Ultrastructure of the Spermathecae of Necturus Beyeri (Amphibia: Proteidae) in Relation to Its Breeding Season Author(S): David M

Home , Necturus

Ultrastructure of the Spermathecae of Necturus beyeri (Amphibia: Proteidae) in Relation to Its Breeding Season Author(s): David M. Sever and Henry L. Bart, Jr. Source: Copeia, Vol. 1996, No. 4 (Dec. 27, 1996), pp. 927-937 Published by: American Society of Ichthyologists and Herpetologists Stable URL: http://www.jstor.org/stable/1447655 . Accessed: 27/12/2010 15:11

Your use of the JSTOR archive indicates your acceptance of JSTOR's Terms and Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp. JSTOR's Terms and Conditions of Use provides, in part, that unless you have obtained prior permission, you may not download an entire issue of a journal or multiple copies of articles, and you may use content in the JSTOR archive only for your personal, non-commercial use.

Please contact the publisher regarding any further use of this work. Publisher contact information may be obtained at . http://www.jstor.org/action/showPublisher?publisherCode=asih. .

Each copy of any part of a JSTOR transmission must contain the same copyright notice that appears on the screen or printed page of such transmission.

JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact [email protected].

American Society of Ichthyologists and Herpetologists is collaborating with JSTOR to digitize, preserve and extend access to Copeia.

http://www.jstor.org Copeia, 1996(4), pp. 927-937

Ultrastructure of the Spermathecae of Necturusbeyeri (Amphibia:Proteidae) in Relation to Its Breeding Season

DAVID M. SEVER AND HENRY L. BART JR.

Sperm occurred in the spermathecae of all female Necturus beyeri collected in a Louisiana stream from Dec. through May, including those taken from nests after oviposition in May. The spermathecae possessed two types of epithelial cells: dark cells, with secretory vacuoles 1-2 jm dia; and light cells, with secretory vacuoles 0.5-1.0 im dia. The vacuoles of both types of cells contained glyco- proteins that were secreted into the lumen and bathed sperm during storage. The vacuoles were most abundant early in the breeding season, and light cells in individuals examined after oviposition were completely depleted whereas dark cells retained vacuoles only along the luminal border. If the secretions help maintain sperm viability, the depletion of secretory product could limit the storage of remnant sperm during the summer and fall months. However, no evidence of sperm degradation was found, and the fate of sperm remaining in the spermathecae following fertilization is unknown. Necturus beyeri can store sperm for at least six months prior to oviposition.

THE Proteidae consists of five species of from Talisheek Creek between the Gulf, Mo- branchiated, aquatic salamanders in the bile, and Ohio Railroad crossing at Talisheek genus Necturus from eastern North America and and the LA Hwy 41 crossing, Saint Tammany Proteus anguinus from the Adriatic coast of Parish, Louisiana (Table 1). Specimens collect- northeastern Italy and Croatia (Frost, 1985). ed in May were postoviposition females re- Like other salamanders in the suborder Sala- moved from nests. Numbers of embryos in each mandroidea, cloacal glands in males produce nest at the time of guardian removal are given spermatophores and cloacal glands, called sper- in Table 1. We collected other specimens with mathecae, in females store sperm (Sever, 1991 a, modified Thiel-Turkay traps baited with earth- 1992b). The actual transfer of a spermatophore worms (Manning, 1986). We sacrificed these and fertilization has never been observed in the specimens before oviposition. Proteidae. Salthe (1967) suggested that sper- We sacrificed specimens 0-2 days after col- matophore transfer in proteids is via cloacal ap- lection by immersion in 10% MS-222. We ex- position. Eggs apparently are fertilized by re- cised cloacal regions and preserved them in a lease of sperm from the spermathecae as the 1:1 solution of 2.5% glutaraldehyde in Millon- eggs pass through the cloaca (Jordan, 1893; ig's phosphate buffer at pH 7.4 and 3.7% form- Boisseau and Joly, 1975). aldehyde buffered to pH 7.2 with monobasic Ultrastructure of sperm storage in the the and dibasic phosphate (NBF). We preserved car- spermathecae of female salamanders has been casses in NBF and cataloged them into the Tu- described in the Ambystomatidae (Sever and lane Museum of Natural History (TU) herpe- Kloepfer, 1993; Sever, 1995; Sever et al., 1995), tology collection (Table 1). Plethodontidae (Pool and Hoage, 1973; Davitt After fixing cloacal tissue in glutaraldehyde: and Larsen, 1988a; Sever and Brunette, 1993), formalin, we subsequently prepared it by par- and Salamandridae (Dent, 1970; Brizzi et al., affin infiltration for light microscopy (LM), or 1995; Sever et al., 1996), but no reports exist for embedding in epoxy resin for semithin (LM) for the Proteidae. In this paper, we report on or ultrathin sections for transmission electron aspects of female sperm storage during the microscopy (TEM). For the paraffin method, we breeding season of Necturus beyeri in a popula- rinsed the tissue in water after fixation, dehy- tion in which certain other aspects of the re- drated it in ethanol, and cleared it in Histosol production were studied by Shoop (1965), and (National Diagnostics, Inc., Manville, NJ) prior we describe the cytology of the spermathecae to embedment in paraffin. We cut sagittal sec- of N. beyeriduring the breeding season. tions (10 ,Lm)with a rotary microtome, affixed them to albuminized slides, and stained alter- MATERIALSAND METHODS nate slides with hematoxylin-eosin (HE) for gen- eral cytology or alcian blue at pH 2.5 (AB) for This study was based on 17 specimens of N. glycosaminoglycans with a counterstain of pe- beyeri collected 13 Dec. 1993 to 8 Jan. 1995 riodic acid and Schiffs reagent (PAS) for "neu-

? 1996 by the American Society of Ichthyologists and Herpetologists 928 COPEIA, 1996, NO. 4

TABLE 1. VOUCHER,SIZE, AND OVARIANDATA FORFEMALE Necturus beyeriUSED IN THIS STUDY.

Meanova CollectionDate TU no. SVL (mm) GSI' Ova Nb size (mm) 13 Dec. 1993 23401 112 0.025 50 2.33 23402 110 0.029 60 2.37 23403 106 0.039 57 2.51 23404 112 0.042 54 2.50 2 Jan. 1994 23405 129 0.044 76 2.91 23406 108 0.045 41 2.99 23407 145 0.025 59 2.42 13 March 1994 23408 116 0.064 28 4.62 23409 129 0.034 36 3.39 23411 104 0.086 38 3.98 23412 127 0.048 37 3.66 22 May 1994 23414 119 nesting 37 23415 116 nesting 26 23416 138 nesting 33 23417 117 nesting 32 4 Dec. 1994 23528 114 0.022 47 2.22 8 Jan. 1995 23538 103 0.041 35 2.90

* Gonadosomaticindex (ovaryweight as proportionof body weight withoutviscera). bTotal numberof ova in left and ovaries or in nest. c enlargedyellow right (= clutch), embryos Meansize (mm)of 10 randomly-selectedova.

tral" carbohydrates such as glucose, galactose, position, but the glands appeared relatively re- mannose, and sialic acids. These staining pro- duced in diameter, especially those glands that cedures followed Kiernan (1990). lacked sperm (Fig. 1D). In the latter glands, After initial fixation for TEM, we rinsed tis- "light cells," whose ultrastructure is described sues in Millonig's buffer, postfixed in 2% os- below, were particularly conspicuous (Fig. 1D). mium tetroxide, dehydrated in ethanol, cleared In paraffin sections, apical portions of the sper- in propylene oxide, and embedded in an epoxy mathecal epithelium were mainly PAS+, al- resin (EMBED-812; Electron Microscopy Sci- though some AB+ areas occurred as well (see ences, Fort Washington, PA). We cut semithin also Sever, 1994). PAS+ substances also oc- sections (0.5-1.0 ium)for light microscopy with curred around clusters of sperm in the lumen. glass knives, placed on them microscope slides, In plastic sections, the secretory material stained and stained them with toluidine blue. We col- darkly with toluidine blue but was not meta- lected ultrathin (70 nm) sections for TEM on chromatic. The staining reactions were most uncoated copper grids and stained them with intense in those specimens sacrificed prior to uranyl acetate and lead citrate. We cut sections oviposition. on RMC XL1000 or RMC MT7 ultramicro- Transmission electron microscopy revealed tomes and viewed ultrathin sections with a Hi- two types of epithelial cells that were distin- tachi H-300 transmission electron microscope. guished based upon the size of their secretory vacuoles and the electron density of their cy- RESULTS toplasm (Figs. 2-3). In "dark cells," the secretory vacuoles were generally 1-2 ,m dia and As reported by Sever (1992b), the sperma- consisted of a central dense area (0.5-0.7 ,um thecae of female N. beyeriwere simple tubular dia) surounded by an outer, more lucent, floc- exocrine glands that opened onto the roof of culent material (Fig. 2B-C). Secretory vacuoles the cloaca. Sperm occurred in the spermathecae also were present in light cells, at least prior to of all specimens examined (Fig. 1). Sperm were oviposition, but these vacuoles were 0.5-1.0 ,m not evenly distributed among the glands, how- dia, with a central dense material that usually ever, and some glands or at least portions of was 0.3-0.5 ,m dia (Fig. 3A-D). Light cells were some glands were devoid of sperm in each spec- interspersed among dark cells, and apical ends imen. When present in a gland, sperm were of both cell types were on the luminal border randomly oriented and clustered in the center (Fig. 3C). The intercellular canaliculi between of the lumen (Fig. 1). Sperm seemed most nu- light cells and dark cells were narrow and in- merous in those specimens collected in March terdigitating, and desmosomes between the cell (Fig. 1C). Much sperm still remained after ovi- types were numerous (Fig. 3B,D). SEVER AND BART-SPERMATHECAE OF NECTURUS 929

. .fl . \ t^l lA4 i

Fi.1' s ' f 0 Fig. 1. Paraffinsections through the spermathecaeof Necturusbeyeri stained with hematoxylin-eosin.(A) TU 23402, a 110 mm SVL gravid specimen collected 13 Dec.; (B) TU 23407, a 145 mm SVL gravidspecimen collected 2 Jan.; (C) TU 23412, a 127 mm SVL gravid specimen collected 13 March;(D) TU 23417, a 117 mm SVL spent specimen collected 22 May. Ep = spermathecalepithelial cell; Lc = light cell; Sp = sperm in lumen of spermatheca;and Tp = tunica propria. 930 COPEIA, 1996, NO. 4

Fig. 2. Spermathecae of TU 23403, a 106 mm SVL Necturus beyericollected gravid on 13 Dec. (A) Plastic semithin section stained with toluidine blue. (B) Ultrathin section of a dark cell. (C) Ultrathin section showing detail of secretory vacuoles and organelles involved in synthesis of the product. Cv = condensing vacuoles; De = desmosome; Dm = dense material; Ep = spermathecal epithelial cells; Fm = flocculent material; Go = Golgi apparatus; Ic = intercellular canaliculus; Lu = lumen; Mf = microfilaments; Nu = nucleus of a spermathecal epithelial cell; Rer = rough endoplasmic reticulum; Sv = secretory vacuoles; and Tp = tunica propria. SEVER AND BART-SPERMATHECAE OF NECTURUS 931

~-' i> yS; ^. {t h 3aJSf1'-^-..~. Jte^"*>^'^*;'C ~~~~~~~~~~::2

" r

A,0,:..'4. {~.:.~ :" . ~ ..:,-..

,, -?,,.. -?,,.. 5I m :P ~~. Lu P- .;- .,.~.~...:~,,,~ .... ~?L '"~ :'~

.;s o.., ';~,?,~,' ,,, ,~:'':'~,.?.t...x~L?~'.""~. ...~-~~ + . / ' .D" ' '~.;-t,.- , ' T, ?; * ... " 'D '. -''? .. 4~'. :,

Fig. 3. Ultrathin sections through the spermathecaeof TU 23403, a 106 mm SVL Necturusbeyeri collected gravid on 13 Dec. (A) Overview of a light cell and adjacentdark cell. (B) Detail of the border between a dark cell and a light cell. (C) Luminal border. (D) Detail of the cytoplasm of a light cell. Dc = dark cell; De = desmosome; Dm = dense material; Fm = flocculent material; Ic = intercellularcanaliculus; Lc = light cell; Lu = lumen; Nu = nucleus of a spermathecalepithelial cell; and Sv = secretory vacuoles. 932 COPEIA, 1996, NO. 4

The secretory vacuoles in the light and dark matids Ambystomaopacum (Sever and Kloepfer, cells probably were responsible for the PAS+ 1993) and A. tigrinum (Sever, 1995). In S. ter- reaction in the cytoplasm since the PAS+ re- digitata and A. opacum, both the dense material action was most intense around the luminal bor- and the flocculent material are exported into der where the secretory vacuoles were most nu- the lumen to bathe the sperm during storage merous. Therefore, release of the product like- (Brizzi et al., 1995; Sever and Klopefer, 1993). ly was responsible for the PAS + reaction around In A. tigrinum, however, Sever (1995) believed sperm in the lumen. Golgi bodies and rough that the flocculent material results from disso- endoplasmic rectiulum (Rer) were associated ciation of the compacted granules, and the vac- with the vacuoles in the cytoplasm (Fig. 2C). A uole is then released into the lumen via exo- PAS+ reaction combined with these synthetic cytosis. The process of release of the secretory organelles indicates the product contained gly- material was not determined in N. beyeri, but coproteins (Kiernan, 1990). the quantity of secretory vacuoles along the api- In specimens collected in May, shortly after cal border decreased during sperm storage, and oviposition, sperm still were numerous in some the release of the product probably is merocrine spermathecae (Figs. 1D, 4A-B). The luminal as reported in other salamanders (Brizzi et al., sperm appeared normal in cytology (Fig. 4B). 1995; Sever, 1991a, 1995). Instead of a wide supranuclear area filled with The secretion bathed the sperm during stor- secretory vacuoles, the apical cytoplasm of spec- age in N. beyeri, as reported in salamandrids imens collected after oviposition contained rel- (Brizzi et al., 1995, Sever et al., 1996) and in A. atively few vacuoles (Fig. 4C). Also, the secre- opacum(Sever and Kloepfer, 1993). Since sperm tory vacuoles appeared more uniform in density are inactive during storage (Hardy and Dent, (Fig. 4C) than those observed in specimens sac- 1986), Sever and Kloepfer (1993) proposed that rificed prior to oviposition. Microvilli seemed this type of secretion helps maintain the envi- relatively more elongate after oviposition (Fig. ronment for sperm viability and quiescence. In 4B-C). No evidence of spermiophagy by the A. tigrinum, however, the secretions are not re- spermathecal epithelium was noted, but struc- leased until oviposition and may serve to flush tures consisting of concentric membranes were the sperm from the spermathecae (Sever, 1995). common especially basally in the cytoplasm of This role for these secretions, hypothesized by specimens sacrificed after oviposition (Figs. 4D, Sever (1995) to be the ancestral function of 5A). These concentric membrane structures of- spermathecal secretions, is associated with short- ten were associated with Golgi bodies (Fig. 4D). term sperm storage (several days or less). Nec- Intercellular canaliculi were wider basally fol- turus beyerican store sperm for at least six months lowing oviposition (Fig. 5A), and desmosomes prior to oviposition (Dec.-May), and the secre- were frequent at the basal borders of adjacent tions apparently were released slowly through- cells (Fig. 5B). Basal globules that appear to be out the period of storage, since some secretory lipid droplets (Sever and Kloepfer, 1993) were vacuoles still remained (at least in the dark cells) seen in individuals sacrificed after oviposition after oviposition. Thus, we hypothesize that the (Fig. 5C). secretions provide the environment for keeping Light cells seemed especially depleted after the sperm viable but inactive during long-term oviposition. Secretory vacuoles and organelles storage in N. beyeri. involved in synthetic activity were not notice- We found two distinct types of spermathecal able in these light cells (Fig. 6). The most con- epithelial cells, dark cells and light cells, in N. spicuous structures were small dense bodies beyeri.Only one other study reported more than (about 0.3 ,tm) that occasionally were seen in one type of epithelial cell in spermathecae of the cytoplasm (Fig. 6C). salamanders. Davitt and Larsen (1990), in an abstract, stated that an unspecified "plethodon- DISCUSSION tid salamander" has five types of cells in its spermathecae. Their cell type II consists of an elec- Secretory vacuoles in the plethodontid Eu- tron dense core surrounded by more lucent ar- rycea cirrigera (Sever, 1991b, 1992a) and in the eas, much like the secretory vacuoles we found salamandrid Notophthalmus viridescens (Sever et in N. beyeri.The other cell types found by Davitt al., 1996) are of uniform density throughout and Larsen (1990) contain vacuoles that differ the period of production. Secretory vacuoles in size and shape as well as the density of the consisting of a central dense area and a sur- product, which is uniform in each cell type. Dav- rounding flocculent material have been noted itt and Larson (1990) proposed that some of the in the salamandrid Salamandrina terdigitata secretions are involved in sperm activation (Brizzi et al. 1989, 1995) and in the ambysto- whereas others, including those in cell type II, SEVER AND BART-SPERMATHECAE OF NECTURUS 933

' ' L' : Lu '~ . .-:u .....

.:,-',. .''~' s ,;f, ;,~ ? ~... ~ ~ - ...... ,c) ~~~~-e f~~~V~~f>:.> I - z/ ' .... "'" i ~'",."~S~'.~i.'

jJf';n~'.", .r '~,.', ,,

' .- . , '~ '~'~';'"'~ C; ~'~~:.~..-:?-:i.,"~~~~~~.1 mu m & , ~~~~ ~ ~ , v ( 4 t~- ;- '

i.f ! : ~ .....''.. .'- I? " 'r A '~,D ,! ::"","~,Q?,;?,,' 'u :,~ri: ", .,' ' :..~, ??=~;. ~,.?.- - ?'?4''~:.

Fig. 4. Spermathecae of TU 23416, a 138 mm SVL Necturus beyericollected spent on 22 May. (A) Plastic semithin section stained with toluidine blue. (B) Ultrathin section showing sperm in the lumen. (C) Luminal border of a dark cell. (D Cytoplasm of a dark cell. Cm = concentric membranes; Ep = spermathecal epithelial cell; Go = Golgi apparatus; Lu = lumen; Mv = microvilli; Nu = nucleus of a spermathecal epithelial cell; Sn = sperm nucleus; Spl = sperm in the lumen; Sv = secretory vacuole; and Tp = tunica propria. 934 COPEIA, 1996, NO. 4

Fig. 5. Ultrathin sections through basal portions of dark cells in the spermathecaeof TU 23416, a 138 mm SVL Necturusbeyeri collected spent on 22 May. (A) Overview. (B) Desmosome between two cells. (C) Presumed lipid droplet and surrounding cytoplasm. Bl = basal lamina; Cm = concentric membranes;De = desmosome;Ic = intercellularcanaliculus; Ld = lipid-likedroplet; and Nu = nucleusof a spermathecalepithelial cell. help modulate continued alterations in sper- collected during Dec. and Jan. from this pop- mathecal function during storage. In N. beyeri, ulation contained motile sperm in the Wolffian both spermathecal cell types release their prod- ducts, and several females collected in Jan. had ucts into the lumen during sperm storage, and the remains of spermatophores in their cloacae. the main difference between the cell types is the Shoop's observations provide additional evi- size of the secretory vacuoles. Also, the light dence that females may store sperm up to 5-6 cells apparently are depleted before the dark months prior to fertilization and oviposition. cells. We could not collect animals from June-Nov., The presence of sperm in all N. beyeri ex- although Shoop and Gunning (1967) had suc- amined raises the question regarding whether cess during this period. sperm storage can continue from one breeding Thus, the fate of sperm left in the sperma- season to the next, as suggested for some other thecae of N. beyerifollowing fertilization is un- salamanders (Baylis, 1939; Massey, 1990) but known. However, the spermathecae showed de- never substantiated (Sever, 1995; Sever et al., pletion of secretory product after oviposition. 1996). Shoop (1965) found that male N. beyeri If the secretions do indeed help maintain the SEVER AND BART-SPERMATHECAE OF NECTURUS 935

.4js~L3~~~~C #j.#'?. 0~ 1? iB ~ ;r~.!e~u,L~

Fig. 6. Sections through light cells in the spermathecaeof 23416, a 138 mm SVL Necturusbeyeri collected spent on 22 May. (A) Plastic semithin section stained with toluidine blue. (B) Ultrathin section showing an overview. (C) Ultrathin section showing detail of the cytoplasm. Db = dense bodies; Dc = dark cell; Lc = light cell; Lu = lumen; Nu = nucleus of a spermathecalepithelial cell; and Tp = tunica propria. = tunica propria. environment for sperm viability, the depletion In many species of salamanders that have been of secretory product could limit the storage of studied, remnant sperm are phagocytized, and remaining sperm during the summer and fall often spermiophagy by the spermathecal epi- months. thelium occurs even prior to oviposition (Davitt 936 COPEIA, 1996, NO. 4 and Larson, 1988b; Sever, 1992a; Sever and HARDY,M. P., ANDJ. N. DENT. 1986. Transport of Kloepfer, 1993). No evidence of spermiophagy sperm within the cloaca of the female red-spotted was found in N. beyeri,a situation also noted in newt. J. Morphol. 190:259-270. E. 1893. The A. tigrinum (Sever, 1995). Perhaps the sperm JORDAN, 0. habitsand development of in the "leak out" the newt (Diemyctylusviridescens). Ibid. 8:269-366. remaining spermathecae simply A. 1990. or are con- KIERNAN,J. Histologicaland histochemical gradually expelled by myoepithelial methods: and 2d ed. as for theory practice. Pergamon tractions, reported sperm remaining in Press, Oxford. the vas deferens of A. macrodactylum and (Zalisko MANNING,R. B. 1986. A small trap for collecting Larsen, 1989). A complete understanding of crustaceans in shallow water. Proc. Biol. Soc. Wash. the entire annual cycle of sperm storage in fe- 99:266-268. male N. beyeriawaits the study of specimens with MASSEY,A. 1990. Notes on the reproductiveecology known breeding histories from June-Nov. of red-spotted newts (Notophthalmusviridescens). J. Herpetol. 24:106-107. ACKNOWLEDGMENTS POOL, T. B., AND T. R. HOAGE. 1973. The ultrastructure of secretion in the spermatheca of the Laboratory work at Saint Mary's College was salamander, Manculus quadridigitatus (Holbrook). Tissue Cell supported by National Science Foundation grant 5:303-313. DEB 9024918 to DMS. We thank D. Miller and SALTHE,S. N. 1967. Courtshippatterns and the phy- E. Moen for their in tissue sam- logeny of the urodeles. Copeia 1967:100-117. help preparing SEVER,D. M. 199 la. and This is number 9 from the Comparativeanatomy phy- ples. publication of the cloacae of salamanders Saint Electron Fa- logeny (Amphibia: Mary's College Microscopy Caudata).I. Evolution at the family level. Herpe- cility. tologica 47:165-193. . 199 b. Sperm storage and degradation in LITERATURE CITED the spermathecaeof the salamanderEurycea cirrigera (Green).J. Morphol. 210:71-84. BAYLIS, H. A. 1939. Delayed reproduction in the . 1992a. Spermiophagyby the spermathecal spotted salamander.Proc. Zool. Lond. 109A:243- epithelium of the salamander Euryceacirrigera. Ibid. 246. 212:281-290. BOISSEAU,C., ANDJ.JOLY. 1975. Transport and sur- 1992b. Comparativeanatomy and phylogeny vival of spermatozoain female Amphibia, p. 94- of the cloacaeof salamanders(Amphibia: Caudata). 104. In: The biology of spermatozoa: transport, V. Proteidae. Herpetologica 48:318-329. survival,and fertilizing ability. E. S. E. Hafez and 1994. Observationson regionalizationof se- C. G. Thibault (eds.). Karger, Basel, Switzerland. cretoryactivity in the spermathecaeof salamanders BRIZZI,R., G. DELFINO,AND C. CALLONI. 1989. Fe- and comments on phylogeny of sperm storage in male cloacalanatomy in the spectacledsalamander, female salamanders.Ibid. 50:383-397. Salamandrina terdigitata (Amphibia: Salamandri- . 1995. Spermathecae of Ambystomatigrinum dae). Herpetologica 45:310-322. (Amphibia Caudata):development and a role for , M. G. SELMI,AND D. M. SEVER.1995. the secretion.J. Herpetol. 29:243-255. The of spermathecae Salamandrina terdigitata (Am- ,AND N. S. BRUNETTE.1993. Regionalization phibia: Salamandridae):patterns of sperm storage of eccrine and spermiophagicactivity in the sper- and degradation.J. Morphol. 223:21-33. mathecae of the salamanderEurycea cirrigera (Am- DAVITT, C. M., AND J. H. LARSEN JR. 1988a. Scan- phibia:Plethodontidae).J. Morphol. 217:161-170. electron ning microscopy of the spermatheca of ,AND N. M. KLOEPFER.1993. Spermathecal Plethodon larselli (Amphibia: Plethodontidae): cytology of Ambystomaopacum (Amphibia: Ambys- in the changes surface morphology of the sper- tomatidae)and the phylogeny of sperm storage or- mathecal tubule prior to ovulation. Scanning Mi- gans in female salamanders.Ibid. 217:115-127. crosc. 2:1805-1812. ,J. D. KRENZ, K. M.JOHNSON, AND L. C. RANIA. , AND . 1988b. Phagocytosis of stored 1995. Morphology and evolutionary implications and spermatozoa cytoplasmicdroplets by the sper- of the annual cycle of secretion and sperm storage mathecal of epithelium the female salamanderRhy- in spermathecaeof the salamanderAmbystoma opa- acotritonolympicus. Am. Zool. 28:30A. cum(Amphibia: Ambystomatidae). Ibid. 223:35-46. ,AND . 1990. Morphology of the prin- , L. C. RANIA, AND J. D. KRENZ. 1996. The cell ciple typesof the plethodontidsalamander sper- annual cycle of sperm storage in the spermathecae matheca following treatment with gonadotropin. of the red-spotted newt, Notophthalmus viridescens Ibid. 30:38A. (Amphiba:Salamandridae). Ibid. 227:145-170. DENT, J. N. 1970. The ultrastructureof the sper- SnooP, C. R. 1965. Aspects of reproductionin Lou- matheca in the red spotted newt. J. Morphol. 132: isiana Necturuspopulations. Amer. Midi. Nat. 74: 397-424. 357-367. FROST,D. R. (ED). 1985. Amphibian species of the , AND G. E. GUNNING. 1967. Seasonal world: a taxonomic and activity geographic reference. Al- and movements of Necturusin Louisiana. Copeia len Press, Inc., and Association of SystematicCol- 1967:732-737. KS. lections, Lawrence, ZALISKO, E. J., AND J. H. LARSEN JR. 1989. Fate of SEVER AND BART-SPERMATHECAE OF NECTURUS 937

unused sperm in post-breedingmale Ambystomama- NATURAL HISTORY, BELLE CHASSE, LOUI- crodactylumcolumbianum. J. Herpetol. 23:463-464. SIANA70037. Send reprint requests to DMS. Submitted: 8 Sept. 1995. Accepted: 23 Jan. (DMS) DEPARTMENTOF BIOLOGY, SAINT MARY'S 1996. Section editor: J. R. Spotila. COLLEGE,NOTRE DAME, INDIANA 46556; AND (HLB) TULANE UNIVERSITY MUSEUM OF

Copeia, 1996(4), pp. 937-944

Gymnotusbahianus sp. nov., a New Gymnotid Fish from Eastern Brazil (Teleostei: Ostariophysi: Gymnotiformes), with Evidence for the Monophyly of the Genus

RICARDO CAMPOS-DA-PAZAND WILSON J. E. M. COSTA

Gymnotusbahianus sp. nov. is described from the Rio Almada basin, near Ilheus, Bahia State, Brazil. It is currently known only from that area, repre- senting the third record of a gymnotid species in eastern Brazilian coastal plain basins, north of the Rio Doce basin, Espirito Santo State. Gymnotusbahianus shares with G. cylindricus LaMonte, G. inaequilabiatus (Valenciennes), and G. pantherinus (Steindachner),a pattern of small dark rounded spots over the body, which distinguish these species from their congeners. The new species is further distinguished by a unique combination of morphological features, including depth of the body and head, length of the head, pectoral fin and maxillary bone, and the number of perforated scales along lateral line. The phylogenetic rela- tionships of the new species are still unknown. New evidence for the monophyly of Gymnotusis presented. Gymnotusbahianus sp. nov. e descrita da bacia do rio Almada, vizinhancas de Ilheus, Estado da Bahia, Brasil. Atualmente, e conhecida apenas daquelas areas, consistindo o terceiro registro de uma especie de Gymnotidae para as bacias costeiras do leste do Brasil, ao norte da bacia do rio Doce, Estado do Espirito Santo. Gymnotusbahianus e similar a G. cylindricusLaMonte, G. inaequilabiatus (Valenciennes) e G. pantherinus (Steindachner), diferindo das demais especies do genero por seu padrio de colorido, o qual consiste de pequenas manchas escuras e arredondadas distribuidas sobre o corpo. A nova espicie e tambem distinta com base numa combinacao unica de caracteristicas morfologicas, com- preendendo as alturas do corpo e da cabeca, comprimentos da cabeca, nadadeira peitoral e osso maxilar, e numero de escamas perfuradas da linha lateral. As relaqies filogeneticas de G. bahianus sio ainda desconhecidas. Nova evidencia para o monofiletismo de Gymnotuse apresentada.

FISHES of the ostariophysan order Gymno- sult, an increasing amount of information con- tiformes attracted the attention of early cerning gymnotiform electrophysiology has be- ichthyologists mainly because of their very spe- come available (e.g., Heiligenberg and Bastian, cialized morphology (e.g., Kaup, 1856). Sub- 1980; Kramer et al., 1981), but taxonomic sequently, Lissmann (1951, 1958) demonstrat- problems at various levels preclude an objective ed that gymnotiform species, in addition to the investigation of those observed patterns. Ellis' electric eel Electrophorus electricus (Linnaeus), monograph (1913) and the recently published possess electrogenic and electroreceptive abili- study of Mago-Leccia (1994) are the most com- ties, resulting in a change in the focus of interest prehensive published studies on gymnotiform to the electrophysiology of the order. As a re- taxonomy, but our understanding of the species

? 1996 by the American Society of Ichthyologists and Herpetologists