Ultrastructure of the Annual Cycle of Female Sperm Storage in Spermathecae of the Torrent Salamander, Rhyacotriton Variegatus (Amphibia: Rhyacotritonidae)

JOURNAL OF MORPHOLOGY 261:1–17 (2004)

Ultrastructure of the Annual Cycle of Female Sperm Storage in Spermathecae of the Torrent Salamander, Rhyacotriton variegatus (Amphibia: Rhyacotritonidae)

David M. Sever,1* Cynthia K. Tait,2 Lowell V. Diller,3 and Laura Burkholder3

1Department of Biology, Saint Mary’s College, Notre Dame, Indiana 46556 2Bureau of Land Management, Vale, Oregon 97918 3Simpson Timber Company, Arcata, California 95521

ABSTRACT This study is the first report on the ultra- spermathecae. The ancestral condition for structure of the sperm storage glands (spermathecae) in salamanders is lack of sperm storage glands, a con- the salamander Rhyacotriton variegatus. The population dition found in three families with external fertili- studied is associated with cold-water, rocky streams of the zation, Sirenidae, Hynobiidae, and Cryptobranchi- redwood (Sequoia) zone in northern California. Males possess sperm in their vasa deferentia and undergo spermi- dae (Sever, 1991b). ation throughout the year, but mating is seasonal. Most The anatomy of sperm storage glands has been females with large, vitellogenic follicles (2.0–3.9 mm extensively studied in salamanders and was most mean dia.) collected from February–June contain sperm in recently reviewed by Sever (2003). Much work has their spermathecae, although some females with large been done on spermathecae at the light microscopy follicles lack sperm. Other mature-size females collected level, but ultrastructural studies using transmission during this period have small ovarian follicles (0.9–1.2 electron microscopy are the best means for studying mm mean dia.) and lack stored sperm. All females col- cytology of sperm/epithelial interactions and the se- lected from September–November have small follicles cretory cycle of spermathecal epithelium. (0.6–1.6 mm mean dia.) and lack sperm, except in one instance in which a female collected in November had a In this article, we present the first ultrastructural small amount of degraded sperm, apparently retained observations on sperm storage in females of a spe- from the previous breeding season. The spermathecae con- cies of the Rhyacotritonidae, using Rhyacotriton var- sist of simple tubulo-alveolar glands in which the neck iegatus (Fig. 1A). Rhyacotriton variegatus is one of tubules produce a mucoid secretory product, and the distal four species in the family Rhyacotritonidae, which is bulbs, where sperm are stored, contain secretory vacuoles restricted to the Pacific Northwest region of the of uniform density that stain positively for glycosamino- United States (Good and Wake, 1992). Sister-group glycans. In specimens containing sperm, some bulbs have relationships of Rhyacotritonidae to other abundant sperm and others lack sperm, but the ultra- salamander families are obscure (Larson et al., structure is similar in both conditions. The acini contain columnar epithelial cells with wide intercellular canalic- 2003). uli, and a merocrine process releases the secretion. Sper- Members of this family occupy springs, seeps, and miophagy occurs. In specimens from spring and summer the edges of swift-flowing, permanent streams with with small ovarian follicles, the neck tubules are similar coarse substrates (Petranka, 1998). In Oregon, ovi- to those of breeding females, but the distal bulbs are position can occur at almost any time, although reduced to cords of cells lacking a discernible lumen. Se- there may be a peak of courtship and egg-laying cretory activity in the distal bulbs is initiated in the fall. activity in spring or early summer (Nussbaum and Spermathecae of R. variegatus are most similar to those of Tait, 1977). The few nests reported for any of the a stream-dwelling plethodontid, Eurycea cirrigera. J. Mor- species were found submerged in rock crevices or phol. 261:1–17, 2004. © 2004 Wiley-Liss, Inc. under gravel in first order streams and springs. In KEY WORDS: Amphibia; Urodela; Rhyacotritonidae; these nests, eggs were apparently laid in summer or Rhyacotriton; reproduction; sperm storage; spermathecae; fall (Nussbaum, 1969; Karraker, 1999; Russell et al., ultrastructure 2002). A description of the cloaca and spermathecae

Sperm storage occurs in all females of the seven *Correspondence to: David M. Sever, Department of Biology, Saint families of salamanders comprising the suborder Mary’s College, Notre Dame, IN 46556. Salamandroidea (Sever, 1991a, 1994). Instead of E-mail: [email protected] oviductal sperm storage, as known in other female Published online in vertebrates that store sperm, sperm storage in fe- Wiley InterScience (www.interscience.wiley.com) male salamanders occurs in cloacal glands called DOI: 10.1002/jmor.10149

Fig. 1. Rhyacotriton variegatus. A: Dorsal view of a 60.8 mm SVL female collected 10 April with ovarian follicles 3.5 mm mean dia. B: Ventral view, dis- sected to show reproductive tract. Od, oviduct; Ov, ovary; St, spermathecae. of Rhyacotriton variegatus at the light microscopy sample from March 2001 was used in a separate study involving level was provided by Sever (1992b). scanning electron microscopy (SEM) of sperm packing in the spermathecae (Sever et al., in prep.). The purpose of this study is three-fold. First, we Specimens were killed by immersion in 10% aqueous solution of provide descriptions of the seasonal variation in ul- MS-222 (3-aminobenzoic acid ethyl ester). The Animal Care and trastructure of the spermathecae of Rhyacotriton Use Committee of Saint Mary’s College approved this procedure. variegatus. Second, we compare our findings on R. After death, snout–vent length (SVL) was measured from the tip variegatus to those on ultrastructural characteris- of the snout to the posterior end of the cloacal orifice. Vitellogenic follicles were counted and their diameters measured to the near- tics of sperm storage in other salamanders. Finally, est 0.1 mm. Based upon the size of the follicles and appearance of we provide preliminary observations on other as- the oviducts, females were separated into three categories: 1) pects of the male and female reproductive cycles of Gravid—females with large yolked follicles (Ͼ2.0 mm mean dia.) R. variegatus in northern California. This study is and thick, convoluted oviducts (Fig. 1); 2) Spent—females with part of a more extensive project coordinated by LVD smaller follicles but flaccid, thick, convoluted oviducts indicative of recent stretching; and 3) First yolkers—females with small on the reproductive biology of R. variegatus. follicles and thin, straight or wavy oviducts. First yolkers are females reaching sexual maturity, beginning to yolk eggs for the first time, but are not yet ready to breed. MATERIALS AND METHODS The cloacal area and an associated oviduct were removed from The specimens of Rhyacotriton variegatus used in this study each specimen. The spermathecal area is pigmented (Fig. 1B), came from the North Fork Mad River watershed on lands owned and this region was bisected. One-half was prepared for TEM and by the Simpson Timber Company in western Humboldt County, the other half for LM. For TEM, a 1-mm block of pigmented tissue California, in the north coast redwood (Sequoia sempervirens) was removed and fixed in a 1:1 solution of 2.5% glutaraldehyde zone. Rhyacotriton variegatus is considered a “species of concern” and 3.7% formaldehyde in cacodylate buffer at pH 7.2. After by the state of California, and permits for the collection of a initial fixation, tissues were rinsed in distilled-deionized water, limited sample were granted to Lowell V. Diller from the Califor- postfixed in 2% osmium tetroxide, dehydrated through a graded nia Department of Fish and Game. Collection dates and other series of ethanol, cleared in propylene oxide, and polymerized in data on the 25 female specimens used in this study are shown in an epoxy resin (Embed 812, Electron Microscopy Sciences, Port Table 1. Animals generally were sacrificed and preserved within Washington, PA). Plastic sections were cut with an RMC MT7 24 h of collection. Collections were made in February 2002, April ultramicrotome (Research and Manufacturing Co., Tucson, AZ) 2000, 2001, June 1999, 2001, September 2001, and November and DiATOME (Biel, Switzerland) diamond knives. Semithin 1999, 2001. The spermathecae of these specimens were examined sections (0.5–1 ␮m) for LM were placed on microscope slides and by light (LM) and transmission electron microscopy (TEM). A stained with toluidine blue. Ultrathin sections (70 nm) for TEM TORRENT SALAMANDER SPERM STORAGE 3

TABLE 1. Female Rhyacotriton variegatus used in this study1 Follicles Date SVL N Range Mean SE Cond2 Sperm 10 Feb 52.2 8 1.5–2.5 2.0 0.11 GD 0 10 Feb 54.2 10 1.6–3.0 2.4 0.11 GD ϩ 10 Feb 54.2 6 2.1–3.1 2.8 0.16 GD ϩ 10 Feb 54.4 7 1.8–2.6 2.1 0.11 GD ϩ 10 Feb 56.8 11 1.9–2.7 2.3 0.08 GD ϩ 1 April 45.3 10 0.8–1.1 0.9 0.03 FY 0 1 April 55.0 9 3.0–3.5 3.2 0.06 GD ϩ 10 April 48.6 8 0.8–1.6 1.1 0.05 FY 0 10 April 60.8 12 3.2–3.9 3.5 0.07 GD ϩ 18 June 45.1 7 0.6–1.2 1.0 0.06 FY 0 18 June 48.1 7 1.0–1.5 1.2 0.07 FY 0 18 June 50.3 6 1.4–3.3 2.8 0.16 GD ϩ 18 June 53.0 9 3.0–4.4 3.5 0.16 GD ϩ 23 Jun 45.8 eggs not yolked; count unreliable FY 0 23 Jun 52.1 7 3.8–4.0 3.9 0.03 GD 0 23 Jun 57.4 8 2.7–3.7 3.1 0.15 GD ϩ3 30 Jun 50.6 6 2.1–3.2 2.7 0.17 GD 0 7 Sep 52.6 11 1.0–1.4 1.2 0.04 ? 0 1 Nov 43.4 5 0.5–0.7 0.6 0.03 FY 0 1 Nov 44.3 5 0.7–1.0 0.9 0.04 FY 0 1 Nov 47.9 16 0.6–1.0 0.8 0.04 FY 0 1 Nov 52.2 9 1.2–1.9 1.6 0.08 ST 0 5 Nov 52.6 14 1.1–1.8 1.4 0.04 ST ϩ 5 Nov 53.7 10 1.0–1.5 1.3 0.06 ST 0 5 Nov 55.0 12 1.1–1.7 1.4 0.07 ST 0

1Measurements for SVL and the range and mean of follicle dia. are in mm. 2Condition ϭ Gr, gravid; FY, first yolking; ST, spent. 3Three eggs were inside the oviduct and too distorted to measure, so n ϭ 5 for calculation of mean and SE. were collected on uncoated copper grids and stained with solu- equally intense in all samples, but did not occur tions of uranyl acetate and lead citrate. Ultrathin sections were uniformly throughout the testes. Each specimen viewed with a Hitachi H-300 TEM (Nissei Sangyo America, Mountain View, CA). contained testicular lobules in which little or no Spermathecal tissue prepared for LM was processed with tes- spermatogenesis was occurring. The testes are sim- tes and vasa deferentia removed from males. Previous studies on ple, not multiply lobed, as known in some the male reproductive cycle of Rhyacotriton variegatus and other salamander families. A caudo-cephalic wave of sper- species in the genus found that males possess sperm in their vasa matogenic activity characteristic of simple testes in deferentia throughout the year (Nussbaum and Tait, 1977; Zalisko and Larsen, 1988). We removed testes and vasa deferen- many salamanders (e.g., Uribe Aranza´bal, 2003) is tia from males collected with females throughout the year, in- not apparent in those of Rhyacotriton variegatus. cluding two males (52.6, 53.0 mm SVL) collected 1 April, three Sperm seem most numerous in the vasa deferentia males (51.1, 53.9, and 56.4 mm SVL) collected 10 April, three of males from the February–April samples (Fig. 2A). males collected 18 June (50.8, 51.9, and 53.6 mm SVL), and four males (44.9, 45.7, 47.7, and 48.9 mm SVL) collected 1 November. In other months, sperm are often associated with Tissues prepared for LM were fixed in 10% neutral buffered colloidal material in the lumina of the vasa deferen- formalin (NBF), rinsed in water, dehydrated in ethanol, cleared tia (Fig. 2D,F). However, the presence of sperm in in toluene, and embedded in paraffin. Paraffin sections (10 ␮m) the vas deferens in all samples indicates that males were cut with a rotary microtome and affixed to albuminized possibly could engage in mating activity throughout slides. Slides were stained with hematoxylin-eosin. In addition, alternate slides from a 54.2 mm SVL female collected 10 Febru- the year. ary and a 55 mm SVL female collected 1 April were stained with Alcian blue 8GX at pH 2.5 (AB, primarily for carboxylated gly- cosaminoglycans) followed by the periodic acid-Schiff’s procedure Female Reproductive Cycle (PAS, for neutral carbohydrates and sialic acids), or stained with bromophenol blue (BB, for proteins). Procedures followed Dawes The largest ovarian follicles (2.0–3.9 mm mean (1979), Humason (1979), and Kiernan (1990). dia.) occur in females from the February–June samples, although some females from these samples pos- RESULTS sess much smaller follicles (0.9–1.2 mm mean dia.; Male Reproductive Cycle Table 1). The females with small follicles could be immature, although the smallest of these (45.1 mm Males from all samples possess testes in which SVL) is within the range of mature body size re- spermiation is actively occurring (Fig. 2A,C,E) and ported by Nussbaum and Tait (1977). Females from have vasa deferentia containing sperm (Fig. the September–November samples all have small 2B,D,F). Spermiogenic activity in the testes seemed follicles (0.6–1.6 mm mean dia.). The females with Fig. 2. Rhyacotriton variegatus. Sagittal paraffin sections stained with hematoxylin-eosin. Scale bar in lower right corner is the same for all micrographs. Testis (A) and vas deferens (B) of a male collected 10 April. Testis (C) and vas deferens (D) of a male collected 18 June. Testis (E) and vas deferens (F) of a male collected 1 November. Co, colloidal material; Sg, spermatogonia; Sp, sperm; Ss, secondary spermatocytes; St, spermatids. TORRENT SALAMANDER SPERM STORAGE 5 larger follicles (1.2–1.6 mm mean dia.) may have Ultrastructure of the spermathecae in Feb- been yolking follicles for the breeding season the ruary and April. With one exception, gravid fe- following spring. A more detailed analysis of the males collected February–April with ovarian folli- reproductive cycle, based upon a much larger sam- cles 2.0–3.9 mm mean dia. contain sperm in their ple (161 specimens), is in progress (Tait et al., in spermathecae, but some distal bulbs lack sperm prep.). (Fig. 4A), whereas others are packed with sperm Females collected February–June with mean fol- (Fig. 4B). The one exception, mentioned previously, licle dia. Ͼ2.0 mm possessed sperm in their sper- has a spermatophore cap in her cloaca, indicating a mathecae except for one specimen collected in Feb- recent mating. Sperm in the spermathecae are not ruary and two specimens from June (Table 1). The aligned in any orderly fashion. The distal bulbs are specimen from February has a spermatophore cap in similar in cytology, however, whether sperm are its cloaca and sperm apparently had not yet mi- present or not. grated into the spermathecae. Females with mean The epithelium is simple columnar with large het- follicle dia. Ͻ1.6 mm lack sperm in their spermathe- erochromatic nuclei aligned with the long axis of the cae, except for a spent specimen collected 11 Novem- cell and occupying the basal halves of the cells (Fig. ber with a mean follicle size of 1.4 mm, which has a 4A,B). Myoepithelial cells occur between the base of few sperm in some tubules, perhaps remnants from the epithelial cells and the basal lamina (Fig. 3A). the previous breeding season (see below). The apical halves of the epithelial cells are filled with irregularly shaped secretory vacuoles of mod- erate but uniform electron density. The product con- Spermathecae tained in the vacuoles is released into the lumen by a merocrine process (Fig. 4C). Golgi complexes and The spermathecae consist of simple tubulo- rough endoplasmic reticulum are found in the pe- alveolar glands that branch from an invagination rinuclear areas (Fig. 4D). Intercellular canaliculi into the roof of the anterior, tubular portion of the narrow to tight junctions at the luminal border (Fig. cloaca (Fig. 3A). This invagination was called the 4C), but in the basal half of the cell wide intercellu- “dorsal tube” by Sever (1992b). The dorsal tube is lar spaces occur. In the widened areas of the inter- similar to the common tube characteristic of the cellular canaliculi the plasma membranes from ad- Plethodontidae, except that the dorsal tube is not as jacent cells interdigitate (Fig. 4D). narrow and spermathecae evaginate from the entire Distal bulbs contain areas where the epithelium is upper half of the dorsal tube, whereas in plethodon- desquamated, so that complete or nearly complete tids narrow neck tubules extend only from the apex gaps exist between the lumen and the stroma of the of the common tube (Sever, 1992a). superficial tunica propria (Fig. 5A). Sperm occur The spermathecae extend into the loose connec- both in the lumen and in the tunica propria, and tive tissue tunica propria of the cloacal sheath (Fig. sperm appear normal in cytology in both areas (Fig. 3B). In individuals in which sperm occur, some sper- 5B,C). In a few instances, portions of sperm are mathecae contain many sperm, whereas others con- found embedded in the cytoplasm or in between tain few or no sperm (Fig. 3B,C). Occasionally, distal epithelial cells in the intercellular canaliculi (Fig. portions of a spermathecal tubule have areas of des- 5D). This is considered evidence of spermiophagy, quamated epithelium, and this condition apparently because sperm trapped in these areas are unlikely to is not artifactual because adjacent tubules are nor- be subsequently released (Sever, 1992a; Sever and mal (Fig. 3B). Hamlett, 1998). The most interesting aspect of the spermathecae Females collected in February and April with and of their relationship to the rest of the cloaca at small ovarian follicles 0.9–1.1 mm mean dia. were the LM level is the variation in the epithelium. The undergoing their first yolking and lack sperm in cloacal cavity is lined with stratified, keratinized their spermathecae. The distal bulbs at the LM level epithelium continuous with the epidermis of the sur- appeared as cords of cells with no discernible lumen rounding skin (Fig. 3A). As the dorsal tube invagi- (Fig. 6A). Ultrastructural observations on the distal nates, this lining changes into simple columnar mu- bulbs reveal that the cells contain large, uniformly cinogenic cells, and this lining continues into the dense nuclei that fill nearly the entire cell (Fig. 6B). proximal neck tubules of the spermathecae (Fig. Intercellular canaliculi are wide, with interdigitat- 3C). The apical mucous product reacts PASϩ for ing plasma membranes. neutral carbohydrates and stains ABϩ for glycos- Neck tubules, however, do not vary much in ap- aminoglycans. This mucoid secretion, however, is pearance between the mated females that possess somewhat different in the distal bulbs of the sper- large ovarian follicles and the unmated females that mathecae, which are simple columnar or cuboidal, have small ovarian follicles. Under both conditions depending on their activity, as described below. The the neck tubules contain numerous secretory vacu- apical cytoplasm of active distal bulbs is also ABϩ oles. A gradation in quantity of secretory vacuoles but is PAS–. All secretory regions of the spermathe- occurs, with fewest in the area adjoining the distal cal epithelium are BB– for proteins. bulb (Fig. 6C) and the most in the region gradating 6 D.M. SEVER ET AL.

Figure 3 TORRENT SALAMANDER SPERM STORAGE 7 into the dorsal tube (Fig. 6D). The secretory vacuoles 1), may have been immature. The epithelial cells of of neck tubules are overall lighter in density than the distal bulbs are nearly squamous and no indica- those of sperm-containing distal bulbs, and the se- tion of secretory activity is apparent (Fig. 8D). cretory vacuoles in the neck tubules often contain an Ultrastructure of the spermathecae in Sep- eccentric denser particle (Fig. 6D). Little secretory tember and November. Only one of the females product is apparent in the lumina of the neck tu- examined from the fall had sperm in its spermathe- bules. cae. This individual and other females collected in Ultrastructure of the spermathecae in June. November with ovarian follicles 1.3–1.6 mm mean The females collected in June with large ovarian dia. and thickened, flaccid oviducts are considered follicles have spermathecae similar in cytology to spent individuals that oviposited the previous the gravid females from February and April. Some of spring or summer and would likely have reached the neck tubules of spermathecae containing sperm, breeding condition again the following spring. In the however, have fewer secretory vacuoles in the apical distal bulbs of these specimens the epithelial cells cytoplasm and have abundant secretory product oc- are columnar, the nuclei are basal and parallel to cluding the lumina (Fig. 7A). Once again, in speci- the long axes of the cells, and the apical halves of the mens containing sperm, distal portions of some sper- cells are filled with secretory vacuoles (Fig. 9A). mathecae are empty, whereas others are packed Some secretory product is found in the lumen (Fig. with irregularly aligned sperm (Fig. 7B). 9A) and Golgi complexes occur in the supranuclear Apical areas of some of the epithelial cells of the cytoplasm (9B). The apical portions of the neck tu- distal bulbs are crowded with large, irregular secre- bules of these individuals are filled with large, irreg- tory vacuoles, many of which contain central dense ular secretory vacuoles that are once again lighter in material not noted in the April specimens (Fig. 7C), overall electron density than those of the distal although others lack secretory vacuoles (Fig. 7D). In bulbs (Fig. 9C). Golgi complexes and smooth endo- the cells that lack secretory vacuoles, numerous plasmic reticulum occur in the supranuclear areas of small vesicles are present and Golgi bodies and neck tubules (Fig. 9D). rough endoplasmic reticulum are often observed Finally, one specimen collected in November with (Fig. 7D). 1.4 mm mean follicle dia. possessed a small number The specimens collected in June that lack sperm of sperm in several distal bulbs, although most and contain small ovarian follicles show somewhat glands are devoid of sperm (Fig. 10A). The sper- dissimilar cytologies (Fig. 8). A female undergoing mathecae are similar to those of other specimens first yolking with ovarian follicles 1.2 mm mean dia. from November except that the intercellular canal- has constricted neck tubules and expanded distal iculi appear wider and membranous structures oc- bulbs (Fig. 8A). The epithelium varies in thickness cur in the lumen (Fig. 10C,D). The sperm do not and nuclei are heterochromatic, basal, and have appear normal. They are uniformly electron-dense their long axes parallel to the basal lamina (Fig. 8B). and show no evidence of plasma membranes, the Secretory vacuoles are numerous in the apical re- axonemes, or mitochondrial rings (Fig. 10D). These gions of some epithelial cells (Fig. 8B) and condens- sperm are considered degrading sperm that remain ing vacuoles and organelles involved in synthesis of from a mating in the previous spring or summer. secretory products occur in the supranuclear areas Females with ovarian follicles 0.6–0.8 mm mean (Fig. 8C). Thus, although this female is not in breed- dia. are considered first yolkers and they could have ing condition for the current season, cytological ev- reached breeding condition the following spring or idence suggests an initiation of secretory activity so summer. Spermathecae of these individuals are sim- that the glands would be ready for the next breeding ilar in cytology to those of females from February– season. June with follicles 0.9–1.1 mm mean dia. Two other non-breeding females examined from June, one with ovarian follicles 1.0 mm mean dia. DISCUSSION and the other with numerous nonyolked eggs (Table Nussbaum and Tait (1977) reported that males of Rhyacotriton variegatus in Oregon are capable of producing spermatophores throughout the year, but Fig. 3. Rhyacotriton variegatus. A: Midsagittal paraffin sec- that males have reduced supplies of mature sperm tion through the cloacal region of a female, stained with in their vasa deferentia from May to August. Zalisko hematoxylin-eosin, and modified from Sever (1992a). B: Semithin and Larsen (1988) reported that the vasa deferentia epoxy section through the distal bulbs of a 60.8 mm SVL specimen collected 10 April, stained with toluidine blue. C: Semithin of R. olympicus from Skamania County, Washing- epoxy section through the neck tubules and distal bulbs of a 53.0 ton, exhibit only minor seasonal variation and that mm SVL specimen collected 18 June, stained with toluidine blue. sperm typically can be found in the vasa deferentia Cc, cloacal chamber; Ct, cloacal tube; Db, distal bulb; De, desqua- of mature males throughout the year. Neither Nuss- mated epithelium; Dt, dorsal tube; Ep, epidermis; Mc, mucous cells; Nsl, no sperm in the lumen; Nt, neck tubules; Pi, posterior baum and Tait (1977) nor Zalisko and Larsen (1988) intestine; Sc, spermatophore cap; Spl, sperm in the lumen; Tp, examined the spermatogenic cycle of the testes. We tunica propria. also found that sperm occur in the vasa deferentia of Fig. 4. Rhyacotriton variegatus. TEM through the spermathecae of a 60.8 mm SVL female collected 10 April that contained sperm and had ovarian follicles 3.5 mm mean dia. A: Portion of a distal bulb lacking sperm in the lumen. B: Distal bulb containing luminal sperm. C: Apical cytoplasm showing a stage in release of secretory product. D: Perinuclear cytoplasm showing synthetic organelles. Go, Golgi apparatus; Ic, intercellular canaliculi; In, interdigitating plasma membranes; Lu, lumen; Me, melanin granules; My, myoepithelial cell; Nsl, no sperm in lumen; Nu, nucleus; Rer, rough endoplasmic reticulum; Se, secretory product; Spl, sperm in the lumen; Sv, secretory vacuoles; Tj, tight junction; Tp, tunica propria. Fig. 5. Rhyacotriton variegatus. TEM through the spermathecae of a 60.8 mm SVL female collected 10 April that contained sperm and had ovarian follicles 3.5 mm mean dia. A: Desquamated area of a spermathecal tubule. B: Luminal border. C: Stromal border. D: Sperm embedded in the epithelium. Bl, basal lamina; Cf, collagen fibers; Ic, intercellular canaliculi; Lu, lumen; Me, melanin granules; Mi, mitochondria; Mpt, middle piece of the tail; Nu, nucleus; Ppt, principle piece of the tail; Sn, sperm nucleus; Sp, sperm; Sv, secretory vacuoles; Tj, tight junction; Tp, tunica propria. Fig. 6. Rhyacotriton variegatus. Semithin epoxy section (A) and TEMs (B–D) through the spermathecae of a 48.6 mm SVL specimen collected 10 April lacking sperm and possessing ovarian follicles 0.05 mm mean dia. A: Overview of the spermathecal region, showing areas (B–D) illustrated in the electron micrographs. B: Distal bulb. C: Neck tubule. D: Junction between proximal portion of neck tubule and dorsal tube of the cloaca. Ic, intercellular canaliculi; Lu, lumen; Nu, nucleus; Se, secretory product; Sv, secretory vacuoles; Tp, tunica propria. Fig. 7. Rhyacotriton variegatus. TEM through the spermathecae of a 53.0 mm SVL female collected 18 June that contained sperm and had ovarian follicles 3.5 mm mean dia. A: Neck tubule. B: Distal bulb. C: Luminal border of distal bulb in an area containing numerous secretory vacuoles. D: Luminal border of distal bulb in an area lacking secretory vacuoles. Go, Golgi apparatus; Ic, intercellular canaliculi; Lu, lumen; Mf, microfilaments; Mi, mitochondria; Mpt, middle piece of the tail; Nu, nucleus; Ppt, principle piece of the tail; Rer, rough endoplasmic reticulum; Se, secretory product; Sn, sperm nucleus; Spl, sperm in the lumen; Ve, vesicles. Fig. 8. Rhyacotriton variegatus. Light (A) and TEM (B–D) through the spermathecae of females that were collected 18 June and lacked sperm. One specimen (A–C) was 48.1 mm SVL and had ovarian follicles 1.2 mm mean dia., and the other (C,D) was 45.1 mm SVL and had ovarian follicles 1.0 mm mean dia. A: Several spermathecae and adjacent mucous cells (Mc) of the dorsal tube. B: Distal bulb epithelium. C: Supranuclear cytoplasm of a distal bulb epithelial cell. D: Epithelium of a distal bulb. Cv, condensing vacuoles; Db, distal bulb; Go, Golgi apparatus; Ic, intercellular canaliculi; Lu, lumen; Mc, mucous cells; Mi, mitochondria; Mv, microvilli; Nsl, no sperm in the lumen; Nt, neck tubule; Nu, nucleus; Rer, rough endoplasmic reticulum; Sv, secretory vacuoles; Tp, tunica propria. Fig. 9. Rhyacotriton variegatus. TEM through the spermathecae of a 52.2 mm SVL female collected 1 November that lacked sperm and had ovarian follicles 1.6 mm mean dia. A: Lumen and apical cytoplasm of a distal bulb. B: Supranuclear cytoplasm of a distal bulb epithelial cell. C: Neck tubule. D: Supranuclear cytoplasm of a neck tubule epithelial cell. Go, Golgi apparatus; Ic, intercellular canaliculi; Lu, lumen; Mi, mitochondria; My, myoepithelial cells; Nu, nucleus; Se, secretory product; Ser, smooth endoplasmic reticulum; Sv, secretory vacuoles; Ve, vesicles. Fig. 10. Rhyacotriton variegatus. Semithin epoxy section (A) and TEMs (B–D) through the spermathecae of a 52.2 mm SVL female collected 11 November that contained sperm and had ovarian follicles 1.0 mm mean dia. A: Overview of spermathecal distal bulbs, showing a few sperm in one tubule. B–C: Apical cytoplasm and adjacent lumen of a distal bulb containing sperm. D: Detail of sperm in the lumen. Ic, intercellular canaliculi; Lu, lumen; Mb, membranous structures; Mv, microvilli; Nsl, no sperm in lumen; Se, secretory product; Sn, sperm nucleus; Sp, sperm; Spl, sperm in lumen; Sv, secretory vacuoles; Tp, tunica propria; Ve, vesicles. TORRENT SALAMANDER SPERM STORAGE 15 our specimens at all times and, furthermore, this the Salamandroidea; 2) some of the differences ob- condition results from active spermatogenic activity served among taxa in spermathecal characters may throughout the year. not be phyletically informative but related to other Males, therefore, seem capable of delivering ma- species-specific reproductive adaptations; 3) sperm ture sperm throughout the year, and females pos- storage is apparently obligatory prior to fertilization sess spermathecae for sperm storage that presum- in salamandroids so that the duration of effective ably could allow mating to occur long before sperm storage must be considered in any study on oviposition. In Oregon, Nussbaum and Tait (1977) the reproduction of these taxa; and 4) storage of reported finding spermatophores in the cloacae of sperm facilitates multiple matings and provides the females in every month except August, September, conditions for sperm competition within the sper- December, and January. In the current study, how- mathecae of salamanders (Halliday, 1998). ever, the only mated females were those with large Sever (2002) performed a phenetic analysis of the vitellogenic follicles in the February, April, and June same dataset used by Sever and Brizzi (1998) with samples, with the exception of one individual from the inclusion of data on Rhyacotriton variegatus. November that apparently retained some residual The simple matching coefficient used by Sever sperm from an earlier mating. Our results indicate (2002) gives a different perspective than a cladistic an annual egg-laying cycle, because we found no analysis. As mentioned previously, in the cladistic mature, large-sized females in the spring with small analysis of Sever and Brizzi (1998) the only charac- eggs, which we would expect if females needed 2 ter found to have phyletic value was complex (pleth- years to reyolk after oviposition. In western Oregon, odontids) vs. simple (other salamanders with sperm Nussbaum and Tait (1977) also found evidence that storage) spermathecae. In the phenetic analysis, most females oviposit in the spring and early sum- other characters dealing with the types and distri- mer on an annual cycle. bution of epithelial cells and their relationship to More is known about the ultrastructure of sperm spermiophagy assume more importance (Sever, storage in salamanders than in any other vertebrate 2002). group. Nevertheless, the ultrastructure of female Three groups were found in the phenetic analysis, sperm storage in salamanders has been studied in and one of these groups was a clustering of Eurycea only 2% of the known species of salamanders (Sever, cirrigera and Rhyacotriton variegatus. This cluster- 2002). Besides the Rhyacotritonidae, the annual cycle ing is most interesting, because these are the of sperm storage has been studied at the ultrastruc- spermathecae-bearing taxa in this sample that tural level in representatives of five of the remaining breed in rocky, swift-flowing streams (Nussbaum, six salamander families in which female sperm stor- 1969; Nussbaum and Tait, 1977; Sever, 1999). Rhya- age occurs. These studies involve two species of Pleth- cotriton variegatus has a limited range in the up- odontidae (Sever, 1991c, 1992a, 1997; Sever and Bru- lands of northwestern California and southwestern nette, 1993), three Salamandridae (Brizzi et al., 1995; Oregon (Petranka, 1998). Eurycea cirrigera occurs Sever et al., 1996a, 1999, 2001), two Ambystomatidae widely in the eastern United States and Canada and (Sever, 1995; Sever and Kloepfer, 1993; Sever et al., has somewhat broader habitat tolerances, but the 1995), one Amphiumidae (Sever et al., 1996b), and one population in which sperm storage has been studied Proteidae (Sever and Bart, 1996). Dicamptodontidae is occurs in rocky, wooded streams in the hilly, ungla- the only family in which the spermathecal ultrastruc- ciated Ohio River valley of Indiana (Sever, 1991c, ture of a representative species has not been studied. 1992a; Sever and Brunette, 1993). Rhyacotriton var- Much diversity exists in reproductive habits and iegatus is in the family Rhyacotritonidae and E. sperm storage characters among the species that have cirrigera is in the Plethodontidae, two families that been studied. lack a close sister-group relationship (Larson and Sever and Brizzi (1998) made an initial attempt to Dimmick, 1993). find phyletic trends in sperm storage characters. These two species share these spermathecal sim- They mapped 14 characters involved with sper- ilarities: secretory vacuoles of uniform density (oth- mathecae and sperm storage on a phylogeny of erwise known only in Notophthalmus viridescens); salamander families taken from Larson and Dim- regionalized differences in secretory activity; differ- mick (1993). The only character with definite phyl- ent types of epithelial cells proximally and distally; etic value is whether sperm storage occurs in a “com- regionalized spermiophagy; and desquamation of plex spermatheca” composed of a single compound epithelial cells into the stroma. The evolution of tubulo-alveolar gland (Plethodontidae) or in “simple some of these characters may be linked within a spermathecae” consisting of numerous simple tubu- taxon. In both species, the proximal ends of the lar glands (other families). The variation in complex spermathecal tubules have greater secretory activ- spermathecae of plethodontids was described by ity than the distal portions, and spermiophagy oc- Sever (2000). curs only in the distal portions. Why these charac- Sever and Brizzi (1998) concluded that: 1) sperm ters have evolved convergently in two species with storage is an ancient trait in salamanders, evolving similar breeding habits and habitat begs even the in the common ancestor of all the extant families in wildest speculation at this time. In other words, do 16 D.M. SEVER ET AL. these characters have some adaptive value to Halliday T. 1998. Sperm competition in amphibians. In: Birkhead salamanders breeding in swift-flowing, rocky TR, Møller AP, editors. Sperm competition and sexual selection. New York: Academic Press. p 465–502. streams, and if so, why are these traits favored? We Humason GL. 1979. Animal tissue techniques, 4th ed. San Fran- are far from understanding the significance of the cisco: WH Freeman. variation of spermathecal characters in the repro- Karraker NE. 1999. Rhyacotriton variegatus (southern torrent ductive activities of Rhyacotriton variegatus or any salamander) nest site. Herpetol Rev 30:160–161. other urodele. Kiernan JA. 1990. Histological and histochemical methods: the- Indeed, we still need to understand why female ory and practice. New York: Pergamon Press. Larson A, Dimmick WW. 1993. Phylogenetic relationships of the Rhyacotriton variegatus store sperm. The mating salamander families: an analysis of congruence among morpho- season is long (at least February–June), and the logical and molecular characters. Herpetol Monogr 7:77–94. species aggregates in breeding sites along streams, Larson A, Weisrock DW, Kozak KH. 2003. Phylogenetic system- so access to mates does not seem limited. Perhaps atics of salamanders (Amphibia, Urodela), a review. In: Sever the possibilities of sperm competition arising from DM, editor. Reproductive biology and phylogeny of Urodela (Amphibia). Enfield, NH: Science Publishers. p 31–108. multiple matings are enhanced through sperm stor- Nussbaum RA. 1969. A nest site of the Olympic salamander, age, as discussed by Sever et al. (2001) and Sever Rhyacotrition olympicus (Gaige). Herpetologica 25:277–278. (2002) for the salamandrid Triturus vulgaris. One Nussbaum RA, Tait CK. 1977. Aspects of the life history and mating is surely sufficient to fertilize the small ecology of the Olympic salamander, Rhyacotriton olympicus clutches of eggs produced by R. variegatus. However, (Gaige). Am Midl Nat 98:176–199. we know nothing about the mating dynamics of R. Petranka JW. 1998. Salamanders of the United States and Can- ada. Washington: Smithsonian Inst Press. variegatus. Of especial interest would be knowledge Russell KR, Gonyaw AA, Strom JD, Diemer KE, Murk KC. 2002. of the duration of sperm storage between mating Three new nests of the Columbia torrent salamander, Rhyaco- and oviposition. Sever (1996) proposed that short- triton kezeri, in Oregon with observations of nesting behavior. term sperm storage (2 days or less) as found in many Northw Nat 83:19–22. Ambystoma is plesiomorphic relative to long-term Sever DM. 1991a. Comparative anatomy and phylogeny of the cloacae of salamanders (Amphibia: Caudata). I. Evolution at sperm storage (weeks or months) documented for the family level. Herpetologica 47:165–193. some species. Perhaps sperm storage in R. variega- Sever DM. 1991b. Comparative anatomy and phylogeny of the tus simply provides a time period for the female to cloacae of salamanders (Amphibia: Caudata). II. Crypto- move from the area of mating activity (unknown, but branchidae, Hynobiidae and Sirenidae. J Morphol 207:283–301. presumably the stream bank) to the place of egg Sever DM. 1991c. Sperm storage and degradation in the sper- deposition (cracks in submerged rocks, under gravel mathecae of the salamander Eurycea cirrigera (Green). J Mor- phol 210:71–84. or boulders in first order streams; Nussbaum, 1969; Sever DM. 1992a. Spermiophagy by the spermathecal epithelium Karraker 1999; Russell et al., 2002). Or perhaps of the salamander Eurycea cirrigera. J Morphol 212:281–290. sperm storage is an atavism of little significance, Sever DM. 1992b. Comparative anatomy and phylogeny of the retained from the ancient origins of the Rhyacotri- cloacae of salamanders (Amphibia: Caudata). VI. Ambystoma- tonidae. Further exploration of these topics will oc- tidae and Dicamptodontidae. J Morphol 212:305–322. Sever DM. 1994. Observations on regionalization of secretory cur in subsequent studies involving SEM of sperm activity in the spermathecae of salamanders and comments on storage (Sever et al., in prep.) and a more detailed phylogeny of sperm storage in female salamanders. Herpeto- analysis of the reproductive cycle of R. variegatus logica 50:383–397. (Tait et al., in prep.). Sever DM. 1995. Spermathecae of Ambystoma tigrinum (Am- phibia: Caudata): development and a role for the secretions. J Herpetol 29:243–255. Sever DM. 1997. Sperm storage in the spermatheca of the red- ACKNOWLEDGMENTS back salamander, Plethodon cinereus (Amphibia: Plethodonti- dae). J Morphol 234:131–146. We thank Elizabeth Ryder and Joel Thompson for Sever DM. 1999. Eurycea cirrigera. Cat Am Amphib Rept 648: aid in the collections, and Adrian Kirby and Emily 1–6. Moriarty for help in the laboratory. This is publica- Sever DM. 2000. Sperm storage in female plethodontids with tion number 25 from the Saint Mary’s College Elec- especial reference to the Desmognathinae. In: Bruce RC, Jaeger R, Houck, LC, editors. The biology of plethodontid salamanders. tron Microscopy Facility. New York: Kluwer Academic/Plenum. p 345–369. Sever DM. 2002. Female sperm storage in amphibians. J Exp Zool 292:165–179. LITERATURE CITED Sever DM. 2003. Courtship and mating glands. In: Sever DM, editor. Reproductive biology and phylogeny of Urodela (Am- Brizzi R, Delfino G, Selmi MG, Sever DM. 1995. The spermathe- phibia). Enfield, NH: Science Publishers. p 323–381. cae of Salamandrina terdigitata (Amphibia: Salamandridae): Sever DM, Bart HL. 1996. The ultrastructure of the spermathe- patterns of sperm storage and degradation. J Morphol 223:21– cae of Necturus beyeri (Amphiba: Proteidae) in relation to its 33. breeding season. Copeia 1996:927–937. Dawes C. 1979. Biological techniques for transmission and scan- Sever DM, Brizzi R. 1998. Comparative biology of sperm storage ning electron microscopy. Burlington, VT: Ladd Research In- in female salamanders. J Exp Zool 282:460–476. dustries. Sever DM, Brunette NS. 1993. Regionalization of eccrine and Good DA, Wake DB. 1992. Geographic variation and speciation in spermiophagic activity in the spermathecae of the salamander the torrent salamanders of the genus Rhyacotriton (Caudata: Eurycea cirrigera (Amphibia: Plethodontidae). J Morphol 217: Rhyacotritonidae). U Calif Publ Zool 126:1–91. 161–170. TORRENT SALAMANDER SPERM STORAGE 17

Sever DM, Hamlett WC. 1998. Sperm aggregations in the sper- Amphiuma tridactylum (Caudata: Amphiumidae). J Morphol mathecae of female desmognathine salamanders. J Morphol 230:79–97. 238:143–155. Sever DM, Halliday T, Waights V, Brown J, Davies HA, Moriarty Sever DM, Kloepfer NM. 1993. Spermathecal cytology of Ambys- EC. 1999. Sperm storage in females of the smooth newt (Tritu- toma opacum (Amphibia: Ambystomatidae) and the phylogeny rus v. vulgaris L.). I. Ultrastructure of the spermathecae during of sperm storage in female salamanders. J Morphol 217:115– the breeding season. J Exp Zool 283:51–70. 127. Sever DM, Halliday T, Moriarty EC, Arano B. 2001. Sperm stor- Sever DM, Krenz JD, Johnson KM, Rania LC. 1995. Morphology age in females of the smooth newt (Triturus v. vulgaris L.). II. and evolutionary implications of the annual cycle of secretion and Ultrastructure of the spermathecae after the breeding season. sperm storage in spermathecae of the salamander Ambystoma Acta Zool 82:49–56. opacum (Amphibia: Ambystomatidae). J Morphol 223:35–46. Uribe Aranza´bal MC. 2003. The testes, spermatogenesis and Sever DM, Rania LC, Krenz JD. 1996a. The annual cycle of sperm male reproductive ducts. In: Sever DM, editor. Reproductive storage in the spermathecae of the red-spotted newt, Notoph- biology and phylogeny of Urodela (Amphibia). Enﬁeld, NH: thalmus viridescens (Amphibia: Salamandridae). J Morphol Science Publishers. p 183–202. 227:155–170. Zalisko EJ, Larsen JH Jr. 1988. Ultrastructure and histochemis- Sever DM, Doody JS, Reddish CA, Wenner MM, Church DR. try of the vas deferens of the salamander Rhyacotriton olympi- 1996b. Sperm storage in spermathecae of the great lamper eel, cus: adaptations for sperm storage. Scan Micros 2:1089–1095.