MICROSCOPY RESEARCH AND TECHNIQUE 41:506–518 (1998)

Species, Sex, and Seasonal Differences in VNO Size

ELLEN M. DAWLEY*
Department of Biology, Ursinus College, Collegeville, Pennsylvania 19426

KEY WORDS dimorphism; salamanders; reptiles; mammals; behavior

ABSTRACT Differences in the sizes of sensory and neural structures are used as an indication of
differences in the function of those structures. Large VNOs often are interpreted to mean that this
sense is particularly important in the life history of the animal. They also are assumed to be
associated with more primitive animals. I examined VNO sizes across mammalian, reptilian, and
amphibian lineages while attempting to account for total body size, because VNO and total body
sizes are related. Most descriptions of VNO size and development are not quantified and often
ambiguous. Large VNOs in a lineage should not be interpreted necessarily as primitive. Compari-
sons across smaller taxonomic ranges are easier to interpret. Plethodontid salamanders are a
diverse set of species for which VNO descriptions show trends in size associated with habitat, sex,
and season. Semiaquatic species tend to have proportionately larger VNOs than terrestrial species,
males have larger organs than females, and VNOs can show increases and decreases in size that
may be associated with seasonal activities. Salamanders may use their VNOs to locate and identify
mates, as part of the courtship sequences, or to identify and assess neighboring territory holders.
Microsc. Res. Tech. 41:506–518, 1998. r 1998 Wiley-Liss, Inc.

INTRODUCTION olfactory bulb probably is the basis for increased olfac-

Although most tetrapods possess olfactory epithelia,
the presence of vomeronasal epithelia is associated
with particular classes and with particular ecological
niches. Because a vomeronasal organ (VNO) is absent
in all classes of fish (Eisthen, 1992), it is thought to be a
tetrapod innovation (Bertmar, 1981). Further, the VNO
is reduced or lost in aquatic reptiles (crocodilians and
marine turtles), some marine mammals, in arboreal
lizards, birds, and some primates (Bertmar, 1981). The
inference is that the VNO is a terrestrial adaptation
and that it is lost in tetrapods that have become
arboreal or secondarily aquatic. If reduction or loss of
the VNO obviously results in loss of function, then,
conversely, a larger than expected VNO implies en-
hanced function (Dawley and Crowder, 1995; Halpern,
1992). This hypothesis of enhanced function rests on
the premise that a larger VNO contains more vomerona-
sal receptor cells. More receptor cells improve chemical
sensing acuity either by increasing the range of odor-
ants that can be detected or by enhancing detection of
odorants at low concentrations (Hildebrand and Shep-
herd, 1997).
The details of how information is encoded in verte-
brate chemoreceptive systems are better known for the
olfactory system than for the vomeronasal system.
Each olfactory receptor cell probably expresses only one
of possibly hundreds of genes for specific receptor
proteins that interact with odorant ligands (Buck,
1996). Although each receptor gene is expressed in only
a fraction of receptor cells, receptor proteins can re-
spond to a range of odorants. Thus, receptor cell
population size, coupled with the size of the gene family,
probably is the basis for the range of odors that can be
discriminated (Hildebrand and Shepherd, 1997; Ngai
et al., 1993). A greater number of receptor cells converg-
ing upon the central relay cells (mitral cells) in the
tory sensitivity at lower odor concentrations (Meisami,
1989). Thus, postnatal growth of rabbit olfactory epithe-
lium resulting in a fivefold increase in number of
olfactory neurons between birth and 30 days may
account for the increased responsiveness of newborn
rabbits to odors associated with suckling (Meisami et
al., 1990). Because olfactory and vomeronasal epithelia
are so similar, the size of putative vomeronasal gene
families and of vomeronasal receptor cell populations
can be expected to similarly affect the range of odorants
and concentration of odorants that can be detected.
Comparing VNO sizes, then, is one component in
comparing function of the vomeronasal system. Clearly,
such comparisons, particularly interspecific compari-
sons, need to be coupled with data on the size of the
gene family coding for odorant receptor proteins. In this
paper, I examine species, sexual, and seasonal differ-
ences in size of the VNO. It must be kept in mind that
comparisons of more distantly related taxa often are
difficult to interpret because size differences are con-
founded by the very different selective histories of the
taxa being compared. In addition, VNO size compari-
sons are only meaningful if the effects of body size are
taken into account, because the size of body components
is related to total body size.
BROAD COMPARISONS
The nasal cavity of tetrapods is subdivided into two
or more diverticulae that may be confluent with one
another; one of these diverticulae is the VNO. In
Contract grant sponsor: Whitehall Foundation; Contract grant sponsor: Howard
Hughes Medical Institute; Contract grant sponsor: Ursinus College.
*Correspondence to: Ellen Dawley, Ursinus College, Department of Biology,
Collegeville, PA 19426.
Received 17 March 1998; accepted in revised form 4 May 1998

r 1998 WILEY-LISS, INC.

 VARIATION IN VNO SIZE
anurans and amniotes, the VNO is in a medial position;
in caudates and most apodans, the VNO is usually
lateral (Bertmar, 1981; Parsons, 1967). With the excep-
tion of some reptiles, the main olfactory organ and bulb
are larger than the VNO and accessory olfactory bulb in
a particular organism. Exact size comparisons usually
are not made, and often the comparisons are made
between main olfactory and accessory olfactory bulbs,
rather than between olfactory and vomeronasal epithe-
lia (e.g., Scalia, 1976; Schmidt et al., 1988). Naturally,
some attempt has been made to wade through these
size comparisons and establish some evolutionary
trends, but such efforts are hindered by lack of preci-
sion in size descriptions. Too often, vague descriptions
of the VNO as ‘‘well developed’’ or conversely ‘‘not well
developed’’ are carried through the literature. For ex-
ample, in the late 1800’s Symington (1891) and Broom
(1895, 1896, 1897) carried out comparative anatomical
studies of mammalian VNOs, partly in an attempt to
clarify phylogenetic relationships of Prototherians, Met-
atherians, and Eutherians. Symington (1891) baldly
states ‘‘further research will show that Jacobson’s
organ attains its highest development in the Proto-
theria,’’ (p. 579) with no actual data. Later, Broom
(1895) disagreed that the VNO reaches its highest
development in Prototheria, although he did describe it
as exceedingly well developed, working with the rule
that the organ is better developed in lower forms than
higher. Parker (1922) repeats that the mammalian
VNO is best developed in the lower forms, such as the
Australian duckbill Ornithorhynchus, apparently based
upon Symington’s statement. Wysocki (1979), in his
summary of vomeronasal development in mammalian
orders, repeats the same assertion. Thus, some care
should be taken when attempting to decipher compari-
sons, and perhaps the quantification of amount of
vomeronasal epithelium (scaled to body size) may be a
starting point for meaningful comparisons.
I attempt to compare VNO sizes, starting with mam-
mals, in Table 1. The mammalian VNO is a blindly
ending tube located in both sides of the nasal septal
mucosa with vomeronasal sensory epithelium re-
stricted to its medial surface; thus, VNO sizes most
often are reported as lengths. Clearly, using length
alone as a measure of organ size is an over simplifica-
tion, because the depth of the sensory epithelium may
vary greatly and sensory epithelium may not cover the
entire length of the organ; volume and/or number of
receptor cells is a better measure of total size. Even if
we assume that length is a reasonable estimate of VNO
size, comparisons across mammalian orders are con-
founded by differences in total body size. Thus, I have
plotted VNO length against body length (minus the
tail) in an attempt to examine variation in VNO size
that is not due to variation in total body size (Fig. 1a).
Because none of the original references report the exact
body sizes of the specimens they examined, I have used
average body sizes (calculated from measurements
reported in Walker, 1983). Not surprisingly, VNO length
increases with increasing total body size (Fig. 1). Assum-
ing that average body size represents the actual speci-
men used in the original study and that VNO lengths
were measured in a comparable way, I conclude that
horses (point 5), African buffaloes (point 6), and camels
(point 7) have larger than expected VNOs because their
507
points depart above the line suggested by the scatter of
points of smaller mammals (including sheep, point 3,
and oxen, point 4). But the extreme range in body sizes
among the mammals depicted in Figure 1a may obscure
more subtle variation, particularly for those mammals
with a body size less than 500 mm.
A comparison of VNO lengths of smaller-bodied mam-
mals is depicted in Figure 1b. Ignoring for the moment
the bats clustered at the smallest body sizes, the larger
scatter of points includes a tree shrew, two primates,
two rodents, a rabbit, a cat, two Marsupials, and two
Prototherians. From the scatter of points I would not
conclude that Prototherians (points 10 and 11) have the
‘‘best-developed’’ VNOs, as has been claimed histori-
cally (see above). But I might be tempted to conclude
that tree shrews (point 1), bushbabies (point 2), and
opossums (point 8) have large VNOs compared to other
similar sized mammals. Clearly more data are needed
to draw conclusions about the trends in VNO size
among mammals. Among primates, for example, Ma-
ier’s (1980) data (some of which is incorporated in Fig.
1b) probably do support the hypothesis that a well
developed VNO is a plesiomorphic character, retained
in Strepsirhines and reduced in Haplorhines (rudimen-
tary in Old World monkeys and apes). But the data in
Figure 1 do not necessarily support the claim that
members of the Order Rodentia (points 4 and 5 in Fig.
1b) possess the most developed VNO of Eutherians
(Broom, 1897; Crosby and Humphrey, 1939; McCotter,
1912; Wysocki, 1979). From Figure 1b I might claim
that the domestic cat (point 7) has a VNO that is
proportionately as large as the VNO found in rats and
rabbits.
Part of the problem that arises from drawing conclu-
sions across broad taxonomic categories is that the
lineages (in this case, mammalian orders) have been
separate for so long that many of their features reflect
this long and separate evolutionary history. Thus, it is
easier to look within a particular lineage, particularly
one that encompasses a smaller range of body sizes, and
make comparisons. For example, among Microchirop-
teran bats (Fig. 1b) the vampire bat (point 12) clearly
has a proportionately larger VNO than the free-tailed
bat (point 13) or a vespertilionid bat (VNO is absent;
Bhatnagar, 1980). Thus, among microchiropterans, the
small size of the VNO may be a plesiomorphic character
associated with the plesiomorphic insectivorous diet
seen in free-tailed bats and vespertilionid bats and a
larger VNO may be a derived (apomorphic) character
associated with the diversification of diet seen in Phyl-
lostomid (additionally frugivorous and nectivorous;
other bat points in Fig. 1b) and Desmodontid (vampire)
bats. Many earlier comparisons of VNO development
have rested on the premise that because a VNO is found
in amphibians and reptiles, it is a plesiomorphic charac-
ter for mammals, and those mammals with the most
plesiomorphic characters (the ‘‘lower’’ mammals) should
have the best developed VNOs. Because this trend is
seen in primates (the VNO is rudimentary among
‘‘higher’’ primates like Old World monkeys, apes, and of
course, humans), its premise has been considered con-
firmed (see also Harvey and Krebs, 1990). However, the
variation in vomeronasal size and development among
the different reptilian lineages further challenges this
premise.
508 E.M. DAWLEY

TABLE 1. Mammalian vomeronasal organ sizes and descriptions1

Body VNO
Species length length Other descriptions Reference
Short-tailed Shrew 88–104 Accessory olfactory bulb is minute Crosby & Humphrey, 1939
Blarina brevicauda (1 or 2 glomer)
Eastern mole 130–175 Accessory olfactory bulb is minute Crosby & Humphrey, 1939
Scalopus aquaticus
Tree shrew 140–230 10 Maier, 1980
Tupaia glis
Mouse lemur 125–150 209,000 receptors/organ Shilling, 1970
Microcebus murinus
Bushbaby 105–123 7–8 surface area 5 5mm2 Maier, 1980
Galago demidovii
Squirrel monkey 260–360 5 Maier, 1980
Saimiri sciureus
Domestic cat 460 10.4 volume—1.2mm3 Negus, 1958
Felis catus
Domestic dog 360–1450 33–40 McCotter, 1912
Canis familiaris
Camel 2250–3450 150–180 Arneutovic et al., 1970
Camelus dromedar
Ox 1500–3100 80–90 short and broad Minett, 1925
Bos taurus
Sheep 1200–1800 52.2 McCotter, 1912
Ovis aries
African buffalo 2100–3000 165–168 Estes, 1972
Syncerus caffer
Horse 2000–2400 120–130 ‘‘comparatively well developed’’ Minett, 1925
Equus caballus
Norway rat 150–235 6–7 McCotter, 1912
Rattus norvegicus Vaccarazza et al., 1981
Guinea pig 200–400 7 McCotter, 1912
Cavia porcellus
European rabbit 350–450 9.6 volume 5 0.96mm3 1,650,000 Negus, 1958
Lepus cuniculus receptor cells
Big-eared bat 50–69 1.39 ‘‘well developed’’ Bhatnagar, 1980
Macrotus sp.
Yellow-shouldered bat 51–101 1.25 ‘‘well developed’’ Bhatnagar, 1980
Sturnira sp.
Long-nosed bat 70–95 2.56 ‘‘highly developed’’ Bhatnagar, 1980
Leptonucteris sp.
Short-tailed bat 48–65 1.76 ‘‘highly developed’’ Bhatnagar, 1980
Carollia sp.
Vampire bat 85 3 ‘‘most spectacular’’ Bhatnagar, 1980
Diaemus youngi
Free-tailed bat 46–66 0.27 ‘‘rudimentary’’ Bhatnagar, 1980
Tadarida sp.
Opossum 381–483 9 McCotter, 1912
?Didelphis
Koala 600–800 10–12 Kratzing, 1984
Phascolarctos cinereus
Bandicoot 250 ‘‘well developed’’ Kratzing, 1978
Isoodon macrourus
Echidna 260–440 5 Broom, 1895
?Tachyglossus
Platypus 200–300 5–7 Broom, 1895
Ornithorhynchus anatinus
1Body length and VNO length expressed in mm.

Size, position, and shape of the VNO varies among
the four extant reptilian lineages, which perhaps has
discouraged simple linear measurements as an esti-
mate for size. A VNO and accessory olfactory bulb are
lacking in Crocodilians and its sister lineage, Aves
(Parsons, 1970). Conversely, VNOs are described as
more highly developed in Squamates (snakes and liz-
ards) than in other animals (Parsons, 1970). Both main
olfactory and accessory olfactory systems are described
as ‘‘well developed’’ in most snakes (sea snakes are an
exception because their main olfactory system is greatly
reduced; Gabe and Saint Girons, 1976; Halpern, 1992).
In contrast to mammals, for whom the area of the main
olfactory epithelium always greatly exceeds that of the
vomeronasal epithelium (Negus, 1958), snake VNOs
are relatively ‘‘better developed’’ than their main olfac-
tory organs (as calculated by Gabe and Saint Girons,
1976 and summarized by Halpern, 1992, based on
percentages of sensory cells in VNO compared to per-
centages of sensory cells in main olfactory organ). The
vomeronasal epithelium has been characterized in colu-
brid snakes as extraordinarily thick (300 µm) as com-
pared to the main olfactory epithelium (60–75 µm), the
accessory olfactory nerve as thicker than the main
olfactory nerve, and the accessory olfactory bulb as
longer than the main olfactory bulb (Crosby and Hum-
 VARIATION IN VNO SIZE
Fig. 1. Mammalian vomeronasal lengths plotted against total body
size (without tail). Points correspond to data in Table 1. A: Larger
mammals: Point 1 5 cat; Point 2 5 dog; Point 3 5 sheep; Point 4 5 ox;
Point 5 5 horse; Point 6 5 African buffalo; Point 7 5 camel. B: Smaller
mammals: Point 1 5 tree shrew; Point 2 5 bushbaby (which is in the
suborder Strepsirhini whose members retain a large number of
plesiomorphic characters); Point 3 5 spider monkey (a New World
monkey in the suborder Haplorhini); Point 4 5 rat; Point 5 5 guinea
pig; Point 6 5 rabbit; Point 7 5 cat; Point 8 5 opposum; Point 9 5
koala; Point 10 5 echidna; Point 11 5 platypus; Point 12 5 vampire
bat; Point 13 5 free-tailed bat. Bats are symbolized by squares and all
other mammals by circles.
phrey, 1939; Halpern, 1980; Wang and Halpern, 1980).
The numerous lizard families show an array of develop-
ment of both chemosensory systems (for a review, see
Halpern, 1992). While explanations of the selection
pressures that increased chemosensory abilities in gen-
eral and increased vomeronasal systems in particular
are not offered for the various snake and lizard families,
those families with reduced chemical senses are associ-
ated with arboreal habitats.
Because turtles diverged from stem reptiles before
any of the other surviving reptilian groups, their nasal
anatomy is quite different and a distinct VNO was not
initially identified (Parsons, 1970). Parsons suggested
that a portion of the nasal chamber is homologous to the
509
vomeronasal epithelium, and there is a distinct and
often large accessory olfactory bulb (see also Cotter,
1917; Crosby and Humphrey, 1939; Graziadei and
Tucker, 1970). The extent of the vomeronasal epithe-
lium was later found to vary among families (Scott,
1979), although the descriptions seem to suggest that
the area of olfactory epithelium always exceeds the
area of vomeronasal epithelium. Both areas are re-
duced in sea turtles. However, Matsuzaki et al. (1980)
counted 1,300,000 olfactory receptor cells/side and
1,800,000 vomeronasal receptor cells/side in one spe-
cies, suggesting that in this species the vomeronasal
epithelium slightly exceeds the main olfactory epithe-
lium.
The fourth reptilian lineage, Rhynchocephalia, in-
cludes only 2 species of Sphenodon, whose VNOs are
moderately long and tubular with sensory epithelium
found only on the dorsal half (Parsons, 1970). Both
chemical senses are poorly developed, and there is less
vomeronasal epithelium than main olfactory epithe-
lium (Gabe and Saint Girons, 1976; Halpern, 1992).
The position and shape of the VNO also differs
greatly among the three amphibian orders, although it
probably is safe to say that for all three orders the main
olfactory epithelium is more extensive than the vomero-
nasal epithelium. Even within orders, vomeronasal
morphology and size varies. Apodan VNOs may be the
most variable in position, and, although sizes are not
calculated, they range from ‘‘small’’ to ‘‘very large’’ (see
figure 1b in Schmidt and Wake, 1990), with correspond-
ing size ranges in accessory olfactory nerve projection
fields on the accessory olfactory bulb. Interestingly, the
smallest VNOs are found in species of the most terres-
trial family (Caecilianidae) and the largest VNOs are
found in species of the most aquatic family (Typhlonec-
tidae). These aquatic apodans also have the smallest
main olfactory organs; a large VNO coupled with
reduced olfactory organ parallels the situation in sea
snakes (Schmidt and Wake, 1990).
Anuran VNOs are found in a small medial recess of
the ventral chamber of the nasal cavity; the accessory
olfactory bulb also is described as small (Parsons, 1967;
Jurgens, 1971). The only measure of absolute size of an
anuran VNO that I found was Burton et al.’s (1990)
count in Rana catesbeiana of 295,000 vomeronasal
receptor cells compared to 19 million olfactory receptor
cells (a ratio of 1:64). The position and extent of the
medial recess housing the vomeronasal epithelium
appears to vary among anuran families (Jurgens, 1971);
I suspect size varies as well. Doving et al. (1993)
proposed that the anuran VNO is positioned to sample
odorants in the water, which suggests that the VNO
should be larger in more aquatic species and smaller in
more terrestrial species. This is precisely what Schmidt
and Wake (1990) propose for aquatic vs. terrestrial
apodans.
Salamander vomeronasal epithelium generally lies
within a ventrolateral diverticulum of the main nasal
cavity, although the extent of that epithelium can vary
(Fig. 2). In some species, only the more posterior and
lateral portions of the diverticulum contain vomerona-
sal epithelium; I have found that the lateral diverticu-
lum is nearly completely covered by vomeronasal epithe-
lium in terrestrial salamanders in the family
Plethodontidae (Dawley and Bass, 1988). This is coupled
510 E.M. DAWLEY
with specific structures (nasolabial grooves) that direct
odorants to the VNO during a specific behavior, nose-
tapping (Dawley and Bass, 1989). Thus, the vomerona-
sal system may be more emphasized (perhaps propor-
tionately larger) in plethodontid salamanders than in
other families. This speculation has led to a series of
observations about the size of VNOs in plethodontid
salamanders that I will focus on for the remainder of
this paper. Before doing so I briefly mention that the
effect of a species and aquatic life style on the size of the
VNO is not consistent among species. Some aquatic
genera (Necturus and Proteus) lack a ventrolateral
diverticulum, and the vomeronasal epithelium is de-
scribed as represented by a lateral row of olfactory buds
(thus, there is no clear separation between main olfac-
tory and VNOs; Jurgens, 1971). Other aquatic species
(Cryptobranchus and Amphiuma) have very reduced
lateral diverticulae and VNOs (Jurgens, 1971). Finally,
the aquatic Siren has a uniquely shaped diverticulum
housing the vomeronasal epithelium (Seydel, 1895).
Conclusions about size and terrestriality are difficult to
make based upon these families, but I will exlore this
issue within the family Plethodontidae. These data are
previously unpublished or are extracted from Dawley,
1992; many conclusions are previously unpublished as
well.
SIZE COMPARISONS WITHIN THE
SALAMANDER FAMILY PLETHODONTIDAE
Plethodontid salamanders are a diverse family inhab-
iting a diverse set of habitats (Wake, 1966). Species
inhabiting semiaquatic habitats and retaining ex-
tended aquatic larval stages are considered plesiomor-
phic, while more derived species forego a larval stage
and undergo direct development. Some of these more
derived species have become completely terrestrial, and
may be arboreal or semifossorial. Other derived species
show some degree of neoteny, retaining an aquatic
larval condition throughout life. This diversity within
one family allows comparisons of adaptations that are
not complicated by differences due to very different
evolutionary histories. I compared the volume of vom-
eronasal epithelia. Given that supporting cell and basal
cell nuclei each form a single layer, the thickness of the
epithelium is directly related to number of sensory
neurons (Mackay-Sim et al., 1988), and the volume is
the sum of consecutive epithelial thicknesses (times
section thickness).
General Methods and Results
(see also Dawley, 1992)
I collected adult salamanders of both sexes in the
eastern United States (New York, Pennsylvania, Mary-
land, Virginia, North Carolina) throughout the year,
whenever weather conditions permitted. Terrestrial
salamanders (Plethodon cinereus, P. glutinosus, and
Desmognathus wrighti ) were collected by turning over
logs and rocks within a forest; stream-dwelling species
(D. quadramaculatus, Eurycea wilderae , and Leurogna-
thus marmoratus) were collected at night when they
crawled up on rocks or on shore. Five neotropical
species were donated by David Wake from the collection
at the University of California, Berkeley. These sala-
manders included two terrestrial species (Thorius macdou-
gali and Pseudoeurycea cephalica) and three arboreal
species (Chiropterotriton multidentatus, Dendrotriton bro-
meliacia, and Bolitoglossa rufescens). Captured sala-
manders were killed within 2 days, decapitated, and fixed
in 2.5% glutaraldehyde in 0.1 M phosphate buffer
overnight, decalcified, and processed for plastic embed-
ding. Prior to decapitation, I measured snout-vent
lengths as a measure of total body size (from tip of snout
to posterior edge of cloaca or vent; this length measure-
ment excludes the tail). Embedded heads were cut
transversely with glass knives at a thickness of 0.5, 2.5,
or 5.0 µm, mounted on glass slides, and stained with 1%
toluidine blue. The vomeronasal and main olfactory
epithelia of each collected section were traced with a
drawing tube, digitized, and the VN or main olfactory
epithelial volumes calculated by summing these areas
with a reconstruction software package (PC3D, Jandel
Scientific, Corte Madera, CA). Only the area of the
sensory epithelia was calculated, not including the area
of the lumen or nonsensory areas.
The VN epithelial data were compared using mul-
tiple regression (Data Desk statistical package for
Macintosh or SAS statistical package on a VAX main-
frame). VN epithelial volume was considered the depen-
dent variable that could be determined by several
factors, including body size, sex, or species.
An ANOVA of the regression of VN epithelial volume
on body size, sex, and species yields a significant F
value (F 5 2181.9, df 5 12, P , 0.0001). Thus, VN
epithelial volume is dependent on all other variables
(body size, sex, and species). The regression equation
explains 65% of the variation in VN epithelial volume.
Each independent variable was examined further for
significant effect on VN epithelial volume. Total body
size, sex, and species significantly affect VN epithelial
volume (P , 0.0001 for each; Fig. 3); as body size
increases, so does VN epithelial volume, and males
have significantly larger VN epithelial volumes than
females (see also below). A Scheffé post-hoc test shows
that only certain species comparisons yield significant
differences at the 0.05 level (Table 2). In general, the
small terrestrial species of eastern North America
(P. cinereus and D. wrighti) have smaller VNOs for their
body size than other plethodontids, including four of
the Neotropical species, which are also terrestrial. The
Neotropical species are all part of a separate lineage,
Bolitoglossini, the only plethodontids that have in-
vaded tropical habitats. The semiaquatic species,
E. wilderae, D. quadramaculatus, and L. marmoratus
all have larger VNOs for their body sizes than do the
terrestrial P. cinereus and D. wrighti, perhaps suggest-
ing that increased terrestriality is associated with
smaller VNOs in nontropical plethodontids. Schmidt et
al. (1988) made a similar conclusion based upon com-
plexity of vomeronasal nerve projections on the acces-
sory olfactory bulb. However, P. glutinosus, a large
terrestrial species, clusters with the semiaquatic spe-
cies with a larger VNO. P. glutinosus and the semi-
aquatic species are significantly larger in total body
size than are P. cinereus or D. wrighti; perhaps VNO
volume does not have a linear relationship with total
body size. For all plethodontid species examined except
one, olfactory epithelial volume is significantly greater
than vomeronasal epithelial volume (for example, P.
cinereus, paired t-test, P 5 0.0001; Fig. 4a). However,
the most aquatic species, Leurognathus marmoratus,
 VARIATION IN VNO SIZE
Fig. 2. Line drawings of representative
transverse sections of the left nasal cavities of
(A) Triturus alperstrix, (B) Ambystoma macu-
latum, and (C) Plethodon cinereus (after Daw-
ley and Bass, 1988). moc 5 main olfactory
chamber; vne 5 vomeronasal epithelium.
has greatly reduced main olfactory epithelia (Fig. 4b).
Thus, selection for an aquatic life style has resulted in a
reduction of the olfactory epithelium but not the vomero-
nasal epithelium in aquatic plethodontid salamanders,
aquatic apodans, and sea snakes.
SEXUAL AND SEASONAL DIMORPHISM
OF THE VNO
There is much scatter among the points in Figure 3,
even within a single species, that is due to sexual
dimorphism of the VNO. This can be more clearly seen
in Figure 5, which focuses on three species and delin-
eates between males and females. Male salamanders of
511
all three species have significantly larger VNOs than
conspecific females (ANOVA, P , 0.0001 for P. cinereus,
P 5 0.0131 for E. wilderae, and P 5 0.0001 for
P. glutinosus; see also Dawley and Crowder, 1995).
Because I have a large data set available for P. cinereus,
I was able to show that some of the additional scatter is
the result of seasonal dimorphism of the VNOs (Dawley
and Crowder, 1995). In a multiple regression of the data
when VNO volume is considered the dependent vari-
able that may be determined by total body size, sex, and
season, male and female salamanders collected in one
season had significantly larger VNOs (Fig. 6). Animals
collected in December were designated Winter, those
512 E.M. DAWLEY

Fig. 3. Vomeronasal organ volumes plot-

ted against a measure of total body size
(snout-vent length) for eleven species of sala-
manders in the family Plethodontiae. Dia-
monds represent P. cinereus; squares repre-
sent D. wrighti; filled circles represent a
single specimen each of five Neotropical spe-
cies; open circles represent D. quadramacula-
tus; X represents E. wilderae; triangles repre-
sent P. glutinosus, squares with diagonal
lines represents L. marmoratus.

TABLE 2. Selected species vomeronasal organ comparisons. That this seasonal difference in VNO volume is
Results of a Scheffe post hoc comparison1
caused by an increased number of cells, rather than by
Species comparison Probability Significance some other factor such as increase in volume of cells, is
T. macdougali: P. cinereus 1.00 2 supported by two different data sets. First, vomerona-
P. cephalica: P. cinereus 0.0000 1 sal epithelial cell densities among the four seasons
P. cephalica: T. macdougali 0.0000 1 were compared by counting cells per square millimeter
D. bromeliacia: P. cinereus 0.0000 1 of vomeronasal epithelium (Dawley and Crowder, 1995).
D. bromeliacia: T. macdougali 0.0004 1
D. bromeliacia: P. cephalica 0.0000 1 There were no significant differences among cell counts
C. multidentatus: P. cinereus 0.023 1 due to season. Second, I examined rate of receptor cell
C. multidentatus: T. macdougali 0.546 2 neurogenesis in spring and summer animals and found
C. multidentatus: P. cephalica 0.0000 1 that neurogenesis is up-regulated in spring collected
C. multidentatus: D. bromeliacia 0.598 2
B. rufescens: P. cinereus 0.134 2 animals, immediately prior to a VN epithelial volume
B. rufescens: T. macdougali 0.800 2 increase in June (Dawley et al., unpublished data).
B. rufescens: P. cephalica 0.0000 1
B. rufescens: D. bromeliacia 0.321 2 General Methods and Results
B. rufescens: C. multidentatus 1.0000 2 (Dawley et al., unpublished data)
P. glutinosus: P. cinereus 0.00000 1
E. wilderae: P. cinereus 0.00000 1 To examine neurogenesis of the vomeronasal epithe-
E. wilderae: P. glutinosus 0.6714 2 lium, we injected salamanders collected in May and
D. wrighti: P. cinereus 1.0000 2
D. wrighti: P. glutinosus 0.00000 1 June with 5-bromo-28-deoxyuridine (BrDU). We se-
D. wrighti: E. wilderae 0.0004 1 lected these months because, as shown above, vomero-
L. marmoratus: P. cinereus 0.00000 1 nasal epithelial volumes increase within this time
L. marmoratus: P. glutinosus 0.1320 2 frame. BrDU is a thymidine analog that is incorporated
L. marmoratus: E. wilderae 0.0001 1
L. marmoratus: D. wrighti 0.0000 1 in the DNA of cells during DNA synthesis. Animals
D. quadramaculatus: P. cinereus 0.0000 1 were injected intraperitoneally on day 1 and day 3 after
D. quadramaculatus: P. glutinosus 0.999 2 collection and sacrificed on day 5. Heads were removed
D. quadramaculatus: E. wilderae 0.78 2 and immersion fixed overnight in Zamboni’s fixative,
D. quadramaculatus: D. wrighti 0.0000 1
D. quadramaculatus: L. marmoratus 0.0029 1 rinsed, cryoprotected in 30% sucrose, and frozen sec-
tioned at 14 µm. Every section was collected and
1Minus sign 5 not significantly different; plus sign 5 significantly different. mounted on gelatin coated slides, and we used immuno-
cytochemistry to locate cells that had incorporated the
collected in April and May were Spring, those collected BrDU and thus had arisen from cell division since
in June and August were Summer, and those collected BrDU injection. (Briefly, after endogenous peroxidase
in September and October were Fall. Summer males activity was blocked by incubation in 0.3% hydrogen
and females have significantly larger VNOs than Fall, peroxide, sections were incubated for 3 days with
Winter, or Spring collected salamanders (P , 0.002). anti-BrDU, followed by incubation in biotinylated anti-
 VARIATION IN VNO SIZE
Fig. 4. Olfactory organ volumes compared
to vomeronasal organ volumes plotted against
snout-vent length of (A) P. cinereus and
(B) L. marmoratus. Open circles represent
main olfactory organs; filled circles represent
vomeronasal organs.
mouse IgG; cells that had incorporated BrDU were
visualized by application of nickel intensified diamino-
benzidine.) We compared 10 salamanders in May and
10 in June by counting number of labeled cells per area
(mm2) of sensory epithelium (for this study, main
olfactory and vomeronasal epithelia were combined) for
one section on each slide. Thus, we counted cells on
every fifth or sixth section, which was equivalent to
every 70 –84µm, and consequently avoided counting
the same cell twice. We counted labeled basal or
receptor cells but not supporting cells and compared
513
numbers of labeled cells among animals using a re-
peated measures ANOVA (Zar, 1996).
Salamanders collected in May and June had labeled
nuclei within olfactory and vomeronasal epithelia in
the basal one-third to one-half of the epithelium (Fig. 7).
Adult and juvenile salamanders collected in May had
significantly more labeled nuclei in their chemosensory
epithelium than adults collected in June (P 5 0.004
when May and June adults were compared and P ,
0.044 when May juveniles were compared to June
adults). However, there were no significant differences
514 E.M. DAWLEY
between juveniles collected in May or June (P 5 0.544)
or between June juveniles and May adults (P 5 0.646)
or between May adults and juveniles (P 5 0.824). Thus,
juveniles maintained the same higher level of BrDU
incorporation (and, thus, cell division) in both months,
whereas this higher rate of cell division was found only
in May adults, but not June adults. This increased
neurogenesis of receptors in May at least partially
accounts for the increased epithelial volumes measur-
ing during the summer. June adults do not show this
increased rate of neurogenesis, and Fall, Winter, and
Spring animals have smaller VNOs than Summer
animals. This suggests, in addition, that, subsequent to
birth of these receptors, a population of cells must die to
account for the decrease of VNO volumes during the
rest of the year.
BEHAVIORAL CORRELATES
The larger question is why males have larger VNOs
and why the size of this organ varies in both sexes
Fig. 5. Vomeronasal organ volumes of male
(open symbols) and female (filled symbols)
salamanders plotted agains snout-vent length
for 3 species of plethodontid salamanders.
Filled triangles represent female P. cinereus;
open triangles represent male P. cinereus;
filled circles represent female E. wilderae;
open circles represent male E. wilderae; filled
squares represent female P. glutinosus; open
squares represent male P. glutinosus.
depending on season. This sexual dimorphism in size
may be linked with sexually dimorphic behaviors. One
outstanding example of this linkage is the sexual
dimorphism of brain nuclei that control song in passer-
ine birds. In canaries and zebra finches, these brain
regions are larger in volume in males than in females
(Nottebohm and Arnold, 1976), and singing is a court-
ship or territorial behavior typical of males but not of
females. A greater number of neurons and differences in
dendritic morphology and number of synapses account
for the larger volume of song control nuclei in males
(DeVoogd et al., 1985; Gurney and Konishi, 1980). In
addition, passerine bird species have one or more
periods of song learning, and during these periods, the
anatomy of the brain regions controlling song changes
radically (Nordeen and Nordeen, 1990). Because canar-
ies retain the ability to modify their song into adult life,
seasonal changes in brain nuclei accompany these
seasonal changes in song repertoire (Nottebohm, 1981).
 VARIATION IN VNO SIZE
Fig. 6. Comparison of vomeronasal organ volumes throughout the
year plotted against snout-vent lengths of females (A) and males (B).
Salamanders collected in the spring are represented by open squares;
those collected in the summer are represented by circles enclosing
crosses; those collected during the fall are represented by filled
diamonds; and those collected during the winter by squares enclosing
crosses.
Is the functional significance of the sexual and seasonal
dimorphism of VNOs in salamanders as clear?
Chemoreception in plethodontid salamanders has
been shown to be associated with a number of behaviors
including mate recognition and courtship (e.g., Dawley,
1986; Houck and Reagan, 1990; Jaeger et al., 1986).
Whether certain behaviors are mediated mainly or
515
solely by the main olfactory or vomeronasal systems
has not been demonstrated. However, stimulation of
the VNO in plethodontid salamanders is associated
with a specific observable behavior, nose-tapping. Pleth-
odontid salamanders touch the bases of the naso-labial
grooves to objects (nose-tap) and these grooves function
as capillary tubes to convey liquids preferentially to the
VNO (Dawley and Bass, 1989). Nose-tapping is associ-
ated with territorial maintenance (Jaeger et al., 1986)
and courtship Arnold (1977).
There have been many suggestions that the vomero-
nasal system has a role in mating and reproduction.
The strongest evidence is that from garter snakes.
Kubie et al. (1978) showed that male garter snakes will
not initiate courtship without an intact vomeronasal
system. Snakes are well known for the tongue-flicking
behavior that specifically delivers odorants to the VNO.
Similarly, courtship in plethodontid salamanders is
initiated by the male, who approaches a female (or any
salamander) and rubs and taps her with his nose before
initiating courtship (Arnold, 1977; Dawley, 1984). Many
plethodontids are solitary and must find appropriate
mates during the breeding season. P. cinereus find and
follow female pheromone trails by nose tapping (Ger-
gits and Jaeger, 1990). During the breeding season,
glands surrounding the nasolabial grooves hypertrophy
in males but not in females (Sever, 1975), although a
function for these nasolabial glands has yet to be
shown. These data are consistent with the idea that
males use their vomeronasal systems to locate mates
and that the VNO is involved in releasing courtship
behaviors in males. This may account for the larger
VNOs in males than in females. How and when are
females using their VNOs?
Pheromones are clearly involved in affecting female
receptivity to and during courtship (Houck, 1986). Male
plethodontid salamanders have large chin glands, whose
secretions are delivered to the female by slapping the
gland on her nares (most Plethodon species) or by
rubbing the secretions into scratches on the female’s
skin created by the male’s premaxillary teeth (other
plethodontid genera). The VNO may be involved in the
former, but nasal chemosensory systems are clearly
bypassed in the latter. Both sexes may use their
vomeronasal systems in territorial or home range main-
tenance.
Many plethodontid species, especially those which
are terrestrial, show dispersal patterns that suggest
they are solitary and perhaps maintain territories
(Jaeger, 1979). P. cinereus, in particular, has a reper-
toire of competitive and aggressive behaviors that
suggest territoriality (Jaeger, 1986). Both male and
female P. cinereus nose-tap substrates and may form a
cognitive map of neighboring territorial pheromones
(Jaeger et al., 1993). Fecal pellets often are nose-tapped
and some researchers contend that feces contain infor-
mation about the sex and body size of the depositor and
quality of diet (Mathis, 1990; Walls et al., 1989). Both
sexes may be chemically advertising territorial owner-
ship.
The role of the vomeronasal system in mating activi-
ties or territoriality also may explain seasonal changes
in VNO size in male and female P. cinereus. In the
P. cinereus yearly cycle, mating begins in the fall, is
interrupted by cold weather during the winter (but
516 E.M. DAWLEY
Fig. 7. A photomicrograph of a portion of the olfactory epithelium
of a salamander injected with BrDU. The nasal lumen is at the top of
the photo. s 5 surface of epithelium; r 5 receptor cell layer; b 5 basal
salamanders can become active during warm spells),
and resumes in milder spring weather (Lofts, 1984;
Sayler, 1966). Mating may occur over this entire period
and females can store sperm in spermathecae (special-
ized cloacal receptacles) for months. Eggs are laid in
June, and salamanders complete spermatogenesis dur-
ing the summer. Therefore, I categorized the yearly life
cycle of P. cinereus into breeding and prebreeding
periods. Summer is the prebreeding period, during
which food foraging is intense but can be limited
because salamanders cannot tolerate hot, dry weather.
Summer is also the period of VNO enlargement.
There are at least three possible hypotheses that may
explain vomeronasal receptor cell neurogenesis that
results in enlargement of the VNO: (1) New vomerona-
sal receptors are generated each year in anticipation of
certain predictable yearly behaviors, perhaps mating or
territorial maintenance. Therefore, new receptors may
be specific to odors involved in mate detection and
courtship, or new receptors may be generated to detect
territorial intruders. Because we do not know how long
it takes for new receptors to mature into functional
receptors (they must migrate away from the basal
layer, develop axonal and dendritic processes, and form
synapses in the accessory olfactory bulb), receptors
born in May may mature to coincide with the beginning
of the courtship season in early Fall. Alternatively,
because P. cinereus becomes more territorial in the
hotter, drier summer, new receptors may coincide with
the beginning of a season of increased territory mainte-
cell layer. Most labeled cells are found in the basal cell layer, but a few
(white arrow) have migrated into the receptor cell layer. Calibration
bar 5 50 µm.
nance. (2) A surge of chemosensory neurogenesis may
be favored prior to the environmentally rugged summer
season in anticipation of a season when receptors are
likely to be damaged and die. The original explanation
for why chemoreceptor epithelia retain the ability to
generate new receptor cells was because receptor cells
in the nasal cavity are vulnerable to damage (Farbman,
1990). Thus, the surge in neurogenesis would establish
a large supply of immature receptors in reserve. (3)
Increased chemosensory neurogenesis may be a by-
product of response to some other physiological event,
and may not, in itself, be adaptive.
OTHER EXAMPLES OF VOMERONASAL
SYSTEM DIMORPHISM
The only other vertebrate for which sexual dimor-
phism of the vomeronasal system has been documented
is rats (reviewed in Segovia and Guillamon, 1993).
Initially, Segovia and Guillamon (1982) described sexual
dimorphism of the VNO; male VNOs are larger in total
volume because they contain more receptor cells. Early
postnatal androgens affect this size dimorphism. Subse-
quently, sexual dimorphism was described in the acces-
sory olfactory bulb; males have larger accessory olfac-
tory bulbs because several layers of the bulb (mitral,
plexiform, and granular layers) are larger in volume
(Segovia et al., 1984). Perinatal androgens also influ-
ence this dimorphism. Secondary and tertiary projec-
tions along the vomeronasal pathway also are sexually
dimorphic such that male nuclei have greater volumes
 VARIATION IN VNO SIZE
and numbers of neurons than females (summarized in
Segovia and Guillamon, 1993). These nuclei also con-
tain sexually dimorphic quantities of certain neurotrans-
mitter producing cells and contain high densities of
cells that express estrogen and androgen receptors
(Simerly, 1990). Segovia and Guillamon suggest that
larger numbers of cells in male vomeronasal system
structures inhibit expression of female behaviors, in-
cluding lordotic behavior (sexual presentation behav-
ior) and maternal behaviors, which are regulated by
structures along the vomeronasal system pathway.
However, none of these studies considered the effect of
body size on vomeronasal organ structures; males may
have larger vomeronasal structures simply because of
the positive allometric relationship between total body
size and sizes of structures within that body. More
directly related to the VNO itself, Mennella and Moltz
(1988) suggested that larger male VNOs are related to
infanticidal tendencies. The VNO may allow adults to
detect the odor of newborn young, which nulliparious
females and males find aversive; the large VNO of
males reflects their greater sensitivity to aversive pup
odors, which leads to infanticide. Other rodent studies
show that vomeronasal receptors are involved in the
release of male sexual behaviors, suggesting that fe-
male odors trigger these behaviors (Wysocki, 1979). For
example, ultrasonic vocalizations in male mice (male
mice vocalize in the presence of a female or her odors)
are reduced or eliminated after VNOs are removed
(Wysocki et al., 1982). Male hamsters may fail to mate
altogether following vomeronasal nerve transection
(Powers and Winans, 1975).
CONCLUSION
Plethodontid salamanders may be an ideal group to
investigate variation in vomeronasal structure and
function. Their simple nasal structure (they lack con-
chae) and small size mean that it is not difficult to look
at the entire nasal epithelia and associated central
nervous system structures. They retain a plesiomor-
phic solitary social structure, which means they have
limited behavioral interactions so that there is a smaller
set of chemosensory mediated behaviors to consider.
They probably have a relatively small gene family
coding for odorant receptor proteins, and thus a limited
set of odorants that they can detect. Additionally, there
is a range of life styles within the family, so that various
ecological interactions with chemoreception can be
investigated. These animals, then, possess a tractable
set of variables that may affect chemosensory structure
and function.
ACKNOWLEDGMENTS
I thank the many students who spent many lab hours
sectioning and digitizing sections, especially Jim Crow-
der, Bea May, and Chuck Webb. This research was
funded by grants from the Whitehall Foundation, the
Howard Hughes Medical Institute, and Ursinus Col-
lege.
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