The adaptive significance of mandibular symphyseal fusion in mammals

doi: 10.1111/j.1420-9101.2012.02457.x The adaptive significance of mandibular symphyseal fusion in mammals J. E. SCOTT*, A. S. HOGUE &M.J.RAVOSAà,, *Department of Biological Sciences, University of Notre Dame, Notre Dame, IN, USA Department of Biological Sciences, Salisbury University, Salisbury, MD, USA àdepartments of Biological Sciences, Aerospace and Mechanical Engineering and Anthropology, University of Notre Dame, Notre Dame, IN, USA Department of Zoology, Mammals Division, Field Museum, Chicago, IL, USA Department of Anatomy and Cell Biology, Indiana University School of Medicine-South Bend, South Bend, IN, USA Keywords: Carnivora; dietary mechanical properties; fusion; jaw; Mammalia; mandibular symphysis; Marsupialia; masticatory biomechanics; phylogenetic comparative methods; Strepsirrhini. Abstract The mandibular symphyseal joint is remarkably variable across major mammalian clades, ranging in adults from unfused (amphiarthrosis) to partially fused (synarthrosis) to completely ossified (synostosis). Experimental work conducted on primates suggests that greater ossification of the symphysis is a response to increased recruitment of the balancing-side (i.e. nonchewing side) jaw-adductor muscles during forceful unilateral biting and chewing, with increased fusion strengthening the symphysis against correspondingly elevated joint stresses. It is thus expected that species with diets composed primarily of foods that require high-magnitude bite forces and or repetitive loading to process will be characterized by greater degrees of symphyseal ossification than species with relatively easy-to-process diets (i.e. food items typified by low toughness and or low stiffness). However, comparative support for this idea is limited. We tested this hypothesis in four dietarily diverse mammalian clades characterized by variation in symphyseal fusion the Strepsirrhini, Marsupialia, Feliformia, and Caniformia. We scored fusion in adult specimens of 292 species, assigned each to a dietary category based on literature accounts, and tested for an association between these two variables using Pagel s test for the correlated evolution of binary characters. Results indicate that greater fusion is associated with diets composed of resistant items in strepsirrhines, marsupials, and feliforms, providing some support for the hypothesis. However, no such relationship was detected in caniforms, suggesting that factors other than dietary mechanical properties influence symphyseal ossification. Future work should focus on such factors, as well as those that favour an unfused mandibular symphysis. Introduction Correspondence: Matthew J. Ravosa, Professor, Department of Biological Sciences, Galvin Life Science Center, University of Notre Dame, Notre Dame, IN 46556, USA. Tel.: +1 574 631 2556; fax: +1 574 631 7413; e-mail: matthew.j.ravosa.1@nd.edu The mandibular symphysis is one of the most interesting and complex articulations in the mammalian body. Anatomically, it varies from (1) the plesiomorphic condition of smooth, opposing dentaries loosely connected by fibrocartilage and ligaments (amphiarthrosis), to (2) a more tightly bound joint with greater sutural complexity consisting of interlocking rugosities and numerous variably calcified ligaments (synarthrosis), to (3) a fully ossified, or fused, joint (synostosis) (Scapino, 1965, 1981; Beecher, 1977, 1979). Such variation characterizes a diverse array of mammalian clades, including marsupials, xenarthrans, carnivorans, cetartiodactyls, perissodactyls, proboscideans, hyracoids, chiropterans, and primates (Scapino, 1965, 1981; Beecher, 1977, 1979, 1983; Ravosa, 1991, 1996, 1999; Ravosa & Hylander, 1994; Ravosa & Simons, 1994; Hogue & Ravosa, 2001; Ravosa & Hogue, 2004; Ravosa et al., 2007b; Williams et al., 2008; Davis et al., 2010; Fitzgerald, 2012). The functional significance of symphyseal fusion has been studied most intensively in the order Primates, where there is a morphological dichotomy between 661

662 J. E. SCOTT ET AL. extant anthropoids with fusion and extant strepsirrhines with mostly unfused or partially fused joints. A large body of experimental work conducted on members of this clade suggests that fusion is an adaptive response to elevated joint stresses associated with increased activity of the balancing-side (i.e. nonchewing-side) jaw-adductor muscles during postcanine biting and chewing (Hylander, 1979a,b, 1984, 1985; Hylander & Johnson, 1994; Ravosa & Hylander, 1994; Hylander et al., 1998, 2000, 2005, 2011; Ravosa & Hogue, 2004; Vinyard et al., 2005, 2006, 2007). In anthropoids, increased muscle forces generated by the balancing-side jaw adductors are transferred across the symphyseal joint to augment bite forces along the working-side mandible during unilateral mastication. Thus, increased ossification strengthens the symphyseal joint against significant bending and shear stresses associated with routinely high levels of balancing-side jaw-muscle recruitment. Because mastication of mechanically demanding foods requires greater jaw-muscle activity and relatively higher levels of balancing-side jaw-muscle force in primates (Luschei & Goodwin, 1974; Hylander et al., 1992, 2000; Hylander & Johnson, 1994), variation in symphyseal fusion in living and fossil primates, including strepsirrhines and basal anthropoids, has been linked to differences in dietary mechanical properties (e.g. Beecher, 1977, 1979, 1983; Ravosa, 1991, 1996, 1999). However, all members of the crown anthropoid radiation which includes extant and fossil apes, humans, and New and Old World monkeys have a fused symphysis but are characterized by remarkable dietary diversity (Ravosa & Hylander, 1994; Ravosa, 1999). For example, fusion is found in anthropoids that feed on foods that are highly resistant to crack initiation or propagation, such as seeds and leaves, which require high-magnitude bite forces and or repetitive loading (Strait, 1997; Hogue, 2004; Lucas, 2004), and in species with diets composed of relatively easy-to-process items, such as tree exudates, insects, and ripe fruits with low toughness and or low stiffness. The ubiquity of fusion in this clade clearly poses a problem in terms of linking greater ossification to increased reliance on mechanically demanding foods. Outside of primates, there have been few attempts to link variation in fusion to dietary mechanical properties. A notable exception is Scapino s (1981) work on fusion in the Carnivora. Although Scapino (1981) failed to detect a consistent dietary signal, he suggested that species that prey on large animals may require greater symphyseal ossification because the tissues of larger animals may be more difficult to break down due to the positive allometry of the cross-sectional area of connective tissues and skeletal elements (Yamada & Evans, 1970; Anderson et al., 1979). According to this argument, species that consume relatively large animals do so by recruiting a greater amount of balancing-side jaw-adductor force or by incurring greater numbers of daily loading cycles. Therefore, to resist the concomitant elevation in symphyseal stresses, such taxa require greater symphyseal ossification (Scapino, 1981; Ravosa & Hogue, 2004). While several studies have concluded that prey size relative to predator size is an important determinant of the forces that must be generated by the jaw adductors and resisted by the skull in carnivorans (Biknevicius & Van Valkenburgh, 1996; Wroe et al., 2005; Christiansen & Wroe, 2007; Wroe & Milne, 2007; Meachen-Samuels & Van Valkenburgh, 2009; Slater & Van Valkenburgh, 2009; Slater et al., 2009), none has explicitly examined the link between relative prey size and symphyseal fusion. Thus, although experimental work in primates implicates food mechanical properties as an important influence on ossification of the mandibular symphysis, the comparative evidence for this hypothesis is limited primarily to the Strepsirrhini (Beecher, 1977; Ravosa, 1991). Even in this case, however, the potentially confounding effect of phylogenetic relatedness on the distribution of fusion has never been adequately addressed. In this study, we take advantage of recent advances in the understanding of phylogenetic relationships within the Strepsirrhini and three other major mammalian clades Marsupialia, Feliformia, and Caniformia to perform a phylogenetically informed comparative test of the link between diet and symphyseal morphology in these four dietarily and morphologically diverse taxa. We show that increased ossification is associated with mechanically challenging diets in three of these four clades. In the fourth clade, the Caniformia, no such relationship was detected, indicating that there are other determinants of symphyseal fusion worthy of consideration in mammals. Materials and methods We scored symphyseal fusion in 2875 adult individuals representing 292 species using a four-state system: (1) unfused with smooth symphyseal plates, (2) partially fused with simple rugosities (simple partial fusion), (3) partially fused with complex, interlocking rugosities (complex partial fusion), and (4) completely ossified (Fig. 1; see also Scapino, 1981; Ravosa & Hogue, 2004). This system recognizes that fusion is a continuous trait, not a dichotomy between fused and unfused, but like all categorical representations of continuous variation, it is an oversimplification. Nevertheless, our categories capture a significant portion of the variation in symphyseal morphology observed in the clades included in our analysis. Sample information is summarized in Table 1 and the mean fusion score for each species is presented in the supplementary information (see Table S1 for full details). Our sample contains a number of recently extinct species, including the giant subfossil lemurs of Madagascar (Pachylemur, Babakotia, Mesopropithecus, Archaeolemur, Hadropithecus, Palaeopropithecus, Archaeoindris, Megaladapis), the Tasmanian tiger (Thylacinus

Diet and mandibular symphyseal fusion 663 (a) (b) (c) (d) Fig. 1 Diagrammatic representations of symphyseal character states in transverse cross section (anterior labial is at top): (a) unfused, (b) simple partial fusion, (c) complex partial fusion, and (d) completely fused. See also Scapino (1981). cynocephalus), and the Caribbean monk seal (Monachus tropicalis). Nearly all of the species in our sample were assigned to dietary categories based on accounts from the literature regarding the foods that are most commonly consumed (Table S1). In a few cases, however, species that are generally thought of as omnivorous (e.g. some ursids and mustelids) were classified based on foods that may not be exploited as frequently as others but which may, because of their mechanical properties, exert strong selection pressure on a species masticatory system (e.g. Rosenberger, 1992; Lambert et al., 2004). This difference in how extant species were classified does not affect the results. In the case of the subfossil lemurs, we relied on Table 1 Summary of samples used in the analysis. Clade Families Genera Species Median n per species Strepsirrhini 10 30 54 17 Marsupialia 18 53 83 8 Feliformia 7 46 70 6 Caniformia 9 64 86 6 See Table S1 for detailed sample information. recent studies of dental wear to characterize them dietarily (Jungers et al., 2002; Rafferty et al., 2002; Godfrey et al., 2004; Scott et al., 2009). This method is obviously inferior to observational studies, but given that living species with different diets exhibit distinct patterns of dental wear, it is considered reliable, if not always precise. Additionally, the studies cited above are in general agreement with each other, meaning that our assignments are not controversial. Finally, using dental wear to infer the diets of extinct lemurs allows us to include this group in our analysis, which is important because some of these species have completely fused symphyses, a condition not observed in extant strepsirrhines. Our dietary categories are necessarily crude, given the broad-scale comparative approach adopted here and the quality of the dietary data available for many species. Species were classified as follows: folivore, seed-predator, frugivore (no seed-predation), exudativore, insectivore, and carnivore. Carnivorous carnivorans were further subdivided based on relative prey size. The latter classifications were taken mainly from Christiansen & Wroe (2007), supplemented with descriptions from the literature for the species in our sample that were not included in Christiansen and Wroe s study. Under this scheme, prey size is defined relative to the predator s body mass: small prey, 20% or less; medium prey, 20 100%; large prey, greater than 100%. Note that medium- and largeprey carnivores are not necessarily restricted to medium and large prey, respectively i.e. they may regularly consume smaller animals. This system was not applied to marsupial carnivores for the statistical analysis due to insufficient data, but we discuss the potential influence of prey size in selected cases. We used Mesquite s (Maddison & Maddison, 2010) version of Pagel s (1994) test for the correlated evolution of binary characters to examine the association between fusion and diet. For strepsirrhines, feliforms, and caniforms, symphyseal fusion was coded as follows: 0 = unfused or simple partial fusion; 1 = complex partial fusion or fused. Because there are no marsupials with complex partial fusion in our sample, the binary coding used for fusion in this clade differs: 0 = unfused; 1 = simple partial fusion or full fusion. This difference in coding does not affect interpretations because analyses were performed primarily within clades and we are interested in relative differences. With respect to diet, we contrasted exudativores, insectivores, frugivores, and small-prey carnivores (code = 0) with folivores, seed-predators, and medium- and large-prey carnivores (code = 1). Thus, our binary codes reflect the following distinctions: for symphyseal form, less fused vs. more fused; for diet, less difficult to process vs. more difficult to process. Reducing our categorical variables to binary traits obviously results in a loss of information. However, this approach is appropriate, in our view, because fusion is expected to be related to dietary mechanical properties (e.g. stiffness,

664 J. E. SCOTT ET AL. toughness) rather than dietary categories per se (e.g. fruit, insects), and our categories are only crude approximations of the properties they are supposed to represent. For example, under our scheme, large-prey carnivores and folivores are both classified as having mechanically challenging diets, but for different reasons i.e. large prey are thought to differentially require higher-magnitude bite forces whereas leaves are challenging because they require greater repetitive loading. We conducted Pagel s test using phylogenetic trees compiled from a variety of molecular studies (see Appendix S1). Because the phylogenetic trees used in our analyses are composites, we ran Pagel s test using the arbitrary branch lengths available in the PDAP:PDTREE module (Midford et al., 2010) of Mesquite: Pagel, Nee, Grafen, and all branch lengths set to one (i.e. unit). The use of arbitrary branch lengths instead of estimated branch lengths is not ideal, but by comparing the outcomes of tests performed using different sets of arbitrary branch lengths we can determine whether our results are robust to how branch lengths were specified (M. Pagel, personal communication; see also Robertson et al., 2010). Fortunately, results for each clade were consistent regardless of the branch lengths used (i.e. Pagel s test was either always significant or always nonsignificant for a given clade, regardless of branch lengths). Significance was assessed using 1000 simulations. Thus, Pagel s test was performed four times on each clade, for a total of 4000 simulations per group. We report P-values for each test and adjust significance levels using the false-discovery-rate procedure described by Curran-Everett (2000) in order to control for multiple comparisons. Results Strepsirrhine primates exhibit a strong association between diet and symphyseal fusion (Fig. 2; Table 2). None of the species classified as frugivorous, insectivorous, or exudativorous exhibits a degree of ossification greater than simple partial fusion, and of the 21 species classified as folivores and seed-predators, 19 exhibit complex partial fusion or complete fusion. The two exceptions are Lepilemur mustelinus (unfused) and Hapalemur griseus (simple partial fusion). These results confirm previous observations made using conventional comparative methods (Beecher, 1977; Ravosa, 1991). Moreover, the studies of dental wear we used to assign subfossil Malagasy lemurs to dietary categories (Jungers et al., 2002; Rafferty et al., 2002; Godfrey et al., 2004; Scott et al., 2009) allow us to confidently associate complete fusion with mechanically demanding diets in these taxa. This condition appears to have evolved three times in the subfossil taxa: once in archaeolemurids (seed-predators), once in the ancestor of the palaeopropithecid genera Palaeopropithecus and Archaeoindris (seedpredators and folivores), and once in megaladapids (folivores). Marsupials present a pattern that is similar to that observed in strepsirrhines, though the signal is not as strong (Fig. 3; Table 2). Only 22 of the 83 species in this clade exhibit some degree of symphyseal ossification, with nineteen being folivores distributed across four clades: the Phalangeroidea, Macropodiformes, Pseudocheiridae, and Vombatiformes. Notably, however, there are sixteen folivores with unfused symphyses; these are mainly concentrated in the Macropodiformes. There Fruit, exudates, or insects Leaves or seeds Cheirogaleidae Lepilemur mustelinus Archaeolemur majori A. edwardsi Hadropithecus stenognathus Babakotia radofilai Mesopropithecus globiceps M. dolichobrachion M. pithecoides Palaeopropithecus ingens P. maximus Archaeoindris fontoynonti Indri indri Avahi laniger Propithecus diadema P. verreauxi P. tattersalli Megaladapis edwardsi M. madagascariensis M. grandidieri Lemur catta Hapalemur griseus Prolemur simus Eulemur Pachylemur Varecia variegata Daubentonia madagascariensis Lorisidae Galagidae Unfused or simple partial Complex partial or fused Archaeolemuridae Palaeopropithecidae Indriidae Megaladapidae Lemuridae Fig. 2 Association between diet and fusion in the Strepsirrhini. Taxa marked with a dagger ( ) are recently extinct. Stars indicate taxa with complete ossification. In some cases, clades that are homogeneous with respect to symphyseal morphology and diet have been collapsed to facilitate presentation. Branch lengths are not to scale.

Diet and mandibular symphyseal fusion 665 Table 2 Likelihood ratios (LR) and P-values for Pagel s test by taxon and branch lengths. Branch lengths Unit Grafen Pagel Nee Taxon LR P LR P LR P LR P Strepsirrhini 20.14 0.014* 17.94 0.001* 57.49 < 0.001* 54.84 < 0.001* Marsupialia 11.61 0.007* 10.52 0.013* 10.52 0.015* 10.41 0.012* Feliformia 15.00 0.002* 13.28 0.004* 13.60 0.001* 11.86 0.011* Caniformia 5.78 0.249 5.45 0.277 4.36 0.452 4.15 0.143 All clades 13.87 < 0.001* 13.01 < 0.001* 13.84 < 0.001* 15.87 < 0.001* Likelihood ratios were computed as: LR = )2(ln L 0 -lnl 1 ), where L 0 is the likelihood for the model of independent evolution and L 1 is the likelihood for the model of dependent evolution. P-values are based on 1000 simulations conducted in Mesquite; those marked with an asterisk are significant after adjusting a for multiple comparisons using the false-discovery-rate procedure (Curran-Everett, 2000). The latter procedure orders the P-values from least to greatest, and then computes a new a for each based on its rank using the following formula: i k 0.05, where i is the rank of the P-value (with rank 1 being the lowest), k is the total number of comparisons, and 0.05 is the error rate for the family of k tests. are only four species with complete fusion, and three of these are the folivorous Vombatiformes: Phascolarctos cinereus, Vombatus ursinus, and Lasiorhinus latifrons. The other taxon with fusion is the carnivore Sarcophilus harrisii. Thus, full fusion appears to have evolved twice in marsupials and in very different ecological contexts. It is also notable that simple partial fusion is not restricted to folivores: the carnivore Thylacinus cynocephalus and the exudativore insectivore Petaurus norfolcensis exhibit this condition as well. The results for feliform carnivorans provide support for a link between fusion and prey size (Fig. 4; Table 2): species classified as medium- and large-prey carnivores tend to have more advanced degrees of fusion primarily complex partial fusion than species that consume small prey, insects, and fruit (note that there are no folivores in this clade). This distinction is mainly between the larger felids (Panthera, Neofelis, Pardofelis, Lynx, and Puma) and other feliforms, but Hyaena brunnea and Cryptoprocta ferox also exhibit the predicted relationship. Note, however, that in contrast to H. brunnea, the other bone-crushing, large-prey hyaenids H. hyaena and Crocuta crocuta exhibit simple partial fusion. Furthermore, C. ferox is one of only four feliform species that exhibit complete (or nearly complete) ossification and is the only one that preys on relatively large animals. The other species with full fusion are the insectivores Proteles cristatus, Suricata suricatta, and Herpestes naso. The relationships between diet and fusion observed in strepsirrhines, marsupials, and feliforms break down in the Caniformia, with Pagel s test failing to detect a relationship between these variables (Fig. 5; Table 2). It is possible that this result is driven partly by the fact that some caniforms rely on a wide variety of foods, making it difficult to assign them to a single dietary category (e.g. ursids and some mustelids, particularly the badgers Mellivora and Meles; Ewer, 1972). However, the distribution of character states among those species for which dietary assignments are more secure indicates that the lack of association between diet and fusion is not entirely an artifact of an inadequate dietary classification scheme. For example, within the Canidae, four species are largeprey carnivores, but only one Speothos venaticus exhibits complete ossification; the rest have unfused joints like other canids with less challenging diets. Similarly, species of the genus Mustela and the closely related Neovison vison all prey on relatively large animals but all have unfused symphyses. The two caniform folivores also present an inconsistent signal: the bambooeating Ailuropoda melanoleuca has a fused symphysis, whereas Ailurus fulgens, which also specializes on bamboo, exhibits less ossification (simple partial fusion) than the frugivore Potos flavus and the insectivore Melursus ursinus (both fused). The lack of association between diet and fusion in caniforms is particularly notable, given that complete ossification appears to have evolved more times (perhaps as many as 11) in this clade than in strepsirrhines, marsupials, and feliforms combined. Given the contrast between Caniformia and the other three clades examined here, we conducted a broader test of the association between diet and symphyseal fusion in the entire sample. For this analysis, the three placental clades were rescored using the marsupial scheme i.e. the split in the binary coding was between, on the one hand, species with unfused symphyses and, on the other hand, species exhibiting simple partial fusion or more advanced degrees of ossification. The results indicate that the association holds at this level as well (Table 2). Discussion The hypothesis that symphyseal fusion is a response to increased stress associated with greater reliance on mechanically demanding foods finds support in three of the four mammalian clades examined in this study; the

666 J. E. SCOTT ET AL. Ailurops ursinus Phalanger Trichosurus vulpecula Buramys parvus Cercartetus Onychogalea unguifera Setonix brachyurus Macropus giganteus M. fuliginosus M. (Osphranter) M. (Notamacropus) Wallabia bicolor Thylogale Petrogale Dendrolagus lumholtzi Potorous tridactylus Bettongia penicillata Aepyprymnus rufescens Gymnobelideus leadbeateri Dactylopsila trivirgata Petaurus breviceps P. norfolcensis P. austalis Acrobates pygmaeus Hemibelideus lemuroides Petauroides volans Pseudochirops cupreus P. archeri Pseudocheirus Pseudochirulus mayeri P. canescens P. forbesi P. herbertensis Phascolarctos cinereus Vombatus ursinus Lasiorhinus latifrons Dromiciops gliroides Dasyurus Sarcophilus harrisii Dasycercus cristicauda Paranthechinus apicalis Phascogale tapoatafa Antechinus Sminthopsis Myrmecobiusfasciatus Thylacinus cynocephalus Notoryctes typhlops Macrotis lagotis Peramelidae Caenolestidae Didelphidae Phalangeroidea Macropodiformes Petauridae Pseudocheiridae Vombatiformes Dasyuridae Fruit, exudates, insects, or meat Leaves Unfused Simple partial or fused Fig. 3 Association between diet and fusion in the Marsupialia. Taxa marked with a dagger ( ) are recently extinct. Stars indicate taxa with complete ossification. In some cases, clades that are homogeneous with respect to symphyseal morphology and diet have been collapsed to facilitate presentation. Branch lengths are not to scale. signal is also apparent when these four clades are examined simultaneously. In strepsirrhines and marsupials, greater joint ossification is found primarily in folivores and seed-predators, and in feliforms, species that prey on relatively large animals tend to be characterized by more advanced degrees of fusion. Although we did not examine marsupial carnivores for a relationship between prey size and fusion, the fully ossified symphysis of Sarcophilus harrisii, which is known to hunt and consume relatively large animals and crush bones (Jones & Barmuta, 1998; Owen & Pemberton, 2005; Wroe et al., 2005; Attard et al., 2011), is consistent with this idea. Our data also indicate that clades can differ from each other in the distribution of symphyseal character states while still exhibiting the same diet-related pattern of relative differences. For example, strepsirrhine folivores are characterized primarily by complex partial fusion or complete ossification, whereas advanced degrees of ossification are rare in marsupial folivores none of the species in this group exhibits complex partial fusion and

Diet and mandibular symphyseal fusion 667 Nandinia binotata Prionodon linsang Felis Prionailurus bengalensis P. viverrinus P. planiceps P. rubiginosa Otocolobus manul Puma yagouaroundi P. concolor Acinonyx jubatus Lynx lynx L. canadensis L. rufus Leopardus Caracal caracal Profelis aurata Leptailurus serval Pardofelis marmorata P. temminckii Neofelis nebulosa Panthera pardus P. leo P. onca P. tigris P. uncia Viverridae Proteles cristatus Crocuta crocuta Hyaena hyaena H. brunnea Fossa fossana Eupleres goudoti Cryptoprocta ferox Galadictis fasciata Galidia elegans Suricata suricatta Liberiictiskuhni Helogale parvula Herpestes fuscus H. edwardsii H. vitticollis H. smithii H. brachyurus H. urva H. naso Atilax paludinosus Ichneumia albicauda Cynictis penicillata Paracynictis selousi Rynchogalemelleri Felidae Hyaenidae Eupleridae Herpestidae Fruit, insects, or small prey Medium and large prey Unfused or simple partial Complex partial or fused Fig. 4 Association between diet and fusion in the Feliformia. Stars indicate taxa with complete ossification. In some cases, clades that are homogeneous with respect to symphyseal morphology and diet have been collapsed to facilitate presentation. Branch lengths are not to scale. only three are characterized by full fusion. Nevertheless, marsupial folivores, as a group, tend to have a greater degree of fusion (i.e. simple partial fusion) than marsupials in other dietary categories. The reason for this difference between marsupials and strepsirrhines is unclear, but it may be related to subtle functional variation in dietary properties and jaw-loading patterns. Alternatively, given that advanced degrees of ossification are also common in the two carnivoran clades, this particular distinction may be related to differences between marsupials and placental mammals in general. Given that these two clades diverged sometime in the Jurassic (more than 150 million years ago; Bininda- Emonds et al., 2007; Luo et al., 2011; Meredith et al., 2011), the existence of such differences should not be surprising. One possible explanation is the fact that the craniofacial apparatus undergoes fundamentally different developmental processes in the two clades (Smith, 2006). These differences are tied to their divergent developmental strategies, with marsupials exhibiting a short intrauterine development and early, prolonged suckling compared to placental mammals (Smith, 2006). This distinction likely imposes different constraints on symphyseal development and form in the two clades. In any case, while our results suggest that direct comparisons between distantly related groups have the potential to obscure adaptive signals, they also indicate that this problem can be overcome, at least in some instances, and such signals can be detected by taking appropriate measures to adjust for interclade differences.

668 J. E. SCOTT ET AL. Canis lupus C. latrans C. aureus Cuon alpinus Lycaon pictus Canis mesomelas Speothos venaticus Chrysocyon brachyurus Lycalopex Cerdocyonthous Vulpes Nyctereutes procyonoides Otocyon megalotis Urocyon cinereoargenteus Ursus arctos U. maritimus U. americanus U. thibetanus Melursus ursinus Helarctos malayanus Tremarctos ornatus Ailuropoda melanoleuca Otariidae Odobenus rosmarus Phocidae Mydaus Mephitis mephitis Spilogale putorius Conepatus chinga Ailurus fulgens Potos flavus Bassaricyon gabbii Nasuella olivacea Procyon lotor P. cancrivorus Bassariscus Taxidea taxus Mellivora capensis Meles meles Arctonyx collaris Pekania pennanti Eira barbara Gulo gulo Martes Melogale moschata Galictis Vormela peregusna Ictonyx Poecilogale albinucha Mustela putorius M. nivalis M. erminea M. frenata Neovison vison Pteronura brasiliensis Lontra canadensis Enhydralutris Lutra lutra Aonyx capensis A. congicus Canidae Ursidae Pinnipedia Mephitidae Procyonidae Mustelidae Fruit, insects, or small prey Leaves, medium and large prey Unfused or simple partial Complex partial or fused Fig. 5 Association between diet and fusion in the Caniformia. Stars indicate taxa with complete ossification. In some cases, clades that are homogeneous with respect to symphyseal morphology and diet have been collapsed to facilitate presentation. Branch lengths are not to scale. It is important to note that a number of taxa violate our predictions, indicating that increased joint ossification is not simply a function of elevated masticatory stress and pointing to the existence of other influences on symphyseal form. Within the Marsupialia, the exudativore Petaurus norfolcensis and the carnivore Thylacinus cynocephalus exhibit simple partial fusion, a relatively advanced degree of ossification in comparison to closely related taxa. The reason for increased fusion in P. norfolcensis is unclear, as this species does not differ substantially in diet from other members of the Petauridae, at least at the resolution provided by currently

Diet and mandibular symphyseal fusion 669 available data (summarized by Hogue, 2004). On the other hand, consumption of relatively large prey is a possible explanation for simple partial fusion in T. cynocephalus. This argument is problematic, however, because although the diet of this recently extinct carnivore is not well-documented, morphological studies suggest that it may have been restricted to small prey (Jones & Stoddart, 1998; Attard et al., 2011). It is also worth noting that some members of the genus Dasyurus the sister taxon of Sarcophilus are known to hunt medium and large prey (Belcher, 1995; Jones, 1997; Wroe & Milne, 2007), yet their symphyses are unfused. The Caniformia present the biggest problem in terms of linking increased joint ossification to predation on relatively large animals. Eleven caniforms included in this analysis were classified as medium- or large-prey carnivores, but only three of them Speothos venaticus, Gulo gulo, and Ursus maritimus exhibit complex partial fusion or full fusion. The contrasting results obtained for caniforms and feliforms may be driven to some extent by differences in killing behaviour. For example, felids are typically solitary hunters that rely on a powerful, sustained bite when killing prey, whereas canids use numerous shallow slashing bites and frequently hunt in packs (e.g. Ewer, 1973; Van Valkenburgh & Ruff, 1987; Biknevicius & Ruff, 1992; Biknevicius & Van Valkenburgh, 1996). Thus, it may be that the hunting style of large-prey felids exposes the mandibular symphysis to greater risk of damage in comparison to that of large-prey canids, because of more prolonged contact time with struggling prey and a concomitant increase in the duration of daily loading events. Other aspects of mandibular form can be invoked to support this idea (Biknevicius & Ruff, 1992; Therrien, 2005), as can differences in canine strength among major carnivoran groups (Van Valkenburgh & Ruff, 1987). However, one problem with this hypothesis is that mustelids that hunt relatively large prey (i.e. species of Mustela and Neovison vison) do so in a manner similar to that of felids (Ewer, 1973; Biknevicius & Van Valkenburgh, 1996) but exhibit unfused symphyses. Thus, despite the fact that our analysis detected a link between prey size and fusion in the Feliformia, more work is needed to understand the relationship between these variables within the Carnivora as a whole. As noted above, three of the four feliform species that exhibit complete ossification are insectivorous. Most of the other species classified as insectivores in this clade have unfused joints (n = 9) or simple partial fusion (n = 7). Moreover, insectivores are common in Strepsirrhini and Marsupialia, but they are uniformly characterized by unfused symphyses. Some researchers make a distinction between insectivores that prey on insects with exoskeletons that are resistant to deformation and crack propagation, such as beetles, and those that prey on insects with exoskeletons that are fragile and pliant and therefore easier to pulverize, such as caterpillars and moths (Freeman, 1979; Strait, 1993, 1997; Strait & Vincent, 1998; Freeman & Lemen, 2007; Friscia et al., 2007). For example, in her study of feeding adaptations in molossid bats, Freeman (1979) linked more frequent predation on beetles (vs. moths) to features related to increased bite-force production and resistance (e.g. deep mandibular corpora, tall mandibular ascending ramus). Detailed comparisons of the mechanical properties of the insects consumed by feliform insectivores with fused symphyses such as Suricata suricatta and Herpestes naso vs. those ingested by closely related insectivores with lesser degrees of ossification may reveal a similar dietary signal. However, this explanation is unlikely to apply to Proteles cristatus, which is large (10 kg) for a dedicated insectivore and has reduced, peglike molars (Anderson et al., 1992), indicating that it does not spend much time masticating the termites that constitute the bulk of its diet. Moreover, it is certainly true that the diet of P. cristatus cannot be described as more mechanically demanding than the diets of the closely related bone-crushing hyaenids (Hyaena, Crocuta), which are characterized by lesser degrees of joint ossification. The Caniformia present a similar contrast: Melursus ursinus is a much larger-bodied insectivore (ranging from 50 kg to well over 100 kg; Joshi et al., 1995; Ratnayeke et al., 2007) with reduced postcanine teeth (Sacco & Van Valkenburgh, 2004), but it is the only ursid other than Ailuropoda melanoleuca characterized by full fusion. Given what is known about the diets of other ursids, it is unlikely that M. ursinus generates greater masticatory stresses than other members of its family (see also Scapino, 1981). Thus, comparison of Proteles and Melursus to closely related taxa indicates that factors other than stress resistance may select for increased joint ossification. We are not the first to suggest that functional inputs into symphyseal fusion vary within or among clades. For example, Williams et al. (2008) argued that the symphyses of camelid artiodactyls are fused because the large, medially positioned mandibular incisor roots of these species would compromise the structural integrity of a partially fused joint. This hypothesis was invoked by Davis et al. (2010) to explain variation in fusion in vampire bats (Desmodontinae). Freeman (1995) linked fusion in nectarivorous chiropterans to tongue protrusion during food acquisition, arguing that this behaviour may require an immobile symphysis. A similar hypothesis was proposed by Holliday et al. (2010) to explain the presence of features that serve to stiffen the symphyseal joint (e.g. fused Meckel s cartilages) in iguanian and gekkotan lepidosaurs. These hypotheses require further testing, but they provide a good starting point in terms of developing a more comprehensive explanation for variation in symphyseal fusion in mammals. Another factor that may affect the distribution of fusion in mammals is suggested by the ubiquity of this condition in crown anthropoid primates. Specifically, the

670 J. E. SCOTT ET AL. fact that there are no decreases in fusion in crown anthropoids in the face of marked ecological diversity raises the possibility that the symphysis is, in some clades, unlikely to become unfused once complete ossification is achieved (Ravosa, 1999), perhaps due to developmental canalization (Lockwood, 2007). In some cases, then, symphyseal fusion may reflect phylogenetic history rather than the diet-related selection pressures currently acting on a population. Thus, it may be that all crown anthropoids have a fused symphysis simply because they inherited it from a common ancestor with a mechanically demanding diet (Beecher, 1977; Ravosa & Hylander, 1994; Ravosa, 1999). This hypothesis deserves further attention, but it is unlikely to apply to the clades included in this study, especially given that complete ossification is relatively uncommon in these taxa. It is also important to recognize that the potential selective advantages of having an unfused symphysis remain largely unexplored (but see Scapino, 1981; Fitzgerald, 2012). Such factors may be relevant to understanding the patent symphyses of taxa that regularly process mechanically challenging objects. For example, Scapino (1981) hypothesized that symphyseal mobility in bone-crushing hyenas and the hard-objectfeeding sea otter (Enhydra lutris) may help protect the postcanine teeth during the forceful tooth-on-tooth impacts that occur when a bone or other hard object suddenly fails, either by allowing the mandibular carnassial to deflect away from its maxillary counterpart or by absorbing some of the energy such impacts engender. Another possibility is that the independent mobility of the left and right sides of the mandible permitted by an unfused symphysis may be important for incisor and postcanine occlusion in some species (Ride, 1959; Scapino, 1965, 1981; Crompton & Hiiemae, 1970; Weijs, 1975; Hylander, 1979a; Gorniak & Gans, 1980). Testing these hypotheses will be difficult, as it will likely require phylogenetically controlled comparisons of feeding behaviour in alert organisms. Finally, one issue not addressed by this study is the mechanism by which increased ossification of the symphyseal joint is achieved in species. Given the adaptive signal recovered by our analysis, natural selection has presumably favoured increased ossification in many of the species included herein. However, another possibility is phenotypic plasticity i.e. an organism-level postnatal response to changes in dietary mechanical properties. Long-term diet-manipulation experiments performed on growing rabbits (Oryctolagus cuniculus) have shown that the symphyses of animals raised from weaning on a diet of intact pellets and fracture-resistant hay cubes differ from those of subjects fed a much less challenging diet of ground pellets. Specifically, rabbits in the former group are characterized by symphyses with longer and wider joint surfaces, greater biomineralization of the hard tissues, and decreased proteoglycan and type II collagen expression in the fibrocartilage pad (Ravosa et al., 2007a, 2008, 2010). Moreover, in some cases, there is evidence that symphyseal rugosities extending from the opposing joint surfaces have fused, which would further affect symphyseal function (Ravosa et al., 2007a, 2008, unpublished data). These experimental results suggest that symphyseal hard tissues respond adaptively to dietary mechanical properties during an individual s lifetime, in part compensating for the load-induced degradation of fibrocartilage due to a diet requiring intensive postcanine processing. Assuming that the rabbit results are fairly representative of mammals in general, it is interesting that the organism-level developmental response to a mechanically challenging diet is similar to the one expected for members of a population or species experiencing natural selection to strengthen the symphyseal joint. In our view, it is likely that both factors variably contribute to the development of adult symphyseal form in those species included in our study. However, it is not possible at present to determine the relative contribution of these processes to the observed interspecific patterns. In this context, it is worth noting that there are interspecific differences in the developmental timing of complete ossification in primates: anthropoid primates are early fusers, achieving complete ossification by weaning, whereas the strepsirrhines that exhibit full fusion do so much later in postnatal life (Ravosa & Simons, 1994; Ravosa, 1996, 1999). (The latter pattern also characterizes many of the carnivorans in our data set; Ravosa, unpublished data.) Such differences may reflect the extent to which ossification is driven by plasticity vs. selection. For example, because all crown anthropoids achieve full ossification prior to adopting an adult diet, we can reject plasticity as an influence on fusion in this group; on the other hand, plasticity may play a greater role in strepsirrhines perhaps the dominant role in some species with complete fusion occurring only after prolonged exposure to mechanically challenging foods. Thus, it may be possible to tease apart the influences of plasticity and selection within species by examining symphyseal development on a case-by-case basis. Such analyses, combined with additional in vivo data on feeding behaviour and symphyseal function, will provide greater resolution to our understanding of this interesting and widely distributed biological phenomenon. Acknowledgments Financial support for this research was provided by the NSF (BCS-1029149, BCS-0924592, BCS-0127915), Thomas J. Dee Fund of the Field Museum, American Philosophical Society, American Society of Mammalogists, Sigma Xi, and the Leakey Foundation. We thank the editors, anonymous referees, Mark Pagel, Wayne Maddison, Justin Lack, Beth Fox, Kate Davis, and the curators and staff at the following institutions: American Museum of Natural History, Australian Museum,

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