Reproduction of the Salamander Siren Intermedia Le Conte with Especial Reference to Oviducal Anatomy and Mode of Fertilization

JOURNAL OF MORPHOLOGY 227:335-348 (1996)

Reproduction of the Salamander Siren intermedia Le Conte With Especial Reference to Oviducal Anatomy and Mode of Fert iI izati on

DAVID M. SEVER, LISA C. RANIA, AND JOHN D. KRENZ Department of Biology, Saint Mary's College, Notre Dame, Indiana 46556 (D.M.S., L.C.R.); Savannah Riuer Ecology Laboratory, Aiken, South Carolina 29802 (L.C.R., J.D.K.); Institute of Ecology, University of Georgia, Athens, Georgia 30602 (J.D.K.)

ABSTRACT Reproduction was studied in a South Carolina population of the paedomorphic salamander Siren intermedia with emphasis on anatomy of the female oviduct. The oviduct forms 67-79% of the snout-vent length in this elongate species and can be divided into three portions. The atrium, 7-13% of oviducal length, is the narrow anteriormost portion, with the ostial opening immediately caudad of the transverse septum. The ampulla, 63-75% of oviducal length, is the highly convoluted, middle portion in which gelatinous coverings are added to the eggs during their passage. Hypertrophy of the oviducal glands in the ampulla causes the ampulla to increase in diameter during the ovipository season. The secretion of the eosinophilic oviducal glands is in- tensely positive following staining with the periodic acid-Schiff procedure and does not react with alcian blue at pH 2.5. This staining reaction, coupled with the presence of abundant rough endoplasmic reticulum and Golgi complexes, indicates that the secretion contains a glycoprotein. The ovisac, 16-25% of oviducal length, is the most posterior portion of the oviduct and holds up to 10-11 eggs prior to oviposition. Oviducal glands similar to those in the ampulla are absent in the ovisac. Oviposition in female sirens occurs during February- April in this population, and male spermiation is concurrent. Entire oviducts were sectioned from three females collected during the ovipository season and from two collected prior to the breeding season, and sperm were not found in the oviducts of these specimens. Thus no evidence was found for internal fertilization or sperm storage in the oviducts of sirens. o 1996 Wiley-Liss, Inc.

The Sirenidae consists of two species each most species of salamanders, ova are fertil- of Siren (S.intermedia and S. lacertina) and ized internally by sperm released from cloa- Pseudobranchus (P. striatus and P. axan- cal glands (spermathecae) as the eggs pass thus) (see Moler and Kezer, '93). Members of through the cloaca during oviposition (Bois- the family are restricted to the southeastern seau and Joly, '75). Cloaca1 glands, which United States and northern Mexico (Duell- function in the production of spermato- man and Trueb, '86). The species are elon- phores (males) and the storage of sperm (fe- gate, aquatic perennibranchs that lack a pel- males), are lacking in the Sirenidae (Sever, vic girdle and hindlimbs (Martof, '74).Within '91b), implying external fertilization. Al- the order Caudata, the Sirenidae is the only though Noble and Marshall ('32) and Godley member of the suborder Sirenoidea (Duell- ('83) stated that Siren intermedia oviposit man and Trueb, '86), although some have their entire clutch in large nests of 206-555 questioned whether sirens are salamanders at all and proposed placing sirenids in their eggs, other studies on S. intermedia (Noble own order, Trachystoma, within the subclass and Richards, '32) and Pseudobranchus stria- Lissamphibia (Cope, 1889; Goin and Goin, tus (Goin and Goin, '62) reported eggs laid in '62). The mating behavior of sirens has never Address reprint requests to Dr. David M. Sever, Department of been observed (Martof, '74). In females of Biology, Saint Mary's College, Notre Dame, IN 46556.

B 1996 WILEY-LISS, INC 336 D.M. SEVER ET AL. small clusters over a period of days. In P. males. For LM, the entire oviduct was pre- striatus, Goin and Goin (’62) found it “diffi- pared for sectioning, except for 1 mm blocks cult to see how such eggs could be fertilized taken from the proximal and distal ends and after they are laid” and suggested that inter- from locations just anterior and posterior to nal fertilization occurs within the family. the middle 10-40 mm of the oviduct, which If internal fertilization occurs in sirens, were prepared for TEM. the oviduct could be the site of sperm storage For LM, the tissue was rinsed in water and/or fertilization. Although the male uro- after fixation, dehydrated in ethanol, cleared genital system has been studied in Siren in Histosol (National Diagnostics, Manville, intermedia (Willet, ’651, the only description NJ), and embedded in paraffin. Sections (10 of the female urogenital system is a brief p,m) were cut with a rotary microtome, af- account by Vaillant (1863) on S. lacertina fixed to albuminized slides, and alternate (which actually may have been S. interme- slides were stained with hematoxylin-eosin dia, see Noble and Richards, ’32). In this (HE, for general cytology) or alcian blue at study, we examine the male and female repro- pH 2.5 (AB,for carboxylated glycosaminogly- ductive cycles ofsirenintermedia from South cans) followed by the periodic acid-Schiff pro- Carolina, with especial emphasis on the cedure (PAS, for “neutral” carbohydrates anatomy of the oviduct. such as glucose, galactose, mannose, and si- alic acids). These staining procedures fol- MATERIALS AND METHODS lowed Kiernan (’90). Siren intermedia were collected using min- After initial fixation for TEM, tissues were now traps in a swampy drainage below the rinsed in Millonig’s buffer, postfixed in 2% spillway of Risher Pond, on the U. S. Depart- osmium tetroxide, dehydrated in ethanol, ment of Energy’s Savannah River Site (Aiken cleared in propylene oxide, and embedded in County, SC). Collections were made on 12-14 an epoxy plastic resin (EMBED-812; Elec- January, 16 February, 24-28 February, 6-7 tron Microscopy Sciences, Fort Washington, March, 7 April, 1 May, 23-27 June, 14 July, PA). Semithin sections (0.5-1.0 p,m) for light 18 September, and 27 October 1994. Some microscopy were cut with glass knives, placed specimens were sacrificed soon after capture, on microscope slides, and stained with tolu- whereas others were maintained in the labo- idine blue. Ultrathin (70 nm) sections for ratory for various periods of time prior to TEM were collected on uncoated copper grids sacrifice. Specimens maintained in the lab and stained with solutions of uranyl acetate were kept in 25 x 75 x 30 cm glass aquaria, and lead citrate. Plastic sections were cut supplied with local well water and a liberal with RMC XLlOOO and RMC MT7 ultrami- diet of bloodworms (Chironomidae). Speci- crotomes and viewed with a Hitachi H-300 mens in aquaria were monitored daily to transmission electron microscope. ascertain whether oviposition occurred. In Stages of spermatogenesis were identified addition to S. intermedia, two S. lacertina using the criteria established in Ambystoma were captured 6-7 March 1994 and main- by Armstrong (’89) and Uribe et al. (’94). tained in the laboratory. Oocytes in ovarian follicles were considered Specimens were sacrificed by immersion in mature and preovulatory when they were 10% MS-222, and snout-vent length (SVL) darkly pigmented and > 2.0 mm in diameter was measured from the tip of the snout to the (Noble and Marshall, ’32). Oocytes were mea- posterior end of the vent. Tissues were ex- sured using an ocular micrometer in a dissect- cised from freshly killed specimens and fixed ing microscope. for preparation by paraffin infiltration for light microscopy (LM), or for embedding in RESULTS epoxy resin for semithin (LM) or ultrathin Seasonal changes in trapping success of sections for transmission electron micros- males and females of Siren intermedia were copy (TEM). The fixatives were 10% neutral- found. The male:female ratio by month was: buffered formalin for LM, and a 1:1 solution January 5:6; February 05; March 3:2; April of 2.5% glutaraldehyde in Millonig’s phos- 0:3; May 51; June 7:O; July 4:O; September phate buffer at pH 7.4 and 3.7% formalde- 4:l; October 50. Thus females were collected hyde-buffered to pH 7.2 with monobasic and commonly only during January-April. dibasic phosphate for TEM. Testes and asso- The reproductive system of male Siren in- ciated urinary structures were removed from termedia matched the description by Willet males, and oviducts were dissected from fe- (’65). In females, we found that the oviduct is OVISAC AMPULLA ATRIUM

cloaca ovaryI I I intestine stomachI I iver A

OV I SAC AMPULLA with eggs B

Fig. 1. Gross anatomy of the oviduct of Siren intermedia. A. Drawing of the oviduct of a 176 mm SVL female collected 6-7 March and sacrificed 10 March 1994, showing the three major regions and relationships to some other major viscera. B. Photograph of the oviduct of a 119 mm SVL female collected 16 February and sacrificed 18 February, showing eggs in the ovisac. divided into three distinct portions (Fig. 1A): 1B). These portions of the reproductive tract (1) an anterior atrium into which eggs pass are described anatomically after consider- from the coelom, (2) a middle ampulla in ation of the male and female reproductive which gelatinous coverings are secreted onto cycles. the eggs, and (3) a posterior ovisac in which Our observations on Siren intermedia and the eggs are held prior to oviposition (Fig. the related S. lacertina indicate that rela- 338 D.M. SEVER ET AL. tively few follicles are ovulated at one time. were lined with secondary spermatogonia. Thus eggs apparently are laid in small groups. Thus maturation and transfer of sperm into An ovisac of S. intermedia probably cannot the reproductive ducts occurred between hold many more than 10-11 eggs, as ob- January and March. served in two individuals (Table 1). In the Females laboratory, eggs were laid separately or in clumps of 2 or 3 eggs, and the eggs adhered Six females (117-182 mm SVL) collected tightly to the substrate. Still, the egg-laying and sacrificed in January had 83-179 season may be relatively short. A captive 349 (mean = 140.3, SD = 25.6) pigmented oo- mm SVL S. lacertina laid 120 eggs, which cytes >2.0 mm in diameter (2.6-2.9 mm were removed between 15-17 March, and 72 mean diameters; Table 1) in their ovaries. more eggs by 21 March. The latter eggs re- These probably represented full clutch mained in the enclosure with others ovipos- complements. In three females (115-156 mm ited later to see if any of the eggs were fertile; SVL) collected and sacrificed in February, on 7 April, 113 eggs were removed, none some oviposition apparently had occurred. fertile. This siren did not deposit any addi- The ovaries contained 25,66, and 68 follicles, tional eggs prior to its sacrifice on 19May. and the mean oocyte diameter for each female was 2.6-2.7 mm. In addition, oviducal Reproductive cycles oocytes (eggs) occurred in the ovisacs of two Males of the specimens. In the 156 mm individual, Testicular lobules from specimens col- five eggs were in the right ovisac and 11 in lected in January were filled largely with the left; in a 119 mm female, 10 eggs were in spermatids indicating the imminent onset of the right and eight in the left ovisac (Fig. 1B; spermiation. No males were present in the Table 1). February sample, but mature spermatozoa A 144 mm SVL female collected on 24 were abundant in the testes and vasa efferen- February oviposited five infertile eggs prior tia of males collected in March, and some to her sacrifice on 10 March. At that time, spermatozoa were present in a specimen ex- only 35 oocytes (2.8 mm mean diameter) amined on 4 May. In late June, testicular remained in the ovaries, whereas the right lobules were filled with primary spermatogo- and left ovisacs each contained five eggs. In nia. In a male from the September collection, 162 mm and 176 mm SVL females collected proliferation of the spermatogonia was evi- and sacrificed in March, the ovaries con- dent, and lumina of the testicular lobules tained 36 (2.7 mm mean diameter) and 16

TABLE 1. Female Siren intermedia data' OD regions4 Oocytes5 Collected Sacrificed SVL2 OD/SVL3 AT/OD AMiOD OS/OD OV ROS LOS DIAM 12-14 Jan 16 Jan 182 .75 .10 .67 .23 179 0 0 2.6 12-14 Jan 16 Jan 166 .72 .ll .67 .22 147 0 0 2.9 12-14 Jan 16 Jan 160 .72 .09 .75 .16 162 0 0 2.6 12-14 Jan 16 Jan 137 .68 .12 .64 .24 151 0 0 2.7 12-14 Jan 16 Jan 117 .74 .09 .75 .16 83 0 0 2.7 12-14 Jan 18 Jan 139 120 0 0 2.6 16 Feh 18 Feh 156 25 5 11 2.6 16 Feb 18 Feb 119 66 10 8 2.7 16 Feb 18 Feb 115 68 0 0 2.6 24 Feb 10 Mar 144 .71 .09 .68 .23 35 5 5 2.@ 6-7 Mar 10 Mar 176 .76 .13 .64 .23 16 0 0 2.4 6-7 Mar 10 Mar 162 .74 .12 .63 .25 36 0 0 2.7 7 Apr 22 Apr 150 .71 .10 .66 .24 20 0 0 2.4 7 Apr 22 Apr 156 .69 .09 .72 .19 90 0 2.1 7 Apr 22 Apr 166 .74 .09 .72 .19 80 0 2.4 16 May 17 May 122 .67 .09 .71 .20 60 0 1.5 18 Sep 20 Sep 150 .69 .09 .72 .19 225 0 0 1.0 'All measurements are in mm; oocytes Jan-Apr refers to pigmented oocytes > 2.0 mm in diameter %nout-vent leneth. "Length of ovid&ISVL. 4ATIOD, length of atriumilength of oviduct; AMIOD, length of ampullailength of oviduct; OSIOD, length of ovisacilength of oviduct. 50V, number oocytes in ovary; ROS, number oocytes in right ovisac; LOS, number oocytes in left ovisac; DIAM, mean diameter of oocvtes~~~ _. 60viposited five infertile eggs in captivity. SIREN REPRODUCTION 339 (2.4 mm mean diameter) pigmented ovarian The ovisac is the posterior 16-25% oocytes, respectively. (mean = 20%,SD = 2.0)of the oviduct. Eggs In three specimens (150-166 mm SVL) are held in the ovisac prior to oviposition; in collected and sacrificed in April, 8-20 ovarian three of our specimens a single ovisac held oocytes > 2.0 mm were found; these oocytes 5-11 eggs. Unlike the “ovisac” of some frogs, generally were smaller (2.1-2.4 mm mean in which gelatinous egg coverings are se- diameter) than those of females collected from creted (Salthe, ’631, the ovisac of sirens is January-March and probably represent fol- simply a holding area. The ovisac is narrow licles undergoing atresia. The specimen (122 and rugose outside of the ovipository season mm SVL) collected and sacrificed in May had but expanded and smooth-walled internally only six pigmented, atretic oocytes (1.5 mm while holding eggs. mean diameter) that were embedded deeply in ovarian tissue. Histology and cytology Finally, a specimen collected and sacrificed in September contained - 225 pigmented and The entire oviducts of five females were unpigmented ovarian oocytes 1.0 mm in mean sectioned, and all lacked sperm. Specimens diameter. This specimen apparently was in included a 119 mm SVL female collected and the act of yolking oocytes for the forthcoming sacrificed in February with only 66 mature breeding season. oocytes remaining in her ovaries and 18 eggs In summary, these specimens indicate that in her ovisac, and 162 mm and 176 mm SVL the height of the ovipository season for fe- females collected and sacrificed in March that males was February and March, although contained only 36 and 16 mature ovarian some oviposition may commence in late Janu- oocytes remaining, respectively (Table 1). ary and the season could extend into early These data indicate that these females were April. This time period corresponds with the in the ovipository and presumed mating pe- period when males undergo spermiation. riod. The other two females, a 139 mm SVL individual collected and sacrificed in Janu- Oviducal anatomy ary, and a 150 mm SVL female collected and Gross anatomy sacrificed in September, represent individu- The oviduct is long, 67-79% of SVL als sacrificed outside of the presumed mating (mean = 73%, SD = 3.3). The anterior end is and ovipository period. proximal to the posterior border of the trans- The oviduct is lined with simple, ciliated verse septum, and the caudal end joins the epithelium that varies from columnar to cu- dorsal roof of the anterior end of the cloaca. A boidal when a particular region is empty or mesentery attaches the oviducts to the dorsal inactive but becomes squamous when the body wall and forms attachments with: (1) region is hypertrophied or contains follicles. adjacent digestive viscera, (2) the lungs, which Oviducal glands occur in the atrium and the extend along almost the entire length of the ampulla (Fig. 2A-C). These glands, when oviducts, and (3) the ovaries, which occur actively secreting, are strongly PAS+, and medial to the posterior end of the oviducts. hypertrophy so that they occupy most of the The oviducts are divided into three regions connective tissue of the tunica propria. Al- (Fig. 1A). though the atrium and ampulla both have The atrium is the anterior 7-13% oviducal glands, the atrium is narrower and (mean = lo%, SD = 1.0) of oviductal length. less convoluted. Oviducal glands are lacking We do not use the more traditional term in the ovisac (Fig. 2D). “infundibulum” for this region because the Superficial to the basal lamina of the ovidu- anterior end of the siren oviduct is not funnel- cal epithelium is a thin connective tissue shaped. The atrium is straight and relatively layer, the tunica propria, in which collagen narrow compared to the middle region of the fibers are abundant. The basal portion of the oviduct. tunica propria contains numerous enlarged The ampulla is the middle 63-75% capillaries, characterized by a diameter suffi- (mean = 69%, SD = 3.0) of the oviduct. This cient to hold several red blood cells in any glandular region is highly convoluted in speci- particular section and by walls composed of mens collected from all seasons and becomes simple squamous endothelium. Superficial to greatly hypertrophied during the ovipository the tunica propria is a sheet of longitudinal season. It is in this section of the oviduct that smooth muscle, the tunica muscularis, which outer gelatinous capsules are added to the is only one or two layers thick in the atrium follicles. and ampulla but many layers thick in the 340 D.M. SEVER ET AL.

Fig. 2. Siren intermedia. Sagittal paraffin sections of A. C. Ampulla of a 119 mm SVL individual collected 16 the oviduct stained with alcian blue at pH 2.5 and the February and sacrificed 18 February. D. Junction be- periodic acid-Scbiff procedure, showing development of tween the ampulla and atrium of the specimen used in A. oviducal glands (Og) during the breeding season. A. Lu, lumen of oviduct; Sm, smooth muscle; Vp, visceral Atrium of a 162 mm SVL specimen collected 6-7 March pleuroperitoneum. and sacrificed 10 March. B. Ampulla of specimen used in SIREN REPRODUCTION 341 ovisac. Superficial to the muscularis is the eggs, the ovisacs are widened and therefore visceral pleuroperitoneum (Fig. 2). smooth-walled internally. Areas of ciliated Seasonal differences occur in the histology epithelium occur among cells that lack cilia and histochemistry of the oviducts. No diag- (Fig. 5A, B). Although oviducal glands are nostic staining was done on the specimens lacking, the cytoplasm contains vacuoles filled collected and sacrificed in January. The ovi- with a flocculent material (Fig. 5B, C) that ducal glands, however, are eosinophilic and probably is responsible for the PAS+ reac- appear to be full of secretory material. In tion of the epithelium. The cytoplasm sur- specimens collected and sacrificed in Febru- rounding these vacuoles contains numerous ary and March, which were in the process of mitochondria with tubular cristae and free laying eggs (the ovaries contained only 16-68 ribosomes (Fig. 5C). Also, the intercellular mature oocytes and the ovisacs of some speci- canaliculi are convoluted more in the ovisac mens contained eggs), the epithelial lining of than in other portions of the oviduct. the atrium is AB+,and a mix of AB+ and In the specimen collected and sacrificed in PAS+ material occurs throughout the cyto- September, the atrium is not greatly hyper- plasm. The ampulla is greatly hypertrophied. trophied. A weak AB+ reaction occurs around The oviducal glands of the ampulla are in- the luminal border, but a PAS+ reaction is tensely PAS+, and an Al3+ reaction is lim- limited to the nuclei in the epithelium. In the ited to the mucosal epithelium. Both the ampulla, scattered AB+ areas occur deep to atrium and ampulla lack eggs, and the inner the luminal border, but an AB+ reaction lining is rugose. In specimens collected in does not occur superficially and oviducal March, the areas of PAS+ activity in the glands are not well developed (Fig. 6A). Cilia atrial and ampullar epithelium are not as are not numerous (Fig. 6B, C) and are most uniform as in oviducts of females collected in highly developed in the posterior portion of February, perhaps indicating depletion of the the ampulla. Clusters of lysosomes are nu- secretory product (Fig. 2B, C). The epithelial merous in many cells in the ampulla (Fig. cytoplasm appears more basophilic in March 6D), and perhaps these lysosomes are involved in degradation of secretory product specimens than in February specimens. left from the previous breeding season. Fi- Cytologically, oviducal glands are simple nally, intercellular spaces deep to the apical invaginations of epithelial cells lining pores junctional complexes are wider (Fig. 6C) than that open into the oviducal lumen. During in specimens collected during the breeding the breeding season, elongate microvilli are season. present along the luminal border of glands (Fig. 3A), although areas also occur that lack DISCUSSION microvilli (Fig. 3B). Several types of secre- Reproductive cycle tory products are present. One type of secre- The number of mature ovarian oocytes we tory vacuole is large (often 2-3 p,m in diam- observed in female Siren intermedia prior to eter), moderately electron dense, and fills oviposition is lower than reported elsewhere. much of the cytoplasm (Figs. 3, 4A), exclu- We found 83-179 darkly pigmented oocytes sive of the basal nucleus. Mucous droplets >2.0 mm in diameter in the ovaries of fe- can be found interspersed among these vacu- males sacrificed during January. Martof ('73) oles in some areas (Fig. 3B). reported that the average complement of eggs In other regions, smaller (1-1.5 Fm), more is "about 200" for S. intermedia. The only electron-dense secretory vacuoles also occur published egg counts are the reports of Noble (Fig. 4A), perhaps representing concentra- and Richards ('32) and Godley ('83) on speci- tions of the secretory product. Associated mens from Florida, and Noble and Marshall with the production of secretory vacuoles are ('32) who examined specimens from Arkan- extensive profiles of rough endoplasmic retic- sas. These observations are somewhat contra- ulum (Rer) and Golgi complexes with associ- dictory. Noble and Richards ('32) found 265 ated condensing vacuoles (Fig. 4B). The com- eggs from the caudal end of the right oviduct bination of a PAS+ reaction with organelles of a female, a number not consistent with our involved in peptide synthesis (Rer) indicates observations. Noble and Richards ('321, how- that the secretions contain neutral carbohy- ever, reported that captive sirens laid small drates conjugated with proteins (i.e., glycopro- numbers of eggs over a period of several days, teins; Kiernan, '90). which would seem to indicate fewer eggs in The ovisac has a thick muscularis and lacks the oviduct at any one time. Noble and Mar- oviducal glands (Fig. 5). When filled with shall ('32) found 299 mature ovarian oocytes 342 D.M. SEVER ET AL.

Fig. 3. Siren intermedia. Ultrastructure of the luminal border of oviducal glands from the ampulla of a 119 mm SVL specimen collected 16 February and sacrificed 18 February. A. Apical border possessing microvilli (Mv). B. Apical border lacking microvilli and possessing mucous droplets (Md) in additional to moderately electron-dense secretory vacuoles (Sv). Lu, lumen of oviducal gland; Md, mucous droplet; Mv, microvilli; Sv, moderately dense secretory vacuoles. SIREN REPRODUCTION 343

Fig. 4. Siren intermedia oviduct. Ultrastructure of secretory products. Cv, condensing vacuoles; Ds, dense the ampulla of a 119 mm SVL specimen collected 16 secretory vacuoles; Go, Golgi complexes; Lu, lumen of February and sacrificed 18 February. A. Relationship of a oviduct; Nu, nuclei of oviducal epithelial cells; Rer, rough gland to the epithelium lining the oviducal lumen (Lu). endoplasmic reticulum; Sv, moderately dense secretory B. Organelles involved in production of the glycoprotein vacuoles. 344 D.M. SEVER ET AL.

Fig. 5. Siren intermedia ovisac. Ultrastructure of the flocculent material (Fm) and associated organelles. Bb, ovisac of a 119 mm SVL female collected 16 February and basal bodies; B1, basal lamina; Cf, collagen fibers; Ci, cilia; sacrificed 18 February. A. Overview of epithelial lining Fm, flocculent material in vacuoles; Lu, lumen of ovi- showing ciliated (Ci) and unciliated areas. B. Detail of duct; Mi, mitochondria; Nu, nuclei of oviducal epithelial ciliated border. C. Perinuclear area showing vacuoles of cells; Ri, ribosomes. SIREN REPRODUCTION 345

Fig. 6. Siren intermedia ampulla in a 150 mm SVL somes (Ly). B1, basal lamina; De, spot desmosomes; Ic, female collected 18 September and sacrificed 20 Septem- intercellular canaliculus; Ld, lipid droplet; Lu, lumen of ber. A. Histology of a paraffin section stained with hema- the oviduct; Mi, mitochondria; Nu, nuclei of oviducal toxylin-eosin. B. Ultrastructure of the epithelial border epithelial cells; Og, oviducal glands; Vp, visceral pleuro- of the ampulla. C.Detail of the apical border. D. Detail of peritoneum; 20, zonula occludens. the basal portion of the oviducal epithelial showing lyso- 346 D.M. SEVER ET AL (139 in the right ovary and 160 in the left) in jelly coats are necessary for successful sperm a S. intermedia measuring 396 mm total penetration during fertilization, the coats length, and they observed “nests” of 260 and block sperm penetration when they become 555 eggs in shallow hollows in the mud at the hydrated after expulsion of the fertilized eggs bottom of ponds. Godley (’83) reported find- from the cloaca (McLaughlin and Humphries, ing two s. intermedia nests in masses of ’78). aquatic vegetation in Florida, and both nests Greven (’80a) reported that the oviduct of were attended by single females. One nest, the ovoviviparous salamandrid Salamandra attended by a 151 mm SVL female contained salamandra has five regions: an anteriorpars 206 eggs and the other nest, attended by a recta, a middle pars convoluta with three 167 mm SVL female, had 362 eggs. divisions, and a posterior uterus where the More observations on the number of ma- fetuses develop. The regions are distinguished ture oocytes produced during a reproductive based upon cell types in the epithelial layer. season (and its relationship to body size), Ciliated epithelium occurs in all regions ex- mode of oviposition (large nests or small clus- cept the uterus (Greven and Ruterbories, ters of eggs), and geographic variation in ’84). Simple gland cells and tubular glands fecundity are needed for S. intermedia. are found in all areas but are especially numerous in the pars convoluta, and products Oviducal anatomy include neutral as well as acid mucosub- Detailed reports on the anatomy of the stances (Greven, ’80a). oviducts of salamanders are few. McCurdy The uterus in S. salamandra produces (’31) found that the oviduct of Triturus toro- small amounts of secretion, probably not sus (Salamandridae) has four parts: an ante- enough to supply nutritional demands for rior, widened ostial portion, a second, highly developing fetuses (Greven, ’80b). Indeed, convoluted region, a third, widened area ion flow is generally from the uterine lumen where jelly coats of the eggs are added, and a into the uterine mucosa, perhaps regulating “uterine” portion extending from the poste- the intrauterine milieu appropriate for devel- rior end of the kidney to the cloaca. The opment of offspring (Greven, ’80b). In the anterior three divisions have thinly muscular viviparous S. atra, however, the anterior end walls, whereas uterine walls are thick. All of the uterus, the “zona trophica,” is highly divisions have villi, simple columnar ciliated glandular and may produce nutrients for the epithelium and secretory cells. No mention fetus in utero (Greven, ’77). In viviparous was made of tubular glands, and some varia- frogs and caecilians, virtually the entire ovi- tion exists in the extent of ciliation and abun- ducal epithelium proliferates and secretes nu- dance of secretory cells. trient material (Wake, ’93). Rodgers and Risley (’38) studied develop- In summary, the oviducal anatomy of Si- ment of the reproductive system in Ambys- ren intermedia at the gross, histological, and toma tigrinum (Ambystomatidae). They cytological levels seems unique among those found two regions, an anterior, convoluted described for other salamanders, regardless oviducal portion and a posterior, straight of mode of fertilization or whether oviducal uterine part. In the oviducal region, the retention of developing fetuses occurs. lamina propria is thin and the epithelium is thick with tubular glands that open into the Implications for internal versus external lumen. The uterus has thinner walls than fertilization the oviduct, and the uterine mucosa has co- Sperm were absent from the oviducts of all lumnar epithelium with a lamina propria females examined histologically. For the that is more abundant and less compact than specimens collected during the ovipository in the oviducal region. From their illustra- period in which some oviposition apparently tion, it seems that tubular glands are absent had occurred, this finding leaves two possibili- in the uterine region. ties: (1) no oviducal sperm storage/fertiliza- The oviduct of the salamandrid Notophthal- tion occurs, or (2) oviducal sperm storage1 mus viridescens is divisible into five regions, fertilization does occur, but these individuals designated A-E, that differ in their polysac- had voided all the sperm from previous mat- charide histochemistry and are involved in ings. Salamanders in the suborder Salaman- secreting jelly coats around the eggs droidea store sperm in specialized cloacal (Humphries, ’66). All areas have a PAS+ glands, spermathecae, that are lacking in si- secretion, and secretions from areas A, B, renids (Sever, ’91a, ’94). Eggs in oviparous and D are alcian blue+ as well. Although the salamandroids are fertilized internally as they SIREN REPRODUCTION 347 pass through the cloaca during oviposition U.S. Department of Energy (D.O.E.) con- (Boisseau and Joly, '75). In salamandroid tract DE-AC05-760R00-033. LCR was sup- species in which detailed studies on the cytol- ported by the Undergraduate Research Pro- ogy of sperm storage and fertilization have gram of the Savannah River Ecology been done, the presence of abundant sperma- Laboratory. JDK was supported by contract tozoa in the spermathecae prior to and after DE-AC09-76SR00-819 between the Univer- oviposition has been documented (Sever, '91c, sity of Georgia and the D.O.E. This paper is '92; Sever and Brunette, '93; Sever and publication number seven from the Saint Kloepfer, '93; Brizzi et al., '95; Sever et al., Mary's College Electron Microscopy Facility. '95). Thus if oviducal sperm storage and/or fertilization occurred in Siren intermedia, LITERATURE CITED one would expect to find some trace of sperm Armstrong, J.B. (1989) Spermatogenesis. In J.B. Arm- in the three animals sacrificed during their strong and G.M. Malacinski (eds): Developmental Biol- ovipository period. The absence of oviducal ogy of the Axolotl. Oxford: Oxford Univ. Press, pp. sperm provides support for the traditional 36-41. view that fertilization in the Sirenidae is Boisseau, C., and J. Joly (1975) Transport and survival of spermatozoa in female Amphibia. In E.S.E. Hafez and external. C.G. Thibault (eds): The Biology of Spermatozoa: TransDort. Survival and Fertilizine" Abilitv. Basel: Implications for phylogeny Karge;, pp: 94-104. Brizzi, R., G. Delfino, M.G. Selmi, and D.M. Sever (1995) The species of Sirenidae have been consid- The spermathecae of Salamandrina terdigitata (Am- ered as either early offshoots from the urode- phibia: Salamandridae): Patterns of spermstorage and lian lineage, one of the most derived sala- deeradation. J. MorDhol. 223.2-33. mander families, or not even salamanders at Cop; E.D. (1889) The Batrachia of North America. U.S. Nat. Mus. Bull. 34:l-525. all (Cope, 1889; Hecht and Edwards, '77; Duellman, W.E., and L. Trueb (1986) Biology of Amphib- Estes, '81; Milner, '83; Duellman and Trueb, ians. New York: McGraw-Hill. '86; Hillis, '91; Larson, '91; Sever, '91a,b, Estes, R. (1981) Handbuch der Palaoherpetolopie. Teil2. '94; Larson and Dimmick, '93). The most Gymnophiona, Caudata. Stuttgart: G. Fischer. Godley, J.S. (1983) Observations on the courtship, nests recent cladistic analysis, based upon a combi- and young of Siren intermedia in southern Florida. nation of molecular and morphological data, her.Midl. Nat. 110:215-219. places the Sirenidae basally as the sister group Goin, C.J., and O.B. Goin (1962) Introduction to Herpe- of all other families of salamanders (Larson tology. San Francisco: W.H. Freeman. Greven, H. (1977) Comparativeultrastructural investiga- and Dimmick, '93). tions of the uterine epithelium in the viviparous Sala- Key characters in the latter analysis are mandra atra Laur. and the ovoviviparous Salamandra the absence of cloacal glands involved in inter- salamandra (L.) (Amphibia, Urodela). Cell Tissue Res. nal fertilization in the Salamandroidea and 181:215-237. Greven, H. (1980a) Licht- und electronenmikroskopische thus the presumption of external fertiliza- Untersuchungen zur Struktur und Histochemie des tion within the Sirenidae. Internal fertiliza- Oviduktepithels (Pars recta und Pars convoluta I, 11, tion resulting from spermatophore produc- 111) von Salamandra salamandra (L.) (Amphibia, tion by males and sperm storage in Urodela). Z. mikrosk.-anat. Forsch., Leipzig 94:387- 429. spermathecae by females is a synapomorphy Greven, H. (1980b) Ultrahistochemical and autoradio- for the Salamandroidea (Sever, '94). Our re- graphic evidence for epithelial transport in the uterus sults support the hypothesis that fertiliza- of the ovoviviparous salamander Salamandra salaman- tion in sirenids is external, representing the dra (L.) (Amphibia, Urodela). Cell Tissue Res. 212t147- ancestral state, and since no evidence exists 162. Greven, H., and H.-J. Ruterbories (1984) Scanning elec- that this condition is a reversal, we must tron microscopy of the oviduct of Salamandra sala- conclude that sirenids have retained the an- mandra (L.) (Amphibia, Urodela). Z. mikrosk.-anat. cestral state for fertilization in the Lissam- Forsch., Leipzig 98:49-62. phibia (Sever, '94). It is apparent, however, Hecht, M.K., and J.L. Edwards (1977) The methodology of phylogenetic inference. In M.K. Hecht, P.C. Goody, that characters involving oviducal anatomy and B.M. Hecht (eds): Major Patterns in Vertebrate may have phylogenetic significance within Evolution, New York Plenum Press, pp. 3-51. the Caudata, and future, comparative re- Hillis, D.M. (1991) The phylogeny of amphibians: Cur- search on the oviducal anatomy of sala- rent knowledge and the role of cytogenetics. In D.M. Green and S.K. Sessions (eds): Amphibian Cytogenet- manders is welcomed. ics and Evolution. New York: Academic Press, pp. 7-31. ACKNOWLEDGMENTS Humphries, A.A., Jr. (1966) Observations on the deposi- tion, structure, and cytochemistry of the jelly enve- DMS received support from National Sci- lopes of the egg of the newt, Triturus uiridescens. ence Foundation Grant DEB 90-24918 and Develop. Biol. 13:214-230. 348 D.M. SEVER ET AL. Kiernan, J.A. (1990) Histological and Histochemical data). I. Evolution at the family level. Herpetologica Methods, 2nd ed. Oxford: Pergamon Press. 47:165-193. Larson, A. (1991) A molecular perspective on the evolu- Sever, D.M. (1991b) Comparative anatomy and phylog- tionary relationships of the salamander families. In eny of the cloacae of salamanders (Amphibia: Cau- M.K. Hecht, B. Wallace, and R.J. MacIntyre (eds): data).11. Cryptobranchidae, Hynobiidae and Sirenidae. Evolutionary Biologv, Vol. 25. New York: Plenum Press, I J. Morphol. 207t283-301. pp. 211-277. Sever, D.M. (1991~)Sperm storage and degradation in Larson. A.. and W.W. Dimmick (1993) Phvlogenetic rela- the spermathecae of the salamander Eurycea cirrigera tionships of the salamander families: "An-analysis of (Green)J. Morphol. 21 03-84. congruence among morphological and molecular characters. Herpetol. Monogr. 7t77-94. Sever, D.M. (1992) Spermiophagy by the spermathecal Martof, B.S. (1973) Siren intermedia. Cat. Amer. Am- epithelium of the salamander Eurycea cirrigera. J. phib. Rept. 127:l-3. Morphol. 212t281-290. Martof, B.S. (1974) Sirenidae. Cat. her.Amphib. Rept. Sever, D.M. 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