Lisa Haitzinger
Behavioural Experiments on Synchronous Chorusing in the Tropical Bushcricket Mecopoda elongata (Tettigoniidae; Orthoptera): Cooperation or Competition?
Masterarbeit zur Erlangung des akademischen Grades eines Master of Science an der Naturwissenschaftlichen Fakultät der Karl-Franzens-Universität Graz
Betreut von: Univ.-Prof. Dr. Heinrich Römer Institut fur Zoologie, AG Neurobiologie und Verhalten
2012 Danksagung
An dieser Stelle möchte ich mich bei den Menschen bedanken, die mich auf dem oft steinigen Weg durch das Studium begleitet haben. Vielen Dank an Herrn Prof. Dr. Römer, der mit seiner außergewöhnlichen Fähigkeit, komplexe Inhalte zu vermitteln, maßgeblich dazu beigetragen hat meine wissenschaftliche Begeisterung zu wecken. Ich bedanke mich auch bei Herrn Dr. Hartbauer für sein intensives Engagement bei der Betreuung meiner Masterarbeit. Ein großes Dankeschön geht an Stefan, Arne und Marina, die mir mit ihren zahlreichen schlauen Tipps oft weiterhelfen konnten. Ganz besonders möchte ich mich bei Marian für seine aufopfernde Hilfsbereitschaft bedanken. Vielen Dank auch an Isi: du warst mir die beste Studienfreundin die man sich nur wünschen kann! Danke für eure charmante Gesellschaft, egal ob im Kino, beim Cocktailtrinken, Beachen, Biken oder Wandern. Ihr habt mir einen großartigen Ausklang meines Studentenlebens beschert. Vielen Dank an meine "Linzer-Freunde", die nie auf mich vergessen haben. Ich danke meinen Eltern Alfred und Helga, meiner Schwester Christina und meiner Oma von Herzen! Sie haben sich immer mit mir über meine Erfolge gefreut und haben es geschafft mich in schwierigen Phasen aufzumuntern. Papa, Mama, danke für die so großzügige finanzielle Unterstützung, danke dafür auch an dich liebe Oma! Danke Stefan, dass du immer für mich da bist, auch wenn uns viele Kilometer voneinander trennen.
I Zusammenfassung
Männchen der Laubheuschrecke Mecopoda elongata (Orthoptera: Tettigoniidae) produzieren akustische Signale (Chirps) um Weibchen anzulocken. Die Männchen bilden Chöre, in denen sie ihre Gesänge synchronisieren. Diese Synchronisation ist jedoch nicht perfekt, da einige Männchen als Leader etwas früher zu singen beginnen als andere (Follower). In einer früheren Studie konnte gezeigt werden, dass Leader von Weibchen bevorzugt gewählt werden. In meiner Masterarbeit untersuchte ich Mechanismen die zur Evolution von synchronem Chorgesang beigetragen haben könnten: (1) Synchroner Chorgesang evolvierte als Ergebnis von Kooperation zwischen Männchen, die aufgrund eines hohen Überlappungsgrades von Signalen eine größere Lautstärke erreichen können ("Beacon-Effekt"). (2) Synchroner Chorgesang evolvierte als Folge der Konkurrenz zwischen Männchen die sich einen Wettstreit um die attraktive Leaderrolle liefern. (3) Die Präferenz von Weibchen für Signale mit einer fixen Signalperiode von 2 Sekunden ist eine treibende Kraft für die Evolution von regelmäßigen Signalperioden. Um zeitliche Interaktionen und Signalamplituden zu analysieren wurde unter Laborbedingungen der Gesang von Chören und isolierten Männchen aufgezeichnet. Folgende Ergebnisse wurden gefunden: Synchroner Chorgesang resultierte in einem Beaconeffekt, bei dem die maximale Signalamplitude synchroner Sänger gegenüber einem isoliert singenden Männchen um 7.3 dB erhöht ist. Durch die Simulation eines Chores durch Playback von exakt synchronen Signalen über vier Lautsprecher konnte ebenfalls ein Anstieg in der maximalen Signalamplitude von 7.5 dB gefunden werden. Die Berechnung des "active space" von synchronen Signalen belegt zudem quantitativ den Vorteil in der Attraktion von Weibchen, den ein solcher "Beacon-Effekt" haben könnte. Ich konnte außerdem zeigen, dass mit einem Anstieg des Signalüberlappungsgrades die Signalamplitude signifikant ansteigt. Die Chirpraten der meisten Männchen waren im Chor höher als in den Sologesängen. Wahlversuche ergaben, dass Weibchen Signale mit einer regelmäßigen Chirpperiode von zwei Sekunden gegenüber Signalen mit variabler Chirpperiode bevorzugten. Die Ergebnisse deuten einerseits darauf hin, dass die Mitglieder eines Chores durch den "Beacon-Effekt" von einer höheren Anzahl an angelockten Weibchen im Vergleich zu Solosängern profitieren könnten, und demnach synchroner Chorgesang durch Kooperation entstanden sein könnte.
II Andererseits liefern die Ergebnisse Grund zur Annahme, dass die Tiere um die attraktive Leaderrolle konkurrieren. Ergebnisse der Wahlversuche deuten darauf hin, dass die Präferenz der Weibchen für Periodendauern von ca. 2s einen großen Einfluss auf die Evolution von regelmäßigen Signalperioden hat.
Abstract
Males of the tropical katydid Mecopoda elongata (Orthoptera: Tettigoniidae) produce acoustic signals in a mating context to attract females from a distance. In their natural habitat males often aggregate and interact acoustically by synchronizing their signals at a fine-scale. However, synchrony is not perfect, and some males (leaders) time their signals in short advance to others (followers). Relative signal timing has a strong influence on a male’s mating success since females show a strong preference for leader signals. In my master thesis I studied possible mechanisms driving the evolution of chorus synchrony: (1) Chorus synchrony may have evolved as an outcome of cooperation between males, since a high degree of signal overlap may lead to an increase of peak signal amplitudes of the combined, synchronous signals ("beacon-effect"). (2) Chorus synchrony may be the result of competition among males trying to time their signals in advance to others. (3) The female recognition mechanism for conspecific signals relies on fixed signal periods of about 2s, which forces males to produce acoustic advertisement signals in regular periods. In order to investigate signal timing and amplitudes I established small choruses of 3-4 males under laboratory conditions, and recorded their individual and chorus signals. Following results were found: There is a "beacon-effect", since the average SPL of synchronous signals recorded in four male choruses showed an increase of 7.3 dB compared to solo singing males. Similarly, simulations of a chorus with four loudspeakers revealed an increase of 7.5 dB when identical signals were broadcast from four speakers in exact synchrony compared to signals broadcast through a single speaker. Moreover, analysis of multichannel recordings of choruses showed that a high degree of signal overlap within a chorus leads to an increase in signal amplitude. A calculation of the active space of synchronous signals compared to isolated ones further demonstrates the advantage in attracting females, when singing in synchrony. Comparing the song of males singing in isolation with males singing within a chorus revealed a significant increase in chirp rate. In choice
III experiments females preferred signals with a fixed chirp period of 2s over signals with variable chirp periods. Although chorus synchrony may be the outcome of a cooperative act between males due to a "beacon effect" it is also suggested that synchrony may have evolved due to competition for the attractive leader role. Results of female choice experiments suggest that female preference may be a driving force for the evolution of regular signals with a rather fixed chirp period of 2 s.
IV Contents
1 Introduction 1
2 Methods 6 2.1 Animals ...... 6 2.2 Experimental design for testing hypothesis 1 ...... 6 2.2.1 Song recordings in a four-male chorus ...... 7 2.2.2 Analysis of sound recordings ...... 9 2.2.3 Simulation of a chorus using 4 loudspeakers ...... 10 2.2.4 Simulation of the active space of a small chorus . . . . 11 2.3 Female choice experiments ...... 12 2.3.1 Acousticstimuli...... 14 2.3.2 Analysis of phonotactic trials ...... 15 2.4 StatisticalAnalysis ...... 15
3 Results 16 3.1 Singing activity in a chorus ...... 16 3.1.1 Leader - follower delays in a chorus ...... 17 3.1.2 Signal timing among males ...... 17 3.2 Beaconeffect ...... 20 3.2.1 Sound intensity in the middle of a chorus ...... 20 3.2.2 Simulation of a chorus (4 loudspeakers) ...... 20 3.2.3 Modelling of the active space of a chorus signal . . . . 22 3.3 Femalechoicetests ...... 22 3.3.1 Handedness of female choice ...... 24 3.3.2 Examples of walking paths of a single female ...... 25
4 Discussion 29 4.1 Femalechoice ...... 29 4.2 Synchrony and signal recognition ...... 32 4.3 The"beaconeffect" ...... 33
V 1 Introduction
Like in other insects, males of crickets and katydids produce acoustic signals in the context of mating and courtship, but also during inter-male aggression. By means of more or less conspicuous signals it is usually the males attracting sexually responsive females from a distance. These signals convey biologically significant information about sender identity as well as about the physiological and behavioral state of signalers which allows females to select high quality males [1, 2]. Endler and Basolo [3] emphasized that signal generation, signal transmission and perception are strongly shaped by physical and environmental constraints. For example, eavesdropping predators can drive the evolution towards less conspicuous advertisement signals [4], whereas female choice usually drives evolution towards exaggerated advertisement signals, such as loud and redundant signals. Therefore, natural selection and sexual selection can have opposing influences on the evolution of advertisement signals [5]. Acoustic signals are often produced in groups of males, with important consequences for signalers if females select individual males based on certain signal traits. Unless signaling in groups provides other benefits, e.g. reduced per capita predation risk, males may increase their mating success by signaling alone, instead of competing with others in a chorus. However, in choice experiments Morris and colleagues [6] revealed that Conocephalus nigropleurum (Orthoptera: Tettigoniidae) females preferred signals typically found in male assemblages over signals of lone singing males. Ultimately, such a preference forces males of this species to join a chorus where they expose themselves to high inter-male competition. No matter which mechanisms are responsible for the evolution of acoustic "leks", aggregation of males often occurs at common feeding and/or breeding sites, but can also be the consequence of a phonotactic response to the calls of competitors [7]. In some male assemblages signals of individuals are precisely timed in relation to those of competitors, leading either to a synchronous or alternating display. One of the most spectacular forms of long-range sexual signaling is the rhythmic flashing of aggregated tropical fireflies. Thousands of males aggregate in trees, where they produce rhythmic flashes in synchrony [8, 9, 10]. Greenfield [2] suggested analogy in selective forces driving the evolution of firefly synchrony and the evolution of chorus synchrony in acoustically communicating insects. Greenfield and Schul [11] compared calling behavior and female preference functions between Neoconocephalus
1 spiza and Neoconocephalus nebrascensis (Orthoptera: Tettigoniidae) and proposed that different evolutionary mechanisms may have led to the same outcome in both species: chorus synchrony. Like in other acoustically communicating insects N. spiza synchrony is not perfect and some males produce their signals slightly in advance to signals of neighbors. Imperfect synchrony has important consequences for a male’s attractiveness because N. spiza females show a strong preference for leader signals [12]. Therefore, synchrony in N. spiza may have evolved as a byproduct of competition between males trying to produce attractive leader signals in acoustic interactions with other males [13]. N. nebrascensis males also synchronize their acoustic signals in male assemblages, but different to N. spiza, females exhibit only a weak preference for leader signals. In contrast, a phonotactic response of N. nebrascensis females depends on a minimum amplitude modulation of 20 dB between verses interrupted by periods of silence [14]. Therefore, males appear to synchronize their signals in order to preserve a species-specific rhythm that requires silent intervals between verses. Synchrony in N. nebrascensis is likely the outcome of inter-male cooperation whereas in N. spiza synchrony emerges as a by-product of inter-male competition for attractive leader signals. Further support for the rhythm-preservation-hypothesis was provided by Walker [15] for a cricket species and by Moiseff and Copeland [16] for the firefly species Photinus carolinus, where females responded more likely to synchronous signals than to nonsynchronic stimuli. The authors suggested that male synchrony facilitates species recognition and leads to the reduction of visual clutter. Another proximate mechanism driving evolution towards synchronous communal signal display was found in various species where females typically respond acoustically to advertisement signals. Assuming that a high number of interacting individuals leads to a high noise-to-signal ratio, it was suggested that synchronous signaling leads to a better perception of female responses within silent intervals and hence may increase the mating probability by reducing the noise level [17, 18]. Evidence for a cooperative function of chorus synchrony was also provided in the synchronizing treefrog species Smilisca sila, where Tuttle and Ryan [19] showed that as a result of chorus synchrony males reduce per capita risk of bat predation. Another potential benefit of chorus synchrony may arise from an increase of peak signal amplitude, which depends on the degree of signal overlap and is likely improving signal-to-noise ratio of signals in distant receivers. This beacon effect was originally suggested by Buck and Buck [8, 9]
2 and explains flash synchrony in fireflies as a mechanism that enhances the conspicuousness of group displays and suggests synchrony as a cooperative act. Mecopoda elongata (Orthoptera: Tettigoniidae) provides a suitable model for studying mechanisms driving the evolution of chorus synchrony, because communal signals are characterized by a high degree of signal overlap and females exhibit a strong preference for leader signals. This species is subject of the present master thesis. M. elongata males produce acoustic signals termed chirps in a highly redundant way leading to song bouts of 5 to 45 min in duration. One chirp consists of ten to fifteen syllables of increasing amplitude and lasts for 200-300 ms. Each syllable consists of a loud hemi-syllable produced during wing opening and is followed by a softer one produced during wind closing [20]. With respect to their carrier frequencies, chirps are broad-band signals with energy peaks around 8, 14-20 and 30 kHz. At an ambient temperature of 27◦ C intrinsic chirp period of males singing in isolation is about 2 s [21] (figure 2). Like in other Orthopterans, acoustic interactions of M. elongata underlie three levels of temporal precision [22]:
• Males interact on a diel basis and start usually singing shortly before or after the onset of the dark period.
• Males produce their signals in unison song bouts.
• Interacting males time their signals at a fine-scale leading either to synchrony or alternation.
The mechanism responsible for the emission of periodic signals and signal timing in acoustic interactions depends on the properties of the signal oscillator, located in the central nervous system [22]. Depending on the phase and intensity of signals produced by other males, the phase of the signal oscillator either shift forwards or backwards in the cycle. In M. elongata a phase advance or phase delay occurs in the disturbed cycle, whereas the cycle following the disturbed cycle remains unaffected depending on phase and intensity of perturbing stimuli [23, 24]. Hartbauer [25] simulated acoustic interactions of M. elongata males in a computer model by coupling individual signal oscillators with phase-response-properties obtained from real males. In such realistic simulations as well as in real male duets
3 synchrony between males is not perfect because some males, called leaders, consistently produce their chirps slightly in advance to other males, the followers [24]. Males with a faster intrinsic signal rate are more likely to attain leadership in acoustic interactions with competitors with a slower intrinsic chirp rate. Relative signal timing has fundamental consequences for males because in a choice situation females exhibit a strong preference for signals leading other, identical signals by 140 ms [26]. Such a preference for leader signals constitutes a precedence effect, which is defined as a receiver preference for the leading signal of two closely timed identical signals presented from different directions and can be found in humans, mammals, birds, frogs and insects (humans: [27, 28]; Mammals, birds, frogs and insects: [29, 30, 31, 32, 33, 34]). A possible neuronal correlate that may underlie a preference for leader signals in M. elongata is given in the form of side-homologous auditory neurons exhibiting a stronger response to leader signals compared to identical follower signals presented with a delay of some tens of milliseconds from the opposite side of the receiver [35, 36]. Such a leader-biased response to imperfectly timed signals originates from contralateral inhibition, which is a mechanism that enhances the contrast between both hemispheres. Since this mechanism improves sound localization, it not necessarily evolved in the context of mating, which suggests the leader preference in M. elongata to be the outcome of a sensory bias [37, 38, 39, 40]. In my master thesis I tested three hypotheses about the evolution of chorus synchrony in M. elongata: (1) Synchronous signaling in M. elongata may have evolved in order to maximize peak signal amplitudes of communal signal displays. Higher peak amplitudes would result in an increase of the active space of the combined signal, which is defined as that area in which a receiver is able to detect conspecific signals. In a noisy habitat, like the native Malaysian rainforest, detection and discrimination of conspecific signals is especially difficult during night, when background noise amounts to high sound pressure levels of more than 60 dB [41, 42, 43, 44]. Under these conditions augmentation of group displays through signal overlap may indeed improve signal-to-noise ratio, if receivers evaluate peak signal amplitude instead of integrating signals over a longer time period. According to this hypothesis chorus synchrony in M. elongata is the outcome of inter-male cooperation. (2) Alternatively, chorus synchrony may be the result of ongoing competition among M. elongata males trying to time their signals in advance to others.
4 This "competitive hypothesis" was tested by analyzing relative signal timing within small male choruses under laboratory conditions. (3) Finally, I hypothesize that female preference for a rather fixed signal period of about two s forces males to display acoustic advertisement signals in regular periods. Therefore, I studied the preference of M. elongata females given the choice between advertisement signals differing in signal periods or signal period variability.
5 2 Methods
2.1 Animals
Figure 1: Mecopoda elongata female
All experiments were conducted with the tropical katydid species Mecopoda elongata (figure 1). These insects where originally collected in the field in Malaysia and reared at the Department of Zoology at the University of Graz. Animals are reared in glass terrariums in the dimension 100 x 50 x 70 cm. A two - color code was used for the identification of individuals. In the breeding room ambient temperature and relative humidity was maintained at 27◦C and 70%, respectively. The circadian rhythm of insects followed a 12:12 h light: dark cycle with the dark phase starting at 11 am. Insects were fed ad libitum with water or water gel, oat flakes, fish food and lettuce. In Southeast Asia there are several sibling species of Mecopoda that are hardly distinguishable morphologically, but easily by means of their song patterns [23]. Here I worked with the chirping species S, defined by Sismondo [23]. For detailed description of signals see introduction and figure 2 - 3.
2.2 Experimental design for testing hypothesis 1 In order to study a possible beacon effect in M. elongata choruses, I recorded the sound pressure level of acoustic signals displayed in groups of 3-4 males under laboratory conditions and compared it with peak signal amplitudes of solo singing males. For control purposes, I mimicked a chorus situation
6 syllable A
0 time [ms] 300
chirp periode B
0 time [s] 5
Figure 2: (A) Oszillogram of a typical chirp produced by M. elongata. (B) Oszillogram of three subsequent chirps with an intrinsic chirp period of about two seconds. with four loudspeakers to measure the maximum possible increase of sound pressure level (SPL) when identical signals overlap perfectly, or slightly differ in timing. In addition, I simulated the increase in active space of males in a chorus compared to solo singing males with the help of a computer model.
2.2.1 Song recordings in a four-male chorus Song recordings were conducted between March and May 2010 (performed by Dipl.-Biol. Marian Siegert) and between June 2011 and January 2012 in the first four hours of the dark phase when males usually sing. Acoustic interactions of four males were recorded in an acoustic chamber (M:Box, Desone Modulare Akoustik, Berlin) with the dimension 280 x 220 x 200 cm. In order to prevent acoustic reflections, inner walls were completely covered with sound absorbing foam (wedge length = 3 cm). Ambient temperature and humidity of this room was controlled using a fan heater (DeLonghi) and
7 delta t
male 1
male 2
male 3
male 4
chorus signal
0 time [s] 5
Figure 3: Sound recording of four M. elongata males singing in synchrony in a chorus. The males were caged and arranged in a square with a nearest-neighbor distance of 2m. Chirps of each male were recorded with a separate closeby microphone, a further microphone recorded the combined chorus signal in the center of the square. Dotted lines indicate the onset of leader chirps in acoustic interactions. The grey bar indicates the time delay between leader male and utmost follower male. For further explanation see text. a humidifier. Temperature and relative humidity in the sound chamber was on average 26◦C ± 1 and 60%, respectively. During experiments males were caged in cylindrical tubes (diameter: 7 cm, height: 24 cm) made of wire mesh (mesh width = 6 mm). Tubes were positioned vertically in a square with a nearest neighbor distance of two meters. In order to avoid acoustic reflections from the floor, insect cages were elevated by 25 cm, by means of cubic wire mesh boxes. Sound of each male was recorded via a closeby microphone (Voltcraft Inc. sound level meter) and digitized using an analogue-digital converter (Power1401 mk 2, Cambridge Electronic Design Limited, Cambridge, UK) operating at a sampling rate of 10 kHz. Although this sampling rate is too low to analyze the full frequency spectrum (up to 80 kHz) of the call, it is sufficient to resolve the temporal relationship between chirps. The peak
8 signal amplitude in the centre of the chorus was recorded using an additional 1 2 " microphone (type 40AC, serial no. 80264; G.R.A.S. Sound & Vibration, 1 Denmark) connected to a 2 " preamplifier (type 26AM, serial no. 86313; G.R.A.S Sound & Vibration, Denmark) which was positioned in the center of the acoustic chamber. Microphone signals were further amplified using a Power Module (type 12AK, serial no. 69498; G.R.A.S Sound & Vibration, Denmark) that was connected to a professional external soundcard (type FA-101, serial no. AW87238 Edirol, Roland Corporation) using a sampling rate of 96 kHz. The microphone pointed with its membrane (0◦-direction) towards the ceiling so that the microphone capsule was elevated about 30 cm from ground, which was at the same height as the singing insects. The directionality of the centre microphone (and the upward microphone orientation) resulted in reduced absolute sound pressure values of the chorus signal, because the angle of sound incidence of the calls of all four males was ◦ ◦ 1 90 instead of 0 (G.R.A.S. Sound and Vibration, 2 -inch Wide-frequency, Free-field Microphone Type 40AC, Product data and Specifications, vers. 24-04-07). Another problem associated with the recording of the chorus signal may result from movements of males while singing during a bout. Considering that sound is loudest when recorded from the dorsal side of a singing male, the position relative to the microphone is a crucial variable in the chorus experiments. Indeed, in field experiments Nityananda & Balakrishnan [45] observed that in another species of M. elongata males sometimes change their position while singing. This was, however, rarely observed in the Malaysian M. elongata species. Therefore, male orientation may be responsible for the high variability of the maximum sound level between choruses, but less likely within a song bout.
2.2.2 Analysis of sound recordings Sound recordings of each microphone were stored in separate channels in Spike2 data files (Version 4.2.3, Cambridge Electronic Design Limited, Cambridge, UK). A custom-made script was written in order to measure the duration of sound signals, time delays between the onset of individual chirps (∆t), the root mean square (rms) amplitude and the peak amplitude of chirps produced by each male. Another custom-written script was used to evaluate root mean square (rms) amplitude and peak signal amplitude in the recording of the microphone that was positioned in the center of the chorus. Rms and peak amplitudes were calculated in time segments in which overlapping chirps
9 were found. Amplitude values of sound signals (mV) were converted into SPL values using the microphone-specific sensitivity of 11.11 mV/Pa. To evaluate the influence of signal overlap on sound intensity, interactions of a single four male chorus where analyzed. For analysis of the variability of signal timing among males, segments with a duration of about 200 seconds were evaluated for each chorus. In order to calculate signal amplitudes of the center microphone, segments were chosen either from one or two different song bouts. These segments were taken from the middle part of the first song bout when four males were singing simultaneously because chirp period variability is highest in the first and last part of a song bout [24]. A third Spike2 script was used to evaluate the percentage of males singing during a song bout. Intrinsic chirp periods of isolated males were recorded in a temperature-controlled incubator (dimension 37 x 50 x 40 cm). Temperature inside this incubator was on average 27±1◦C. Walls were covered with sound absorbing foam in order to minimize acoustic reflections. A custom-assembled tie pin microphone was positioned close to the insect (distance = 15 cm). Solo songs were recorded on a laptop (Dell, M1530) using Cool Edit Pro 2.1 (Syntrilium Inc.) for the control of recordings. Intrinsic chirp periods of males were measured by averaging five randomly chosen chirps in the middle section of the first song bout.
2.2.3 Simulation of a chorus using 4 loudspeakers By manipulating signal timing in a playback experiment using a conspecific male chirp broadcast from multiple speakers, it is possible to reveal the influence of signal timing on the maximum SPL in a chorus. Acoustic simulation of a chorus consisting of four males was obtained by playback of a conspecific signal (a representative chirp of a solo singing male) from four loudspeakers, arranged in a square (string tweeter, Leaf type EAS-10TH400A). The following equipment was used in this experiment: C272 stereo power amplifier (NAD Electronics International, Ontario, Canada), PA5 programmable attenuator (Tucker-Davis technologies, Florida, USA), FA-101 soundcard ( Edirol, Roland Corporation). The sound pressure level of each speaker was calibrated to 60 dB SPL (rms fast mode) / 71 dB SPL (peak mode). Three different chorus situations (figure 4) were simulated and average SPL and peak SPL was measured using a Larson Davis 2540 microphone (Larson Davis, Inc., New York, USA) that was positioned in
10 the center of the acoustic chamber, pointing towards the ceiling and a CEL (CEL, Bedford, U. K.) 414 Precision Impulse Sound Level Meter. In the first situation four identical signals were played back in exact synchrony in order to obtain the maximum increase in sound amplitude of overlapping signals relative to non-overlapping signals (for four signals: theoretically 6 dB). In the other chorus situations two of four identical signals were time shifted relative to the others so that syllables either interleaved (∆t= 8ms) or the onset of chirps exhibited a time lag of 70ms (∆t= 70 ms; see figure 4).
t = 0 t = 8 t = 70
speaker 1
speaker 2
speaker 3
speaker 4
Figure 4: Schematic representation of four chirps (representing four different singing males) under three different timings of chirps relative to other chirps in a chorus situation simulated by four speakers. Time delays (∆t) are given in ms.
2.2.4 Simulation of the active space of a small chorus In addition to sound recordings performed in the center of four-male choruses, a numerical computer model (Net Logo) was developed (courtesy of M. Hartbauer) to simulate the active space of males producing overlapping signals in a chorus versus lone singing males. Simulations are based on realistic properties of sound propagation in the field and take into account the increase in SPL with more than one male singing, and a hearing threshold of receivers of 40 dB SPL (typical for katydids). The active space of one to four equally loud signalers was calculated in three different scenarios: A signal intensity of 81 dB SPL (average SPL of M. elongata males at a distance of one meter) and a signal attenuation found for pure tones of 5
11 kHz or 10 kHz (for attenuation values which strongly depend on distance see [46]). Attenuation of acoustic signals does not only depend on carrier frequency, but also on the position of the sender relative to ground. A pure tone with a carrier frequency of 10 kHz broadcast at a height of 2 m suffers from similar excessive attenuation values compared to a pure tone with a carrier frequency of 5 kHz broadcast on ground. In the frequency range between 10 and 20 kHz the SPL of M. elongata males is on average 61dB SPL (measured at a distance of 1 m). Therefore, multiple simulation runs were performed with senders broadcasting either 81 or 61 dB SPL signals based on the signal propagation of a pure tone with a carrier frequency of 5 kHz or 10 kHz according to attenuation values measured in a katydid habitat [46]. The attenuation was calculated using equations 1-2. Summation of sound pressure levels of coherent signals was calculated using equation 3:
5kHz : I = I0 − (10.054 ∗ ln(x) − 0.8649) (1)
10kHz : I = I0 − (14.179 ∗ ln(x) − 0.9809) (2)
I = Sound level of a sender, x = distance to the sender
10 ∗ (log(10(sumIntensity/10) + (10(level/10)) (3)
2.3 Female choice experiments In two-choice experiments I studied the preference of females for one of two stimuli differing either in chirp timing, chirp period or chirp period variability. Females used for choice experiments were spatially separated from males as last instar larvae. Isolation should prevent fertilization of eggs and hence a potential reduction of female’s motivation to perform phonotaxis. All experiments were performed as phonotactic arena trials in an acoustically isolated chamber (see above). Two loudspeakers were positioned in the back of the arena opposite to the release site, at a distance of 155 cm to each other. The speakers were elevated 8 cm above a soft carpet covering the floor of the arena. Females were released at a distance of 210 cm relative to each speaker, so that the angle separating both speakers at the release site was 44◦ (figure 5). Air temperature and relative humidity was controlled by
12 L1 210
R 196 α=22° 155
L2
Figure 5: Arrangement of the experimental setup for female choice tests. R: release point of females, L 1-2: two loudspeakers in the left and right corner of the acoustic chamber, a: separation angle of both speakers at the release point, grey hemi circles: area with a radius of 30 cm. Distances are given in cm. means of an air humidifier and a fan heater. The average temperature during phonotaxis experiments was 26◦C and relative humidity was 53%. Before the start of a choice experiment females were placed in tubes made of wire mesh (diameter: 7 cm, height: 24 cm) and placed inside an incubator maintaining a constant temperature and relative air humidity of 25◦C and 70%, respectively. At the beginning of each trial a single caged female was positioned at the release point and was allowed to adapt to the experimental conditions for five to ten minutes, without any acoustic playback. Then the cage was manually opened and acoustic stimulation was turned on. If the female remained inside the tube, or left the tube without approaching one of the two speakers within 15 minutes, the trial was aborted and treated as negative. A trial was considered as positive phonotaxis towards one sound source when females entered a hemicircle of 30 cm surrounding each speaker. Females were tested up to 4 times in the first 7 hours of the dark cycle. Habituation of female response was prevented by interrupting subsequent trials by pauses lasting at least 1 hour. Each female was tested at least one time in a control situation in which stimuli have been switched between speakers.
13 2.3.1 Acoustic stimuli All playback stimuli used the same chirp originally recorded and digitized from one solo singing male. This representative chirp consists of 16 syllables increasing in amplitude with a total duration of 308 ms. The signal was 1 recorded using a 2 " microphone (type 40AC, serial no. 80264; G.R.A.S. 1 Sound & Vibration, Denmark) connected with a 2 " preamplifier (type 26AM, serial no. 86313; G.R.A.S Sound & Vibration, Denmark) and a Power Module (type 12AK, serial no. 69498; G.R.A.S Sound & Vibration, Denmark). The signal was digitized using an external soundcard (type FA-101, serial no.: AW87238 Edirol, Roland Corporation) operating at a sampling rate of 96 kHz. Playback of stimuli was controlled using Cool Edit Pro 2.1 (Syntrillium Inc.). Songs with different chirp periods were generated by inserting silent intervals between successive chirps in order to obtain different chirp periods: 1, 1.5, 2 and 2.5 s. The chirps in simultaneously broadcast stimuli were not phase-locked. A fixed timing of chirps resulting in leader-follower situations was produced by inserting a delay of 70ms or 140ms between chirps broadcast in each channel. In order to control for a bias towards the right or left speaker stimuli were switched between both loudspeakers. Additionally, I generated a song in which chirp period varied ’randomly’, which means that an average chirp period of two seconds was maintained over the whole stimulation period of 2 minutes, but a variation was introduced where minimum and maximum chirp periods were limited to one and three seconds, respectively. The playback of sound signals was controlled in Cool Edit Pro using an external multi-channel soundcard (type FA-101, Edirol, Roland Corporation). Playback signals were first attenuated using a two-channel programmable attenuator (PA5, Tucker-Davis technologies, Florida, USA) and amplified by means of a stereo power amplifier (C272, NAD Electronics International, Ontario, Canada). Signals were broadcast through string tweeters with almost identical frequency response properties (Leaf Tweeter EAS-10TH400A). The loudest 3 syllables of playback signals were calibrated to 70 dB SPL at the release point using a CEL 414 sound level meter (microphone: type 2540, Larson Davis, Depew, NY, USA, serial: 1898). This value corresponds to the average SPL of males (81±2.8 dB SPL) measured at a distance of about 3 m.
14 2.3.2 Analysis of phonotactic trials Phonotactic walking paths of females were reconstructed on the basis of still images recorded with an infrared video camera (GKB CB-38075) providing a bird’s eye view of the arena. Two infrared spotlights (EuroTECH LED, 850 nm) were used to obtain a rather uniform illumination of the arena. Single frames were digitized by means of a video frame grabber card (Pixel Smart Inc., USA) taking 12 frames per minute. Owing to a wide field lens of the video camera, frames were distorted, a bias that was widely removed by applying a lens correction and a lens distortion filter in Adobe Photoshop (version 12.0.4). The manual tracking tool MtrackJ (ImageJ plugin) was used to reconstruct walking paths of individual females. Frame-by-frame positions of the female W11 were analyzed for all choice situations. In addition, I reconstructed walking paths of 10 females obtained during the leader-follower experiment with a time delay of 70 ms.
2.4 Statistical Analysis All statistical analyses were performed using Sigmaplot (version 12.0). The significance of female choice for one signal in a choice situation was tested in R (version 2.13.1) by application of a generalized linear mixed model fit by Laplace approximation. In this binomial model female ID was used as random intercept.
15 3 Results
3.1 Singing activity in a chorus Chirp timing and chorus attendance in small choruses of four males (N=19) was analyzed in the first song bout of dark periods. Results shown in figure 6 represent the percentage of total singing activity relative to the duration of the first song bout, in cases when one to four males were singing. The duration of four males spent singing together was significantly higher compared to the duration of one male singing alone (p < 0.05, ANOVA). A similar result was obtained with duets were males were singing for longer periods of time compared to solo singing. * * 100
80
60
40 proportion of 20
synchronized singing [%] 0
1 2 3 4 number of simultaneously singing males
Figure 6: Relative duration of time spent singing at the same time for two, three or four males compared to solo singing. Data from the first song bout recorded in a dark period. Horizontal lines represent median values. Boxes cover 25-75% of the data distribution. Outliners are shown as black dots. Kruskal-Wallis One Way Analysis of Variance on Ranks followed by a Tukey’s post hoc Test, * indicates p < 0.05, N= 19.
16 3.1.1 Leader - follower delays in a chorus In 18 different four-male choruses the time difference between leader and follower signals (∆t) was on average 73ms ±34 ms (figure 7). Only in four choruses (chorus 14-17) the average time delays of single males were quite high and thus signal overlap low.
300
250
200
150
100
mean delta T [ms] 50
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Choruses
Figure 7: Mean time delay (∆t) between males in chorus. The vertical line indicates the mean ∆t obtained from 72 males, respectively 18 choruses.
3.1.2 Signal timing among males Multichannel recording of the singing activity of a four-male chorus allowed quantifying chirp timing of males relative to the chirps of other males in the chorus. In 68% of recorded choruses (N= 19) a single male attained leadership (∆t = 0ms) in more than 50% of signal interactions. Figure 8 shows an example of a representative chorus in which one male attained leadership in most signal interactions. Analysis of signal timing in 11 choruses with more than one singing bout revealed that in 64% of choruses one male attained leadership in at least two subsequent song bouts. Furthermore, 71% of males (N= 7) retained their leader/follower roles in at least two different chorus situations through interacting with other chorus participants. Males contributing to a chorus decreased their average chirp period from 2.1 s to 1.9 s significantly compared to solo singing (figure 9). However, there was no significant difference
17 between leaders and followers with respect to this decrease in chirp period (figure 10).
M17 M18 M19 M20 100
relative number
of leader chirps [%]
0 1 2 3 4 5 songbouts
Figure 8: Example of a male chorus in which one male maintained the leadership more often compared to his competitors. The percentage of leader chirps of four acoustically interacting males (M17-M20) is shown for five subsequent song bouts (N= 106). Note that male M20 attains leadership in all song bouts.
18 2.8 * 2.6
2.4
2.2
2.0
intrinsic CP [s] 1.8
1.6
1.4 solo chorus
Figure 9: Chirp periods (CP) of males singing in isolation (solo) and singing in a chorus situation (chorus). (*) paired t-test, p = < 0 001, N=70 (53 males).
25 20 15 10 5 0 -5
of solo and chorus [%] relative indifference CP -10 -15 leader follower
Figure 10: Difference in CPs between solo singing and during interaction with males in a small chorus (3-4 males). Values are separately analyzed for leaders (N= 13) and followers (N= 39). Positive values indicate a reduction of CPs in a chorus relative to solo singing, whereas negative values indicate the opposite. There was no significant difference between leader and follower males (Mann-Whitney Rank Sum Test, p= 0.465).
19 In a total of 11 choruses the intrinsic CPs of all four males where known from analysis of solo singing. In 36% of these choruses the male leading for more than 50% of time exhibited the lowest intrinsic CP of all chorus members.
3.2 Beacon effect 3.2.1 Sound intensity in the middle of a chorus An important aim of this master thesis was to show, if signal overlap in a chorus causes a beacon effect. Figure 11 illustrates with an original oscillogram of a recording the difference in amplitude between overlapping signals recorded in the centre of a chorus and chirps produced by a lone singing male using the same chorus. Signals produced in small choruses (N=12) formed by three to four males resulted in an average increase of the maximum amplitude of 7.3± 3 dB compared to solo singing males (*) paired t-test, p =< 0.001, (containing 3 three-male choruses). A similar increase of 7.5± 2 dB was found when the rms amplitude of communal signals was compared with solo chirps. The influence of the temporal overlap on the increase in the combined signal amplitude is shown for one chorus in figure 12. There is a significant correlation between the amount of signal overlap and the signal amplitude, with a maximum in increase of about 2 dB. This appears to contradict with results obtained with real male choruses, and simulated choruses (speaker signals), where a maximum increase of about 7.5 dB was measured as a difference between one male and four males singing. But note that the results in figure 12 were obtained in a chorus where permanently four males were singing, so that the SPL increase as a result of additional chirps do not occur in this chorus. Thus the additional 2 dB are only due to the precision of temporal overlap.
3.2.2 Simulation of a chorus (4 loudspeakers) In addition to the analysis of a beacon effect in choruses of real interacting males, I also simulated such choruses by using four speakers broadcasting identical chirps at different degrees of temporal overlap. Such simulations revealed an increase in the maximum sound pressure level of 7.5 dB when identical signals were broadcast in exact synchrony compared to signals broadcast through a single speaker. Delaying two of the four signals by 8 ms
20 0.4 A B
0.2
0
amplitude [mV] -0.2
-0.4 0 6 0 6 time [s]
Figure 11: Example of signals recorded in the middle of a chorus, when four males were singing in synchrony (A) or only one male was active (B).
2.5 2 1.5
1 0.5
rms amplitude [dB] 0 0 20 40 60 80 100 120 140 160 180
mean deltaT [ms]
Figure 12: Influence of the degree of signal overlap on amplitude of a chorus signal within a four male chorus (no. 17). Low values of the time delay between follower signals (∆t) indicate high signal overlap. dB values are given relative to the minimum value of rms amplitude. Each dot represents an interaction between chorus members (N= 93). R2= 0.52. reduced this increase by 2 dB, i.e. in this setting four speakers increased the peak SPL by 5.5 dB compared to a signal broadcast by a single speaker.A delay of 70 ms of two speakers relative to the others led to an increase in peak SPL of 4.5 dB compared to single speaker playbacks. Similar, but slightly
21 lower values were obtained when calculating SPLs with an integration time constant of 200 ms (fast mode) (table1).
SPL 1 speaker [dB SPL] SPL 4 speakers [dB SPL] ∆t [ms] average peak average peak 0 60 71 66.5 78.5 8- - 66 76.5 70 - - 63.5 75.5 Table 1: Focal sound pressure levels in a chorus simulated by four speakers broadcasting a conspecific signal in exact synchrony, or with a delay of 8 and 70 ms, or by a single speaker.
3.2.3 Modelling of the active space of a chorus signal An increased SPL of the synchronous chorus signal should result in an increase in the active space of the chorus signal. This was simulated in a model under three different assumptions: (1) a signal level of 81 dB SPL at 1 m, and an excess attenuation found for pure tones with a frequency of 5 kH (figure 13A), (2) same signal level, but excess attenuation found for pure tones with a frequency of 10 kH (figure 13B), and (3) a signal level of 61 dB SPL at 1 m and excess attenuation found for pure tones with a frequency of 5 kH (figure 13C) (For excess attenuation values see [46]).In all cases the synchronous signals in a chorus resulted in a strong increase in the active space compared to solo singing. The strongest increase in the active space was found when two, instead of only one male, were singing at the same time. Comparing the active space of a single sender with the active space produced by four males revealed an increase between 112% to 220%. Interestingly, a nearest-neighbor distance of 2, 5 or 10 m did not affect the active space significantly.
3.3 Female choice tests When females was given the choice between two song models differing in chirp period or its variability, or the timing of chirps, the motivation to approach one of the song models was different. Generally, females approached one of two speakers more frequently (positive trials) in the choice situations CP 2 vs. 2.5 (59%), CP variable vs. 2 (56%), L/F ∆t140 ms (65%) and L/F ∆t70
22 2m 5m 10m 40 30 20 10 0
]
2 3
2
1
active space [km active 0
0.8 0.6 0.4 0.2 0 1 2 3 4 number of males
Figure 13: Simulated active space of the signal of one male, or the synchronous signals of 2 – 4 males (arranged in a square) assuming perfect synchrony, i.e. total signal overlap. Assuming excessive attenuation of a (A) 5kH pure tone and a SPL of 81 dB at a distance of 1m, (B) 10 kHz pure tone with a SPL of 81 dB and (C) 5 kHz pure tone and a SPL of 61 dB. Simulations were repeated with different nearest neighbor distances (see color code). ms (69%) In contrast, only 33% of females showed a positive response in the choice situation CP 2 vs. 1.5 and only 11% in the choice situation CP 1 vs. 1.5 (figure 14) In addition to a L/F situation in which chirps exhibited a delay of 140 ms, I tested a time delay of only 70 ms because this is about the average time
23 CP 1 vs. 1.5 N=51 * CP 2 vs. 1.5 N=172
CP 2 vs. 2.5 N=101 *
CP variable vs. 2 N=90
L/F 140 N=78
L/F 70 N=74
0 50 100 relative number of positive trials [%]
Figure 14: Motivation of females to approach a speaker in various two-choice situations. Values represent the percentage of positive trials in six different choice situations. N = number of all trials, * indicates p < 0.01 obtained with a z- test. lag between leader and follower chirps found in real choruses (see above). Females preferred the leader signal significantly more often than the follower with time delays of 70 ms and 140 ms (figure 15). Females also preferred the stimulus with a fixed chirp period of two seconds compared to a stimulus in which the average CP was the same, but varied randomly between 1 and 3s. Females also selected a stimulus with a chirp period of two seconds over a stimulus with a CP of 2.5 seconds, whereas there was no significant choice between CPs of 2s and 1.5s. As shown in the previous figure, the motivation of females to approach a speaker in this choice situation was generally low (figure 14).
3.3.1 Handedness of female choice In order to exclude a female bias towards one side of the arena, stimuli were regularly switched between speakers. Among a total of 21 females only 3 persistently chose one speaker in the arena, irrespective of the presented stimulus.
24 ** (70 ms) leader follower
** (140 ms) leader follower
* 2 variable
** 2 2.5
2 1.5
100 50 0 50 100
relative number of choices [%]
Figure 15: Preferences of females in various two-choice situations, as indicated, given as percentage of positive phonotaxis in all trials. (**) p = < 0.01, (*) p = < 0.05, N = 46 from at least 12 individuals. See methods for statistics.
3.3.2 Examples of walking paths of a single female In addition to the decision of females to approach one signal alternative in a choice situation the actual path taken by a female can also be quite informative for the decision making mechanism. Figure 16 shows an example of phonotactic approaches of one representative individual (W11) in various choice situations, where the choice task was identical in the respective left and right part of the figure, but the stimuli were switched between speakers. In some cases (e.g. choice between a fixed and variable stimulus period) the female did choose the same stimulus, irrespective of the broadcast side, whereas in other choice situation the female preferred not the same stimulus. Furthermore, there were large differences concerning the actual path, which was rather straight towards one speaker in some trials whereas in others even contained loops. Interestingly, in some approaches (e.g. L/F-situation with a delay of 140; right part of the figure) the female had already closely approached one speaker without entering the 30cm zone, but then finally decided to walk to the other speaker.
25 Such a strong variation in the actual approach is also evident when comparing ten different females in the same choice situation (figure 17). Again, most walking paths strongly deviated from a straight line, with remarkable variations between individuals. Interestingly, some females circled and some selected the other speaker after passing by the competing one.
26 1.5s 2s
X X
50 cm 2s 1.5s
2.5s 2s
X X
2s 2.5s
var 2s
X X
2s var
L140 F140
X X
F140 L140
L70 F70
X X
F70 L70
Figure 16: Positive tracks performed by one individual (W11) in 5 choice situations (left row). The right row presents the same choice situation, but stimuli were switched between speakers. The border of the arena is represented by a black rectangle. Semicircles illustrate a radius of 30 cm surrounding each loudspeaker. Black crosses represent the release point. Part of the walking path outside the rectangle represent wall climbing acts. Different colors represent walking paths obtained in different trials. Leader
Follower 50cm
Follower
Leader 50cm
Figure 17: Positive walking paths of 10 individuals in the choice situation leader / follower ∆t 70 ms. Leader and follower speakers were switched between panels. The border of the arena is represented by a black rectangle. Walking paths of different females are represented by different colors. Semicircles illustrate a radius of 30 cm surrounding loudspeakers. Large dots indicate the chosen loudspeaker.
28 4 Discussion
In my master thesis I studied signal timing of males interacting in a small chorus of three to four males, and the resulting increase in SPL of the combined signal output caused by overlapping chirps. Additionally, I investigated the preference of females for signals differing in timing and signal period. Results of my experiments reveal new insights for the discussion on the evolution of signal synchrony in acoustic insects. Previous behavioral studies investigated the acoustic interactions of males in duets thereby revealing imperfect synchrony [23, 24]. Similarly, in my experiments with small choruses consisting of 3-4 males, individual males consistently timed their signals on average 73 ms in advance to others. Interestingly, the persistency of leader and follower roles in two different nights is 71%, despite the fact that males were interacting with different chorus participants. Thus the likelihood that males can be distinguished into leader and follower males under different "social" conditions is rather high. This contrasts results in an Indian Mecopoda "chirper" where males singing as followers in one night produced leader chirps in the next night, and the disadvantage of being a follower in one night would be compensated by being a leader in other nights [45]. Although timing of chirps in small choruses is imperfect, and leader males on average time their signals 73 ms in advance to competitors, a high degree of signal overlap was found among males, which favors a "beacon effect". In the following I discuss possible causes driving the evolution towards chorus synchrony before discussing the observed beacon effect accompanying this phenomenon.
4.1 Female choice It has been suggested that chorus synchrony is an epiphenomenon originating from the competition of males for the attractive leader role [31, 13]. A similar ultimate explanation may also hold true for M. elongata where choice experiments revealed that females prefer a stimulus in which chirps were constantly timed 140 ms in advance to otherwise identical chirps presented from a different direction [26]. My own results confirm such a preference and show in addition, that the preference does also exist with a time lag between chirps of only 70 ms (figures 15 and 16). Although phonotactic walking paths strongly deviate from straight lines in this stimulus situation,
29 this result indicates that females likely prefer leader males in small choruses as well because there the average time lag between leader and follower signals is 73 ms (figure 7). A leader preference in females may originate from a bias in the sensory system. In a neurophysiologic approach Römer and colleagues [35] revealed an asymmetry in neural representations of leader and follower signals presented from opposite sides in a pair of local prothoracic neurons (omega cells), which is highest when the follower signal is presented with a time delay of 70 - 140 ms. A similar asymmetry in the neuronal representation of leader and follower signals was found in another pair of ascending auditory neurons (TN1) [36]. Both interneurons have in common that they receive excitatory input from the ipsilateral side and inhibitory input from the contralateral side. Owing to these response characteristics of side-homologous auditory neurons, leader males may exploit this sensory bias in females when timing their chirps 70 - 100 ms in advance to competitors. If this is the case, synchrony in a M. elongata chorus emerges as a by-product of inter-male competition for the leader role, which inevitably leads to imperfect synchrony (e.g. [47, 16, 1, 22, 18, 48, 12]). A bias in the sensory system of receivers can be the product of sensory mechanisms that evolved in a non-sexual context [49, 50, 51, 37, 38, 39, 40] and is termed "sensory bias" when it already exists before signallers evolved traits exploiting it ("sensory exploitation hypothesis") [52, 53, 40]. The asymmetrical representation of leader and follower signals in the omega- and TN1 neurons of M. elongata was similar in males and females and appears to result from contralateral inhibition, i.e. to be a consequence of directional hearing, and thus a property of the nervous system that is not necessarily related to mate choice [35, 36]. However, evidence against the "sensory bias" hypothesis in M. elongata was provided in a recent phylogenetic study conducted in the genus Neoconocephalus. There, discontinuously calling species synchronize their calls (except one species) [54, 11],(J. Schul, unpublished observations), but females did not show a leader preference in choice experiments [55, 11]. Whatever the mechanism(s) for the evolution of acoustic synchrony in Mecopoda may have been, they must explain the persistency of leader and follower roles found in duets and choruses, and thus the apparent disadvantage of followers. Males producing follower chirps likely suffer from a lower mating success, and therefore it was suggested that less attractive follower males may use certain strategies to compensate their unattractive role [26]. Indeed, time-intensity-trading experiments revealed
30 that a preference for leader signals can be compensated by increasing the loudness of follower signals by at least 6 dB. However, my own sound recordings of follower males do not indicate that they increase their SPL when contributing to a chorus (data not shown). In addition, a slight increase in the signal amplitude of follower signals caused by signal plasticity [20] may be too weak to compensate the leader advantage found at a delay of 70 ms. However, loud syllables of followers often precisely overlap in time and followers may indeed compensate a leader advantage. In field experiments performed with an Indian Mecopoda species, two other compensatory strategies may allow follower males to compensate the advantage of leader males: Strategic spacing and singing when leaders are quiet [45]. About 60% of follower males in this species called more often when leaders were not calling and spacing enabled quieter males to gain areas where they were the loudest among all males in a chorus. These results demonstrate that followers in the genus M. elongata make use of compenstory strategies in order to increase their attractiveness. It remains to be tested whether such compenstory strategies also exist in the Malaysian M. elongata species. In acoustic interactions, intrinsically slower signalling males have to increase their signal rate in order to produce signals at the same, fast rate compared to leader males [24]. On average follower males decreased signal period by 7% when singing in a chourus compared to solo singing (figure 10). Therefore, followers not only suffer from being unattractive to females but also suffer higher energetic expenditures during acoustic interactions with intrinsically faster signalling males. However, a recent study showed that M. elongata males shortened their follower chirps and in doing so males save energy in acoustic interactions with intrinsically faster singing males [24]. Nevertheless, consistently timing signals as follower seems to be a maladaptive strategy considering the strong preference for leader signals in this species. Therefore, one needs to consider that followers may accrue other benefits, e.g. an increased per capita mating succes caused by a beacon effect, or that there exist trade-offs with signalling as leaders (see below). It is well known that predators and parasitoids may exert a strong selection on the evolution of signalling and signal timing in male aggregations [19, 56, 5]. Captured M. elongata males in their natural habitat sometimes contained fly puparia (personal communication M. Hartbauer). The emerged flies were exemplars of tachinids, a fly family that parasitizes insects. Females of these flies locate singing males by eavesdropping on their acoustic signals. In a related tachnid fly species (Ormia ochracea) a leader preference, similar
31 to that of M. elongata females, was found [32], which points to the intriguing possibility that follower males in synchronous interactions benefit from a lower parasitation rate compared to leader males. Ultimately, a convergent preference of leader signals between M. elongata females and parasitoid flies may result in frequency-dependent selection on signal timing (see also [56]).
4.2 Synchrony and signal recognition In many insect species females recognize conspecific calling songs by evaluation of signal parameters regarded as static (e.g. pulse repetition rate) [1], whereas mate choice is often based on dynamic signal parameters (e.g. signal intensity, chirp rate) [57, 58, 59]. For example, females usually prefer males calling at higher chirp rates (review in Gerhard & Huber [1]), thus select males that are able to invest much in advertisement signals. Extreme values of dynamic signal parameters are regarded as "condition-dependent handicap" due to their correlation with sender quality [60, 61]. In the choice experiments females preferred shorter signal periods in the situation 2 vs 2.5 s, but not in the choice situation 1.5 vs 2 s (figure 15). Additionally, the general motivation to approach any speaker was very low in the choice situations were shorter CPs were used (figure 14). These results are surprising given the preference for higher signal rates in many insect species, where the timing of chirps is more or less random [1]. My results suggest that female preference in M. elongata exerts a stabilizing selection on CPs in the range of 1.5 to 2.0 s. This preference function counteracts a runaway selection towards faster signal periods that has to be expected as the result of a leader preference, thus a selection of males exhibiting intrinsically faster signal rates [24]. Therefore, chirp period mainly conveys information about species identity in M. elongata rather than about the quality of the sender. This would also corroborate results of a diet study where even males in poor condition were able to signal as fast as males set on a high quality diet [21]. The increased SPL of the synchronous display of chirps in a male chorus leads to a preservation of the species-specific signal period, which is especially important for M. elongata females preferring calling songs with a conspecific signal period of 2.0 s over identical, but randomly timed signals with a similar average signal period (figure 15). This result parallels those of behavioral experiments with females of the rhythmically flashing firefly Photinus carolinus, where males in aggregations synchronize their signals in order to reduce visual clutter thereby increasing the chance of a female
32 flash response [16]. In a noisy habitat like the nocturnal rainforest receivers likely benefit from a better detectability of synchronous signals repeated in a fixed interval. Males of M. elongata produce their chirps in synchrony with a conspecific stimulus even at rather low signal-to-noise ratios [62]. This receiver performance seems to be based on the detection of periodic signals, which is an easier task compared to the evaluation of other signal features, such as the syllable rate within the chirp, or other fine-scale temporal features of aperiodic signals.
4.3 The "beacon effect" A major objective of my thesis was to investigate a potential cooperative aspect of chorus synchrony in M. elongata that arises from an increase in peak signal amplitude of synchronous signals. The effect is termed "beacon effect" and was originally proposed for rhythmically flashing fireflies [9]. Indeed, the average SPL of communal acoustic displays recorded in four male choruses showed an increased loudness of about 7.5 dB compared to solo singing males. This increase in SPL was confirmed in the "four loudspeakers experiment", when signals were broadcast in perfect synchrony. Given the syllable structure of Mecopoda signals, the probability of perfect syllable overlap between imperfectly timed chirps is rather low and may contradict the observed beacon effect found in real choruses. However, even a 70 ms delay between chirps broadcast from two loudspeakers relative to the other ones yet caused a 4 dB increase in loudness (table 1). Since followers usually time their chirps in almost perfect synchrony after perceiving the onset of leader signals and produce loud syllables in a shorter period of time (due to signal plasticity) [20], the probability of signal overlap between loud syllables of leader and follower signals in acoustic interactions of M. elongata males is likely to be high. In M. elongata it remains to be examined whether males in a group may increase their individual mating success via females prefering communal signals. Choice experiments performed with Conocephalus nigropleurum revealed that females of this katydid indeed preferred the combined song of two males over the song of a solo singing male [6]. Similarly, Doolan & MacNally [63] showed that males of the cicada Cystosoma saundersii with a high mating success are singing significantly closer to their nearest singing neighbors compared to the average inter-male distance. At the ultimate level, such female preference forces males to signal in aggregations, where they
33 may benefit from increased per capita mating success and lower per capita predation risk due to a predator dilution effect. Both of these group benefits were recently discovered in the lekking wax moth Achroia grisella though signal overlap is a rare event in this species [64]. The SPL of the broadcast signal is only one of several other parameters determining the success in attracting a female. The so-called active space of a signal defines the area over which a signal can be detected by receivers, and the detection threshold of receivers as well as the attenuation of the signal have to be taken into account. Simulating the active space of males in a computer model by implementing realistic values for these parameters from previous studies showed that an increase of chorus participants leads to a strong increase in the active space of a chorus signal when signal overlap is perfect (figure 13). Chirps recorded in a distance of 1 m apart from singing M. elongata males contained signal amplitudes in a frequency range between 10 and 20 kHz that correspond to a SPL of 61 dB. This frequency range is most relevant for katydids because ears are less sensitive to frequencies below 9 kHz and excessive attenuation for ultrasound restricts communication range strongly. Therefore, results shown in figure 13C are most realistic, though based on ideal conditions for signal transmission and signal perception, i.e. little influence of vegetation and background noise. Assuming a random distribution of females, even small groups of males may attract a significantly higher number of females compared to lone singing males. Although this has not been quantified in my thesis, a beacon effect probably leads to an increased per capita mating success of M. elongata males synchronizing their signals in a chorus. We also have to consider the environmental conditions under which communication in M. elongata takes place. The nocturnal rainforest is a noisy habitat, and a beacon effect caused by signal overlap likely increases the conspicuousness of communal mating displays due to an increased signal-to-noise-ratio. Thus, possible group benefits accrued through a beacon effect suggest chorusing in M. elongata to be the outcome of a cooperative act between acoustically interacting males. However, a slight increase in signal rates of males in a chorus also suggests that chorus synchrony is the result of ongoing competition for the attractive leader role (see also [65]) since intrinsically faster singing males would improve signal overlap in a chorus by reducing their signal rate. Therefore, the beacon effect found in small choruses was mainly caused by overlapping follower chirps that focuses signal energy in a short time interval. A beacon effect may originate from an inter-male competition for the attractive leader role and may help to attract a higher
34 number of females to a chorus. Although groups of males may be more attractive to distant females compared to lone singing males, males in groups suffer from higher inter-male competition when females mate only once in a while. As a consequence, a dilution of per capita mating success in a chorus can be expected, and hence to signal as follower not to be the best signaling strategy. However, two counteracting factors may provide a relative advantage for followers: a strong beacon effect of males overlapping their signals may reduce the attractiveness of leaders in a chorus. In addition, parasitoid flies with a similar preference for leaders as females do may stabilize follower roles in evolutionary terms.
35 References
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