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Cloacal Anatomy of Male Salamanders in the Families Ambystomatidae, Salamandridae, and Plethodontidae Author(s): David M. Sever Source: Herpetologica, Vol. 37, No. 3 (Sep., 1981), pp. 142-155Published by: Herpetologists' LeagueStable URL: http://www.jstor.org/stable/3891884Accessed: 30-06-2015 16:16 UTC

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Herpetologica, 37(3), 1981, 142-155 ? 1981 by The Herpetologists' League, Inc.

CLOACAL ANATOMY OF MALE SALAMANDERS IN THE FAMILIES AMBYSTOMATIDAE, SALAMANDRIDAE,

AND PLETHODONTIDAE

DAVID M. SEVER

ABSTRACT: This paper is the first interfamilial study of cloacal anatomy of male salamanders since 1895. Cloacae from representatives of the Salamandridae (Notophthalmus viridescens), Ambystomatidae (Ambystoma tigrinum), and Plethodontidae (Eurycea bislineata) were examined by light microscopy. Homologies and standarized names for cloacal glands are proposed. Four cloacal glands occur in N. viridescens. These are the pelvic gland, dorsal gland, anterior ventral-gland, and posterior ventral-gland. The dorsal gland has no homologue in A. tigrinum or E. bislineata, but the other glands are present. However, the pelvic gland has two divisions in A. tigrinum and three in E. bislineata. In addition, A. tigrinum and E. bislineata possess Kingsbury's gland and the vent gland, neither of which is found in N. viridescens.

Key words: Amphibia; Caudata; Anatomy; Cloaca; Glands

VARIOUS names have been given to the exocrine glands lining the cloaca of male salamanders (Sever, 1980). The same names should be used for homologous glands, but there are few studies on the comparative anatomy of cloacal glands to serve as a basis for determining homologies (Benson, 1965; Heidenhain, 1890; Kingsbury, 1895; Sever, 1978, 1980; Wil- liams, 1976). This paper is the first comparative study since Kingsbury (1895) of the cloacal anatomy of male salamanders in the families Salamandridae, Ambysto- matidae, and Plethodontidae. These are the only families of salamanders whose members are known to possess at least three well-developed types of cloacal glands (Sever, 1978). I propose homologies and standarized names for the cloacal glands.

MATERIALS AND METHODS

The species examined were Noto- phthalmus viridescens (Salamandridae), Ambystoma tigrinum (Ambystomatidae), and Eurycea bislineata (Plethodontidae). The cloacal glands were in a hypertro- phied, breeding condition in all specimens except for one N. viridescens in nonbreeding condition. The latter specimen was collected in July in Macon County, North Carolina, while the remaining N. viridescens were collected in

April in Monongalia County, West Vir- ginia, or in January in Murray County, Georgia. Ambystoma tigrinum were collected in March in Saint Joseph County, Indiana, and Eurycea bislineata were collected in October in Montgomery and Brown counties, Indiana. All specimens were originally preserved in 10% form- aldehyde. Seven specimens of N. viridescens and five specimens of both A. tigrinum and E. bislineata were examined by light microscopy following histological preparation. An additional 16 specimens of E. bislineata were examined by light microscopy in a previous study (Sever, 1980). The cloacae of at least six specimens of each species were examined by gross dissection.

Following fixation, the distance from snout to posterior end of the vent (SVL) was measured, and the cloacal region was dissected from the body. For the specimens prepared for histological examination, this tissue was dehydrated in etha- nol, cleared in toluene, and embedded in paraffin for sectioning. Representative transverse sections from the cloacae of three individuals of each species were stained with the following: hematoxylin- eosin (general cytology), Mallory's triple stain (connective tissue), periodic acid/ Schiff reagent (PAS, for general carbohydrates), toluidine blue (mucin), and al-

142



September 1981] HERPETOLOGICA 143

cian blue at pH 2.5 (acid mucopolysaccharides). These stains were used primarily as an aid in identification of gland clusters. The secretions of these glands are complex enough that more controlled procedures would be needed to analyze secretory products fully.

For N. viridescens and A. tigrinum, the cloaca of one specimen was sectioned frontally and that of another was sectioned sagittally. These sections were stained with Mallory's triple stain. All remaining specimens were sectioned trans- versely, and these sections were stained with hematoxylin-eosin. Staining procedures followed Humason (1978).

RESULTS

The appearance of the posterior end of the intestine is the same in all three species. The simple epithelium there is unciliated, glandular (PAS positive), and formed into small longitudinal folds (ru- gae) when the lumen is devoid of food. The mucous cells cease prior to the union of the urogenital ducts and urinary blad- der to form the anterior end of the cloaca. The cloaca is divided into an anterior tu- bular portion, the cloacal tube, and a posterior region surrounding the vent, the cloacal chamber. The cloacal anatomy of each species is described below.

Notophthalmus viridescens.-The anterior one-fourth of the cloacal tube is a small, roundish cavity bordered by simple, unciliated epithelium arranged in a series of small, longitudinal folds (Fig. IA). Posterior to this region is the ventral papilla, a broad ciliated protuberance that evaginates from the ventral floor (Fig. 1B). Immediately caudad of the ventral papilla, the ventral floor abruptly invaginates towards the vent, and dorsally there is a broad, ciliated, medial evagination from the roof of the cloacal tube, the dorsal papilla (Fig. IC).

In the dorsal papilla there is a medial, ciliated groove, the dorsal groove. At the superior apex of the dorsal groove, it branches into lateral recesses giving the

groove a T-shaped appearance in transverse sections. Ventral to the dorsal papilla at the posterior end of the cloacal tube, the cavity is narrow and lined with 4 or 5 pairs of short folds that are directed ventrally (Fig. 1D). These are the cloacal ridges. At this point, the entire cloacal tube is ciliated except for a few cells in the epithelium lateral to the dorsal papilla.

At the anterior end of the cloacal chamber, a small medial fold evaginates into the superior apex of the dorsal groove so that the groove assumes a Y-shaped in- stead of a T-shaped appearance in transverse sections (Fig. ID). The dorsal papilla becomes more narrow and elongate dorsoventrally. The dorsal half of the anterior end of the cloacal chamber is thus a narrow cavity composed of the dorsal papilla, the dorsal groove, and the cloacal ridges (Fig. ID).

The ventral half of the anterior end of the cloacal chamber is the cavity surrounding the cloacal orifice. This cavity is relatively wide although it is largely filled with numerous relatively long, thick papillae that radiate from the cloacal walls (Fig. ID). These vent papillae circumscribe the entire cloacal orifice, but there are relatively few around the posterior angle. The most caudal vent papillae are not evaginations from the caudal angle of the vent but represent pro- jections that pass posteriorly from more anterior portions of the cloacal wall.

The most anterior vent papillae are lightly ciliated. Otherwise, ciliation in the anterior end of the cloacal chamber is limited to the upper portions of the dorsal groove and the epithelium of the cloacal ridges. This condition continues through the anterior one-third or one- fourth of the cloacal chamber.

At this point, the lateral walls of the dorsal papilla unite with surrounding walls of the cloacal chamber (Fig. 2A). Just posterior to this, the cloacal ridges cease and the medial fold in the dorsal groove ends. Posterior to this area, ciliation is absent in the cloacal chamber ex-



144 HERPETOLOGICA [Vol. 37, No. 3

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September 19811 HERPETOLOGICA 145

cept for proximal portions of the vent papillae.

About midway through the cloacal chamber, there begins a gradual, caudal, dorsoventral shortening of the cloacal chamber. Also, the cavity dorsal to the cloacal orifice is at its widest point. Viewed by frontal sections, the ventral half of the cloacal chamber is circular. The anterior two-thirds of the circle's circumference consists of vent papillae. The posterior one-third of the circumference is lined with much smaller papillae as shown in transverse section in Figure 2B.

There are four distinct glands in the cloaca of male N. viridescens. These are the anterior ventral-gland, posterior ventral-gland, pelvic gland, and dorsal gland.

The anterior ventral-gland is the largest gland and is responsible for the swol- len cloacal region characteristic of male newts in breeding condition. Anterior ventral-glands secrete on the cloacal ridges and on the vent papillae (Figs. IC, ID 2A). These glands are relatively long, and their distal ends radiate primarily in an anterior or anterolateral direction.

Posterior ventral-glands comprise a small group of tubules in the lateral areas of the anterior end of the cloacal chamber. They appear most anteriorly when the dorsal papilla has elongated and the dorsal groove has assumed a Y-shaped appearance in transverse sections. In this region, posterior ventral-glands secrete onto the ciliated epithelium lining the base of the dorsal groove (Fig. 1C). More posteriorly, as the lateral borders of the dorsal papilla merge with lateral walls of the cloacal chamber, the posterior ven-

tral-glands secrete along the entire ventral half or two-thirds of the dorsal groove and on the most posterior portions of the cloacal ridges (Fig. 2A). The posterior ventral-glands are relatively short, and distal ends mostly pass anteroventrally and become entwined with adjacent tubules of the anterior ventral-gland.

The cytoplasm and luminal contents of both types of ventral glands are basophilic, but the luminal contents of the posterior ventral-glands give a much lighter reaction in hematoxylin-eosin. Also, the anterior ventral-glands give a strong positive test with toluidine blue while the posterior ventral-glands are negative. Using Mallory's triple stain, the luminal contents of anterior ventral- glands stain brown or gray. The posterior ventral-glands stain gray or pale blue in Mallory's, and red-staining globules often occur in the luminal contents. Such globules are absent in anterior ventral- glands.

Both anterior and posterior ventral- glands give strong positive reactions with alcian blue at pH 2.5. Proximal portions of the anterior ventral-gland are strongly PAS positive, but distal portions are negative. All parts of the posterior ventral- gland are strongly PAS positive.

The pelvic glands are relatively short. They secrete into the roof of the cloacal tube just cranial to the dorsal papilla and into the superior portion of the dorsal groove in the cloacal tube and anterior cloacal chamber (Fig. 1B-D). Pelvic glands secrete into the medial fold of the dorsal groove (Fig. ID). The most posterior of the pelvic glands are found at

FIG. 1.-Transverse sections from anterior (A) to posterior (D) through the cloaca of a male Notophthal- mus viridescens (SVL = 51 mm) collected 9 January 1980 in Murray County, Georgia. Bar in lower right corner = 500 ,um. (A) Anteriormost end of the cloacal tube. (B) Anterior appearance of the ventral papilla of the cloacal tube. (C) Dorsal papilla of the cloacal tube. (D) Anteriormost end of the cloacal chamber. AV = anterior ventral-glands; CR = cloacal ridges; CT = cloacal tube; D = dorsal papilla; DG = dorsal glands; G = dorsal groove; MF = medial fold; P = vent papillae; PG = pelvic glands PV = posterior ventral-glands; VP = ventral papilla.




FIG. 2.-Continuation of transverse sections through the cloaca of the N. viridescens shown in Fig. 1. Bar in lower right corner = 500 ,um. (A) Middle of the cloacal chamber. (B) Posterior end of the cloacal chamber. Abbreviations same as for Fig. 1 plus CC = widened ventral area of the cloacal chamber and CP = papillae lining caudal end of the cloacal chamber.

the apex of the dorsal groove at the mid- point of the cloacal chamber just caudad of the posterior end of the medial fold. Distal ends of the pelvic glands radiate from the roof of the cloaca and largely pass anteriorly or anterolaterally.

Overall, the pelvic gland is eosinophilic, although the luminal contents and especially the cytoplasm of the more dorsomedial tubules are often decidedly basophilic. The luminal contents of the dorsomedial tubules are also more granular than those of the tubules secreting along the dorsal groove.

Dorsal glands are relatively long glands that branch extensively at their distal ends. Most of the dorsal glands secrete on the tips of the small papillae lining the caudal end of the vent (Fig. 2B). Distal ends of the dorsal glands pass anteriorly. Some pass lateral or ventral to the anterior ventral-gland, but most pass between the pelvic gland superiorly and the ventral gland inferiorly to the poste-

rior end of the peritoneal coelom where tubules form a large mass lateral and ventral to the rectum.

Dorsal glands are easily distinguished from pelvic glands by cytology and staining reactions with PAS. As noted by Kingsbury (1895), dorsal glands have numerous small globules in their epithelium. These globules are especially numerous apically, and they stain intensely with PAS. In contrast, the cytoplasm of pelvic glands lacks these globules and gives a very weak reaction to PAS. The luminal contents of both types of glands are strongly PAS positive.

Other stains show only minor differences between pelvic and dorsal glands. Like the pelvic glands, the cytoplasm and luminal contents of dorsal glands are usually eosinophilic in hematoxylin-eosin. Luminal contents of the pelvic and dorsal glands generally stain orange or red in Mallory's triple stain, although dorsomedial portions of the pelvic gland also




have a granular, blue-staining component. The reaction of the pelvic and dorsal glands to alcian blue is weak and re- stricted to localized areas of the luminal contents. The reaction of the pelvic gland with alcian blue is stronger than that of the dorsal gland. Neither pelvic nor dorsal glands give positive tests with tolu- dine blue.

Ambystoma tigrinum.-The anterior end of the cloacal tube is laterally nar- rowed dorsally and laterally widened ventrally (Fig. 3A). A prominent ventral evagination, the ventral papilla, composed of ciliated epithelium occurs in the anterior one-fourth of the cloacal tube (Fig. 3A). Posterior to this, the ventral floor of of the posterior half of the cloacal tube invaginates abruptly towards the in- vaginating epidermis of the cloacal slit. The ventral epithelium is then formed into about 12 pairs of narrow, ventrally projecting folds, the cloacal ridges, covered with ciliated epithelium.

Concurrent with the ventral invagination, the cloacal tube widens dorsally. The epithelium of the dorsal half becomes ciliated, and two pairs of narrow folds that pass longitudinally appear dor- solaterally (Fig. 3B). The more medial pair is the primary lateral folds and the more lateral pair, the secondary lateral folds. Both lateral and medial surfaces of the primary lateral folds are ciliated, but only the medial surfaces of the secondary lateral folds are ciliated. Distal edges of both types of folds consist of unciliated papillae. Recesses of the cloacal tube lateral to the secondary lateral folds remain unciliated.

Just posterior to the appearance of the lateral folds, in the caudal one-third of the cloacal tube, there is a middorsal evagination, the dorsal papilla. As in N. viridescens, a medial invagination, the dorsal groove, appears in the dorsal papilla. However, the dorsal groove of N. viridescens is lined with ciliated epithelium while that of A. tigrinum is unciliated.

At the anterior end of the cloacal chamber, the dorsal papilla is an elongate,

grossly conspicuous structure, and the dorsal groove extends the vertical length of the papilla (Fig. 4A). The ventral one- third of the cloacal chamber (inferior to the medial tips of the cloacal ridges) is a widened cavity dorsal to the narrow slit in the epidermis forming the cloacal orifice (Fig. 4). In N. viridescens, this space is filled by the large vent papillae. In A. tigrinum, the walls of this area are lined with relatively short papillae covered by simple, lightly ciliated epithelium con- tinuous with the epidermis of the cloacal orifice.

One-third of the way posterior through the cloacal chamber, the epithelium of the entire chamber becomes unciliated. The dorsal half of the cloacal chamber narrows, and the lateral folds and lateral walls of the dorsal papilla merge with those of the dorsal half of the cloacal chamber (Fig. 4B). The cloacal ridges ter- minate posteriorly in the roof of the cavity dorsal to the cloacal orifice (Fig. 4B). This cavity is at its widest point, and short, unciliated papillae are numerous on its lateral walls.

Further posteriorly, there is a gradual shortening of the cloacal chamber and a narrowing of the cavity dorsal to the cloacal orifice (Fig. 5A). The extent of the epidermal lining increases dorsally (Fig. 5B). The posterior one-fourth of the cloacal chamber is a narrow slit lined en- tirely with epidermis.

Basophilic glands homologous to the anterior and posterior ventral-glands of N. viridescens are readily recognized in A. tigrinum. The anterior ventral-gland again comprises the the largest group of tubules and forms the bulk of the anterior mass of glands. Anterior ventral-glands secrete onto the cloacal ridges and papillae lining the anteroventral portion of the cloacal chamber (Figs. 3B, 4). Distal ends pass anteriorly and anterolaterally, and the most cephalic ends form large masses lateral and ventral to the cloacal tube and lateral to the posterior ends of the intestine and opisthonephric masses.

The posterior ventral-gland is much




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FIG. 3.-Transverse sections through the cloaca of a male AmbyJstoma tigrinum (SVL = 101 mm) col- lectd 1 Marh 180 n Sant oseh Conty Iniana Ba inlower right corner = 500 ,utm. (A) Anterior-

most end of the cloacal tube. (B) Middle of the cloacal tube. Abbreviations same as for Fig. 1 plus DP =

dorsal pelvic-glands; KG = Kingsbury's glands; LP = lateral pelvic-glands; PF = primary lateral folds; SF = secondary lateral folds.



Septetmtber 1981] HERPETOLOGICA 149

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FIG. 4.-Continuation of transverse sections through the cloaca of the A. tigrinum shown in Fig. 3. Bar in lower right corner = 500 ,um. (A) Anteriormnost end of the cloacal chamber. (B) Area about one-third of the way posterior through the cloacal chamber. Abbreviations same as for Figs. 1-3.

larger in A. tigrinum than in N. viridescens. Most anteriorly, posterior ventral- glands secrete into the dorsal groove between more medial and lateral eosinophilic clusters of gland tubules. More caudad, posterior ventral-glands contin- ue to secrete into this groove, and after

the lateral walls of the dorsal papilla merge with the dorsolateral walls of the cloacal chamber, posterior ventral-glands come to line the entire dorsal half of the cloacal chamber except for the apex of the dorsal groove where some medial, eosinophilic glands still occur (Fig. 4B).




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FIG. 5.-Continuation of transverse sections through the cloaca of the A. tigrinum shown in Figs. 3, 4. Bar in lower right corner = 500 gin. (A) Area about two-thirds of the way posterior through the cloacal chamber. (B) Posterior end of the cloacal chamber. Abbreviations same as used in Figs. 1-3 plus VG =

vent glands.

Distal ends of the more cephalic posterior ventral-glands pass anteriorly and can be seen lateral to the opisthonephric masses in some of the first transverse sections of the cloaca. However, distal ends of posterior ventral-glands secreting on

more caudad portions of the dorsal papilla pass posteriorly.

After the posterior termination of the cloacal ridges in the cloacal chamber of A. tigrinum, posterior ventral-glands secrete along the entire dorsal groove and



September 1981] HIERPETOLOGICA 151

on the short, unciliated papillae lining the posterior portion of the widened cavity dorsal to the cloacal orifice. The posterior ventral-glands secreting on these short papillae are the widest in diameter of any of the cloacal glands and are much convoluted. Their distal ends pass posteriorly beyond the caudal end of the vent and then curve and pass anteriorly to lie lateral to the caudad portions of the anterior ventral-gland.

As the widened cavity narrows and the extent of the epidermis increases dorsally, posterior ventral-glands secrete only on the unciliated papillae remaining in the dorsal half of the cloacal chamber (Fig. 5A). After the caudal angle of the cloacal chamber becomes a narrow slit completely lined with epidermis, no cloacal glands secrete into the cloaca (Fig. 5B).

As in N. viridescens, the posterior ventral-glands of A. tigrinum have a lighter basophilic staining reaction with hematoxylin-eosin than do the anterior ventral- glands. The more anterior and dorsal groups of posterior ventral-glands stain darker than more caudal groups, but these "darker" posterior ventral-glands are still lighter than the anterior ventral- glands. Unlike N. viridescens, both anterior and posterior ventral-glands give a positive reaction with toluidine blue. However, the reaction of the posterior ventral-glands is weaker, especially in proximal regions of tubules secreting along the dorsal groove.

Another group of basophilic glands occurs in A. tigrinum, and I call these Kingsbury's glands because they are similar in anatomy and staining reactions to glands described under that name by Sever (1978, 1980) in the plethodontid genera Plethodon and Eurycea. In A. tigrinum, Kingsbury's gland consists of a relatively large number of short, narrow tubules that mostly secrete into the primary lateral folds of the posterior cloacal tube and anterior cloacal chamber, although some secrete into the dorsal portion of the cloacal tube just anterior to the

dorsal papilla (Fig. 3B). Distal ends of Kingsbury's glands generally pass anteriorly to lie ventral to the posterior ends of the opisthonephric masses (Fig. 3A).

Kingsbury's glands rarely have much stainable luminal material, but the cytoplasm gives a strong positive reaction with toluidine blue although this reaction is not as strong as for the anterior ventral-glands in A. tigrinum. Kings- bury's glands stain as darkly as anterior ventral-glands in hematoxylin-eosin.

All the basophilic gland clusters of A. tigrinum give strong, positive staining reactions with alcian blue at pH 2.5 and with PAS.

The pelvic gland has two divisions in A. tigrinum. One portion, the dorsal pelvic-gland, secretes into the superior apex of the dorsal groove from the anterior appearance of the dorsal groove in the cloacal tube until the dorsal papilla merges with the dorsolateral walls of the cloacal chamber (Fig. 4A, B). Distal ends of these small glands pass anteriorly and anterolaterally. The other part of the pelvic gland, the lateral pelvic-gland, secretes onto the medial tips of the secondary lateral folds (Figs. 3B, 4A). Distal ends of these glands pass anterodorsally and are lateral to the cephalic tubules of the posterior ventral-glands. The dorsal pelvic- gland is homologous to the pelvic gland in N. viridescens, but the lateral pelvic- gland has no clear homologue in N. viridescens, which lacks the secondary lateral folds.

Both types of pelvic glands have copious amounts of secretory products in their lumina, and this material stains orange or red in Mallory's triple stain. Nei- ther of the pelvic glands gives a positive reaction with toluidine blue or alcian blue. The luminal contents of both parts of the pelvic gland are PAS positive, and PAS positive granules occur in the cytoplasm. These granules are most pro- nounced in the lateral pelvic-gland.

Another eosinophilic gland occurs in A. tigrinum that I call the vent gland, a name applied to groups of small eosino-




philic tubules secreting into the caudal end of the vent in plethodontids (Sever, 1978, 1980). In A. tigrinum, the vent gland consists of 4-5 pairs of relatively elongate, thick tubules that secrete into the most posterior end of the widened area of the cloacal chamber dorsal to the cloacal orifice (Fig. 5A). This is also the most caudad area for secretion by posterior ventral-glands. Distal ends of the vent glands pass anteriorly in a single layer inferior to the posterior ventral- gland, and the most distal tips extend anteriorly into inferior layers of the anterior ventral-gland. There is great variation in epithelial height of the vent gland re- sulting in a festooned appearance not seen in any other gland.

Vent glands rarely have much secretory material in the lumina, but when present, it stains gray, brown, or purple in Mallory's triple stain. The luminal contents are weakly positive with PAS, but cytoplasmic granules occur that are mod- erately PAS positive. Neither the cytoplasm nor the luminal contents give positive reactions with toluidine blue or alcian blue.

Eurycea bislineata.-Information is given here on staining characteristics and gland anatomy not reported for E. bislineata by Sever (1980).

The same three basophilic glands that occur in A. tigrinum are found in E. bislineata. Staining reactions of the basophilic glands of E. bislineata are similar to those of A. tigrinum except that the posterior ventral-glands are negative with toluidine blue, as in N. viridescens.

The eosinophilic glands of E. bislineata are divided into four groups. The vent glands are homologous to those of A. tigrinum and have the same cytology and staining reactions. The other eosinophilic glands in E. bislineata are portions of the pelvic gland. These parts are the dorsal pelvic-gland, dorsolateral pelvic- gland, and caudal pelvic-gland. Sever (1980) did not describe the difference in position between the dorsal and dorso-

lateral pelvic-glands, and he referred to the caudal pelvic-gland as the dorsal gland.

The dorsal pelvic-gland secretes onto ciliated epithelium lining the anterior roof of the cloacal tube, and its tubules pass cephalically, although distal ends may fold back and appear to pass caudal- ly. The dorsolateral pelvic-gland opens into small, ciliated papillae of the dorsal half of the anterior end of the cloacal chamber. Distal ends of the dorsolateral pelvic-gland pass anteriorly ventral to the dorsal pelvic-gland. The caudal pelvic- gland secretes on unciliated papillae lining the dorsal half of the cloacal chamber posterior to the region where ciliated papillae occur. Distal ends of the caudal pelvic-gland pass posteriorly and laterally.

The staining reactions of the three portions of the pelvic gland are similar. In Mallory's triple stain, the luminal contents generally appear red or orange but browns and purples are also common. The reaction with toluidine blue is negative. Weak reactions occur with alcian blue. In the dorsal and dorsolateral pelvic-glands, positive alcian blue material is localized around the luminal borders; in the caudal pelvic-gland, the response is more diffuse throughout the luminal contents.

The pelvic glands of E. bislineata are only slightly PAS positive. In the dorsal pelvic-gland, a positive PAS response is limited to areas around the luminal bor- der. The dorsolateral pelvic-gland gives the weakest response to PAS, but when a positive response is present, it is more diffuse than for the dorsal pelvic-gland. The cytoplasm of the dorsolateral pelvic- gland contains many small globules like the dorsal gland of N. viridescens. Unlike those in the newt dorsal gland, the globules in the dorsolateral pelvic-gland do not react at all with PAS. The caudal pelvic-gland gives a light PAS positive reaction throughout the luminal contents. Except for the PAS reaction, the staining



September 19811 HERPETOLOGICA 153

of the pelvic glands of E. bislineata is similar to that obtained for N. viridescens and A. tigrinum.

DISCUSSION There is no eosinophilic cluster of

glands in A. tigrinum that is directly comparable in position or staining characteristics to the dorsal gland in N. viridescens. In N. viridescens, most dorsal glands secrete into short papillae lining the caudal end of the vent. Tubules of the dorsal gland then pass anteriorly between the pelvic and ventral glands. The lateral pelvic-gland of A. tigrinum secretes only into the secondary lateral folds (lacking in N. viridescens) and does not occur caudad of the anterior end of the cloacal chamber. Vent glands secrete with the most caudad of the posterior ventral-glands in A. tigrinum, but the vent glands do not pass anteriorly between pelvic and ventral glands. Also, the vent glands are only weakly PAS positive while the newt dorsal gland is strongly PAS positive. Thus, I do not con- sider a direct homologue of the dorsal gland to occur in A. tigrinum.

The homology of Kingsbury's gland and the vent gland between A. tigrinum and E. bislineata is based upon similarity in position, cytology, and staining reactions. In A. tigrinum, Kingsbury's glands are more numerous and the vent glands are relatively longer than has been reported for any plethodontid (Sever, 1978, 1980). These glands have no apparent homologue in N. viridescens.

The dorsal pelvic-gland of E. bislineata is probably homologous to the pelvic gland of N. viridescens and the dorsal pelvic-gland of A. tigrinum. These glands are all in the same relative position and are similar in most staining characteristics.

The dorsolateral pelvic-gland of E. bislineata has no clear homologue in either N. viridescens or A. tigrinum. The anterior mass of the dorsal gland of N. viridescens occupies a similar position, but

most dorsal glands in N. viridescens secrete into the caudal end of the vent while the dorsolateral pelvic-glands of E. bislineata secrete onto papillae lining the dorsal half of the anterior end of the cloacal chamber. The lateral pelvic-gland of A. tigrinum secretes into the secondary folds, and its posterior tubules are inferior to the most cephalic tubules of the posterior ventral-gland. Secondary folds are absent in E. bislineata, and the dorsolateral pelvic-gland is superior to the most cephalic tubules of the posterior ventral-gland.

The caudal pelvic-gland of E. bislineata also has no obvious homologue in N. viridescens and A. tigrinum. There is no eosinophilic gland in the cloacal chamber of either of these two species that has tubules whose distal ends pass posteriorly like those of the caudal pelvic-gland of E. bislineata. Sever (1980) was incorrect in assigning the name dorsal gland to this group of tubules in plethodontids.

As in A. tigrinum, I conclude that no single gland in E. bislineata is a direct homologue of the dorsal gland of N. viridescens. As noted above, none of the glands of E. bislineata has tubules in the same relative position as those of the newt dorsal gland. Also, none of the eosinophilic glands of E. bislineata pos- sesses the highly PAS positive cytoplasmic globules or even the strongly PAS positive luminal substance characteristic of the dorsal gland of N. viridescens.

Sever (1978) referred to the posterior ventral-glands of Plethodon cinereus and P. dorsalis as part of the dorsal gland. The posterior ventral-gland of these species is a group of tubules opening on papillae in the caudal end of the cloaca as does the dorsal gland of N. viridescens. Thus, homology was proposed on similarities in relative area of secretion. However, these glands are definitely not homologous as they differ greatly in cytology, staining characteristics, and direction of tubules. Also, as reported herein, N. viridescens has a posterior ventral-




gland similar in all properties to that of E. bislineata. To my knowledge, this paper represents the first time the posterior ventral-gland has been described as a separate gland in the Salamandridae and Ambystomatidae. Previously, the posterior ventral-gland had not been distinguished from the anterior ventral-gland.

Otherwise, the anatomy of the cloacal glands of N. viridescens is similar to that reported for Triturus cristatus, the first salamander for which cloacal anatomy was fully described (Heidenhain, 1890). As noted by Kingsbury (1895), there is no difficulty in assigning names to the cloacal glands of N. viridescens based upon those given by Heidenhain (1890) to the cloacal glands of T. cristatus. Heiden- hain (1890) divided the cloacal glands into three groups, "Beckendruse" (pelvic gland), "Kloakendruse" (cloacal gland), and "Bauchdruse" (abdominal gland). Following Wahlert (1953), I use the name ventral gland for the cloacal gland and dorsal gland for the abdominal gland. Al- though the cloacal glands of T. cristatus and N. viridescens are much alike in anatomy and relative position, there are some striking differences between the two species in cloacal conformation (Sev- er, unpublished). Discussion of these differences must await further research.

Little has been previously published on cloacal glands in Ambystomatidae. Kingsbury (1895) simply stated that he was able to recognize three types of glands in A. maculatum and that the cloacal lips of this species did not possess the fringe of papillae seen in N. viridescens. Benson (1965) never described the cloacal anatomy of A. maculatum, but he noted that histochemical tests on their cloacal glands were in complete accord with his results for N. viridescens. In both species, he noted that the "lateral gland" (=anterior ventral-gland) was PAS negative. Although I found the distal tubules of the anterior ventral-gland to be PAS negative, proximal tubules of this gland were strongly PAS positive as was

the entire anterior ventral-gland of A. tigrinum.

Staining tests reported by Williams (1976) indicate that the cloacal glands secrete complex carbohydrates. Such tests show that the nature of these substances may differ significantly among the glands. For example, the basophilic glands generally give strongly positive results with alcian blue at pH 2.5, and this reaction is usually weaker or negative for the eosinophilic glands. This means that an acid mucopolysaccharide component is more copious in the basophilic than in the eosinophilic glands. However, the positive alcian blue tests may be the result of quite different acid mucopolysaccharides in the various glands. The staining reactions indicate broad categories of similarities for the gland secretions and point the way for future research. More de- tailed biochemical assays are needed be- fore the phylogenetic and functional as- pects of the gland secretions can be determined.

In conclusion, this paper represents the first interfamilial study of comparative anatomy of cloacal glands of male salamanders since 1895. The species I examined are usually considered quite generalized and are among the most widely distributed members of their families. Much more comparative work is needed to see how applicable my proposed homologies are to the cloacal anatomy of other salamanders.

Acknowledgments.-I thank T. Halliday for pro- viding me with some male Triturus cristatus for comparative study. Examination of these specimens reinforced my admiration for the precisian of Hei- denhain's observations and gave me confidence that I was assigning correctly his names for the cloacal glands.

LITERATURE CITED

BENSON, D. G., JR. 1965. Chemical and morpho- logical changes in the cloacal glands of the newt, Triturus viridescens, as induced by hormonal al- terations. Ph.D. Dissertation, Univ. Virginia (Diss. Abstr. No. 66-3163).

HEIDENHAIN, M. 1890. Beitrage zur Kenntniss der Topographic und Histologie der Kloake und ihrer




driisigen Adnexa bei den einheimischen Trito- nen. Arch. Mikrosk. Anat. 35:173-274.

HUMASON, G. L. 1979. Animal Tissue Techniques. Fourth edition. W. H. Freeman and Co., San Francisco.

KINGSBURY, B. F. 1895. The spermatheca and methods of fertilization in some American newts and salamanders. Trans. Am. Microsc. Soc. 17:261-304.

SEVER, D. M. 1978. Male cloacal glands of Pleth- odon cinereus and Plethodon dorsalis (Amphibia: Plethodontidae). Herpetologica 34:1-20.

. 1980. Cloacal anatomy of male brook salamanders (Eurycea). Herpetologica 36:51-60.

WAHLERT, G. VON. 1953. Eileiter, Laich, und Kloake der Salamandriden. Zool. Jahrb. 73:276- 324.

WILLIAMS, A. A. 1976. Morphology and histochem- istry of the cloacal glands and reproductive tract in male Eurycea lucifuga Rafinesque and Eury- cea longicauda (Green). Ph.D. Dissertation, Southern Illinois Univ. (Diss. Abstr. No. 77- 6273).

Accepted: 19 May 1981

Department of Biology, Saint Mary's College, Notre Dame, IN 46556, USA

Herpetologica, 37(3), 1981, 155-160 ? 1981 by The Herpetologists' League, Inc.

FIELD EVIDENCE FOR A RELATIONSHIP BETWEEN COLOR AND DEVELOPMENTAL RATE IN THE NORTHERN

LEOPARD FROG (RANA PIPIENS)

PAUL STEPHEN CORN

ABSTRACT: A stable genetic polymorphism for color (green or brown dorsum) exists in Rana pipiens Schreber. Developmental rates of tadpoles were studied at two sites in Larimer County, Colorado. Synchronous breeding in both populations allowed mean dates of egg deposition to be estimated. The length of the larval period was estimated as the number of days between the mean date of egg deposition and the date of capture of an incompletely metamorphosed frog. In both populations the brown morph had a shorter larval period than the green morph. The proportion of the brown morph was higher in samples taken early in the emergence period, sup- porting the interpretation that tadpoles of the brown morph developed more rapidly. Both morphs, however, had similar rates of development during the metamorphic climax. The frequency of the brown morph was highest at ponds presumed to have higher tadpole mortality due to increased predation by vertebrates, which supports the hypothesis that the brown morph has a selective advantage during the tadpole stage.

Key words: Amphibia; Salientia; Ranidae; Colorado; Developmental rate; Polymorphism

FORD (1965) defined genetic polymorphism as ". . . the occurrence together in the same habitat of two or more discon- tinuous forms or phases of a species in such proportions that the rarest of them cannot be maintained merely by recur- rent mutation." The minimum proportion that cannot be maintained by mutation is somewhat arbitrary, because the value

varies with the intensity of selection (Merrell, 1969). However, Ford (1965) suggested that a character determined at a single locus be considered polymorphic if the character state having the lowest frequency occurs in one percent or more of a large population. Ford drew a dis- tinction between transient and balanced polymorphism, the latter being variation



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