THE JOURNAL OF EXPERIMENTAL ZOOLOGY 282:460-476 (1998)

Comparative Biology of Sperm Storage in Female Salamanders

DAVID M. SEVERN* AND ROSSANA BRIZZI' 'Department of Biology, Saint Mary's College, Notre Dame, Indiana 46556 ^Dipartimento di Biologia Animale e Genetica, Universita di Firenze, 50125 Firenze, Italia

ABSTRACT Females in seven of the ten families of salamanders possess cloacal glands called spermathecae that store sperm. The annual cycle of sperm storage has been studied by light and electron microscopy in eight species representing five families. In these taxa, we recognized 14 characters associated with the spermathecae and traced their evolution on a phylogeny of sala• manders based upon other characters. The plasticity and phyletic significance of the spermathecal characters varied greatly. Plethodontids have complex spermathecae while other families possess simple spermathecae; thus, this character has phyletic value as well as being highly conserved within the Salamandroidea. Other characters, such as carbohydrate histochemistry, are highly plastic and show no obvious phyletic trends. The significance of some of these variable characters, such as duration of sperm storage, is apparent only after including in the analysis other aspects of the reproductive cycle, such as length of the mating season. Additional comparative studies, em• ploying the protocol used in this paper, will help further clarify the relationships between phyletic and functional variability in sperm storage mechanisms in salamanders. J. Exp. Zool. 282:460- 4 76, 1998. © 1998 Wiley-Liss, Inc.

In amphibians with external fertilization (most sperm from those involved in oogenesis, ovula• frogs, some salamanders), close timing must oc• tion, and fertilization. cur among: maturation and transfer of male and In the past decade, we have conducted exten• female gametes (mature, jelly-coated eggs in the sive studies on the annual cycle of sperm storage oviducts and mature, capacitated sperm in the in various salamanders selected with especial em• Wolffian ducts); maximal development of male phasis on their phylogenetic relationships and re• and female secondary sexual characters; male and productive adaptations (Brizzi et al., '89, '95; female sexual urges leading to migration and mat• Sever, '91b, '92a, '94a, '95, '97; Sever and Bru• ing behaviors; and existence of appropriate envi• nette, '93; Sever and Kloepfer, '93; Sever et al., ronmental surroundings. '95, '96a, '96b; Sever and Bart, '96). We believe Female salamanders in the seven families sufficient data exist on a diverse assemblage of composing the suborder Salamandroidea store taxa to allow a first attempt at a synthesis of sperm in exocrine glands, the spermathecae, in the comparative biology of sperm storage in fe• the dorsal roof of the cloaca (Sever, '91a). In male salamanders. Hence, one purpose of this oviparous species, sperm are released on eggs paper is to produce a procedural and phyletic as they pass through the cloaca, so fertilization framework for further studies on sperm storage is internal. The ability to store sperm for ex• in salamanders. We also report our initial find• tended periods before fertilization allows de• ings on the plasticity of spermathecal characters coupling of mating activity from oogenesis, among the urodeles, and analyze the relation• ovulation, and fertilization/oviposition; allows ships of this variability to species-specific repro• delay of fertilization until environmental condi• ductive adaptations. tions are most favorable; allows extension of the mating period; facilitates multiple matings; and provides the conditions for sperm competition (i.e., meeting the criteria established by Parker, Grant sponsor: National Science Foundation; Grant number: DEB 90-24918; Grant sponsor: Ministero DeH'Universita e della Ricerca '84). From a regulatory standpoint, sperm stor• Scientifica e Tecnologica. age permits segregation of hormones involved in *Correspondence to: Dr. David M. Sever, Department of Biology, Saint Mary's College, Notre Dame, IN 46556. E-mail: dsever@saint stimulating mating behavior and storage of marys.edu

© 1998 WILEY-LISS, INC. SPERM STORAGE IN FEMALE SALAMANDERS 461

MATERIALS AND METHODS TABLE 2. Spermathecal characters Determination of the annual cycle of sperm 1. Cloacal glands: A = absent; A^ = present storage in the spermathecae requires examina• 2. Spermathecae: B = absent; B^ = present tion of females: sexually active but unmated; 3. Type of spermathecae: C - simple; C^ = complex 4. Histochemistry: D = PAS+; = alcian blue+; D' = both mated, but prior to ovulation, fertilization, and PAS+ and alcian blue+ oviposition; immediately after oviposition; and 5. Secretory vacuoles: E = mixed density; = uniform from various periods following oviposition, ex• density tending into a period when females are not 6. Secretory activity: F = uniform; F^ = regionalized sexually active (Sever, '97). Tissue from the 7. Timing of release of secretion: G = storage; G"*^ = mating; G' = oviposition; G^ = degradation spermathecal area was taken from freshly sac• 8. Mode of release of secretion: H = merocrine; = rificed individuals representing each of these apocrine periods, and the tissue prepared by similar 9. Types of epithelial cells: I = one type; I^ = two types methods (cf., Brizzi et al., '95; Sever, '97) for 10. Spermiophagy by the epithelium: J = absent; = examination by light and transmission electron present throughout; j' = present but regionalized microscopy (TEM). 11. Sperm degradation in the lumen: K = present; K'^ = absent The species that have been studied using this 12. Desquamation of the epithelium: L = epithelium remains protocol are: Ambystoma tigrinum and A. opa- intact; = desquamation into the lumen; 1? = cum in the Ambystomatidae; Amphiuma trida- desquamation into the stroma; = desquamation into ctylum in the Amphiumidae; Eurycea cirrigera both the lumen and the stroma 13. Intraepithelial leukocytes: M = absent; = present and Plethodon cinereus in the Plethodontiade; 14. Duration of sperm storage: N = <1 month; = 1-2 Necturus beyeri in the Proteidae, and Notoph• months; N' = 2-3 months; = 4-5 months; N* = 6 thalmus viridescens and Salamandrina terdigit- months; = >6 months ata in the Salamandridae (Table 1). Our critical review of these studies led to the recognition of a number of characters relating specifically to RESULTS the spermathecae (Table 2). Different states of Fig. 1 shows the result of tracing character evo• these characters were coded numerically with no lution in this manner. Where the nodes for par• prejudice concerning ancestral or derived condi• ticular states were equivocal, one of the equally tion (Tables 3, 4). Using McClade 3.0 (Maddison parsimonious solutions was chosen, except for and Maddison, '92), these characters were map• character A, where the ancestral state for cau- ped on a phylogeny of the taxa based upon the dates was left equivocal. The consistency indices preferred cladogram of interfamilial relation• for the characters range from 0.60 to 1.00, with ships of salamanders proposed by Larson and an overall index of 0.80. Dimmick ('93). Character A: Presence of cloacal glands

TABLE 1. Species for which the ultrastructure of the annual The ancestral state is equivocal, but cloacal cycle of spermathecal sperm storage has been studied using glands of at least one type are found in all sala• the protocol described in the text mander families except Sirenidae (Figs. 1, 2A; Family Sever, '91a,c). Species Reference Character B: Presence or absence Ambystomatidae of spermathecae Ambystoma opacum Sever and Kloepher, '93 Sever etal.,'95 Hynobiids and cryptobranchids have cloacal Ambystoma tigrinum Sever, '95 glands but lack those that produce spermato- Amphiumidae phores and store sperm (Fig. 2B-D; Sever, '91c). Amphiuma tridactylum Sever et al.,'96b Plethodontiade This condition is considered the ancestral state Eurycea cirrigera Sever, '91b, '92b for females in all families of salamanders except Sever and Brunette, '93 Sirenidae (Fig. 1). Plethodon cinereus Sever, '97 Proteidae Character C: Type of spermathecae Necturus beyeri Sever and Bart, '96 Salamandridae Simple spermathecae characterize six families Notophthalmus viridescens Sever etal,'96a and consist of simple tubuloalveolar glands open• Salamandrina terdigitata Brizzi et al., '89, '95 ing individually into the roof and lateral walls of 462 D.M. SEVER AND R. BRIZZI

TABLE 3. Character matrix

Character

Taxon A B C D E F G H I J K L M N

Sirenidae A 0 0 0 0 0 0 0 0 0 0 0 0 0 Cryptobranchidae Al B 0 0 0 0 0 0 0 0 0 0 0 0 Hynobiidae Al B 0 0 0 0 0 0 0 0 0 0 0 0 Ambystoma opacum Al Bi C Di E F G H I J K Li Ml Ni Ambystoma tigrinum Al Bi C D^ E F G' H I J K L M N Amphiuma tridactylum Al Bi C D E F G^ Hi I J K Li M N3 Eurycea cirrigera Al Bi ci D El pi G H I J2 K 1} M N' Plethodon cinereus Al Bi Ci D' E F G H I Jl K L M Necturus beyeri Al Bi c D E F G H II J K L M Notophthalmus viridescens Al Bi c D El F Gi J I Jl K L M Salamandrina terdigitata Al Bi c Di E F G H I Jl K Li Ml the anterior half of the cloaca (Fig. 3A; Sever, tion of the states within the Salamandridae and '94a). Complex spermathecae, found only in the Ambystomatidae are equivocal (Fig. 1). Plethodontidae (the largest family of salaman• ders) consist of compound tubuloalveolar glands Character E: Ultrastructure of the (Fig. SB; Sever, '87, '94a,b). A single common duct secretory vacuoles connects distal acini to the middorsal roof of the This character refers to whether secretory vacu• anterior end of the cloaca. As pointed out by Sever oles have a mixed density (an electron dense core ('94a), the ancestral state is equivocal, and it is surrounded by flocculent material. Fig. 4C), or equally parsimonious to consider either type an• whether they have a uniform electron density, as cestral to the other or to hypothesize that the found in Eurycea cirrigera and Notophthalmus character is polyphyletic. In Fig. 1, the ancestral viridescens (Fig. 4D; Sever, '95; Sever et al., '96a). state for the Salamandroidea is mapped as pres• The ancestral state is considered secretory vacu• ence of simple spermathecae. oles of mixed density (Fig. 1).

Character D: Carbohydrate histochemistry Character F: Whether secretory This character (Fig. 4A, B) concerns whether activity is regionalized the epithelium stains with the periodic-acid Schiff Regionalization of secretory activity occurs when procedure for neutral carbohydrates (D), alcian secretions are absent in some areas. Only one spe• blue at pH 2.5 for carboxylated glycosaminogly- cies in which the complete annual cycle of the sper• cans (D^), or equally for neutral and acidic mathecae has been studied with transmission mucosubstances (D^). Mapping the character onto electron microscopy (TEM) shows regionalization, the cladogram of salamander relationships re• Eurycea cirrigera (Fig. 4B; Sever and Brunette, veals by parsimony that the ancestral state is '93). Parsimony analysis indicates that the ances• presence of PAS+ secretions, although the evolu• tral state is uniform secretory activity (Fig. 1).

TABLE 4. Data matrix

Character Taxon A B c D E F G H I J K L M N

Sirenidae 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Cryptobranchidae 1 0 0 0 0 0 0 0 0 0 0 0 0 0 Hynobiidae 1 0 0 0 0 0 0 0 0 0 0 0 0 0 Ambystoma opacum 1 1 1 2 1 1 1 1 1 2 1 2 2 2 Ambystoma tigrinum 1 1 1 3 1 1 3 1 1 1 1 1 1 1 Amphiuma tridactylum 1 1 1 1 1 1 4 2 1 1 1 2 1 4 Eurycea cirrigera 1 1 2 1 2 2 1 1 1 3 1 3 1 3 Plethodon cinereus 1 1 2 3 1 1 1 1 1 2 1 1 1 4 Necturus beyeri 1 1 1 1 1 1 1 1 2 1 1 1 1 5 Notophthalmus viridescens 1 1 1 1 2 1 2 1 1 2 1 1 1 5 Salamandrina terdigitata 1 1 1 2 1 1 1 1 1 2 1 2 2 5 SPERM STORAGE IN FEMALE SALAMANDERS 463

SIREN SP.

CRYPTOBRANCHUS SP.

HYNOBIUSSP.

EURYCEA CIRRIGERA

PLETHODON CINEREUS

AMPHIUMA TRIDACTYLUM

NECTURUS BEYERI

NOTOPHTHALMUS VIRIDESCENS

SALAMANDRINA TERDIGITATA

AMBYSTOMA TIGRINUM

AMBYSTOMA OPACUM

Fig. 1. Cloacal characters from Tables 2-4 mapped us- a phylogeny of salamander taxa based upon Larson and ing McClade version 3 (Maddison and Maddison, '92) upon Dimmick ('93).

Character G: When the secretion is released Character H: Mode of release The ancestral state appears to be release dur• This character state codes whether release of ing sperm storage, state G (Figs. 1, 5A). found secretion is merocrine (exocytosis after membrane in Notophthalmus viridescens is secretion during fusion of the vacuoles and the apical epithelium. mating (Fig. 5B); G^ found in Ambystoma tigrinum Fig. 6A) or apocrine (desquamation of apical cy• is release during oviposition (Fig. 5C); and G^, se• toplasm containing secretory vacuoles. Fig. 6B). cretion during luminal degradation of sperm, is At the TEM level, an apocrine secretion has been found in Amphiuma tridactylum (Fig. 5D). reported only for Amphiuma tridactylum (Fig. 6B; 464 D.M. SEVER R. BRIZZI

Fig. 2. Characters A and B. Transverse paraffin sections B). (B) Cryptobranchus alleganiensis. (C) Batrachuperus through the cloaca stained with hematoxylin-eosin (Sever, tibetanus. (D) Onychodactylus japonicus. Cc, cloacal cham• '91c). Scale bars are in microns. (A) Siren intermedia, which ber; Gg, granular gland; Sk, skeletal muscle; Tp, tunica pro• lacks cloacal glands (character A). In B, C, and D, ventral pria; Vg, ventral gland. glands are present but spermathecae are lacking (character

Sever et al., '96b), and the ancestral state resolves with TEM in only one species, Necturus beyeri as merocrine (Fig. lA). (Fig. 6C, D; Sever and Bart, '96).

Character I: Types of spermathecal Character J: Whether spermiophagy occurs epithelium in the spermathecal epithelium The ancestral state is one type of epithelial cell The ancestral condition is equivocal, but it is in the spermathecae (Fig. 1). So far, more than mapped here as being absent (Fig. 1). Sper• one type of epithelial cell has been documented miophagy by the spermathecal epithelium occurs SPERM STORAGE IN FEMALE SALAMANDERS 465

mimm

Fig. 3. Character C, type of spermathecae. Transverse plex spermatheca autapomorphic for plethodontids (charac• paraffin sections through the cloaca stained with hematoxy• ter C^; Sever, '87). Cc, cloacal chamber; Ct, common tube of lin-eosin. Scale bars are in microns. (A) Ambystoma opacum, the spermatheca; Db, distal bulbs; Nt, neck tubules; Op, showing simple spermathecae (character C; Sever and opisthonephros; Sk, skeletal muscle; St, spermathecae; Tp, Kloepfer, '93). (B) Hemidactylium scutatum, showing a com• tunica propria.

in Ambystoma opacum (Fig. 7A), in both sala- often accompanies desquamation (Fig. 9B, C). The mandrids (Fig. 7B, C), and in both plethodontids ancestral condition is absence of desquamation. (Figs. 7D and 8). In Eurycea cirrigera, however, spermiophagy is regionalized and occurs only in Figure 4 appears on the next page. the distal bulbs where secretory activity is ab• Fig. 4. Characters D, E, and F. Scale bars are in microns. sent (Fig. 8; Sever and Brunette, '93). (A) Transverse parafiHn section through the cloaca of Ambystoma opacum with spermathecae stained positively with alcian blue Character L: Epithelial desquamation 8GX (character D; Sever, '94a). (B) Transverse paraffin section through the cloaca oi Eurycea cirrigera with spermatheca stained In Salamandrina terdigitata (Fig. 9A) and Am• positively with PAS (character D^; Sever and Brunette, '93). Note bystoma opacum (Fig. 9B), after depletion of the also limitation of positive staining reaction to common tubes secretory product by merocrine processes, portions and neck tubules (character F^). (C) Transmission electron mi• crograph of apical spermathecal cytoplasm of Ambystoma of the apical cytoplasm desquamate into the lu• tigrinum prior to oviposition, showing secretory vacuoles of mixed men where the epithelium degenerates (Sever and density (character E; Sever, '95). (D) Transmission electron mi• Kloepfer, '93; Brizzi et al., '95). In Amphiuma crograph of apical spermathecal C3rtoplasm of Notophthalmus tridactylum, portions of apical cytoplasm desqua• viridescens prior to mating, showing secretory vacuoles with uni• mate as a result of the apocrine secretion pro• form density (character E^; Sever et al., '96a). AB, alcian blue positive reaction; Clt, cloacal tube; Db, distal bulbs; Dm, dense cess (Fig. 9C; Sever et al., '96b). In Eurycea material; Fm, flocculent material; Lu, lumen; Nt, neck tubules; cirrigera, epithelial desquamation occurs also, but Nu, epithelial cell nucleus; PAS, periodic acid/Schiflf's reagent the cytoplasm desquamates into the surrounding positive reaction; Set, sperm in the common tube; St, spermath• tunica propria (Sever, '91a). Sperm degeneration ecae; Sv, secretory vacuoles. 466 D.M. SEVER AND R. BRIZZI

Figure 4. SPERM STORAGE IN FEMALE SALAMANDERS 467

Figure 5. (Caption on next page.) 468 D.M. SEVER AND R. BRIZZI

Character M: Presence of In the diversification of the Salamandroidea intraepithelial leukocytes into seven families, we feel it is highly signifi• In both Ambystoma opacum (Fig. lOA) and cant that a similar spermathecal type, the simple spermatheca, was conserved in six of the fami• Salamandrina terdigitata (Fig. lOB), white lies. Only in the Plethodontidae did a distinct blood cells have been reported in the intercel• spermathecal anatomy evolve, the complex sper• lular canaliculi between spermathecal epithe• matheca, and this character helps support phy- lial cells (Sever and Kloepfer, '93; Brizzi et al., logenies that hypothesize that the Plethodontidae '95). This condition resolves as convergent in forms the sister taxon to the rest of the Sala• the two taxa (Fig. 1). mandroidea (Larson and Dimmick, '93). Character N: Duration of effective Certain spermathecal characters are found only sperm storage in plethodontids and therefore may be dependent upon the possession of complex spermathecae. When mapped without prejudice on the cla• Regionalization of secretory activity (character F^) dogram (Fig. 1), the ancestral state is resolved was noted only in Eurycea cirrigera among spe• as N^, 4-5 months as seen in Plethodon cinereus cies in which the annual cycle of sperm storage and Amphiuma tridactylum. in spermathecae has been studied using TEM. In DISCUSSION a survey done at the light microscopy level, how• ever, Sever ('94a) reported regionalization of secre• The results so far show some interesting tory activity in 10 of 41 plethodontids examined trends, some of which indicate unique adapta• (Sever, '94a). Thus, some plasticity in this char• tions in certain taxa and others that imply that acter is apparent among plethodontids. A goal of homoplasy may be as rampant in spermathecal future studies will be to determine whether other characters as in many other morphological char• plethodontids that possess regionalized secretory acters of salamanders. No doubt additional re• activity also possess regionalized spermiophagy search will reveal more characters and clarify (character J'), as in E. cirrigera. some of the current ones that possess three or more states. No regionalization of secretory activity was found in any of the non-plethodontids examined Conserved and plastic characters for this paper or in 30 species that possess simple spermathecae that were examined at the light mi• As discussed by Sever ('94a), the mechanism croscopy level by Sever ('94a). The lack of region• for internally inserting sperm in the female alized specializations in the simple tubuloalveolar cloaca, the spermatophore, must have evolved be• glands that compose simple spermathecae may fore the mechanism for sperm storage, the sper• in turn limit some of their functional/physiologi• mathecae. The glands involved in spermatophore cal capabilities, as compared to the compound production, and indeed the basic structure of the tubuloalveolar glands of plethodontids. spermatophore itself, are similar among the vari• ous families in the Salamandroidea, and it makes The significance of several other characters sense from an anatomical as well as a parsimony that were noted in only one taxon (characters standpoint to accept the hypothesis that the sper• matophore and spermathecae are sjmapomorphies Fig. 6. Characters H and I. Scale bars are in microns. for the Salamandroidea (Sever, '91b). (A) Transmission electron micrograph of spermathecal lu• minal border in Ambystoma tigrinum showing merocrine re• lease of secretion (character H; Sever, '95). (B) Transmission Fig. 5. Character G, timing of the release of the secre• electron micrograph of spermathecal luminal border in tion. Transmission electron micrographs of the apical cyto• Amphiuma tridactylum showing apocrine mode of secretion plasm and adjacent lumen of spermathecae. Scale bars are (character H^; Sever et al., '96b). (C) Light micrograph of a in microns. (A) Eurycea cirrigera, showing release during stor• plastic section stained with toluidine blue through the sper• age (character G; Sever and Brunette, '93). (B) Notophthalmus mathecae of Necturus beyeri showing two types of epithelial viridescens, showing release after mating (character G^; Sever cells (character I^; Sever and Bart, '96). (D) Transmission et al., '96a). (C) Ambystoma tigrinum, showing release dur• electron micrograph of the two cell types in A^. beyeri (Sever ing oviposition (character G^; Sever, '95). (D) Amphiuma and Bart, '96). Ac, apical cytoplasm; Dc, dark cells; Dml, tridactylum, showing release during sperm degradation (char• dense material in the lumen; Eac, evagination of apical cy• acter G^; Sever et al., '96b). Ac, apical cytoplasm; Dsp, de• toplasm; Ev, exocytotic vacuole; Fml, flocculent material free graded sperm; Ic, intercellular canaliculi; Lu, lumen; Nu, in the lumen; Lc, light cells; Lu, lumen; Iv, luminal vacu• epithelial cell nucleus; Se, secretory products; Sp, sperm; Sv, oles; Mv, microvilli; Nu, epithelial cell nucleus; Sv, secre• secretory vacuoles. tory vacuoles; Tp, tunica propria. SPERM STORAGE IN FEMALE SALAMANDERS 469

Figure 6. 470 D.M. SEVER AND R. BRIZZI

H\\, and L^) and characters found in two experimental evidence exist that such sperm could taxa from different clades (characters and M^) still fertilize eggs. simply must wait examination of additional taxa. For example, in Amphiuma, 15 months of Data from some limited studies on other species, spermathecal sperm storage has been reported however, indicate that some of these characters (Baker, '62). However, we found that A. trida• may not be so unique. For example, among the ctylum in southeastern Louisiana initiates mat• species used for Fig. 1, only Amphiuma trida• ing in February and nesting in June, so five ctylum was found to have an apocrine secretion months of effective sperm storage may occur, al• (character H^). In a study using scanning elec• though we don't know whether sperm that were tron microscopy (SEM), however, Davitt and stored in February are actually used in fertiliza• Larsen ('88) report an apocrine product in the tion five months later or whether sperm from plethodontid Plethodon larselli, and studies in later matings are used (Sever et al., '96b). Sperm progress on the newt Triturus vulgaris indicate persist after the nesting season, but the sper• an apocrine secretion may occur in that species mathecae are inactive and the sperm are degrad• also (Sever and Brizzi, unpublished). We found ing. By May of the following year, a few sperm two types of epithelial cells (character I^) only may be left, but these are remnants that we be• in Necturus beyeri. But Davitt and Larsen ('88) lieve are too few and damaged to fertilize eggs. recognized two epithelial cell types with SEM The apparent "record" for sperm storage of 2.5 in Plethodon larselli, and, in an abstract, they years reported by Baylis ('39) is based upon an reported five types in an unspecified pletho• isolated, captive specimen of Salamandra sala• dontid (Davitt and Larsen, '90). mandra. As pointed out by Sever ('95), this record One of the most plastic characters appears to must be viewed with suspicion due to uncertain• be carbohydrate histochemistry (character D). ties among relationships between mating, sperm The significance of this character is unclear, but storage, fertilization, and gestation period in that consistently the secretory products of the sper• ovoviviparous species. In the related S. atra, fer• mathecae of different species react predomi• tilization occurs within three months after mat• nately to one type of carbohydrate stain or the ing, but gestation lasts 2-3 years (Hafeli, '71). other (Fig. 4A, B; Sever, '94a). In other verte• Sever ('95) hypothesized that short-term sperm brates, oviducal sperm storage glands often are storage (two days or less), as seen in Ambystoma PAS+ (Howarth, '74). tigrinum and many other ambystomatids, should The most conserved character is K, degrada• tion of sperm in the lumen. Indeed, an alterna• tive character state has not been observed as yet. Figure 8 appears on p. 472. We believe it is highly significant that the even• Fig. 7. Character J^, spermiophagy. Transmission electron tual fate of sperm remaining in the lumen after micrographs showing degradation of sperm in the spermathecal the breeding season is rapid degeneration, with epithelium. Scale bars are in microns. (A) Ambystoma opacum (Sever and Kloepfer, '93). (B) Salamandrina terdigitata (Brizzi no carry-over of cytologically normal sperm from et al., '95). (C) Notophthalmus viridescens (Sever et al., '96a). one breeding season to the next. This observa• (D) Plethodon cinereus (Sever, '97). Af, axial filament; Fl, fda- tion obviously has implications as well for char• mentous material; Ld, lipid droplet; Mpt, middle piece of the acter N, duration of effective sperm storage, which tail; Nr, nuclear ridge; PI, primary lysosome; Ppt, principle piece of the tail; Pv, phagocytic vacuole; Sn, sperm nucleus; is highly plastic. Sv, secretory vacuole. Periods of effective sperm storage in spermath• Fig. 8. Character J^, regionalized spermiophagy. Trans• ecae of female salamanders have been reported mission electron micrographs through the spermathecal epi• from several days (Ambystoma) to several years thelium of Eurycea cirrigera. Scale bars are in microns. (A) (Salamandra) (Baylis, '39; Benson, '68; Marynick, Neck tubules showing secretory activity and absence of spermiophagy (Sever and Brunette, '93). (B) Detail of sper• '71; Pool and Hoage, '73; Boisseau and Joly, '75; miophagy in the distal bulbs showing various stages of lyso• Trauth, '83; Brizzi et al., '89; Pecio, '92; Sever, some formation (Sever, '92b). (C) Spermathecal epithelium '92b), and this topic has been reviewed rather ex• and adjacent lumen in the distal bulbs showing absence of tensively in several recent papers (Sever, '95; secretory vacuoles and intense spermiophagic activity (Sever, Sever et al., '96a,b). As noted by Sever ('95), the '92b). Ac, apical cytoplasm; Af, axial filament; Dsl, degener• ating sperm in the lumen; Fl, filamentous material; Ic, in• reports of long-term spermathecal sperm storage tercellular canaliculi; Lu, lumen; Nu, epithelial cell nucleus; (>6 months) are not based upon an ultrastruc- PI, primary lysosome; Pp, pseudopodia; Ppt, principle piece tural analysis of the annual cycle of sperm stor• of the tail; Ps, phagosome; SI, secondary lysosome; Sv, secre• age in the spermathecae nor does verifiable tory vacuoles. SPERM STORAGE IN FEMALE SALAMANDERS 471

Figure 7. Figure 8. SPERM STORAGE IN FEMALE SALAMANDERS 473

Figure 9. (Caption on next page.) 474 D.M. SEVER AND R. BRIZZI

Fig. 10. Character M , presence of intraepithehal leuko• al., '95). Ic, intercellular canaliculi; Nu, epithelial cell cytes in the spermathecae. Transmission electron micro• nucleus; Pmn, polymorphonuclear leukocyte nucleus; SI, sec• graphs; scales are in microns. (A) Ambystoma opacum (Sever ondary lysosomes; Sv, secretory vacuoles. and Kloepfer, '93). (B) Salamandrina terdigitata (Brizzi et be regarded as more plesiomorphic than long-term significance above the generic level. This factor sperm storage (weeks or months). This conclu• may also explain the plasticity and apparent phyl• sion seems logical, but one cannot obtain such a etic irrelevance of character G, timing of release result when the character is traced by parsimony of spermathecal secretions. on Fig. 1. We believe this results from the fact In Notophthalmus viridescens, sperm are im• that duration of sperm storage has co-evolved mobile during storage (Hardy and Dent, '86), and with other species-specific reproductive adapta• spermathecal secretions are released upon mat• tions and therefore has little or no phylogenetic ing (character G^). The mating season is long (De• cember-March at the study site used by Sever et al., '96a) and so is the period of effective sperm Fig. 9. Characters K and L. Transmission electron mi• crographs A, B, and C show desquamation of epithelium into storage (December-May, six months, character the spermathecal lumen (character L^); B also shows sperm N''). Oviposition occurs from February to May. The degradation in the lumen (character K). (A) Salamandrina function of the secretions in A^. viridescens there• terdigitata (Brizzi et al., '96). (B) Ambystoma opacum (Sever fore is maintenance of sperm for up to six months. et al., '95). (C) Amphiuma tridactylum (Sever et al., '96b). Maintenance does not involve nourishing the (D) Eurycea cirrigera, showing desquamation of sperma• thecal epithelium into the surrounding connective tissue sperm; inactive sperm will not require metabolic stroma (character L^; Sever, '91b). Aaf, abnormal axial fila• fuel. Instead, the secretions provide sperm with : ment; Dbl, debris in the lumen; Dmi, detached mitochon• the chemical/osmotic environment for sperm qui• dria; Ds, desquamated cell; Gp, gap in the epithelial cells; escence (Sever and Kloepfer, '93). Lu, lumen; Mg, melanin granules; Ms, mitochondrial sheath; Nu, epithelial cell nucleus; Spl, sperm in the lumen; Stp, In Ambystoma tigrinum, females migrate to sperm in the tunica propria; Tfl, tail fragments in the lu• breeding ponds, mate, and usually lay their men; Tp, tunica propria. clutch within a few days, depending upon am- SPERM STORAGE IN FEMALE SALAMANDERS 475

bient conditions (Sever, '95). The sperm are held CONCLUSION so briefly in the spermathecae that essentially Sperm storage is an ancient trait, evolving in they act as cloacal containers allowing females the common ancestor of all the current families to carry sperm from the mating leks in the cen• in the Salamandroidea (i.e., before the splitting ter of the pond to egg-laying areas among of any of the lineages). These families show a wide - bushes around the periphery of the pond. Sever array of other, oftentimes unique, reproductive ('95) reported that release of secretion from the adaptations. Some of the differences we observe spermathecae in A. tigrinum is not concomitant among taxa in spermathecal characters may not with the appearance of sperm in the spermath• be phyletically informative but related to other ecae but with the act of oviposition. The func• species-specific reproductive adaptations. tion of the secretions therefore is to flush sperm However, it is worthwhile to try to separate from the spermathecae as eggs pass through the those characters that elucidate phylogeny from cloaca. those that obfuscate it. Only by conducting more Thus, the duration of sperm storage and the comparative studies that consider the complete function of the secretions are two factors that annual cycle of spermathecal sperm storage in cannot be considered apart from other aspects various taxa will we resolve any of the questions of the reproductive biology of the species, such about the significance of variability in sperm stor• as extent of the mating period and length of age mechanisms in female salamanders. the ovipository period. The frequency of mul• tiple matings interspersed with bouts of egg- ACKNOWLEDGMENTS laying, separated by days or weeks, as known in many newts (Halliday, '98), also are factors Much of the work by D.M.S. was supported by that may influence the effective period of stor• National Science Foundation grant DEB 90- age of sperm from a particular male. Charac• 24918. The research of R.B. was supported by ters G and N may have phyletic significance for grants from the Ministero Dell'Universita e della assemblages of species within a particular fam• Ricerca Scientifica e Tecnologica. R.B. expresses ily or genus when analyzed with other repro• her gratitude for the invaluable assistance of ductive activities, but data are insufficient at Giovanni Delfino. this time to make such an analysis for any group of salamanders. LITERATURE CITED Finally, spermiophagy by the spermathecal epi• Baker, C.L. (1962) Spermatozoa of Amphiumae: Sperma- teleosis, helical motility and reversibility. J. Tenn. Acad. Sci., thelium (character J) has been observed in many 37:23-37. taxa but seemingly is absent in others (Fig. 1). BayHs, H.A. (1939) Delayed reproduction in the spotted sala• One purpose of spermiophagy may be to rid the mander. Proc. Zool. Soc. London, 109A:243-246. spermathecae of unused sperm. However, sper• Benson, D.G. (1968) Reproduction in urodeles II. Observa• miophagy has been observed in Eurycea cirrigera tions on the spermatheca. Experientia, 24.-206-218. (Sever, '92a), Ambystoma opacum (Sever and Boisseau, C, and J. Joly (1975) Transport and survival of spermatozoa in female Amphibia. In: The Biology of Sper• Kloepfer, '93), and Notophthalmus viridescens matozoa: Transport, Survival, and Fertilizing Ability. E.S.E. (Sever et al., '96a) prior to any oviposition. In Hafez and C.G. Thibault, eds. Karger, Basel, Switzerland, Plethodon cinereus, the spermiophagy observed pp. 94-104. after oviposition did not seem of sufficient magni• Brizzi, R., G. Delfino, and C. Calloni (1989) Female cloacal tude to account for the huge decrease in number anatomy in the spectacled salamander, Salamandrina terdigitata (Amphibia: Salamandridae). Herpetologica, of sperm observed over two months (Sever, '97). 45.-310-322. It seems apparent to us that degradation is Brizzi, R., G. Delfino, M.G. Selmi, and D.M. Sever (1995) The the fate of any sperm cell that contacts or be• spermathecae of Salamandrina terdigitata (Amphibia: comes embedded in the spermathecal epithelium. Salamandridae): Patterns of sperm storage and degrada• tion. J. MorphoL, 223:21-33. In Ambystoma tigrinum (Sever, '95), Amphiuma Davitt, CM., and J.H. Larsen, Jr. (1988) Scanning electron tridactylum (Sever et al., '96b), and Necturus microscopy of the spermathecae of Plethodon larselli (Am• beyeri (Sever and Bart, '96), sperm were never phibia: Plethodontidae): Changes in the surface morphol• found embedded in the spermathecal epithelium, ogy of the spermathecal tubule prior to ovulation. Scanning although sperm did degrade in the lumen. Much Microsc, 2.-1805-1812. still remains to be learned about the phenom• Davitt, CM., and J.H. Larsen, Jr. (1990) Morphology of the principle cell types of the plethodontid salamander sper• enon of spermiophagy and its role in the repro• mathecae following treatment with gonadotropin. Am. Zool., ductive biology of salamanders. 5a-38A. 476 D.M. SEVER AND R. BRIZZI

Hafeli, H. (1971) Zur Fortpflanzungsbiologie des Alpen- thelium of the salamander Eurycea cirrigera. J. Morphol., salamanders {Salamandra atra Laur.). Rev. Suisse Zool., 212:281-290. 7&-235-293. Sever, D.M. (1992b) Comparative anatomy and phylogeny of Halliday, T.R. (1998) Sperm competition in amphibians. the cloacae of salamanders (Amphibia: Caudata). III. In: Sperm Competition and Sexual Selection. T.R. Ambystomatidae and Dicamptodontidae. J. Morphol., Birkhead and A.R Holler, eds. Academic Press, London 212:305-322. (in press). Sever, D.M. (1994a) Observations on regionalization of sec• Hardy, M.P., and J.N. Dent (1986) Regulation of motility of retory activity in the spermathecae of salamanders and sperm of the red-spotted newt. J. Exp. Zool, 240:385-396. comments on phylogeny of sperm storage in female sala• Howarth, B., Jr. (1974) Sperm storage: As a function of the manders. Herpetologica, 5a-383-397. female reproductive tract. In: The Oviduct and its Func• Sever, D.M. (1994b) Comparative anatomy and phylogeny of tions. A.D. Johnson and C.W. Foley, eds. Academic Press, the cloacae of salamanders (Amphibia: Caudata). VII. New York, pp. 237-270. Plethodontidae. Herpetol. Monogr., &-276-337. Larson, A., and W.W. Dimmick (1993) Phylogenetic relation• Sever, D.M. (1995) Spermathecae of Ambystoma tigrinum ships of the salamander families: An analysis of congru• (Amphibia Caudata): Development and a role for the secre• ence among morphological and molecular characters. tion. J. Herpetol., 29:243-255. Herpetol. Monogr., 7.77-93. Sever, D.M. (1997) Sperm storage in the spermathecae of the Maddison, W.R, and D.R. Maddison (1992) McClade Analysis red-back salamander, Plethodon cinereus (Amphibia: Pletho• of Phylogeny and Character Evolution Version 3. Sinauer dontidae). J. Morphol., 254:131-146. Assoc., Inc., Sunderland, MA. Sever, D.M., and H.L. Bart, Jr. (1996) Ultrastructure of the Marynick, S.P. (1971) Long term storage of sperm in Des- spermathecae of Necturus beyeri (Amphibia: Proteidae) in mognathus fuscus from Louisiana. Copeia i57i.'345-347. relation to its breeding season. Copeia, 1996:927-937. Parker, G.A. (1984) Sperm competition and the evolution of Sever, D.M., and N.S. Brunette (1993) Regionahzation of ec- animal mating systems. In: Sperm Competition and the Evo• crine and spermiophagic activity in the spermathecae of the lution of Mating Systems. R.L. Smith, ed. Academic Press, salamander Eurycea cirrigera (Amphibia: Plethodontidae). New York, pp. 1-60. J. Morphol., 2i 7.161-170. Pecio, A. (1992) Insemination and egg-laying dynamics in the Sever, D.M., J.S. Doody, C.A. Reddish, M.M. Wenner, and D.R. smooth newt, Triturus vulgaris, in the laboratory. Herpetol. Church (1996b) Sperm storage in spermathecae of the great J., 2:5-7. lamper eel, Amphiuma tridactylum (Caudata: Amphiu• Pool, T.B., and T.R. Hoage (1973) The ultrastructure of se• midae). J. Morphol, 230:79-97. cretion in the spermathecae of the salamander, Manculus Sever, D.M., and N.M. Kloepfer (1993) Spermathecal cytol• quadridigitatus (Holbrook). Tissue Cell, 5.-303-313. ogy of Ambystoma opacum (Armphibia: Ambystomatidae) Sever, D.M. (1987) Hemidactylium scutatum and the phylog• and the phylogeny of sperm storage organs in female sala• eny of cloacal anatomy in female salamanders. Herpe• manders. J. Morphol., 217:115-127. tologica, 43:105-116. Sever, D.M., J.D. Krenz, KM. Johnson, and L.C. Rania (1995) Sever, D.M. (1991a) Comparative anatomy and phylogeny of Morphology and evolutionary implications of the annual the cloacae of salamanders (Amphibia: Caudata). I. Evolu• cycle of secretion and sperm storage in spermathecae of the tion at the family level. Herpetologica, 47.'165-193. salamander Ambystoma opacum (Amphibia: Ambysto• Sever, D.M. (1991b) Sperm storage and degradation in the matidae). J. Morphol, 223:35-46. spermathecae of the salamander Eurycea cirrigera (Green). Sever, D.M., L.C. Rania, and J.D. Krenz (1996a) The annual J. Morphol., 210:71-84. cycle of sperm storage in the spermathecae of the red-spot• Sever, D.M. (1991c) Comparative anatomy and phylogeny of ted newt, Notophthalmus viridescens (Amphibia: Caudata). the cloacae of salamanders (Amphibia: Caudata). II. J. Morphol, 227:155-170. Cryptobranchidae, Hynobiidae, and Sirenidae. J. MorphoL, Trauth, S.E. (1983) Reproductive biology and spermathecal 207:283-301. anatomy of the dwarf salamander {Eurycea quadridigitata) Sever, D.M. (1992a) Spermiophagy by the spermathecal epi• in Alabama. Herpetologica, 55.-9-15.