The Vomeronasal Organ of New World Monkeys - Wiley Online Library

THE ANATOMICAL RECORD 294:2158–2178 (2011)

The Vomeronasal Organ of New World Monkeys (Platyrrhini) TIMOTHY D. SMITH,1,2* EVA C. GARRETT,3,4 KUNWAR P. BHATNAGAR,5 CHRISTOPHER J. BONAR,6 AMANDA E. BRUENING,7 JOHN C. DENNIS,8 JONATHAN H. KINZNGER,1 EDWARD W. JOHNSON,9 8 AND EDWARD E. MORRISON 1 School of Physical Therapy, Slippery Rock University, Slippery Rock, Pennsylvania 2 Department of Anthropology, University of Pittsburgh, Pittsburgh, Pennsylvania 3 Department of Anthropology, The Graduate Center at the City University of New York, New York 4 New York Consortium in Evolutionary Primatology (NYCEP), New York, New York 5 Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, Kentucky 6 Dallas World Aquarium, Dallas, Texas 7 Department of Biology, Slippery Rock University, Slippery Rock, Pennsylvania 8 Department of Anatomy, Physiology, and Pharmacology, College of Veterinary Medicine, Auburn University, Auburn, Alabama 9 Central Oregon Community College, 2600 N.W. College Way, Bend, Oregon

ABSTRACT Although all platyrrhine primates possess a vomeronasal organ (VNO), few species have been studied in detail. Here, we revisit the microanatomy of the VNO and related features in serially sectioned samples from 41 platyrrhine cadavers (14 species) of mixed age. Procedures to identify terminally differentiated vomeronasal sensory neurons (VSNs) via immunolabeling of olfactory marker protein (OMP) were used on selected specimens. The VNO varies from an elongated epithelial tube (e.g., Ateles fusciceps) to a dorsoventrally expanded sac (e.g., Saguinus spp.). The cartilage that surrounds the VNO is J-shaped or U-shaped in most species, and articulates with a groove on the bony palate. Preliminary results indicate a significant correlation between the length of this groove and length of the VNO neuroepithelium, indicating this feature may serve as a skeletal correlate. The VNO neuroepithelium could be identified in all adult primates except Alouatta, in which poor preservation prevented determination. The VNO of Ateles, described in detail for the first time, had several rows of VSNs and nerves in the surrounding lamina propria. Patterns of OMP-reactivity in the VNO of perinatal platyrrhines indicate that few or no terminally differentiated VSNs are present at birth, thus supporting the hypothesis that some platyrrhines may have delayed maturation of the VNO. From a functional perspective, all platyrrhines studied possess structures required for chemoreception (VSNs, vomeronasal nerves). However, some microanatomical findings, such as limited reactivity to OMP in some species, indicate that some lineages of New World monkeys may have a reduced or vestigial vomeronasal C 2011 Wiley Periodicals, Inc. system. Anat Rec, 294:2158–2178, 2011. V

Grant sponsor: National Science Foundation; Grant number: BCS 0820751; Grant sponsor: Department of Homeland Security; Grant number: 01-G-022. *Correspondence to: Timothy D. Smith, School of Physical Therapy, Slippery Rock University, Slippery Rock, PA. Tel: 724738-2885. E-mail: [email protected] C 2011 WILEY PERIODICALS, INC. V

Received 15 September 2011; Accepted 16 September 2011 DOI 10.1002/ar.21509 Published online 1 November 2011 in Wiley Online Library (wileyonlinelibrary.com).

VNO AND RELATED STRUCTURES IN PLATYRRHINI

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Key words: olfaction; olfactory epithelium; primates; vomeronasal neuroepithelium; VNO; platyrrhines

The vomeronasal organ (VNO) is the peripheral receptor organ for the vomeronasal, or accessory, olfactory system. Experimental work has demonstrated that this chemosensory system is an important mediator of sociosexual behaviors in many mammals. Disrupting the vomeronasal system (VNS), through removal of the VNO or lesions of vomeronasal nerves, reduces some sexual and aggressive behaviors in many rodents (Powers and Winans, 1975; Beauchamp et al., 1982; Clancy et al, 1984; Lepri and Wysocki, 1987; Wysocki et al., 1991), opossums (Jackson and Harder, 1996), and at least one primate (Aujard, 1997). Yet, it has become clear that the VNO is not a critical mediator of these behaviors in all mammals (Barrett et al., 1993; Woodley et al., 2004). One fundamental difference between Old World monkeys and New World monkeys is the complete absence of the VNO in the former (Frets, 1912). The VNO, comprising a neuroepithelial tube in most mammals, disappears during fetal development in Old World monkeys (Hendrickx, 1971; Wilson and Hendrickx, 1977). Although a vestigial VNO has been described in some other catarrhines (e.g., humans and chimpanzees—Smith et al., 2001b, 2002; Witt et al., 2002), not even a trace of the VNO neuroepithelium has been found in postnatal Old World monkeys (Jordan, 1972; Smith et al., 2001a,b). In contrast, the VNO of at least some New World monkeys (platyrrhines) possesses bipolar neurons and vomeronasal nerves, characteristics in keeping with a functional neuroepithelium (Taniguchi et al., 1992; Mendoza et al., 1994; Dennis et al., 2004). Regarding differences in olfactory anatomy, Frets (1912) observed that in monkeys ‘‘ : : : nature has made an experiment’’ (p. 137). For Frets, this experiment could potentially reveal the function of the VNS, since it is present in one group and absent in the other. Nearly a century later, the function of the VNO continues to be debated (Halpern and Martıńez-Marcos, 2003; Doty, 2010). Among primates, the platyrrhine–catarrhine dichotomy has generated debate concerning evolution of olfactory abilities (Liman and Innan, 2003; Zhang and Webb, 2003; Gilad et al., 2004; Wang et al., 2010; Matsui et al., 2010; Young et al., 2010), yet has yielded little insight into the function of the VNO itself. Attempts to experimentally ablate the VNO have produced no evidence, as of yet, for VNS function in platyrrhines (Barrett et al., 1993). The present state of knowledge on the VNS of primates, then, is incomplete. Aside from quantitative studies of the accessory olfactory bulb (Stephan et al., 1981), the site of first order vomeronasal sensory neuron synapses, the VNS has been studied in only a few platyrrhine species. Anatomical knowledge of the VNO, the peripheral receptor organ for this neural system, is especially lacking in platyrrhines. Only small-bodied species have provided the basis for detailed microscopic descriptions (Taniguchi et al., 1992; Mendoza et al., 1994; Dennis et al., 2004).

Herein, we present new findings on the VNO and related structures in 14 species of platyrrhines. The aims of this report are two-fold. First, this is the broadest taxonomic survey of VNO morphology in platyrrhines to date. Very few species have been described regarding neuroepithelial organization. The present report includes observations on the vomeronasal neuroepithelium (VNNE) morphology in 14 species, including three for which VNNE has never been described using adult specimens. New and existing information is then summarized in an effort to reconcile previous descriptions of the VNO in platyrrhines, which have included vague descriptors such as ‘‘well developed’’ (Hershkovitz, 1977; Bhatnagar and Meisami, 1998; Brennan, 2001; Cartmill and Smith, 2009), or as showing ‘‘signs of reduction’’ (Maier, 1980; p. 229). A second aim is to assess maturational characteristics of the VNNE in perinatal monkeys. Specifically, the present study seeks to investigate the organization of the VNNE and histochemistry and immunoreactivity of vomeronasal sensory neurons (VSNs) and extraepithelial neuronal bodies surrounding the VNO. The hypothesis that platyrrhine primates exhibit delayed maturation of the VNO (Evans and Grigorieva, 1994; Shimp et al., 2003) is tested using the perinatal sample.

MATERIALS AND METHODS Sample Characteristics The present study is based on observations on 41 adult, juvenile, perinatal, and fetal specimens from 14 species of primates (Table 1). The sample includes some specimens that were used previously (Smith et al., 2002, 2003a, 2004; Dennis et al., 2004). Included are three species that have never been subjected to detailed examination using adult samples, Ateles fusciceps, Cebuella pygmaea, and Leontopithecus rosalia. Tables 2–4 present a compilation of new and previously published observations (e.g., VNO measurements, immunoreactivity to neuronal markers, or conventional histochemical procedures), and includes an update on metric and non-metric aspects of the VNO in platyrrhini. All specimens were acquired from cadaveric remains preserved in 10% formalin. This opportunistic sample included animals that were stillborn or died postnatally in captivity at various research centers and zoos (Table 1). No animal was euthanized specifically for the present study. Whenever possible, both males and females of each genus were studied.

Sample Preparation Procedures for sectioning material used in earlier studies are described elsewhere (see Smith et al., 2003a and Dennis et al., 2004 for details). Newly prepared histological specimens were processed similarly, described briefly as follows. Before embedding, cranial length

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TABLE 1. Species, sample size, and source of primates sectioned Species (common name) Aotus trivirgatus (Northern owl monkey) Alouatta caraya (black howler monkey) Alouatta seniculus (red howler monkey) Ateles fusciceps (brown-headed spider monkey) Callithrix jacchus (common marmoset) Cebuella pygmaea (3) (pygmy marmoset) Leontopithecus rosalia (golden lion tamarin) Pithecia pithecia (2) (White-faced saki) Saimiri boliviensiss (Bolivean squirrel monkey) Saguinus spp

Sample size, age (sex)

Source

2 adults (m, f) 1 adult (m) 1 fetus (f) 1 adult (f) 2 adults; 6 perinatal (m, f) 3 adults (3f); 1 41-day-old (f); 1 perinatal (f) 2 adults (m,f); 1 juvenile; 1 perinatal 1 adult (m), 1 perinatal (u) 1 adult (f); 6 perinatal (m, u) 6 adults (m,f); 2 juveniles; 2 perinatal (m,f)

USAPRC CMZ DWA CMZ WRPC CMZ, PZ NMNH CMZ USA, MKCMR NEPC, CMZ, GPZ

CMZ, Cleveland Metroparks Zoo; DWA, Dallas World Aquarium; GPZ, Gladys Porter Zoo; MKCMR, Michael E Keeling Center for Comparative Medicine and Research ; NEPC; New England Primate Center; NMNH, National Museum of Natural History; PZ, Philadelphia Zoo; USAPRC, University of South Alabama Primate Research Center; WRPC, Wisconsin Regional Primate Center.

TABLE 2. Summary of characteristics of the vomeronasal complex in Platyrrhines: Duct communications and cartilaginous capsule Species Aotus trivirgatus Alouatta caraya Ateles fusciceps Callithrix jacchus Cebuella pygmaea Cebus spp Leontopithecus spp Pithecia pithecia Saimiri spp. Saguinus spp.

VNO

VNO duct communica.

VNC

References

dorsoventrally elongate dorsoventrally elongate? round or dorsoventrally round round round or dorsoventrally irregular/ dorsoventr. elongate round? round irregular/ dorsoventr. elongate

dorsal NPD dorsal NPD dorsal NPD mid NPD mid NPD mid NPD mid to dorsal NPD dorsal NPD mid NPD mid NPD

‘‘closed’’ J bar/rod-shaped ‘‘open’’ J compressed closed J closed J open J compressed closed J open J open J closed J

1, 2 1 1 1, 3 1 4, 5 1, 3 1 2, 3

NPD, nasopalatine duct; VNC, vomeronasal cartilage; VNO, vomeronasal organ; References: 1, this study; 2, Hunter et al., 1984; 3, Smith et al., 2003a; 4, Frets, 1912; 5, Jordan, 1968 TABLE 3. Summary of characteristics of the vomeronasal complex in Platyrrhines: Lamina propria Species Aotus trivirgatus Alouatta caraya Ateles fusciceps Callithrix jacchus Cebuella pygmaea Cebus spp Leontopithecus spp Pithecia pithecia Saimiri spp. Saguinus spp.

Neural elements

Glands, commun.

Glands, histochem.

Reference

large and small branches ? smaller branches large and small branches, PVNG large and small branches ‘‘nerve bundle’’ small branches ? small branches small branches, PVNG

A, P, D,V A, P, R,V, L A, P, D, V, M, L ciliated ducts A,P,D,V ? A, P, D, M, L ? ciliated ducts ?

? ABþ/PASþ ? ABþ/PASþ ? ? ? ABþ/PASþ ? ?

1 2 1 2 1 3 1, 2, 4 2 1 4

PVNG, paravomeronasal ganglia; Glands, commun. (refers to VNO surface at which glands communicate, and other comments): A, anterior; P, posterior: D, dorsal; V, ventral; M, medial; L, lateral. References: 1, this study; 2, Smith et al., 2002; 3, Frets (1912) describes and shows a drawing of Cebus with a large nerve bundle near the VNO; 4, Smith et al., 2003a.

(prosthion-inion) and palatal length (prosthion-posterior mid-palatal point) were measured with digital calipers to the nearest 0.01 mm. In some specimens, damage during necropsy prevented acquisition of these measurements. All paraffin embedded heads were serially sectioned at 10–12 lm and stained for histomorphometric analysis using ImageJ software (NIH). At least every tenth section was mounted on glass slides with serial numbers and stained with hematoxylin–eosin and Gomori trichrome procedures. Intervening sections were saved for alternative procedures.

Histochemical and Immunohistochemical Procedures for the Study of Subadults A subset of the perinatal sample (Leontopithecus, Saguinus) was studied to elucidate characteristics of cell bodies associated with nerves adjacent to the VNO. Since similar cells were previously demonstrated to react to the lectin Ulex europaeus-1 (UEA-1) (Evans and Grigorieva, 1994) as well as neuron-specific markers (Dennis et al., 2004; Smith et al., 2004), we selected closely adjacent unstained sections of tamarin VNOs to characterize

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TABLE 4. Measurements and microanatomical characteristics of the vomeronasal neuroepithelium in adult Platyrrhines Species

Morphology

VNNEL (mm)

VNEA (mm2)

OMP

Lumen/VNO ratio

Microvilli

References

Aotus trivirgatus Alouatta caraya Ateles fusciceps Callithrix jacchus Cebuella pygmaea Leontopithecus rosalia Pithecia pithecia Saimiri spp. Saguinus spp.

SEO ? SEO? SEO SEO ISE SEO? SEO ISE

2.4 ? 10.1 2.6, 2.8 1.4–1.7 2.7, 3.1 ? 3.2 3.0–4.0

0.095 ? 0.054 0.04, 0.066 0.038, 0.04 0.136 ? 0.051 0.16–0.27

(þ) ? (þþ) (þþ) () ? ? () (þ)

0.62 ? 0.49 0.40, 0.49 0.45, 0.49 0.47 ? 0.74 0.45–0.69

P (LM) ? P (LM) P (TEM) ? P (LM) ? ? P (LM, TEM)

1, 2 – 1, 2 3, 4 1, 5 1 1 1, 2 1, 2, 3, 4

VNNEL, anteroposterior length of VNNE; VNEA, cross-sectional area of VNNE (in mm2) ¼ an average of the cross-sectional area at 25th, 50th, and 75th percentile of VNNE length; SEO, sensory epithelium only; ISE, interrupted sensory epithelium; ?, unclear from available specimens; P, present; LM, light microscopic observation; TEM, transmission electron microscopic observations. () indicates that no OMP-reactive cells are visible in the VNNE (only one specimen of Saimiri boliviensis was studied by Smith et al., 2011); (þ), indicates sparse, individual OMP-reactive cells visible in the VNNE (see Smith et al., 2011 for details); (þþ), indicates multiple rows of OMP-reactive cells visible in the VNNE (see Smith et al., 2011 for details). References: 1, This study; 2, Smith et al., 2011; 3, Taniguchi et al., 1992; 4, Smith et al., 2004; 5, unpublished observations (on Cebuella only).

groups of cells. In addition, we examined VNNE immunoreactivity to olfactory marker protein (OMP), a marker of terminally differentiated neurons. These specimens were examined to see whether a broad range of platyrrhines resemble Saguinus spp. (Dennis et al., 2004; Smith et al., 2011) in having few or no mature VSNs at birth. Lectin binding was performed using the avidin-biotin peroxidase complex (ABC) method following specified procedures. Protocols were adopted and modified accordingly from Vector literature and previous studies (Nakajima et al., 1998). All procedures were done at room temperature and solutions were mixed daily just prior to and/or during experimental procedures. After sections were deparaffinized and hydrated in graded ethanols, they were rinsed in running tap water for 5 min, incubated in 1% bovine serum albumin (BSA) for 30 min, rinsed in 0.05 M tris buffered saline (TBS, pH ¼ 7.2) for 5 min, and incubated in UEA-1 (Vector Inc, Burlingame, CA) for 30 min. Following incubation, sections were washed in 0.05 M TBS for 5 min, incubated with ABC reagent for 30 min, washed in 0.05 M TBS for 5 min, reacted with diaminobenzidine (DAB) labeling kit (Vector Inc.) for 5 min, and washed in distilled water for 15 min. Sections were dehydrated and cleared with xylene in the reverse order of that noted above and mounted with Permount (Fisher Scientific, Pittsburgh, PA). Several slides were lightly counterstained with hemotoxylin prior to this step for delineation and confirmation of adjacent cell populations. Two control conditions were examined to confirm lectin specificity: (1) omission of lectin which was replaced by 0.05 M TBS for 30 min; and, (2) introduction of 0.4 M of inhibitory sugar (L-Fucose, from Ferro Pfanstiehl, Waukegan, IL), premixed with lectin, for 30 min. For neuron-specific beta tubulin (BT) (Covance Inc.,Princeton, NJ) immunohistochemistry, sections were deparaffinized in Hemo-D (Scientific Safety Products, Fort Lauderdale, FL) and hydrated to distilled H2O (dH2O). Endogenous peroxidase-like activity was blocked by holding the slides for 20 min in 0.9% H2O2 made in absolute methanol. After washing in dH2O, the slides

were washed 3 3 min each in phosphate buffered saline (PBS, pH 7.4). Subsequently, the slides were placed in humidified chambers and incubated 30 min in blocker solution of 5% normal horse serum and 2.5% BSA. The slides were then rinsed briefly in PBS and returned to their chambers. Anti-BT (Covance Inc.), diluted 1:16,000 in blocker, was applied to the tissues and the slides were left overnight at 23*C. Next morning, the slides were washed 3 3 min each in PBS, and the tissues were incubated 1 hr in biotinylated horse anti-mouse IgGs, (Vector, Inc.), diluted 1:200 in blocker. Following incubation in the secondary antibody, the tissues were washed 2 3 min each in PBS and incubated 30 min in ABC reagent (Vector Inc.). After 2 3 min washes in PBS, antibody binding was detected with a DAB labeling kit. Secondary antibody binding specificity was tested by running tissue sections of each species through all the steps above except that the sections were incubated in blocker without primary antibody. Sections selected for double labeling with BT and PGP 9.5 (Chemicon, Temecula, CA) were prepared as above except that the incubation in methanol and H2O2 was omitted. Anti-BT (1:2,000) and anti-PGP (1:1,000) antibodies were applied as a cocktail and treated as described above. Secondary antibodies conjugated to Alexa fluorochromes (Molecular Probes, Carlsbad, CA) were also applied as a cocktail. The slides were mounted with Vectashield (Vector, Inc.) and viewed with a Nikon E600 microscope equipped with epifluoresence optics. Images were made with a Spot Slider digital camera and Spot Advanced (4.0.1) software (Diagnostic Instruments, Sterling Heights, MI). For OMP immunohistochemistry, sections were deparaffinized in Hemo-D (Scientific Safety Products, Fort Lauderdale, FL) and hydrated to distilled H2O (dH2O). Endogenous peroxidase-like activity was blocked by holding the slides for 20 min in 0.9% H2O2 made in absolute methanol. After washing the slides in dH2O, the slides were washed 3 3 min each in PBS (pH 7.4). Subsequently, the slides were placed in humidified chambers and incubated overnight in anti-OMP (Wako

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Chemicals USA, Inc, Richmond, VA) diluted appropriately in blocker (5% normal rabbit serum and 2.5% BSA in PBS). The next morning, slides were washed 3 3 min each in PBS, and the tissues were incubated 1 hr in biotinylated rabbit anti-goat IgG (Vector Inc.), diluted 1:200 in blocker. Following incubation in the secondary antibody, the tissues were washed 2 3 min each in PBS and incubated 30 min in ABC reagent. After 2 3 min washes in PBS, antibody binding was detected with a DAB labeling kit. Secondary antibody binding specificity was tested by running tissue sections of each species through all the steps above, except that the sections were incubated in blocker without primary antibody. Slides were examined using light microscopy with a Leica DMLB photomicroscope at 100 to 630. VNOs were studied for reactivity in apical processes, presumptive receptor (bipolar) cells, supporting cells, and basal cells.

The Vomeronasal Groove Posterior to its communication to the nasopalatine duct (Fig. 1A), the VNO lies in a cartilaginous capsule that articulates with the maxillary bone (Fig. 1B). A groove forming from the articulation of the vomeronasal cartilage and the hard palate provides a good approximation of VNO gross dimensions such as length and width, based on studies of primates including strepsirrhines and platyrrhines (Garrett et al., 2009; Garrett, 2010). In the present study, we examined the relationship between the vomeronasal groove (VNG) and the VNO in platyrrhines only. In particular, VNG length and width were regressed against a measurement of the sensory part of the VNO, length of the VNNE. Linear measurements were collected on photographs of previously sectioned platyrrhine nasal fossae using ImageJ software. For length of the VNG, the number of slides in which the VNNE and VNG were observed in contact were counted and multiplied by slice thickness. Width measurements were taken of the VNG (Fig. 1B) at the 25th, 50th, and 75th percentiles of the length of the VNO using the line tool in ImageJ. This regression analysis was necessarily preliminary, since our sample was limited to specimens with a VNNE that was identifiable across anteroposterior limits. Fifteen adult specimens including nine species were suitable for the analysis.

RESULTS General Observations on Adult Sample In all adult specimens, except where freezing artifact prevented assessment, three cell types are identifiable in the VNNE. Although ultrastructural confirmation of cells types was not made, these matched previous descriptions of supporting, basal, and receptor cells in the VNNE. A row of presumptive supporting cells with abundant apical cytoplasm is seen adjacent to the lumen. Presumptive basal cells are visible as small, flat, or triangular cells with scant cytoplasm that are sparsely distributed along the basement membrane. Presumptive VSNs are visible in all adult specimens (except in Alouatta caraya and Pithecia pithecia due to freezing damage). In previous studies, the affinity of cells within the VNNE to neuron-specific markers was described in

Fig. 1. Vomeronasal complex of Cebuella pygmaea. A: View of the ductal communication (*) of the VNO to the nasopalatine duct (npd). B: Location of the VNO within a scroll of cartilage (vnc) that articulates with the maxillary bone (m). The cartilage rests in a groove (width indicated by yellow arrows) in the maxilla. C: View of the VNO, showing a prominent large bundle of vomeronasal nerves (double arrows). Note the proportionally small VNO lumen (L). D: Higher magnification view of the VNO neuroepithelium (vnne), showing the multiple rows of VSNs (arrows). Gomori trichrome. gd, gland duct; nf, nasal fossa. Scale bars: A, 400 lm; B, 1 mm; C, 200 lm; D, 30 lm.

some adult callitrichines (Dennis et al., 2004; Smith et al., 2004), verifying that a neuronal complement of cells exists in VNNE of at least some species.

Specific Observations on Adult Sample, by Species Cebuella. The VNO of Cebuella pygmaea communicates with the nasopalatine duct at a mid-palatal level (Fig. 1A). The VNO itself is round or oval in cross-section, with large bundles of vomeronasal nerves connected dorsally (Fig. 1B,C). The VNNE has rows of three or more VSN nuclei on all sides of the VNO in cross-section (Fig. 1C). Glands communicate to the VNO lumen, from anterior, posterior, medial, lateral, and dorsal sides of the VNO (Fig. 1C,D). Preliminary observations using OMP immunohistochemistry indicate the VNNE of adult Cebuella is OMP (), whereas olfactory neuroepithelium


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Fig. 2. Vomeronasal complex of Leontopithecus rosalia. A–D: Anteroposterior views of the VNO, its ductal communication (*) to the nasopalatine duct (npd), its cartilaginous capsule (vnc), and communicating glands (gl). E: The dorsoventrally elongate VNO, with a large lumen (L). Numerous venous sinuses surround it. F: The VNO neuroepithelium (vnne) is visible on both sides of the organ, although there appear to be gaps (brackets) where receptor neurons are lacking. G–

F: Higher magnification views showing that one to two rows of VSNs (arrows). Small bundles of nerves (double arrows) connect to the base of the VNNE (2G, I). Microvilli (open arrows, 2H) are visible. Basal cell (bc) and supporting cell (sc) nuclei are also apparent. A: Hematoxylin– eosin; B–I: Gomori trichrome. Scale bars: A–D, 400 lm; E, 200 lm; F, 50 lm; G, 30 lm; H, 20 lm; I, 20 lm.

of the same specimens is OMP (þ) (unpublished findings).

times irregular and always dorsoventrally elongated sac (Fig. 2C–E). Anteriorly, the irregular border has fingerlike projections in all directions, where glands communicate from all sides (Fig. 2C). Posterior to the VNO, more glands are found (Fig. 2D), connecting to the VNO via multiple ducts. Numerous venous sinuses surround the VNO (Fig. 2E). The walls of the VNO are neuroepithelial on both sides (Fig. 2F), although only one to two rows of receptor nuclei are visible (Fig. 2G–I). VSNs are sparsely

Leontopithecus. The VNO, lined with neuroepithelium, extends anterior to its communication with the nasopalatine duct (Fig. 2A, also showing anterior glands on the contralateral side). The VNO opens into the nasopalatine duct via a short, laterally projecting duct (Fig. 2B). Posterior to this, the VNO becomes a large, some-

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Fig. 3. Vomeronasal complex of Aotus trivirgatus. A–D: Anteroposterior views of the VNO, its ductal communication (*) to the nasopalatine duct (npd), its cartilaginous capsule (vnc), and communicating glands (gl). E: The dorsoventrally elongate VNO, with a large lumen (L). Numerous venous sinuses (vs) surround it. F: The VNO neuroepithelium (vnne)

is visible on both sides of the organ. E: Shows gland ducts (gd) entering the VNO dorsally and ventrally. F: Higher magnification view showing rows of VSNs (arrows). Small bundles of nerves (double arrows) connect to the base of the VNNE. A–D: Gomori trichrome; E,F: Hematoxylin eosin. Scale bars: A–D, 500 lm; E, 125 lm; F, 30 lm.

distributed in some regions, with some isolated patches having no apparent VSNs at all (Fig. 2F,H). Long microvilli are visible at the apical surface of the VNNE (Fig. 2H). Vomeronasal nerves are visible in the lamina propria (Fig. 2G,I).

VNNE (Fig. 3F). Nuclei of VSNs typically are arranged in rows of three.

Aotus. In Aotus, glands located anterior to the VNO and in the region of its communication to the nasopalatine duct (Fig. 3A) open into the VNO. The VNO is dorsoventrally elongate in its cross-sectional contour (Fig. 3B,C) and ends posteriorly in communication with glands (Fig. 3D). Throughout its length, numerous glands communicate at dorsal and ventral poles (Fig. 3E). Posteriorly, the vomeronasal cartilage is partially ossified, especially at posterior levels (Fig. 3C,D). The VNNE is present at all surfaces in the coronal plane, and nerve bundles are visible in contact with the

Saimiri. The VNO communicates with the nasopalatine duct at the mid-level of the palate in the single adult specimen studied (Fig. 4A), among adjacent glands that open into the VNO. The VNO is round in cross-section (Fig. 4B), ending in communication with posterior clusters of glands (Fig. 4C). In the lamina propria, numerous venous sinuses are present (Fig. 4D) and small bundles of nerve fibers are seen at high magnification (Fig. 4E). The VNNE has one to three rows of VSNs (Fig. 4E). At intermediate and posterior levels, gland ducts are ciliated (Fig. 4F,G). Ateles. Anterior to the VNO communication with the nasopalatine duct, the vomeronasal cartilage mostly


Fig. 4. Vomeronasal complex of Saimiri sciureus. A–C: Anteroposterior views of the VNO, its ductal communication (*) to the nasopalatine duct (npd), its cartilaginous capsule (vnc), and communicating gland ducts (gd). D: The round VNO, with a large lumen (L). Numerous venous sinuses (vs) surround it. Note the uniform thickness of the wall of the VNO. E: The VNO neuroepithelium has several rows of VSNs

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and also shows a gland duct (gd) entering the VNO. Very small nerves bundles (nn), containing few axons each, are visible in the lamina propria (E). F,G: Show ciliated (open arrows) gland ducts that entered the VNO at approximate mid-levels as well as posteriorly. All stained with Gomori trichrome except (F) (hematoxylin–eosin). Scale bars: A– C, 400 lm; D, 200 lm; E, 20 lm; F, 40 lm; G, 20 lm.

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Fig. 5. Vomeronasal complex of Ateles fusciceps. A–C: Anteroposterior views of the VNO, its ductal communication (*) to the nasopalatine duct (npd), its cartilaginous capsule (vnc), and communicating gland ducts (gd). D: The VNO is dorsoventrally compressed (C) and ends as a large duct receiving gland ducts (gd) from all sides (D,E) as

well as posteriorly. The lumen (L) is large but varies in relation to thickness of the VNO wall. F: The VNO neuroepithelium (vnne) has three or more rows of VSNs (arrows). Small bundles of axons (nn) are visible in the lamina propria. A,B,F: Gomori trichrome; (C–E) hematoxylin–eosin. Scale bars: A–C, 800 lm; D, 100 lm; E, 50 lm; F, 30 lm.

encircled small glands (Fig. 5A), which communicated by ducts to the VNO lumen. A stratified cuboidal duct extended for a short distance from the upper (nasal) extent of the nasopalatine duct to the VNO itself (Fig. 5B), here defined as beginning where a neuroepithelium was present. The VNO is dorsoventrally compressed through most of its extent (Fig. 5C), with glands entering posteriorly, superiorly, inferiorly, medially, and laterally through the wall of the VNO (e.g, Fig. 5D,E). In the single adult Ateles specimen, the VNNE appears to have distinct junctions with non-sensory epithelium (Fig. 6B,C). Long microvilli are visible (Fig. 6C). Some surfaces of the VNNE are covered with a fine poorly defined meshwork (Fig. 6A). These may represent apical cell surface projections, but could not be identified at 1000 levels of light microscopy.

ing artifact) in these specimens was noted, but only the surrounding lamina propria was described in detail. In A. caraya, the VNO is dorsoventrally elongate in crosssection and communicates with the NPD at about the mid-palate (Fig. 7). Compared to all other specimens, this specimen’s vomeronasal cartilage is atypical because it remains superior to the nasal septum’s base and has a discontinuous bar-shaped form at some cross-sectional intervals (Fig. 7B–D). Glands communicate with the VNO at multiple sites (Fig. 7A–E). In P. pithecia (not figured), the VNO is round in cross-section and the opening of the VNO occurs in the nasopalatine duct, but very close to the base of the nasal cavity.

Observations on Subadult Samples

Alouatta and Pithecia. The general morphology of

General observations on perinatal and fetal samples. Perinatal or fetal platyrrhine primates have

adult Alouatta caraya and Pithecia pithecia were previously described. In the work by Smith et al. (2002), the poor preservation of the VNO epithelium (due to freez-

in common a relatively thin VNNE (Fig. 8A,C,E,G). The VNNE of perinatal marmosets (e.g., Callithrix, Fig. 8B), has no discernable differentiation of VSN and supporting


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cell nuclei. The VNNE is only one to two nuclei in depth. In Saimiri boliviensis and Pithecia pithecia, most of the VNNE is two to three nuclear rows in depth, but a row of cells with abundant apical cytoplasm (presumptive supporting cells) borders the lumen (Fig. 8D,F). Some patches of thicker epithelium are sparsely distributed in each of these species and these patches contain VSN-like cell clusters (Fig. 8D,F). Preservation of the fetal Alouatta seniculus was poorer than the perinatal specimens. However, existing tissues indicate a similarity to Saimiri and Pithecia, and it is possible that a more discrete VNNE exists in this specimen (Fig. 8G,H).

Lectin and immunohistochemical results on subadult primates. No OMP reactivity is observed in the VNO of perinatal monkeys, including marmosets (Callithrix jacchus and Cebuella pygmaea) (Fig. 9) or the Saki monkey (Pithecia pithecia, not shown). The olfactory neuroepithelium of these perinatal monkeys is OMP (þ) (Fig. 9). In one perinatal squirrel monkey, no OMP (þ) VSNs are visible. In a second Saimiri specimen, which is older (10 days postnatal), a single layer of OMP (þ) cells is seen in the VNNE. The olfactory neuroepithelium of this specimen is also OMP (þ), with rows of reactive olfactory sensory neurons (Fig. 10). In subadult Saguinus geoffroyi (Fig. 11) and Leontopithecus rosalia (not shown), nearly the entire VNO epithelium is intensely UEA-1 (þ) (Fig. 11C,E), but BT (þ) cells in adjacent sections are sparse (Fig. 11B,G). In L. rosalia, more numerous BT (þ) cells are seen (Fig. 12b,e) compared to S. geoffroyi. Double labeling with BT/ PGP 9.5 revealed that neonatal S. geoffroyi had mostly BT()/ PGP 9.5(þ) cells in the VNO epithelium (Fig. 11D) while in L. rosalia colabeling was seen at perinatal and juvenile ages (Fig. 12c,f). Extraepithelial PGP 9.5 (þ) or BT(þ) cell bodies are found in association with the VNO or nerves to the VNO in both species of tamarin at perinatal and older subadult ages (Figs. 11D,G and 12b,e). The lectin procedure reveals that cell bodies found in vomeronasal nerves are either UEA-1() or weakly UEA-1(þ). In both species of tamarin, some small UEA-1(þ) cell bodies are found within nerves connected to the VNO (e.g., Fig. 11E). Double labeling with BT/PGP 9.5 in L. rosalia reveals that populations of cells clustered within the VNN are BT(þ)/PGP 9.5(þ) (Fig. 12c,f). Control sections in which UEA-1 was preabsorbed with 0.4 M of L-fucose showed minimal to no reactivity. Additionally, controls where no lectin was introduced (0.05 M TBS replaced lectin) demonstrated no VNO staining specificity.

Vomeronasal Groove

Fig. 6. Higher magnification views of the VNNE in Ateles fusciceps. A: Rows of up to four nuclei of VSNs (arrows) are shown. Also note a layer of stained structures at the surface of the VNNE, which could be short microvilli (double arrows). B: A transition from VNNE to a thinner epithelium is shown. C: At some locations fine, long microvilli (long arrows) were visible at the apical surface of the VNO epithelium. A,B: Gomori trichrome; (C) hematoxylin–eosin. bc, basal cell nucleus; sc, supporting cell nucleus. Scale bars: 20 lm.

Dimensions of the VNG are significantly correlated with VNNE length, despite the small sample size (Fig. 13). VNG width is significantly correlated with VNNE length (r2 ¼ 0.613, P < 0.05). The correlation is stronger if Ateles fusciceps is removed from the analysis (r2 ¼ 0.798, P < 0.01). The location of Ateles in the plot distribution indicates this species falls below the regression line for the remaining, smaller species (Fig. 13). Further analysis may determine whether the relationship of VNG width to VNNE width differs in larger relative to smaller platyrrhines. There is a stronger association of

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Fig. 7. Vomeronasal complex of Alouatta caraya. A–E: An adult A. caraya. A–E: Anteroposterior views of the VNO, its ductal communication (*) to the nasopalatine duct (npd), its cartilaginous capsule (vnc), and communicating gland ducts (gd) are shown. Poor preservation

prevented a description of epithelial structure, but a dorsoventrally tall contour is apparent in this specimen (E). Also note the diminutive VNC. All stained with Gomori trichrome except C (hematoxylin–eosin). Scale bars: A–D, 500 lm; E, 250 lm.

Fig. 8. Vomeronasal complex of perinatal and fetal platyrrhines. The bottom row shows an enlargement of boxed regions in the top row. Perinatal Callithrix jacchus (A,B), Saimiri sciureus (C,D), and Pithecia pithecia (E,F) are shown. G,H: Fetal specimen of Alouatta seniculus. In all species, VSNs (arrows) were sparse, or in discontinuous

clusters. B: Shows a cluster of cells dorsal to the VNO that may represent a paravomeronasal ganglia. A,B,G,H: Gomori trichrome; (C–F) hematoxylin–eosin. gd, gland duct; L, lumen of VNO; vncC, vomeronasal cartilage; vno, vomeronasal organ. Scale bars: A, C, E 500 lm; G, 200 lm; B, D, F, H, 30 lm.

VNG length with VNNE length (r2 ¼ 0.772, P < 0.01; Fig. 13).

across the order, rather than a single evolutionary trajectory for all primates (Smith and Rossie, 2006; Smith et al., 2007). Taken together, anatomical and genetic data provide one coherent evolutionary picture, supporting a hypothesis that the catarrhine VNS became nonfunctional prior to the divergence of the cercopithecoid and hominoid lineages (Smith et al., 2001a; Liman and Innan, 2003; Zhang and Webb, 2003; Rossie, 2005; Bhatnagar and Smith, 2006). For the main olfactory system (MOS), different lines of evidence support remarkably different evolutionary views on primate olfaction (Cave,

DISCUSSION The two olfactory systems (main olfactory system and VNS) are generally considered reduced in primates compared to other mammals (Stephan et al., 1981; Barton, 2006), yet recent findings on the VNO and other olfactory structures indicate a complex pattern of reduction


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Fig. 9. Expression of OMP in perinatal callitrichines. A: VNO of Callithrix, and (B) Cebuella, showing lack of OMP expression in the wall of the VNO. C,D: OMP expression in the olfactory neuroepithelium (OE) of Callithrix (same specimen as Fig. 11A) showing reactivity of ol-

factory receptor neurons (arrows) and olfactory nerves (ON). E: OMP expression in the olfactory neuroepithelium (OE) of Cebuella (same specimen as Fig. 11B) showing reactivity of olfactory neuroepithelium (OE) and olfactory bulb (OB). Scale bars: A–D, 20 lm; E, 250 lm.

1973; Rouquier et al., 2000; Whinnett and Mundy, 2003; Laska et al., 2005, 2006). For example, data on relative volume of the main olfactory bulb indicate that primates may vary in the extent that they rely on the MOS and VNS for dietary versus social functions (Alport, 2004). Anatomical and genetic evidence have generated several evolutionary scenarios for the primate VNS (Fig. 14) that hinge entirely on extant taxa. Smith et al. (2007) suggested that details of evolutionary change may be gleaned from anatomic variations of olfactory structures in extant primates, especially since these var-

iations may bear on interpretations of fossil primate morphology and behavior. However, the VNO of platyrrhines, a major radiation and one that is generally more primitive than cercopithecoids and hominoids in numerous features, has not been described in similar terms in all previous investigations. Maier (1980) provided a functional assessment of the VNS in platyrrhines primarily based on Saimiri and callitrichines (species not specified). He described a basic similarity of the VNO epithelium to olfactory neuroepithelium (i.e., the presence of supporting, receptor, and

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Fig. 10. Expression of OMP in a 10-day-old Saimiri boliviensis. A: VNO, and (B) olfactory neuroepithelium. A single row of OMP(þ) cells is seen along most of the VNO wall [arrows, (A)]. Multiple rows of OMP(þ) cells are seen in the olfactory neuroepithelium [arrows, (B)] on, olfactory nerves. Scale bars: 100 lm.

basal cells). Subsequently, this arrangement was confirmed in Aotus trivirgatus (Hunter et al., 1984), Saguinus fuscicollis (Hunter et al., 1984; Mendoza et al., 1994), and Callithrix jacchus (Taniguchi et al., 1992; Dennis et al., 2004). Transmission electron microscopy specifically confirmed that receptor neurons are present in two species of callitrichines (Taniguchi et al., 1992; Mendoza et al., 1994). Based on the presence of a neuroepithelium, some authors concluded that the VNO of certain platyrrhines may be functional (Maier, 1980; Taniguchi et al., 1992). However, potential signs of evolutionary regression in the platyrrhine VNS have also been observed. In comparison to Tupaia and strepsirrhines, Maier (1980) discussed ‘‘signs of reduction’’ in the VNO of Saimiri and, to a greater degree, in unspecified callitrichines. In this regard, he noted the lack of large venous sinuses, the lack of a distinction between non-sensory and sensory portions of the VNO, and a poor distinction of cell types within some portions of the VNO epithelium. Smith et al. (2003a) noted that the VNO of Saguinus geoffroyi comprises a neuroepithelium interrupted by patches of nonsensory epithelium; sparse distribution of VSNs was confirmed by immunohistochemical studies on this species (Dennis et al., 2004; Smith et al., 2004). Hunter et al. described the VNO of Ateles geoffroyi as ‘‘similar to that lining the nasopalatine ducts’’ (p. 223, 1984) without making reference to sensory cells. Thus, it remains uncertain whether all platyrrhines possess a neuroepithelial VNO and to what extent it varies among taxa. Below, we integrate data from our present report with existing literature on the primate VNNE.

General Characteristics of the Vomeronasal Complex in Platyrrhines VNO communication with the oral and nasal cavities. The basic route of stimulus access to the VNO, via the nasopalatine duct, has been established previously (Jordan, 1972; Hunter et al., 1984; Shimp et al., 2003). In this study, we found that the specific point of communication with the nasopalatine duct varies. In Leontopithecus, Aotus, Ateles, Alouatta, and especially in Pithecia, the VNO communicates with the dorsal part of the nasopalatine duct. A vomeronasal duct, which is a non-sensory stratified cuboidal or stratified squamous epithelial conduit connecting the sensory part of the VNO to the nasopalatine duct (see Shimp et al., 2003), is barely discernable as a distinct conduit in most species. Only larger species, such as Ateles fusciceps, have a vomeronasal duct that was distinct from the VNNE for multiple serial sections. In most species, a neuroepithelium is present near the communication point of the VNO with the nasopalatine duct.

VNO shape. The cross-sectional contour of the VNO is round or oval in some small platyrrhines (Cebuella, Callithrix and Saimiri), and dorsoventrally elongate in Saguinus, Leontopithecus, and Aotus (Table 2, and references therein). In two larger platyrrhines, Cebus (Jordan, 1972) and Ateles (Table 2), the VNO is round or dorsoventrally compressed. In one adult Alouatta caraya, the VNO is dorsoventrally elongated (although freezing may have distorted the region).


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Fig. 11. In neonatal Saguinus geoffroyi (A–C), hematoxylin and eosin stained sections [(A), inset with position indicated by open arrow] showed an epithelium of the VNO that was nearly homogeneous, showing patches of non-sensory epithelium (nse), and thicker, possibly sensory epithelium (double arrows). B: BT reactivity was restricted to single (double arrows) or sparsely scattered cells in neonates in each section of the VNO. C: UEA-1 reactivity was ubiquitously present throughout the VNO epithelium. No BT or UEA-1 axons were seen in the lamina propria adjacent to the VNO. D: Shows a neonatal S. geoffroyi prepared with PGP 9.5 (green)/BT (red), showing the numerous PGP 9.5þ cells in the VNO epithelium (double arrows) and one extrae-

pithelial reactive cell (open arrow). The VNO of 1-month-old (D–F) Saguinus geoffroyi has ubiquitous UEA-1 reactivity throughout the VNO epithelium, except for scattered weakly or non-reactive cells (white arrows). The vomeronasal nerves (VNN) are non-reactive but sometime contain UEA-1þ cells (open arrows). A more posterior section of the VNO is shown in (E) (Gomori Trichrome) in which the VNN can be seen superiorly to the VNO. F: Shows a higher magnification of an adjacent section, in which extraepithelial BTþ neuronal bodies of different sizes and morphology can be seen near the VNN. L ¼ lumen. Scale bars, A–C and inset, 100 lm; D, 20 lm (inset, 500 lm); E, G ¼ 50 lm; F ¼ 100 lm.

Relative size of the lumen varies. Marmosets have the smallest proportional size of the lumen while Aotus and Saguinus spp. have the largest (see lumen/VNO ratios, Table 4). Tamarins in particular have proportionally large VNOs, in part because the lumen is voluminous. The VNNE of tamarins is also more superoinferiorly elongated compared to other platyrrhines. Because of that, it has a greater cross-sectional area in the coronal plane (Table 4). The platyrrhine VN varies from an elongated tube (Ateles) to a mediolaterally compressed sac (Saguinus, Leontopithecus, and Aotus).

marsupials, insectivores, most bats, and many primates (Broom, 1897; Cooper and Bhatnagar, 1976; Sańchez Villagra, 2001; Wo¨hrmann-Repenning and Bergmann, 2001). Here, we refer to this morphology as an open J configuration (Figs. 1–3; Table 2). Some mammals possess more complete, laterally enclosed capsules, which may be mostly or entirely osseous (Salazar and Sanchez Quintero, 1998). This specialization of the vomeronasal capsule, in which the vomeronasal cartilage is mostly replaced by extensions of the vomer bone (Salazar and Sanchez Quinteiro, 1998), may maximize efficiency of the vasomotor vomeronasal pump mechanism, which delivers stimuli to the lumen of the VNO (Meredith and O’Connell, 1979). The nearly complete enclosure of the VNO by cartilage in Saguinus (Maier, 1980; Hunter et al., 1984) and Aotus (Hunter et al., 1984) has been noted previously. Here we document this closed J morphology as being characterstic of Aotus and most callitrichine genera (Table 2; note Callimico has not been

VNO capsule and glandular apparatus. The vomeronasal capsule shows two variants. In one morph, the cartilage is open laterally, that is, there is no cartilaginous (or osseous) wall between the dorsolateral side of the VNO and the respiratory mucosa (Fig. 4). This morphology is fairly typical of many mammals, including

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Fig. 12. The vomeronasal organ (VNO) of a neonatal (a–c) and juvenile (d–f) L. rosalia, illustrating populations of extraepithelial cell bodies at both ages [open arrows, (a,b,e)]. Adjacent sections prepared with BT/PGP 9.5 are shown in (c) and (f). These double-labeled fluores-

cence (BTþ ¼ red; PGP 9.5þ ¼ green; co-labeled ¼ yellow) images indicated that such cells were BTþ/PGPþ (open arrows). L ¼ lumen; VNC ¼ vomeronasal cartilage; VNN ¼ vomeronasal nerves. Scale bars: a, b, 100 lm; c, e, f, 50 lm; d, 200 lm.

Fig. 13. Plots of VNG width and VNG length against VNNE Length. Linear regression line is indicated, although the largest species (Ateles fusciceps) indicates some scaling phenomena require further investigation. Af ¼ Ateles fusciceps, At ¼ Aotus trivirgatus, Cj ¼ Callithrix

jacchus, Cp ¼ Cebuella pygmaeus, Lr ¼ Leontopithecus rosalia, Sb ¼ Saguinus bicolor, Sg ¼ Saguinus geoffroyi, So ¼ Saguinus oedipus, Saim ¼ Saimiri boliviensis.


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Fig. 14. Cladogram showing major primate groups with detailed relationships amongst the living platyrrhini (constructed based on information from Steiper and Ruvolo (2003), Ray et al. (2005), and Hodgson et al., (2009)). Cladogram also traces character history for VNO character states using parsimony analysis (Mesquite software). Groups with bullet have known morphology. A VNO with well-defined

neuroepithelial and non-sensory parts was likely primitive for Euprimates. A VNO with sensory-epithelium only was most likely primitive for haplorhines based on its extant distribution. The ancestral VNO state of catarrhines is equivocal, with a SEO, displaced, or absent VNO being equally likely.

studied). The capsule appears to ossify in Aotus, which has also been reported for ‘‘tamarins’’ by Maier (1980, taxa not specified). The platyrrhine VNO receives glandular ducts at multiple points along its anteroposterior, mediolateral, and dorsoventral axes (Table 3). All species studied here have multiple entry points for glands at more than two surfaces, as described previously for some individual species (Jordan, 1972; Mendoza et al., 1984). Glands at the dorsal poles extend out of the vomeronasal capsule in all platyrrhines. This is common among mammals (Roslinski et al., 2000), and may represent a primitive mammalian innovation in which some septal glands are co-opted into the VNS (Smith and Bhatnagar, 2009). All species also receive gland ducts posteriorly. The anterior communicating glands in Alouatta are not unique, as once written by Smith et al. (2002), and may be a similarly widely distributed trait. At least one other gland entry point (medial, lateral, and or ventral) was observed in every species examined in this study. In many mammals, the VNO comprises two tissues: a sensory epithelium and a receptor-free (non-sensory) epithelium. Gland duct entry points appear to occur commonly at the junction of these two epithelia (Roslinski et al., 2000). Since all platyrrhines possess a VNO with multiple glandular inputs, this may well be a primitive

arrangement for a haplorhine common ancestor. This is in keeping with the vestigial VNOs of some (possibly all) hominoids, in which the VNO itself may serve mainly as a glandular duct (Smith et al., 2002). This vestige, which is ciliated (like some of the gland ducts of platyrrhines) may well have evolved from the primitive haplorhine VNO as a remnant duct that delivers secretions to the proportionally large homninoid nasal fossa.

Structure of the Vomeronasal Epithelium Based on prominent adjacent and apparently communicating vomeronasal nerves, Frets (1912) suspected the VNO in adult Cebus was neurosensory in nature. The results of the present study, combined with previous findings, indicate that a neuroepithelial VNO exists in most, if not all, platyrrhines (Table 2). Some previous reports could not confirm the presence of neuroepithelium, but this very likely relates to artifactual phenomena. One methodological consideration that arises in histological studies of rare samples, such as non-human primates, is that it is difficult to obtain adequately prepared samples for study. Thus, postmortem decay or freezing have interfered with assessments in some studies (Smith et al., 2002). In addition, Hunter et al. (1984) suggested prolonged decalcification may have distorted

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the epithelial structure in one specimen of Ateles, a dilemma that is particular to larger species. We have achieved our best results with a formic acid–sodium citrate solution (see above) which has been effective in decalcifying very large tissue blocks from hominoids (Smith et al., 2002) and relatively large monkeys such as Ateles. This decalcifier works slowly (taking more than two months in some cases) and appears to leave epithelial tissue undistorted. Most evidence indicates that a more or less homogeneous epithelial wall is the primitive VNO epithelial morphology for anthropoids (Fig. 14). This is in contrast to the ventromedially restricted VNNE seen in most mammals, including strepsirrhine primates (Hunter et al., 1984; Smith and Bhatnagar, 2009; Smith et al., 2011). A uniform sensory epithelium was described previously for most New World primates (Maier, 1980; Hunter et al., 1984; Taniguchi et al., 1992; Mendoza et al., 1994). This morphology typifies adult tarsiers as well (Starck, 1975; Wo¨hrmann-Repenning and Bergmann, 2001), raising the possibility that this represents a haplorhine synapomorphy. Non-sensory epithelium has been described in the VNO of some platyrrhines, but in no case have descriptions matched the morphology of receptor-free epithelium. Receptor-free epithelium (Breipohl et al., 1979) is widespread among therians. It is often ciliated, and may therefore play a role in transporting secretions within the VNO. To date, two types of distribution of non-sensory epithelium have been reported in adult platyrrhines, neither of which matches the descriptions of receptor-free epithelium by Breipohl et al. (1979). First, large patches of non-sensory tissues have been described in Cebus (Jordan, 1972) and Ateles (this study). In Cebus capuchinus, Jordan (1972) described some medial to lateral differences of the VNO wall. Medially, sensory epithelium was observed. Laterally, sensory epithelium was more limited and instead a stratified, non-sensory epithelium predominated. In Ateles fusisceps, a ventral patch of non-sensory epithelium is described here. Based on such small samples, it is unclear whether these represent species characteristics or aberrations. Transient stages of VNO development have been observed in which the VNO of haplorhines has a non-sensory component (e.g., tamarins, Dennis et al., 2004; tarsiers and squirrel monkeys, Smith et al., 2003b). Thus, the morphology could be atavistic. A second example of non-sensory epithelium pertains to the so-called ‘‘interrupted sensory epithelium’’ described in Saguinus geoffroyi by Smith et al. (2003a). In this case, the VNO wall has numerous interruptions of the neuroepithelium by intervening patches of non-sensory epithelium. Immunohistochemical studies using neuron-specific markers indicate that the basis for this morphology may be a reduced density of VSNs, which occur in clusters (Dennis et al., 2004; Smith et al., 2004). Leontopithecus may also possess this phenotype. Of all species examined in this study, adult Leontopithecus exhibits the fewest rows of VSNs (one to two). A single specimen of Leontopithecus chrysomelas similarly was observed to have only few VSNs that react to antibodies for neuron-specific BT, when compared to marmosets or a strepsirrhine species (Smith et al., 2004). Taken together, these results suggest that the VSN population is small in Saguinus and Leontopithecus, and patches of non-sensory epithelium may simply reflect a regional dearth of VSNs.

Based on available evidence, therefore, the occurrence of non-sensory components does not constitute a functional receptor-free epithelium (Breiphol et al., 1979) as observed in other mammals. If not an atavistic trait, such non-sensory patches could be construed as a byproduct of neural reduction. Marmoset only possesses one type of G-protein in its accessory olfactory bulb (Takagami et al., 2004). In rodents and marsupials, two distinct populations of G-protein-expressing cells (Go and Gi2) are found in the VNNE and AOB (Halpern and Martıńez-Marcos, 2003). Therefore, it is possible that in marmosets an entire subset of VSNs is absent. If this applies to platyrrhines broadly, it may explain the relatively small number of rows of VSNs that are found in the VNNE compared to strepsirrhines (Smith et al., 2004) and other mammals (Halpern and Martıńez-Marcos, 2003). Previous findings showed that some tamarins (Saguinus spp.) have a very sparse distribution of VSNs, as detected by neuronal markers such as PGP 9.5 and neuron-specific BT (Dennis et al., 2004; Smith et al., 2004). Since no estimates of total neuronal numbers have been provided to date, it is not known whether Saguinus spp. have fewer as opposed to more widely dispersed VSNs. However, recent work has shown primate VNOs vary in reactivity to an additional neuronal marker, OMP (Dennis et al., 2004; Smith et al., 2005, 2011). Since this marker is expressed in terminally differentiated olfactory or VSNs (Farbman and Margolis, 1980; Weiler and Benali, 2005), the limited reactivity to OMP in the VNO of some platyrrhines indicates the presence of only few mature sensory neurons (Dennis et al., 2004; Smith et al., 2011). Smith et al. (2011) examined five species of Saguinus, and found this genus has few mature vomeronasal neurons at any age when compared to strepsirrhines. Although other platyrrhine species have only been investigated using one or two specimens, some resemble Saguinus (e.g., Aotus and Saimiri) while others appear to have a VNNE with multiple rows of OMP(þ) VSNs (Dennis et al., 2004; Smith et al., 2011). In possessing relatively small numbers of terminally differentiated VSNs, Saguinus spp (and possibly other platyrrhines as well) differ from a diverse array of mammals, including some other primates (Halpern and Martıńez-Marcos, 2003; Smith et al., 2005, 2011). Observations on a larger sample of platyrrhines are still much needed; available observations of atelines are extremely limited and no detailed information exists regarding the VNNE of adult pitheciines.

Ontogenetic Characteristics of the Vomeronasal Epithelium Delayed maturation of the VNNE. Perinatal morphology of the VNO is, to date, documented in callitrichines alone. Evans and Grigorieva (1994) suggested tamarins (Saguinus labiatus) and marmosets (Cebuella pygmaea) have delayed VNNE maturation. This hypothesis, based on lectin histochemistry (Evans and Grigorieva, 1994), was supported in subsequent studies using neuron-specific markers. In Leontopithecus rosalia and Saguinus geoffroyi, the density of VSNs detected by PGP 9.5, neuron-specific BT, or OMP is lower than in lemurs at birth (Dennis et al., 2004; Smith et al., 2004). In our sample, we detected no OMP reactivity in the VNOs of


marmosets and Pithecia. Our limited sample suggests that infant Saimiri boliviensis may have mature VSNs, as also observed in juvenile Leontopithecus rosalia. However, this is difficult to interpret without additional specimens of other ages, especially since an adult of this species was observed to lack OMP(þ) VSNs (Smith et al., 2011). For most species, the lack of perinatal expression of OMP provides support to the hypothesis of delayed maturation of the VNNE. However, if the VNNE is poorly developed in adults of certain species, as suggested for Saguinus spp. and possibly other primates (Aotus and Saimiri) (Smith et al., 2004, 2011; this study) delayed maturation appears to be a misnomer. In such species, the neuroepithelial part of the VNS may be postnatally reduced or even vestigial, unless OMP is not required for VNO function (Smith et al., 2011). In callitrichines, in particular, the hypothesis of delayed maturation may be applied to Callithrix, which has a thin OMP() VNNE at birth, and later develops a thick VNNE with terminally differentiated VSNs (Taniguchi et al., 1992; Dennis et al., 2004). Saguinus has a different postnatal developmental trend, in which the density of VSNs may actually decrease postnatally. In S. geoffroyi, OMP(þ) VSNs are scarce in both newborns and adults (Dennis et al., 2004; Smith et al., 2011). The density of BT(þ) VSNs decreases postnatally, based on a small sample of Saguinus spp. (Smith et al., 2004). Although previous reports indicate OMP reactivity is greater in a juvenile L. rosalia compared to perinatal specimens, observations in this study suggest VSNs may become sparsely distributed in adult L. rosalia. The possibility that this reflects a lifelong comparatively low rate of neurogenesis in Saguinus and Leontopithecus could be investigated with certain neuronal markers in the future (e.g., Gap43). Another goal of the present study was to examine late fetal/perinatal representatives across subfamilies to complement the published data on tamarins. Though our sample was limited for some taxa (with no Callicebus or Aotus), a coherent pattern among all specimens was a thin VNO epithelium with a patchy distribution of VSNlike cells. The available specimens suggested two distinct groups. First, callitrichines appear to have especially poorly differentiated VNOs at birth compared to perinatal Pithecia, Saimiri and fetal Alouatta. In all callitrichines, there is no distinction (at least by light microscopy and the procedures used here) between supporting cells and other types at birth; VSN-like cells are distributed as isolated cells (as confirmed based on the distribution of BT(þ) cells—Smith et al., 2004). P. pithecia and S. boliviensis have groupings of VSN-like cells scattered in clusters. Although its preservation was not optimal, the fetal A. seniculus appeared to have larger patches of neuroepithelium as well.

Extraepithelial neuronal bodies adjacent to the VNNE. Evans and Grigorieva (1994) suggested that neuron-like cells in the lamina propria surrounding the VNO of callitrichines were evidence for prolonged postnatal neurogenesis. These authors speculated that some of these cells were migratory, possibly luteinizing hormone releasing hormone (LHRH) neurons. Subsequent reports identified neuronal bodies in the lamina

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propria surrounding the VNO of Saguinus spp., Leontopithecus rosalia, and Callithrix jacchus (Dennis et al., 2004; Smith et al., 2004). Using Saguinus and Leontopithecus, we confirm here that cell bodies associated with nerves adjacent to the VNO are both UEA (þ) and reactive to neuronal markers. Although it is not clear that these cells are migratory or ganglionic, their neuronal identity is certain. In morphology, these PGP 9.5 and/or BT reactive cells varied from relatively large-bodied neurons, as described for the paravomeronasal ganglia of bats (Bhatnagar and Kallen, 1974, Bhatnagar et al., 2006) to relatively fusiform neurons with processes of varying length, as described in LHRH neurons of mice (Wu et al., 1997). Our observation of UEA-1 (þ) cell bodies within the vomeronasal nerves also is noteworthy since such cells have been observed in close association with LHRH neurons in other mammals (Tobet et al., 1997), and UEA-1 is thought to identify one of the neural cell adhesion molecules (Pestean et al., 1995). Nonetheless, the presence of a migratory population of neurons would seem extremely prolonged in callitrichines compared to other mammals described to date. At least some neuroblastic cell migration from olfactory epithelium occurs postnatally in rodents (Monti-Graziadei, 1992). Postnatal migration also may occur along the vomeronasal nerves of rodents, yet it is clear that there is a marked postnatal reduction of LHRH reactive cells found in the nasal cavity from about mid-gestation (77.6% in nasal region/22.4% in CNS) to birth (7.9% in nasal region/ 92.1% in CNS) (Wu et al., 1997). Similarly, large numbers of extraepithelial neurons associated with the VNO have not previously been described in primates, except during embryonic and fetal stages (Boehm and Gasser, 1993; Kjær and Fischer Hansen, 1996; Smith et al., 2003b). A second explanation is that these neurons represent aberrant ganglia, as previously described for cranial nerves (Satomi & Takahashi, 1986). OMP expression was reported before in subepithelial nasal ganglia (Storan and Key, 2006). In this regard, the frequent presence of paravomeronasal ganglia in adult bats, another taxonomic group with extreme variations in the VNO (Bhatnagar and Kallen, 1974; Bhatnagar and Meisami, 1998), is noteworthy. Silver-stained extraepithelial neuronal bodies also were described outside of the VNO in human infants (Brookover, 1917). The significance of such ganglia is presently unclear. A possible explanation may lie in putatively lost, aberrant LHRH neurons located outside typical migratory paths of vomeronasal or terminal nerves described in mice (Wu et al., 1997). While clusters of paravomeronasal neurons are common in fetal haplorhines that possess a VNO (Smith et al., 2003b), their ubiquity in perinatal callitrichines is unique. At least some callitrichines have a protracted period of postnatal brain growth (Leigh, 2004). If a subset of the paravomeronasal cells is migratory, it is possible that their relatively late occurrence is a reflection of other developmental delays in the central nervous system.

Implications for Evolution of the VNS in Platyrrhine Primates Because of the complex role of olfactory communication in platyrrhines (Dobroruka, 1972; Epple, 1972;

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1974; Milton, 1975; 1985; Boinski, 1987; Smith et al. 1997; Miller et al., 2003; Saltzman, 2003; Difiore et al., 2006; Campos et al., 2007; Jones and Van Cantfort, 2007; Wolovich and Evans, 2007), this group of primates presents a unique opportunity to understand the evolution of the VNS. Olfactory communication via scent marking in platyrrhines is hypothesized to be important for mediation of territoriality, social and/or reproductive dominance, or intrasexual competition and mate attraction (Heymann, 2006), all of which have been hypothesized to be mediated by the VNS in other mammals (see Halpern and Martıńez-Marcos, 2003 for review). Since genetic data suggest the common ancestor of platyrrhines had a functional VNS (Liman and Innan, 2003; Zhang and Webb, 2003), there is reason to believe a sociosexual function played some part in the evolutionary history of the platyrrhine VNS. The present study suggests this ancestral stock would eventually yield a great morphological diversity, leaving much room for interpretation of its function in extant platyrrhines. Our morphological survey of the platyrrhine VNO indicates that some platyrrhines are similar to other mammals (including at least some strepsirrhines) in certain respects. Notably, Ateles and both genera of marmosets have a VNO neuroepithelium with numerous rows of VSNs. In addition, Callithrix and Ateles (but perhaps not Cebuella) have terminally differentiated VSNs within the VNNE, in keeping with observations on several rodents. Thus, Maier’s interpretation (1980) that some platyrrhines exhibit ‘‘signs of reduction’’ (such as the relatively thin VNNE and regions of poorly organized VNNE) does not denote a uniform characteristic of platyrrhines. Maier’s statement is more evocative of Saguinus spp, and perhaps Saimiri spp, those with the thinnest VNNE in terms of nuclear rows (Smith et al., 2004; and see Figs. 2 and 4) and also the smallest population of terminally differentiated VSNs (Dennis et al., 2004; Smith et al., 2011). Although disagreements exist about platyrrhine phylogenetic relationships (e.g., Steiper and Ruvolo, 2003; Ray et al., 2005; Hodgeson et al., 2009; Rosenberger et al., 2009), those platyrrhines with the thickest VNNE in terms of nuclear rows (marmosets and Ateles) or those that appear to have fewer VSNs (Saguinus and Saimiri) are not regarded as especially close, as sister taxa (Fig. 14). Thus, no clear phylogenetic pattern is apparent. Our observations are generally consistent with the assessment of Liman and Innan (2003), who suggested selection pressure on a signal transduction channel related to VNO function is reduced (but present) in all platyrrhines. Further, these authors suggested ‘‘that in some species of the NW [New World] monkeys the VNO may be vestigial or redundant’’ (p. 3331). Further work on platyrrhines may clarify the persisting functional roles of the VNS, as well as the complex manner in which sociosexual behaviors rely on the VNS as opposed to other special senses. If some lineages of platyrrhines are undergoing evolutionary regression of the VNS, by what means do they detect social chemical signals? In mammals, the MOS appears to have a synergistic overlap with the VNS (Powers and Winans, 1975; Beauchamp et al., 1982). Thus it is unsurprising that sociosexual functions that are strongly related to the VNS of some mammals are taken up by the MOS in taxa where the VNS is vestigial or absent (Wysocki and

Preti, 2004). If the MOS is mediating chemoreception in some platyrrhines with reduced (if not vestigial) VNOs, as in Saguinus spp, this may have implications for olfactory communication in other anthropoid primates like Old World monkeys, apes, and humans. The results of the present study isolate several lineages of living platyrrhines that may shed light on VNNE morphological diversity (Fig. 14). In addition, our preliminary findings suggest subtle osteological features, previously identified on primates (Garrett et al., 2009), may be used to draw inferences from fossil platyrrhines on the evolutionary history of the VNS.

ACKNOWLEDGEMENTS This study was made possible, in large part, by efforts of numerous individuals who made well preserved tissues of rare specimens available to us over a ten year period. A partial list of these individuals includes H. Kafka and L. Gordon at the National Museum of Natural History, E. Less of the Cleveland Metroparks Zoo, E. Curran of the New England Primate Center, L. Williams at the Michael E. Keeling Center for Comparative Medicine and Research, J. Chatfield and T. deMaar of the Gladys Porter Zoo, J. Trupkiewicz of the Philadelphia Zoo, and S. Gibson of the University of South Alabama Primate Research Center. In addition, authors thank all other veterinary, research, or technical staff at these institutions who took time to ensure that these specimens were preserved in formalin. Authors are grateful to C. Vinyard and A. Taylor, who have shared cadaveric tissue samples with them over the years. Some of the specimens used in this study were sectioned by K. L. Shimp and L. Maico-Tan.

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