Behavioral Ecology Vol. 11 No. 1: 115-124
© 2000 International Society for Behavioral Ecology
Female choice, male interference, and sperm precedence in the red-spotted newt
a Section of Integrative Biology, University of Texas, Austin, TX 78712, USA, b Division of Biological Sciences, University of Missouri, Columbia, MO 65211, USA, c Department of Biology and Institute of Cognitive Science, University of Louisiana, Lafayette, LA 70504, USA
Address correspondence to C. R. Gabor. E-mail: gabor{at}uts.cc.utexas.edu .
Received 27 May 1998; revised 7 February 1999; accepted 23 June 1999.
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ABSTRACT |
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 TOPFOOTNOTES ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Darwin first identified female choice and male—male competition as forms of sexual selection resulting in the evolution of conspicuous sexual dimorphism, but it has proven challenging to separate their effects. Their effects on sexual selection become even more complicated when sperm competition occurs because sperm precedence may be either a form of cryptic female choice or a form of male—male competition. We examined the effects of tail height on male—male competition and female choice using the sexually dimorphic red-spotted newt (Notophthalmus viridescens viridescens). Experiment 1 examined whether male tail height influenced male mating success. Males with deep tails were more successful at mating with females than those with shallow tails. Successful, deep-tailed males also were bigger (snout-vent length; SVL) than unsuccessful, shallow-tailed males, but they did not vary in tail length or body condition. Of these, only tail height and tail length are sexually dimorphic traits. Experiment 2 tested the hypothesis that the differential success of males with deeper tails was due to female choice by examining both simultaneous female preference for association and sequential female choice. We found no evidence of female choice. When males were not competing to mate with females, tail height did not influence male mating success. Successful males did not have different SVL and tail lengths than unsuccessful males. Thus, tail height in male red-spotted newts appears to be an intrasexually selected secondary sexual characteristic. Experiment 3 used paternity exclusion analyses based on molecular genetic markers to examine the effect of sperm precedence on sperm competition in doubly-mated females. Sperm precedence likely does not have a pervasive and consistent effect on fertilization success because we found evidence of first, last, and mixed sperm usage.
Key words: female choice, male—male competition, molecular markers, newts, Notophthalmus viridescens, paternity exclusion analysis, sexual selection, sperm competition.
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INTRODUCTION |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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A primary goal of sexual selection theory is to explain the evolution of conspicuous sexual dimorphism (Andersson, 1994
). Often female
choice or male—male competition has been implicated in the evolution of
secondary sexual dimorphism, and both can occur in nature
(Andersson, 1994). However,
separating the effects of female choice and male—male competition is
difficult. Female choice and male—male competition might favor the same
characters or select for different characters
(Moore, 1990). For example, in
swordtails, Xiphophorus nigrensis, male—male competition and
female choice both contribute to the greater reproductive success of larger
males (Morris et al., 1992).
In contrast, in eastern tiger salamanders, Ambystoma tigrinum
tigrinum, male—male competition favors increased male body length,
while mate choice selects for greater male tail length
(Howard et al., 1997).
Therefore, the impact of male—male competition and female choice should
be distinguished when examining the relative importance of each on the
evolution of secondary sexual traits
(Moore, 1990).
The interaction of male—male competition and female choice on sexual
selection becomes more complicated and subtle when females mate multiple
times, thus fostering sperm competition. Although the influence of sperm
precedence on male reproductive success is not obvious, this form of cryptic
female choice can be important in a mating system
(Eberhard, 1996). Sperm
precedence can be defined as a nonrandom likelihood of fertilization by sperm
from a male that depends on the order of matings by that male and other males
with a particular female. The precedence effect, if present, can be positive
or negative. According to Wade and Arnold
(1980), the intensity of
sexual selection on a male may be related to sperm precedence, the duration of
sperm competition, and the number (or fraction) of sperm that are not used in
fertilization. Wade and Arnold
(1980) found that both cases
of absolute advantage (either the first male or the second male always fathers
all of the offspring) are equivalent in their effects on the intensity of
sexual selection, whereas mixed paternity can reduce the variation among males
in reproductive success but increase the intensity of sexual selection on
males. In a multiply mating species, sperm precedence effects can influence
sequential female choice. Halliday
(1983) proposed that for
species that mate multiply and have last-male sperm advantage, a female could
ensure fertilization of her eggs by mating with the first male that she
encounters; thereafter, she can maximize the quality of her progeny by
sampling additional males and by mating only with males of higher quality than
those with which she previously mated. Smooth newts, Triturus vulgaris
vulgaris, use this type of mate choice
(Gabor and Halliday, 1997).
Thus, it is also important to examine the outcome of sperm competition in such
systems. In the present study, we examined the impact of male traits on
male—male competition, on female choice, and on paternity in the
red-spotted newt, Notophthalmus viridescens viridescens.
The mating system of newts (Salamandridae) offers an opportunity to
distinguish between hypotheses of female choice, male—male competition,
and the subsequent influence of sperm competition. Males of the North American
red-spotted newt (N. v. viridescens) develop deeply keeled tails
during the courtship season. In addition to having deeper tails than females,
male red-spotted newts also have longer tails (nmales =
276, mean ± SE = 44.9 ± 0.2 mm; nfemales =
157, mean = 42.7 ± 0.2 mm; t' = 2.30, df = 342.5,
p =.01; pooled for 1995-1997). The impact of only one of these
sexually dimorphic characters (tail depth) has been previously studied
(Able, 1999). Able found that
in the field, male tail depth was positively correlated with variation in male
amplexus frequency. In the laboratory Able found that males with deeper tails
had greater success at capturing females. The operational sex ratio
(Emlen and Oring, 1977) in this
species is usually male biased (1.75:1 male:female;
Massey, 1990) because not all
females are receptive at any given time
(Halliday and Verrell, 1984).
Males do not provide any known material resources, such as paternal care or
oviposition sites (Verrell and McCabe,
1988), and female assessment of males is limited to aspects of
male morphology, courtship vigor, and chemical cues
(Halliday, 1977;
Verrell, 1982). Female
red-spotted newts mate two to three times in a season
(Halliday and Verrell, 1984)
and store viable sperm throughout the mating season but not between seasons
(Sever et al., 1996). Sperm
competition has not been examined in red-spotted newts and has rarely been
studied in salamanders (but see Houck et
al., 1985).
Courtship in red-spotted newts consists of either amplexus or a lateral
display described as the "hula"
(Verrell, 1982). Amplexus
consists of a male clasping a female for an average of 3 h. Following
amplexus, the male must dismount the female to deposit his spermatophores,
thus providing the female an opportunity to acquire his gametes, thereby
choosing to accept or reject him as a mate. Following dismount, if the female
continues to follow the male and touches the base of his tail, he may deposit
a spermatophore and then turn 90° while leading the female over the
spermatophore (Verrell, 1982).
Sperm transfer occurs when the sperm cap of the spermatophore attaches to the
conically shaped cloaca of the female. Alternatively, when a female is
initially responsive to a male's approach, amplexus may be bypassed (observed
in the laboratory only) and replaced with the hula. The hula usually lasts for
a few minutes and is followed by spermatophore deposition and perhaps
transfer. Thus, the hula may be a lowcost display (energetically) compared to
amplexus, but it is more susceptible to sperm transfer failure
(Verrell, 1982). This led
Verrell (1989) to predict that
males of species with external sperm transfer but internal fertilization will
be vulnerable to interference from other males and will adjust their mating
strategies accordingly. He suggested that amplexus evolved as a form of sexual
defense against sexual interference (as defined by
Arnold, 1976) by rival
males.
We examined the effect of tail height on female choice and male—male
competition in N. v. viridescens. In addition, we examined the
effects of tail length, snout-vent length (SVL), and body condition (1995 data
only) on male mating success. In experiment 1, "Male—male
competition," we examined the hypothesis that male tail height
influences male mating success. In this experiment, we conducted breeding
encounters, each involving two males of varying tail depths and a female.
These encounters were allowed to proceed fully to the time of insemination. In
experiment 2, "Female choice," we tested the hypothesis that the
differential success of males with deeper tails was due to female choice. We
performed two experiments to examine female choice. In experiment 2a,
"Simultaneous female preference," we examined the preference of
females to associate with deep-tailed or shallow-tailed males. However, we did
not allow the encounters to progress to insemination so as to separate the
influence of female choice from the outcome of the male—male competition
experiment. In experiment 2b, "Sequential female choice," we
examined female preference to mate in a sequence of encounters with individual
males, with male tail height either differing in each subsequent encounter or
remaining the same. A shortcoming inherent in many previous simultaneous
female preference experiments is that females were not allowed to mate with
the males. We conducted our sequential female choice experiment (sperm
transfer allowed) to correct for this problem and to test Halliday's
(1983) prediction that females
will become progressively more choosy.
Finally, in experiment 3, "Sperm precedence," we performed
paternity exclusion analyses based on molecular genetic markers to gain a more
complete understanding of the red-spotted newt's mating system. Specifically,
we tested for multiple paternity by assigning paternity for individual
offspring within clutches of females. We examined whether sperm precedence
depended on the length of time between mating with different males. This
experiment was also designed to complement the sequential female choice
experiment and to gather evidence that might be relevant to the supposition on
which Halliday's (1983)
prediction is based—namely, the existence of "last-male
advantage" or at least the absence of "first-male
advantage."
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METHODS |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Experiment 1: Male—male competition
We collected unmated female red-spotted newts, N. v. viridescens, from a drift fence that completely encircled the pond at Pond Ridge in George Washington National Forest, Rockingham County, Virginia, USA, 21-24 March and 11 April 1995. The use of a complete drift fence precluded the chance that females entered the pond, mated, left the pond, and then were captured. This assertion is supported by the fact that our unmated females did not lay fertile eggs (see experiment 3). Males and females were maintained temporarily in separate containers in the laboratory in Virginia on a 14:10 h light: dark photoperiod until they were brought to the laboratory in Louisiana. The newts were then separated by sex and maintained in aquaria (50.8 x 25.4 x 30.5 cm) on a 14:10 h photoperiod with 25 newts per aquarium at 18°C. The photoperiod was gradually changed to a 16:8 h light:dark cycle. We filled the tanks with aged tap water, lined the bottom with gravel, and placed two bricks in each tank to allow individuals to emerge from the water. We fed the newts earthworms (Lumbricus terrestris) and crickets (Acheta domestica) ad libitum. Water was partially changed weekly. We individually identified newts based on drawings of their spot patterns (Gill, 1978
). Before testing,
we measured the mass of each salamander using a Mettler PL-300 balance, SVL to
the anterior end of the cloaca of each male and female, tail length from the
anterior end of the cloaca to the tail tip, and the tail height of each male
at the deepest section of the tail using a metric ruler. Body condition was
determined for each male by using the residuals of the regression of the
cube-root of mass against SVL (Baker,
1992; Howard et al.,
1997).
We staged 30 encounters to examine male—male competition. In each
encounter, a female (n = 30 females) was matched with a deep-tailed
and a shallow-tailed male. Testing was performed 6-27 April 1995 in the
laboratory at 16°-19°C. Test chambers (61.0 x 30.5 x 30.5
cm) were divided into three sections (left and right = 15.5 cm, center = 30.0
cm) by clear Plexiglas perforated with small holes that potentially permitted
individuals to gain visual and chemical information but prevented matings.
Test chambers were floored with gravel and were filled with aged tap water to
a depth of 11 cm. We placed each female into the central compartment of a test
chamber for a 15-min habituation period. A deep-tailed male (8.5-10 mm) and a
shallow-tailed male (6.5-8 mm) were randomly placed on the right or left sides
of the aquarium, one male per side. The difference in tail height between
males within a pair was always 
2 mm. Males were chosen based on tail
height, but otherwise random pairs (based on a random numbers table) were
selected. Tail depth was within ± 1 SD of the population mean. Some of
the females used in this experiment had been previously used in experiment 2a.
After 15 min of habituation, we carefully lifted the dividers and observed the
behavioral interactions between the two males and between the males and the
female. Often males began to court the female from behind the dividers. If no
mating occurred within 90 min, we considered the encounter unsuccessful.
In each test, we recorded the form of courtship (amplexus or hula) used by
a male, instances of sexual interference, and time to successful sperm
transfer by a male. Sexual interference consists of a rival male: (1)
wrestling and attempting to dislodge the courting male during amplexus, (2)
displaying hula to the courted female, or (3) using female mimicry, which
consists of the rival male moving between the courting male and female during
the spermatophore deposition stage and eliciting spermatophore deposition by
the courting male (Verrell,
1982).
We used a two-tailed binomial test to determine whether males with deep or
shallow tail heights were more successful at mating with females and whether
sexual interference affected sperm transfer, with 
= 0.05
(Siegel and Castellan, 1988).
We subsequently used Student's t tests to examine the relationship
between SVL, tail length, and body condition between successfully versus
unsuccessfully mated males to examine the influence of other traits on male
mating success.
Experiment 2a: Simultaneous female preference
Procedures were as in experiment 1. We examined female preference to
associate with deep or shallow-tailed males to determine whether the results
of the male—male competition experiment arose purely due to mate
competition or whether mate choice by females was also important. We imposed
three treatments with each female (n = 55) tested in all three
treatments. Treatment orders were randomized for each female. In treatment 1
(control), females were matched with two males with shallow tails (6-7 mm). In
treatment 2 (control), females were tested with two males with deep tails
(8.5-11 mm). In treatment 3 (experimental), females were tested with one male
with a shallow tail and one with a deep tail ( 2 mm difference between
shallow and deep). Tail heights were within ±1 SD of the population
mean. The control treatments were used as points of comparison for the
experimental treatment.
In treatments 1-3, we used the same aquarium setup as in experiment 1. We placed the test female in the center chamber and one male in each outer compartment. Females and males were habituated in their separate compartments for 15 min. We placed a 12.7-cm mesh net over the females during habituation so that females could potentially observe both males. Females were then observed for 15 min while we recorded the amount of time spent on each side of the center line. We also observed courtship by males, but we did not use this in our analyses.
We used a Friedman two-way analysis of variance by ranks test to compare the time that females associated with the deep-tailed male in treatment 3 (experimental condition) with the time that they spent on the same side of the aquarium (randomly determined) when they were tested in treatment 1 (shallow-tailed males) and when they were tested in treatment 2 (deep-tailed males). For example, if in treatment 3 a female was tested with a deep-tailed male on the left and a shallow-tailed male on the right, then we compared the time she spent on the left side of the chamber among the three treatments.
Experiment 2b: Sequential female choice
We collected unmated males and females of N. v. viridescens from a
drift fence around the same pond as in experiment 1 from 1-17 April 1996. The
research was performed in Virginia so that the newts could be maintained
outdoors in natural conditions, as newts remain in breeding condition for a
longer period of time when maintained in natural conditions than when
maintained in the laboratory (C. Gabor, personal observation; T. Halliday,
personal communication). We maintained the newts in outdoor aquaria (50.8
x 25.4 x 30.5 cm) on a natural photoperiod and water temperature
regime (0°-12°C). The tanks were filled with aged tap water and the
bottom was covered with small, tan gravel. A perch was placed in each tank to
provide shelter and a dry surface. We fed the newts live zooplankton,
Tubifex, and minced earthworms ad libitum. We housed males and
females in separate aquaria. Before the experiments, we recorded individual
spot patterns and measured SVL, tail length, and the tail height of each male
at the deepest section of the tail.
We examined female preference to mate in a sequence of encounters with
individual males, with male tail height either differing in each subsequent
encounter or remaining the same. The protocol for this experiment was
essentially the same as used for T. vulgaris vulgaris in Gabor and
Halliday (1997). A female was
paired in the initial pairing with a shallow-tailed male (6.5-7.5 mm in
treatments 1 and 2) or a deep-tailed male (9.0-10.5 mm in treatments 3 and 4;
Table 1). Females (n =
18-20 females for each treatment) and males were habituated for 15 min on
separate sides of an aquarium (as described in experiment 1) before mating was
permitted. We lifted the Plexiglas divider after the habituation period and
allowed courtship to occur. We permitted only one sperm transfer in the
initial pairing by briefly placing a 12.7-cm net between pairs just before a
male deposited a second spermatophore. After another 15-min habituation
period, each female was then re-paired in the second pairing with a male
bearing the opposite extreme in tail height (3 m difference) from the male
with which she first had mated in either treatment 1 or 3 (experimental). In
treatments 2 and 4 (controls), each female was re-paired in the second pairing
with a male of the same tail height with which she first had been paired
(Table 1). Females were
re-paired in the second pairing regardless of whether they had mated in the
initial pairing.
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If sperm transfer did not occur in the second pairing, we attempted to
determine whether the female was receptive by re-pairing her with an extremely
deep-tailed male (10-13 mm, 2 mm deeper than the previous male) in the
third pairing. However, so few matings occurred in the third pairing that
these results are not presented. Encounters were considered unsuccessful if
sperm transfer did not occur within 1 h. Male tail heights were within
±1 SD of the mean of the males collected.
We performed the experiments 15 March-4 May 1996, during the middle of the
breeding season, in a laboratory (24°-27°C) at James Madison
University, Virginia. All encounters within each treatment were randomized
based on the assigned number for each male and female. We recorded time to
sperm transfer for each pairing. We used a two-tailed Wilcoxon signed-ranks
test to compare latency to sperm transfer within a treatment between the
initial pairing and the second pairing. Comparisons within the initial
pairings and the second pairings for treatments 1 and 3 (experimental
treatments), and for all four treatments were analyzed using a 2 x 2
contingency test (Siegel and Castellan,
1988). We subsequently used Student's t tests to examine
the relationship between SVL and tail length of successfully and
unsuccessfully mated males to examine the influence of other traits on male
mating success. We used a two-tailed Wilcoxon-Mann-Whitney test to compare the
latency to sperm transfer between dyadic and triadic encounters to test the
hypothesis that sexual interference increases time invested in courtship. All
analyses were significant at = 0.05.
Experiment 3: Sperm precedence
We collected unmated males and females of N. v. viridescens from
the same ponds as in experiments 1 and 2 using the same complete drift fence
to ensure that females were seasonally unmated virgins, 26-27 March 1997. We
maintained newts in the same manner as in the procedures for experiment
2b.
We paired females (n = 22) with one male at a time using the same aquaria as in experiments 1 and 2. The order of testing females was randomly determined. Adult genotypes were determined before the matings and trios were selected such that each of two males would contribute unique alleles to offspring. Females were paired with one male and sperm transfer was observed. We used the individual spot patterns of the newts to identify each individual uniquely. We noted the chronological order of spermatophores deposited and which ones were successfully transferred to the females. After sperm transfer, the first-male was removed and a second male was immediately placed in the aquarium. Again we observed matings, but if no sperm transfer occurred within 3 h we left the pair together overnight and examined the aquarium within 15 h for spermatophores. If sperm caps were missing from spermatophores, we concluded that sperm were transferred during this time period. We measured the elapsed time between matings as the time between the first sperm transfer and the second sperm transfer. If the pair was left overnight, then we used the general term "overnight" to indicate an elapsed time greater than 3 h and less than 15 h between matings.
After females mated doubly (n = 8), they were individually housed in an egg-laying chamber (27 x 16 x 11.5 mm deep) with aged tap water, gravel, live zooplankton, and live plants. Females were also fed live worms (Tubifex) weekly. The chambers were placed at the base of a large open window with a natural photoperiod. We searched for eggs every 24 h, removed pieces of plants that contained eggs, and placed the pieces into separate containers uniquely identified for each female. As a control, to provide evidence that the experimental females were unmated before the experiment, we placed unmated females (n = 6) into chambers to determine whether the females would lay fertilized eggs. We collected hatched young from all females that laid eggs (n = 6 females) and at age 3 days, these were stored at -80°C.
We used genetic markers to determine paternity for offspring from females
that were mated to two males. We determined the genotypes of each adult and
offspring using standard horizontal starch-gel electrophoresis of proteins
(Selander et al., 1971).
Adults were genotyped from toes excised at capture and hatchlings were killed
and genotyped whole. Four polymorphic loci that resolved consistently were
used to exclude putative fathers from paternity: lactate dehydrogenase-2
(Ldh-2), malate dehydrogenase-1 and -2 (Mdh-1, Mdh-2), and
phosphoglucose isomerase (Pgi;
Table 2).
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We used a chi-square test to determine, in cases of double paternity, whether paternity sharing was equal among the two sires. We used a two-tailed Wilcoxon-Mann-Whitney test to determine whether SVL and tail height were significantly different between males that successfully sired most of the offspring and those that did not.
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RESULTS |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Male—male competition
Eleven matings occurred in 30 trio encounters. Deep-tailed males (n = 10) were significantly more successful at mating with females than were shallow-tailed males (n = 1; two-tailed binomial test, p =.012). Significantly more mating attempts (amplexus or hula displays) were performed by deep-tailed males (n = 19) than were performed by shallow-tailed males (n = 2) in 30 total encounters (two-tailed, binomial test, p <.01). In the 11 successful encounters (two male pairs were used twice with different females), we found that successful males did not have significantly greater tail lengths than unsuccessful males (Table 3). Successfully mated males had significantly greater SVLs than unsuccessful males (Table 3). Males from successful encounters were not in significantly better condition than unsuccessful males (Table 3).
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Sexual interference by the rival male occurred in 9 of the 11 successful encounters (82%), and 9 courting males used amplexus as opposed to hula in courtship. Four cases of sexual interference consisted of the rival male performing female mimicry, two of wrestling, two of displaying (hula), and one of approaching the courting pair. However, sexual interference did not significantly prevent successful sperm transfer by the courting male (nno transfer = 4, ntransfer = 7, two-tailed binomial test p =.27). In 1 case out of 11 (9%), a shallow-tailed male successfully performed female mimicry by moving between the larger tailed courting male (the latter having just dismounted the female after amplexus) and the female and depositing a spermatophore that was transferred successfully to the female. Subsequently the female picked up two more sperm caps from the deep-tailed male.
Simultaneous female preference
Females spent a similar amount of time with deep-tailed males (mean
± SE = 452.78 ± 23.08 s, n = 55) and shallow-tailed
males (447.22 ± 23.08 s) in experimental treatment 3. Females
demonstrated no significant difference in the time that they associated with
deep-tailed males in the experimental condition (treatment 3) as compared with
the time the same females spent with shallow-tailed males, treatment 1, and
deep-tailed males, treatment 2, on the same side of the aquarium (Friedman
test; n = 55, df = 2, 
2 = 2.76, p =.25;
Figure 1).
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Sequential female choice
Females demonstrated no significant preference to mate with deep or
shallow-tailed males whether the females had mated once or twice. First, we
examined female preference for deep-tailed or shallow-tailed males for all
four treatments, and there was no significant difference in frequency of
females mating or not mating with shallow-tailed males (treatments 1 and 2)
versus with deep-tailed males (treatments 3 and 4) in the initial pairing
(Table 4). There was also no
significant difference in frequency of females mating or not mating with
shallow-tailed males versus deep-tailed males for the second pairing
(treatments 2 and 3 versus treatments 1 and 4;
Table 4). Second, we examined
this preference for only the experimental treatments, and there was no
significant difference in the frequency of females mating or not mating with
shallow-tailed males (treatment 1) versus deep-tailed males (treatment 3) in
the initial pairing. There was also no significant difference between females
mating and not mating with shallow versus deep-tailed males in the second
pairing for the experimental treatments (treatment 3 versus treatment 1;
Table 4). Third, we examined
Halliday's (1983) prediction
that females would become more choosy in subsequent matings. We found that for
those females that mated twice, there was no significant difference in
frequency of mating or not mating initially with shallow-tailed males and
subsequently with deep-tailed males (treatment 1) versus with deep- to
shallow-tailed males (treatment 3; Tables
1 and
4).
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There was no significant difference between the latency to sperm transfer (from the start of the test) in the initial pairing and the second pairing when females mated twice in the experimental treatments (treatment 1; two-tailed, Wilcoxon signed ranks, n = 10, z = -.26, T+ = 30, p =.80: treatment 3; n = 9, z = -0.54, T+ = 18.0, p =.59; Figure 2). In the control treatments, there was also no significant difference between the latency to sperm transfer (from the start of the test) in the initial pairing and in the second pairing (treatment 2; n = 11, z = -1.92, T+ = 54.5, p =.06: treatment 4; n = 11, z = -1.21, T+ = 46.5, p =.23; Figure 2).
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There was no significant difference between the mean SVL of males that successfully (sperm transfer) mated with females and of unsuccessful (no sperm transfer) males in the initial pairing (Table 3) and in the second pairing (Table 3). There was also no significant difference between the tail lengths of males that successfully mated with females and of unsuccessful males in the initial pairing (Table 3) and in the second pairing (Table 3).
When the results of the trials from the male—male competition experiment (triadic) were compared with those from the sequential female choice experiment (dyadic), we found that there was a trend for the latency to sperm transfer to increase in the male—male competition trials, but this was not significant (triadic: n = 11; dyadic: n = 54; two-tailed Mann-Whitney test; U' = 395.5, z' = -1.72, p =.08; Figure 3).
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Sperm precedence
From the six doubly-mated females that laid eggs, five of the six clutches
displayed multiple paternity (see Figure
4 for paternity determinations for the offspring of a doubly-mated
female). We found statistically significant deviations from equal (shared)
paternity in four of six clutches (Table
5). Overnight matings appeared to produce a second-male mating
advantage, whereas those females that mated twice within a shorter time period
(<3 h) had clutches exhibiting a first-male advantage. Thus, the time
interval between mating and egg laying may have an effect on whether there is
first- or second-male advantage (Table
5). These results were not confounded by the possibility that
females were mated before being brought to the laboratory because none of the
unmated females laid eggs (n = 6) in the laboratory.
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There was no significant difference in the SVLs of males that successfully sired most of the offspring (mean ± SE, 47.3 ± 0.60 mm) as compared to those that did not (46.7 ± 0.40 mm; n = 6, two-tailed Mann Whitney test; U = 24.5, z = -1.07, p =.28). There was also no significant difference in the tail height of males that successfully sired most of the offspring (8.75 ± 0.44 mm) as compared to those that did not (9.42 ± 0.71 mm; n = 6, U = 22.5, z = -0.70, p =.46).
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DISCUSSION |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Male-male competition
Deep-tailed males were more successful at mating with females than shallower tailed males. These deep-tailed males had longer SVLs but were not necessarily in better body condition than shallow-tailed males. Tail length, the other sexually dimorphic trait, did not affect a male's success or lack of success in courtship encounters when another male was present.
Verrell (1986) found that
bigger males (greater SVL) were more successful at preventing sexual
interference during wrestling matches with an intruding rival male. We, too,
found that bigger males with deep tails were significantly more successful at
mating with females than were smaller males with shallow tails. Our results
support the inference that male-male competition is a major factor in the
mating system of red-spotted newts
(Massey, 1988; Verrell,
1982,
1983,
1986). Eighty-two percent of
the courtship attempts were affected by sexual interference at the
spermatophore deposition stage. Sexual interference did appear to influence
the form of courtship used by deep-tailed males, as courtship in most
encounters consisted of amplexus instead of the more vulnerable hula display.
This is consistent with prior results for red-spotted newts
(Verrell, 1983). However,
sexual interference did not prevent successful sperm transfer by the courting
male. The ineffectiveness of sexual interference may be an incidental factor
of testing in the laboratory. In ponds, females usually swim away when two
males are competing (Massey,
1988), but deep-tailed males may be more likely to follow and
capture these females (Able,
1999).
Verrell (1989) hypothesized
that competition between males for access to reproductive females provides a
strong selective pressure in the evolution of male behavior. In red-spotted
newts, male—male competition appears to be a strong selective force that
has influenced the evolution of amplexus in the courtship of this species, as
Verrell (1989) predicted. The
greater amount of amplexus used versus hula by males in this experiment lends
support to Verrell's (1989)
hypothesis that amplexus is a form of sexual defense that has coevolved with
sexual interference.
There was a trend for male—male competition and sexual interference
to increase the length of time invested in courtship for males of N.
viridescens that were in competitive encounters as compared to encounters
with only one male and female, but it was not significant. This scenario may
be similar to that found for the small-mouthed salamander, Ambystoma
texanum, which significantly increased the total duration of courtship as
more conspecific males were present
(McWilliams, 1992).
Female choice
Females demonstrated no significant preference to associate with deep- or
shallow-tailed males. They also did not demonstrate a significant preference
to mate with shallow- or deep-tailed males in the first or second matings.
When no male-male competition was involved, there was also no significant
difference in the tail height, SVL, or tail length of successful or
unsuccessful males. These results provide no evidence that sequential or
simultaneous comparison processes affect mate choice of female red-spotted
newts. These results are in contrast to other studies of salamanders where
female choice has been shown (i.e., Triturus vulgaris,
Gabor and Halliday, 1997;
Ambystoma tigrinum, Howard et
al., 1997; Desmognathus ocrophaeus;
Houck and Reagan, 1990).
Choosing mates from sequential visits with males may be more difficult than
simultaneous choice and requires the female's ability to remember previously
visited males to make comparisons (Bakker
and Milinski, 1991; Real,
1990). Although the mating system of smooth newts (T.
vulgaris) supported Halliday's
(1983) sequential choice
hypothesis (Gabor and Halliday,
1997), our results for red-spotted newts did not.
Our results, when combined with the male-male competition experiment,
indicate that tail height is an intrasexually selected characteristic. Within
the scope of our study, these results suggest that tail length is not intra-
or intersexually selected. Female choice does not appear to occur for these
traits in red-spotted newts. Our results are further supported by Able
(1999), who found that males of
N. viridescens with deeper tails were more successful at capturing
females in the pond and in laboratory aquariums. Our study also concurs with
that of Howard et al. (1997)
in which they found that male-male competition in Ambystoma t.
tigrinum favored larger male body length. However, Howard et al.
(1997) also found that female
mate choice selected for greater male tail length; in our study, this was not
evident for red-spotted newts.
Males of the European smooth newt (T. v. vulgaris) develop a
dorsal crest during the courtship season, and females prefer males with larger
crests (Gabor and Halliday,
1997; Hosie,
1992). Crest height has been demonstrated to be an indicator of a
male's foraging success (Green,
1991). Gabor and Halliday
(1997) found that in smooth
newts, where there is no male amplexus of females during courtship, female
choice of mates plays a major role during courtship
(Halliday, 1990). However,
red-spotted newts, in which males amplex females during part of courtship,
appear to have a sexual strategy that induces male-male competition for mates
with little evidence of female choice playing a major role. Furthermore, male
body condition does not seem to influence a male's mating success, so females
may not benefit from being choosy.
Sperm precedence
While multiple paternity occurs in red-spotted newts in the laboratory (and
it probably occurs in the field as well), the results from our sperm
precedence and our sequential mate choice experiment suggest that cryptic
female choice (Eberhard, 1996)
through the selective use of stored sperm does not occur. In clutches of
double paternity, significantly greater numbers of offspring were sired by the
first male in some clutches and by the second male in others. In two clutches,
no significant departure from shared paternity occurred. Because of the small
number of clutches examined, we cannot infer that either first or last males
will tend to have a fitness advantage. Our main result is that sperm
precedence likely does not have a pervasive and consistent effect on
fertilization success. However, to the extent that the range in latency of
sperm transfer in our experiments may be unnatural, sperm precedence effects
may be more consistent. For example, if double matings tend to occur within a
few hours, then our observations suggest that first-male advantage may occur
more frequently. Thus, females may show cryptic choice by controlling the
magnitude of time between successive matings.
Our sperm precedence results, when combined with the sequential
female-choice study, suggest that females are not controlling sperm usage and
that they are not controlling matings based on sperm precedence patterns. This
explains why our data did not support Halliday's
(1983) prediction for
sequential mate choice by females that capitalize on last-male advantage.
Smooth newts, on the other hand, may have last-male sperm precedence (A. Pecio
and J. Rafinski, unpublished data), hence their mating system supports
Halliday's hypothesis (Gabor and Halliday,
1997).
Another study has found evidence for multiple paternity in salamanders, but
little is known about possible manipulation of sperm by the female or about
the mode of sperm competition among competing sperm populations. Houck et al.
(1985), studying
Desmognathus ochrophaeus (Plethodontidae), used sires from two
different populations and females that may had been previously mated. They
found evidence of mixed male paternity based on allozyme analyses, but they
did not address the precise insemination process that was responsible for
these results. We were able to examine both the number and rate of
inseminations for female red-spotted newts, and it appears that second-male
paternity was more likely to occur when there was >3 h between matings. In
birds, last-male sperm precedence is achieved through passive stratification
of stored sperm with the first male's sperm overlaid by sperm from subsequent
matings. In birds if matings are <4 h apart, then sperm mixes; if matings
are >4 h apart, then the last male's sperm overlays the first
(Birkhead and Hunter, 1990),
thus giving the last male an advantage. We did not find this pattern in
red-spotted newts.
General inferences
In red-spotted newts, during the male-male competition experiment, males
with greater SVL and deep tails were more successful at mating with females
than those with smaller SVL and shallow tails. When males were not competing
with each other to mate with females, tail height, SVL, and tail length did
not appear to influence the success of mating with females. Moreover, there
was no evidence for female choice in red-spotted newts (simultaneous female
preference experiment). However, female choice may operate only when males are
in overt contact, but this could not be differentiated from our data. Thus,
male tail height in red-spotted newts appears to be an intrasexually selected
secondary sexual characteristic. Although tail length is also a sexually
dimorphic trait, it does not appear to be under either intra- or intersexual
selection. Snout-vent length, on the other hand, is not a sexually dimorphic
trait.
Within the power of our experiments, male-male competition appears to be
the dominant force in the red-spotted newt's mating system. Although there was
no evidence of female choice in our experiments, this cannot be ruled out
entirely. For example, females may be controlling the timing between matings
to control sperm precedence. Moreover, some other component of female choice,
beyond the parameters of this study, may have selected for secondary sexual
characters in males. If female mate choice occurs, it may be for some other
characteristic than that measured here. For example, spermatophore deposition
rate has been demonstrated as being important for female choice in smooth
newts (Halliday and Houston,
1978). This, however, could not be measured in our experiment
because we were also attempting to examine sperm precedence using the
offspring from the sequential female choice experiment, so we had to limit the
number of spermatophores that were deposited by males. If females are unable
to judge differences among males, then they may be gaining indirect benefits
from polyandry. For example, mating with multiple males may be a form of
genetic bet-hedging. Watson
(1991) suggested that in
sierra dome spiders, Linyphia litigiosa, polyandry enables a female
to distribute fertilizations among several mates, thus reducing the impact of
occasional flawed evaluations of male quality. Thus, in newts, mating multiple
times may reduce a female's risk of all her offspring being of low genetic
quality because she was unable to differentiate between males of differing
quality.
Two questions arise from the lack of evidence for female choice in our
system. First, does amplexus impede female mate choice in newts? Second, how
unusual is it for amplexus to impede female choice? In frogs, where amplexus
also occurs, female choice does not appear to be limited. For example,
Andersson (1994) reviewed 25
studies of frogs that examined sexual selection. In 10 studies, there was
evidence of female choice, and in 12 studies, there was evidence of both
female choice and male-male competition. In three other studies, there was
only evidence of some form of male-male competition but no female choice.
However, in two of these studies, the evidence was only observational and not
experimental. Thus, amplexus in frogs generally does not limit female choice.
However, the mating system of newts is different because there is internal
fertilization and multiple mating, unlike most species of frogs. The lack of
female choice in newts may be balanced by the potential for females to mate
multiple times as a form of genetic bet-hedging.
One way to resolve the two questions above is to trace the phylogenetic
history of courtship involving female capture (amplexus) within the newt
family Salamandridae. This, however, is difficult because courtship behavior
patterns of some important genera are lacking
(Titus and Larson, 1995).
Titus and Larson (1995) were
unable to determine whether the absence of amplexus is the ancestral state. We
propose that if amplexus were the ancestral state, then the absence of
amplexus in T. v. vulgaris would be derived and a consequence of
female choice precluding male capture of females. Alternatively, if the
ancestral state were the lack of amplexus, then the presence of amplexus in
N. v. viridescens is derived and is a consequence of female capture
by males (and male-male competition) impeding female choice. Alternatively,
the evolution of amplexus to nonamplexus and of nonamplexus to amplexus may
have evolved independently in each scenario
(Halliday, 1990). We concur
with Titus and Larson's (1995)
suggestion that further examination is needed of the adaptive status of female
capture in the selective regimes of newts.
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ACKNOWLEDGEMENTS |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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We thank L. Higgins, A. Mathis, G. Rosenthal, M. J. Ryan, P. Verrell, and two anonymous reviewers for helpful comments on the manuscript. We are grateful to R. Harris for use of the newts from his ponds and for laboratory space, S. Babcock for housing, C. Carreno, H. Mansfield, and C. Wells for aid in collecting newts, and J. Bosch for statistical help. C.R.G. was partially supported by funds from the Graduate Student Organization at The University of Southwestern Louisiana, by a grant from Sigma Xi, by Louisiana Board of Regents Doctoral Fellowship grant LEQSF (1993-97)-GF-20 through R.G.J., and by National Science Foundation Post-doctoral Research Fellowship grant DBI-9750278. R.G.J. was supported by National Science Foundation grant DEB-9314081 and National Geographic Society grants 5108-93 and 5721-96. J.D.K. was supported by a postdoctoral Research Fellowship in the Molecular Biology Program at The University of Missouri, and by National Science Foundation grant IBN-9278495.
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FOOTNOTES |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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J. D. Krenz is now at the Department of Biological Sciences, Minnesota State University, Mankato, MN 56002-8400, USA.
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