THE ANATOMICAL RECORD PART A 281A:1157–1172 (2004)

Olfactory Fossa of Tremacebus harringtoni (Platyrrhini, Early , Sacanana, Argentina): Implications for Activity Pattern

RICHARD F. KAY,1* VICTORIA M. CAMPBELL,1 JAMES B. ROSSIE,2 3 3 MATTHEW W. COLBERT AND TIM B. ROWE 1Department of Biological Anthropology and Anatomy, Duke University, Durham, North Carolina 2Section of Vertebrate Paleontology, Carnegie Museum, Pittsburgh, Pennsylvania 3Jackson School of Geosciences, University of Texas, Austin, Texas

ABSTRACT CT imaging was undertaken on the skull of ϳ 20-Myr-old Miocene Tremacebus har- ringtoni. Here we report our observations on the relative size of the olfactory fossa and its implications for the behavior of Tremacebus. The endocranial surface of Tremacebus is incomplete, making precise estimate of brain size and olfactory fossa size imprecise. How- ever, olfactory fossa breadth and maximum endocranial breadth measured from CT images of one catarrhine species and eight platyrrhine species for which volumes of the olfactory bulb and brain are known show that the osteological proxies give a reasonably accurate indication of relative olfactory bulb size. Nocturnal Aotus has the largest relative olfactory fossa breadth and the largest olfactory bulb volume compared to brain volume among extant anthropoids. Tremacebus had a much smaller olfactory fossa breadth and, by inference, bulb volume— within the range of our sample of diurnal anthropoids. Variations in the relative size of the olfactory bulbs in platyrrhines appear to relate to the importance of olfaction in daily behaviors. Aotus has the largest olfactory bulbs among platyrrhines and relies more on olfactory cues when foraging than Cebus, Callicebus,orSaguinus. As in other examples of nocturnal versus diurnal , nocturnality may have been the environmental factor that selected for this difference in Aotus, although communication and other behaviors are also likely to select for olfactory variation in diurnal anthropoids. Considering the olfactory fossa size of Tremacebus, olfactory ability of this Miocene was probably not as sensitive as in Aotus and counts against the hypothesis that Tremacebus was nocturnal. This finding accords well with previous observations that the orbits of Tremacebus are not as large as nocturnal Aotus. © 2004 Wiley-Liss, Inc.

Key words: nocturnality; olfaction; Platyrrhini; Anthropoidea; Miocene

Among extant Anthropoidea, the platyrrhine Aotus 2000; Kirk and Kay, 2004). This hypertrophy stems from stands apart as the only wholly or partially nocturnal the absence in Aotus and Tarsius of a tapetum lucidum,a species (Wright, 1996). Anatomical and genetic evidence reflective layer behind the retina (Kirk and Kay, 2004). In from the visual system suggests that crown Anthropoidea were diurnal and that the nocturnal habits of Aotus are secondarily acquired (Martin, 1973, 1979, 1990; Cartmill, 1980; Jacobs et al., 1996; Ross, 1996, 2000). The antiquity *Correspondence to: Richard F. Kay, Department of Biological of the diurnal to nocturnal transition in Aotus is not well Anthropology and Anatomy, Box 3170, Duke University Medical understood. A key anatomical marker for nocturnality is Center, Durham, NC 27710. Fax: 919-684-8542. an enlarged orbit. Nocturnal primates in general have E-mail: [email protected] relatively large orbits but nocturnal haplorhines (Tarsius Received 20 May 2004; Accepted 1 July 2004 and Aotus) have comparatively enormous ones, propor- DOI 10.1002/ar.a.20121 tionately larger than nocturnal strepsirrhines of compa- Published online 12 October 2004 in Wiley InterScience rable size (Cartmill, 1980; Martin, 1990; Kay and Kirk, (www.interscience.wiley.com).

© 2004 WILEY-LISS, INC. 1158 KAY ET AL. nocturnal strepsirrhines, the tapetum reflects photons 6–10 million of these cells. Cell bodies of OSNs’ send back onto the retina, giving its photoreceptors a second single dendrites to the surface of the neuroepithelium opportunity to trigger the retinal photoreceptor array. The (investing portions of the nasal septum and ethnoturbi- loss of a tapetum is hypothesized to have evolved as part of nals, of the ethmoid and the nasal septum) and terminate a package of adaptations for high visual acuity in stem in a knoblike swelling. Arising from the swellings are haplorhines. In the absence of a tapetum, reacquisition of olfactory ciliae that contain the sensory receptors for smell nocturnality in Tarsius and Aotus is thought to have se- (Young, 1966; Firestein, 2001). Leading away from the lected for the evolution of a much larger eye and a larger OSN cell bodies are single axons that project to the main array of photoreceptors than typically found in nocturnal olfactory bulb (MOB) of the central nervous system. The forms. Therefore, the anatomical hallmark of nocturnality MOB contains glomeruli. Over the entire array of the in an extinct anthropoid should be the presence of a very neuroepithelium, all of the neurons expressing the same large orbit—larger than in nocturnal strepsirrhines. odorant receptor gene converge on the same glomeruli in Documenting the evolution of nocturnality in platyr- the MOB (Mombaerts, 1999; Rubin and Katz, 1999; rhines is hampered by the extreme rarity of skulls. Firestein, 2001). In the glomerulus, OSN axons meet den- Only five crania of Cenozoic platyrrhines have been de- drites of mitral and tufted cells that form the main pro- scribed: Tremacebus, Dolichocebus, Chilecebus, Homuncu- jection cells from the olfactory bulb to higher-order olfac- lus, and Lagonimico. Of these, one (Lagonimico)istoo tory structures (e.g., the piriform lobe) and other brain crushed to allow a reliable estimate of orbit size and centers. The relay system from neuroepithelium to the another (Chilecebus) is not yet fully described. The ϳ mitral and tufted cells is modulated by intrabullar inputs 20-Myr-old platyrrhine Tremacebus harringtoni (Fig. 1) is and inputs from other parts of the brain (Shipley et al., noted for its orbital enlargement (Rusconi, 1935; Hersh- 1995). kovitz, 1974), a feature that led Rosenberger (1979, 1984) Mammalian odor receptors (ORs) comprise a single and Szalay and Delson (1979) to conclude that it has structural family of G-protein-coupled receptors (Firest- phylogenetic affinities with Aotus and that nocturnality in ein, 2001; Godfrey et al., 2004; Malnic et al., 2004). In the Aotus clade extends back at least to the early Miocene. mice, ϳ 1,000 genes code for ORs, making this the largest However, though relative orbit size in Tremacebus falls family of genes in the mammalian genome (Godfrey et al., within the range of nocturnal strepsirrhines, it is rela- 2004). Each OR codes for the recognition of a particular tively smaller than the orbits of nocturnal haplorhines feature of a ligand (odorant molecule; mostly low-molecu- Aotus and Tarsius (Kay and Kirk, 2000). This led Kay and lar-mass organic compounds) (Firestein, 2001). An odor- Kirk to conclude that the activity pattern of Tremacebus ant molecule may have more than one recognizable part (a may not have been nocturnal. The only possible, if implau- determinant), and a particular OR can detect a common sible, alternative interpretation is that Tremacebus was chemical structure on a variety of odorant molecules. Fur- nocturnal and evolved a tapetum. Mohanamico hershko- ther, each OR can have its response to a particular deter- vitzi (Aotus dindensis) (Luchterhand et al., 1986) from minant reduced by an antagonist responding to a related 12.8-Myr-old levels in Colombia probably had large orbits structural compound. Peripheral coding is based on acti- also and therefore could have been nocturnal, although vation of arrays of olfactory receptor cells with overlap- only a small part of the orbit is preserved (Setoguchi and ping tuning profiles (Duchamp-Viret et al., 1999). If odor- Rosenberger, 1987). ant molecules are recognized by more than one receptor, What additional evidence can be brought to bear on the and if this response can be modulated by the presence of a question of the antiquity of nocturnality in extinct platyr- structurally related molecule, it is easy to see why the rhines? One possibility is to examine the olfactory system. olfactory system is capable of such fine sensitivity and In primates, the olfactory bulbs are relatively larger in discrimination (Firestein, 2001). nocturnal species than in their diurnal close relatives Many psychophysical tests have been designed to test (Barton et al., 1995), and this trend is particularly obvious odor sensitivity in where sensitivity refers to in Aotus.IfTremacebus had a particularly large olfactory the threshold of stimulus at which response occurs more bulb compared with other platyrrhines, this would provide often than chance. A challenge for such sensitivity tests is some support for the hypothesis that it was nocturnal. that individual ORs cannot really measure concentration. Fortunately, the braincase of Tremacebus, when CT-im- As concentration of an odor is increased, additional glo- aged, reveals a partial natural endocast of the olfactory meruli are recruited (Rubin and Katz, 1999). A further fossa, which housed the olfactory bulbs. In this article, we implication of the olfactory system’s structural design is pose the question of whether the proportions of the endo- that different individuals or species may be better at dis- cast of the brain and of the olfactory fossa can prove useful criminating or detecting odor not only due to differences in in determining the relative proportions of the brain and the number of ORs, but also to what kinds of ORs and, olfactory bulb in an extinct anthropoid. If so, and if Trema- downstream, how many different kinds of glomeruli they cebus were found to have a relatively large fossa (and possess. bulb), this would be evidence for nocturnality. A second olfactory detection system, the vomeronasal system (VNS), is separate from the main olfactory system. BACKGROUND It responds to pheromones generally (but not always) pro- Before we examine the evidence pertaining to the size of duced and emitted by conspecifics. Pheromones act as the olfactory bulbs in Tremacebus, first we summarize important chemical signals in mating behavior and other some important aspects of the anatomy and genetics of social interactions. One critical difference is that in the olfaction and the relative olfactory abilities in haplo- VNS, the sensory lumen of the vomeronasal organ (VNO) rhines. is filled with fluids of the vomeronasal gland and receptive Olfactory sensory neurons (OSNs) are the primary sen- to the dispersal of odorant molecules through fluid, sory cells of the olfactory system. In mammals, there are whereas the main olfactory epithelium is receptive to OLFACTORY FOSSA OF TREMACEBUS 1159

Fig. 1. Type specimen of Tremacebus harringtoni. A partial skull in lateral, frontal, and dorsal view. 1160 KAY ET AL. odorant molecule dispersal through air; also, the VNO Morrison, 2003). This means that the surface area of the epithelium is nonciliated (Keverne, 1999). The VNO is turbinals as a whole is unlikely to provide any precise surrounded by an erectile tissue that functions as a phys- information about olfactory sensitivity, although the eth- iological pump (Salazar et al., 1997) to suck into its fluid- moturbinals may do so. filled lumen high-molecular-weight odorant molecules The volumes of the main and accessory olfactory bulbs such as pheromones (Del Punta et al., 2002). The VNO and other brain components related to the olfactory sys- olfactory neuron axons project both to the accessory olfac- tem have been reported in a broad spectrum of mammals, tory bulb and toward the hippocampus, but not to the including many primates (Stephan et al., 1981, 1984; main olfactory bulb. Its glomeruli are located caudal to the Baron et al., 1983; Frahm et al., 1984). The relative size of main olfactory bulb. Projections from the accessory olfac- the main olfactory bulbs relative to brain size or body tory bulb in turn mediate responses in the endocrine sys- weight (Fig. 2) is smaller in haplorhines than in strepsir- tem. Two families (and more than 200 receptors) of G- rhines but does not differ in a systematic way between protein-coupled receptors, unrelated to OR families, have platyrrhine and catarrhine primates. Indeed, in the region been identified in the VNO of mice (Firestein, 2001). As of brain size overlap, MOB size in platyrrhines and ca- noted above, all ORs expressing a particular receptor over tarrhines overlaps extensively. Among diurnal anthro- the entire array of nasal neuroepithelium converge on a poids, the only significant outlier is Homo sapiens (not single target, one or at most three glomeruli, in the olfac- shown in Fig. 2), a species that has exceptionally small tory bulb. In contrast, VNO receptors disburse to as many olfactory bulbs (Baron et al., 1983). as 10–30 glomeruli in the accessory olfactory bulb. Sexual The volume of the olfactory bulb in platyrrhines is dimorphism of the VNS has been established in several highly correlated with overall brain volume and body vertebrate species, and often certain structures of this weight (Fig. 3). Scaling coefficients, slopes of least-squares system will be larger in males, while others will be larger regressions on logged data, are less than 1.0, indicating a in females (Halpren and Martinez-Marcos, 2003). How- negative allometry between olfactory bulb size and either ever, both sexes appear to have almost the same expres- brain volume or body weight. Among haplorhines, the sion of VNO receptor genes, suggesting that they may nocturnal species Tarsius and Aotus have proportionally detect similar pheromones and that differences in behav- larger olfactory bulbs than diurnal anthropoids. This is ioral response between sexes are the result of higher brain obvious comparing a lateral view of the brains of two functions in the VNS (Firestein, 2001). similar-sized platyrrhines, Aotus and Callicebus (Fig. 4). What evidence is there for variations in the odor-detect- In an allometrically and phylogenetically controlled ing abilities in the olfactory system and vomeronasal sys- comparison of the visual and olfactory components of the tem of primates? Several lines of evidence contribute a brain, Barton et al. (1995) conclude that across all pri- partial answer to this question but not all lines of evidence mates, there has been an evolutionary trade-off between are concordant. specializations in the olfactory and visual systems. Thus, Anatomical Evidence they report that the sizes of structures of the brain that process olfactory information are negatively correlated Two (linked or nonindependent) aspects of the nervous with the sizes of brain structures related to vision. For system are thought to relate in some way to the odor- example, species with large olfactory bulbs have relatively detecting abilities of primates: the degree of development smaller striate visual cortices. (surface area and number of odorant receptors) of the The relative size of olfactory and visual components sensory neuroepithelium within the nasal cavity where varies with respect to activity pattern (Barton et al., olfactory receptors reside, and the size of the olfactory 1995). Nocturnal clades tend to have larger olfactory bulb and other brain components that relay and integrate structures and smaller visual structures than do diurnal olfactory information (e.g., the piriform lobe). By analogy, or cathemeral clades. Within primates, using the method two anatomical components of the vomeronasal system of independent contrasts (Felsenstein, 1985; Purvis and are the size of the vomeronasal organ and the size of the Rambaut, 1995), Barton et al. (1995) identify four evolu- accessory olfactory bulb. An ancillary measure of the im- tionary shifts (transitions) between nocturnality and di- portance of either system would be the presence of scent urnality: anthropoids became diurnal from a nocturnal glands or other scent-related structures that use ancestor; the nocturnal platyrrhine Aotus had a to communicate with conspecifics or other species. diurnal ancestor; the diurnal clade of Propithecus ϩ Indri Olfactory epithelium and MOB. The olfactory epi- evolved from a nocturnal ancestor; and the diurnal/cath- thelium is located on the ethmoid turbinals and nasal emeral clade of Eulemur ϩ Varecia evolved from a noctur- septum. Turbinals of strepsirrhines generally are more nal ancestor. They report that olfactory bulbs and other elaborate than those of haplorhines (Paulli, 1900; Cave, olfactory components become larger in the nocturnal tran- 1973), but we know of no mensurational data comparing sitions and smaller in the diurnal transitions. The reverse the development of the turbinals of strepsirrhines or hap- pattern occurs with respect to the visual system. Visual lorhines. Also, the surface area of the turbinals includes brain components become smaller in nocturnal transitions both the respiratory and the olfactory epithelium. The and larger in diurnal transitions. main function of the turbinal bones is believed to be that Folivorous and frugivorous anthropoids also show dif- of warming and moistening incoming air (Young, 1966; ferences in the visual system when the effects of phylog- Van Valkenburgh et al., 2004), and the olfactory epithe- eny are controlled for. Folivorous diurnal anthropoids lium only covers the more posterior turbinals, formed by have smaller visual cortices than frugivorous diurnal spe- the ethmoid bone, toward the back of the nasal cavity. The cies. However, independent contrasts in dietary prefer- olfactory epithelium also extends onto the nasal septum ence are not significantly correlated with contrasts in the (Young, 1966; Ewer, 1973; Koppe et al., 1999; Menco and size of the olfactory system (Barton et al., 1995). OLFACTORY FOSSA OF TREMACEBUS 1161

Fig. 3. A: Log volumes of the main plus accessory olfactory bulbs versus log brain volume for catarrhine and platyrrhine species. B: Log Fig. 2. A: Log volumes of main and accessory olfactory bulbs versus volumes of the main plus accessory olfactory bulbs versus log body log brain volume. Slope of the least-squares regressions for all strepsir- mass. Envelopes are drawn around the catarrhine and platyrrhine scatter rhines and diurnal haplorhines are 0.67 and 0.74, respectively. B: Log of species. Data from Stephan et al. (1981). volumes of main plus accessory olfactory bulbs versus log body weight. Slope of the least-squares regressions for all strepsirrhines and diurnal haplorhines are 0.44 and 0.51, respectively. Within the strepsirrhine The vomeronasal organ and anterior accessory bulb are envelope, the filled circles are nocturnal species and the open circles are well developed in strepsirrhines (Stephan et al., 1981, nocturnal species. Nocturnal haplorhines Aotus and Tarsius are sepa- rated from the envelope of diurnal haplorhines. Data from Stephan et al., 1984; Baron et al., 1983). The organ and bulb are smaller (1981). and extremely variable in platyrrhines (Stephan et al., 1981, 1984; Baron et al., 1983; Dennis et al., 2004) and rudimentary in adult catarrhines (Stephan et al., 1981, 1984; Baron et al., 1983). This suggests that pheromonal Vomeronasal organ and accessory olfactory communication may not be as important to platyrrhines bulb. In mice and many primates, chemoreceptors located compared to strepsirrhines and may be even less impor- in the vomeronasal organ are exposed to nonvolatile odor- tant in catarrhines than in platyrrhines. However, it is ant molecules that diffuse through a fluid medium, via the important to note that the dissociation of the vomeronasal engagement of a physiologically regulated pump mecha- system and main olfactory system is not as absolute as nism (Firestein, 2001). Axons of the vomeronasal neurons generally thought. Recent research suggests that in cer- project to the accessory olfactory bulb located at the dor- tain species, the main olfactory system (MOS) can detect solateral limit of the main olfactory bulb. pheromones, and the VNS can detect volatile chemicals 1162 KAY ET AL. provide some challenging discrepancies from expectations based on the anatomy of the brain. Genetic Evidence Genes that code for olfactory receptors in mice and other mammals can be subdivided into subfamilies. OR genes are defined as belonging to the same subfamily if they code for amino acid sequences with Ն 60% identity. This cutoff has functional significance because olfactory receptors that are 60% or more identical tend to recognize odorants with related chemical structures (Firestein, 2001). Mouse and human OR genes have been studied exten- sively (Young and Trask, 2002; Godfrey et al., 2004; Malnic et al., 2004). Most recently, it has been reported that mice have 913 intact OR genes divisible among 241 subfamilies (Godfrey et al., 2004), whereas humans have 339 intact OR genes divided among 172 subfamilies (Malnic et al., 2004). Thus, mice have approximately 2.7 times as many ORs as humans but only 1.4 times as many subfamilies. Mice and humans share a substantial num- ber of subfamilies but in most cases (89%) the number of ORs in a shared subfamily is greater in mouse than in human (Godfrey et al., 2004). It is possible that the num- ber of subfamilies may be a good predictor of the diversity of odorant features that a species can detect (Godfrey et al., 2004), and the number of genes in each subfamily may well indicate the acuteness of that species’ ability to detect odorants coded by that subfamily. It appears that intact humans possess genes from most subfamilies, which means that humans may be able to detect as wide a range of odorants as mice, but that mice may be more sensitive to odors and perhaps are better able to distinguish closely related odors. However, one complication in using the number of intact OR genes to predict olfactory sensitivity is that intact OR genes may not imply gene expression and, therefore functionality. Fig. 4. Lateral views of the brains of nocturnal Aotus (top) and diurnal Finally, Godfrey et al. (2004) note that while mice and Callicebus (bottom) showing that Aotus has a proportionately larger humans share 150 OR subfamilies, 13% of human OR olfactory bulb than Callicebus. Scale bar ϭ 10 cm. Photos modified from subfamilies are not found in mice and 35% of mouse OR original images on Web site (http://brainmuseum.org/index.html). subfamilies are not found in humans (Fig. 5A). This raises the possibility that these species-specific OR subfamilies can detect odorants that are sensed by humans or by mice, but not by both. (Doving and Troiter, 1998; Dulac, 2000; Rodriguez et al., One indirect way to compare olfactory abilities between 2000; Smith et al., 2001; Zhang and Webb, 2003). This species might be to compile the complete set of intact may mean that catarrhines, while lacking a VNO, may genes for each species. However, because the full set of OR still rely to some extent on pheromonal communication genes is presently known only for a small number of mediated by the main olfactory epithelium. species (e.g., humans, mice, rats, and dogs), calculating A final word of caution. After an extensive literature the number of intact OR genes must rely at the moment on review, we find ambiguous data to support the hypothesis a sampling approach. Rouquier et al. (2000) hypothesize that species with well-developed olfactory bulbs (either that reduction in the sense of smell observed in primates relatively or absolutely) are necessarily endowed with bet- could parallel the reduction in the percentage (not the ter olfactory sensitivity and/or discrimination. While com- absolute number) of intact OR genes. Therefore, compar- mon sense suggests that the relative or absolute size of the ative approaches to the study of ORs in primates so far sensory nasoepithelium or olfactory bulbs must be related have relied on the demonstration that a variable percent- to differences in olfactory abilities, the literature offers age of ORs carry one or more disruptions in the coding contradictory evidence. In addition, glomeruli in the olfac- regions and are therefore noncoding, or pseudogenes (Rou- tory bulbs may vary in size within and across species (K.B. quier et al., 2000). Rouquier et al. (2000) report that the Doving, personal communication); this indicates that it percentage of pseudogenes in the OR gene repertoire var- may be inaccurate to rely on bulb size comparisons across ies considerably in primates. In their sample, the percent- species to indicate functional capabilities and the range of age of intact OR genes in the platyrrhines Callithrix and odorants detected in those species. As discussed below, Saimiri ranges from 93% to 100%. In cercopithecids (Pa- only detailed behavioral tests may begin to answer these pio and Macaca), it is 65–81%; in nonhuman hominoids questions. Also, as will be apparent in the next section, (Hylobates, Pongo, Gorilla, and Pan), it ranges from 50% newly emerging data on the genetics of olfactory receptors to 61%; and in humans, it is just 30%. They hypothesize OLFACTORY FOSSA OF TREMACEBUS 1163 with, as noted above, it is unclear whether intact OR genes imply functionality. A second problem relates to the reliability of using OR gene sampling to assess the num- ber of intact OR genes in a species. The percentage of pseudogenes is hypothesized by Gilad et al. (2004) to give an unbiased estimate of the size of the intact OR gene family. An underlying assumption is that all species have roughly the same number of OR genes. However, they may not, which means that the percentages may not accurately represent the number of distinct kinds of OR genes within and across species. To illustrate this, suppose that mice have 82.6% intact genes while humans have 49% intact genes [supplemental data from Gilad et al. (2004)]. If each had the same number of OR genes, then mice would have 1.69 times as many intact OR genes as humans. But there are 636 OR genes in humans and 1,209 OR genes in mice (Fig. 5B) (Godfrey et al., 2004; Malnic et al., 2004). Using the percentages given by Gilad et al. (2003) gives us 999 intact OR genes in mice but only 312 intact OR genes in humans, with mice having 3.2 times as many intact OR genes as humans. In short, because we do not know exactly how many OR genes, either intact or pseudogenes, exist in any nonhu- man primate, the percentages given by Gilad et al. (2004) may underestimate the actual number of intact genes by an unknown and possibly large factor. The distribution of these intact genes into gene families is also unknown and, as discussed above, olfactory ability and sensitivity may rely not only on the number of intact OR genes, but also on Fig. 5. A: Schematic diagram illustrating the number of subfamilies of olfactory receptor genes in humans and mice. Data from Godfrey et al. how these intact genes are distributed into OR gene sub- (2004) and Malnic et al. (2004). The gray area corresponds to the sub- families. Thus, olfactory ability may not simply be a mat- families shared by the two species. The shaded area of the human bar ter of the number of intact genes a species possesses, but is proportional to the uniquely human olfactory subfamilies. The shaded also how these intact genes are distributed across subfam- area of the mouse bar is proportional to the uniquely mouse olfactory ilies (Fig. 5). subfamilies. B: Total number of OR genes and percentage of functional Another troubling feature of the genetic findings is the OR genes (shaded) in mouse (left) and human (right). The word “func- apparent lack of correlation with the relative size of the tional” as used here does not mean that there is evidence that the gene olfactory apparatus. The data from Rouquier et al. (2000) is expressed. indicate that humans have the lowest percentage of intact OR genes. The other taxa examined are arranged relative that the sense of smell varies as a function of the fraction to humans as follows: humans Ͻ other hominoids ϭ Eu- of intact OR genes in the genome. lemur Ͻ papionins Ͻ platyrrhines ϭ mice. But the rela- Additional attempts have been made to examine intact tive size of the olfactory bulb (from small to large) is versus pseudogene percentages. Gilad et al. (2003) se- human Ͻ hominoids ϭ papionins ϭ platyrrhines Ͻ strep- quenced a random sample of 50 OR genes in different sirrhines Ͻ mice. It is surprising that the strepsirrhine primates and determined the percentage of these genes Eulemur should have comparable percentages to Macaca that are intact. The percentage ranged from 65% to 72% in and that both should exceed platyrrhines, but no more great apes and Macaca and 48% in Homo. Gilad et al. surprising than that the platyrrhine Callithrix and the (2004) estimate the proportion of OR pseudogenes in 19 mouse are similar despite the vast difference in the rela- primate species. Using degenerate primers and PCR am- tive sizes of MOBs in these species. This means that if the plification, they assembled a random sample of 100 OR sampling paradigm of Rouquier et al. (2000) is valid [a genes in each species and calculated the percentage of the point challenged by Gilad et al. (2004)] and if intact OR sample that is composed of pseudogenes. They report 49% genes translate into olfactory ability mediated by the intact OR genes in humans. The greater and lesser apes MOB, some strepsirrhines have a less well developed range from 66% to 68%; cercopithecids range from 69% to MOS sense of smell than either platyrrhines or mice, both 74%; and all but one platyrrhine fall between 83% and of which would seem to be similarly endowed. 85%. The exception among platyrrhines is Alouatta, which The genetic data from Gilad et al. (2004) is also discrep- has 69% intact OR genes, comparable to catarrhines. The ant with anatomical observations. In their data, humans authors suggest that Alouatta resembles catarrhines be- have the lowest percentage of intact genes (relative to cause of its greater reliance on color vision compared with humans): humans Ͻ other hominoids Յ cercopithecids Ͻ other platyrrhines. Alouatta and catarrhines have trichro- platyrrhines (except Alouatta) ϭ Eulemur ϭ mouse. But matic color vision, whereas in other platyrrhines males platyrrhines and catarrhines have similar-sized olfactory are dichromatic and females are variably trichromatic or bulbs and both have much smaller bulbs than strepsir- dichromatic. rhines. Likewise, the proportion of OR psuedogenes in the Several things need to be resolved before the functional mouse and in the strepsirrhine Eulemur falls within the meaning of this genetic data can be understood. To begin range of platyrrhines despite having far larger olfactory 1164 KAY ET AL. bulbs. Finally, among platyrrhines, Gilad et al. (2004) stand the importance of these findings in a species-specific identify Alouatta as an outlier but Aotus as being similar functional way by examining olfactory behaviors in the to other platyrrhines, whereas the anatomical data have species of interest. If the importance of olfactory ability to Alouatta as being unremarkable in terms of olfactory sys- behavior in a given species can be correlated with a high tem size while nocturnal Aotus as having large bulbs. The number of intact olfactory genes or with large OBs (or the two sets of genetic data conflict with one another and with lack of olfactory ability and behavior with small OBs and the relative size of MOB. It should be noted that in the many pseudogenes), then researchers may begin to use species mentioned here, the anatomical evidence appears bulb size or genetic repertoires to predict olfactory ability to correlate more precisely with admittedly sparse behav- in a given extant, or even extinct, species. ioral data on olfactory ability and reliance. Perhaps the Before going further, it is important to keep in mind discrepancy is related to the way the comparisons are that olfactory ability or the sense of smell in a given expressed, i.e., as percentages rather than absolute num- species is most probably controlled by both the MOS, bers. Until we know how many OR genes (pseudogenes which senses odorants, and the VNS, which senses pher- plus intact genes) each taxon has, the percentages of pseu- omones. Individual species will undoubtedly vary in the dogenes may be misleading. extent to which each system is used in their species- Lastly, as touched on above, we know very little about specific sense of smell. the expression of OR genes. Clearly, a single OR gene In platyrrhines, olfactory ability plays an important role must code for many copies of a particular type of OR, but in behavioral responses and social interactions (Epple, there may be differences in the number of copies in vari- 1986). This is especially true in behavior associated with ous species with attendant differences in olfactory acuity. reproduction, such as in the assessment of reproductive With 1,000 OR genes, Firestein (2001) notes, the number status (Hennessy et al., 1978; Boinski, 1992; Ziegler et al., of possible combinations of scents that could be recognized 1993; Converse et al., 1995; Heymann, 1998; Smith and amounts to billions. The genetic evidence brought forward Abbott, 1998). Chemical stimuli have also been imple- so far appears to have no bearing on the ways OR signals mented in the suppression of subordinate female’s ovarian are mediated or recombined in the olfactory bulb or in cycles (Abbott, 1984; Epple and Katz, 1984; French et al., higher brain centers. Clearly, we have a long way to go 1984; Heistermann et al., 1989; Barrett et al., 1990). Scent before either the anatomical or the genetic data can be delivered via scent marks or urine may also be used in the used with confidence for comparisons of the olfactory abil- recognition of conspecifics (Laska and Hudson, 1995), to ities among primate species. communicate dominance, mediate interactions among in- We end this section with a final short note concerning dividuals of different ranks (Epple et al., 1986), and may genes coding for receptors of the vomeronasal system. be important in interactions between infants and other Recent comparative studies indicate that the genes for group members (Kaplan and Russell, 1974; Epple et al., expression of VNO receptors are present in strepsirrhines 1986), as well as in infant-mother bonding (Kaplan et al., and platyrrhines but have mostly become pseudogenes in 1979). Scent also appears to be important in territorial catarrhines. In the human genome with the exception of defense, in intergroup relations, and for maintaining in- just a few genes, all putative genes that code for vomero- tergroup spacing (Epple, 1974; Ueno, 1994b, 1994c; Smith nasal receptors are pseudogenes (Meredith, 2001; Liman et al., 1997; Lazaro-Perea et al., 1999), as well as for the and Innan, 2003). The loss of intact VNS genes is sug- discrimination of food items (Ueno, 1994a). gested to have occurred in synchrony with a greater reli- Several lines of evidence suggest that platyrrhines de- ance on vision and visual cues in communicating with pend on olfaction more than catarrhines. For example, conspecifics (Liman and Innan, 2003; Zhang and Webb, there is ample anatomical evidence in the form of scent 2003). As noted by Gilad et al. (2004), Alouatta’s higher glands as well as behavioral evidence of animals respond- percentage of OR pseudogenes is hypothesized to corre- ing to urine and scent marks left by conspecifics (Epple, spond to the acquisition of full trichromatic vision to aid in 1974; Uenor 1994a, 1994b, 1994c; Smith et al., 1997; Laz- foraging; however, Alouatta resembles other platyrrhines aro-Perea et al., 1999). Though it has yet to be tested in having a functional VNO system (Zhang and Webb, systematically, these behaviors are likely to depend on the 2003; Webb et al., 2004). Webb et al. (2004) point out that VNS and are probably mediated via pheromones. These this evidence indicates that the acquisition of color vision observations are consistent with the presence of VNO and may not necessarily be the only variable responsible for AOB in platyrrhines, whereas these structures appear to the reduction of the VNS in primates. They suggest that be rudimentary or absent in catarrhines. additional ecological variables may have resulted in the While these examples establish the importance of the reduction of this system. Certain environments may make VNS in the behavioral repertoires of platyrrhines, there is leaving pheromonal scent marks difficult. However, if also evidence that platyrrhines rely on odorants encoded pheromone detection has been taken over by MOS in by the main olfactory system. For example, the recogni- catarrhines, then perhaps like Alouatta, these species use tion of food odorants is likely to be a good indicator of the color vision primarily for foraging tasks rather than con- reliance on, and the sensitivity of, a species’ main olfactory specific communication. It is also possible that color vision system. By examining behaviors related to foraging in the may be used and perceived in different ways across spe- wild and by undertaking laboratory studies examining cies and in combination with signals from other modali- sensitivity to food-related odorants, researchers may be- ties. gin to determine to what extent platyrrhines and other primates rely on the MOS. Recent studies show that an- Behavioral Evidence thropoids discriminate and remember different odorants, While there has been much written on anatomical dif- especially in the detection of food (Laska and Hudson, ferences of olfactory bulb (OB) size and on interspecific 1993a, 1993b; Ueno, 1994a, 1994b, 1994c; Garber and olfactory genetic repertoires, it is only possible to under- Paciulli, 1997; Laska et al., 2003b). Laska and Hudson OLFACTORY FOSSA OF TREMACEBUS 1165 (1993b) even found that in the squirrel monkey, different components in odor mixtures interact and can have com- plex relationships so that small changes in the composi- tion of an odorant may have significant consequences on how that odor is perceived and interpreted as a signal. A handful of comparative behavioral studies using ol- factory cues mediated provide additional information on functionality. Evidence indicates that dietary preference rather than phylogeny determines how well both platyr- rhines and catarrhines perform on olfactory tests; and these results appear to correlate with some of the genetic and anatomical observations. Laska et al. (1993b) found that both the platyrrhine Ateles geoffroyi and the ca- tarrhine Macaca nemestrina can discriminate between ob- jects on the basis of odor cues, can transfer their selections to new positive and negative stimuli, and can remember the significance of previously learned odor stimuli over prolonged intervals. However, with regard to the speed of Fig. 6. Tremacebus harringtoni. Surface-rendered CT image of type initial task acquisition and the ability to master transfer skull. Note the clear separation between fossil bone and rock matrix. tasks, A. geoffroyi outperformed M. nemestrina. This dif- ference in behavioral ability in the platyrrhine Ateles cor- relates only weakly with OB size. The two have similar- sized bulbs, although in Ateles the AOB comprises 2.5% of Province, Argentina. Rusconi named the specimen a spe- the total size of the olfactory bulb, whereas in Macaca the cies of Homunculus (Rusconi, 1933) and provided an ex- percentage is zero. On the other hand, Laska et al. (2003a) tended description of the fossil (Rusconi, 1935). Hershko- found that the catarrhine M. nemestrina generally outper- vitz (1974) proposed a new for this skull. At some formed the platyrrhine Saimiri sciureus in detecting ali- point after Rusconi’s original description, a substantial phatic aldehydes, a class of odorants that is presumed to amount of plaster was added to reconstruct missing parts. indicate a fruit’s degree of ripeness. This is in agreement Later, Hershkovitz and others made an effort to have the with the findings of Laska (2001) that these two species plaster removed but succeeded only partially because the exhibited different food preferences; compared with plaster is cleverly tinted to match the color of the fossil Siamiri, Macaca prefers foods with a higher carbohydrate bone. In an effort to determine more precisely the limits of and fructose content. This behavioral evidence correlates bone, matrix, and plaster, and to better appreciate the more clearly with anatomical evidence available for these structural details of the interior of the skull, one of the two species, with Macaca having relatively much larger authors (R.F.K.) borrowed the specimen from Tucuman bulbs than Saimiri. and had it CT-imaged. Details of the preservation pro- In addition to dietary preference, MOS ability has also vided by Hershkovitz (1974) are substantially correct ex- been linked to activity pattern. Bolen and Green (1997) cept that the apex of the orbit and optic foramen are not found the nocturnal Aotus monkey to be more adept at preserved as Hershkovitz claimed, and the lateral ptery- locating baited sites than the diurnal Cebus. Bicca- goid plate figured by Hershkovitz consists of plaster. Her- Marques and Garber (2004) also found that in foraging shkovitz’s claim that there was a large inferior orbital tasks Aotus relies more on olfactory cues than Callicebus, fissure in life cannot be confirmed. or most Saguinus. These differences are hypothesized to A sample of CT images of extant platyrrhines was the relate to the reduced availability of visual cues in noctur- source of comparative measurements from the interior of nal species, which leads to a greater reliance on olfactory the cranium (Table 1). Data for brain size and the size of ability. The postulated differences in olfactory abilities the main olfactory bulb and accessory olfactory bulb come goes along with the larger size of the olfactory bulbs in from previously published information (Stephan et al., Aotus but has no corresponding difference in the percent- 1981, 1984; Baron et al., 1983; Frahm et al., 1984). age of pseudogenes. By gathering behavioral evidence in careful studies, CT Scanning such as in the ones mentioned here, researchers may The Tremacebus specimen was scanned at the High- begin to understand exactly what olfactory genes and Resolution X-Ray Computed Tomography Facility at the olfactory bulb size can tell us about how a species may University of Texas at Austin, which is described by have lived, e.g., whether it was nocturnal or diurnal, and Ketcham and Carlson (2001). X-ray energies were set to even whether it ate fruit or leaves (although the latter has 150 kV and 0.16 mA using a FeinFocus X-ray source. not yet been demonstrated) (Barton et al., 1995). X-ray intensities were measured using an Image Intensi- ϫ MATERIALS AND METHODS fier detector employing a 1,024 1,024 video camera. Each slice was acquired using 1,000 views (angular orien- Specimens tations), with four samples taken per view. The specimen, The type specimen of Tremacebus harringtoni consists mounted in a plastic cylinder, was scanned with a cen- of a partial skull (Figs. 1 and 6). Carlos Rusconi received tered axis of rotation (Ketcham and Carlson, 2001) with a the specimen in 1932 from Thoma´s Harrington, who col- source-to-object distance of 135 mm (Ketcham and Carl- lected it together with other fossil remains of son, 2001). Slice thickness and interslice spacing was Colhuehuapian age (ϳ 20 Ma) from approximately 12 km 0.0466 mm (one video line). The image field was recon- southwest of Cerro Sacanana, in north central Chubut structed to 43 mm, based on a maximum field of view of 1166 KAY ET AL. TABLE 1. Osteological measurements* Sample size/specimen Olfactory fossa Greatest endocranial Taxon number breadth breadth Olfactory fossa index Callicebus moloch 3 2.95 32.89 Ϫ12.2 Pithecia sp. 1 4.51 40.69 13.3 Aotus trivirgatus 3 4.31 36.10 19.1 Lagothrix lagotricha 2 5.27 57.74 0.5 Callimico goeldii 1 3.28 28.38 9.8 Saguinus oedipus 3 2.90 26.60 2.2 Alouatta seniculus 3 5.01 47.88 10.8 Cebus apella 2 4.24 49.21 Ϫ8.3 Saimiri sp. 3 3.21 36.82 Ϫ12.5 Miopithecus talapoin 1 3.51 44.42 Ϫ17.7 Tremacebus harrringtoni Rusconi Collection 3.83 33.18 12.3 *Measurements are in millimeters. Olfactory fossa breadth and maximum endocranial breadth are illustrated in Figure 7. The estimate of relative size of the olfactory fossa is based on measurements of olfactory fossa breadth and maximum endocranial breadth. A least-squares regression was fit to In maximum endocranial breadth (MEB; independent variable) versus In olfactory fossa breadth (OFB; dependent variable) for eight species of diurnal platyrrhines. The equation expressing this line is In OFB ϭ 0.794 (In MEB) Ϫ 1.557. For each taxon, the expected OFB was calculated from this equation. The observed (measured) OFB for each species was compared with the expected and expressed as a residual (OFI): OFI ϭ 100 ϫ (observed Ϫ expected)/(expected). The Rusconi Collection at Museo de Fundacio´n Miguel Lillo, Tucuman, Argentina. The collection label and catalog entry for the skull of Tremacebus in the Rusconi collection and the number painted on the specimen gives the number as 619, but Rusconi (1933, 1935) refers to it as number 661.

44.164 mm, yielding an interpixel spacing of 0.042 mm. specimen: one at the point where the internal surface of Reconstruction parameters were calibrated to maximize the neurocranium corresponding to the maximum breadth usage of the 16 bit range of grayscales available in the of the fossa containing the olfactory bulbs, and a second at output images. Twenty-seven slices were acquired for the level where the brain would have attained its maxi- each rotation of the turntable, with a resulting acquisi- mum breadth, usually in the parietal region. The mea- tion time of about 11.1 sec per slice. The entire scan took surements are illustrated in the platyrrhine Pithecia sp. about 4.75 hr, including calibrating the scanner and in Figure 7. Each section was imported into Photoshop, mounting the specimen, which took about 1 hr. The data version 7.0 for Macintosh, and the maximum breadth of comprises 1,177 slices, from the front to the back of the the fossa (OFB) and parietal endocranium (PB) were mea- skull. sured in pixels using the measurement tool, then cor- The coronal slice-by-slice animation may be viewed on rected for the number of pixels per mm. The breadth the DigiMorph Web site (http://www.digimorph.org/ across the neurocranium in Tremacebus (Fig. 8) was esti- index.phtml). The coronal movie (COR) begins at the tip mated by mirroring missing parts of the right side onto of the left premaxilla; the slices are in anterior view. the right. The horizontal movie (HOR) starts dorsally and passes ventrally; the slices are oriented in dorsal view. The RESULTS sagittal movie (SAG) proceeds from left to right through Endocranial Dimensions the specimen; the slices are in left lateral view. Our objective is to draw inferences about the relative The platyrrhine skulls that comprise the comparative size of the olfactory bulb and brain size of Tremacebus, but database were scanned with various protocols, depending all we have are osteological measurements of the interior on skull size, in order to maximize resolution for each of the cranium. The first step is to see whether olfactory specimen. Resolution ranged from 0.088 to 0.176 mm in fossa breadth and maximum endocranial breadth can be the coronal plane (interpixel spacing) and 0.097 to 0.189 used as proxies for olfactory bulb volume and brain vol- mm in the Z-axis (interslice spacing). For further details, ume. see Rossie (2003). Species means of endocranial dimensions for extant and fossil taxa are presented in Table 1. For the two osteolog- Measurements ical measurements to serve as a reliable surrogates for the Perhaps the most comparable osteological measure- brain volumes, it must be shown first that each osteolog- ment of the size of the olfactory bulbs would be to deter- ical measurement is highly correlated with its soft-tissue mine the volume of the olfactory fossa. However, in surrogate (i.e., olfactory bulb volume with olfactory fossa Tremacebus only the dorsal portion of the natural endo- breadth, and brain volume with maximum endocranial cast of the olfactory fossa is preserved and some portions breadth) and second, that the deviations away from the of the braincase are missing and cannot be mirror-imaged. general trend are matched. For our sample of eight diur- Therefore, we used the breadth of the fossa as our mea- nal anthropoid species, ln (natural log) olfactory bulb vol- sure of olfactory fossa size and estimated maximum ume and ln brain volume are tightly correlated: 67% of the breadth of the endocranium (in the parietal region) as a variance in ln olfactory bulb volume is explained by ln measure of brain size. The validity of this approach is olfactory fossa breadth in a group that includes platyr- examined below. Serial CT cross-sections in the coronal rhines and Miopithecus (the only catarrhine in our sam- plane were examined and two images selected for each ple; P Ͻ 0.007). Notably, the olfactory bulb volume of OLFACTORY FOSSA OF TREMACEBUS 1167 of its olfactory bulbs suggests that Tremacebus was diur- nal.

DISCUSSION The anatomical and genetic distinctiveness of the vom- eronasal system from the main olfactory system is well known. Though there is evidence that in some species the MOS may detect pheromones and the VNS may detect odorants (Doving and Troiter, 1998; Dulac, 2000; Rodri- guez et al., 2000; Smith et al., 2001; Zhang and Webb, 2003), in general the two systems appear functionally distinct, with the former playing a role in the detection of nonvolatile pheromones and the latter detecting volatile compounds. While foraging, animals appear to rely on volatile cues and therefore on the main olfactory system. Thus far, the anatomical evidence of OB size in platyr- rhines and catarrhines appears to correlate with olfactory behavioral differences mediated by the MOS (e.g., forag- ing activity). As touched on in this article, with respect to foraging, Fig. 7. Example of the measurements made in this study. Pithecia sp. there is conflicting evidence as to whether catarrhines or Coronal CT slices representing the maximum breadth of the olfactory platyrrhines rely more on the olfactory sensory modality fossa and the interior of the braincase in the parietal region. (Laska et al., 2003a, 2003b). Therefore, it is not surprising that there are no consistent differences in the relative sizes of the olfactory bulbs between platyrrhines and ca- tarrhines (Fig. 2). It appears that dietary preferences (per- Aotus is larger than other anthropoids (Fig. 9A). The same haps the importance of fruits with high levels of sugar) trends are apparent in the osteological measurements. may be an important selective determinant of olfactory Among diurnal anthropoids, ln olfactory fossa breadth and abilities (Laska and Hudson, 1993b; Laska, 2001). Activ- ln maximum endocranial breadth are also correlated: 75% ity pattern also plays an important, perhaps even a pre- of the variance in brain volume is explained by maximum ponderant, selective role in shaping the main olfactory endocranial breadth (P Ͻ 0.002). Again, olfactory fossa system. Behavioral observations on nocturnal Aotus (an breadth of Aotus is larger than other platyrrhines (Fig. outlier in having a proportionally larger olfactory bulb 9B). than other platyrrhines) suggest this species relies more Residuals (a percentage of expected) are calculated from heavily on olfactory cues for foraging than its diurnal the least-squares regression of ln brain volume (indepen- platyrrhine relatives (Bolen and Green, 1997; Bicca- dent variable) versus ln olfactory bulb volume (dependent Marques and Garber, 2004). variable). Similar residuals (called olfactory fossa index; Whereas anatomical evidence (the size of MOB) ap- OFI) are calculated between ln maximum endocranial pears to agree with expectations from available behav- breadth (independent variable) and ln olfactory fossa ioral evidence, genetic data, as currently interpreted, breadth (dependent variable). The residuals of the two are discrepant. Based on percentages of intact genes, it comparisons are significantly correlated: P Ͻ 0.03 [Fig. has been inferred that there is little or no difference in 10; the Spearman’s rank correlation (rho) between the the size of the intact olfactory receptor gene repertoire residuals is 0.77 with a P value of 0.016; the Kendall between platyrrhines and strepsirrhines but large dif- rank correlation (tau) is 0.556 with a P value of 0.037]. ferences between platyrrhines and catarrhines (Gilad et From this we conclude that the osteological measure- al., 2003, 2004). Likewise, the data of Roquier et al. ments provide reasonable surrogates for the brain mea- (2000) appear to indicate that strepsirrhines and ca- surements: species with relatively large olfactory bulb tarrhines have higher percentages of pseudogenes than volumes also have relatively large olfactory fossa platyrrhines. We offer several ideas as to why the ge- breadths. Thus, an inference about relative olfactory netic evidence may be misleading, including uncer- bulb size from endocranial dimensions is warranted in tainty about the absolute number of OR genes in each fossil platyrrhine skulls. species and a lack of knowledge about how (and From the cranial dimensions of Tremacebus, it is prob- whether) intact OR genes are expressed as functional able that the olfactory bulb of this species does not depart receptors. Moreover, single intact OR gene must code from that of most extant platyrrhines of similar size. The for many copies of a particular OR and this is likely an OFI is similar to that for Callimico (Fig. 10). This gives an important variable in olfactory ability. estimated olfactory bulb size residual of ϳϩ12, most Before commenting on the significance of our findings resembling Pithecia in our sample and unlike Aotus which for , we should make one caveat: neither osteological has comparatively large olfactory bulb (a residual of ϩ19). measurement nor volumetrically determined gross size of Admittedly, the samples are small, but on the available the olfactory bulbs of living species can distinguish rela- evidence it seems reasonable to infer that Tremacebus did tive or absolute sizes of the main versus accessory olfac- not have as well developed olfactory bulbs as Aotus. More tory bulbs. This is because AOB, while recognized func- specifically, given that Aotus is nocturnal whereas other tionally and histologically, cannot be visualized as a platyrrhines are diurnal, the smaller inferred relative size discrete structure distinct from the olfactory bulb on the 1168 KAY ET AL.

Fig. 8. Tremacebus harringtoni. Coronal CT slices representing the maximum breadth of the olfactory fossa and the interior of the braincase in the parietal region. region.

external surface of the brain. What is commonly referred contribution of the AOB to the total volume of the ol- to as the olfactory bulb, and its manifestation of the factory bulb in primates, any measure of the olfactory internal surface of the braincase, includes volumetri- fossa size is likely tracking MOB size. cally both the main and the accessory olfactory struc- Many have proposed that Tremacebus was a noctur- tures. Moreover, in living platyrrhines and strepsir- nal primate (Rosenberger, 1979; Szalay and Delson, rhines, the AOB amounts to such a very small 1979; Martin, 1990). However, our findings about the percentage of the volume of the gross anatomical olfac- size of the olfactory fossa complement previous reports tory bulb and as a whole the difference between the about the size of the orbits in suggesting that Tremace- volumes of MOB versus MOB plus AOB is not apparent bus was diurnal or at least cathemeral. In relative orbit in comparisons of total overall bulb volume in platyr- size, Tremacebus overlaps the distributions of extant rhines versus catarrhines (Stephan et al., 1981, 1984; nocturnal strepsirrhines but it also overlaps the distri- Baron et al., 1983). To summarize, it is unlikely that butions of extant diurnal platyrrhines (Fig. 11). More- there are anatomical features in a fossil that would over, its orbits are far smaller than those of nocturnal allow us to distinguish the accessory olfactory bulb from haplorhines Aotus and Tarsius. The extreme orbital the size of the olfactory bulb as a whole. Moreover, the hypertrophy in Aotus and Tarsius stems from the as- primate vomeronasal organ is not surrounded by an sumption of a nocturnal activity pattern in the absence osseous structure. It leaves no obvious osteological (and of a tapetum lucidum (Kirk and Kay, 2004). If so, therefore fossil) signature. Thus, given the very small Tremacebus could have effectively exploited a nocturnal OLFACTORY FOSSA OF TREMACEBUS 1169

Fig. 10. Scatter plot of the residuals of olfactory bulb volume and olfactory fossa breadth (Table 1). Correlation between the two variables for diurnal species has a P value of 0.0324. Subset of taxa as for Figure 9. Tremacebus (for which only the olfactory fossa quotient is known) is projected onto the line of best fit.

cebus. We demonstrate that olfactory fossa of Tremacebus harringtoni (and by inference its main olfactory bulb) is proportionally smaller than that of the only nocturnal anthropoid Aotus and is comparable in relative size to that of extant diurnal platyrrhines. This implies that Trema- cebus’ sense of smell was no different from diurnal platyr- rhines but less acute than in nocturnal Aotus, again sup- porting the inference of a diurnal activity pattern for Tremacebus. Our results have implications for the antiquity of the nocturnal activity pattern in anthropoids. If 20-millon- year-old diurnal Tremacebus is a relative of Aotus, noctur- nality must have evolved subsequent to the Tremacebus- Aotus node. Indeed, the hypothesis of a Tremacebus-Aotus clade should be reevaluated since the only evidence sup- porting such a clade is the supposed presence of equiva- lently enlarged orbits in the two (Horovitz, 1999). The next oldest possible sister taxon to Aotus is Aotus dinden- sis ϭ Mohanamico hershkovitzi (middle Miocene, Colom- bia). This species probably had enlarged orbits (Setoguchi and Rosenberger, 1987). On the other hand, if Mohana- mico hershkovitzi (Luchterhand et al., 1986) and “Aotus dindensis” are conspecific and related to pitheciines as argued by Kay (1990), then possibly nocturnality arose Fig. 9. A: Adult-specific ln-ln scatter plot of olfactory bulb volume (OBV) versus brain volume (with OBV subtracted). B: Adult-specific ln-ln independently twice in platyrrhine evolution: once in the scatter plots of olfactory fossa breadth versus maximum endocranial Mohanamico hershkovitzi lineage and a second time inde- breadth. Filled squares, diurnal anthropoids; open circles, Aotus. The pendently in the Aotus lineage. In short, much remains to only taxa included in the analysis are those for which both the brain and be learned about the origins of nocturnality in platyr- osteological measurements are available. Correlation between the two rhines. variables is significant at P Ͻ 0.003. ACKNOWLEDGMENTS niche only by reevolving a tapetum lucidum. We con- The authors thank Dr. Jaime Powell, curator of the sider this an unlikely possibility. Rusconi Collection at Museo de Fundacio´n Miguel Lillo, The size of the olfactory fossa reveals additional data Tucuman, Argentina, for graciously loaning the skull of pertinent to the question of the activity pattern of Trema- Tremacebus. They have especially profited from discus- 1170 KAY ET AL.

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