Behav Ecol Sociobiol (2006) 59: 796?804
DOI 10.1007/s00265-005-0125-5
ORIGINAL ARTICLE
Kathrin P. Lampert . Ximena E. Bernal .
A. Stanley Rand . Ulrich G. Mueller .
Michael J. Ryan
No evidence for female mate choice based on genetic similarity
in the t?ngara frog Physalaemus pustulosus
Received: 15 April 2005 / Revised: 7 November 2005 / Accepted: 7 November 2005 / Published online: 17 December 2005
# Springer-Verlag 2005
Abstract In most sexually reproducing animals, the
behavior of one or both sexes during courtship critically
influences the success at mating of the opposite sex. This
behavior is often interpreted as ?mate choice,? and there is
great interest in why such choices are exercised. The ex-
planation for the evolution of mate choice that has received
the most attention and generated the most controversy is
based on assumed genetic effects. In this study, we in-
vestigated whether female t?ngara frogs, which choose
mates based on acoustic cues, have a preference for
genetically less related males. Specifically, we determine if
there is disassortive mating based on microsatellite mark-
ers, if there is information in the advertisement call that
could be used to assess genetic similarity, and if females
exhibit acoustic-based mating preferences that would pro-
mote choice for genetic diversity. Using seven micro-
satellite markers, we found no correlation of male call
similarity and male genetic relatedness. Female choice ex-
periments showed no female preference for calls of less
related males, and there was no evidence for inbreeding
avoidance in the field. Our results do not support the hy-
pothesis of mate choice based on information about genetic
relatedness conveyed by acoustic signals in t?ngara frogs.
Keywords Animal communication . Mate choice .
Relatedness . Microsatellite marker . Sexual selection
Introduction
In most sexually reproducing animals, the behavior of one
or both sexes during courtship critically influences the
success at mating of the opposite sex. This behavior is
often interpreted as a ?mate choice,? and there is great
interest in why such choices are exercised (Kirkpatrick and
Ryan 1991; Andersson 1994; Ryan 1997; Kokko et al.
2003). The competing, often not mutually exclusive, hy-
potheses can be classified in several categories. In many
cases, females gain direct benefits in fecundity due to the
chosen male providing superior paternal care or having
access to superior resources (Andersson 1994; Ryan 1997;
Kokko et al. 2003). A second explanation for female choice
is that current mate-choice patterns are incidental con-
sequences of behavior that evolved in a different context.
Female preferences for conspecific vs heterospecific males,
for example, can incidentally influence their preferences
among conspecific males (Gerhardt 1994; Pfennig 1998;
Hankison and Morris 2003). Similarly, sensory biases that
exist for reasons outside of the context of conspecific mate
choice or mating behavior in general can influence female
preferences for novel cues and allow males to evolve traits
that exploit these preexisting biases (Ryan 1990, 1998;
Endler 1992; Shaw 1995; Endler and Basolo 1998). A third
explanation for female choice evolution is the hypothesis
of Fisher (1930) of runaway sexual selection. He suggested
that male traits and female preferences become genetically
correlated, and that female preferences evolve not because
they provide any fecundity advantage to the females, but as
an incidental effect of their correlation to the male trait (see
also Lande 1981; Kirkpatrick 1982).
Communicated by J. Christensen-Dalsgaard
K. P. Lampert (*) . X. E. Bernal .
U. G. Mueller . M. J. Ryan
Section of Integrative Biology C0930,
University of Texas,
Austin, TX 78712, USA
e-mail: kathrin.lampert@biozentrum.uni-wuerzburg.de
Tel.: +49-931-8884104
Fax: +49-931-8884150
Present Address:
K. P. Lampert
Department of Physiological Chemistry I,
University of Wuerzburg,
Biozentrum, Am Hubland,
97074 Wuerzburg, Germany
A. S. Rand . U. G. Mueller . M. J. Ryan
Smithsonian Tropical Research Institute,
Apd. 2012, Balboa, Panama
The explanation for the evolution of female mate choice
that has received greatest attention and generated the most
controversy is based on assumed heritable survival effects
in the offspring. Originating with the handicap principle of
Zahavi (1975) and culminating in a series of revisions and
emendations (e.g., Grafen 1990; Pomiankowski 1988;
Zahavi and Zahavi 1997; Hamilton and Zuk 1982; re-
viewed in Maynard Smith and Harper 2003), this hypoth-
esis posits that female choice evolved under selection to
choose males with superior genotypes for survivorship.
This is a difficult hypothesis to support unequivocally
because it requires the demonstration of a paternal effect on
juvenile survivorship. Nevertheless, some studies have
provided strong support when measuring various compo-
nents of survivorship (Petrie 1994; Raberg et al. 2003;
Welch et al. 1998). However, there is one form of female
choice based on genetic quality that has been well dem-
onstrated, and that is mate choice that results in species
recognition.
It is well known that interspecific mating often results in
decreased fecundity or in offspring with decreased viability
due to the genomes of the two hybridizing species not being
compatible (Dobzhansky 1975). However, mate choice for
genetic compatibility is not restricted to contrasts between
species. Since the demonstration of Yamazaki et al. (1976)
that female mate choice in mice is influenced by variation in
the major histocompatibility complex (MHC), a series of
studies have extended these inquiries to a diverse number of
taxa such as other rodents (Penn and Potts 1999) and humans
(Wedekind et al. 1995; Wedekind and Furi 1997) as well as
nonmammalian taxa such as fishes (Landry et al. 2001;
Milinski 2003) and birds (Zelano and Edwards 2002;
reviewed in Bernatchez and Landry 2003). In addition,
some other studies have documented mate choice for
genetically less similar individuals based on factors other
than MHC-related odor cues (e.g., Waldman et al. 1992;
Waldman and Tocher 1998) or odor cues related to the t
complex in rodents (Lenington et al. 1994).
In this study, we ask if female mate preferences in
t?ngara frogs, Physalaemus pustulosus, which are based
primarily on acoustic cues, result in choice of males that are
genetically dissimilar. Specifically, we investigated micro-
satellite markers to determine if there is disassortative
mating, if there is information in the advertisement call that
could be used to assess genetic similarity, and if females
exhibit acoustic-based mating preferences that could pro-
mote choice for genetic complementarity. If this hypothesis
can be substantiated for P. pustulosus, it would parallel a
similar phenomenon described for American toads, Bufo
americanus, by Waldman et al. (1992) and Waldman and
Tocher (1998), and would encourage the study of choice
for genetic complementarity in other systems that appear
not to be mediated by MHC. If the hypothesis is not
supported, it might encourage comparative studies to
understand the evolutionary histories that do and do not
produce choice for genetic complementarity in sexual
communication systems.
Materials and methods
T?ngara frogs (P. pustulosus) are abundant and widespread
in Middle America. They have a lek mating system in
which males aggregate in temporary ponds and advertise
for mates by calling. The male mating call consists of two
components. The initial component is a frequency sweep
(whine) that can be produced alone and is necessary and
sufficient for species recognition (simple call). The whine
can be followed by one or more much shorter components
(chucks), and together, the whine and chuck constitute the
complex call (Ryan 1985).
Females visit male choruses to select a mate. All recordings
of male advertisement calls in this study were made at such
choruses, and females used in phonotaxis experiments were
also collected at these sites, usually after they had chosen a
mate and were in amplexus. All frogs used in this study were
captured in a 10-km range around Gamboa, Panama (09?07?
0?N, 79?41?53?W; Lampert et al. 2003). They were released
immediately after being marked individually (toe clips) at the
same place where they had been found.
Relatedness estimates
All toe clips were stored in a 20% EDTA/sarcosyl buffer
for microsatellite marker analysis in Austin, TX. DNA
extractions were performed with a DNeasy extraction tis-
sue kit (Qiagen). Seven highly polymorphic microsatellite
loci (CA 120, CA 298, A #3.11, A #19.11, C #30.11, ATG
159, and ATG 263; Pr?hl et al. 2002) were used to indi-
vidually genotype all animals. We used fluorescent poly-
merase chain reaction (PCR) and an ABI Prism 3100
capillary sequencer (Applied Biosystems) as described in
Lampert et al. (2003) to estimate allele sizes. Individual
pairwise relatedness was estimated using the program
Relatedness 5.0.8 (Goodnight and Queller 1995). This
measure estimates pairwise relatedness based on allele
frequencies (Queller and Goodnight 1989). Estimated re-
latedness values vary between ?1 and +1. Full siblings are
expected to have a mean relatedness of 0.5; half siblings are,
on average, 0.25, and unrelated individuals are 0.0 related
(Blouin et al. 1995). We used the simulation function in the
program Kinship 1.3.1 (Goodnight and Queller 1996) to
estimate the expected relatedness of 1,000 full siblings,
1,000 half-siblings, and 1,000 unrelated individuals to
evaluate the reliability of our microsatellite loci and to create
reference points on the Queller and Goodnight R scale.
Mating call recording and analyses
In July 1996, we recorded 48 males from one population in
Gamboa, Panama. Calls were recorded with a Marantz
PMD 420 recorder and a Sennheiser ME 80 microphone
(details on recording and analysis in Ryan and Rand 2003).
At least five complex calls (whine plus one chuck) per male
797
were recorded. The calls were digitized at a sampling rate
of 20 kHz and analyzed using the program Signal. Fifteen
call parameters were measured (abbreviations): duration of
the entire complex call (calldur), dominant frequency of the
entire call (callhz), duration of the chuck (ckdur), dominant
frequency of the chuck (ckdomhz), duration of the whine
(whdur), initial frequency of the fundamental frequency of
the whine (inithz), final frequency of the fundamental
frequency of the whine (endhz), time to mid-frequency of
the fundamental frequency of the whine (hfhz), falltime of
the whine (fall), time to half amplitude of the fall from the
call?s end (hffall), rise time of the whine (rise), time to half
amplitude of the rise (hfrise), maximum frequency of
fundamental frequency of the call (maxhz), time to max-
imum frequency of the whine (timmxhz), and peak
amplitude of chuck divided by peak amplitude of the
whine (relamp). For the analysis of call similarities, we
arbitrarily selected one call per male (the third call in the
recorded sequence) of all the recorded calls. We trans-
formed call characters into Z scores, and we calculated the
Fig. 1 Histograms of pairwise
relatedness values (x-axis, relat-
edness value following Queller
and Goodnight 1989) for
a Gamboa males to each other
(malemale), b females vs males
in the female choice tests (fem-
males), c couples found in the
field (couples), d randomly
generated couples (random)
Table 1 Descriptive statistics of pairwise relatedness values (Queller and Goodnight 1989) for males vs males in the call?genetic
relatedness comparison, females from the female choice tests toward males from the call?genetic relatedness comparison, field couples
(couples), randomly generated pairs (random), and females vs. females in the female choice tests
Male/male Female/male Couples Random Female/female
Number of cases 1,128 960 153 153 190
Minimum ?0.378 ?0.402 ?0.346 ?0.499 ?0.347
Maximum 0.630 0.563 0.460 0.511 0.515
Median ?0.002 ?0.023 0.036 ?0.021 ?0.005
Mean 0.015 ?0.015 0.049 0.000 ?0.003
Standard deviation 0.172 0.166 0.185 0.183 0.180
798
Euclidean distances as a measure of overall call similarity
and similarity of each call character among males. As the
data matched the assumptions of Mantel tests (e.g., linear
correlation between variables, same scales within matrices,
and independent observation pairs) call similarities were
compared to the genetic similarity with Mantel tests
(Mantel Version 2.0; Liedloff 1999). In addition, we used
Ritland?s Marq program (h2 only model; Lynch and Ritland
(1999) measure of relatedness) to estimate heritability of
mating call characteristics in this field population (Ritland
and Ritland 1996; Mousseau et al. 1998). To enable a direct
comparison between this study and the results of the
studies of Waldman et al. (1992), we additionally cal-
culated a simple allele-sharing index (Sxy) between in-
dividual males (Sxy = number of shared alleles divided by
the average number of alleles scored).
Female phonotaxis
Phonotaxis experiments were conducted in an acoustic
chamber (1.8?2.7 m) with two small speakers (Cambridge
Soundworks) placed in the center of the shorter walls op-
posite one another along the longer axis of the chamber
(distance between the speakers about 2.5 m). The females
were released at the center of the chamber. Each call used in
the tests consisted of a whine plus one chuck. The calls
were chosen from a set of 300 natural calls from this
population that were recorded and analyzed for a previous
study (Ryan and Rand 2003). Calls were adjusted to a
maximum whine amplitude of 82 dB sound pressure level
(SPL) (re. 20 ?Pa) at the release point of the females, which
mimics the male?s call at a distance of approximately 1.0 m.
The chuck amplitude was maintained at the original
relative amplitude to the whine. Calls were broadcasted
alternately from the two speakers on either side of the test
arena. The females were observed remotely using a video
camera and infrared light. A choice was considered valid if
the female approached a speaker within 10 cm without
following the chamber walls (details on the testing rules in
Ryan and Rand 2003).
Twenty-five mated females were collected in August
2003 from the same location where we had recorded the
males? calls. Each female was presented with a different
pair of calls that were randomly chosen from the 48
males used in the previous call-relatedness study (test 1).
In addition, each female was presented with one of four
male pairs that were found to be rather unrelated to
enhance the relatedness bias between the two males and
the female (test 2). To control for the females? general
mating motivation, the females were presented with a
choice between a synthetic whine and whine?chuck
before and after the relatedness tests. Only if she made a
choice in both whine/whine?chuck tests were the choices
that she made in the intervening relatedness tests counted
valid. Females were toe-clipped after the behavioral tests
to prevent retesting and released within the next 12 h. All
frogs were handled and toe-clipped in accordance with
the guide for use of live amphibians and reptiles in field
research by the American Society of Ichthyologists and Her-
petologists (ASIH; http://www.asih.org/pubs/herpcoll.html).
Table 2 Mantel test results for the correlation of male call traits and
male individual pairwise relatedness
g Z r p
Overall 0.380 3112.664 0.024 0.36
Calldur 0.0162 2601.006 0.001 0.49
Callhz ?0.357 2636.119 0.050 0.26
Ckdur ?0.102 2475.117 ?0.006 0.41
Ckdomhz 1.143 2309.975 0.065 0.17
Whdur 0.526 2621.100 0.282 0.26
Inithz ?0.056 2495.560 ?0.003 0.43
Endhz ?0.916 32434.64 ?0.029 0.13
Hfhz 0.061 2396.638 0.004 0.45
Fall ?0.412 2614.435 ?0.020 0.35
Hffall 2.28 2689.023 0.110 0.01
Rise 0.244 2300.576 0.015 0.45
Hfrise 0.645 2005.490 0.045 0.28
Maxhz ?0.834 2579.371 0.045 0.24
Timmxhz 1.202 2318.979 0.053 0.08
Relamp ?1.384 2502.253 ?0.081 0.13
p values were derived from 1,000 randomizations. Significant
correlations previous to Bonferroni correction are given in bold,
italic typeface. After Bonferroni correction, none of the correlations
was significant (abbreviations of call parameters are given in the
text)
Table 3 Mantel test results for a correlation of genetic relatedness
and the hffall in all other recorded calls (1, 2, 4, and 5)
Hffall for all calls
Call number g Z r p
1 0.843 1,933.43 0.060 0.214
2 1.931 2,634.62 0.108 0.018
3 2.280 2,689.02 0.110 0.010
4 2.199 2,676.34 0.121 0.010
5 0.642 2,662.44 0.029 0.230
No results were significant after Bonferroni correction (p<0.01)
0
2
4
6
8
10
12
test 1 test 2
nu
m
be
r o
f f
em
al
es
closer related
less related
Fig. 2 Results of the female choice tests: test 1, random pairs of
males; test 2, one out of four chosen to be the least related male pairs
799
The females? relatedness toward the males was estimated
only after the behavior tests were done.
Relatedness of couples in the field
To estimate the relatedness of mated frogs in the field, we
collected amplectant pairs from 15 different breeding sites
around Gamboa. All frogs were collected between June
and August 2002 between 2000 and 0100 hours.
Whenever possible, we collected all other t?ngara frogs
present at the ponds at that time to estimate the full range
of relatedness and the number of males available to the
females.
Results
Male relatedness and heritability
of male call characteristics
Males captured and recorded in 1996 in Gamboa were not
closely related (Fig. 1a, Table 1). The average relatedness of
males recorded and captured in Gamboa in 1996 was 0.015,
qualifying them as ?unrelated.? The kinship simulations of
relatedness of half-siblings, full siblings, and unrelated in-
dividuals based on the allele frequencies found in the popu-
lation confirmed that the males? relatedness was within the
range of unrelated individuals [given are mean?standard de-
viation full sibling, R=0.501?0.174; half-siblings, R=0.251?
0.180; and unrelated, R=0.011?0.164].
Correlations between the matrices generated for male
genetic relatedness (Relatedness 5.0.8) and male call sim-
ilarity revealed no significant relationship between
individual relatedness and single call parameters (Bon-
ferroni correction for multiple testing) (Tables 2 and 3).
The heritability estimates by Ritland and Ritland (1996)
showed no significant heritability for any call character
(data not shown). The pairwise relatedness between males
calculated with an unweighted allele-sharing index, Sxy,
varied between 0 and 0.9. Genetic distances obtained with
the Sxy measure were highly correlated with the genetic
distance obtained with the Queller and Goodnight method
(r=0.766, p<0.001). Consequently, none of the tested call
parameters was significantly correlated with the genetic
relatedness calculated as Sxy (all r values <0.08, all p
values >0.07).
Female choice in phonotaxis
The females in the choice tests were not closely related to
each other or to the males involved (Table 1, Fig. 1b). The
majority of all females chose in all four behavioral tests
(80%). The choosing females showed no significant pref-
erence for the mating calls of the less related males (test 1:
0
1
2
3
4
5
A vs B C vs D A vs E F vs G
pairs of males
nu
m
be
r o
f f
em
al
es
Fig. 3 Choices by females for
the calls of individuals males of
the four pairs of males used in
test 2. Letters represent the
different males (A?G). Five
females were tested with each
pair of males
-0.5
-0.3
-0.1
0.1
0.3
0.5
0.7
0.9
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75
re
la
te
dn
es
s
Fig. 4 Mate choices of 76
females (x-axis). Bars indicate
the range of relatedness (y-axis)
of available partners during that
night. Squares indicate the mean
relatedness of the males towards
this particular female. Crosses
indicate the relatedness of the
actually chosen male
800
X2=0.100; df=1; p=0.75; test 2: X2=0.000; df=1; p=1.0). In
the first experiment (mean male relatedness 0.0147?
0.186), where the females were presented with randomly
chosen pairs of male calls, 55% (lower 95% confidence
interval limit 31.5%; upper 95% confidence interval limit
76.9%) of the females preferred the more closely related
male?s call (Fig. 2). In the second experiment, in which
females were tested with calls of males (A?G) that were
preselected to enhance differences in relatedness (mean
male relatedness ?0.2365?0.035), 50% (lower 95% con-
fidence interval limit 27.2%; upper 95% confidence
interval limit 72.8%) of the females chose the call of the
more related males (Fig. 2). In addition, in test 2, there was
no single unanimously preferred call in a pair, as would be
expected if females had a preference for a specific genotype
(Fig. 3). Unfortunately, sample size was too low to test for
any statistically significant preference.
Relatedness of couples in the field
A total number of 153 couples were found at the 15 different
breeding sites. The average pairwise relatedness of couples
varied between ?0.346 and +0.460, with a mean value of
0.049?0.185. Randomly generated couples had an average
relatedness of 0.000 (Table 1). T?ngara frog couples did not
show a significantly different distribution of individual
relatedness than randomly generated pairs (Kolmogorov
Smirnov test, two-sided p=0.16; Fig. 1c,d). Out of the 76
matings investigated in more detail (all other males present at
the pond during that night were toe-clipped as well) 33
females mated with males that were more closely related to
them than the average of all males available (Fig. 4).
Statistically, there was no difference between the chosen
mates relatedness toward the female and the rest of the males
available [chosen partner relatedness 0.034?0.175; rest of
males 0.023?0.070; t test: T=0.511, df=150, two-sided
p=0.610]. A greater number of males available during the
night did not seem to alter the females? mating decisions
toward less related males (Pearson correlation of number of
males available and relatedness of males: r=0.02, N=76,
p=0.887). Unmated and mated males did not differ in their
level of overall heterozygosity [mean heterozygosity for seven
loci (%): unmated Ho=0.722?0.108; mated Ho=0.718?
0.113].
Discussion
Our study finds no evidence that mate choice based on
genetic relatedness, as estimated by microsatellite varia-
tion, influences mating patterns in the population of
t?ngara frogs we studied. There is no pattern of negative
assortative mating by relatedness. It is possible, however,
that females have preferences for less related individuals,
but that these preferences are compromised in the wild; that
is, mating success might not be indicative of mate choice.
Contrary to this expectation, we find no evidence for any
information in the advertisement call that could be used to
assess relatedness; in t?ngara frogs, as in most frogs, the
call is the predominant male advertisement signal. Of
course, it is possible that such information exists and is
merely not captured by the signal parameters that we
analyzed. Contrary to this expectation, females show no
patterns of phonotactic preferences between calls of males
with different relatedness to themselves. Thus, in three
separate data sets, mating success, mating call variation,
and phonotaxis preferences, we find no hint that female
mate preferences and male mating success are influenced in
any way by considerations of genetic relatedness.
Comparison to other studies
The aim of our study was to primarily understand factors
that influence the evolution of sexual communication
systems. More specifically, we were motivated by a series
of studies by Waldman et al. (1992) and Waldman and
Tocher (1998) presenting strong evidence that female
toads, B. americanus, preferentially choose to mate with
males that are genetically less related to them. Waldman et
al. showed that B. americanus exhibited negative
assortative mating by mitochondrial haplotype in a series
of ponds. Further, they showed a significant positive
correlation between similarity in several individual
parameters of the mating call and the genetic relatedness
of males as estimated by shared bands in DNA
fingerprints. Finally, Waldman et al. tested phonotaxis
preferences of females for pairs of mating calls and showed
very strong preferences for calls of males that were
genetically less similar to the test females. The main
components of our study were purposefully designed to
parallel those of Waldman et al.
It is instructive to consider potential differences between
the two studies. Some differences are methodological. Our
sample sizes were larger in measures of mating success and
call-relatedness correlations. Even with a sample size three
times larger than theirs, we could not detect any correlation
between male genetic similarity and male call character
similarity (Waldman et al. found significant correlations
within populations with only 15 or 16 individuals per pop-
ulation.). Our approach to measuring mating success and
potential call-related correlations were similar with a few
exceptions. Waldman et al. used mitochondrial haplotypes
to assess relatedness among mating individuals and shared
DNA fingerprint bands as a genetic marker in testing the
hypothesis that calls indicated relatedness. In both com-
ponents of our studies, we used seven highly polymorphic
microsatellite loci previously developed for studies of
t?ngara frogs? by Pr?hl et al. (2002). Microsatellite analyses
utilize a different approach to estimating genetic diversity
than mtDNA sequencing. Since microsatellites are bipa-
rentally inherited codominant markers, they resolve
relatedness at a much finer scale than maternally inherited
801
mtDNA. Using mtDNA, Waldman et al. were able to show
high levels of population differentiation at a small geo-
graphical scale (1?2 km). They also showed significantly
fewer matings than expected between individuals sharing
the same mtDNA haplotype (maternal siblings). Although
we found statistically significant levels of genetic differ-
entiation between breeding sites at a scale of 3?4 km and a
slight male bias in dispersal in a previous study, Fst values
[fixation index (subpopulation compared to total)] still
suggested substantial levels of gene flow between popula-
tions of t?ngara frogs (Lampert et al. 2003). In this study,
microsatellite estimates did not reveal any evidence for
inbreeding avoidance in field populations of t?ngara frogs.
Thus, we are unable to detect genetically biased patterns of
mate choice despite larger sample sizes and more sensitive
tools than those used by Waldman et al.
While in the observation of mating success, our
estimated relatedness values were much finer-scaled than
those utilized by Waldman et al., his estimates of relat-
edness in male call character heritability and female choice
tests based on multilocus DNA fingerprints should allow a
higher resolution of relatedness than our seven micro-
satellite loci. Multilocus fingerprints, however, are prone to
some error in relatedness tests due to the uncertainty of
bands of the same size actually representing alleles at the
same locus. To facilitate a direct comparison of the results
of Waldman et al. and our microsatellite data, we estimated
unweighted allele-sharing distances (Sxy). As expected,
the multilocus DNA fingerprints of Waldman et al. re-
solved pairwise relatedness at a finer scale than the
microsatellites. The range of individual relatedness re-
vealed by the microsatellites, however, was larger (0?0.8)
than the range of individual relatedness revealed by
Waldman et al. (0?0.5). Although our study included
more individuals (48 for one site compared to 15 per site)
and covered a wider range of individual relatedness than
those covered by Waldman et al., we did not find a
significant correlation between call similarity and genetic
similarity in t?ngara frogs. This leads to the conclusion that
in B. americanus, the correlation of call similarity and
genetic relatedness must be much stronger than in P.
pustulosus, and that call trait inheritance in toads might be
based on a rather simple genetic mechanism.
Our phonotaxis studies differed in some ways from those
of Waldman et al. We randomly selected pairs of calls from
the population, and each of a set of the 20 responding
females was tested with a single pair of calls. We feel this is
a better assessment of how female choice could be in-
fluenced by genetic relatedness in nature, but it is not the
strongest test to reveal any potential for such an effect.
Therefore, we also tested the same set of females with a
pair of calls from males that showed a relatively high
difference in genetic relatedness. This last experiment
parallels the approach taken by Waldman et al. In neither
case, however, were female t?ngara frogs? preferences for
calls influenced by genetic relatedness.
It is possible that the different results of these two studies
could be due to critical differences in the study species?
biology. There are, for example, important differences in
mating systems among these species. Toads have a rel-
atively explosive breeding system (Wells 1977), in which
the opportunity for female choice should be less than in the
longer-breeding t?ngara frog. Such a difference, however,
would predict a greater, not a lesser, opportunity for female
choice to assess genetic differences among males. Another
possibility is that female preferences are under selection by
other forces that constrain their opportunity for genetic-
based mate choice. For example, female t?ngara frogs
choose larger males who tend to fertilize relatively more
eggs, and females rely, at least in part, on the spectral
characteristics of the male?s chuck to assess male size
(Ryan 1980, 1983, 1985). Such an immediate benefit to
mate choice could be contrary to and override benefits from
genetic-based mate choice (Kirkpatrick and Barton 1997).
Another possibility is that the need to discriminate against
heterospecifics compromises the use of cues that might be
useful in mate choice within the species, as has been shown
in spadefoot toads by Pfennig (1998). We have no strong
evidence that would support or reject such a view.
A final set of explanations for the differences between
these studies could derive from differences in population
structure. Depending on patterns of philopatry and sur-
vivorship, the distribution of relatedness among individuals
can differ drastically and significantly constrain or promote
the opportunity for genetic-based mate choice. The Amer-
ican toad is a rather long-lived species with high levels of
philopatry in both sexes (Wells 1977), which should pro-
mote a higher probability of close kin encounters. T?ngara
frogs seem to suffer high mortality rates during all life
stages and are likely to only survive one breeding season.
They also breed in temporary ponds and are known to be
rather flexible in the choice of their breeding habitat (Marsh
et al. 2000). In a former study (Lampert et al. 2003), we
found high levels of gene flow between sites and a male
bias in dispersal. All these factors might make breeding-
pond encounters with close relatives rather unlikely. Low
probabilities of encounters with close kin could explain
why females did not evolve a genetically based mechanism
for adult kin recognition. Unfortunately, we are not able to
estimate the costs of inbreeding in these frogs, nor is it
known in the American toad.
There is one additional possibility that could explain the
difference between these species in the use of genetics in
mate choice. American toads exhibit sibling preferences in
the tadpole stage (e.g., Waldman and Adler 1979). It is
possible that bias against closely related individuals in the
adult stage is necessary to counter kin preferences that
might be adaptive earlier in life but might be maladaptive
later in life. Despite a wealth of information on the natural
history of t?ngara frogs, their propensity for sibling
preferences has not been explored.
Genetic-based mate choice and olfactory cues
As mentioned previously, preferences for conspecific vs
heterospecific mates are a phenomenon thought to result
from strong selection on females to avoid mates with
802
genomes that are not complementary (Dobzhansky 1975;
Coyne and Orr 1998; Mays and Hill 2004). When mate
choice is restricted to comparisons among conspecifics,
however, such evidence for genetic-based mate choice is
less common. It seems to be most widespread in mate
choice influenced by variation in MHC and associated odor
cues. Differences in the antigen-binding site of class I
MHC molecules result in distinctive odors (Yamazaki et al.
1991; Penn 2002), but as of now, we have no evidence that
variation in vocalizations correlate with variation in MHC
(Zelano and Edwards 2002). At this point, it is difficult to
posit a mechanism for MHC heterozygosity recognition
mediated by mating calls. We cannot, however, exclude the
possibility that female t?ngara frogs attend to male MHC
type. Although it might be unlikely that MHC variation is
encoded in the male mating calls, females could detect
olfactory cues when coming into close proximity of their
potential mate (Waldman and Bishop 2004). Female
t?ngara frogs, for example, approach several males closely
before choosing a mate (Ryan 1985). It might be possible
that t?ngara frogs choose mates based on MHC compat-
ibility rather than based on overall genetic relatedness
(Landry et al. 2001).
Acknowledgements We wish to thank Autoridad National del
Ambiente of the Republic of Panama and the Smithsonian Tropical
Research Institute (STRI) for research and export permits. STRI
provided invaluable logistic support. We are especially indebted to
H. Pr?hl, who developed the microsatellite primers used in this study.
The critical comments of T. Juenger and B. Waldman greatly
improved the manuscript. This work was supported by the German
Science Foundation (DFG) LA 1382/2-1 (KPL), by National Science
Foundation (NSF) grants IBN-0078184 to M.J.R., D. Cannatella, and
W. Wilczynski, and IBN-9816564 to M.J.R., and by an NSF-
CAREER award DEB-9983879 to U.G.M. The research presented
complies with the current laws of the countries in which it was
performed.
References
Andersson M (1994) Sexual selection. Princeton University Press,
Princeton
Bernatchez L, Landry C (2003) MHC studies in nonmodel
vertebrates: What have we learned about natural selection in
15 years? J Evol Biol 16:363?377
Blouin MS, Parsons M, Lacaille V, Lotz S (1995) Use of
microsatellite loci to classify individuals by relatedness. Mol
Ecol 5:393?401
Coyne JA, Orr HA (1998) The evolutionary genetics of speciation.
Philos Trans R Soc Lond B Biol Sci 353:287?305
Dobzhansky T (1975) Analysis of incipient reproductive isolation
within a species of Drosophila. Proc Natl Acad Sci U S A
72:3638?3641
Endler JA (1992) Signals, signal conditions and the direction of
evolution. Am Nat 139:S125?S153
Endler JA, Basolo AL (1998) Sensory ecology, receiver biases and
sexual selection. Trends Ecol Evol 13:415?420
Fisher RA (1930) The genetical theory of natural selection.
Clarendon, Oxford
Gerhardt HC (1994) Reproductive character displacement of female
mate choice in the grey treefrog, Hyla chrysoscelis. Anim
Behav 47:959?969
Goodnight KF, Queller DC (1995) Relatedness, version 5.0.8. http://
gsoftnet.us/GSoft.html
Goodnight KF, Queller DC (1996) Kinship 1.3.1. http://gsoftnet.us/
GSoft.html
Grafen A (1990) Sexual selection unhandicapped by the Fisher
process. J Theor Biol 144:473?516
Hamilton WD, Zuk M (1982) Heritable true fitness and bright birds
a role for parasites? Science 218:384?387
Hankison SJ, Morris MR (2003) Avoiding a compromise between
sexual selection and species recognition: female swordtail fish
assess multiple species-specific cues. Behav Ecol 14:282?287
Kirkpatrick M (1982) Sexual selection and the evolution of female
choice. Evolution 36:1?12
Kirkpatrick M, Barton NH (1997) The strength of indirect selection
on female mating preferences. Proc Natl Acad Sci U S A
94:1282?1286
Kirkpatrick M, Ryan MJ (1991) The paradox of the lek and the
evolution of mating preferences. Nature 350:33?38
Kokko H, Brooks R, Jennions MD, Morley J (2003) The evolution
of mate choice and mating biases. Proc Biol Sci 270:653?664
Lampert KP, Rand AS, Mueller UG, Ryan MJ (2003) Fine-scale
genetic pattern and evidence for sex-biased dispersal in the
t?ngara frog, Physalaemus pustulosus. Mol Ecol 12:3325?3334
Lande R (1981) Models of speciation by sexual selection on
polygenic traits. Proc Natl Acad Sci U S A 78:3721?3725
Landry C, Garant D, Duchesne P, Bernatchez L (2001) Good genes
as heterozygosity: the major histocompatibility complex and
mate choice in Atlantic salmon (Salmo salar). Proc Biol Sci
268:1279?1285
Lenington S, Coopersmith CB, Erhart M (1994) Female preference
and variability among t-haplotypes in wild house mice. Am Nat
143:766?784
Liedloff A (1999) Mantel version 2.0, Mantel nonparametric test
calculator. http://www.real.sci.qut.edu.au/NRS/mantel.htm
Lynch M, Ritland K (1999) Estimation of pairwise relatedness with
molecular markers. Genetics 157:1753?1766
Marsh D, Rand AS, Ryan MJ (2000) Effects of inter-pond distance
on the inbreeding ecology of t?ngara frogs. Oecologia
122:505?513
Maynard Smith J, Harper D (2003) Animal signals. Oxford
University Press, Oxford
Mays HL Jr, Hill GE (2004) Choosing mates: good genes versus
genes that are a good fit. Trends Ecol Evol 19:554?559
Milinski M (2003) The function of mate choice in sticklebacks:
optimizing MHC genetics. J Fish Biol 63(Suppl A):1?16
Mousseau TA, Ritland K, Heath DD (1998) A novel method for
estimating heritability using molecular markers. Heredity
80:218?224
Penn DJ (2002) The scent of genetic compatibility: sexual selection
and the major histocompatibility complex. Ethology 108:1?21
Penn DJ, Potts WK (1999) The evolution of mating preferences and
major histocompatibiliy complex genes. Am Nat 153:145?169
Petrie M (1994) Improved growth and survival of offspring of
peacocks with more elaborate trains. Nature 371:598?599
Pfennig KS (1998) The evolution of mate choice and the potential
for conflict between species and mate-quality recognition. Proc
R Soc Lond B Biol Sci 265:1742?1748
Pomiankowski AN (1988) The evolution of female mate preferences
for male genetic quality. Oxf Surv Evol Biol 5:136?184
Pr?hl H, Adams RMM, Mueller UG, Rand AS, Ryan MJ (2002)
Polymerase chain reaction primers for polymorphic micro-
satellite loci from the t?ngara frog Physalaemus pustulosus.
Mol Ecol Notes 2:341?343
Queller DC, Goodnight KF (1989) Estimating relatedness using
genetic markers. Evolution 43:258?275
Raberg L, Stjernman M, Hasselquist D (2003) Immune responsive-
ness in adult blue tits: heritability and effects of nutritional
status during ontogeny. Oecologia 136:360?364
Ritland K, Ritland C (1996) Inferences about quantitative inheri-
tance based on natural population structure in the yellow
monkeyflower, Mimulus guttanus. Evolution 50:1074?1082
Ryan MJ (1980) Female mate choice in a neotropical frog. Science
209:523?525
803
Ryan MJ (1983) Sexual selection and communication in a
neotropical frog, Physalaemus pustulosus. Evolution 39:261?
272
Ryan MJ (1985) The t?ngara frog: a study in sexual selection and
communication. University of Chicago Press, Chicago
Ryan MJ (1990) Sensory systems, sexual selection, and sensory
exploitation. Oxf Surv Evol Biol 7:157?195
Ryan MJ (1997) Sexual selection and mate choice. In: Krebs JR,
Davies NB (eds) Sexual selection and mate choice. Blackwell,
Oxford, pp 179?202
Ryan MJ (1998) Receiver biases, sexual selection and the evolution
of sex differences. Science 281:1999?2003
Ryan MJ, Rand AS (2003) Sexual selection in female perceptual
space: how female t?ngara frogs perceive and respond to
complex populations variation in acoustic mating signals.
Evolution 57:2608?2618
Shaw K (1995) Phylogenetic tests of the sensory exploitation model
of sexual selection. Trends Ecol Evol 10:117?120
Waldman B, Adler K (1979) Toad tadpoles associate preferentially
with siblings. Nature 282:611?613
Waldman B, Bishop PJ (2004) Chemical communication in an
archaic anuran amphibian. Behav Ecol 15:88?93
Waldman B, Tocher M (1998) Behavioral ecology, genetic diversity,
and declining amphibian populations. In: Caro T (ed)
Behavioral ecology and conservation biology. Oxford Uni-
versity Press, New York, pp 394?443
Waldman B, Rice JE, Honeycutt RL (1992) Kin recognition and
incest avoidance in toads. Am Zool 32:18?30
Wedekind C, Furi S (1997) Body odour preferences in men and
women: do they aim for specific MHC combinations or simply
heterozygosity? Proc Biol Sci 264:1471?1479
Wedekind C, Seebeck T, Bettens F, Paepke AJ (1995) MHC-
dependent mate preferences in humans. Proc Biol Sci 260:245?
249
Welch AM, Semlitsch RD, Gerhardt HC (1998) Call duration as an
indicator of genetic quality in male gray tree frogs. Science
280:1928?1930
Wells KD (1977) The social behavior of anuran amphibians. Anim
Behav 25:666?693
Yamazaki K, Boyse EA, Mike V, Thaler HT, Mathieson BJ, Abbott
J, Boyse J, Zayas ZA, Thomas L (1976) Control of mating
preferences in mice by genes in the major histocompatibility
complex. J Exp Med 144:1324?1335
Yamazaki K, Beauchamp GK, Shen F, Bard J, Boyse JA (1991) A
distinctive change in odor type determined by H-2D/L muta-
tion. Immunogenetics 34:120?131
Zahavi A (1975) Mate selection: a selection for a handicap. J Theor
Biol 53 205?214
Zahavi A, Zahavi A (1997) The handicap principle: a missing piece
of Darwin?'s puzzle. Oxford University Press, Oxford
Zelano B, Edwards SV (2002) An MHC component to kin
recognition and mate choice in birds: predictions, progress,
and prospects. Am Nat 160:S225?S237
804