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The Journal of Experimental Biology 209, 2182-2198 Published by The Company of Biologists 2006 doi:10.1242/jeb.02239 Time-domain signal divergence and discrimination without receptor modification in sympatric morphs of electric fishes
Matthew E. Arnegard*, B. Scott Jackson and Carl D. Hopkins
Department of Neurobiology and Behavior, Cornell University, Ithaca, NY 14853, USA
*Author for correspondence (e-mail: mea21@cornell.edu) Accepted 22 March 2006 Summary heterotypic EODs in our experimental paradigm. Part of Polymorphism in an animal communication channel this behavioral asymmetry may have resulted from a provides a framework for studying proximate rules of previously undetected difference in adult size, which may signal design as well as ultimate mechanisms of signal have enhanced apparent discrimination by the smaller diversification. Reproductively isolated mormyrid fishes morph (type II) due to a relatively higher risk of injury from Gabon's Brienomyrus species flock emit distinctive from the larger morph (type I). Knollenorgan receptors, electric organ discharges (EODs) thought to function in which mediate electrical communication in mormyrids, species and sex recognition. Species boundaries and EODs showed similar spectral tuning in type I and type II. These appear congruent in these fishes, with the notable electroreceptors coded temporal features of any single exception of three morphs designated types I, II and III. magnostipes-complex EOD with similar patterns of timeWithin the species flock, these morphs compose a locked spikes in both morphs. By contrast, Knollenorgans monophyletic group that has recently been called the exhibited distinctive responses to different EOD magnostipes complex. Co-occurring morphs of this waveforms. These results suggest that discrete EOD complex express distinctive EODs, yet they appear variation in this rapidly diversifying complex is functional genetically indistinguishable at several nuclear loci. In this in terms of morph-specific advertisement and recognition. study, we investigated EOD discrimination by these Time-domain signal divergence has outpaced frequencymorphs using both behavioral and physiological domain divergence between sympatric morphs, requiring experiments. During the breeding season, wild-caught little to no change in receptor response properties. We type I and type II males showed evidence that they can discuss our findings in light of a model for EOD timediscriminate their own morph's EOD waveform from that coding by the Knollenorgan pathway, as well as of a sympatric and genetically distinct reference species. evolutionary hypotheses concerning sympatric signal However, we found that type I and type II males exhibited diversification in the magnostipes complex. an asymmetry in unconditioned responses to paired playback of EODs recorded from type I versus type II females. Males of the type II morph responded Key words: Mormyridae, playback experiment, Knollenorgan preferentially to EODs of type II females, whereas type I receptor, tuning curve, temporal coding, signal design, multiple EOD dimorphisms. males did not appear to discriminate homotypic and Introduction Variation in courtship signals among closely related species can arise when signals display a potential mate's genetic lineage, quality and/or offspring mating potential. While receptor tuning is often optimized for the detection of such signals (Gerhardt and Huber, 2002), the sensory side of these signaling systems also involves mate recognition and preference. For example, intersexual selection by mate choice can play an important role in driving signal divergence and speciation among populations (Masta and Maddison, 2002; Allender et al., 2003). It has also been proposed that pre-existing biases in preferences are involved in the origin of signals within some lineages (Ryan, 1990; Endler and Basolo, 1998; Wilczynski et al., 2001). A widespread yet currently underappreciated cause of interspecific diversification in sexually isolating signals and mating preferences involves species recognition in conjunction with deleterious fitness consequences of heterotypic or hybrid matings (Servedio and Noor, 2003). Regardless of the combination of influences that affects signal divergence, important interactions between the production and reception sides of communication are expected during the evolution of signaling systems. A recently discovered species flock of African mormyrid THE JOURNAL OF EXPERIMENTAL BIOLOGY Mormyrid EOD discrimination and coding 2183
fishes (Sullivan et al., 2002) is well suited for studying signal diversification because its constituent species exhibit interspecific variation in a relatively simple component of an electrical communication system. All mormyrid fishes generate brief, pulse-like electric organ discharges (EODs) by means of an electric organ in their caudal peduncle (Bennett, 1971a). Different classes of electroreceptors underlie active electrolocation and communication by detecting the distortions in self-generated electric fields or the EODs of other individuals, respectively (Hopkins, 1986; Kramer, 1990; Moller, 1995; von der Emde, 1999). Within individuals, the sequence of pulse intervals (SPI) between successive EODs varies rapidly in different electrolocating or social contexts (e.g. Arnegard and Carlson, 2005). By contrast, the comparatively fixed waveform of each EOD pulse is shaped by much slower modifications in the anatomy and physiology of the electric organ (Bass and Hopkins, 1983; Bass, 1986; Caputi et al., 2005). Despite EOD elongation in breeding males, mormyrids living in speciesrich assemblages are characterized by rather stereotyped, species-typical EODs that are amenable to quantitative comparison (Hopkins, 1999; Arnegard and Hopkins, 2003; Lavoué et al., 2004; Feulner et al., 2006). The largest known radiation of EODs among closely related species occurs in a riverine species flock from Gabon (Central Africa), members of which have been assigned to the genus Brienomyrus (Sullivan et al., 2002). Sympatric populations of Brienomyrus that differ in appearance and EOD waveform are reproductively isolated from one another (Arnegard et al., 2005). Extensive interspecific signal diversification motivates the hypothesis that EOD discrimination contributes to species recognition in this group of fishes. Several behavioral studies have provided strong evidence that mormyrids can recognize species, sex and/or individuals based on EOD variation (Hopkins and Bass, 1981; Graff and Kramer, 1992; Paintner and Kramer, 2003; Hanika and Kramer, 2005). Among the different kinds of electroreceptors possessed by mormyrids, Knollenorgans are responsible for communication (Bennett, 1965; Moller and Szabo, 1981; Hopkins, 1986; Bell and Grant, 1989; Paintner and Kramer, 2003). The Knollenorgan pathway is briefly inhibited each time a fish fires its own electric organ, resulting in a selective responsiveness to EODs produced by other individuals rather than to selfgenerated EODs (Zipser and Bennett, 1976; Mugnaini and Maler, 1987; Bell and Grant, 1989). Knollenorgan cells fire spike-like receptor potentials that are time locked to outside negative-to-positive (NrP) voltage transients (Bennett, 1971b; Szabo and Fessard, 1974). When receptors are activated in nature by an EOD from another fish, Knollenorgans on opposite sides of an individual's body respond to different phases of the stimulus. NrP transients in one polarity of an EOD waveform experienced on one side of the body become PrN transients in the opposite polarity waveform experienced on the other side of the body. The result is fixed latencies between Knollenorgan spiking on opposite sides of the receiver's body in response to a single EOD. In this way, mormyrids are thought to distinguish EOD features, such as overall duration, by comparing spike latencies arising from opposing body regions (Hopkins and Bass, 1981). Such a comparison apparently takes place within the midbrain torus semicircularis (Xu-Friedman and Hopkins, 1999). An emerging model system for studying sympatric signal diversification Our study focuses on the magnostipes complex (Sullivan et al., 2004), which is nested within Gabon's Brienomyrus species flock and contains three morphs called types I, II and III (Arnegard et al., 2005). Similarly sized individuals of cooccurring morphs cannot be distinguished on the basis of external appearance. Rather, the morphs are defined by their characteristic EOD waveforms (Fig.·1). One or two morphs can be found at each of several sites throughout Gabon (Fig.·2). Collections made from the Ivindo River between the middle of September and early December (i.e. at the beginning of a long, bimodal rainy season) have revealed that mature type I and type II males are more likely to display elongated EODs during this period of breeding activity (Fig.·1). At other sites, males and females of a type III morph have also been collected, although sampling has been insufficient to ascertain the degree of seasonal waveform elongation by type III males. Wherever the morphs co-occur, one is always type I; the other can be either type II or type III (Fig.·2). Measurable differences in power spectra exist among EODs of the magnostipes complex (Table·1), yet it is the time-domain waveform that differs most obviously between sympatric morphs (Fig.·1). For example, the first of two major waveform peaks is head-positive in the EOD of type II. Head-positivity corresponds to current inside the animal flowing in the direction of the head (i.e. an electrode in the environment near the head is positive relative to an electrode near the tail). In the case of type I, the first major peak in the EOD is headnegative. In contrast to other sympatric mormyrids exhibiting distinctive EODs, reproductive isolation has not been genetically demonstrated between magnostipes-complex morphs. No robust differences at five microsatellite loci occur between sympatric morphs despite their strikingly different EODs. Nevertheless, allopatric populations of these morphs exhibit signs of strong genetic isolation from one another (Arnegard et al., 2005). The magnostipes complex is a particularly promising system with which to study sympatric signal divergence due to the genetic similarity of co-occurring morphs and the natural replication that exists in the form of multiple dimorphic populations. Knowledge of whether these morphs respond to differences in the EODs they produce is critical to understanding the origins and significance of polymorphism in their electric signals. Here, we report on two sets of experiments in which we tested whether EODs can potentially mediate morph recognition: (1) paired playbacks of female EODs to breeding males; and (2) electrophysiological THE JOURNAL OF EXPERIMENTAL BIOLOGY 2184 M. E. Arnegard, B. S. Jackson and C. D. Hopkins A
Type I (Ivindo River) females & non-breeding males (N=18) Type I (Ivindo River) breeding males (N=18) Type II (Ivindo River) females & non-breeding males (N=18) Type II (Ivindo River) breeding males (N=23) Type III (Okano River) females & males combined (N=14) 1 ms 0 ­20 ­40 ­60 ­80 ­100 ­120 ­140 B dB 102 103 104 102 103 104 102 103 104 Frequency (Hz) 102 103 104 102 103 104 Fig.·1. Examples of electric pulses produced by three morphs of the magnostipes complex. (A) Time-domain EOD waveforms of multiple individuals. All voltage traces are standardized to the same peak-to-peak amplitude, plotted as overlays of waveforms recorded from N unique individuals and aligned by their head-positive peaks (with head-positivity up). Type III waveforms (all from a site near the village Na) were recorded in previous studies (Sullivan et al., 2004; Arnegard et al., 2005). (B) Power spectra calculated for the same EODs. Red voltage traces and power spectra correspond to DC recordings (bandwidth 0­50·kHz). All other recordings in black were AC-coupled (bandwidth 0.1·Hz­50·kHz). AC-coupling did not detectably alter type I and type II female-like EODs relative to DC recordings. Some measurements of EOD power spectra (also extremely similar between morphs) were previously provided for Ivindo River type I and type II females and nonbreeding males by Hopkins, who referred to them as Hippopotamyrus batesii `reverse polarity' and H. batesii `triphasic', respectively (Hopkins, 1981). Note also that Hopkins shows a breeding male EOD of the type I morph in the right column of his fig.·2 (Hopkins, 1981). characterization of Knollenorgan responses to different EOD waveforms. Materials and methods Field playback experiments during the breeding season In 2002, we collected adult specimens of the magnostipes complex in the Makokou region of the Ivindo River from September to November, a period when heavy rain causes floodplain inundation and triggers breeding in many mormyrid species (Fig.·2, inset). Individuals were obtained from the main channel of the Ivindo River (up to ~500·m wide) and several small tributaries (