Cloacal Anatomy of Paedomorphic Female Ambystoma Talpoideum (Caudata: Ambystomatidae), with Comments on Intermorph Mating and Sperm Storage

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2147 Cloacal anatomy of paedomorphic female Ambystoma talpoideum (Caudata: Ambystomatidae), with comments on intermorph mating and sperm storage

STANLEY E. TRAUTH Department of Biological Sciences, Arkansas State University, State University, AR 72467, U.S.A.

DAVID M. SEVER Department of Biology, Saint Mary's College, Notre Dame, IN 46556, U.S.A.

AND

RAYMOND D. SEMLITSCH Division of Biological Sciences, University of Missouri, Columbia, MO 65211, U.S.A. Received January 26, 1994 Accepted August 12, 1994

TRAUTH, S.E., SEVER, D.M., and SEMLITSCH, R.D. 1994. Cloacal anatomy of paedomorphic female Ambystoma talpoideum (Caudata: Ambystomatidae), with comments on intermorph mating and sperm storage. Can. J. Zooi. 72: 2147 — 2157. The cloacal anatomy of paedomorphic itmd\t Ambystoma talpoideum was examined using light microscopy and histochemical techniques. Three types of cloacal glands (anterior ventral glands, dorsal glands, and spermathecae) were observed. Glandular morphology and secretory activity were documented from seasonal samples collected in South Carolina. Maximal development of cloacal glands corresponded to a peak in reproductive activity. Sperm-storage glands, the spermathecae, con• tained sperm in specimens collected from November through February. Although greatly hypertrophied during the breeding season, the anterior ventral glands continue to release secretory material throughout the year. This suggests that these glands could function in capacities other than those related to reproduction. The rudimentary dorsal glands also contain sperm during the breeding season; however, the occurrence of sperm in these glands is considered to be incidental. The cloacal glands of paedomorphs are similar to those of metamorphs. Interbreeding between morphs may occur and thus facilitate the main• tenance of both morphs in nature.

TRAUTH, S.E., SEVER, D.M., et SEMLITSCH, R.D. 1994. Cloacal anatomy of paedomorphic female Ambystoma talpoideum (Caudata: Ambystomatidae), with comments on intermorph mating and sperm storage. Can. J. Zool. 72 : 2147-2157. L'anatomie du cloaque des femelles pedomorphes A'Ambystoma talpoideum a ete etudiee par microscopic photonique et au moyen de techniques histochimiques. Trois types de glandes cloacales (glandes ventrales anterieures, glandes dorsales et spermatheques) ont ete observes. La morphologic des glandes et leur activite secretrice ont ete etudiees sur des echantillons saisonniers de Caroline du Sud. Le developpement maximal des glandes cloacales correspond a une recrudescence de I'activite reproductrice. Les glandes de reserve de spermatozoides, les spermatheques, contiennent du sperme chez les femelles recueillies entre novembre et la fin de fevrier. Tres hypertrophiees au cours de la saison de reproduction, les glandes ventrales anterieures font une secretion continue durant toute I'annee, ce qui indique que ces glandes ne sont pas strictement reliees a la reproduction. 11 y a egalement des spermatozoides dans les glandes dorsales rudimentaires pendant la saison de reproduc• tion, mais leur presence dans ces glandes parait etre accidentelle. Les glandes cloacales des individus pedomorphes sont semblables a celles des salamandres qui se metamorphosent. Les salamandres pedomorphes et les salamandres a metamor• phose peuvent se reproduire entre elles, ce qui assure la survie des deux formes en nature. [Traduit par la Redaction]

Introduction over a 6-year period, with a nearly 1:1 sex ratio each year Ambystoma talpoideum is a common species of salamander (Semlitsch 1987). After the breeding season is finished in in the Atlantic Coastal Plain and Mississippi Valley of the March, metamorphic adults migrate to surrounding terrestrial United States, ranging from Virginia to northern Florida and habitats (a 100- to 300-m distance), where they remain fos- west to Texas, Arkansas, and Tennessee (Shoop 1960; Conant sorial until the next reproductive season. Individuals can breed and Collins 1991). It is a facultatively paedomorphic species more than once in their lifetime (1—6 times) and typically with populations consisting of both terrestrial metamorphic return to the same pond each season (Semlitsch et al. 1993). adults and aquatic paedomorphic adults (Patterson 1978; Female A. talpoideum, like most salamanders, have sperm- Semlitsch 1985a). Both morphs utilize ponds for breeding; storage glands in the walls of their cloaca (Duellman and the terrestrial morphs migrate to sites in South Carolina during Trueb 1986; Sever 1991a). Eggs are fertilized in the cloaca by the winter months (November-January), whereas the paedo• sperm released from the spermathecae during oviposition morphic adults are present in ponds all year (Semlitsch (Boisseau and Joly 1975). Few observations are available on 1985^). Courtship and oviposition by the terrestrial morph the duration of sperm storage both before and after mating. generally occur during December, January, and February Because most ambystomatids migrate from terrestrial habitats (Semlitsch 1985a), but some paedomorphic individuals are to breeding ponds over a period of several weeks, a relatively known to breed earlier in some years (Scott 1993). Females short period of sperm storage probably occurs between mating oviposit up to 1000 eggs {x = 441), which are scattered singly and egg laying. Although retention of sperm in the sperma• across the pond bottom on leaf litter, twigs, and debris thecae after oviposition has been reported for some sala• (Semlitsch 1985(2), but geographic variation in egg-laying manders (Baylis 1939; Houck and Schwenk 1984; Verrell and strategy has also been reported (Semlitsch and Walls 1990). Sever 1988; Massey 1990; Sever 1991^; Sever and Kloepfer Breeding population size at one pond ranged from 333 to 2596 1993), most reports have included no evidence of storage of

Primed in Canada / Imprime au Canada 2148 CAN. J. ZOOL. VOL. 72, 1994 residual sperm between breeding seasons in other species of blue at pH 2.5 (AB, for acid mucosubstances). Strong positive stain• salamanders (Verrell and Sever 1988; Peccio 1992; Sever ing reactions using PTS and AB on cloacal gland secretions appeared 1992a; Sever and Brunette 1993; Sever et al. 1995). Indeed, deep red for mucins and light blue for acid mucopolysaccharides, the spermathecal epithelium may be actively spermiophagic respectively, whereas alternate or neutral staining reactions were light blue and pink, respectively. Some sections from each specimen (Sever 1992a; Sever and Brunette 1993; Sever and Kloepfer were stained with AB at pH 2.5 and then counterstained with periodic 1993). Baylis (1939), however, reported production of suc• acid — Schiff's reagent (PAS, indicative of neutral carbohydrates). In cessive broods of young nearly 2 years apart by an isolated this procedure, PAS-positive substances stain magenta, AB-positive Salamandm salamandra whose last mating was at least 2 — substances stain turquoise-blue, and secretory material that is both 2.5 years before the birth of her second brood. Massey (1990) PAS- and AB-positive is purple (Sheehan and Hrapchak 1980; also reported that female Notophthalmus viridescens retain Kiernan 1990). sperm between successive breeding seasons, although the via• Identification and assignment of cloacal glandular clusters were bility of such sperm was unknown. The only ambystomatid according to Sever (I991fl, \992b). For each specimen, 10 or more species in which the seasonal cycle of sperm storage has been diameters of the largest alveolar portions of each gland were mea• studied is Ambystoma opacum (Sever and Kloepfer 1993; sured (to the nearest 0.1 /xm) using an ocular micrometer. In the case Sever et al. 1995). This species is unique in the family in that of dorsal glands, which are structurally rudimentary and are of breeding occurs on land, and mating may occur before arrival unknown function in many salamanders (Sever 1991a), only selected glandular clusters were examined or measured. The reproductive to the pond and nesting area (Sever et al. 1995). Still, no evi• condition (presence or absence of vitellogenic ova) as well as the dence of mating > 1 month prior to oviposition has been pre• presence or absence of sperm within spermathecae and dorsal glands sented for this species, and sperm persist no longer than were recorded for each female. 6 months after oviposition (Sever et al. 1995). The objectives of the present study were to describe the seasonal variation in cloacal anatomy of female paedomorphic Results A. talpoideum and to determine if paedomorphic females store Gross anatomy sperm for more than one breeding season. The length of sperm We describe the cloacal anatomy of a 51 mm SVL paedo• storage for individual females was not determined; thus, the morphic female Ambystoma talpoideum collected on 25 Novem• length of time sperm were present is a cumulative estimate for ber 1981 from experimental pond A. A midventral incision the population. Our test of sperm storage between breeding (extending from the vent into the cloacal chamber and anter• seasons is conservative and maximizes the opportunity for iorly onto the chest cavity) exposed the preovulatory config• sperm storage because females were collected from the field uration of the internal organs of this gravid specimen. and had had ample opportunity to mate several times or late Upon lifting and reflecting the urinary bladder from its in the breeding season. expanded position (approximately 10 mm anteriorly from its juncture with the cloaca), a highly constricted rectum of the Materials and methods large intestine was observed. The rectum (in this case mosdy Periodic monthly samples of paedomorphic Ambystoma talpoideum devoid of fecal material) was narrowly squeezed (and pushed were taken from October 1980 through November 1983 at four sites ventrally) by enlarged vitellogenic ovarian follicles, whereas on the U.S. Department of Energy's Savannah River Site in Aiken greatly expanded and convoluted glandular oviducts occupied and Barnwell counties. South Carolina (Gibbons and Semlitsch the region distally on either side (posterolaterally). (All speci• 1991). Two additional samples of salamanders were taken in August mens from late November through February possessed vitello• 1985. Collections were made at two permanent natural ponds (Ellen- genic ovarian follicles >2 mm in diameter.) The most posterior ton Bay and Flamingo Bay) known to have large breeding populations segments of the oviducts are narrowed as they extend dorsally of paedomorphic individuals (Patterson 1978; Semlitsch 1985a) and and medially to lie above the rectum before each empties into at two permanent experimental ponds, A and B, also containing the cloacal tube through genital papillae. Mesonephric (opistho- breeding populations of paedomorphic individuals originating from nephric) kidneys reside along the dorsal body wall in close Ellenton Bay and Sun Bay, respectively (Semlitsch and Wilbur 1989). Samples were taken using a dip net or unbailed wire minnow traps. proximity to the oviducts. Salamanders were sacrificed in dilute Chloretone solution in the field Microscopic anatomy and immediately fixed in a solution of 10% neutral buffered formalin. Snout-vent length (SVL; from the tip of the snout to the anterior The major anatomical structures within the cloacal region margin of the vent) of specimens was measured following preserva• (as mentioned above) can also be viewed microscopically via tion and storage in 70% ethanol. a sagittal section as illustrated in Fig. 1, showing the positions Cloacae from 29 females were prepared for histological examination of the three types of cloacal glands relative to one another. The using light microscopy. Blocks of cloacal tissue, including portions following is a descriptive summary detailing the morphologi• of the digestive and urogenital tracts, were dissected from the body cal features of the cloacal complex shown in Figs. 2 and 3 of each female. The tissue blocks were then subjected to routine (from a late-November specimen, No. 58; Table 1), starting histotechniques as outlined in Humason (1979). In brief, tissues were at the level of the juncture between the neck of the urinary dehydrated in a graded series of ethanol and cleared with xylene; this bladder (Ub) and the large intestine (In). was followed by trimming of tissues and infiltration in paraffin for As the Ub and In merge, the anteriormost clusters of 10 h. Paraffin embedding in plastic molds allowed for the orientation anterior ventral glands (Av) are found squeezed between of tissue masses to enhance both transverse and sagittal sectioning of paraffin blocks. Blocks were trimmed of excess paraffin, and ribbons bands of abdominal musculature (Mu) and the abdominal of material, serially sectioned using a rotary microtome, were affixed cavity (Fig. 2A). Secretions fill the lumina of most of these to slides using Haupt's adhesive. Slides were then alternately stained glands (see Cloacal glands and their secretions). Dorsolateral with hematoxylin and eosin (H&E, for general cytology), Pollac to the Ub are the narrowed posterior segments of the oviducts trichrome stain (PTS, for connective tissues and mucins), and Alcian (Ov*; Figs. 2A, 2D). The internal lining of these mostly circu- TRAUTH ET AL. 2149

FIG. 1. Cloacal anatomy (sagittal section) of a paedomorphic female Ambystoma talpoideum collected in late November, showing the cloacal glands at their maximum development (using Pollac trichrome stain). Av, anterior ventral glands; Ct, cloacal tube; Dg, dorsal glands; Gp, genital papillae; In, large intestine; Lf, lateral fold of the cloacal tube; Me, mesonephric kidney; Ov, oviduct (glandular portion); St, spermathecae; Ub, neck of urinary bladder; V, vent. The asterisk indicates the opening of the oviduct into the cloacal chamber. lar tubes takes on a rugose configuration compared with the peared within the abdominal cavity (Ac). The cloacal tube (Ct) anterior-gland-laden portions of the oviducts (Ov). A ciliated lacks the profusion of goblet cells that characterizes the In, but columnar epithelium characterizes the lining of the rugae. retains the convoluted walls. The anterior ventral glands are Paired archinephric ducts (Ad) of the mesonephric kidney now more conspicuous beneath the Ct. (Me), are positioned dorsally and dorsolaterally, respectively, The pair of genital papillae (Gp) hangs downward into the to the Ov*. The intestinal epithelium exhibits a pseudostrati- Ct (Figs. 2C-2F, starting approximately 100 /tm posterior to fied columnar cell layer dominated by numerous round goblet the end of the section in Fig. 2B). The papillae open separately cells. into the Ct; the left side (Fig. 2D) empties anterior to the right In Fig. 2B, taken at a point 320 /im posterior to the section (Fig. 2F). The passageway between the Ov* and the Ct (as shown in Fig. 2A, the glandular oviducts have mostly disap• shown in Figs. 2D and 2E) is lined with a ciliated, pseudo- 2150 CAN. J. ZOOL. VOL. 72, 1994

FIG. 2. Anterior to posterior transverse sections through the cloacal region of a paedomorphic female Ambystoma talpoideum collected in late November. See the text for an explanation of abbreviations and symbols. Scale bar (A-F) = 1 mm. (A) Section through the anterior region of the cloacal complex, showing the structures at the juncture between the urinary bladder and the large intestine. H&E stain. (B) Section showing the beginning of the cloacal tube. AB stain. (C) Section showing a genital papilla inside the cloacal tube. AB stain. (D) Section in the region of the genital papillae, showing a common pathway betwen the left oviduct and the cloacal tube. H&E stain. (E) Section stained with PTS, showing both genital papillae at the level of the anterior groove of the vent. (F) Section revealing the beginning of the cloacal chamber above the vent opening. AB stain. stratified columnar epithelium, as is most of the midventral exhibits a stratified cuboidal epithelium three or four cell floor of the Ct. In Fig. 2E, the more lateral surfaces of the layers in thickness. Ct are mostly composed of nonciliated, low, simple columnar In Fig. 2F (a region approximately 0.5 mm posterior to the cells. On the other hand, the external surfaces of the Gp end of the section in Fig. 2D), the anterior cloacal chamber TRAUTH ET AL. 2151

FIG. 3. Anterior to posterior transverse sections through the cloacal region of a paedomorphic female Ambystoma talpoideum. See the text for an explanation of abbreviations. Scale bar (A-E) = 1 mm. (A-E) Continuation of Fig. 2. (A) In this section the right archinephric duct empties into the cloacal tube. At the same time, the anterior ventral glands separate into lateral clusters within the cloacal lips. AB stain. (B) Section showing lateral diverticula and folds of the cloacal tube at the same level when spermathecae initially appear above the cloacal tabe. PTS stain. (C) In this section the cloacal tube and cloacal chamber merge, resulting in a continuous growth from the vent to the openings of the spermathecal tubules. PTS stain. (D) Section taken in the posterior region of the cloaca, showing the anterior ventral glands opening into the cloacal chamber. AB stain. (E) Section near the posterior wall of the cloaca, showing large numbers of spermathecae and the lack of anterior ventral glands. Note the presence of a bulging mass of tissue within the cloacal chamber; this bulge possesses openings into the dorsal glands. H&E stain. (F) Sagittal section of the cloacal complex of a late-November specimen, showing the relative positions of the three types of cloacal glands. In this section, the dorsal glands contain sperm. PTS stain. Scale bar = 300 fim. 2152 CAN. J. ZOOL. VOL. 72, 1994

TABLE 1. Seasonal variation in diameter (/tm) of cloacal glands in paedomorphic female Ambystoma talpoideum

Spermathecae Dorsal glands Anterior ventral Date of Specimen SVL Sperm glands Sperm collection No. (mm) J ± 2 SE present? (J + 2 SE) J + 2 SE present? 23 Jan. 1981 2 49 129.9 + 19.4 Yes 168.4 + 11.1 — Yes 24 Jan. 1982 4 42 127.3+9.8 Yes 145.3 + 12.9 — — 21 Feb. 1981 5 39 101.5±6.8 Yes 111.5 + 10.3 — — 21 Feb. 1981 7 49 — Yes 127.9+29.0 — — 21 Feb. 1981 8 46 102.3 + 15.0 Yes 149.2+17.3 — — 19 Apr. 1981 13 43 70.4+7.9 No 78.1+9.4 — — 4 May 1983 18 37 55.0 + 8.0 No 67.3±5.2 — — 15 May 1981 20 39 44.1±3.3 No 54.2+5.2 32.7+4.3 No 15 May 1981 22 42 33.4+4.8 No 46.1+4.3 — — 9 June 1983 25 40 — 59.2+9.3 — — 9 June 1983 28 42 30.7+2.3 No 46.5±4.5 — — 9 June 1983 30 41 36.1+3.7 No — — — 26 July 1983 34 48 38.8+4.2 No 34.6 + 3.1 — — 26 July 1983 37 46 37.7 + 3.0 No 58.1+3.7 — — 18 Aug. 1983 38 43 43.4+4.7 No 30.8 + 3.0 — — 18 Aug. 1983 39 45 42.7 + 3.3 No 34.6+5.6 — — 6 Sept. 1983 47 47 50.0+3.8 No 43.1+3.9 — — 6 Sept. 1983 48 44 45.4 + 3.0 No 60.4+3.4 — — 6 Sept. 1983 50 52 72.7+7.7 No 70.7+4.3 — — 18 Oct. 1980 52 39 59.2+3.7 No 77.7±6.6 — — 17 Nov. 1981 54 42 93.8+15.0 No 89.2+4.6 40.8±3.5 No 25 Nov. 1983 57 49 130.8+14.7 Yes 141.9 + 18.1 57.3+5.1 Yes 25 Nov. 1983 58 55 116.1 + 11.0 Yes 116.9+11.7 — 25 Nov. 1983 59 52 106.9 + 19.1 Yes 140.2 + 17.4 — — 25 Nov. 1983 60 52 136.5 + 14.1 Yes 145.0+16.7 56.7+5.0 Yes 25 Nov. 1983 63 48 115.4+19.8 Yes 122.7 + 11.0 50.8+7.7 Yes 9 Dec. 1980 65 50 105.4+9.7 Yes 141.9+11.6 49.2+6.7 Yes 9 Dec. 1980 69 42 109.2 + 15.8 Yes 113.8 + 16.7 45.4+5.6 Yes 16 Dec. 1981 70 45 75.8 + 8.0 Yes 136.5 + 17.4 62.3+7.5 Yes

(Cc) makes its first appearance just above the anterior margin marked by the presence of numerous ductal openings into dor• of the vent (V). The Cc, lined with stratified squamous cells, sal glands (Dg; see Fig. 1). The Av, so prevalent within the receives secretions from the Av. anterior ventral cloacal wall and the anterior cloacal lips, have The right Ad can be seen emptying into the Ct in Fig. 3A all but disappeared. Although the St are quite numerous where (a continuation of Fig. 2 at a point approximately 450 ^m they are embedded in the roof of the posterior cloacal cham• posterior to Fig. 2F). In Fig. 3B, the first spermathecae (St) ber, none of their ducts open into this region of the cavity. The appear at around 180 fim posterior to Fig. 3A. The floor of the separation between the Dg and the St is revealed in Fig. 3F; Ct has now begun to expand laterally to form distinct folds and sperm are present in the Dg of Figs. 1, 3E, and 3F. The entire recesses. When the latter occurs, as in Fig. 3C, the Av are cloacal complex, measured from Figs. 2A through 3E, is divided into two separate lateral clusters within the cloacal roughly 2.2 mm in length. lips. The Cc also exhibit conspicuous foldings as this cavity begins to push dorsally to meet the Ct (Fig. 3B). The Ct retains Cloacal glands and their secretions prominent lateral folds (Lf) as well as a mid-dorsal central In terms of structural design, two of the three types of cloacal groove (Cg), as shown in Fig. 3C. This region communicates glands (i.e., Av and St) of paedomorphic A. talpoideum are of with all of the St (also shown in Fig. 3D). The Ct and Cc the simple, tubuloalveolar, exocrine variety (Sever 1992^); merge soon after the appearance of the St. each exhibited tubules having an expanded secretory distal The paired lateral folds of the cloacal tube (Lf; as viewed portion and a narrowed proximal duct. The Dg differ slightly in Fig. 3D at a point 300 /xm posterior to Fig. 3C) are retained from these two by generally lacking expanded alveolar laterally within the posterior cloacal walls. Each Lf continues regions. The method of secretion is similar in all three types; to possess a ciliated, pseudostratified columnar epithelium, Av liberate in a more or less continuous manner (merocrine whereas both the Cg (lined with a stratified transitional mode of secretion), and their secretions accumulate as globu• epithelium of low columnar cells) and the Cc (lined with strati• lar masses within the distal lumina. Secretions by the St are fied squamous epithelium) lack cilia. Within the posterior also of the merocrine type; however, these substances were cloacal chamber in Fig. 3E (approximately 300 iim posterior discharged in a manner that resulted in distal segments having to Fig. 3D), the Cg has been lost and the grooves within the a thin-walled, evacuated, vesicular appearance (Figs. 1 and 4). Lf have extended ventrally to surround an outpocketing of the When the St exhibited an extremely festooned morphology, posterior cloacal wall. This bulge can be observed lying their secretions may have been absent or may have only par- directly beneath the Lf in Fig. 1. Moreover, this region is fially filled portions of the distal regions, yet their proximal TRAUTH ET AL. 2153

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FIG. 4. Cloacal anatomy (transverse section) of a paedomorphic female Ambystoma talpoideum collected in late November showing well- developed spermathecae and anterior ventral glands. Cc, cloacal chamber; Cg, central groove (for explanations of other abbreviations see Fig. 1). 2154 CAN. J. ZOOL. VOL. 72, 1994 TRAUTH ET AL. 2155 duct regions usually possessed secretory materials throughout morphs does not occur on the basis of cloacal anatomy. More• their entire length. The Dg have no secretory products within over, this suggests that any reproductive isolation can occur the lumina. only through other mechanisms such as assortative mating or At the peak of reproductive activity, which corresponds to temporal isolation. The ability to interbreed also has important maximal cloacal gland development, a gravid January speci• ecological implications if offspring fitness is increased by men (Table 1; No. 2) possessed Av that extended through occasional interbreeding of morphs. For example, if a paedo• about 2.3 mm of cloacal tissue. Approximately 80-100 glands morphic female produces more metamorphic offspring by composed the anteriormost Av cluster of distal segments mating with a metamorphic male rather than with a paedo• (Fig. 2D); the majority of these segments contained a globular morphic male, she increases the probability that offspring will secretion (H&E neutral, PTS- and PAS-positive, AB-negative), survive in a drying pond during a drought year. Thus, similar• whereas the lumina of the proximal ducts were devoid of ity in cloacal anatomy enhances the possibility of interbreeding materials. Droplets within the cytoplasm of the simple cuboidal of morphs under certain evironmental conditions and could epithelium lining the distal segments revealed a combination facilitate the maintenance of both morphs through alternating of staining reactions from faintly red to mostly blue—green, selection on mate choice. whereas the ductal epithelium appeared purple with PTS. The The continuation of secretory activity in the anterior ventral 100—120 St with sperm amassed within their secretions (H&E glands may indicate their importance in activities other than neutral, PTS- and PAS-negative, AB-positive) were observed breeding. Sever (1988) hypothesized that the ventral glands emptying into the Cg. Both the cuboidal epithelium and the secrete a pheromone and that this may be the ancestral func• secretions of the St were blue, whereas in the same region Av tion of the cloacal glands. We speculate that the anterior ven• secretions were light pink. Within the posterolateral wall of tral glands of A. talpoideum may be involved in territorial the cloaca, there was a sharp demarcation line between the marking or individual recognition throughout the year. The narrow proximal ducts of the St and the Dg based upon their continued secretory activity, even at a reduced level, may be staining reactions. About 100 St resided above the 75—90 Dg. what is expected of glands of general importance in daily Although the Dg also exhibited sperm, their epithelial linings activity, but could have added significance (as when they were PTS-positive, PAS-positive (slight), and AB-negative. hypertrophy) during a certain time of the year. Recent behav• The PAS reaction in Av was very strong. During the ioral studies of aggression and defense of resident sites (e.g., November-February period, when the glands were hyper• refugia, burrows) by metamorphic ambystomatid salamanders trophied, the apical cytoplasm as well as the lumen contained suggest that chemical cues may be important (Ducey and much secretory material, and the luminal masses were in the Ritsema 1988; Walls 1990). Walls and Roudebush (1991) also form of globules (Fig. 5). But from March to August there was demonstrated kin recognition in A. opacum, which also sug• a less intense reaction in the apical cytoplasm (a spotty appear• gests that secretory substances might be important for behav• ance), whereas the globules were absent; the lumina were nar• ioral interactions of larvae during foraging. row. The St were intensely AB-positive in specimens collected We found that some sperm also occur in the dorsal glands. from November to February, with positively staining materials The only other report of sperm occurring in cloacal glands in either the cytoplasm or the lumen (Fig. 5). From March to other than spermathecae was by Hardy and Dent (1986), who July, only a slight AB-positive reaction occurred in the apical found sperm in the ventral glands of the newt N. viridescens cytoplasm. The Dg gave a scant (slight) reaction with PAS (which lacks dorsal glands). They proposed that sperm move around the luminal borders. There was very little change in thigmotactically along the cloacal epithelium and, upon secretory activity of the Dg throughout the year. encountering the openings of a gland, are carried through these pores by a continuation of the thigmotactic response. Few sperm were found in the ventral glands of N. viridescens, Discussion however, possibly because their lumina were occluded with The overall cloacal anatomy of paedomorphic Ambystoma secretory products (Hardy and Dent 1986). In A. talpoideum, talpoideum was similar to that of a metamorphosed female the dorsal glands were largely posterior to the spermathecae, briefly described by Sever (1992^), except that the central and some sperm would encounter the openings of these glands groove in the paedomorphs extended farther into the cloacal before those of the spermathecae. Entry into the dorsal glands chamber. The staining reactions and cytology of the glands might be restricted, however, by their more minute pores and were also similar, suggesting similar functions. Licht and narrow lumina. No one has provided evidence that the sperma• Sever (1991) reported no difference in cloacal anatomy between thecae secrete a substance that attracts sperm. metamorphosed and paedomorphic Ambystoma gracile and Our results show that sperm are present in the spermathecae concluded that the two morphs of that species should be cap• of all females during the breeding season (November-March) able of interbreeding. Our data suggest that this is true for and that sperm storage may occur for a period up to 5 months. A. talpoideum also, and that reproductive isolation between No females contained sperm during the nonbreeding period

FIG. 5. Histosections showing seasonal variation in the spermathecae (A-E) and anterior ventral glands (F—J) in paedomorphic female Ambystoma talpoideum. Scale bar in A and F are the same for A-E and F-J, respectively. (A) Late-November specimen exhibiting masses of sperm (Sp) within tubules. Mc, melanocyte. PTS stain. (B) Relatively few sperm (Sp) are present in a mid-February specimen. AB stain. (C) Regressed glands of an early May specimen; no sperm are present. PTS stain. (D) Hypertrophy of epithelial cell nuclei in a September specimen. H&E stain. (E) Glandular activity in a specimen collected in mid-October reveals cytological preparation for the reception of sperm. AB stain. (F) Maximal secretory activity in late-November specimen, showing globular masses (arrowheads) within the lumina of glands. PTS stain. (G) Highly regressed condition of the glands in early May. H&E stain. (H) Total regression in August specimen; numerous melanocytes (Mc) are interspersed among the glands. H&E stain. (I) Recrudescence of glands in September; epithelial cell hypertrophy is evident. PTS stain. (J) September specimen exhibiting increased cytological activity within the glands. AB stain. 2156 CAN. J. ZOOL. VOL. 72, 1994

(May—October). This is a maximal estimate for sperm storage Baylis, H.A. 1939. Delayed reproduction in the spotted salamander. because individual females were not followed over the breed• Proc. Zool. Lond. Sect. A, 109: 243-246. -"^ ing season. We suspect that the actual time for which individ• Birkhead, T.R., and Moller, A.P. 1982. Sperm competition in birds: uals can store sperm is considerably less. A recent study of a evolutionary causes and consequences. Academic Press, London. Boisseau, C., and Joly, J. 1975. Transports and survival of sper• closely related species, A. opacum, indicates that sperm begin matozoa in female Amphibia. In The biology of spermatozoa: to degenerate within a month, and none persist for more than transport, survival and fertilizing ability. Edited by E.S.E. Hafex 6 months after oviposition (Sever et al. 1995). and CG. Thibault. Karger, Basel, pp. 94-104. The presence of sperm storage for even a short time has Conant, R., and Collins, J.P. 1991. A field guide to reptiles and direct implications concerning sperm competition and sexual amphibians of eastern and central North America. Houghton selection. Sperm competition is highly probable if females Mifflin Co., Boston. (Parker 1970; Houck and Schwenk 1984) (i) mate more than Ducey, P.K., and Ritsema, P. 1988. Intraspecific aggression and once before the eggs are fertilized, (//) store inseminated responses to marked substrates in Ambystoma maculatum sperm, (///) retain inseminated and viable sperm for longer (Caudata: Ambystomatidae). Copeia, 1988: 1008-1013. than one breeding season, or (iv) use sperm sparingly. For Duellman, W.E., and Trueb, L. 1986. Biology of amphibians. McGraw-Hill, New York. birds (Birkhead and Moller 1992), it has been proposed that Gibbons, J.W., and Semlitsch, R.D. 1991. Guide to the reptiles and the presence of sperm storage may compensate for the lack of amphibians of the Savannah River Site. University of Georgia synchrony between copulation and fertilization in the mating Press, Athens. system, or may simply be the phylogenetic consequence of a Hardy, M.P., and Dent, J.N. 1986. Transport of sperm within the particular ancestry (i.e., reptilian in the case of birds). For cloaca of the female red-spotted newt. J. Morphol. 190: 259-270. salamanders, the presence of sperm storage could be advan• Harris, R.N. 1980. The consequences of within-year timing of breed• tageous when the probability of finding a mate each year is ing m Ambystoma maculatum. Copeia, 1980: 719 — 922. low, as in fossorial, nonmigrating species (i.e., some pletho- Humason, G.L. 1979. Animal tissue techniques. 4th ed. W.H. dontid salamanders). Sperm storage in A. talpoideum and Freeman, San Francisco. other migrating species is probably not necessary to ensure Houck, L.D., and Schwenk, K. 1984. The potential for long-term fertilization each year because of the high concentration of sperm competition in a plethodontid salamander. Herpetologica, 40: 410-415. individuals at breeding ponds. We suggest that short-term Kiernan, J.A. 1990. Histological and histochemical methods. 2nd ed. sperm storage like that found in ambystomatid salamanders Pergamon Press, Oxford, U.K. may be advantageous in allowing environmentally dependent Licht, L.E., and Sever, D.M. 1991. Cloacal anatomy of metamor• oviposition in aquatic habitats. Once inseminated, females can phosed and neotenic salamanders. Can. J. Zool. 69: 2230-2233. wait until aquatic conditions are optimal to maximize the sur• Massey, A. 1990. Notes on the reproductive energy of red-spotted ^ vival of embryos and larvae in the pond. For instance, ovi• newts (Notophthalmus viridescens). Copeia, 1990: 106—107. , positing too early in the season can result in the loss of eggs Parker, G.A. 1970. Sperm competition and its evolutionary conse• due to pond freeze (Harris 1980; R.D. Semlitsch, unpublished quences in the insects. Biol. Rev. Camb. Philos. Soc. 45: 525- data). The decoupling of insemination from oviposition facili• 568. tates reproductive success in variable aquatic environments such Patterson, K.K. 1978. Life history aspects of paedogenic populations as ponds. of the mole salamander, Ambystoma talpoideum. Copeia, 1978: 649-655. However, as has been found in other species, any sperm Peccio, A. 1992. Insemination and egg laying dynamics in the storage increases the probability of sperm competition (Birk• smooth newt, Triturus vulgaris, in the laboratory. Herpetol. J. 2: head and Moller 1982). At present, it is unknown whether 5-7. female A. talpoideum pick up more than one spermatophore or Scott, D.E. 1993. Timing of reproduction of paedomorphic and mate with more than one male, thereby producing clutches fer• mtXwiovphic Ambystoma talpoideum. Am. Midi. Nat. 129: 397- tilized by more than one male (but see Arnold 1977). Research 402. on these and related topics of sperm release from the sperma• Semlitsch, R.D. 1985a. Reproductive strategy of a facultatively thecae or dorsal glands should increase our understanding of paedomorphic salamander ^mi^yifoma talpoideum. Oecologia, 65: the mating system and sexual selection in this facultatively 305-313. Semlitsch, R.D. 1985/). Analysis of climatic factors influencing paedomorphic species. migrations of the salamander Ambystoma talpoideum. Copeia, 1985: 477-489. Acknowledgements Semlitsch, R.D. 1987. Relationship of pond drying to the reproduc• tive success of the salamander Ambystoma talpoideum. Copeia, We thank J.W. Gibbons for his help and permission to col• 1987: 61-69. lect specimens at Flamingo Bay and Ellenton Bay. Collection Semlitsch, R.D., and Walls, S.C. 1990. Geographic variation in the of salamanders was supported by Department of Energy con• egg-laying strategy of the mole salamander, Ambystoma tal• tract DE-AC09-76SR00-819 with the University of Georgia's poideum. Herpetol. Rev. 21: 14-15. Savannah River Ecology Laboratory. Support was provided to Semlitsch, R.D., and Wilbur, H.M. 1989. Artificial selection for S.E.T. by the Department of Biological Sciences, Arkansas paedmorphosis in the salamander Ambystoma talpoideum. Evolu• tion, 43: 105-112. State University, and to D.M.S. by National Science Founda• Semlitsch, R.D., Scott, D.E., Pechmann, J.H.K., and Gibbons, J.W. tion grant DEB-9024918. 1993. Phenotypic variation in the arrival time of breeding ^ salamanders: individual repeatability and environmental influ- ^ ences. J. Anim. Ecol. 62: 334—340. Arnold, S.A. 1977. The evolution of courtship behavior in New Sever, D.M. 1988. The ventral gland in female salamander Eurycea World salamanders with some comments on Old World sala• bislineata (Amphibia: Plethodontidae). Copeia, 1988: 572-579. manders. In The reproductive biology of amphibians. Edited by Sever, D.M. 1991a. Comparative anatomy and phylogeny of the D.H. Taylor and S.I. Guttman. Plenum Press, New York, cloacae of salamanders (Amphibia: Caudata). I. Evolution at the pp. 141-183. family level. Herpetologica, 47: 165 — 193. TRAUTH ET AL. 2157

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