in some key taxa, such reconstruction of the ancestral shapes reinforces the as representatives of the most carnivorous category (V), results of the canonical phylogenetic analysis, which it can be seen how the length and position of the showed that most of the molar shape variance was ex- metacrista (one of the main indicators of the carnivo- plained by phylogeny, while diet category and size ex- rous diet) is influenced by the position of taxa in the plained only a small proportion of that variance.

Nonetheless, it is important to keep in mind that the Catalano SA, Goloboff PA. 2012. Simultaneously mapping tribosphenic molar is very versatile, and allowed and superimposing landmark configurations with parsimo- members of the Didelphidae to develop different diets ny as optimality criterion. Systematic Biology 61: 392–400. (although the differences might not be as strong as in Caumul R, Polly PD. 2005. Phylogenetic and environmen- other groups of mammals) without the need for dra- tal components of morphological variation: skull, mandible, matic shape change in the molars. and molar shape in marmots (Marmota, Rodentia). Evolu- In summary, the analyses here indicated a weak re- tion 59: 2460–2472. lationship between molar shape and diet. On the one Crompton AW, Hiiemae KM. 1969. Functional occlusion in tribosphenic molars. Nature 222: 678–679. hand, this could be related to the lack of strong dietary Crompton AW, Hiiemae KM. 1970. Molar occlusion and man- specializations, where maintaining a molar able to cope dibular movements during occlusion in the American opossum, with mixed diets is highly advantageous. On the other Didelphis marsupialis L. Zoological Journal of the Linnean hand, the strong correlation found between the posi- Society 49: 21–47. tion of the taxa on the phylogeny and molar shape in- Dray S, Dufour AB. 2007. The ade4 package: implementing dicates that the shape exhibited by each taxon is highly the duality diagram for ecologists. Journal of Statistical influenced by phylogenetic legacy. This was clearly seen Software 22: 1–20. in the optimizations, where although some conver- Dumont ER, Strait SG, Friscia AR. 2000. Abderitid gences were observed, we also found that taxa with marsupials from the Miocene of Patagonia: an assessment similar diets showed molars with different shapes, with of form, function, and evolution. Journal of Paleontology 74: a strong influence of the shape of sister taxa. 1161–1172. Evans AR. 2005. Connecting morphology, function and tooth wear in microchiropterans. Biological Journal of the Linnean ACKNOWLEDGEMENTS Society 85: 81–96. We thank J. Faivovich and M. Pereira for allowing Evans AR, Sanson GD. 2006. Spatial and functional mod- use of the microscope and for technical help, A. Kramarz eling of Carnivore and Insectivore molariform teeth. Journal (MACN-PV) and M. Reguero (MLP-PV) for access to of Morphology 267: 649–662. specimens under their care, G. Cassini for discussion Evans AR, Wilson GP, Fortelius M, Jernvall J. 2007. High- of results and help with the R scripts, and D. Astúa level similarity of dentitions in carnivorans and rodents. Nature 445: 78–81. and an anonymous reviewer for help with the manu- Felsenstein J. 1985. Phylogenies and the comparative method. script. G.M. thanks E. Watkins and M. Simeon for American Naturalist 125: 1–15. financial support. 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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1. First lower molar (m1) shape variation along the first two principal components from the PCA of the Procrustes coordinates, showing the distribution of diet categories. Deformation grids show the extreme shape of each PC. Figure S2. First upper molar (M1) shape variation along the first two principal components from the PCA of the Procrustes coordinates, showing the distribution of diet categories. Deformation grids show the extreme shape of each PC. Figure S3. Third lower molar (M1) shape variation along the first two principal components from the PCA of the Procrustes coordinates, showing the distribution of diet categories. Deformation grids show the extreme shape of each PC. Figure S4. Third upper molar (M1) shape variation along the first two principal components from the PCA of the Procrustes coordinates, showing the distribution of diet categories. Deformation grids show the extreme shape of each PC. Figure S5. Scatterplots resulting from the between-group PCA of the first lower molar (m1), summarizing differences between the five diet categories. White squares with the Roman numeral of each diet category represent the centroid of the distribution for that category. Deformation grids show the extreme shape of each PC. Figure S6. Scatterplots resulting from the between-group PCA of the first upper molar (M1), summarizing differences between the five diet categories. White squares with the Roman numeral of each diet category represent the centroid of the distribution for that category. Deformation grids show the extreme shape of each PC. Figure S7. Phylogeny showing the optimization of the third upper molar. Numbers on branches indicate node number. Green dots represent landmarks. Red lines represent the transformation from the last ancestor of the taxa/node. Figure S8. Phylogeny showing the optimization of the first upper molar. Numbers on branches indicate node number. Green dots represent landmarks. Red lines represent the transformation from the last ancestor of the taxa/node. Figure S9. Phylogeny showing the optimization of the third lower molar. Numbers on branches indicate node number. Green dots represent landmarks. Red lines represent the transformation from the last ancestor of the taxa/node. Figure S10. Phylogeny showing the optimization of the first lower molar. Numbers on branches indicate node number. Green dots represent landmarks. Red lines represent the transformation from the last ancestor of the taxa/node. Table S1. Sampled specimens for each molar, including collection data. Data S1. TPS file with the unaligned specimens. The file contains the data of the four molars analysed here.