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Sperm Storage in the Oviduct of the Internal Fertilizing
Frog Ascaphus truei
David M. Sever,
1
* Emily C. Moriarty,
1
Lisa C. Rania,
1
and William C. Hamlett
2
1
Department of Biology, Saint Mary’s College, Notre Dame, Indiana
2
Indiana University School of Medicine, South Bend Center for Medical Education, Notre Dame, Indiana
ABSTRACT This study provides the first descriptions of
sperm storage at the tissue and cellular levels in a female
frog or toad. Oviducal anatomy was studied by light and
electron microscopy in Ascaphus truei from north coastal
California. Ascaphus truei is one of the few species of
anurans in which fertilization is internal. Unlike other
anurans with internal fertilization, however, mating in A.
truei consists of a unique combination of amplectic and
copulatory mechanisms that we term “copulexus.” Poste-
rior to a short, aglandular infundibular region, the oviduct
possesses: 1) a proximal, convoluted ampullary region
where intrinsic tubular glands secrete gelatinous enve-
lopes around eggs; 2) a middle ovisac region where fertil-
ization occurs; and 3) a distal oviducal sinus formed by
medial junction of the ovisacs. Sperm storage tubules
(SSTs) occur in the anterior portions of the ovisacs and
consist of simple tubular glands. SSTs and the rest of the
oviducal lining stain positively with the periodic acid-
Schiff’s procedure for neutral carbohydrates and this re-
action is especially intense in reproductively active fe-
males. Sperm were found in the SSTs of gravid females as
well as some nonvitellogenic females. The sperm are in
orderly bundles in the SSTs, and although occasionally
sperm nuclei were embedded in the epithelium, no evi-
dence for spermiophagy was found. Oviducal sperm stor-
age in A. truei is homoplastic, with closest structural sim-
ilarities to squamate reptiles. Oviduct/sperm design
constraints appear to limit the options for expression of
features associated with oviducal sperm storage. J. Mor-
phol. 248:1–21, 2001. © 2001 Wiley-Liss, Inc.
KEY WORDS: Anura; Ascaphus truei; reproduction;
sperm storage; ultrastructure
The tailed frog Ascaphus truei Stejneger (1899) is
the sole member of the family Ascaphidae and is
generally considered the sister taxon of all other
anurans (Ford and Cannatella, 1993). Ascaphus
truei is associated with cold, clear mountain streams
in disjunct populations in the Cascade Mountains
west to the coast from southern British Columbia to
northwest California, in the Blue Mountains of
southwestern Washington and northeastern Ore-
gon, and in the Rocky Mountains of northern Idaho
and western Montana (Metter, 1968). Of the nearly
5,000 species of anurans, A. truei is the only species
known to engage in copulation that includes intro-
mission. The male possesses a “tail” that, when en-
gorged, forms a sulcus for passage of sperm and is
inserted in the cloaca of the female (Noble, 1925;
Noble and Putnam, 1931; Slater, 1931). Copulation
has been assumed to be an adaptation that ensures
fertilization in fast-moving water (Stebbins and Co-
hen, 1995).
The presence of sperm in the lumen of the ovi-
ducts and in oviducal glands of female Ascaphus
truei was first reported by Noble (1925). Noble
(1925) apparently examined sections of the oviduct
prepared for light microscopy but the histology of
the oviduct and stored sperm was not described in
any detail. Metter (1964a,b) suggested periods of
1–2 years of sperm storage in female A. truei and
briefly described the gross anatomy of the oviduct
and location of sperm storage, but once again did not
provide any histological details. In this article we
provide the first descriptions using light and elec-
tron microscopy of the oviduct and sperm storage in
A. truei, the only frog in which female sperm storage
is known to occur.
MATERIALS AND METHODS
All specimens of Ascaphus truei were collected
from the North Fork Mad River watershed on lands
owned by the Simpson Timber Company in western
Humboldt County, California, in the north coast red-
wood (Sequoia sempervirens) zone (Diller and Wal-
lace, 1994). Although A. truei is not listed under
either the federal or state of California endangered
species acts, it is considered of special concern due to
its habitat requirements and sensitivity to land
management activities. Thus, we sought to mini-
mize the number of animals collected for this study.
Permits for the collection of specimens were granted
*Correspondence to: Dr. David M. Sever, Department of Biology,
Saint Mary’s College, Notre Dame, Indiana 46556.
E-mail: dsever@saintmarys.edu
JOURNAL OF MORPHOLOGY 248:1–21 (2001)
©2001 WILEY-LISS, INC.
to L.V. Diller from the California Department of
Fish and Game.
Females of Ascaphus truei were collected during
four periods: 8 –15 June, 5– 6 July, 24 October 1999,
and 22–29 March 2000. Specimens were collected at
night from along the border of streams. The frogs
were shipped to Saint Mary’s College where the
specimens were killed within a week of receipt (Ta-
ble 1) and oviducal tissue was removed and prepared
for microscopic examination.
Specimens were killed by immersion in 10% MS-
222 (3-aminobenzoic acid ethyl ester), and snout–
vent length (SVL) was measured from the tip of the
snout to the posterior end of the cloacal orifice. The
reproductive tracts and cloacae were removed from
freshly killed specimens and prepared for light mi-
croscopy (LM) and transmission electron microscopy
(TEM). Carcasses of all specimens were preserved in
10% neutral buffered formalin (NBF) and are
housed in the research collections at Saint Mary’s
College.
For LM examination, some tissues were initially
fixed in 10% NBF, rinsed in water, dehydrated in
ethanol, cleared in toluene, and embedded in paraf-
fin or glycol methacrylate (JB-4 Plus; Electron Mi-
croscopy Sciences, Fort Washington, PA) plastic
resin. Paraffin sections (10 m) were cut with a
rotary microtome and affixed to albuminized slides.
Alternate paraffin slides from each specimen were
stained with hematoxylin-eosin (general histology),
brilliant indocyanine 6B (BB, for proteins), and Al-
cian blue 8GX at pH 2.5 (AB, for primarily carbox-
ylated glycosaminoglycans), followed by the periodic
acid-Schiff’s procedure (PAS, for neutral carbohy-
drates and sialic acids). Sections (2 m) from tissues
embedded in JB4 were stained with methylene blue
and basic fuchsin. Sections also were cut from tis-
sues frozen at – 60°C after NFB fixation. The frozen
sections (15 m) were cut with an AO Cryo-Cut II
(American Optics, Buffalo, NY) and stained with
Sudan black (SB). Procedures followed Dawes
(1979), Humason (1979), and Kiernan (1990).
Tissue for TEM was trimmed into 1-mm blocks
and fixed in a 1:1 solution of 2.5% glutaraldehyde in
Millonig’s phosphate and 3.7% formaldehyde in ca-
codylate buffer, pH 7.2. After initial fixation, tissues
were rinsed in distilled-deionized water, postfixed in
2% osmium tetroxide, dehydrated through a graded
series of ethanol, cleared in propylene oxide, and
polymerized in an epoxy resin (Embed 812; Electron
Microscopy Sciences). Plastic sections were cut with
an RMC MT7 ultramicrotome (Research and Manu-
facturing Co., Tucson, AZ) and DiATOME (Biel,
Switzerland) diamond knives. Semithin sections
(0.5–1 mm) for LM were placed on microscope slides
and stained with toluidine blue. Ultra-thin sections
(70 nm) for TEM were collected on uncoated copper
grids and stained with solutions of uranyl acetate
and lead citrate. Ultrathin sections were viewed
with a Hitachi H-300 transmission electron micro-
scope (Nissei Sangyo America, Mountain View, CA).
RESULTS
Regions of the Oviduct
Oviducal terminology, except where noted, follows
Sever et al. (1996a) and Wake and Dickie (1998).
Regions of the oviduct of Ascaphus truei are illus-
trated in Figures 1 and 2. The ostial opening leads
into the infundibulum, which is a narrow, thin-
walled tube that extends from posterior to the trans-
verse septum to the anterior border of the kidney.
The infundibulum connects to the ampulla, which is
lateral to the anterior portion of the kidneys. The
ampulla is thick-walled, sinuous, and contains exo-
crine glands involved in the deposition of outer ge-
latinous egg envelopes (Wake and Dickie, 1998).
Distal to the ampulla is the straight ovisac, called
the “uterus” by Metter (1964b). In the anterior por-
tion of the ovisac are exocrine glands that serve as
sperm storage tubules (SSTs). The ampulla and the
ovisac are the only regions of the oviduct that pos-
sess intrinsic tubular exocrine glands (Fig. 2). The
two ovisacs join medially dorsal to the urinary blad-
der to form an oviducal sinus (Fig. 1). The only other
anuran in which an oviducal sinus has been noted is
the viviparous African bufonid Nimbaphrynoides oc-
cidentalis by Xavier (1973). This area is clearly an-
terior to the cloaca, which is dorsal to the pubic
symphysis. The oviducal sinus is not “urogenital,”
because the Wolffian ducts and urinary bladder
empty into the cloaca, along with wastes from the
colon.
Reproductive Condition of the Sample
Mating in this population has been observed in
the field only during May, although males with
highly developed secondary sex characters have
been observed in June and July. In addition, males
captured and brought into captivity in April were
TABLE 1. Specimens utilized in this study
1
Date SVL
Follicles Sperm
2
N Range Mean SE Am Ov Os
10 April 47.4 47 2.3–3.0 2.6 0.07 0 0 0
10 April 47.1 67 2.3–2.9 2.6 0.06 0 0 0
10 April 42.3 25 0.8–0.9 0.8 0.01 0 0 0
18 June 52.5 64 2.0–3.7 2.8 0.06 0 ⫹⫹
18 June 49.4 71 0.5–1.6 1.2 0.03 0 ⫹0
12 July 48.1 15 1.1–1.5 1.4 0.04 0 ⫹0
12 July 47.2 63 2.8–3.9 3.4 0.08 0 ⫹0
7 Nov 43.2 70 1.0–1.6 1.3 0.05 0 0 0
7 Nov 38.5 42 0.6–0.8 0.6 0.02 0 0 0
1
All measurements are in mm.
2
Absence (0) or presence (⫹) of sperm in the ampulla (Am), ovisac
(Ov), or oviducal sinus (Os).
2 D.M. SEVER ET AL.
observed to grasp females in amplexus and unsuc-
cessfully attempt copulation.
Two of the four females collected in June and July
for this study have large, vitellogenic eggs (2.8 –3.4
mm mean dia.; Table 1), and one female was killed
while in amplexus and copulation with a male. The
combination of amplectic and copulatory mecha-
nisms resulting in internal fertilization in Ascaphus
truei is unique among anurans, and we term this act
“copulexus.” The ampullae of these females are hy-
pertrophied and highly convoluted. These females
are considered to be in active reproductive condition
for the current breeding season. The other two fe-
males have small ovarian follicles (1.2–1.4 mm
mean dia.) and their ampullae are relatively narrow
and unconvoluted. These females clearly are not in
the same stage of active reproductive readiness as
the two females with large ovarian follicles. All four
females from June and July, however, have sperm in
their SSTs, and the female in copulexus also has
sperm in the oviducal sinus (Table 1).
The two females examined from the November
collection appear reproductively inactive, with small
ovarian follicles (0.6 –1.3 mm mean dia.) and nar-
row, unconvoluted oviducts. Two of the females sac-
rificed in April have numerous vitellogenic follicles
nearly as large (2.6 mm mean dia.) as those of gravid
females from June and July (Table 1). These females
also possess hypertrophied ampullary regions of the
oviduct. The other female from the April sample has
small ovarian follicles (0.8 mm mean dia.) and re-
gressed oviducts. None of the females sacrificed in
November and April possess sperm in their oviducts.
Females Sacrificed in Summer in Active
Breeding Condition
As noted above, two females sacrificed in summer
have large ovarian follicles (2.8 –3.4 mm mean dia.)
and are considered reproductively active in a post-
mating and preovulatory condition. One of the fe-
Fig. 1. Female Ascaphus
truei. Oviduct of a 52.5 mm SVL
specimen sacrificed 18 June.
Right ovary removed.
3SPERM STORAGE IN ASCAPHUS TRUEI
males was killed while in copulexus. These females
both possess sperm in SSTs in the ovisac and are
similar in other aspects of oviducal anatomy except
for some features of the oviducal sinus. In the female
in copulexus, sperm also are present in the oviducal
sinus and epithelial sloughing occurs in the oviducal
sinus (absent in the other female). Sections through
the infundibulum are illustrated in Figure 3, the
ampulla in Figure 4, the ovisac in Figures 5– 6, and
the oviducal sinus in Figures 7– 8.
Infundibulum. The infundibulum is relatively
thin-walled; the lining is rather smooth anteriorly
but becomes rugose posteriorly toward the ampulla
(Fig. 3A). The infundibulum possesses three basic
layers found throughout the oviduct. The most su-
perficial layer is the visceral pleuroperitoneum com-
Fig. 2. Female Ascaphus truei. Drawing of the oviduct of the same specimen used in Figure 1,
illustrating the histology of the ampulla and ovisac.
4 D.M. SEVER ET AL.
Fig. 3. Female Ascaphus truei. Light micrograph (A) and transmission electron micrographs of the infundibulum of a 47.2 mm SVL
vitellogenic female sacrificed 12 July. A: Tissue layers of the infundibulum. B: Visceral pleuroperitoneum and muscularis. C: Luminal
border. D: Adjacent ciliated and secretory cells. Bb, basal bodies; Cf, collagen fibers; Ci, cilia; Ep, epithelium; Fm, flocculent material;
Ic, intercellular canaliculus; Lp, lamina propria; Lu, lumen; Me, mesothelium; Mi, mitochondria; Ms, muscularis; Nu, nuclei; Sv,
secretory vacuoles; Vp, visceral pleuroperitoneum.
Figure 4.
posed of simple squamous mesothelium and a thin,
subsurface tunica propria of connective tissue
proper that features a dense submesothelial layer of
collagen fibers (Fig. 3B). The middle muscularis
layer is thin in the infundibulum and is composed
primarily of longitudinal smooth muscle fibers. Else-
where in the oviduct both a superficial circular and
a deep longitudinal layer are apparent, with these
most prominent in the ovisac (Fig. 5A) and oviducal
sinus (Fig. 12A). The deepest layer is the mucosa,
composed of the epithelium lining the inner walls of
the oviduct and the subsurface connective tissue of
the lamina propria. The mucosa, especially the epi-
thelium and intrinsic exocrine glands derived from
the epithelium, shows the most variation through-
out the oviduct and is the focus of the ultrastruc-
tural observations.
In the infundibulum, the mucosal epithelium is
simple squamous to cuboidal, with irregular,
densely staining nuclei (Fig. 3C). Cells with elongate
cilia are numerous and interspersed among the cil-
iated cells are secretory cells characterized by apical
microvilli. Ciliated cells often appear pyramidal in
shape, with the broadest aspect in a luminal position
and the truncated region projecting away from the
lumen. The ciliated cells possess numerous supranu-
clear mitochondria and contain vacuoles, some of
which appear empty, as well as others that contain
a flocculent material (Fig. 3C,D). Intercellular canal-
iculi exhibit broad gaps between cells (Fig. 3D) that
narrow to tight junctions apically and become laby-
rinthine basally. The secretory cells also contain the
flocculent inclusions as well as dark, uniformly
electron-dense secretory vacuoles (Fig. 3C,D) that
stain intensely PAS⫹and are AB⫺and BB⫺in
paraffin sections.
Ampulla. The ampulla of the reproductively ac-
tive but preovulatory females contain greatly hyper-
trophied oviducal glands (Fig. 4). These exocrine
glands are so numerous, crowded, and enlarged that
they obliterate any gaps between one another and
compress the lamina propria (Fig. 4B) and muscu-
laris into thin layers barely discernible with light
microscopy.
Electron microscopy, however, reveals that two
types of epithelial cells are still present: secretory
and ciliated cells. The secretory cells are pyriform,
with the base being the widest (Figs. 2, 4A). They
have densely staining, irregular basal nuclei (Fig.
4B). The secretory vacuoles also are irregular in
outline and their contents usually are uniform and
moderately electron-dense, although occasionally a
darker, eccentric circular area occurs in the vacuole
(Fig. 4B,C). The vacuoles stain intensely PAS⫹and
are AB⫺and BB⫺. The apices of the secretory cells
have microvilli and the unit membrane of the secre-
tory vacuoles appears in places to be in contact with
the plasmalemma of the apical epithelium (Fig. 4C),
perhaps indicating the initial phase of merocrine
secretion of the contents. No eggs, however, were in
the ampulla and secretory products were not being
elaborated from the vacuoles. The ciliated cells are
small squamous cells squeezed between the apices of
the secretory cells (Fig. 4D). The ciliated cells have
irregular euchromatic nuclei that occupy much of
the cytoplasm and numerous supranuclear mito-
chondria.
Ovisac. The upper portion of the ovisac is also
characterized by the presence of intrinsic exocrine
glands, but these glands are not packed with the
irregular, moderately electron-dense secretory vacu-
oles found in the ampulla. The glands are simple
tubular or simple branched tubular (Fig. 5A) and
serve as SSTs. The lower portion of the ovisac lacks
tubular glands. The lining of the SSTs and the lu-
minal epithelium of the oviduct is simple columnar
epithelium and consists of secretory cells with mi-
crovilli interspersed among ciliated cells (Fig. 5B,C).
Intercellular canaliculi are wide, especially among
groups of ciliated cells, and tortuous basally (Fig.
5B). Circular vacuoles of varying sizes and electron
densities occur in secretory cells and are especially
numerous and variable in size in the distal portions
of the SSTs (Fig. 5D). These vacuoles are intensely
PAS⫹and are AB⫺and BB⫺. Both ciliated and
secretory cells often possess vacuoles containing a
flocculent material, especially in the epithelium of
the oviducal lining (Figs. 5B, 6A).
Sperm are present in the lumen of the oviduct and
the SSTs (Figs. 5, 6). Sperm in the lumen usually
were loosely aggregated (Figs. 5B, 6A) and small
clusters of sperm show similar orientations (i.e.,
similar sections through nuclei, tails aligned). Occa-
sionally groups of sperm in the oviducal lumen are
found embedded in an acellular matrix composed of
a uniformly electron-dense substance and small ves-
icles (Fig. 6B). Sperm are also frequently found em-
bedded in the secretory epithelial cells, especially in
the SSTs (Fig. 6C,D). Sperm nuclei sometimes are
found deep in these cells, in the infranuclear cyto-
plasm bordering the basal lamina (Fig. 6C). The
nuclei are not vacuolated, appear normal in cytol-
ogy, and often are in close proximity to secretory
vacuoles (Fig. 6D).
Oviducal sinus. The epithelium of the oviducal
sinus is rugose and consists of stratified cuboidal
epithelium and lacks cilia and intrinsic tubular
glands (Fig. 7A). A thick layer of collagen fibers
occurs in the lamina propria superficial to the epi-
thelium (Fig. 7A), and the muscularis is relatively
wider than in other regions. In the female that was
not in the act of mating when sacrificed, the apical
Fig. 4. Female Ascaphus truei. Electron micrographs of the
ampulla of 47.2 mm SVL vitellogenic female sacrificed 12 July.
A: Luminal border. B: Basal border. C: Secretory cell. D: Ciliated
cell. Bb, basal bodies; Ci, cilia; Lp, lamina propria; Lu, lumen; Mi,
mitochondria; Mv, microvilli; Nucc, nucleus of a ciliated cell;
Nusc, nucleus of a secretory cell; Sv, secretory vacuoles.
7SPERM STORAGE IN ASCAPHUS TRUEI
Fig. 5. Female Ascaphus truei. Light micrograph (A) and transmission electron micrographs of the ovisac of a 47.2 mm SVL
vitellogenic female sacrificed 12 July. A: Sperm storage tubules (Sst) in the anterior ovisac. B: Epithelial border of the ovisac. C: Orifice
of an Sst. D: Distal portion of an Sst. Ci, cilia; Fm, flocculent material; Ic, intercellular cannalculi; Lp, lamina propria; Lu, lumen; Ms,
muscularis; Nu, nucleus; Sn, sperm nucleus; Sp, sperm; Splu, sperm in the lumen; Sst, sperm storage tubule; Sv, secretory vacuole.
Fig. 6. Female Ascaphus truei. Transmission electron micrographs of the ovisac of a 47.2 mm SVL vitellogenic female sacrificed 12
July. A: Luminal border. B: Sperm in luminal matrix (Lm). C: Basal border of an Sst. D. Sperm nucleus embedded in an Sst. Bl, basal
lamina; Ci, cilia; De, desmosome; Fm, flocculent material; Ic, intercellular canaliculus; Lm, luminal matrix; Mv, microvilli; Sn, sperm
nucleus; Sp, sperm; Sv, secretroy vacuole; Tj, tight junction.
Fig. 7. Female Ascaphus truei. Light micrograph (A) and transmission electron micrographs of the oviducal sinus of a 47.2 mm SVL
vitellogenic female sacrificed 12 July. A: Mucosa and submucosal collagen layer. B: Apical cytoplasm. C: Degenerating epithelial cell.
D: Merocrine secretory process. Cf, collagen fibers; Ep, epithelium; Ic, intercellular canaliculus; Lf, lipofuscin particles; Lp, lamina
propria; Lu, lumen; Ly, lysosomes; Mi, mitochondria; Nu, nucleus; Sm, secretory material; Sv, secretory vacuoles.
Fig. 8. Female Ascaphus truei. Light micrograph (A) and transmission electron micrographs of the oviducal sinus of a 52.5 mm SVL
vitellogenic female sacrificed in copulexus on 18 June. A: Mucosa. B: Luminal border. C: Desquamating cell. D: Secretory material and
sperm in the lumen. Cl, cleavage line; Ds, desquamating cells; Ep, epithelium; Lp, lamina propria; Lu, lumen; Sm, secretory material;
Sn, sperm nucleus; Sp, sperm; Splu, sperm in the lumen; Sv, secretory vacuoles.
cytoplasm is packed with electron-lucent secretory
vacuoles (Fig. 7B) that are PAS⫹and AB⫺and BB⫺
in paraffin sections. A few cells bordering the lumen
lack secretory vacuoles and have numerous mito-
chondria in the apical cytoplasm (Fig. 7B). Some
sloughing of the epithelium is apparent, as cells
with apparently deteriorating cytoplasm are occa-
sionally found among normal cells (Fig. 7B,C). Com-
plex interdigitations occur in the intercellular
spaces between adjacent epithelial cells (Fig. 7B,C).
In some areas, a product is observed during release
from these vacuoles into the oviducal lumen by the
merocrine process (Fig. 7D).
In the female sacrificed while in copulexus, sperm
occur in the oviducal lumen (Fig. 8A) including the
folds of the rugae. Some sloughing of the apical
epithelium is apparent (Fig. 8A–C), and this process
results in an abundance of secretory material that is
associated with sperm in the lumen (Fig. 8D).
Nonvitellogenic Females Sacrificed in
Summer
Two females examined from June–July contain
small ovarian follicles (1.2–1.4 mm mean dia) and
narrow, relatively unconvoluted ampullae of the ovi-
duct. These individuals obviously are not in the
same stage of reproductive readiness as those fe-
males with larger follicles and hypertrophied ovi-
ducts. Both of the females with small follicles, how-
ever, contain sperm in their SSTs.
Infundibulum. The infundibulum (Fig. 9A) did
not appear significantly different from that of fe-
males with larger ovarian follicles (Fig. 3A). Ciliated
and secretory cells occur in the epithelium and the
secretory cells contain dark, uniformly electron-
dense vacuoles that stain PAS⫹in paraffin sections.
A minor difference is that the secretory vacuoles are
more irregular in outline than those in the infundib-
ulum of reproductively active females (compare
Figs. 3D, 9A). The epithelium contains numerous
clear vacuoles as noted in reproductively active fe-
males.
Ampulla. The oviducal glands in the ampulla in
females with small ovarian follicles (Fig. 9B–D) are
much reduced relative to the condition found in fe-
males with large ovarian follicles (Fig. 4). Ciliated
cells are not prominent. The secretory cells of the
glands lack the large, moderately electron-dense se-
cretory vacuoles and the pyramidal shape of active
glands. Instead, the secretory cells are columnar
and contain numerous elongate secretory vacuoles
that vary in electron density (Fig. 9B–D) but are
generally darker than those in active glands. The
secretory vacuoles observed in this condition may be
precursors to those in glands ready to form egg cap-
sules. Debris is frequently found in the lumina of the
glands (Fig. 9C). Like ampullary glands in reproduc-
tively active females, the secretory cells of the ovid-
ucal glands in nonreproductive females are charac-
terized by elongate microvilli (compare Figs. 4A,C,
9C,D).
Ovisac. Sperm occur in the oviducal lumen and
the SSTs of the anterior ovisac (Figs. 10, 11) of the
nonvitellogenic females examined from June and
July (Table 1). Sperm are especially numerous and
closely packed in the SSTs and clusters of sperm
often exhibit the same orientation of their axes (Fig.
10B–D, 11A). Occasional sperm nuclei are embed-
ded in the apical cytoplasm of SSTs and these
sperm, like those in the lumen, appear normal in
cytology (Fig. 11B). No evidence for spermiophagy
was observed. Ciliated cells were numerous. Many
secretory cells lack extensive clusters of secretory
vacuoles, but other cells contain numerous, electron-
dense, PAS⫹secretory vacuoles (Fig. 11A,C). Vacu-
oles with a lighter density as noted in females with
large ovarian follicles (Figs. 5D, 6D) are lacking.
Occasional lipid droplets (Fig. 11B,C) occur in the
cytoplasm of secretory cells and these lipids stain
Sudan Black⫹in frozen sections. Capillaries closely
abut the basal lamina (Figs. 10B, 11A). In some
secretory cells that lack large vacuoles, mitochon-
dria and Golgi complexes are prominent in the pe-
rinuclear cytoplasm (Fig. 11D).
Oviducal sinus. The epithelial lining of the ovid-
ucal sinus in females with small ovarian follicles
(Fig. 12) is similar to that of the vitellogenic female
that was not sacrificed while in copulexus (Fig. 7).
Most cells bordering the oviducal lumen are filled
with large secretory vacuoles. In females with small
ovarian follicles, however, these vacuoles are of
varying densities rather than the more uniform den-
sities observed in females with large ovarian folli-
cles. Interdigitations and junctions between adja-
cent epithelial cells are complex (Fig. 12B,C). Some
apical cells lack secretory vacuoles and contain nu-
merous mitochondria with tubular cristae (Fig.
12D).
Females Sacrificed in November and April
None of the females sacrificed in November or
April possess sperm in their oviducts and observa-
tions were limited to light microscopy. Both of the
females sacrificed in November and one of the three
sacrificed in April contain only small ovarian folli-
cles (0.6 –1.3 mm mean dia.) and have relatively
undeveloped oviducts. Oviducal histology is similar
to that of nonvitellogenic females examined from
June and July. Two females from the April sample,
however, have large, vitellogenic ovarian follicles
(2.6 mm mean dia.) and hypertrophied, sinuous ovi-
ducts. In these two females, oviducal histology re-
sembles that of gravid females from June and July,
with the oviducal glands in the ampulla filled with
large secretory vacuoles. Whether the oviduct is hy-
pertrophied or not, the lining is PAS⫹,AB⫺, and
BB⫺.
12 D.M. SEVER ET AL.
Fig. 9. Female Ascaphus truei. Transmission electron micrographs of the infundibulum (A) and ampulla (B–D) of a 48.1 mm SVL
nonvitellogenic female sacrificed 12 July. A: Mucosa of the infundibulum. B: Muscoa and oviducal gland in the ampulla. C: Oviducal
gland in the ampulla. D: Luminal border of an oviducal gland in the ampulla. Cf, collagen fibers; Db, debris; Ic, intercellular
canaliculus; Lu, lumen; Mi, mitochondria; Mv, microvilli; Nu, nucleus; Sv, secretory vacuoles.
Fig. 10. Female Ascaphus truei. Light micrograph (A) and transmission electron micrographs of the ovisac of a 48.1 mm SVL
nonvitellogenic female sacrificed 12 July. A: Tissue layers in the Sst region. B,C: Mucosa and Ssts. D: Distal portion of an Sst. Ci, cilia;
Cp, capillary; Ep, epithelium; Lp, lamina propria; Lu, lumen; Ms, muscularis; Nu, nucleus; Ppt, principal piece of the tail; Sn, sperm
nucleus; Sp, sperm; Sst, sperm storage tubule.
Fig. 11. Female Ascaphus truei. Transmission electron micrographs of the ovisac of a 48.1 mm SVL nonvitellogenic female
sacrificed 12 July. A: Sperm storage tubule (Sst). B: Sperm nucleus embedded in the epithelium. C: Apical cyctoplasm of an Sst,
showing secretory vacuoles. D: Apical cytoplasm of an Sst, showing synthetic organelles. Cp, capillary; De, desmosome; Go, Golgi
complex; Ic, intercellular canaliculus; Ld, lipid droplet; Lu, lumen; Mf, microfilaments; Mi, mitochondria; Nu, nucleus; Ppt, principal
piece of the tail; Rb, ribosomes; Rbc, red blood cell; Sn, sperm nucleus; Sv, secretory vacuoles; Tj, tight junction.
Fig. 12. Female Ascaphus truei. Light micrograph (A) and transmission electron micrographs of the oviducal sinus of a 48.1 mm
SVL nonvitellogenic female sacrificed 12 July. A: Tissue layers. B: Epithelium. C: Luminal border. D: Adjacent secretory and
nonsecretory cells. Cr, cristae; Ep, epithelium; Ic, intercellular canaliculus; Lp, lamina propria; Lu, lumen; Mi, mitochondria; Ms,
muscularis; Mv, microvilli; Nu, nucleus; Sv, secretory vacuoles; Tj, tight junction; Vp, visceral pleuroperitoneum.
DISCUSSION
Reproduction and Reproductive Cycle
The original description of Ascaphus truei by Stej-
neger (1897) was based on a single specimen that
evidently was a female (Gaige, 1920:259), and the
first observations of the male tail were not reported
until Van Denburgh (1912:261), who suggested the
tail may be a sexual organ. Gaige (1920) presented
the first illustration of the tail and reported on the
breeding habits of A. truei near Lake Cushman at
the base of Mount Rose, Washington. She noted that
many females collected 27 July – 24 August had
large eggs, whereas others were “normal,” which she
believed indicated an extended breeding season
(Gaige, 1920:5). Further support of this notion was
provided by: variation in male breeding condition, by
finding larvae of all stages of development on the
same date, and Slater’s (1931) observation of a pair
copulating on 17 May in the Carbon River Valley of
Mount Rainier.
Noble (1925:17) suggested that the tail is pressed
against the cloaca of the female in copulation and
was the first to report sperm in the oviducts females.
Noble stated that his sections of the urinogenital
organs of breeding females revealed great masses of
spermatozoa in the lumen of the oviducts and par-
ticularly in the glands along the posterior part of the
oviducts. Sections of the oviducts of females taken
after their eggs had been laid show many of the
glands of the posterior oviduct still filled with sper-
matozoa.
The actual intromission of the tail in copulation
was first described by Noble and Putnam (1931),
using specimens from the Lake Cushman locale fre-
quented by Gaige (1920). They also reported finding
pairs in the field in copulo from 12 June – 6 July,
although a longer breeding season was apparent as
females possessed large follicles throughout July,
and a male collected 4 September still exhibited
amplectic behavior. In the latter regard, however, it
is interesting to note that later Metter (1967) re-
ported that males, regardless of development of
their secondary sexual characters, will clasp other
individuals (male or female) at any time of year.
Van Dijk (1955:65) reported that the turgid tail
can only be applied to the cloacal orifice of the female
and not inserted into it. This statement is contrary
to our observations of the entire turgid member com-
pletely inserted into the female. The structure of the
flaccid and turgid intromittant organ is the subject
of an article in preparation by Hamlett and Sever.
We introduce the term “copulexus” to describe the
unique form of mating that results in internal fer-
tilization in this species. Amplexus is the term that
describes the act of the male grasping the female to
effect external fertilization. Copulation is the act of
sexual intercourse and implies internal fertilization.
Copulexus is characterized by both mechanisms oc-
curring simultaneously to effect internal fertiliza-
tion with a unique copulatory organ.
In a subsequent article, Van Dijk (1959:222) illus-
trated a transverse section through the oviduct of
Ascaphus truei, “showing spermia.” However, no ex-
planation or description of the plate was presented.
Metter (1964a) studied the reproductive biology of
two geographically close but isolated populations of
Ascaphus truei in northern Idaho and the Blue
Mountains of western Washington. Females appar-
ently have a biennial breeding season (see also Met-
ter, 1967). Secondary sexual characters reach their
peak in early fall and copulation was observed be-
tween 4 September – 3 October. Oviposition, how-
ever, occurs from late June – early August. Thus,
sperm for fertilization are retained from breeding
the previous fall.
Metter (1964b) subsequently studied the occur-
rence of sperm in 19 females sacrificed in November.
Ten of these females were in a group with large
follicles (2.9 –3.3 mm dia.) and would have ovipos-
ited the following summer while the others had
small follicles (0.8 –1.3 mm dia.) and presumably
would have delayed fertilization and ovulation for
almost 2 years. Metter (1964b) reported that 15 of
the females contained sperm, although he was un-
clear how this number was divided among vitello-
genic and nonvitellogenic females except to note (p.
711), “Three females collected in September had
eggs in the smaller size class. . . [and] contained
sperm. This would indicate the sperm can remain
viable for 2 years.” He also mentioned that one fe-
male with large vitellogenic eggs contained no
sperm despite repeated copulations with males over
2 weeks.
Our observations indicate that the height of the
mating season is June and July in northern Califor-
nia. Our limited sample of two vitellogenic and two
nonvitellogenic females from this period all con-
tained sperm in their SSTs, even though the ovi-
ducts of the nonvitellogenic females were not as
hypertrophied and actively secretory as those of
vitellogenic females. Among salamanders (the other
amphibians known to store sperm), Sever et al.
(1996b) found sperm storage in biennially breeding
female Amphiuma tridactylum that were not ex-
pected to oviposit in the forthcoming year. Whether
these findings indicate that these females can store
viable sperm through successive breeding seasons
requires experimental verification.
We must consider, however, the possibility that
sperm storage could occur for approximately a year
in the population we studied. However, storage for 2
years, as suggested by Metter (1964a,b) for Idaho
and Washington populations would appear unlikely.
Metter found mating in the fall so that even vitello-
genic females must wait nearly a year until the
subsequent summer to oviposit, while nonvitello-
genic females mating in the fall were hypothesized
to wait almost 2 years. In our population, sperm
17SPERM STORAGE IN ASCAPHUS TRUEI
were not found in any females sacrificed in fall or
spring, even though two of the females sacrificed in
April contained vitellogenic follicles nearly as large
as those found in gravid females during June and
July. Although our sample is small, we suggest that
oviposition generally follows summer mating in
vitellogenic females. Thus, nonvitellogenic females
mating at the same time would presumably yolk
follicles and oviposit the following year, resulting in
a maximum of 1 year of sperm storage. The Califor-
nia coastal populations also differ from inland pop-
ulations in having a 1–2 year larval period (Wallace
and Diller, 1998) rather than 2– 4 years as reported
by Metter (1964a) in Idaho and southwest Washing-
ton. Thus, considerable geographic variation occurs
in the reproductive biology of this species.
Metter (1964b) reported that sperm are stored in
the lower, straight portion of the oviduct with none
in the upper coiled portion. We found SSTs limited
to the anterior portion of the ovisac, the “straight
portion” of the oviduct. The coiled portion, the am-
pulla, is the area where the gelatinous coats are
applied to the eggs after they enter the oviduct.
Fertilization probably occurs as the jelly-coated eggs
pass through the ovisac. It is well known in anurans
that application of the jelly coat is necessary for
successful fertilization of the eggs (Wake and Dickie,
1998).
Metter (1964b) also noted that sperm taken from
the oviducts of Ascaphus truei were highly motile
when placed in saline and that secretions from the
oviduct may provide “nutrients” for the sperm. This
supposition has also been made for sperm storage in
spermathecae of salamanders, but no evidence ex-
ists that nourishment of stored sperm occurs (Sever
and Kloepfer, 1993). In contrast, Hardy and Dent
(1986) found that sperm stored in salamander sper-
mathecae are quiescent during storage, and sper-
mathecal secretions may therefore provide the
chemical/osmotic environment for sperm quiescence
(Sever and Kloepfer, 1993). In our sample, sperm in
both vitellogenic and nonvitellogenic females of A.
truei appear normal and are similar in abundance
and distribution. Occasionally, sperm are embedded
in the SST epithelium, but we found no evidence of
involvement of the epithelium in either nourishment
of sperm or spermiophagy.
Seasonal Variation of the Oviduct in
Ascaphus truei
We find it noteworthy that all regions of the ovi-
duct have a PAS⫹secretory product, although the
vacuoles differ somewhat in cytology. The lack of an
AB⫹or BB⫹reaction indicates that the product
contains neutral carbohydrates. The reaction is
most intense when glands are most hypertrophied,
but a PAS⫹reaction can be found throughout the
year. The only lipid droplets that were observed
were in the oviducal epithelium of the ovisac of
nonvitellogenic females.
The infundibulum shows little seasonal variation.
In the oviducal sinus, the variation is limited to
increased uniform density of the secretory vacuoles
in reproductively active females and the sloughing
of epithelium in the female in copulexus. We believe
this sloughing of the epithelium may be associated
with insertion of the male intromittent organ. Vari-
ation in the ampulla is associated with hypertrophy
of the oviducal glands that provide gelatinous enve-
lopes as eggs pass through this region. Possibly
some differences in secretion occur in different re-
gions of the ampulla that may correlate with differ-
ences in the jelly layers around the eggs, as known
in other amphibians (McLaughlin and Humphries,
1978). However, whether different layers exist in
the jelly coats of the eggs of Ascaphus truei is un-
known, and our observations indicate that the am-
pullary region is homogeneous in structure and se-
cretory activity.
The condition of the SSTs in the specimens con-
taining sperm is most interesting, because several
females contain sperm despite having regressed ovi-
ducts and small follicles, i.e., they are not in a con-
dition where oviposition seemed likely in the current
breeding season. The SSTs of the nonvitellogenic
females exhibit less secretory activity than those of
the vitellogenic females. The nonvitellogenic fe-
males, however, have lipid droplets in the oviducal
epithelium, and lipid was not noted in other regions
or other seasons. Despite these differences in secre-
tory activity of the SSTs, sperm appear normal in
both vitellogenic and nonvitellogenic females. Thus,
secretions of SSTs may not be important in sperm
maintenance. On the contrary, sperm in the non-
vitellogenic females could be the result of a recent
mating, and lack of sustenance from the secretions
was not yet a factor in sperm maintenance.
Comparative Biology
To date, Ascaphus truei is the only amphibian in
which oviducal sperm storage has been reported.
Indeed, the only other anamniotes in which oviducal
sperm storage is known are elasmobranchs (Pratt,
1993; Hamlett et al., 1998, 1999; Hamlett and Koob,
1999) in the class Chondrichthyes, which is not con-
sidered the sister taxon of Amphibia. Females of
some teleosts in the Osteichthyes store sperm
(Howarth, 1974), but they lack homologs to the ovi-
duct (Kardong, 1995). Instead, sperm are stored in
the ovary or a gonaduct (ovarian duct) formed from
ovarian tissue (Howarth, 1974; Constanz, 1989).
The extant representatives of Actinistia and Dip-
noi, descendant taxa of sarcopterygiian sister groups
of amphibians (Schultze, 1994), possess oviducts
(Millot and Anthony, 1960; Wake, 1987) and Latim-
eria is viviparous (Smith et al., 1975), indicating
that fertilization is internal (Fig. 13). Sperm stor-
18 D.M. SEVER ET AL.
age, however, has not been reported in Latimeria or
any of the extant lungfish.
The Lissamphibia is generally considered mono-
phyletic and consists of three groups, the Anura,
Caudata, and Apoda. Most evidence supports a
frog ⫹salamander clade (Pough et al., 1998) (Fig.
13). Sperm storage is unknown in female apodans,
even though internal fertilization apparently occurs
in all taxa, and many caecilians are viviparous
(Wilkinson and Nussbaum, 1998). Aside from Asca-
phus, only a few anurans have internal fertilization,
with sperm transfer accomplished by cloacal appo-
sition. These species include Mertensophryne mi-
cranotis (Grandison and Ashe, 1983) and four spe-
cies of Nectophrynoides (Wake, 1980) within the
Bufonidae from Africa, and Eleurodactylus jasperi
(Wake, 1978) and E. coqui (Townsend et al., 1981)
within the Leptodactylidae from Puerto Rica. Recent
work, however, has indicated that Nectophrynoides
may not be monophyletic (Dubois, 1986; Graybeal
and Cannatella, 1995). Two internally fertilizing
species remain in the genus, N. tornieri and N.
viviparus, while the other such taxa have been des-
ignated Altiphrynoides malcolmi and Nim-
baphrynoides occidentalis (Dubois, 1986). Necto-
phrynoides, Altiphrynoides, and Nimbaphrynoides
may not be closely related (Graybeal and Can-
natella, 1995).
Obviously, research needs to be done to determine
whether oviducal sperm storage occurs in caecilians
and internal-fertilizing bufonids and leptodactylids.
Ascaphus truei, however, is not the sister taxon of
any caecilian or of the other internal-fertilizing frogs
(Fig. 13), so oviducal sperm storage must be consid-
ered independently derived in A. truei.
Thus, oviducal sperm storage in Ascaphus truei is
a classic example of homoplasy through convergence
(Sanderson and Hufford, 1996). Structural and func-
tional similarities in sperm storage between A. truei
and other vertebrates with oviducal sperm storage
therefore are not based on direct descent but related
either to similar functional adaptations and/or to
internal design restraints (Wake, 1991). In the lat-
ter case, structural and physiological constraints on
the basic vertebrate oviduct and sperm morpholo-
gies (the “bauplans”) may limit the options for ex-
pression of oviducal sperm storage.
The group of anamniotes phyletically closest to
frogs and with which frogs share the most develop-
mental similarities (the closest generative system;
Wake, 1996) is the Caudata. Sperm storage occurs in
females of all seven families of salamanders that
compose the suborder Salamandroidea (Sever, 1991,
1994). Instead of oviducal sperm storage, however,
sperm are stored in cloacal glands (spermathecae)
which consist of a single compound tubulo-alveolar
gland (Plethodontidae) or numerous simple tubular
glands (other families). The ancestral condition for
salamanders is lack of sperm storage glands, a con-
dition found in three families (Sever, 1994). The
ultrastructure of sperm storage in salamanders has
been studied extensively and was recently reviewed
by Sever and Brizzi (1998).
Numerous differences occur between the sper-
mathecae of salamanders and the SSTs of Ascaphus
truei. The distal portions of the spermathecae of
salamanders are typically alveolar, lack cilia, and
possess basal myoepithelium (Sever and Brizzi,
1998). Secretory activity in salamander spermathe-
cae is sometimes regionalized and seasonal, depend-
ing on the taxa (Sever, 1994). A great deal of varia-
tion also occurs in reaction to carbohydrate stains
with, however, most species showing AB⫹reactions
for carboxylated glycosaminoglycans (Sever, 1994).
In some forms the sperm are in orderly arrays in
the spermathecae (Sever and Hamlett, 1998),
whereas in others sperm are in tangled masses (Sev-
er et al., 1999). Alignment of sperm may depend to
some degree on the anatomy of the spermatheca
(more orderly in compound glands than simple tu-
Fig. 13. “Scenariogram” (see Wake and Larson, 1987) showing
distribution of internal fertilization and sperm storage in the
Lissamphibia and extant sarcopterygiians (descendant taxa of
piscine ancestors to amphibians). Only relevant and otherwise
most inclusive taxa are shown. Two of the four species of Necto-
phrynoides reported by Wake (1980) to have internal fertilization
are now designated Altiphrynoides malcolmi and Nim-
baphrynoides occidentalis by some workers (Dubois, 1986; Gray-
beal and Cannatella, 1995). Independent origin of internal fertil-
ization, as illustrated here, would not be most parsimonious in
this tree. Oviducal sperm storage is an autapomorphy for Asca-
phus truei within the Amphibia and sister taxa, but evidence for
sperm storage should be sought in other taxa with internal fer-
tilization.
19SPERM STORAGE IN ASCAPHUS TRUEI
bular). Spermiophagy by the spermathecal epithe-
lium has been described in various taxa of
salamanders (Sever and Brizzi, 1998).
The SSTs of Ascaphus truei more closely resemble
those of squamate reptiles (Fox, 1956; Girling et al.,
1997; Sever and Ryan, 1999). Oviducal sperm stor-
age glands are known from all groups in the reptile–
bird clade except Crocodilia, Amphisbaenia (in
which they likely occur) and Rhynocephalia (Gist
and Jones, 1987). Like reptiles, the SSTs of A. truei
are simply continuations of the oviducal lining and
contain ciliated nonsecretory cells and nonciliated
secretory cells. Myoepithelium is absent, but the
oviduct possesses layers of smooth muscle (tunica
muscularis) superficial to the mucosa. The linings
and glands of reptilian oviducts are generally de-
scribed as PAS⫹, like those of A. truei, with little
reaction to acidic mucosubstances. Sperm in the
SSTs of A. truei are generally in close alignment,
although in squamates this condition varies (Fox,
1956; Sever and Ryan, 1999). Although sperm nuclei
are sometimes found embedded in SSTs of reptiles
(Sever and Ryan, 1999) and of A. truei, no evidence
exists for spermiophagy in these taxa.
Thus, oviducal SSTs in distantly related taxa
show more similarities than SSTs in Ascaphus truei
and spermathecae in salamanders, members of sis-
ter taxa. The basic structure of the vertebrate ovi-
duct, therefore, may limit the range of features as-
sociated with oviducal sperm storage.
Limits to Further Studies
The fact that we describe individual specimens
rather than summarize observations on large sam-
ples is a result of our concern for conservation of this
unique species. We realize, however, that we did not
address some critical aspects of the reproductive
anatomy of Ascaphus truei, and that we have per-
haps engendered more questions than we have an-
swered. For example, we would like to sample fe-
males with eggs passing through the ampulla,
ovisacs, and oviducal sinuses in order to study the
processes of formation of egg envelopes and fertili-
zation. We need to dissect or examine sections of a
male and female fixed in copulexus to confirm our
suspicion that the engorged tail extends through the
cloaca into the oviducal sinus. We would like to
determine the fate of sperm remaining in the ovi-
duct after oviposition (a condition noted by Noble,
1925). Observations of these and various other in-
ternal reproductive phenomena obviously result in
the killing of gravid females, but we will continue to
limit the number of animals killed to the minimum
necessary for such studies.
ACKNOWLEDGMENTS
We thank Lowell V. Diller of the Simpson Timber
Company for overseeing the collection and shipment
of specimens, critically reading the manuscript, and
offering many insights into the biology of Ascaphus
truei. We thank Laura Burkholder, Elizabeth Ryder,
and Joel Thompson for aid in the collections and
Chris Hysell for help in the laboratory. This is pub-
lication number 17 from the Saint Mary’s College
Electron Microscopy Facility.
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21SPERM STORAGE IN ASCAPHUS TRUEI
