Histology and ultrastructure of the caudal courtship glands of the red-backed salamander, Plethodon cinereus (Amphibia: Plethodontidae)

Histology and Ultrastructure of the Caudal CourtshipGlands of the Red-Backed Salamander, Plethodoncinereus (Amphibia: Plethodontidae)

David M. Sever1* and Dustin S. Siegel2

1Department of Biological Sciences, Southeastern Louisiana University, Hammond, Louisiana 704022Department of Biology, Southeast Missouri State University, Cape Girardeau, Missouri 63701

ABSTRACT Caudal courtship glands (CCGs) are sex-ually dimorphic glands described in the skin of the dor-sal tail base of some male salamanders in the generaDesmognathus, Eurycea, and Plethodon in the familyPlethodontidae. These glands are believed to deliverpheromones to females during courtship, when thefemale rests her chin on the dorsal tail base during thestereotypic tail straddling walk unique to plethodon-tids. Although CCGs have been studied histologically,no investigations of their ultrastructure have beenmade. This article presents the first study on the finestructure and seasonal variation of CCGs, using theplethodontid Plethodon cinereus. The CCGs vary sea-sonally in height and secretory activity. The maturesecretory granules observed in males collected in Octo-ber and April consist of oval, biphasic granules that areeosinophilic and give positive reactions to periodic acid-Schiff for neutral carbohydrates but do not stain foracidic mucosusbtances or proteins with alcian blue andbromphenol blue, respectively. Granular glands, someof which contain mucous demilunes, are twice as largeas CCGs, are syncytial (unlike CCGs), and stain forproteins. Mucous glands are similar in size to CCGs,but are basophilic, show no seasonal variation in secre-tory activity, and stain positive for acidic mucosubstan-ces. CCGs do not resemble cytologically the sexuallydimorphic mental glands of some plethodontids, whichcontain round or oval granules filled with an electron-dense amorphous substance. The CCGs are similar his-tologically to sexually dimorphic skin glands describedin some anurans, but more comparative work isneeded. J. Morphol. 276:319–330, 2015. VC 2014 Wiley

Periodicals, Inc.

KEY WORDS: microanatomy; Urodela; sexual dimor-phism; skin glands; microanatomy

INTRODUCTION

Sexually dimorphic skin glands (SDSGs; Thomaset al., 1993) occur in many salamanders (Sever,2003) and anurans (Brunetti et al., 2012) and arefound singly or in clusters with morphologies thatvary among taxa. Noble (1929) reported that malesof Eurycea bislineata possess a cluster of skinglands in the dorsal base of the tail that serve as a“source of attraction” for females. Since then,SDSGs have been reported in the skin of the dorsal

tail base in other Eurycea (Sweet, 1977; Sever,1985, 1989; Trauth et al., 1993; Hamlett et al.,1998), Plethodon (Newman, 1954; Mary andTrauth, 2006), and Desmognathus (Noble, 1929;Mary and Trauth, 2006). These glands are desig-nated caudal courtship glands (CCGs) because oftheir proposed role in the courtship behavior calledthe tail straddle walk (TSW), stereotypic for ple-thodontids, which precedes spermatophore deposi-tion (Noble. 1929). During TSW, the snout of thefemale is placed directly on the CCG area, and thenasolabial grooves of the female could carry phero-mones from the CCG into the nasal cavity (Sever,1989).

Hecker et al. (2003) studied the dermal glandsin the tail base of Plethodon cinereus, a commonterrestrial salamander in the eastern UnitedStates. They recognized mucous glands and twotypes of serous or granular glands, termed S1 andS2. S1, which were largest ventrally, secreted aprotein 1 carbohydrate and was proposed to beinvolved in scent marking, whereas S2 were morenumerous and had a protein secretion, which wasproposed to function in defense and nutrient stor-age. Although both males and females were exam-ined, no mention of SDSGs in the skin of the tailbase was given. Brizzi et al. (1991) also failed tofind SDSGs in the skin of the tail base in theirexamination of four species of Hydromantes.

CCGs have been studied histologically withlight microscopy (LM), but no ultrastructuralstudies using electron microscopy exist. Indeed

Contract grant sponsor: Major Dyson Endowed Professorship atSoutheastern Louisiana University (D.M.S.)

*Correspondence to: David M. Sever, Department of BiologicalSciences, Southeastern Louisiana University, Hammond, LA 70402.E-mail: [email protected]

Received 29 August 2014; Revised 21 October 2014;Accepted 2 November 2014.

Published online 13 November 2014 inWiley Online Library (wileyonlinelibrary.com).DOI 10.1002/jmor.20342

VC 2014 WILEY PERIODICALS, INC.

JOURNAL OF MORPHOLOGY 276:319–330 (2015)

the only sexually dimorphic salamander glandsthat have been studied cytologically are thegenial (cheek) glands of Notophthalmus virides-cens (Salamandridae; Pool and Dent, 1977), themental (chin) glands of the plethodontids Euryceaquadridigitata (Sever, 1975) and Hydromantesgenei (Borgioli, 1977), and the cloacal glands of P.cinereus (Sever, 2014).

In this article, the histology and ultrastructureof the skin glands of the dorsal tail base aredescribed in P. cinereus using seasonally collectedsamples from northern Indiana, United States.This same sample was used in the recent study oncloacal glands (Sever, 2014) and will be used in afuture investigation on mental glands. The goal ofthis study was to see if CCGs exist in P. cinereus,and if so, to compare them using light and electronmicroscopy to other skin glands found in this spe-cies and the SDSGs of other amphibians.

MATERIALS AND METHODS

Specimens of P. cinereus (Green, 1818) were collected in2002 at the Swamp Rose Area, Potato Creek State Park, SaintJoseph County, Indiana, under a scientific collecting permitissued by the Indiana Division of Fish and Wildlife. A total of22 specimens, 37.5–50.4 mm snout-vent length (SVL), wereused from samples collected on16 April (5 males, two females),16 June (4 males, 1 female), 14 August (3 males, 1 female), and16 October (5 males, 1 female). Courtship and mating have notbeen observed in this population, but sperm have been reportedin the vasa deferentia of males collected in October and Marchand are absent in May and August (Sever, 1978).

Specimens were euthanized in 10% MS-222 in 50% ethanol.This procedure was approved by the Institutional Animal Careand Use Committee of Saint Mary’s College, Notre Dame, IN.After death, the tail posterior to the cloaca was excised fromeach specimen. For one male from each sample and a femalefrom April, the tails were fixed in neutral buffered 10% forma-lin (NBF) for light microscopy (Table 1). Tails of the remainingspecimens were fixed in Trump’s fixative, 2.5% glutaraldehydeand 2.5% formaldehyde in 0.1 mol l21 sodium cacodylate bufferat pH 7.4 (Electron Microscopy Sciences, Hatfield, PA), fortransmission electron microscopy (TEM).

For LM, after fixation for 24 h in NBF, tissues were rinsedin water, dehydrated in a graded series of ethanol (70, 95, and100% two changes), cleared in toluene, and embedded in paraf-fin. Sections 10 mm thick were cut with a rotary microtome,and placed on albuminized slides. Alternate slides were stainedor treated with hematoxylin-eosin (general cytology), periodicacid and Schiff (PAS) reagent (neutral carbohydrates) counter-stained with alcian blue at pH 2.5 (primarily carboxylated gly-cosaminoglycans), and bromphenol blue (also called Coomassiebrilliant blue R, for proteins). A Leica DM2000 compoundmicroscope was used for viewing slides, and photographs weretaken with a Leica DF420 attached digital camera (LeicaMicrosystems, Wetzlar, Germany). The heights of 15 glands ofeach type (serous, mucous, and CCG, if present) were measuredusing the manual measuring module in the Leica camera soft-ware (Table 1). Maximum height was determined after follow-ing individual glands in serial sections to find the sectionshowing maximal height of each gland measured from the epi-dermis to the basal end of the gland.

Tissues for TEM were rinsed in deionized water, postfixed in2% osmium tetroxide, dehydrated through a graded series ofethanol (same as LM), cleared in propylene oxide, and embed-ded in epoxy resin (Embed 812, Electron Microscopy Sciences,Hatfield, PA). Plastic sections were cut using a Reichert

ultramicrotome (Reichert Microscope Services, Depew, NY) andDiATOME (Biel, Switzerland) diamond knives at 1 mm (semi-thin) and 70 nm (ultrathin). Toluidine blue was used to stain 1mm semithin sections that were heat affixed to microscopeslides, which were examined in the same manner as paraffin-prepared sections, to pinpoint areas of interest for TEM. Sec-tions 70 nm in thickness were placed on uncoated 200 meshcopper grids (Electron Microscopy Sciences, Hatfield, PA) andstained with uranyl acetate and lead citrate. Grids were viewedusing a JEOL 100 transmission electron microscope (JEOL,Tokyo, Japan) and photographed using a L3C CCD digital cam-era (Scientific Instruments and Applications, Duluth, GA).Gland heights were not measured in semithin and ultrathinsections as sample size was too small.

Plates were prepared using Adobe Photoshop CS6 (www.adobe.com/Photoshop). Statistics were calculated using Excel2010 (www.microsoft.com).

RESULTS

Four types of multicellular, simple alveolarglands occur in the skin of the dorsal tail base ofmale P. cinereus, with narrow ducts that passthrough the epidermis so the secretory productsexit onto the surface of the skin: 1) mucous glands,2) granular glands, 3) mixed glands, and 4) CCGs,which are lacking in females.

The granular glands are the largest and mostnumerous glands. Although some granular glandsare mixed, with mucous demilunes near the excur-rent duct, most granular glands seem to lack themucoid area. Mixed glands are lumped with othergranular glands when counting numbers and com-paring gland heights. Total counts of the threebasic types of glands in four sections of dorsal skinseparated by 50–70 mm in a 42.8 mm malecollected 16 October are 68 granular glands, 12mucous glands, and 18 CCGs.

The three types of glands show differences inreactions to various stains. Granular glands andCCGs are both eosinophilic whereas mucous glandsare basophilic in hematoxylin-eosin (Fig. 1A,D).With PAS for neutral carbohydrates, granularglands give a weak positive reaction, CCGs have astrong positive reaction, and any reaction of mucousglands is masked by their strong positive reactionto alcian blue at pH 2.5 (for acidic mucosubstances),

TABLE 1. Mean Gland Heights From the Dorsal Tail Base of P.cinereus Serially Sectioned in Which 15 Glands of Each Type

Were Measured

Date SexSVL(mm)

GRG MUG CCG

Mean SD Mean SD Mean SD

16 April F 47.7 232.4 32.9 84.5 12.3M 41.7 230.0 29.2 94.1 11.2 99.1 9.6

16 June M 43.9 212.7 21.8 95.5 13.3 79.6 11.314 August M 42.1 193.8 12.0 80.7 8.1 64.6 9.916 October M 42.8 266.4 41.8 89.7 13.5 134.3 24.4

MUG, mucous gland, GRG, granular gland, CCG, caudal court-ship gland.Gland measurements in microns.

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which is lacking in the other glands (Fig. 1B,E,F).With bromphenol blue for proteins, granular glandsgive a positive reaction, which is lacking in theother glands (Fig. 1C). Although secretory activity,as noted below, varies seasonally, the reactions are

consistent throughout the year (Fig. 1D–F). Themucous demilunes found in mixed glands stain likeseparate mucous glands.

Measurement data reveal that granular glands aredefinitively larger than mucous glands and CCGs no

Fig. 1. Plethodon cinereus, transverse paraffin sections through the dorsal tail base of a male. A,B,C. Male 41.7 mm SVL collected16 April. A. Hematoxylin-eosin stain. B. PAS and alcian blue at pH 2.5. C. Bromphenol blue. D. Male 43.9 mm SVL collected 16June stained with hematoxylin-eosin. E. Male 42.1 mm SVL collected 14 August stained with PAS and alcian blue at pH 2.5. F.Male 42.8 mm SVL collected16 October stained with PAS and alcian blue at pH 2.5. Ccg, caudal courtship gland; Gg, granular gland;Mg, mucous gland; Skm, skeletal muscle.

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matter which individual was measured (Fig. 2 top).However, on average, in June and August mucousglands are larger than CCGs in males, whereas inApril and October CCGs are larger (Fig. 2 bottom). Acommon trend in all gland sizes throughout the yearwas a decrease in gland size from April to Augustwith an increase in gland size in October (Fig. 2bottom). Because the glands of only one male weremeasured from each month sampled, statistical com-parisons among males were not attempted.

For April, the female whose glands were meas-ured is larger (47.7 mm SVL) in body size thanthe male (41.7 mm SVL), and a Student’s unpairedt-Test was used to compare heights of the sametypes of glands, with a critical value of t 5 2.048 atP 5 0.05 (two-tailed). The granular glands of themale and female did not differ significantly inheight (t 5 0.216), but the mucous glands of themale are significantly larger than those of thefemale (t 5 2.232). Measurement data should beinterpreted with caution, as individual variationmay play a role in gland size comparisons.

In the following sections, ultrastructure ofmucous glands and granular glands, neither of

which vary seasonally in secretory activity, aredescribed (Figs. 3 and 4). Ultrastructure of CCGs,which do show seasonal variation not only in glandheight but also secretory activity, are subsequentlydescribed for each month sampled (Figs. 4–8).

Mucous Glands

Cells of mucous glands, unlike those of granularglands, are usually quite distinct (Fig. 3A,B)although occasional interruptions occur apically(Fig. 3C), perhaps associated with release of secre-tory product. Plasma membranes between cells,however, are intact and secretory products of adja-cent cells abut closely to one another (Fig. 3C,D).No junctional complexes occur, or at least nonethat can be observed with abundant mucoid mate-rial pressing against the lateral membranes ofeach adjacent cell. Nuclei are basal, flattened andsurrounded by a dense cytoplasm in which feworganelles are easily discerned except for Golgibodies (Fig. 3D). The bulk of the cytoplasm is filledwith secretory products, which consists of a finelygranular flocculent material and large, irregular,electron-dense granules. Completely depletedmucous glands are not found, although width ofthe lumen varies, perhaps indicating release ofproduct (compare Figs. 3A, 4A, and 7A).

Granular and Mixed Glands

Granular glands are syncytial and filled withgranules of various sizes (Figs. 3A, 4A, 5A, and6A). Nuclei in the syncytial areas are scattered,with some basal and others deeper in the cyto-plasm. In mixed glands, mucous demilunes arealways located at the junction of the gland withthe epidermis and occupy a corner of the gland(Fig. 4A). Mucous demilunes have numerousbasal nuclei, and the cells, like those of singlemucous glands, have distinct borders. Theelectron-dense bodies of the mucoid demilunes,however are round and not as irregular as in nor-mal mucous glands (Fig. 4B). In the syncytium,the cytoplasm surrounding the granules is, initself, finely granular (Fig. 4C). Membranous pro-files (Fig. 4C), which on closer inspection arerevealed as rough endoplasmic reticulum (Rer,Fig. 4D), can be found scattered in the syncytium.Small mitochondria are also scattered throughoutthe cytoplasm (Fig. 4D).

April Caudal Courtship Glands

The CCGs in April specimens are filled withsecretory granules and have narrow lumina (Fig.5A). Nuclei are basal, irregularly oblong, and het-erochromatic. The secretory granules of CCGs areeasily distinguished cytologically from those ofadjacent granular glands (Fig. 5B). The secretorygranules of CCGs are oblong and biphasic, con-sisting of an electron-dense core surrounded by

Fig. 2. Top graph illustrates combined data on the mean heightsize (mm) of mucous glands (grey bar), granular glands (whitebar), and CCGs (black bar) from the four males sacrificed overthe sampling period. Bottom graph shows mean height size ofglands in each individual. Error bars represent one standarddeviation. The left y-axis provides scale for mucous and CCGs.The right y-axis provides scale for granular glands. CCGs, caudalcourtship glands; GGGs, granular glands; MUGs, mucous glands.

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lighter peripheral substance (Fig. 5C,D). The cen-tral core is not crystalline. The secretory granulesare so crowded that plasma membranes betweencells are not discernible. Lipid droplets occuradjacent to some nuclei (Fig. 5C). Where a lumenis visible, microvilli occur on the apical cytoplasm(Fig. 5D). Secretory granules abut on the luminalborder, indicating a merocrine process of release(Fig. 5D).

June Caudal Courtship Glands

In June, some variability occurs in the CCGs(Fig. 6). Some CCGs resemble those from theApril specimens (Fig. 6A,B). The lumina arewider in June than April (Fig. 6A), but the cyto-plasm is still filled with biphasic granules that

obscure any other cellular organelles (Fig. 6B).Intercellular canaliculi, obscured in April, arevisible between adjacent cells in June. Also, thenuclei of myoepithelial cells are more obvious(Fig. 6B). In swollen glands of any of the threetypes, the myoepithelium is more difficult to dis-cern because of its stretched nature. Other glandsin the same specimen appear to be largelydepleted of secretory product (Fig. 6C). In thecytoplasm of cells of depleted glands, sparselygranulated cisternae of Rer are numerous(Fig. 6D).

August Caudal Courtship Glands

In August, the CCGs seem to be replenishingsecretory product, but the product does not have the

Fig. 3. Plethodon cinereus, mucous glands. A. Semithin section through a mucous gland and adjacent granular glands in a female44.0 mm SVL collected 16 April and stained with toluidine blue. B–D. TEM micrographs of a mucous gland of a 37.5 mm SVL malecollected 14 August. B. Overview of epithelial cells. C. Apical border. D. Basal border. FM, flocculent material; Gg, granular gland;Go, Golgi bodies; Gr, granule; Int, interruption in plasma membrane; Lu, lumen; Mg, mucous gland; Nu, epithelial cell nucleus; Pm,plasma membrane; Tp, tunica propria.

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appearance of the mature granules in April andJune (Fig. 7). Some epithelial cells in several CCGsseem disrupted from others (Fig. 7A), with wideintercellular spaces. The significance of this couldnot be determined, as otherwise, the disrupted cellsseemed normal in cytology. Intercellular canaliculiare apparent between more closely associated cells,but no junctional complexes occur basally (Fig. 7B),apically or elsewhere along adjacent plasma mem-branes (Fig. 7C). As they reach the lumen, the mem-branes seem to coalesce with surrounding cytoplasm

(Fig. 7C). Secretory granules are general spherical,vary in size, and have different degrees of electron-density, but none are biphasic (Fig. 7B–D). Thesupranuclear cytoplasm is very dense and packedwith thin strands of Rer that are not easily dis-cerned from the background cytosol (Fig. 7D).

October Caudal Courtship Glands

Similar to specimens from June, the CCGs varyin cytology in the October sample. Some have

Fig. 4. Plethodon cinereus, granular and mixed glands. A. Semithin section through a mixed gland and adjacent muocus gland in a37.5 mm SVL male collected 14 August and stained with toluidine blue. B. TEM of the mucoid portion of a mixed gland of a48.1 mm SVL male collected 16 October. C,D. TEM micrographs of a granular gland of a 43.1 mm SVL male collected 16 April. C.Basal cytoplasm. D. Cytoplasm adjacent to a nucleus. Ep, epidermis; Fm, flocculent material; Fn, fibroblast nucleus; Gg, granulargland; Lu, lumen; Md, mucous demilune; Mg, mucous gland; Mi, mitochondria; Ms, membranous structures; Nu, epithelial cellnucleus; Rer, rough endoplasmic reticulum; Sg, secretory granules.

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columnar epithelial cells that are filled withmature biphasic secretory granules such as thosefound in March specimens (Fig. 8A,B). The majordifference is that the lumina are larger in Octoberthan in April. The appearance of granules abut-ting on the luminal lining again indicates a mero-crine release process. Like the April CCGs, themature secretory granules are so numerous thatplasma membranes are not apparent betweenadjacent cells. Other CCGs have the appearance ofthose from the August specimens, with sphericalgranules of different electron densities but mono-phasic (Fig. 8D). These secretory granules, how-ever, seem more densely packed than in August.

Intercellular canaliculi are narrow but clearbetween adjacent epithelial cells, and, once again,junctional complexes are absent.

DISCUSSION

The CCGs of P. cinereus are clearly SDSGsbecause they do not occur in females. The height-ened secretory activity in October and April, whenthe vasa deferentia are hypertrophied with sperm,is supportive evidence for a function of CCGs incourtship activity. Dyal (2006) observed courtshipin captive P. cinereus from Virginia from October tomid-December. She reported that during courtship,

Fig. 5. CCGs of a 43.1 mm SVL male (A,B) and a 45.4 mm SVL male (C,D) P. cinereus collected 16 April. A. Semithin section of acaudal courtship and adjacent granular gland stained with toluidine blue. B. TEM micrograph of basal cytoplasm of a CCG and adja-cent granular gland. C. TEM micrograph of supranuclear cytoplasm of a CCG. D. TEM micrograph of the luminal cytoplasm of aCCG. Ccg, caudal courtship gland; Gg, granular gland; Ld, lipid droplet; Lu, lumen; Md, mucous demilunes; Mv, microvilli; Nu, epi-thelial cell nucleus; Sg, secretory granules; Tp, tunica propia.

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males walk under the female’s chin until her chin isat the base of his tail. The male may straighten hislimbs to lift the female’s chin, and if she is receptive,she will enter TSW from this position.

The CCGs of males can be easily distinguishedfrom granular, mixed and mucous glands by stain-ing characteristics, cytology, and by undergoingseasonal variation in secretory activity. The heightand secretory activity of the CCGs is greatest inthe breeding season (April) and prebreedingseason (October), and replenishment of secretoryproduct in depleted glands occurs in summer, asrepresented in June and August specimens.

The dermal glands of all three orders of livingamphibians (Gymnophiona, Urodela, and Anura)

include mucous glands, granular glands, and mixedglands (Fox, 1983; Clarke, 1997; Delfino et al.,2014). The literature on amphibian skin glands isimmense, and two types of skin glands in amphib-ians were recognized as early as Ascherson (1840).Esterly (1904) conducted the first study on skinglands in the dorsal tail base of a plethodontid,using Ensatina eschscholtzii (5 Plethodon oregonen-sis in his paper) from California. He did not describeSDSGs in the skin of the tail base, which was domi-nated by large granular glands. Esterly (1904)described mucous demilunes in granular glands andproposed that these functioned in replenishment ofdischarged secretion from the granular glands.Later authors, however, found that all dermal

Fig. 6. CCGs of a 37.5 mm SVL male (A) and 45.5 mm SVL male Plethdon cinereus (B–D) collected 16 June. A. Semithin sectionof an active gland stained with toluidine blue. B. TEM micrograph of cytoplasm of an active gland. C. Semithin section of a nearlydepleted CCG stained with toluidine blue. D. TEM microgrpah of the cytoplasm of a nearly depleted gland. Ccg, caudal courtshipgland; Gg, granular gland; Ic, intercellular canaliculi; Lu, lumen; My, myoepithelial cell nucleus; Nu, epithelial cell nucleus; Rer,rough endoplasmic reticulum; Sg, secretory granules.

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glands arise from nests of sphere-shaped intraepi-dermal cells that subsequently organize along amucous, serous or mixed line of development (Del-fino et al., 1982). Perhaps the best histologicaldescription of the mucous, granular and mixedglands of a salamander is the study on Necturusmaculosus by Dawson (1920), who also provided athorough review of the earlier literature.

Granular glands have received the most atten-tion because they form “poison glands” in somespecies. Toxicity of amphibian granular glands,however, varies greatly, from atoxic to mildly toxicto deadly (Noble and Noble, 1944). Literature onthe toxic granular glands and their pharmacologi-

cal effects is extensive (Esparmer, 1994; Clarke,1997). Neuwirth et al. (1979) gave a thoroughdescription of the ultrastructure of granularglands in several species of poison dart frogs. Ari-fulova et al. (2007) and Delfino et al. (2014) pro-vide excellent reviews on cytology of granularglands, primarily in anurans.

Our observations on the histology and ultrastruc-ture of the granular, mucous, and mixed glands ofP. cinereus are consistent with the literature,including descriptions of granular glands from skinposterior to the cloacal orifice described by Simonset al. (1999). We did not, however, find a distinctionof the granular glands into an S1

Fig. 7. CCGs of a 37.5 mm SVL male P. cinereus collected 14 August. A. Semithin section of a CCG and adjacent mucous glandsstained with toluidine blue. B. TEM micrograph of basal cytoplasm of a CCG and an adjacent mucous gland. C. TEM micrograph ofapical cytoplasm. D. Supranuclear cytoplasm. Ccg, caudal courtship gland; Ic, intercellular canaliculi; Lu, lumen; Mg, mucous gland;Nu, epithelial cell nucleus; Rer, rough endoplasmic reticulum; Sg, secretory granules.

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protein 1 carbohydrate secreting and S2 proteinonly secreting glands as reported in P. cinereus byHecker et al. (2003). Granular glands are variable,and perhaps the differences in the secretory prod-ucts in S1 and S2 glands escaped my attentionbecause my cytological examinations were limitedto the dorsal skin, and S1 glands are most numer-ous ventrally (Hecker et al., 2003). S2 glands,smaller and more numerous than S1 glands, aremost numerous dorsally. S1 glands have a strong

proteinaceous component and do not stain positivewith PAS like CCGs.

Noble (1929), as noted in the introduction, wasthe first to describe CCGs in plethodontids andpropose a function for them in the TSW. In E.bislineata, he found these glands, which he called“hedonic” glands, differed from mucous glands andgranular glands in staining and appearance of thesecretory product, as herein reported for P. ciner-eus. The term “hedonic” is no longer used because

Fig. 8. CCGs of a 48.1 mm SVL male P. cinereus collected 16 October. A. Semithin section stained with toluidine blue. B. TEMmicrograph showing overview of a portion of a gland. C. Luminal border. D. Another gland in which the secretory vacuoles do nothave the mature appearance. Ccg, caudal courtship gland; Du, duct through the epidermis; Ic, intercellular canaliculi; Lu, lumen;Nu, epithelial cell nucleus; Sg, secretory granules; Tp, tunica propria.

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observations cannot reveal whether the secretionsare pleasure-giving as the term implies (Arnold,1977). Noble (1927) did not illustrate his newlydescribed glands.

Noble (1929) reported that similar hedonicglands occur in scattered areas on the body andeyelids of E. bislineata. Also, he found hedonic(courtship) glands in males of E. multiplicata, Des-mognathus fuscus, D. carolinensis (5 D. ochro-phaeus), D. marmoratus, P. cinereus, P. jordani,and Hydromantes italicus, but not in females ofany species. With the exception of E. multiplicata,however, he did not specify that the SDSGs inthese species included a cluster in the dorsal tailbase.

The next mention of CCGs was by Newman(1954), who, in his description of the taxon Pletho-don jacksoni (5 P. wehrlei) mentioned that “largemales frequently exhibit a hedonic swelling abovethe base of the tail.” Thurow (1968) stated thatskin hedonic glands occur over much of the bodyof plethodontids in the genus Plethodon, includingfeet and ventral surface of the trunk, but suchglands have not been demonstrated histologically.Sweet (1977) reported a “dorsal glandular com-plex” in the tail base of a metamorphosed Euryceaneotenes. Sever (1985) found small groups of CCGsin the dorsal tail base of paedomorphic E. neotenes(4–6 pairs of glands) and E. nana (3–4 pairs) aswell as in the area of the lateral septum of thesespecies. These glands were the only multicellulardermal glands in these paedomorphs that other-wise had larval skin.

Sever (1989) described and illustrated CCGs infour species of the E. bislineata complex: E. bisli-neata, E. cirrigera, E. junaluska, and E. wilderae.The CCGs consisted of rather extensive, oftenclosely packed alveolar glands that showedmarked reduction in size outside of the breedingseason. Granular glands do not seem to occur inthe area where CCGs are found in these species,although mucous glands sometimes occur. Histo-chemistry of mucous, granular and CCGs was sim-ilar to that reported here for P. cinereus.

Trauth et al. (1993) studied the CCGs in maleEurycea longicauda, typically a larger animalthan members of the E. bislineata complex. Theyfound the hypertrophied CCGs were much greaterin length and width than either mucous or granu-lar glands. The CCGs were also more flask-shapedthan those of the E. bislineata complex. Granularglands were four times more numerous thanCCGs, which, in turn were nearly twice as numer-ous as mucous glands. Studies on Eurycea contin-ued with a report on the CCGs of anotherrelatively large species, E. lucifuga by Hamlettet al. (1998). Like E. longicauda, the CCGs of E.lucifuga are flask-shaped. Histochemistry of theskin glands of both E. longicauda and E. lucifugaagree with that of other Eurycea and P. cinereus.

Finally, Mary and Trauth (2006) studied histol-ogy of CCGs in two large Plethodon, P. albagulaand P. ouachitae, and in Desmognathus brimleyo-rum. In all of these species, just as in P. cinereus,mucous glands were alcian blue positive whereasCCGs were negative with alcian blue but positivewith PAS, easily distinguishing these glands.Granular glands gave little reaction to either ofthe carbohydrate stains. Active glands of Pletho-don seemed closely packed in their micrographsand barrel-shaped in appearance. The CCG of D.brimleyorum that was illustrated was a singlegland among granular and mucous glands, waslarger than those glands, and was round in out-line. Mary and Trauth (2006) state, however, thatin D. brimleyorum “the large caudal courtshipglands occupied such an overall volume during thebreeding season that they displaced the adjacentmucous glands in the tail base region.”

As mentioned in the introduction, the otherwell-known SDSG of plethodontids is the mentalgland. Mental glands at the gross and LM levelsshow considerable variation among plethodon-tids, but often are more tubular and elongatethan CCGs (Sever, 1976). The mental gland of P.cinereus has not been described and is the sub-ject of a future study by DMS. Histochemically,mental glands are similar to CCGs (Sever, 1989),but proteinaceous pheromones have been isolatedfrom P. shermani. Plethodontid receptivity factorhas been shown to increase female receptivity,and plethodontid modulating factor decreasesfemale receptivity, although the combined effectof both increases receptivity (Houck et al., 2007,2008).

The ultrastructure of the CCGs of P. cinereusbears little resemblance to that of the mentalglands of E. quadridigitata (Sever, 1975) and H.italicus (Borgiolo, 1977). The secretions of themental glands of these species consist of an oval orround granule filled with an electron-dense amor-phous material. Plasma membranes are distinctbetween cells, and apical junctional complexesexist, unlike the active CCGs of P. cinereus.

The CCGs of salamanders possess similarities inhistology and histochemistry with some of theSDSGs described in anurans (Thomas, 1993,Brizzi et al., 2003), but much more comparativework is needed to understand the phyletic andfunctional significance of the variation observed sofar in these glands. For example, in the treefrogHypsiboas punctatus, SDSGs of two types occur inmental region and lateral flanks of the body(Brunetti et al., 2012).

One type is a specialized mucous gland (SMG)and the other a specialized granular gland (SGG).The histology and ultrastructure of the SMG ismore similar to that of CCG than that of the SGG.Both SMGs and CCGs have biphasic secretorygranules (although those of the SMG are round

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and the CCG are oval), merocrine secretion, andare PAS positive. SGG are syncytial with spongy-appearing granules that do not react with PAS.

We believe that SDSGs are widespread in anu-rans and urodeles, and caecilians should be exam-ined more carefully for SDSGs as well. Wehypothesize that SDSGs are symplesiomorphic forthe Lissamphibia, and the testing of this hypothe-sis requires much more comparative research.

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