HARVARD UNIVERSITY

^

Ernst Mayr

Library

of the Museum of

Comparative

Zoology

^^^ 4 2006

MCZ
LIBRARY^

AUG 2 2 1991 PROCEEDINGS

HARVAPn "f'*^^

UNiv^an Diego Society of Natural History

Founded 1874

Number? 31 January 1991

Post-Mating Selection of Hybrid Toads (Scaphiopus miiltiplicatiis

and Scaphiopus bombifrons)

Marie A. Simovich

Biology Department. University of San Diego. San Diego. California 92110 and San Diego Natural History Museum.

San Diego. California 92112

Clay A. Sassaman

Biology Department. University of California. Riverside. California 92521

Amy Chovnick

Pharmacia LKB Biotechnolgy. Inc.. 1025 Atlantic Ave.. Suite 101 .Alameda. California 94501

ABSTRACT. — High frequencies of F, hybrids and the offspring of baclicrosses occur in a hybrid zone between the spadefoot toads Scaphiopus
muliiplicatus and 5. bombifrons in southeastern Arizona. To assess potential post-mating isolating mechanisms between these species, reproductive
characteristics, tadpole growth rates, and survivorship in the laboratory of the parental species and hybrids were measured and compared.

Hybrid males are sterile; hybrid females, although fertile, produce only about half as many eggs as either parental species and are thus at a
selective disadvantage. On a heterogeneous diet including naturally occurring foods, tadpoles of all types develop faster and are larger than those fed
a trout-food diet. Hybrid tadpoles have higher survival rate on both diets and develop faster on a mixed diet than does either parental species. At
metamorphosis hybrids are intermediate in size between the parental species. These developmental advantages may afford hybrids some
compensation for adverse fertility and fecundity selection.

INTRODUCTION genotypes are often present. This suggests that other forces are at

work. For example, heterotic effects in the development or

Investigations of hybridization and hybrid zones have been survivorship of hybrid offspring, such as have been reported in

important in the development of our understanding of the processes other systems (Wasserman, 1937, 1963; Thornton, 1933; Volpe,

of evolution (Barton and Hewitt, 1981; Templeton, 1981; Hewitt, I960; Pasdaret al., 1984; Samollow and Soule, 1983), might buffer

1988). The hybrid systems that are most informative are those for other fitness reductions .

which we can evaluate not only the conditions under which inter- This paper reports on laboratory measurement of several as-
breeding occurs but also the factors affecting the hybrid offspring pects of post-mating selection in Scaphiopus. including adult fer-
produced and the way in which they respond to their environment. tility and fecundity, egg viability, and tadpole survivorship and

An area of hybridization between the spadefoot toads developmental rate.
Scaphiopus muliiplicatus and Scaphiopus bombifrons (subgenus

Spea) in the San Simon Valley of southeastern Arizona was inves- MATERIALS AND METHODS
tigated over a four-year period (Simovich, 1985, in press). In this

area hybridization and introgression allow the frequencies of hybrid Pairs in amplexus and unmated females were collected from

tadpoles to vary spatially and temporally from 0 to 40%, forming a ponds and cattle tanks in the San Simon Valley of southeastern

dynamic mosaic. A portion of this variability can be accounted for Arizona. The identity of individuals was determined by protein

by the observation that mating in large ponds is species-specific, electrophoresis methods described by Simovich and Sassaman

but in small (crowded) ponds more heterotypic matings occur and (1986). The toads were identified to one of six classes: S.

hybrid tadpoles are more frequent (Simovich. 1983). multiplicatus (M), 5. bombifrons (B), F, hybrids (H), backcross-M

Significant pre-mating isolating mechanisms in at least the large (BKM), the offspring of a mating between a female hybrid and a
ponds might indicate that interbreeding lowers fitness, and that male S. mii/n/j/Zcofiw, backcross-B (BKB), the offspring of a mat-
hybrid offspring are at a disadvantage relative to non-hybrid off- ing between a female hybrid and a male S. bombifrons, or double-
spring. However, frequencies of adult hybrids are as high as 31% in backcross (DBK), the offspring of matings involving these back-
some breeding choruses, and substantial numbers of introgressed crosses. This procedure uses four independent loci and can cor-

Marie A. Siniovich. Clay A. Sassaman, and Amy Chovnick

rectly identify pure species, F, hybrids, and 879^ of individuals re-
sulting from backcrosses (Simovich and Sassaman, 1986).

Fertility and Fecundity. — Amplexed pairs were allowed to
breed in buckets in the laboratory. The number of eggs laid was
counted, and random samples (averaging 160) were transferred to
plastic trays of aerated water. Embryos were raised to the point
where they could swim freely (about four days old), at which time
fertility and the percent successful hatch was determined. Com-
parisons of fertility and successful hatch were made for the three
most frequent crosses (the two intraspecific crosses and the back-
cross of F| hybrid females to S. multiplicatus males).

To estimate fecundity in unmated females, egg counts of a
weighed subsample of ovary were extrapolated to the total ovarian
weight. Comparisons were limited to the three most common
classes of females (S. multiplicatus. S. honihifrons, and F, hybrid).
The ovaries from some of the females that had bred were also
examined, and since none of these had significant numbers of
mature eggs remaining, the numbers of eggs laid by mated females
in the fertility experiments were also used to estimate fecundity.

Sunival and Developmental Rale. — Tadpoles from selected
crosses were combined in an experiment designed to determine if
the developmental patterns of the classes differed and if diet or
density had significant effects on these patterns. For this, mixtures
of tadpoles were kept under two density (high and low) and two
food (trout food and mixed diet) regimes. Unfortunately, raccoons
destroyed the low-density treatment, so only early, qualitative com-
parisons are available.

In the high-density treatment, groups of swimming tadpoles
(which had hatched on the same day during the fertility experi-
ments) were placed in four 10-liter buckets. Each group consisted
of 60 S. muliiplicatu.'! (M) (sampled from three clutches), 60 S.
homhifrons (B) (also sampled from three clutches), and 59 F,
hybrids (H) (from the one clutch that was available).

The buckets were kept outdoors in a screened enclosure that
received late morning and afternoon sunlight. Water temperatures
were taken daily, and buckets were rotated so that similar tempera-
tures could be maintained. Two replicates of each food treatment
were used. The first was a trout-food treatment, in which tadpoles
were fed ad lihilum with commercial high-protein trout chow. The
second was a mixed-diet treatment, in which the ad lihitum trout
diet was supplemented daily with live fairy shrimp (an important
component of the natural diet) in excess of demand.

Toadlets that had completed metamorphosis were collected and
frozen at two-day intervals. Those that died in the process were frozen
as found. The experiment was terminated while some tadpoles still
remained, and these were also frozen. All individuals were identified
by electrophoresis, and three aspects of performance were deter-
mined: survivorship (the proportion of the initial sample surviving at
the end of the experiment), successful metamorphosis (proportion of
survivors completing metamorphosis by the end of the experiment),
and developmental rate (average number of days to metamorphosis).
Size at metamorphosis was also recorded for each individual.

RESULTS

Fertility. Fecundity, and Halchini; Succe.'i.i. — Female S.
multiplicatus and S. honihifrons did not differ from each other in
fecundity, but hybrid females produced about 45'7c of the number of
eggs that pure females produced (Table 1 ). Analysis of a subset of
females for which somatic weights were available showed the same
fecundity differences despite the intermediate weight of hybrid
females (.i = 11.66 g, « = 5) and a significant difference in the
weights of pure females, with S. homhifrons ( .v = 13.68 g. n = 11)
heavier than S. multiplicatus ( .v = 10.34 g, /i = 7) (ANOVA, F = 5.06,
df 2/20, and multiple range tests, p < 0.05). There was no correla-
tion between somatic weight and number of eggs produced within
each species for this subset of data. Although Woodward (1987)
found that in New Mexico 5. homhifrons produced more eggs than
did S. multiplicatus. we found no significant difference. If there is
any bias, it is not in favor of the heavier S. homhifrons.

Hybrid males from the San Simon Valley are apparently sterile
(Table 2). Of eight amplexed pairs involving hybrid males, seven

Table 1 . Fecundity of female Scaphiopus toads.

S. Diuliipliciiiiis

S- homhifrons

F hybrids

No. of clutches

24

15

9

Mean clutch size

1139"

1042"

447

Standard error

69

108

83

F,,,,,= 12.925''

p < 0.05

"Not significantly different
''F of ANOVA

Table 2. Fertility and hatch success of natural matings of Scaphiopus toads.

Number of

Percentage

Percentage

Number

Pairs

of

of

Cross

of

with

Clutches

Egg

(Male X Female)"

Pairs

Eggs

Fertilized

Hatching'"

MxM

43

36

97

91

(11)'

BxB

25

25

100

85

(il)'

MxH

8

8

100

82

(3)'

HxH

7

7

0

—

—

MxB

5

1

100

88

(1)

BxM

2

0

—

—

—

HxM

0

—

—

—

BxH

1

100

8

(1)

MxBKM

I

100

98

(1)

BxDBK

I

100

99

(1)

DBKxB

1

100

96

(1)

"M. S. mulliplicalus: B, S. homhifrons: H, F, hybrids: BKM, backcross-M; DBK, double

backcross

''Number of crosses on which percent halch is based m parentheses
'No significant heterogeneity in the percent halch for the three most common crosses

(arcsin translomied: one-way ANOVA and multiple range tests; F = 1.197 ,„|,/7 > 0.05.

Posl-Mating Selection of Hybrid Toads

were hybrids crossed inter se. Although eggs were laid after all of
these matings, none cleaved. This failure of hybrid-hybrid crosses
was not caused by female sterility because hybrid females were
fertile in backerosses. (The remaining mating involving a male
hybrid was with a female 5. midlipUcLilus that did not lay eggs. ) The
only male other than a hybrid that failed to fertilize eggs was one of
46 S. miiltipluatus. Even some eggs from a mating of S. coiuhi (a
member of the other subgenus, Scaphiopits) with S. muluplkatus
were fertilized and developed partially beyond gastrulation.

The three most common types of crosses (two pure species and
the hybrid S. multiplkatiis) did not differ in the percentage of eggs
that hatched (Table 2). The percent hatch of eggs was also high for
most of the other crosses, with the exception of the one cross of a
male S. homhifrnns to a female hybrid. Of the seven S. honihifrons-
S. multipliciitus pairs collected, six did not produce eggs; however,
the one that did showed 88% hatching success (Table 2).

Survival and Developmental Rate. — In this experiment, both
differences between genotypic classes and the effect of diet on
development were compared. (Within-treatment replicates gener-
ally did not differ [contingency X"] and were pooled.) Diet had a
significant effect on the development but not on the survival of each
class when compared to itself. Within each class more survivors
metamorphosed by the end of the experiment (contingency /-, /; <
0.03) and the average number of days to metamorphosis was lower
(/ test, /' < 0.05) on a mixed diet than on a trout-food diet. (One
replicate within .9. nndtiplicatus indicated a possible effect of food
on survival but the other did not.)

When the various classes on the two diet regimes were com-
pared against each other, significant differences were also seen
(Table 3). On both food regimes hybrids showed the highest sur-
vival, S. homhifrons was intermediate, and S. midtiplicatus showed
the lowest survival. The classes also differed in the average number
of days to metamorphosis. On a mixed diet, hybrids metamorphosed
sooner than did either parental class, and on a trout-food diet they
metamorphosed sooner than did S. homhifrons.

On a mixed diet, hybrids began and completed metamorphosis
about four days earlier than did either parental class, as illustrated
by the cumulative proportion of survivors metamorphosing over
time (Fig. I ). In contrast, on a trout-food diet, although hybrids may
have had a slight advantage, differences between classes were not
as clear. Furthermore, none of the three classes had completed

c

(0

o
a
o

E

CD

^-'
0)

.4 -

(A

o

> 1.0

3

t; .8 -

c
o

o
a
o

0)

>

w 2 -

_2

3

E

3

o

.6 -

.4 -

trout diet

55

Days

Figure 1 . Cumulative proportion of survivors successfully completing
metamorphosis under two food treatments. Replicates have been pooled.
Triangles, Scaphiopiis bomhifrons; circles, S. multiplicatiis; diamonds,
hybrids.

Table 3. Survival and development of Scaphiopii.'! tadpoles."

S. niiilri-
plicanis

F,
hybrids

S.
homhifrons

Comparison
Procedure''

Initial number of individuals

120

118

120

Mixed Diet

Proportion surviving
to the end of experiment

0.14*

0.67'

0.44*

A

Proportion of

survivors metamorphosing

0.94*

1.00*

1.00**

A

Mean day to metamorphosis

43.94*

38.67*

43.81*

B

Trout Diet

Proportion surviving to the end
of experiment

0.36*

0.70*

0.54*

A

Proportion of survivors
metamorphosing

0.54*

0.56*

0.45*

A

Mean day to

metamorphosis (underestimates)

48.91*

47.91*

50.79*

B

"Common superscripts indicate values that do not differ significantly {p > 0.05).
''A, Ryan's procedure for chi square; B, one-way ANOVA with multiple-range test.

Marie A. Simovich, Clay A. Sassamun. and Amy Chovnick

E
E

c

c
0)

>

o

c
CO

trout 1 trout 2 mixed 1 mixed 2
Diet

Figure 2. Average size at metamorphosis of toads under two food
treatments. Vertical bars indicate the 95% confidence interval. Replicate
experiments are plotted separately. Multiple-range tests from ANOVA indi-
cate significant differences between all categories within replicates except
M and B of trout-food replicate 1 and M and H of trout-food replicate 2. B.
Scaphiopiii htimhifwns; M, S. miilupliciiluy. H, hybrids.

metamorphosis by the end of the experiment on the trout-food diet,
yielding an underestimate of the average time to metamorphosis.

In addition, before their loss to raccoons, animals in the concur-
rent low-density replicates were larger and developing faster than
those at high density. This could have been expected from similar
findings in other studies on density effects (see. for example, Travis
II984J). Furtheniiore. of the low-density groups, the mixed-food
treatment groups were larger than those on trout food and were
developing more rapidly.

Size at metamorphosis was a function of both genotypic class
and food regime (ANOVA) (Fig. 2). Diet affected the size of all
classes, and the differences among classes were accentuated on a
mixed diet (ANOVA with multiple range tests). On a trout food
diet, 5. homhifrons toadlets were always significantly larger than
hybrids, but the ranking of 5. midliplicatus differed in the two rep-
licates. All classes were larger on a mixed diet (93% confidence
interval) than on trout food (Fig. 2). and on a mixed diet, all classes
differed significantly in size with S. hamhifrons being largest, hy-
brids intermediate, and .S'. nmllipliccitiis smallest in both replicates.

DISCUSSION

In the San Simon Valley zone of sympatry. Scciphiopns hybrids
exhibit low reproductive potential relative to the parental species.
Females are fertile but exhibit considerably reduced fecundity, and
males are sterile. This pattern of the reproductive potential of male
hybrids being lower than that of females is not uncommon in toads
(e.g.. Volpe, I960; Wassemian. 196."?). However, the observation of
male sterility in this area is interesting, since fertile hybrid males

are reported elsewhere.

One source of this heterogeneity in males may be geographical '
differentiation within S. homhifrons. Two call types of S.
homhifrons are known, a "slow call" type in the eastern portion of
the species' range and a "fast call" type in the southwest (Pierce,
1976). Although the two call types differ little in electrophoretic
characters (Sattler. 1980). it may be (as noted by Pierce in
Wassemian 1 1 970] ) that crosses involving the "fast call" type of S.
homhifrons (as found in the San Simon Valley) inherently produce
sterile males, while at least some hybrid males from areas where the
"slow call" form is found are fertile (Forester. 1969. 1975). A
second possibility is that the fertility of the offspring of reciprocal
crosses differs, as has commonly been shown in other anurans
including Rana, Bufo, and Scaphiopits (e.g.. Frost, 1982; Thornton,
1955; Volpe. 1952; Wassemian, 1957, 1963). Because in the San
Simon Valley most of the direct interspecific crosses appear to be of
S. mtiltipliaitiis males with 5. homhifrons females (Simovich,
1985). we could not address this possibility with our data from
field-captured pairs.

The interfertility levels of crosses between the two parental
species were comparable to or higher than those previously reported
by Brown ( 1967). Forester ( 1969. 1975). Littlejohn (1959). Sattler
(1978), or Wassemian (1964). Any variation in hatching success
could be due to differences in rearing or breeding conditions.
Discrepancies could also result from inherent differences in the
genetic compatabilily of the two call types of 5. homhifnms (Pierce,
1976) with S. miilliphcatiis. This geographic variability under-
scores the importance of quantifying such components of selection
as interfertility for specific areas in any effort to quantify the
dynamics of a hybrid interaction.

Hybrid females, when backcrossed to S. multiplicaius males, do
not suffer any additional loss of fitness due to reduced fertility or
offspring survival under controlled, noncompetitive conditions. The
alternative hybrid x 5. homhifrons cross is rare and more data are
needed. The results suggest that direct interspecific F, (M x B)
crosses, although highly fertile, may produce eggs less frequently
than pure crosses, but we have no indication as to how frequently
this occurs in nature or if it is simply that S. midtiplicatiis females do
not always lay eggs.

It is of considerable interest that despite low reproductive fitness
due to reduced fecundity and fertility, hybrids may have higher
fitness for later components of selection such as tadpole survival
and developmental rate. The data indicate that on a diet including
live food (as in most natural ponds) and at fairly high densities,
several important differences between genotypic classes appear.
First, emerging 5. homhifrons toadlets are largest, hybrids are in-
termediate, and S. multipUcatiis are smallest. This ranking is the
same as found in toadlets emerging from natural ponds (Simovich,
1985). Second, hybrids show significantly higher survival and de-
velop significantly faster than either parental species. In the field,
however, S. homhifrons metamorphoses sooner than does S.
multiphvatiis (Simovich. 1985). Possibly the dietary needs of 5.
homhifrons are more exacting than those of 5. mii/tiplicatus or hy-
brids, and laboratory conditions did not pemiit optimum develop-
ment. Our laboratory, for example, was at a higher elevation and
thus at lower temperatures than the desert where these toads (espe-
cially S. homhifrons) are found.

The differences we found among genotypic classes in their
responsiveness to diet and density are consistent with some but not
all previous investigations of anuran development. Our results do
seem to agree with Travis et al. (1985) and Alford (1986) that
variable ecological conditions can alter the relative fitness of in-
traspecific sibships and that competition can affect growth and
survival even if food is not limited. However, the details of these
responses are not in accordance with tho.se found in other aniphib-

Post-Mating Selection of Hybnd Toads

ians. Wilbur and Collins (1973), Collins (1973, 1979), and Travis
( 1980. 1983. 1984) have discussed an intraspecific model in which
some species exhibit a size/rale tradeoff, with slower developers
being larger at metamorphosis above a minimum threshold size (see
also Newman, 1988a, and Crump, 1989). In Hyla. furthermore.
Crump (1989) has shown faster development and smaller size in
rapidly drying environments. In contrast, in Biifn Woodward ( 1987)
has shown environment to affect growth rate but not size at meta-
morphosis. Our study indicates a different relationship. Slow devel-
opers were actually smaller at metamorphosis both within and
between species. Most studies in which decreased growth rate was
related to smaller size at metamorphosis involved increased densi-
ties of intra- and/or interspecific competitors (Travis, 1980, 1983,
1984: Travis et al., 1985; Wilbur, 1977; Wilbur and Collins, 1973:
Collins, 1979). We do not have the density comparisons with which
to confront this issue. It does appear that size, survival, and devel-
opmental characters differ between the parental species and hybrids
and that the full expression of these differences is dependent on the
developmental environment, including diet.

Interestingly, the genotypic classes that were largest and fastest
in development in this experiment, hybrids and S. homhifrons. also
expressed the carnivore morphology (characteristic of some species
of spadefoot toads) most often in the field (Simovich, 1985, in
press). The expression of extreme carnivore morphology has been
tied to a diet of live food, and since carnivores tend to develop faster
and be larger than conspecific omnivores (Pomeroy, 1981), there
may be a correspondence between the tendency to develop carni-
vore morphs and the other developmental differences between ge-
notypic classes. Because we cannot develop extreme carnivore
morphs in the laboratory, we do not know the genetic basis of the
morphologies or if other dietary requirements are involved in the
full expression of the morph.

All things considered, the ephemeral ponds in which these
species breed and develop can present extremely harsh selective
conditions. Life cycles must be precisely timed to the availability of
a temporary and frequently unpredictable resource. The toads must
emerge and breed on the first night of summer rains (Bragg, 1965;
Ruibal et al., 1969; Dimmitt and Ruibal, 1980) so that the tadpoles
can develop and metamorphose in the short time that the ponds
remain. If rainfall is sparse, these small ponds dry very quickly,
creating crowded conditions for all inhabitants. Owing to aquatic
predators and desiccation, tadpole mortality can be quite high
(Mayhew, 1965: Licht, 1974; Creuser and Whitford, 1976; Wilbur,
1977; Caldwell et al., 1980: Travis, 1980, 1983; Alford. 1986;
Woodward, 1987).

There is thus a premium on fast growth, rapid development, and
early metamorphosis (Wilbur and Collins, 1973; Licht, 1974;
Wilbur, 1977; Cadwell et al„ 1980; Travis. 1980, 1983: Smith.
1983). Reduction of the time animals are exposed to aquatic
predators should be advantageous (Travis et al., 1985). Fast-devel-
oping tadpoles should be favored in years when ponds dry quickly.
Furthermore, eariy emergence may allow a longer period of terres-
trial feeding to build up fat reserves for overwintering. Larger size
at metamorphosis may also decrease the risk of post-metamorphic
desiccation (Martof, 1956) and afford later advantages should those
individuals continue to grow faster and reach breeding size sooner
(Collins, 1975; Wilbur et al., 1978). In tradeoff models, these pre-
and post-metamorphic attributes are alternatives, either fast devel-
opment/small size or slow development/large size, and plasticity is
retained by fluctuating selection (i.e., variation in pond longevity)
(Wilbur, 1977; Travis. 1983, 1984; Newman, 1988b).

In the Scaphiopiis hybrids, rapid development occurs without a
concomitant reduction in size at metamorphosis. Unfortunately, the
genetic basis of the success of hybrids is still unclear. Several
possibilities exist, including heterosis, maternal influence, and

simple dominance. Laboratory evidence points to intermediate ex-
pression of size characters but heterosis in developmental rate.
Heterosis has been seen in other hybrid spadefoot toads
(Wasserman, 1957, 1963) and other anurans (Thornton, 1955:
Volpe, 1960). However, maternal influence in development ( Volpe,
1952) and temperature tolerance have also been documented
(Brown, 1967). Also, in fish reciprocal hybrids have been shown to
differ in growth rate (Pasdar et al., 1984). Since most hybrids in the
San Simon Valley appear to result from crosses in one direction,
these possibilities remain to be addressed. Further tests evaluating
the development of hybrids generated from both reciprocal F
crosses are needed in order to determine which model is most
applicable.

ACKNOWLEDGMENTS

We thank R. Tinsley and his wonderful co-workers, G. Bell, and
the numerous researchers at the Southwestern Research Station for
their invaluable help catching toads in the pouring rain and counting
thousands of eggs. We also thank L. Nunney, V. Shoemaker, S.
Morey, R. Dingman. and J. Graves for reviewing this manuscript.
This work was funded in part by grants from the National Science
Foundation, the Theodore Roosevelt Memorial Fund, Sigma Xi,
and the Chancellor's Patent Fund (University of California at Riv-
erside).

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