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 ovidu- cal length, is the highly convoluted, middle portion in which gelatinous cover- ings 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.