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Sperm Aggregations in the Spermathecae of Southern Torrent Salamanders, Rhyacotriton variegatus Author(s): David M. Sever, Cynthia K. Tait, Lowell V. Diller and Laura Burkholder Source: Journal of Herpetology, Vol. 38, No. 1 (Mar., 2004), pp. 61-72Published by: Society for the Study of Amphibians and ReptilesStable URL: http://www.jstor.org/stable/1566087Accessed: 30-06-2015 15:31 UTC

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Journal of Herpetology, Vol. 38, No. 1, pp. 61-72, 2004 Copyright 2004 Society for the Study of Amphibians and Reptiles

Sperm Aggregations in the Spermathecae of Southern Torrent Salamanders, Rhyacotriton variegatus

DAVID M. SEVER,"2 CYNTHIA K. TAIT, LOWELL V. DILLER,4 AND LAURA BURKHOLDER4

tDepartment of Biology, Saint Mary's College, Notre Dame, Indiana 46556, USA 3Bureau of Land Management, Vale, Oregon 97918, USA

4Simpson Timber Company Arcata, California 95521, USA

ABSTRACT.-Female salamanders in the suborder Salamandroidea store sperm in cloacal glands called spermathecae. Scanning electron microscopy was used to study these glands in females of the Southern Torrent Salamander, Rhyacotriton variegatus (Rhyacotritonidae), from northern California. Sperm initially enter the cloaca in a tangled mass from the spermatophore cap, but within the spermathecal tubules, small groups become aligned along their long axes. Sperm nuclei typically are embedded in apical microvilli of the secretory cells forming the distal acini of the spermathecae. Junctional complexes between apices of adjacent epithelial cells are occasionally broached or otherwise absent, and sperm can be found in the intercellular canaliculi between such cells. Spermathecae of R. variegatus are simple glands that branch off a tube

extending dorsally into cloacal tissue. This spermathecal anatomy is in-between that of the Plethodontidae, which have a compound tubuloaveolar gland as the single spermatheca, and other salamandroids, in which sperm storage occurs in numerous simple tubular glands, each one a spermatheca. Sperm associations in the simple spermathecae of R. variegatus, however, are most similar to those reported for plethodontids.

Rhyacotriton variegatus is one of four species in the salamander family Rhyacotritonidae, which is restricted to the Pacific Northwest region of the United States (Good and Wake, 1992). Sister- group relationships to other salamander families are obscure (Larson et al., 2003). The common name "torrent salamander" is appropriate because these animals typically occupy the edges of swift-flowing, permanent streams (Petranka, 1998). Like the other six families in the suborder Salamandroidea, females of the Rhyacotritonidae store sperm in specialized cloacal glands called spermathecae (Fig. 1), and the literature on this subject was most recently reviewed by Sever (2003). The spermathecae of R. variegatus were studied using light microscopy (LM) by Sever (1992) and with transmission electron microscopy (TEM) by Sever et al. (2004).

The spermathecae of R. variegatus consist of simple tubuloalevolar glands that branch from an invagination, called the dorsal tube, into the roof of the anterior cloaca (Fig. 1B). The cloacal cavity is lined with stratified, keratinized epithelium continuous with the epidermis of the surrounding skin. As the dorsal tube invaginates, this lining changes into simple columnar muci- nogenic cells, and this lining continues into the proximal neck tubules. The distal bulbs, where sperm are stored, are columnar when active and cuboidal when quiescent (after the breeding

2 Corresponding Author. E-mail: dsever@saintmarys.

edu

season) and produce a carbohydrate secretion that differs histochemically from the more proximal tubules (Sever et al., 2004).

In their TEM study, Sever et al. (2004) examined 25 specimens from Humboldt County, California, that were collected in February, April, June, September, and November 1999-2002. Most females collected from February to June with large vitellogenic follicles (2.0-3.9 mm diameter) contained sperm in their spermathecae. Mature-size females with small ovarian follicles (0.9-1.2) lacked stored sperm and were considered "first yolkers," females reaching sexual maturity and yolking eggs for the first time but not yet ready to breed. In females storing sperm, some distal bulbs lacked sperm, whereas others were packed. Thin and thick sections did not appear to show that the sperm were aligned in any orderly fashion. In a few instances, sperm were observed embedded in the apical cytoplasm or between epithelial cells in the intercellular canaliculi, and this was considered evidence of spermiophagy (Sever et al., 2004).

The present study extends the observations of Sever et al. (2004) by using scanning electron microscopy (SEM) to examine the spermathecae of six females (three gravid and three first yolkers) collected in March 2001 from the same locality used in our previous study. SEM may reveal patterns of organization within a sperm mass that are not easily discernible with thin or thick sections. Previous SEM studies on sperm storage in salamanders have shown that some species store sperm in spaghetti-like tangles,



D. M. SEVER ET AL.

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Od

10 mm

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FIG. 1. Rhyacotriton variegatus. (A) Ventral view showing the reproductive tract of a 60.8 mm SVL female collected 10 April 2001 with ovarian follicles 3.5 mm mean diameter. Modified from Sever et al. (2004). (B) Midsagittal paraffin section through the cloacal region of a female showing general histology of the spermathecae

!

62



SPERMATHECAE OF TORRENT SALAMANDERS

whereas in others, sperm occur in small clusters aligned along their longitudinal axes (Sever, 2003). Finally, SEM could help resolve the issue of sperm sections that appear embedded in the apical cytoplasm or in intercellular canaliculi, that is, are sperm actually penetrating cellular junctions?

MATERIALS AND METHODS

The six specimens of R. variegatus used specifically in this study were collected on 21 March 2001 and sacrificed and preserved within 24 h of collection. The locality for this sample was a tributary of the North Fork Mad River on lands owned by the Simpson Timber Company in western Humboldt County, California. A permit for the collection was granted to LVD from the California Department of Fish and Game. Speci- mens were killed by immersion in 10% aqueous solution of MS-222 (3-aminobenzoic acid ethyl ester). The Animal Care and Use Committee of Saint Mary's College approved the protocol.

The specimens included three individuals with large, vitellogenic follicles and three first yolkers (Table 1). After excising the spermathecae and surrounding tissue, this area was either cut frontally, transversely or sagittally in one female from each group. One of the halves was prepared for examination by light microscopy (LM) using paraffin infiltration, and the other half was prepared for examination by SEM. We used procedures established in Sever's lab at Saint Mary's College over the past 30 years and did not consult references for additional techniques.

For LM, the tissue was fixed in neutral buffered 10% formalin, rinsed in water, dehydrated in ethanol, cleared in toluene, and embedded in paraffin. Sections (10 plm thickness) were cut with a rotary microtome, affixed to albuminized slides, and stained with hematoxylin-eosin.

For SEM, tissue was fixed in a 1:1 solution of 2.5% glutaraldehyde and 3.7% formaldehyde in a cacodylate buffer at pH 7.2. After initial fixation, tissues were rinsed in distilled-deion- ized water, postfixed in 2% osmium tetroxide and subjected to critical point drying. The tissue was then mounted on a metal stub with adhesive tape and sputter-coated with gold in a Denton Desk II. The specimens were examined with a JEOL JSM-T300 scanning electron microscope.

Some LM and TEM results from Sever et al. (2004) have been incorporated into the present study because they complement the SEM obser-

TABLE 1. Female Rhyacotriton variegatus used in this

study. Measurements of SVL and the range and mean of follicle diameter are in mm. N = number of yolked follicles; Cond = condition, either G, gravid or FY, first

yolking; Sperm are either present (+) or absent (-).

Follicles

SVL N Range Mean SE Cond Sperm 46.1 5 1.1-1.3 1.1 0.05 FY + 46.6 8 1.2-1.7 1.4 0.05 FY 46.7 8 2.1-3.0 2.8 0.11 G + 47.0 5 1.0-1.3 1.1 0.05 FY + 47.1 7 2.8-3.2 2.9 0.06 G + 50.1 9 1.2-2.8 1.9 0.22 G

vations. See that paper for details on specimen preparation for TEM.

RESULTS

Two of the gravid females possessed stored sperm, and one did not. The latter specimen contains yolked eggs and flaccid, convoluted oviducts and is considered in breeding condition but apparently had not yet mated. This is not unusual; several gravid females from the study by Sever et al. (2004) were also gravid but unmated. However, two of the three first yolkers also had some sperm in their spermathecae, and sperm were not found in any of the first yolkers examined by Sever et al. (2004). In this study, no sperm were observed in the 46.1 mm SVL first yolker by SEM examination, but a small amount was found in some tubules in sections prepared for LM. Sperm were seen by SEM examination in the other first yolker that had stored sperm, but sperm in neither of the first yolkers are as numerous as in the gravid females.

Ultrastructure of the Spermathecal Epithelium.- Figure 2 shows an overview of the spermathecal region and the branching of the distal bulbs from the neck tubules in first yolkers that contained some sperm. The radiating pattern of the neck tubules from the dorsal tube provides numerous options for migrating sperm (Fig. 2A). The cornified epidermis of the lower dorsal tube and cloacal orifice lacked any surface features and became reduced to one layer prior to the branching of the neck tubules (Fig. 2B).

The epithelium of all regions of the spermathecae produce a mucinous secretory product (Sever et al., 2004). The layer of secretory products on the luminal surface of the neck

and surrounding cloacal structures. Section stained with hematoxylin-eosin and modified from Sever (1992) and Sever et al. (2004). Ct, cloacal tube; Co, cloacal orifice; Cs, cloacal sheath; Db, distal bulb; Dt, dorsal tube; Nt, neck tubules; Od, oviduct; Ov, ovary; Sc, spermatophore cap; Sp, sperm; St, spermathecae.

63



D. M. SEVER ET AL.

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tubules often appeared more uniform and thicker than in the distal bulbs, where thin layers of

secretory material conformed closely to the outline of the columnar cells (Fig. 3A). Indeed, as reported by Sever et al. (2004), the lumen of some neck tubules may be occluded with secretory products (Sm, Fig. 3B). In the distal bulbs of one first yolker, separate globules of secretory material were prominent and in some instances were associated with sperm (Fig. 3C). With TEM, small groups of sperm were often observed associated with the merocrine blebs (Sb, Fig. 3D) that are involved in the release of the product (Sever et al., 2004).

Sperm in the Cloacal Orifice.-A 46.7 mm gravid female had the remnants of a spermatophore in her cloacal orifice, indicating a recent mating (Fig. 4). Secretory material formed globs in the sperm mass (Fig. 4A-B). This material was apparently from the spermatophore cap, because the epidermis does not produce a secretion, and there is no evidence that any material passed down from the spermathecae into the cloacal orifice at this time. The sperm were in wavy masses that did not show a consistent alignment, as nuclear ends and tail pieces were intertwined together (Fig. 4C).

Sperm in the Distal Bulb.-TEM indicated that sperm do not exhibit an orderly alignment in the distal bulbs, although small groups of sperm are aligned in parallel along their axes (Sever et al., 2004). This condition was evidenced by thin sections that showed regions of the lumen containing clusters of sperm nuclei or tail pieces (Fig. 5A). As also found with TEM, SEM revealed that one area of a distal bulb may possess a sperm mass whereas other areas were empty (Fig. 5B). An overview of the sperm mass with SEM also did not reveal much order (Fig. 5B), but closer examination revealed that smaller groups of sperm within the mass exhibited similar orienta- tions (Fig. 5C). Clusters of sperm nuclei were frequently found embedded in the microvilli of the spermathecal epithelium (Fig. 5D).

TEM studies have revealed portions of sperm in the intercellular canaliculi and, in several cases, in the tunica propria superficial to the spermathecae, apparently after desquamation of epithelial cells (Fig. 6A-B; Sever et al., 2004). With SEM, we observed an instance where portions of three sperm cells were extended into an in-

tercellular gap between several epithelial cells

(Fig. 6C). Thus apical junctional complexes were absent or leaky between these cells. In this area, secretory blebs were numerous, but microvilli were not prominent.

DISCUSSION

In the salamander families Ambystomatidae, Amphiumidae, Dicamptodontidae, Proteidae, and Salamandridae, sperm storage occurs in numerous simple tubular cloacal glands, each one of which is a spermatheca. Females in these groups possess what are termed "simple spermathecae" (Sever, 1994). In the Plethodontidae, sperm storage occurs in a single compound tubuloalevolar gland, designated the "complex spermatheca." In a complex spermatheca, a rela- tive long and narrow common tube of stratified epithelium extends from a middorsal papilla into the dorsal roof of the cloaca and distally branches into narrow neck tubules that expand into alveolar distal bulbs (Sever, 2003).

The cloacal sperm storage glands in females of the Rhyacotritonidae are considered simple spermathecae (Sever, 2003). The dorsal tube is not as narrow as the common tube of plethodontids, and spermathecae evaginate from the entire upper half of the dorsal tube, whereas in plethodontids, neck tubules extend only from the apex of the common tube (Sever, 1992). Nevertheless, the spermathecae of rhyacotritonids are the most specialized in salamanders except for the Pletho- dontidae. Rhyacotritonid spermathecae can be considered "in-between" the anatomical complexity of the two main types of spermathecae.

Salamanders with internal fertilization consti- tute the suborder Salamandroidea. The presence of cloacal sperm storage in females is considered a synapomorphy for the Salamandroidea (Larson et al., 2003; Sever, 2003), but the phylogeny of subsequent diversification of spermathecae is unknown (Sever, 1994). However, any similari- ties between the complex spermatheca of plethodontids and the spermathecae of R. variegatus are most likely convergent. Rhyacotritonidae is an ancient clade that has no close relatives (Larson et al., 2003).

Sperm aggregations in simple spermathecae previously have been studied by ultrastructure in four families. In Ambystoma tigrinum of the Ambystomatidae (Sever, 1995), Necturus beyeri

FIG. 2. Rhyacotriton variegatus. SEM of spermathecae of two first yolkers in similar reproductive condition. Both had five ovarian follicles 1.1 mm mean diameter and contained some stored sperm in the spermathecae. (A) Overview of the spermathecal region showing tubular arrangement in a 46.1 mm SVL female. (B) The epidermal area of the dorsal tube is reduced to one layer in 47.0 mm SVL female. Db, distal bulb; Dt, dorsal tube; Nt, neck tube; Ep, epidermis.

65



D. M. SEVER ET AL.

FIG. 3. Rhyacotriton variegatus. SEM (A, C) and TEM (B, D) of the spermathecae. (A) Neck tubules and distal bulbs in the same specimen used in Figure 2B. (B) Neck tubules occulded with secretory material in spermathecae of a 53.0 mm SVL female collected 18 June with 9 ovarian follicles 3.5 mm mean diameter. (C) Secretoy material and sperm in the distal bulb of the female used in Figure 2A. (D) Distal bulb showing sperm in relation to secretory material in a female collected 10 April with 12 ovarian follicles 3.5 mm mean diameter. (B, D) modified from Sever et al. (2004). Ecn, epithelial cell nucleus; Db, distal bulb; Ic, intercellular canaliculi; Lu, lumen; Nt, neck tubules; Sb, secretory bleb; Sm, secretory material; Sp, sperm; Sv, secretory vacuoles.

66




of the Proteidae (Sever and Bart, 1996), and Triturus vulgaris (Dent, 1970; Brizzi et al., 1995; Sever et al., 1999) and Notophthalmus viridescens (Sever et al., 1996a) of the Salamandridae, sperm occur in tangled masses from spermatophore pick-up to fertilization. Sever et al. (1998) hypothesized that these clusters are an adapta- tion to reduce the effectiveness of sperm competition from the ejaculates of rival males. In Ambystoma opacum of the Ambystomatidae (Sever and Kloepfer, 1993; Sever et al., 1995), Amphiuma tridactylum from the Amphiumidae (Sever et al., 1996b), and Salamandrina terdigitata from the Salamandridae (Brizzi et al., 1995), luminal areas where sperm are arranged in parallel arrays and show the same orientation have been noted. This character appears to lack phyletic significance among salamanders with simple spermathecae (Sever and Brizzi, 1998), and clues for the adaptive importance await the unraveling of species-specific mating dynamics.

In females of the Plethodontidae that have been studied, areas of the lumina of the complex spermathecae invariably have been noted in which clusters of sperm are aligned along their entire lengths in parallel arrays (Marynik, 1971; Pool and Hoage, 1973; Davitt and Larsen, 1988; Sever and Brunette, 1993; Sever, 1997; Sever and Hamlett, 1998). In the most detailed study, concerning Desmognathus ocoee, Sever and Ham- lett (1998) found that sperm form tangled masses at the branches of the neck tubules and the walls of the distal bulb where their movement becomes restricted, but close examination always reveals smaller groups of sperm aligned along their long axes. The aggregations of sperm we noted in female R. variegatus clearly fit this pattern.

The relationship between sperm nuclei and microvilli lining the epithelium of the storage area has been noted in other salamanders as well as other vertebrates. For example, in the bat Pipistrellus pipistrellus, Racy and Potts (1970) found sperm nuclei embedded in PAS-positive uterine microvilli and suggested that this situa- tion provides a pathway by which nutrient material from the endometrium is transferred to sperm, accounting for the prolonged survival of sperm in this species. In isthmic sperm reservoirs in the pig, Tipfer-Petersen et al. (2002) demon- strated that mannosyl-oligosaccharides exposed by the epithelial cells are high-affinity ligands for

sperm-associated lectins. Sperm adherence results in prolonged viability. The lectins are believed to originate in the male seminal vesicle and become associated with sperm during ejaculation. It will be interesting to discover whether carbohydrates in the male spermatophore cap of salamanders act in a similar fashion.

Sever and Hamlett (1998) proposed that sperm move thigmotactically along the cloacal epithelium into the spermathecal tubules (vide Hardy and Dent, 1986) and keep moving until the forward-most tubules hit the luminal border of the distal bulbs. This results in some sperm becoming embedded in the secretory material or epithelium. They postulated that these sperm do not escape but undergo degradation in the spermathecae. The occurrence of spermiophagy in the spermathecae of some species of salamanders is well established, but this phenomenon has not been observed in others (Sever and Brizzi, 1998; Sever, 2003).

In R. variegatus, sperm are occasionally observed embedded directly in the apical cytoplasm, but more often, as illustrated here and by Sever et al. (2004), embedded sperm are found in the intercellular canaliculi. This condition has not previously been reported in salamanders, although it has been observed in sperm storage tubules in the ground skink, Scincella laterale (D. M. Sever and W. A. Hopkins, unpubl.). Sperm also can be found in the tunica propria surrounding spermathecae of R. variegatus, presumably having passed completely through the intercellular areas. Passage into the intercellular areas requires leakage or elimination of the junctional complexes between epithelial cells (Fig. 6C). The junctional complexes may be broached by the bombardment of sperm or by the natural desquamation of epithelium that apparently occurs (Sever et al., 2004). The adaptive significance, if any, of this invasion of the intercellular spaces by sperm is unclear, but it seems likely to us that these sperm are degraded in the spermathecae.

Little is known about the mating dynamics of R. variegatus (e.g., how many times do females mate, how frequently, etc.). Thus, any consideration of postcopulatory sexual selection, either involving sperm competition (Parker, 1984) or cryptic female choice (Eberhard, 1996) is pure specula- tion. In other animals, however, the complexity of sperm storage structures can influence these

FIG. 4. Rhyacotriton variegatus. SEM showing sperm inside the cloacal orifice of a recently mated gravid female 46.7 mm SVL with eight ovarian follicles 2.8 mm mean diameter. (A) Overview, showing remnants of the spermatophore cap, tangles of sperm associated with secretory material, and tubules of the spermathecae. (B) A sperm mass adjacent top the epidermal lining and spermatophore cap remnants. (C) View of intertwined sperm nuclei and tail pieces in the tangled sperm mass. Db, distal bulb; Ep, epidermis; Scm, spermatophore cap material; Sm, secretory material; Sn, sperm nucleus; Sp, sperm; Tp, tail piece.

67



D. M. SEVER ET AL.




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FIG. 5. Rhyacotriton variegatus. (A) TEM of apical epithelium and lumen of a distal bulb showing small clusters of sperm with similar alignment in a 60.8 mm SVL female collected 10 April with 12 ovarian follicles 3.5 mm mean diameter. Specimen used by Sever et al. (2004), but micrograph not previously published. (B-D) SEM of the distal bulbs of a 47.1 mm SVL gravid female with ovarian follicles 2.9 mm mean diameter. (B) Overview of distal bulb showing a sperm mass restricted to one area. (C) The sperm mass and adjacent epithelium that possesses numerous secretory blebs. The mass appears tangled although groups of sperm are in parallel arrays. (D) Sperm nuclei apparently embedded in microvilli on the epithelial surface of the distal bulb. Db, distal bulb; Ic, intercellular canaliculi; Mv, microvilli; Ppt, principle piece of the tail; Sb, secretory blebs; Sn, sperm nuclei; Sp, sperm; Sv, secretory vacuoles.

69



D. M. SEVER ET AL.

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FIG. 6. Rhyacotriton variegatus. (A-B) TEM through the distal bulb of a 60.8 mm SVL female collected 10 April that had 12 ovarian follicles 3.5 mm mean diameter modified from Sever et al. (2004). (A) Sperm are found in the intercellular canaliculi. (B) Some sperm have passed through the epithelium and are found in the surrounding tunica propria. (C) SEM of a gravid 47.1 mm SVL female with seven ovarian follicles 2.9 mm mean diameter. Sperm are observed with portions extended into intercellular canaliculi. Bl, basal lamina; Ecn, epithelial cell nucleus; Ic, intercellular canaliculi; Ld, lipid droplet; Me, melanocytes; Ppt, principle piece of the tail; Sb, secretory blebs; Sn, sperm nucleus; Tp, tunica propria.

70




factors (e.g., Hellriegel and Ward, 1998; Uhl and Vollrath, 1998; Miller and Pitnick, 2002), and the relationship between spermathecal structure and patterns of fertilization success by males has been considered in some salamanders (Halliday, 1998; Sever, 2002). Briefly, mixed paternity from multiple matings has been reported in the complex spermathecae of plethodontids, and last male prevalence has usually been found in species with simple spermathecae (Sever, 2002).

Sever (2003) proposed that, in a complex spermatheca, interactions among different male ejaculates are more important in determining paternity than in simple spermathecae, in which the distribution of sperm in the female's cloaca effectively separates male ejaculates. Because R. variegatus possesses spermathecae that are in many ways structurally in-between complex and simple spermathecae, this species would make an interesting model to use in studying the relationships between multiple matings and male fertilization success.

Finally, the occurrence of small numbers of sperm in two first yolkers needs some consideration. These individuals were yolking their first clutch of eggs but would not be ready to oviposit during the current season. The observation that these females contain relatively small amounts of sperm compared to mated gravid females could indicate that they picked-up fewer spermato- phores and/or did not incorporate many of the sperm for storage.

Among other amphibians, stored sperm have been reported in mature but nonvitellogenic Amphiuma tridactylum (Sever et al., 1996b) and in the internally fertilizing frog Ascaphus truei (Sever et al., 2001). These individuals, such as the R. variegatus first yolkers, were not expected to oviposit in the current breeding season and were deemed unlikely to be storing the sperm for the next breeding season. No strong evidence exists for between-season sperm storage in any female amphibian with storage tubules (Sever, 2003).

Perhaps these matings are simply a result of the dynamics of male-female interactions during the frenzy of the breeding season. Males may pursue and attempt to mate with any mature- sized female, irrespective of the state of development of eggs (e.g., Metter, 1964). In the case of the R. variegatus first yolkers, the fact that they were yolking eggs indicates that gonadotropin levels were significantly elevated in these animals. Sexually undeveloped newts (Cynops pyrrhogaster) treated with prolactin and gonadotropin have been shown to react significantly to attracting pheromones (sodefrin) produced by mate-seeking males (Kikuyama et al., 2003).

Acknowledgments.-We thank E. Ryder and J. Thompson for aid in the collections. We thank

W. Archer for his help with scanning electron microscopy. This is publication 26 from the Saint Mary's College Electron Microscopy Facility.

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DAVITT, C. M., AND J. H. LARSEN JR. 1988. Scanning electron microscopy of the spermatheca of Pletho- don larselli (Amphibia: Plethodontidae): changes in the surface morphology of the spermathecal tubule prior to ovulation. Scanning Microscopy 2:1805- 1812.

DENT, J. N. 1970. The ultrastructure of the spermatheca in the Red Spotted Newt. Journal of Morphology 132:397-424.

EBERHARD, W. G. 1996. Female Control: Sexual Selection by Cryptic Female Choice. Princeton Univ. Press, Princeton, NJ.

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KIKUYAMA, S., S. TANAKA, AND F MOORE. 2003. Endocri- nology of reproduction. In D. M. Sever (ed.), Reproductive Biology and Phylogeny of Urodela (Amphibia), pp. 225-321. Science Publishers, En- field, NH.

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MARYNICK, S. P. 1971. Long term storage of sperm in Desmognathus fuscus from Louisiana. Copeia 1971: 345-347.

METTER, D. E. 1964. On breeding and sperm retention in Ascaphus. Copeia 1964:181-195.

MILLER, G. T, AND S. PITNICK. 2002. Sperm-female coevolution in Drosophila. Science 298:1230-1233.

PARKER, G. A. 1984. Sperm competition and the evolution of animal mating strategies. In R. L. Smith (ed.), Sperm Competition and the Evolution of Animal Mating Systems, pp. 1-60. Academic Press, New York.

PETRANKA, J. W. 1998. Salamanders of the United States and Canada. Smithsonian Institution Press, Wash- ington, DC.

POOL, T. B., AND T. R. HOAGE. 1973. The ultrastructure of secretion in the spermatheca of the salamander,

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Accepted: 13 November 2003.

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