Behavioral Ecology Vol. 13 No. 2: 149–153
Female preference function related to
precedence effect in an amphibian anuran
(Alytes cisternasii): tests with non-overlapping
calls
Jaime Boscha and Rafael Márqueza,b
Museo Nacional de Ciencias Naturales, CSIC, José Gutiérrez Abascal 2, 28006 Madrid, Spain, and
bCentro de Biologia Ambiental, Faculdade de Ciências, Universidade de Lisboa, Bloco C2, Campo
Grande, P-1700 Lisboa, Portugal
a
We quantified precedence effect (measured as female preference for the leading calls in an acoustic interaction between two
males) with non-overlapping, simulated male calls presented with various phase relationships to female Iberian midwife toads
(Alytes cisternasii) in two-speaker phonotaxis playback tests. The resulting information determines the shape of the female
preference function for intercall delays. Females preferentially approached leading callers for most tested phase angles. We
found a gradation in the degree to which females selected the leader. They tended to exert a strong preference for the leader
in 30⬚ test, and at higher phase angles, the overall preference was weaker and graded (the higher the phase angle, the lower
the preference). Other parameters of female preference (latency and repeatability) also had a graded relationship with phase
angle value. The sharp difference in probability of approach between 30⬚ and 60⬚ is consistent with a mechanism of male calling
inhibition immediately after hearing a competitor’s call previously described in other taxa. In natural interactions, male A.
cisternasii adjust the timing of their calls to a phase angle that provides a slight but significant advantage to the leading caller.
Key words: Alytes cisternasii, anurans, calling, female preference, midwife toads, precedence effect. [Behav Ecol 13:149–153
(2002)]
T
he precedence effect has been defined as the phenomenon that ‘‘when two binaural sounds are presented with
a brief delay between them, and are perceived as a single auditory event, the localization of that event is determined largely by the directional cues carried by the earlier sound’’ (Zurek, 1987: 85). The precedence effect has also been used to
describe female preference for leading calls in multimale interactions in species with acoustic or visual signals for sexual
advertisement (Backwell et al., 1998; Vencl and Carlson, 1998;
Wallach et al., 1949; Wyttenbach and Hoy, 1993; Zurek, 1980).
It has even been proposed that this perceptual bias or the
resulting difference in attractiveness to females may result in
the synchronization of calls between neighboring males
(Greenfield et al., 1997).
In insects and amphibians, a preference for the leading call
has been observed when the calls of competing males overlap
in time (i.e., when masking between the two signals occurs;
see review in Klump and Gerhardt, 1992, also Greenfield and
Rand, 2000). This effect may result from the relative importance of the initial transient (raise time) in localization, and
the fact that the initial transient of the second signal is masked
by the leading signal (Zurek, 1987). However, precedence effect in the absence of masking between the signals has also
been described for very short sounds in humans (see review
in Zurek, 1987) and in insects (Minckley and Greenfield,
1995; Snedden and Greenfield, 1998). To date, only Minckley
and Greenfield (1995) have explored the changes in strength
of female preference with several call delays between males in
Address correspondence to J. Bosch. E-mail: [email protected].
Received 29 November 2000; revised 23 March 2001; accepted 23
March 2001.
2002 International Society for Behavioral Ecology
a study of the acridic orthopteran Ligurotettix planum. The
authors showed that only males emitting calls immediately following another male’s calls would be discriminated against by
females, the effect being nonsignificant for males responding
with longer delays. This result pointed toward the determination of the length of the delay that would trigger this female
preference for leaders and the associated strategies that males
had to follow to avoid this critical delay affecting the mechanisms regulating call timing.
Among amphibians it is more difficult to find examples of
studies of mechanisms regulating call timing that include the
study of the effect of call exchanges without call overlap (but
see Dyson and Passmore, 1988a,b; Grafe, 1996, 1999; Narins,
1982; Zelick and Narins, 1983), and there are even fewer examples of studies of non-overlapping acoustic interactions between males with different phase angle relationships (Bosch
and Márquez, 2001a). We studied female preference in relation to the precedence effect in the Iberian midwife toad Alytes cisternasii, (Discoglossidae), a species in which males have
short, simple tonal advertisement calls and relatively long intercall intervals (Márquez and Bosch, 1995). Female reciprocal calling has been reported in A. cisternasii (Márquez and
Verrell, 1991), and female calls have been described in Bosch
and Márquez (2001b). This behavior has been related to advertising receptivity in Alytes muletensis (Bush et al., 1996).
Previous studies of A. cisternasii determined that larger
males emitted advertisement calls with lower frequencies, that
females were preferentially attracted to lower frequency calls
(Márquez, 1995a,b), and that larger males obtained more
matings per season (Márquez, 1993). Concerning the acoustic
interaction among males, additional published studies have
shown that pairs of males interacting naturally have extremely
limited overlap between their calls, that they tend to time
their calls with a phase angle of 78⬚ (⫾13.6), and that acous-
Behavioral Ecology Vol. 13 No. 2
150
tically interacting males alternate the role of leading and following in natural interactions (Bosch and Márquez, 2000).
We aimed to determine whether a precedence effect exists
in simulated two-male interactions without call overlap in A.
cisternasii. Furthermore, we sought to determine the shape of
the female preference function based on the relative timing
of male calls (phase angle, sensu Klump and Gerhardt, 1992).
Thus, we attempted to quantify the relative importance of a
precedence effect with simulated male calls presented with
various phase relationships. We tested whether this shape adjusts to a shelf model (the advantage for the leading call is
flat and drops to nonsignificance after a given delay) or if it
is a graded, gradual loss of advantage. This information not
only will illuminate aspects of the mechanism of the precedence effect, but it will also contribute to our understanding
of the evolutionary significance of timing in intermale acoustic interactions (see Bosch and Márquez, 2001a; Greenfield
and Rand, 2000).
METHODS
We studied A. cisternasii in October 1998 and 1999 in a liveoak forest or ‘‘dehesa’’ near the city of Mérida (235 m above
sea level, Extremadura, west-central Spain, 38⬚59⬘ N, 31⬚24⬘
W). Gravid females (with mature eggs visible through the lower part of the abdomen) were collected at the beginning of
the reproductive period and kept in captivity for 1–2 weeks
under controlled conditions and fed ad libitum with fly larvae.
We synthesized synthetic calls at a sampling rate of 44.1 kHz
and 16-bit resolution with Sound Maker 1.0.4 software. A digital tone generator produced a pure tone sinusoid, and the
intensity envelope was visually adjusted to fit the envelope of
a representative call of the population. We adjusted the duration (175 ms) and the dominant frequency (1491 Hz) of
the synthetic calls to the population average (Márquez and
Bosch, 1995).
We tested six different phase angles: Two speakers were
placed behind opposite sides of a square 1.8 ⫻ 1.8 m arena
under low-intensity light. The walls of the arena were formed
by thin, black cloth hanging down from a semi-rigid frame.
The speakers emitted the synthetic calls alternately. Synthetic
calls were produced directly by an Apple PowerBook G3 and
broadcast through a custom-made amplifier and speaker system. The speakers and channels were switched between trials.
Sound level of the synthetic calls was adjusted to 70 dB in the
middle of the arena with a digital Realistic Sound Pressure
Meter (fast response, A weighting). We placed each female
under a cylinder of plastic mesh in the center of the arena
for 30 s while the stimuli were emitted. Subsequently, the cylinder was lifted and the female’s movements were monitored.
We scored a choice only if the female reached an area within
10 cm of the base of a speaker. A trial provided no data when
the female reached the edge of the arena away from the
speakers or when no edge was reached 2 min after we freed
the female. Each speaker emitted calls of one male at regular
intervals (calling rate of 29 calls/min, equivalent to an intercall interval of 1884 ms), a calling rate which is within the
range found in the natural population (intercall interval
range: 819–6939 ms; Márquez and Bosch, 1995).
The speakers broadcast the calls in pairs so that one of the
speakers (leader) emitted its call before the other (follower).
We tested six different phase angles: 30⬚, 60⬚, 90⬚, 120⬚, 150⬚,
and 180⬚ (therefore 0, 168, 339, 508, 678, and 747 ms of delay
between the end of the leader call in one channel and the
onset of the follower call in the opposite channel). We used
a phase angle of 180⬚ as a control, and before the trial, we
selected randomly one channel and assigned it as the leader
speaker.
We tested each female in a total of four trials for each treatment on different nights. The probability of approach to the
leader speaker was calculated for each female. Thus, if a female approached the leader speaker twice and the follower
speaker once and exhibited no response in the fourth trial,
she would be assigned a probability of 2/3. Only females responding twice or more in the four trials of any of two-choice
experiments were considered for the statistical analyses.
We used several measures to assess the strength of female
preference for each experiment. We measured the latency of
the choice for each female (averaged among trials), as well as
the probability of calling for each female. We also calculated
the repeatability of the choice for each female. For each female, a single measure of repeatability and probability of call
was obtained for the four trials in a similar fashion as for the
probability of approach. These probabilities were compared
with the expected probability of 0.5 with one-sample sign tests.
We compared trends between the tests with different phase
angles using the Jonckheere test for ordered alternatives (Siegel and Castellan, 1988).
RESULTS
Females preferentially approached speakers initiating the
acoustical exchanges (leaders) for most tested phase angles
(30⬚: n ⫽ 18, p ⬍ .0001; 60⬚: n ⫽ 18, p ⫽ .0352; 90⬚: n ⫽ 14,
p ⫽ .0386; 120⬚: n ⫽ 23, p ⫽ .0768; 150⬚: n ⫽ 24, p ⫽ .0213;
Figure 1A). For the 180⬚ phase angle, the females chose randomly between the speakers, as we expected (n ⫽ 23, probability of approach ⫽ 0.558, p ⫽ .4545). We found a gradation
in the degree to which females selected the leader ( Jonckheere test for ordered alternatives, J* ⫽ 7.98, p ⬍ .0001, and
the preference found in the 30⬚ test was higher than in the
rest (Friedman test, ␹2 ⫽ 19.67, p ⫽ .0014). If the 30⬚ test is
omitted for the analyses, the graded trend still remains significant, but the differences between individual tests are no
longer significant (Friedman test, ␹2 ⫽ 8.76, p ⫽ .0675). Thus,
we can conclude that female A. cisternasii tend to exert a
strong preference for the leader in 30⬚ test, and that at higher
phase angles, the overall preference is weaker and graded
(the higher the angle the lower the preference).
The latency of the choice also had a graded (positive) relationship with phase angle value (Figure 1B; latency: Jonckheere test for ordered alternatives, J* ⫽ 2.399, p ⬍ .0084).
The latency of the choice was short for low values of phase
angle and long for high values of phase angle. Similarly, the
probability of female calling also had a graded relationship
with phase angle value (Figure 1C; Jonckheere test for ordered alternatives, J* ⫽ 3.556, p ⬍ .0023), although the trend
was in the opposite direction—the higher the phase angle,
the lower the probability of female call.
Finally, the repeatability was higher in experiments with low
values of phase angle than in experiments with high values of
phase angle (Figure 1D; Jonckheere test for ordered alternatives, J* ⫽ 8.428, p ⬍ .0001).
DISCUSSION
The female preference function in our study is a populationlevel preference function, and not enough repeated tests were
performed on individual females to adequately describe individual preference functions (see Murphy and Gerhardt,
2000). The female preference function obtained is similar to
some extent to that presented by Minckley and Greenfield
(1995) for the acridic orthopteran Ligurotettix planum, in
which males emitting calls immediately following another
male’s calls were strongly discriminated against by females.
Our female preference function, though, allows for an addi-
Bosch and Márquez • Female precedence function in midwife toads
151
Figure 1
(A) Probability of approach to the leader speaker for the six experiments performed. Asterisk indicate significant differences from random
choice (*p ⬍ .05, ***p ⬍ .001). (B) Time taken to make the choice. (C) Probability of call in response to the stimulus. (D) Repeatability of
the choice of the leader speaker. Means ⫾ SEs are shown. The mean ⫾ SE of phase angle found in natural acoustical interactions between
two males are shaded in gray.
tional level of resolution, as five higher phase angles (60–
180⬚) were tested. The graded degree of attractiveness of
these additional five values is a feature of the female preference function of A. cisternasii, which has not been described
for either anurans or orthopterans to the best of our knowledge.
The sharp difference in probability of approach between
the values of 30⬚ and 60⬚ is consistent with a mechanism of
male calling inhibition that occurs immediately after hearing
a competitor’s call, which has previously been described for
some orthopterans (Greenfield, 1994; Greenfield and Minckley, 1993; Minckley et al., 1995). The results also blend in
nicely with the model suggested by Moore et al. (1989) and
discussed by Klump and Gerhardt (1992), whereby an absolute refractory period [defined by Klump and Gerhardt (1992:
158) as ‘‘during this period an external stimulus cannot elicit
a call with a short response latency’’] would occur immediately after hearing the call of another male, and this would
be followed by a relative refractory period [‘‘period during
which fewer calls are given than expected from a random
placement of calls with reference to the acoustic stimulus’’
(Klump and Gerhardt, 1992: 158; see also Zelick and Narins,
1983)]. Curiously enough, if we apply the trend of female
preference observed in our tests and if the relative refractory
period coincided with the higher values of phase angles (60–
180⬚), female preference would favor males that delay their
response as much as possible, thus approaching 180⬚. This
would favor males that delayed their calls, potentially reaching
values above 180⬚ and thus becoming leaders in the following
interaction. This would be consistent with the observation of
Márquez and Bosch (2001), who found that A. cisternasii
males alternate the role of leader and follower in the same
calling session (same night). Márquez and Bosch failed to find
a significant correlation between percentage of interactions as
leader and other male parameters (size and temperature).
In this species, when two males engage in interactions in
nature, the average phase angle is 78⬚ (⫾13.6) (Márquez and
Bosch, 2001). This suggests that males are adjusting the timing of their calls to a phase angle that provides a slight but
significant advantage to the leading caller. The graded trend
found for the 60–180⬚ tests is less sharp. In probability of approach, preference for the leading call is not significant (but
near significance) and is significant again for 150⬚.
Although the phase angle value found in natural acoustic
exchanges between males is similar in another congeneric
species (A. obstetricans), female preference for followers has
been described in this species. However, the differences in
results may be related to the effect of the experimental temperature because the test with A. obstetricans was performed
at relatively low temperatures (average 10.4⬚C; Bosch and Már-
152
quez, 2001a). Furthermore, for A. cisternasii, a decrease in
the strength of female preference for leading callers in acoustical interactions between males has been observed with decreasing temperature, to the point that a reversal of preferences (i.e., preference for follower) has been found at low
temperatures (Bosch and Márquez, unpublished data). Further testing is necessary to determine if these observations
may be as relevant as other effects of temperature on call
characteristics and female preferences (e.g., Gerhardt, 1978).
The graded effect found for all four aspects of female preference contrasts with the trend described for the katydid othopteran Neoconocephalus spiza by Greenfield and Roizen
(1993), in which a sharp, nearly shelflike change in female
preference function was found for changes in phase angle
between 0⬚ and 45⬚. However, the lack of gradation observed
may possibly be due to the fact that the authors did not test
enough phase angles in the critical zone. Our results are more
in line with the data presented by Minckley and Greenfield
(1995), who found that females were attracted to the leading
call, and that the preference was strong for a short intermale
interval and progressively weaker as the interval between calls
increased.
An explanation for the observed precedence effect (without call overlap) may be related to mechanisms to avoid the
confounding information about the location of sound source
from reflected sounds (echoes). The best information about
the source of the sound will be provided by the beginning of
the sound, which is likely to reach the subject through the
most direct (fastest) route. This is a particularly serious problem for the species studied, for which finding the precise location of the calling male may be a challenge for females
(Bosch and Márquez, 2000). Under some circumstances, in
our recordings echoes appear with substantial power in the
call of males and up to 50 ms after the end of the call of the
male (unpublished data). A female preference function biased toward the beginning of the call may thus prove adaptive
for an expedient location of the caller. The general application of such a hypothesis remains to be tested.
It is interesting to note that, from a mechanistic perspective,
there is a marked difference between precedence effect with
overlapping calls and non-overlapping calls. When the leading
call overlaps the beginning of the following call, the leading
one may physically mask the transients at the beginning of
the following call. When there is no overlap, some sort of
central mechanism is likely. For example, a leading call heard
on one side may lead to desensitization of the contralateral
ear for some period into the future, a form of forward masking (Greenfield MD, personal communication). Further experiments are necessary to determine the nature of the underlying mechanism.
J. Falagán helped in the laboratory. M.D. Greenfield and U. Grafe
revised the manuscript. The Agencia de Medio Ambiente of Junta de
Extremadura extended permits for field work. Partial funding was
provided by project PB 97-1147 Ministerio de Educación y Cultura of
Spain (P.I: I. De la Riva).
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