Comparative Anatomy and Phylogeny of the Cloacae of Salamanders (Amphibia: Caudata)

JOURNAL OF MORPHOLOGY 212~305-322 (1992)

Comparative Anatomy and Phylogeny of the Cloacae of Salamanders (Amphibia: Caudata). Vi. Ambystomatidae and Dicamptodontidae DAVID M. SEVER Department of Biology, Saint Mary's College, Notre Dame, Indiana 46556 ABSTRACT Histology of the cloacae of Rhyacotriton olympicus and represen- tative species from the genera Ambystoma and Dicamptodon was examined by light microscopy. Females of Ambystoma possess sperm storage glands, the spermathecae, as well as ventral glands and dorsal glands, both of uncertain function. Females of Ambystoma examined from the subgenus Linguaelapsus differ from those in the subgenus Ambystoma by possessing more extensive ventral gland clusters and a shorter cloacal tube. Females of Dicamptodon possess spermathecae and ventral glands, but differ in cloacal conformation from females of Ambystoma and lack the dorsal glands. Females of R. olympicus possess more extensive epidermal lining in the cloaca than that found in females of Ambystoma and Dicarnptodon, and the only glands present are spermathecae, which cluster around a tube in the dorsal roof. Males of Ambystoma, Dicamptodon, and R. olympicus possess five types of cloacal glands (dorsal pelvic glands, lateral pelvic glands, anterior ventral glands, posterior ventral glands, and Kingsbury's glands) that function in spermatophore formation, and vent glands that may produce a courtship pheromone. In Ambystoma and Dicamptodon, vent glands secrete along the medial borders of the cloacal orifice. Males of A. opacum and A. talpoideum differ from males of other species examined from the two genera by possessing more extensive vent glands. Males of R. olympicus possess unique vent glands in which tubules secrete onto the surface of vent lobes lateral to the posterior end of the cloacal orifice, and distal ends of the glands pass anteriorly, superficial to the fascia enclosing the other cloacal glands. The results from analysis of cloacal anatomy support other data indicating that Ambystoma and Dicamptodon are sister groups, and that Rhyacotriton olympicus is not closely related to either of the other two genera and merits placement in a separate family. o 1992 Wiley-Liss, Inc.

This paper reviews existing literature and Trueb ('86) illustrate the external appear- presents additional descriptions of cloacal ance of the cloacal region of male and female anatomy for the North American salamander A.jeffersonianum in breeding condition, and families Ambystomatidae, containing the gen- the relationships of the cloacal cavities to era Ambystoma (27 species) and Rhyacosire- urogenital and digestive structures of sala- don (4 species), and Dicamptodontidae, con- manders are shown by Sever ('92b). In the sisting of Dicamptodon (4 species) and present paper, the female cloacal anatomy of monotypic Rhyacotriton (Frost, '85). Descrip- three species of Dicamptodon and seven spe- tions exist for the male cloacal anatomy of cies of Ambystoma is described for the first Dicamptodon tenebrosus and Rhyacotriton time. This paper is the sixth in a series on the olympicus (Sever, '88a) and for eight species comparative anatomy and evolution of the of Ambystoma (Sever, '81, '87, '88a; Sever et cloacal region of salamanders (Sever, '91a,b; al., '89; Licht and Sever, '91). Descriptions of '92a-c). female cloacal anatomy, however, are limited to brief reports on A, tremblayi and R. olym- MATERIALS AND METHODS picus (Sever, '87) and more detailed studies Snout-vent length (SVL) refers to the dis- on A. maculatum (Kingsbury, 1895) and A. tance from the tip of the snout to the poste- gracile (Licht and Sever, '91). Duellman and rior edge of the vent. Cloacal anatomy was o 1992 WILEY-LISS, INC. 306 D.M. SEVER examined following excision of the cloacal and/or presence of sperm in the spermathe- region and its histological preparation for cae. Stebbins ('54) reported that mature ova light microscopy. Specimens examined (Ap- of D. ensatus are 5-6 mm in diameter, and pendix I) were initially preserved in 10% neu- Nussbaum ('85, '87) reported mean (ovipos- tral-buffered formalin, and were stored in ited) ova sizes of 5.5 mm for D. copei and 6.6 60% isopropanol or 65% ethanol before re- mm €or D. ensatus. moval of cloacal tissue. In one Dicamptodon copei (UMMZ Cloacal tissue was rinsed in water, dehy- 134960.1, 101.5 mm SVL), ovarian follicles drated in ethanol, cleared in toluene or Histo- are only 0.75-1.25 mm in diameter, but ovi- sol (National Diagnostics, Inc., Manville, New ducts are convoluted, and some sperm are Jersey), and embedded in paraffin; 10-pm present in the spermathecae; thus this speci- sections were then cut with a rotary micro- men is mature. The other D. copei (UMMZ tome. Some sections from each species were 134961) had the ovaries removed and lacks stained with hematoxylin-eosin (for general sperm in the spermathecae, but it is consid- cytology), and others were stained or treated ered mature because the oviducts are convo- with Mallory's triple stain (connective tis- luted and the specimen is even larger (110.0 sue), ninhydrin Schiff/fast green (proteins), mm SVL) than other mature specimens. periodic acid-Schiff s (PAS) reagentlfast The only Dicamptodon aterrimus exam- green FCF (neutral carbohydrates), and/or ined (UMMZ 134671, 135 mm SVL) is meta- alcian blue/nuclear fast red at pH 2.5, or morphosed, has large ovarian follicles (4.5- toluidine blue (acid mucopolysaccharides). 5.25 mm in diameter), and is thus considered Staining procedures followed Humason ('79). mature; however, it has not recently mated Three-dimensional reconstructions were since the spermathecae lack sperm. Of the performed on transverse sections through three D. tenebrosus used in the present study, the cloacae of a male Ambystoma laterale, only one is metamorphosed and definitely and male and female A. opacum, Dicampto- mature. This individual (UMMZ 134955,279 don copei, and Rhyacotriton olympicus. These mm SVL)has ovarian follicles about 5 mm in reconstructions were done using PC3D soft- diameter and some sperm in the spermathe- ware (Jandel Scientific, Corte Madera, CAI cae. Neither branchiate D. tenebrosus exam- and a Jandel digitizing tablet with a Zenith ined possesses spermathecal sperm; both were ZF-248 microcomputer. Every 4th to 15th collected September 13, 1975. One (UMMZ section was digitized for image reconstruc- 137462.1, 130 mm SVL) has ovarian follicles tion following procedures outlined by Sever 1.5-1.75 mm in diameter and is probably ('91a). immature, while the other (UMMZ 137462.2, Sever ('91a) used a ratio of cloacal tube 141 mm SVL) has ovarian follicles 4.0-5.0 length divided by total cloacal length (CTL/ mm in diameter and may be mature. TCL) to express the relative size of the cloacal tube. The cloacal tube is cylindrical and RESULTS extends caudally to the cloacal chamber, a Female cloacal anatomy cavity dorsal to the vent. To determine Ambystomatidae lengths, the number of transverse sections of Reconstructions based on serial sections the cloacal tube and cloacal chamber was through the cloaca of a female Ambystoma counted. The section immediately posterior opacum are shown in Figure 1. Histological to the junction of the Wolffian ducts with the sections through cloacae are illustrated for A. hindgut was considered the most anterior opacum, A. gracile, A. maculatum, and A. section of the cloacal tube. tremblayi in Figure 2, and €or A. annulatum Dicamptodon copei normally is an obligate and A. barbouri in Figure 3. The three types paedomorph, and D. aterrimus and D. tene- of glands Kingsbury (1895) reported for A. brosus are facultative paedomorphs (Nuss- maculatum occur in all species. Interspecific baum, '70, '83; Good, '89). Dicamptodon ater- variation occurs in cloacal conformation and rimus and D. tenebrosus prior to 1989 were anatomy of the ventral gland. considered conspecific with D. ensatus, a species now restricted to parts of northern Cali- Cloacal conformation. The cloacal tube is fornia (Good, '89) and not examined for the present in all specimens examined except that present study. Maturity of branchiate fe- of Ambystoma annulatum, in which the males was determined by large size of ovar- Wolffian ducts do not join until the anterior ian follicles, convolution of the oviducts, end of the cloacal tube, so that a definite SALAMANDER CLOACAE 307 A

Fig. 1. Three-dimensional reconstructions of the clo- sections of the cloacal walls represents approximately aca of a female Ambystoma opacum (UMMZ 187373). 120 pm. Av, anterior ventral glands; Cc, cloacal chamber; Right lateral view with sections rotated 25“ clockwise. A: Cg, central groove; Ct, cloacal tube; Dg, dorsal glands; St, Walls of the cloacal cavities. B: Spermathecae. C: Ante- spermathecae. rior ventral gland. D: Dorsal gland. The distance between cloacal tube is not present (Fig. 3A). In the Posteriorly, the large dorsolateral foldsjoin other forms, the CTL/TCL quotient ranges medially, obliterating the central groove. In from 0.17 (A.texanum) to 0.38 (A.laterale). Ambystoma barbouri, A. jeffersonianum, A. In the species with a cloacal tube, the ante- laterale, A. opacum, A. platineum, and A. rior half is narrowed dorsally and widened tremblayi, obliteration of the central groove ventrally. The pseudostratified epithelium is occurs at the anterior end of the cloacal cham- formed into thick longitudinal folds or rugae ber (Figs. IA, 2B,C). In A. gracile, A. macula- (Fig. 2A) that are ciliated. At the midpoint of tum, A. talpoideum, and A. tigrinum, the the cloacal tube, the dorsal half widens. In central groove ends prior to the claacal cham- the posterior half of the cloacal tube, a pair of ber, and the posterior fourth of the cloacal large folds appears in the lateral walls, form- tube is a flattened slit (Fig. 2D). Since the ing a ‘‘central groove” between them (Figs. cloacal tube is absent in A. annulatum, the lA, 2A). Cilia occur on the epithelium of the central groove occurs in the anterior end of cloacal walls lateral to the folds but not on the cloacal chamber (Fig. 3A). The apex of the lining of the folds or the central groove. the central groove is slanted posteriorly, so a 308 D.M. SEVER

Fig. 2. Transverse sections through the cloacae of maculatum (UMMZ 187377), posterior cloacal chamber. female Ambystomu. Sections stained with hematoxylin- F: A. trembluyi (UMMZ 122783), spermathecae around eosin except F, stained with Mallory’s triple stain. A-C: dorsal end of the cloacal tube. Ru, rugae; Sp, sperm; other A. opucum (UMMZ 187373). A Cloacal tube. B: Anterior labels as for Figure 1. Scale bar = 275 pm for A-C,E, 350 cloacal chamber. C: Posterior cloacal chamber. D: A. pm for D, and 70 pm for F. gracile (UMMZ 133156), posterior cloacal tube. E: A.

small recess of the dorsal end still occurs in the cloacal orifice, the lips of the cloacal ori- transverse sections after disappearance of fice are lined with epidermis. As the chamber the ventral end. decreases in height posteriorly, the extent of The cloacal chamber basically is a simple the epidermal lining increases dorsally, The slit that gradually shortens posteriorly until posterior third or fourth of the cloacal cham- it is even with the epidermis of the ventral ber is entirely lined with epidermis. tail wall. Posterior to the central groove, the dorsal portion of the cloacal chamber is wid- Cloacal glands. Cloacal glands are sim- ened, and the roof has short, blunt folds that ple, tubular exocrine glands. The three types lack cilia (Figs. lA, 2C,E). Ciliated rugae present are spermathecae, anterior ventral from the lateral and ventral walls of the glands, and dorsal glands. cloacal tube terminate in the anterior half of Spermathecae are glands for sperm stor- the cloacal chamber. At the anterior end of age. Sperm occur in the spermathecae of the SALAMANDER CLOACAE 309

Fig. 3. Transverse sections through the cloacae of female Ambystoma annulaturn, UMMZ 187374 (A,B) and A. barbouri, UMMZ 187376 (C,D). Sections stained with hematoxylin-eosin. A Anterior cloacal chamber. B: Posterior cloacal chamber. C: Posterior cloacal tube. D Pos- terior cloacal chamber. Labels as for Figure 1. Scale bar = 375 wm for A-D.

specimens examined except for Ambystoma posterior to the central groove (Figs. lB, 2D). laterale, A. talpoideum, A. platineum, and Melanophores are more abundant in the con- some A. tremblayi. Whether spermatozoa oc- nective tissue around the spermathecae than cur or not, the spermathecal epithelium is other cloacal glands. alcian blue + ,indicating presence of acid mu- Ventral glands secrete onto the tips of the copolysaccharides. Spermathecae are short ciliated rugae lining the lateral walls of the glands that are concentrated around the cen- anterior cloacal chamber (Figs. lC, 2A,B,D). tral groove, and most open into the central Ventral glands are long and convoluted. Their groove (Figs. lB, 2A,B, 3A,B). However, other distal ends extend laterally and anteriorly. spermathecae open onto the walls of the clo- Anterior ends of some ventral glands are aca anterior, lateral, and ventral to the cen- inferior to the posterior end of the intestine. tral groove, and some spermathecae open Ventral gland lumina contain large globules, upon the dorsal and lateral walls of the cloaca and granules occur in the cytoplasm. Glob- 310 D.M. SEVER ules and granules are PAS+, alcian blue-, Dicamptodontidae and ninhydrin Schiff+ ,indicating a mucopro- No differences occur in cloacal conforma- tein. Much variation occurs from specimen to tion or the types of cloacal glands present in specimen in the height of the ventral gland the female Dicamptodon examined. Dicampt- epithelium; the lumina of ventral glands in odon and Rhyacotriton, however, differ some specimens are obscured by the hypertro- greatly in female cloacal anatomy. phy of the cells. Ventral gland hypertrophy probably is related to a particular stage of Cloacal conformation of Dicamptodon. reproductive activity (Sever, '88b). The CTL/TCL quotient in D. atterimus is Both the Ambystoma annulatum and A. 0.21, in the 130-mm SVL D. tenebrosus 0.39, texanum examined have enlarged ovarian fol- and in the 110.0-mm SVL D. copei 0.24. The licles, but no spermatozoa in the spermathe- cloacal conformation of dicamptodontids is cae, indicating their capture prior to breed- simple. Anteriorly, a dorsomedial fold ap- ing. In both species, the ventral glands are pears subsequent to the junction of the uro- enlarged and convoluted. Anteriorly, distal genital ducts with the cloaca, and this fold ends of the ventral glands extend dorsally continues until a widening of the dorsal por- along the lateral borders of the cloacal cavi- tion in the posterior half of the cloacal tube ties to the region just inferior to the posterior (Figs. 4A, 5A-C). Additional folds appear in end of the kidneys (Fig. 3A,C). the dorsal roof of the anterior end of the Dorsal glands, in all species but Ambystoma cloacal chamber, and merge into a second maculatum, are similar in size to spermathe- dorsomedial fold that persists into the poste- cae. The glands appear most anteriorly in the rior end of the chamber (Figs. 4A, 5C-D). roof of the cloacal chamber just posterior to Thick dorsolateral folds with a central groove the most caudad spermathecae (Figs. lD, 2C, like that found in female ambystomatids are 3C,D). Further posteriorly, dorsal glands absent. Laterally, the walls of the cloacal come to line the entire dorsal and dorsolat- tube are rugose, and the rugae persist into eral walls of the cloacal chamber, persisting the anterior half of the cloacal chamber (Fig until the caudal angle of the vent, into which 4A). Ventrally, a medial evagination of the no cloacal glands secrete. Luminal contents floor of the cloacal tube occurs just anterior are scant in most specimens, but the apical to its abrupt junction with the cloacal cham- cytoplasm generally is PAS+, indicating pres- ber (Figs. 4A, 5B). The epithelium is pseudo- ence of neutral carbohydrates. stratified in the cloacal tube and is lightly In an Ambystoma maculatum collected just ciliated. Except for the most anterior end, after oviposition, the dorsal glands are more cilia are absent in the cloacal chamber. Poste- elongate and hypertrophied, and luminal con- riorly, the cloacal chamber simply decreases tents are copious (Fig. 2E). The contents are in height concordant with an increase in the globular, PAS +, and ninhydrin Schiff + . extent of epidermis into the cloaca. The walls Thus, the dorsal glands in A. maculatum are of the entire posterior fourth of the cloacal similar to the ventral glands, and the two chamber are smooth and lined completely clusters are difficult to distinguish except by with epidermis. position. Cloacal glands of Dicamptodon. Sperma- Cloacae were examined from specimens of thecae and ventral glands occur in female Ambystoma tremblayi and A. platineum used Dicamptodon. The spermathecae are short, by Uzzell ('64) to characterize these all- tubular glands that occur around the cloacal female forms of hybrid origin. Ovaries had tube and around the dorsal and dorsolateral been removed from these specimens, but the borders of the anterior cloacal chamber (Figs. oviducts were convoluted. Spermatozoa oc- 4B, 5A-C,E,F). The apical cytoplasm of the cur in the spermathecae of two A. tremblayi spermathecae in the D. copei containing sper- collected in Delaware County, Indiana (Fig. matozoa is PAS+ and alcian blue+. 2F). None is apparent in the other speci- Ventral glands are numerous, elongate, mens. A specimen (DMS 3439) of A. (2) lat- thick, and convoluted. These glands secrete erale-jeffersonianum (using the nomencla- into the lateral borders of the cloacal cham- ture of Lowcock et al., '87) from Cass County, ber and, posterior to the spermathecae, into Michigan, has an egg in the cloaca, but no the dorsal portion of the cloacal chamber sperm are evident in the spermathecae (see (Figs. 4C, 5). Distal ends of the glands pass Fig. 2D in Sever, '87). anteriorly, and those secreting into the dor- SALAMANDER CLOACAE 311 has a CTL/TCL quotient of 0.125. The short cloacal tube is rugose and lined with pseudostratified epithelium. The rugae cease in the anterior cloacal chamber, which is a simple cavity almost completely lined with epidermis. Cilia are lacking in the cloaca. The most remarkable conformational feature is an abrupt invagination into the dorsal wall of the posterior cloacal tube and anterior cloacal chamber, forming a “dorsal tube’’ from which the spermathecae radiate (Figs. 6, 7A,C). Cloacal glands of Rhyacotriton olympicus. The only glands are spermathecae. Individu- als collected on May 29, 1983 (Multnomah County, Oregon) have large ovarian follicles (3.5-3.7 mm) and possess abundant spermatozoa in their spermathecae. Examined specimens from June 19, 1981 (Bentou County, Oregon; Sever, ’87) have small ovarian follicles and lack sperm in the spermathecae, which appear inactive. No diagnostic staining was done on female R. olympicus. The C Av spermathecae are elongate, and radiate from sides and the apex of the dorsal tube (Figs. 6B, 7A-C). Sever (’87:lll) noted a “. . . gland cluster of uncertain homology. . .” in the posterior cloacal region of female Rhyaco- triton olympicus. At the caudal end of the cloaca, some short, tubular glands secrete onto the surface of the epidermis lateral and posterior to the cloacal orifice (Fig. 7D,E). These glands therefore are skin glands, but Fig. 4. Three-dimensional reconstructions of the clo- they are different from the mucous and gran- aca of a female Dicumptodon copei (UMMZ 134981). ular glands found elsewhere in the skin but Right lateral view with sections rotated 25” clockwise. A absent in this area. Walls of the cloacal cavities. B: Spermathecae. C: Ante- rior ventral gland. The distance between sections of the cloacal walls represents approximately 260 pm. Labels as Male cloacal anatomy for Figure 1. Ambystomatidae Cloacal glands present include anterior and lateral pelvic glands, anterior and posterior sal portion of the posterior cloacal chamber ventral glands, Kingsbury’s glands, and the lie superior to the most caudad spermathecae vent gland (Licht and Sever, ’91; Sever, ’81, (Fig. 5C). The ventral glands have granules ’88a, ’91a; Sever et al., ’89). In Ambystoma in the cytoplasm, and the luminal product opacum, Noble and Brady (’33)reported that usually appears as a large globule. The secre- lateral to the pelvic gland is an eosinophilic tory products are PAS+ and ninhydrin “scaffolding” gland with a homogeneous se- Schiff + . cretion. This describes the lateral pelvic gland. In the 130-mm SVL, branchiate Dicampt- Male ambystomatids that have been exam- odon tenebrosus, the spermathecae and ven- ined are similar in cloacal anatomy. Sever tral glands have narrow lumina and appear (’88a), however, found the vent gland better inactive or poorly developed, perhaps a sign developed in Ambystoma opacum and A. tal- of immaturity, since the glands in the poideum than in the other species examined. 141-mm SVL specimen are hypertrophied. Reconstructions through the cloacae of Am- Cloacal conformation of Rhyacotriton bystoma opacum and A. laterale are shown in olympicus. One individual, 54.2 mm SVL, Figure 8 and transverse sections through the 312 D.M. SEVER

Fig. 5. Transverse sections through the cloacae of phosed D. aterrimus (UMMZ 1346711, anterior cloaca1 female Dicamptodon. Sections stained with hematoxylin- tube. F: Paedomorphic D. tenebrosus (UMMZ 137462.1), eosin. A-D: D. copei (UMMZ 134961). A: Anterior cloa- anterior cloacal tube. Labels as for Figure 1. Scale bar = cal tube. B: Posterior cloacal tube. C: Anterior cloacal 250 pm for A-D,F, and 320 pm for E. chamber. D Posterior cloacal chamber. E: Metamor- cloacae of these species are shown in Fig- anterior fourth of the cloacal tube. Posteri- ure 9. orly, the floor of the cavity invaginates to- ward the cloacal orifice, with the walls com- Cloaca1 conformation. The anterior end posed of ciliated rugae that extend into the of the cloacal tube is narrow dorsally and anterior third of the cloacal chamber (Figs. wide ventrally (Fig. 8A,F), and is lined with 8A, 9A-D). Concurrently, the dorsal portion unciliated, pseudostratified epithelium. A of the cloacal tube widens, and two pairs of ventral, ciliated evagination occurs in the dorsolateral folds appear. These are the pri- SALAMANDER CLOACAE 313 responsible for the swelling of the cloacal region apparent in sexually active males. An- terior ventral glands form the large mass of glands surrounding the cloacal orifice, except for the posterolateral portions where posterior ventral glands occur (Figs. BC,D, 9). An- terior ventral glands secrete onto the tips of the rugae forming the lateral walls of the posterior cloacal tube and anterior cloacal chamber. Posterior ventral glands secrete into the dorsal end of the cloaca caudal to the medial papilla and replace the anterior ventral glands caudad to the rugae. No glands secrete into the most posterior angle of the vent. Posterior ventral glands are larger in diameter and stain less intensely than anterior ventral glands. In addition to the basophilic fibrous secretion, posterior ventral glands possess eosinophilic globules in their Fig. 6. Three-dimensionalreconstructions of the clo- lumina, especially in the more anterior and aca of a female Rhyacotriton olympicus (UMMZ dorsal tubules. 135501.2). Right lateral view with sections rotated 25" clockwise.A Walls of the cloacal cavities. B: Spermathe- Additional eosinophilic glands with globu- cae. The distance between sections of the cloacal walls lar secretions are the dorsal pelvic, lateral represents approximately 160 pm. Dt, dorsal tube; other pelvic, and vent glands. Dorsal pelvic glands labels as for Figure 1. and vent glands also often contain some granular, basophilic secretion similar in appear- ance and staining to that of the ventral mary lateral folds (medially) and secondary glands. The globular secretions of the eosino- lateral folds (Fig. 9A-D). philic glands are PAS+ and ninhydrin At the posterior end of the cloacal tube, a Schiff+, while granular material in the lu- middorsal papilla appears with a medial mina and cytoplasm of dorsal pelvic glands groove (Fig. 9C,D). At this point, the entire and vent glands is alcian blue + . Dorsal pelvic cavity is ciliated except for the medial groove, glands secrete along the medial groove and which is ciliated only at the inferior end, and superior portions of the anterior cloacal cham- the lateral borders of the secondary folds. ber and are replaced caudally by posterior In the anterior third of the cloacal cham- ventral glands. Posterior tubules possess ber, the primary and secondary lateral folds more alcian blue+ granular secretion than end, and the lateral walls of the dorsal papilla anterior ones. Lateral pelvic glands occur merge with the walls of the dorsal cloacal between Kingsbury's glands and anterior ven- chamber as that chamber shortens posteri- tral glands, and secrete onto the secondary orly (Figs. BA,F, 9E,F). The cloacal chamber lateral folds. Except in Ambystoma opacum widens ventrally in the anterior end, but this and A. talpoideum, vent glands are few in area narrows posteriorly concordant with the number, and consist of thick, elongate tu- absence of rugae. In the posterior two-thirds bules buried in the ventral gland masses (Figs. of the cloacal chamber, cilia are lacking. The 8E-F, 9). In all species, vent glands secrete walls of the middle third of the cloacal cham- along the edges of the cloacal orifice and ber are papillose. The walls of the most poste- distal ends pass laterally. rior angle of the vent are smooth and lined In Ambystoma opacum and A. talpoideum, with epidermis. vent glands appear relatively more numerous Cloaca1 glands. The basophilic glands and elongate. The vent glands of these spe- present are Kingsbury's glands, anterior ven- cies also extend laterally from the cloacal tral glands, and posterior ventral glands. orifice, but the distal ends often curve anteri- These glands contain alcian blue+ granular orly or posteriorly, resulting in transverse secretions. Kingsbury's glands secrete into sections of vent glands appearing in some of the dorsal, narrowed portion of the cloacal the most cephalad as well as caudad sections tube and onto the tips of the primary lateral of the cloaca (Figs. 8E, 9A,E). In A. talpoi- folds (Figs. BD, 9A,B). Ventral glands are deum, some of the anteriorly passing vent 314 D.M. SEVER

Fig. 7. Cloacae of female Rhyacotriton olympicus, through area posterior to the cloaca, showing rudimen- showing transverse sections of UMMZ 135501.2 in A,B, tary skin glands. E: Detail of skin glands shown in D. Rg, and sagittal sections of UMMZ 135501.1 in C-E. Sec- rudimentary glands; Sc, spermatophore cap; other labels tions stained with hematoxylin-eosin. A Posterior cloa- same as for Figures 1 and 6. Scale bar = 150 km for A,B, eal tube. B: Anterior cloacal chamber. C: Mid-sagittal 250 pm for C, 200 pm for D, and 70 pm for E. section through the cloaca. D: Parasagittal section

glands are so elongate that the distal ends As noted by Sever ('88a), Dicamptodon is curve dorsally to lie anterior to the pelvic similar to Ambystoma in cloacal anatomy (cf. gland cluster. In a 56.9-mm SVL A. texanum Figs. 8 and 10). The vent gland of Dicampt- (UMMZ 184008) collected in western Louisi- odon is not like that of A. opacum and A. ana and also examined by Sever ('88a), the talpoideum, but is more like other Am- vent gland cluster is like that of A. opacum bystoma. Thus, vent glands in Dicamptodon rather than A. texanum from Pelee Island, are limited to several layers of tubules infe- Lake Erie, in which the vent gland cluster rior to the ventral glands (Figs. 10B, 12B-D). resembles A. laterale (Sever et al., '89). The area of secretion of vent glands is along the cloacal orifice, except for the most poste- Dicamptodontidae rior end. Kingsbury's gland is a relatively Sever ('88a) described the cloacal anatomy smaller cluster of tubules in Dicamptodon of Dicamptodon and Rhyacotriton olympi- than in Ambystoma. cus. However, three-dimensional reconstruc- Sever ('88a) noted that the posterior ven- tions of the cloacae of male dicamptodontids tral glands are the only cloacal glands hyper- have not been presented previously (Figs. 10, trophied in a 122-mm SVL branchiate D. 11). Transverse sections in Figure 12 empha- tenebrosus (UMMZ 137462.3; Fig. 12D), and size variation noted in vent gland anatomy. a 106.0-mm SVL D. copei (UMMZ 134960). Histochemical reactions of the cloacal glands Meiotic divisions are found in the testes of of dicamDtodontids are similar to those of these individuals, but no soermatozoa are male ambystomatids (Sever, '88a). present in the testes or vaia deferentia, as SALAMANDER CLOACAE 315

A 6

C m D

Fig. 8. Three-dimensional reconstructions through showing cloacal walls and vent glands. Distance between the cloacae of male Ambystornu. Right lateral view with sections of the cloacal walls represents approximately sections rotated 25" clockwise. A-E: A. opacum (UMMZ 200 +m for A. opacum (A-E) and 300 pm for A. luterule 184009,50.8 mm SVL, Fall 1983, Florida; specimen also (F).Av, anterior ventral glands; Dg, dorsal glands; Dp, used by Sever, '88a). A: Walls of the cloacal cavities. B: dorsal pelvic glands; Kg, Kingsbury's glands; Lp, lateral Dorsal pelvic glands. C: Anterior ventral glands. D: Kings- pelvic glands; Pv, posterior ventral glands; Vg, vent bury's glands and posterior ventral glands. E: Lateral glands; other labels as for Figure 1. pelvic glands and vent glands. FA. luterule (DMS 6803) 316 D.M. SEVER

Fig. 9. Transverse sections through the cloacae of the C,D: Anterior third of the cloacal chamber. E,F Poste- maleAmbystoma opucum (A,C,E)and A. laterule (B,D,F) rior third of the cloacal chamber. Mg, middorsal groove; used in Figure 8, showing sections through equivalent Mp, middorsal papilla; Pf, primary lateral folds; Sf, regions of the two species in horizontal rows. Sections secondary lateral folds; other labels as for Figures 1,2,8. stained with hematoxylin-eosin. A Posterior end of the Scale bar = 325 Fm for A-F. cloacal tube. B: Anterior end of the cloacal chamber.

opposed to specimens in which all the cloacal cloacal conformation and the anatomy of the glands are hypertrophied. pelvic glands and ventral glands are similar As in Dicamptodon, Kingsbury’s gland in to the ambystomatid pattern (Fig. 11). Rhyacotriton olympicus is a relatively small The major difference in cloacal anatomy cluster. The primary and secondary folds of between Rhyacotriton olympicus and the R. olympicus are not as well developed as in other species is in the vent gland. Rhyacotri- Dicamptodon and Ambystoma. Otherwise, the ton olympicus possesses an autapomorphic SALAMANDER CLOACAE 317 A

C Av D Pv

Fig. 10. Three-dimensional reconstructions through sal pelvic glands. B: Lateral pelvic glands and vent the cloaca of a male Dicamptodon copei (UMMZ 134960.3, glands. C: Anterior ventral glands. D: Kingsbury's glands 107.9 mm SVL, August 24, 1969, Skamania County, and posterior ventral glands. Distance between sections Washington; specimen also used by Sever, '88a). Right of the cloacal walls represents approximately 260 Frn. lateral view with sections rotated 25" clockwise. A. Dor- Labels as for Figures 1 and 8. vent gland structure in which the glands glands are present is the Plethodontidae, and secrete at the posterior tips of squarish vent parsimony analyses of the evolution of cloa- lobes dorsolateral to the cloacal orifice (Sever, cal characters support the hypothesis that '88a; Fig. 12A). The vent gland cluster of R. possession of this character in the two fami- olympicus is relatively large in comparison to lies is due to convergence (Sever, '91a). Such that of other species, even Ambystoma analyses of cloacal characters show a sister opacum and A. talpoideum. Vent glands pass group relationship between Ambystoma and from the area of secretion to the anterior end Dicamptodon, a conclusion also reached from of the cloaca in large masses lateral to the analysis of aligned nucleotide sequences of musculature surrounding the cloacal cavities ribosomal RNA (Larson, '91). The sister and other cloacal glands (Fig. 11F; Figs. 1 group relationship revealed by analyses of and 2 of Sever, '88a). Thus, in R. olympicus, cloacal characters, however, is due largely to the vent glands technically are not even cloa- symplesiomorphies (Sever, '91a). Therefore, cal glands, since they secrete onto the skin the cloacal data provide equivocal additional lateral to the cloaca, and vent gland tubules evidence for the current view of separate are superficial to the fascia enclosing glands familial status for Ambystoma and Dicampt- that do secrete into the cloaca. odon. The familial relationships of Rhyacotriton DISCUSSION olympicus are another matter. Female R. Male Dicamptodon are similar in cloacal olympicus differ significantly from Am- anatomy to male Ambystoma except for A. bystoma and Dicamptodon in cloacal anat- opacum and A. talpoideum, which differ from omy. Specifically, in R. olympicus the epider- Dicamptodon as well as other Ambystoma by mal lining of the cloacal chamber is more possessing larger vent gland clusters. Fe- extensive, the only cloacal glands present are males of the two genera, however, are easily the spermathecae, and the spermathecae are distinguished by cloacal anatomy in that all clustered around a "dorsal tube" that is sim- female Ambystoma examined possess dorsal ilar to the common tube characteristic of the glands and the central groove, which are Plethodontidae. The dorsal tube in female R. lacking in female Dicamptodon. The only olympicus, however, is not as narrow as the other family of salamanders in which dorsal common tube of plethodontids. The sper- 318 D.M. SEVER

Fig. 11. Three-dimensional reconstructions through Lateral pelvic glands. D: Anterior ventral glands. E: the cloaca of a male Rhyacotriton olympicus (UMMZ Kingsbury's glands and posterior ventral glands. F Vent 135501.4, 52.5 mm SVL, May 29, 1983, Multnomah glands. Distance between sections of the cloacal walls County, Oregon; specimen also used by Sever, '88a). represents approximately 160 pm. Labels as for Figures Right lateral view with sections rotated 25" clockwise. A 1and 8. Walls of the cloacal cavities. B: Dorsal pelvic glands. C: mathecae of R. olympicus evaginate from the known only from male Ambystoma and Di- entire upper half of the dorsal tube, whereas camptodon among salamanders. The phy- in plethodontids narrow neck tubules extend letic relationships among Ambystoma, Di- from the apex of the common tube and ex- camptodon, and R. olympicus found by pand distally into spermathecal bulbs (Sever, parsimony analyses of cloacal characters '87). differ by whether these conspicuous folds are Males of Rhyacotriton olympicus possess a symplesiomorphic for the genera or conver- unique vent gland (Sever, '88a). Males also gent in Ambystoma + Dicamptodon and in R. possess primary and secondary lateral folds olympicus. The Dicamptodontidae, contain- in the cloacal tube, characters otherwise ing both Dicamptodon and R. olympicus, is SALAMANDER CLOACAE 319

Fig. 12. Transverse sections through the cloacae of of a D. copei (same specimen used for Fig. 10). C: male Dicamptodon and Rhyacotriton olympicus showing Anterior end of a metamorphosed D. tenebrosus (UMMZ sites of secretion and extent of the vent glands in the 128006, 147 mm SVL, October 8, 1967, Benton County, posterior cloacal chamber; specimens also used by Sever Oregon). D: Posterior end of the cloacal chamber of a ('88a). Sections stained with hematoxylin-eosin. A: Poste- paedomorphic D. tenebrosus (UMMZ 137462.3, 122 mm rior end of the cloacal chamber of an R. olympicus (UMMZ SVL, September 13, 1975, Linn County, Oregon). Or, 13550.5, 50.8 mm SVL, May 29, 1983, Multnomah orifices; VI, lateral vent lobes. Other labels as for Figures County, Oregon). B: Anterior end of the cloacal chamber 1and 8. Scale bar = 250 km for A-D.

paraphyletic if the character is symplesiomor- and R. olympicus in the same subfamily (Di- phic, and polyphyletic if the character is con- camptodontinae) based upon similarities in vergent (Sever, '91a). Neither choice sup- feeding specializations. Worthington and ports the placement of R. olympicus within Wake ('71) found no derived characters com- the Dicamptodontidae. mon to Dicamptodon and R. olympicus, and In the original description, Rhyacotriton preferred the classification of Tihen ('58). olympicus was placed in the genus Ranodon Edwards ("761, in an analysis of spinal nerve and assigned to the Hynobiidae, although patterns and osteological data, proposed the little justification for this action was given family Dicamptodontidae, with the extant (Gaige, '17). Dunn ('20) erected the new ge- subfamilies Dicamptodontinae and Rhyaco- nus Rhyacotriton and stated that it was most tritoninae, and this is the classification most closely related to Dicamptodon. Based upon often followed currently (Estes, '81; Frost, jaw anatomy, however, Eaton ('34) believed '85; Duellman and Trueb, '87). Milner ('83), R. olympicus was derived from Ambystoma however, considered Rhyacotriton olympicus and not at all related to Dicamptodon. Tihen more closely related to plethodontids and ('58) described many osteological differences amphiumids than to dicamptodontines, al- between Ambystoma, Dicamptodon, and R. though he did not find this conclusion wholly olympicus and placed the genera in different satisfactory. Duellman and Trueb ('86)noted subfamilies in the Ambystomatidae. Regal that the similarities between R. olympicus ('66) subsequently placed both Dicamptodon and plethodontids may be the result of conver- 320 D.M. SEVER gence of paedomorphic characters, but that The clash between results of phylogenetic synapomorphies linking R. olympicus and analyses of morphological and molecular data Dicamptodon are weak, and that Dicampt- also affects interpretation of vent gland evolu- odontidae may be polyphyletic. Good et al. tion in male Ambystoma opacum and A. tal- ('87) found that neither Ambystoma nor Di- poideum, which have more extensive vent camptodon is similar enough in its allozymes gland clusters than other Ambystoma. Based to make outgroup comparison with R. olym- upon analyses of other morphological charac- picus feasible. Beneski and Larsen ('89), how- ters, Kraus ('84) found that A. opacum and ever, felt that similarities in a stage of tooth A. talpoideum are sister-taxa, but results development might indicate a close relation- from analyses of allozyme data propose that ship between Dicamptodon and R. olympicus. A. opacum and A. talpoideum are in different Finally, Larson ('91), using an analysis of ri- clades (Shaffer et al., '91). Thus, when map- bosomal RNA sequences, found that R. olym- ping the character on existing phylogenies, picus is not closely related to any other sala- the vent gland morphology of A. opacum and mander genus. In summary, the evidence for A. talpoideum is either a synapomorphy monophyly at the family level between Di- (Kraus, '84) or convergent (Shaffer et al., camptodon and Rhyacotriton is poor, and the '91). Cloaca1 characters may provide indepen- genera should be placed in separate families. dent sources of data for constructing future Two of the Ambystoma examined, A. annu- phylogenetic hypotheses. latum and A. texanum, were placed in the Few direct observational or experimental subgenus Linguaelapsus by Tihen ('58), and data are available on the function of female A. barbouri, recently described by Kraus and cloacal glands in Ambystoma, Dicamptodon, Petranka ('891, was considered a liguaelapsid and Rhyacotriton. Spermathecae are used in by Shaffer et al. ('91). Other species of Am- sperm storage, but no detailed cytological bystoma examined here are members of the study on this exists in these genera except for subgenus Ambystoma (Tihen, '58). Linguae- some observations reported in abstracts by lapsus is the more highly derived group, and Davitt and Larsen ('88a,b) on Rhyacotriton differs from the Ambystoma species in shape olympicus. Kingsbury (1895) reported that the secretion of the female ventral glands is of the pterygoid and otoglossal of the hyoid, used in adherence of the egg mass. No data narrowness of the junction between the oto- exist to support or refute this idea. Sever glossal and the second radials, and anatomy ('88b) presented evidence that the ventral of the tongue plicae (Kraus, '88). Although gland secretes a courtship pheromone in the morphological analyses provide unambigu- females of the plethodontid Eurycea cirrig- ous evidence for the monophyly of Linguae- era. The function of the dorsal glands found lapsus, analyses of allozyme data furnish in female Ambystoma is unknown. Dorsal equally compelling evidence that Linguaelap- glands are better developed in Ambystoma sus is polyphyletic (Shaffer et al., '91). than in some genera in the Plethodontidae The female linquaelapsids examined here, that also possess glands in this region. A. annulatum and A. barbouri, share the Much literature exists on the overall ap- same ventral gland morphology, in which pearance and histology of spermatophores of these glands are more highly convoluted an- male Ambystoma (see Russell et al., '81, and teriorly and posteriorly than in other Am- Zalisko et al., '84, for reviews), but the rela- bystoma. Therefore this character may be a tion between parts of the spermatophore and synapomorphy for female Linquaelapsus. particular cloacal gland secretions has not However, the results from allozyme analyses been studied experimentally. In plethodon- (Shaffer et al., '91) hypothesize that A. annu- tids, the pelvic, ventral, and Kingsbury's latum and A. barbouri are in different clades, glands in the male cloaca are involved in making the ventral gland morphology conver- spermatophore production (Sever and Houck, gent in the two species. Variation in ventral '85). Vent glands in male salamanders are gland morphology, especially in female lin- assumed to produce a courtship pheromone quaelaspsids not examined here (A. mabeei, (Sever, '91a); it is at least certain in Rhyaco- A. cingulatum, and A. texanum), merits fur- triton olympicus that the vent glands, which ther study to see if this character can help secrete onto the lateral surface of the body, resolve conflicts in phylogenetic hypotheses cannot be involved in spermatophore forma- concerning the status of Linquaelapsus, tion (Sever, '88a). As with females, experi- SALAMANDEIR CLOACAE 321 mental data are needed for the roles of the Evolutionary Biology. Vol. 25. New York: Plenum Press, various male cloacal glands in spermato- pp. 211-217. Licht, L.E., and D.M. Sever (1991) Cloacal anatomy of phore formation andlor pheromone produc- metamorphosed and neotenic salamanders. Can. J. Zool. tion. 69:2230-2233. Lowcock, L.A., L.E. Licht, and J.P. Bogart (1987)Nomen- clature in hybrid complexes of Ambystoma (Urodela: ACKNOWLEDGMENTS Ambystomatidae): no case for the erection of hybrid This work was supported by National Sci- “species.” Syst. Zool. 36:328-336. Milner, A.R. (1983) The biogeography of salamanders in ence Foundation grants BSR-8715341 and the Mesozoic and early Cenozoic: a cladistic-vicariance BSR-9024918. For the loan or gift of speci- model. In R.W. Sims, J.H. Price, and P.E.S. Whalley mens and/or aid in collecting, I thank A.L. (eds): Evolution, Time and Space: The Emergence of Braswell, K. Mierzwa, R.A. Nussbaum, C.D. the Biosphere. London: Academic Press, pp. 431468. Noble, G.K., and M.K. Brady (1933) Observations on the Sullivan, and T. Williams. I thank J.P. Bog- life history of the marbled salamander, Ambystoma art and L.E. Licht for identification of Am- opacum Gravenhorst. Zoologica 11:89-132. bystoma from Cass County, Michigan. Nussbaum, R.A. (1970) Dicamptodon copei, n. sp., from the Pacific northwest (Amphibia: Caudata: Ambystoma- tidae). Copeia 1970:505-514. LITERATURE CITED Nussbaum, R.A. (1983) Dicamptodon copei Nussbaum. Cat. Am. Amphib. Rept 334:l-2. Beneski, J.T., Jr., and J.H. Larsen, Jr. (1989) Ontoge- Nussbaum, R.A. (1985) The evolution of parental care in netic alterations in the gross tooth morphology of Di- salamanders. Univ. Mich. Misc. Publ. Mus. Zool. L69:l- camptodon and Rhyacotriton (Amphibia: Urodela and 50. Dicamptodontidae). J. Morphol. 199:165-174. Nussbaum, R.A. (1987) Parental care and egg size in Davitt, C.M., and J.H. Larsen, Jr. (1988a) Ultrastructure salamanders: an examination ofthe safe harbor hypoth- and fate of spermatozoan cytoplasmic droplets within esis. Res. Popul. Ecol. 29:27-44. the spermathecal tubule of Rhyacotriton olympicus. Regal, P.J. (1966) Feeding specializations and the classi- FASEB J. 2.1367 (Abstract). fication of terrestrial salamanders. Evolution 20t392- Davitt, C.M., and J.H. Larsen, Jr. (1988b) Phagocytosis 407. of stored spermatozoa and cytoplasmic droplets by the Russell, L.D., R.A. Brandon, E.J. Zalisko, and J. Martan spermathecal epithelium of the female salamander Rhy- (1981) Spermatophores of the salamander Ambystoma acotriton olympicus. Am. Zool. 28:30A (Abstract). tezunum. Tissue Cell 13t609-621. Duellman, W.E., and L. Trueb (1986) Biology ofAmphib- Sever, D.M. (1981) Cloacal anatomy of male salamanders ians. New York McGraw-Hill. in the families Ambystomatidae, Salamandridae and Dunn, E.R. (1920)Notes on two Pacific coast Ambystomi- Plethodontidae. Herpetologica 37: 142-155. dae. Proc. N. Engl. 2001. Club 7:55-59. Sever, D.M. (1987) Hemidactylium scutatum and the Eaton, T.H., Jr. (1934)The affinities ofDicamptodon and phylogeny of cloacal anatomy in female salamanders. Rhyacotriton. Copeia 1934:182. Herpetologica 43:105-116. Edwards, J.L. (1976) Spinal nerves and their bearing on Sever, D.M. (1988a) Male Rhyacotriton olympicus (Di- salamander phylogeny. J. Morphol. 148:305-327. camptodontidae: Urodela) has a unique cloacal vent Estes, R. (1981) Handbuch der Palaoherpetologie. Teil2. gland. Herpetologica 44:274-280. Gymnophiona, Caudata. Stuttgart: G. Fischer. Sever, D.M. (1988b) The ventral gland in female Euryceu Frost, D.R. (1985) Amphibian Species of the World: A bislineata (Amphibia: Plethodontidae). Copeia 1988: Taxonomic and Geographic Reference. Lawrence, Kan- 572-579. sas: Allen Press, Inc., and The Association of System- Sever, D.M. (1991a) Comparative anatomy and phylog- atic Collections. eny of the cloacae of salamanders (Amphibia: Cau- Gaige, H.T. (1917)Description of a new salamander from data). I. Evolution at the family level. Herpetologica Washington. Occ. Pap. Mus. Zool. Univ. Mich. 4O:l-4. 47:165-193. Good, D.A. (1989) Hybridization and cryptic species in Sever, D.M. (1991b) Comparative anatomy and phylog- Dicamptodon (Caudata: Dicamptodontidae). Evolution eny of the cloacae of salamanders (Amphibia: Cau- 43: 728-744. data). 11. Cryptobranchidae,Hynobiidae, and Sirenidae. Good, D.A., G.Z. Wurst, and D.B. Wake (1987) Patterns J. Morphol. 207t283-301. of geographic variation in allozymes of the Olympic Sever, D.M. (1992a) Comparative anatomy and phylog- salamander, Rhyacotriton olympicus (Caudata: Di- eny of the cloacae of salamanders (Amphibia: Cau- camptodontidae).Fieldiana 2001. New Ser. 321-15. data). 111. Amphiumidae. J. Morphol. 211:63-72. Humason, G.L. (1979) Animal Tissue Techniques. 4th Sever, D.M. (1992b) Comparative anatomy and phylog- ed. San Francisco: W.H. Freeman. eny of the cloacae of salamanders (Amphibia: Cau- Kingsbury, B.F. (1895) The spermatheca and methods of data). IV. Salamandridae. Anat. Rec. 233:(In press). fertilization in some American newts and salamanders. Sever, D.M. (1992~)Comparative anatomy and phylog- Trans. Am. Microsc. SOC.17:261-304. eny of the cloacae of salamanders (Amphibia: Cau- Kraus, F. (1988)An empirical evaluation of the use of the data). V. Proteidae. Herpetologica 48:318-329. ontogeny polarization criterion in phylogenetic infer- Sever, D.M., and L.D. Houck (1985) Spermatophore for- ence. Syst. Zool. 37tlO6-141. mation in Desmognathus ochrophaeus (Amphibia: Kraus, F., and J.W. Petranka (1989) A new sibling spe- Plethodontidae). Copeia 1985:394402. cies ofAmbystoma from the Ohio River drainage. Copeia Sever, D.M., L.E. Licht, and J.P. Bogart (1989) Male 1989:94-110. cloacal anatomy in a hybrid population of Ambystoma Larson, A. (1991) A molecular perspective on the evolu- (Amphibia: Caudata). Herpetologica 45161-167. tionary relationships of the salamander families. In Shaffer, H.B., J.M. Clark, and F. Kraus (1991) When M.K. Hecht, B. Wallace, and R.J. MacIntyre (eds): molecules and morphology clash: a phylogenetic analy- 322 D.M. SEVER sis of the North American ambystomatid salamanders 79.1 mm SVL, 1961, Franklin County, Mas- (Caudata: Ambystomatidae). Syst. Zool. 4Or284-303. sachusetts; A. talpoideum-UMMZ 187378, Stebbins, R.C. (1954)Amphibians and Reptiles of West- ern North America. New York McGraw-Hill. 55.9 mm SVL, February 22, 1982, Hender- Tihen, J.A.(1958) Comments on the osteology and phy- son County, North Carolina; A. tigrinum- logeny of ambystomatid salamanders. Bull. Florida UMMZ 187375, 110.7 mm SVL, March 18, StateMus. Bio. Sci. 3:l-50. Worthington, R.D., andD.B. Wake (1971)Larvalmorphol- 1980, St. Joseph County, Indiana; A. trem- ogy and ontogeny of the ambystomatid salamander, blayi-UMMZ 122736, 64.8 mm SVL, April Rhyacotriton olympicus.Am. Midl. Nat. 85:349-365. 15, 1960, Kent County, Michigan; UMMZ Zalisko, E.J., R.A.Brandon, and J. Martan (1984)Micro- structure and histochemistry of salamander spermato- 122783.1, 74.2 mm SVL, UMMZ 122783.2, phores (Ambystomatidae, Salamandridae, and Pleth- 80.0 mm SVL, and UMMZ 122783.3, 81.3 odontidae). Copeia 1984r739-747. mm SVL, February 23, 1961, Delaware Uzzell, T.M., Jr. (1964) Relations of the diploid and triploid species of the Ambystoma jeffersonianum com- County, Indiana. plex (Amphibia, Caudata). Copeia 1964257-300. Dicamptodontidae: Dicamptodon aterrimus-metamorph, UMMZ 134671, 135 mm APPENDIX I SVL, May 10,1967, Benewah County, Idaho. Specimens examined include those listed D. copei-UMMZ 134960.1, 101.5 mm SVL, in Sever ('81, '87, '88a), Sever et al. ('89), and and UMMZ 134961,110.0 mm SVL, August Licht and Sever ('91) plus the following fe- 24, 1969, Skamania County, Washington; D. male sDecimens. all in the collections of the tenebrosus-metamorph, UMMZ 134955, University of Michigan Museum of Zoology 279 mm SVL, Linn County, Oregon; paedo- mWMZ)..~ morphs, UMMZ 137462.1,130 mm SVL, and Ambystomatidae: Ambystoma annulatum-UMMZ 187374, 89.0 mm SVL, no col- UMMZ 137462.2, 123 mm SVl, September lecting data; A. barbouri-UMMZ 187376, 13,1975, Linn County Oregon; Rhyacotriton 83.5 mm SVL, February 22, 1970, Hamilton olympicus-UMMZ 135501.1,52.2 mm SVL, County, Ohio; A. laterale-UMMZ 187371, UMMZ 135501.2,54.2 mm SVL, and UMMZ 57.1 mm SVL, September 13, 1975, Manito- 135501.3,50.4 mm SVL, May 29,1983, Mult- WOC County, Wisconsin; A. rnaculatum- nomah County, Oregon. UMMZ 187377,89.5 mm SVL, April 11,1980, In addition, a male A. annulaturn [UMMZ Cass County, Michigan; A. opacum-UMMZ 187372,91.4 mm SVL, August 1,1978 (sacri- 187373, 68.3 mm SVL, Fall 1983, Missouri; ficed July 25,19791, Stone County, Missouri] A. platineum-UMMZ 122856, 78.4 mm and a male A. laterale in my possession (DMS SVL, UMMZ 122857, 79.0 mm SVL, UMMZ 6803, 64.1 mm SVL, March 13, 1988, Cook 122859, 85.0 mm SVL, and UMMZ 122860, County, Illinois) were examined.