Differentiation of morphology, genetics and electric signals in a region of sympatry between sister species of African electric fish (Mormyridae).

Mormyrid fishes produce and sense weak electric organ discharges (EODs) for object detection and communication, and they have been increasingly recognized as useful model organisms for studying signal evolution and speciation. EOD waveform variation can provide important clues to sympatric species boundaries between otherwise similar or morphologically cryptic forms. Endemic to the watersheds of Gabon (Central Africa), Ivindomyrus marchei and Ivindomyrus opdenboschi are morphologically similar to one another. Using morphometric, electrophysiological and molecular characters [cytochrome b sequences and amplified fragment length polymorphism (AFLP) genotypes], we investigated to what extent these nominal mormyrid species have diverged into biological species. Our sampling covered the known distribution of each species with a focus on the Ivindo River, where the two taxa co-occur. An overall pattern of congruence among datasets suggests that I. opdenboschi and I. marchei are mostly distinct. Electric signal analysis showed that EODs of I. opdenboschi tend to have a smaller initial head-positive peak than those of I. marchei, and they often possess a small third waveform peak that is typically absent in EODs of I. marchei. Analysis of sympatric I. opdenboschi and I. marchei populations revealed slight, but significant, genetic partitioning between populations based on AFLP data (F(ST) approximately 0.04). Taken separately, however, none of the characters we evaluated allowed us to discriminate two completely distinct or monophyletic groups. Lack of robust separation on the basis of any single character set may be a consequence of incomplete lineage sorting due to recent ancestry and/or introgressive hybridization. Incongruence between genetic datasets in one individual, which exhibited a mitochondrial haplotype characteristic of I. marchei but nevertheless fell within a genetic cluster of I. opdenboschi based on AFLP genotypes, suggests that a low level of recent hybridization may also be contributing to patterns of character variation in sympatry. Nevertheless, despite less than perfect separability based on any one dataset and inconclusive evidence for complete reproductive isolation between them in the Ivindo River, we find sufficient evidence to support the existence of two distinctive species, I. opdenboschi and I. marchei, even if not 'biological species' in the Mayrian sense.

Androgen correlates of socially induced changes in the electric organ discharge waveform of a mormyrid fish.

Weakly electric fish from the family Mormyridae produce pulsatile electric organ discharges (EODs) for use in communication. For many species, male EODs are seasonally longer in duration than those of females, and among males, there are also individual differences in EOD duration. While EOD elongation can be induced by the administration of exogenous androgens, androgen levels have never before been assessed under natural or seminatural conditions. By simulating the conditions occurring during the breeding season in the laboratory, we provide evidence of a sex difference in EOD duration as well as document levels of circulating androgens in males. In this study, we analyzed the nature of social influences on male EOD duration and plasma androgen levels in Brienomyrus brachyistius. Individual males, first housed with a single female and then placed into social groups consisting of three males and three females, showed status-dependent changes in EOD duration. Top-ranking males experienced a relatively large increase in EOD duration. Second-ranking males experienced a more modest increase, and low-ranking males experienced a decrease in EOD duration. These changes were paralleled by differences in circulating levels of plasma 11-ketotestosterone (11-KT), but not testosterone, suggesting that the changes in EOD duration may have been mediated by changes in plasma 11-KT levels. Thus, it appears that EOD duration is an accurate indicator of male status, which is under social and hormonal control.

Molecular systematics of the African electric fishes (Mormyroidea: teleostei) and a model for the evolution of their electric organs.

We present a new molecular phylogeny for 41 species of African mormyroid electric fishes derived from the 12S, 16S and cytochrome b genes and the nuclear RAG2 gene. From this, we reconstruct the evolution of the complex electric organs of these fishes. Phylogenetic results are generally concordant with earlier preliminary molecular studies of a smaller group of species and with the osteology-based classification of Taverne, which divides the group into the Gymnarchidae and the Mormyridae, with the latter including the subfamilies Petrocephalinae (Petrocephalus) and Mormyrinae (all remaining taxa). However, we find that several genera previously recognized by Taverne are non-monophyletic. Within the Mormyrinae, the genus Myomyrus is the sister group to all the remaining taxa. Other well-supported clades within this group are recovered. A reconstruction of electrocyte evolution on the basis of our best-supported topology suggests that electrocytes with penetrating stalks evolved once early in the history of the mormyrids followed by multiple paedomorphic reversals to electrocytes with non-penetrating stalks.

Design features for electric communication.

How do the communication discharges produced by electric fish evolve to accommodate the unique design features for the modality? Two design features are considered: first, the limited range of signaling imposed on the electric modality by the physics of signal transmission from dipole sources; and second, the absence of signal echoes and reverberations for electric discharges, which are non-propagating electrostatic fields. Electrostatic theory predicts that electric discharges from fish will have a short range because of the inverse cube law of geometric spreading around an electrostatic dipole. From this, one predicts that the costs of signaling will be high when fish attempt to signal over a large distance. Electric fish may economize in signal production whenever possible. For example, some gymnotiform fish appear to be impedance-matched to the resistivity of the water; others modulate the amplitude of their discharge seasonally and diurnally. The fact that electric signals do not propagate, but exist as electrostatic fields, means that, unlike sound signals, electric organ discharges produce no echoes or reverberations. Because temporal information is preserved during signal transmission, receivers may pay close attention to the temporal details of electric signals. As a consequence, electric organs have evolved with mechanisms for controlling the fine structure of electric discharge waveforms.

Time coding in the midbrain of mormyrid electric fish. I. Physiology and anatomy of cells in the nucleus exterolateralis pars anterior.

In mormyrid electric fish, species-specific electric organ discharge waveforms are thought to be analyzed by the Knollenorgan electroreceptor subsystem. The midbrain anterior and posterior exterolateral nuclei (ELa and ELp) are thought to be the sites of this analysis. This paper is an electrophysiological study of the properties of the neurons in ELa. We recorded intracellularly from three classes of cells within ELa: the afferent axons from the nucleus of the electrosensory lateral line lobe (NELL), the large interstitial cells of ELa and an unidentified cell type. The large cells and the NELL axons were identified by intracellular injection of biocytin and are physiologically similar. Cells in ELa responded to square pulse stimuli with one or more time-locked action potentials with 2.8-3.0 ms latency. Both large cells and NELL axons arborized extensively in ELa and contacted numerous small cells. Based on the pattern of arborizations, we constructed a counter-current flow model of temporal coding by the small cells of ELa. We postulate that individual small cells are not selectively tuned for specific stimulus durations, but rather, the firing patterns of groups of small cells must be analyzed by neurons further up in the sensory hierarchy to determine the stimulus duration.

Neural substrates for species recognition in the time-coding electrosensory pathway of mormyrid electric fish.

Mormyrid electric fish have species- and sex-typical electric organ discharges (EODs). One class of tuberous electroreceptors, the knollenorgans, plays a critical role in electric communication; one function is species recognition of EOD waveforms. In this paper, we describe cell types in the knollenorgan central pathway, which appear responsible for analysis of the temporal patterns of spikes encoded by the knollenorgans in response to EOD stimuli. Secondary sensory neurons in the nucleus of the electrosensory lateral line lobe (NELL) act as relays of peripheral responses. They fire a single phase-locked spike to an outside positive-going voltage step. Axons from the NELL project to the toral nucleus exterolateralis pars anterior (ELa). Immediately after they enter the ELa, they send collaterals to terminate on one to three ELa large cells and then continue in a lengthy neuronal pathway that traverses the ELa several times. After a path length of up to 5 mm, the NELL axon terminates on as many as 70 ELa small cells. Thus the large cells appear to be excited first, followed by the small cells, with the intervening length of the axon serving as a delay line. The large cells also respond with phase-locked spikes to voltage steps. Large cell axons extend for approximately 1 mm and terminate on several small cells within the ELa. The terminals are known to be GABAergic inputs and are presumed inhibitory. We propose that small cells receive direct inhibition from large cells and delayed excitation from NELL axons. The small cells may act as anti-co-incidence detectors to analyze the temporal structure of the EOD waveform.

Molecular insights into the phylogeny of mormyriform fishes and the evolution of their electric organs.

In this report we generate a partial phylogeny of the mormyriform fishes using mitochondrial DNA sequences from twelve species of mormyriforms belonging to five genera. Electric organs and electric organ discharges are also examined. We have sequenced and aligned 373 bases from the mitochondrial 12S rRNA and 559 bases from the 16s rRNA from fourteen species of the superorder Osteoglossomorpha. Two non-mormyriform genera were used as outgroups. Three phylogenetic methods generated concordant partial phylogenies for these fish. Our analysis focuses on the genus Brienomyrus, which is a heterogeneous clade with at least eleven nominal species. Six morphs from Gabon had distinctive EODs but were morphologically 'cryptic' in that they all had the brachyistius-like body morphology. DNA analysis fully supports the EOD data that the six morphs represent distinct clades. The group from Gabon is monophyletic, while B. brachyistius from West Africa is a separate lineage. B. niger, a second distinct lineage, is a sister group to the six species from Gabon. Petrocephalus is the sister group of all the genera of the subfamily Mormyrinae so far analyzed, thereby confirming previous osteological results. Gymnarchus niloticus is the sister group of the family Mormyridae, also confirming an earlier phylogenetic hypothesis based on morphology. The molecular data adds polarity to electric organ characteristics. Stalkless electrocytes appear to be primitive. Petrocephalus, with non-penetrating stalked electrocytes innervated on the posterior side, represents an ancestral state for the Mormyridae, while Marcusenius. Brienomyrus and Gnathonemus with penetrating-stalked electrocytes, represent the apomorphic condition. Two species with doubly-penetrating electrocytes innervated on the posterior side may represent a transitional stage. At least two species of Brienomyrus appear to have reverted to non-penetrating stalked electrocytes, possibly through paedomorphosis.

A quantitative analysis of passive electrolocation behavior in electric fish.

Weakly electric fish of the families Gymnotidae and Hypopomidae (Gymnotiformes) are able to locate the electric discharges from conspecifics or from dipole electrodes, and they demonstrate this by making rapid, well-directed approaches toward these electrical sources. A video tracking system was used to follow the movements of electric fish in a large tank and an analytic method was used for computing the direction and magnitude of the electric field anywhere within the cylindrical test tank. Using a static analysis method, we describe the posture of test fish relative to the electric fields during their approaches to stationary or moving electrical stimuli. Using a dynamic analysis, we examine the movements of the fish including the sign and magnitude of velocity and bending in response to electric fields. Electric fish seek to maintain a zero error angle between their body orientation and the local electric field. They do so by bending their body in the direction of the local electric field. The response has a delay of approximately 0.5 s. Swimming in reverse inverts the direction of the bend. These fish also use 'V-turns' to redirect their swim directions when encountering rapidly-changing electric fields.

Short-range orientation in electric fish: an experimental study of passive electrolocation.

Gymnotiform electric fish are capable of locating and approaching an electrically discharging conspecific over a range of 1-2 m in a behavior called passive electrolocation. This paper investigates the movements of two species in experiments with approaches to stationary dipoles that are either silenced or jumped to a new direction during an approach. Gymnotus carapo fail to find an electrode source in trials in which the dipole electrode is switched off in mid-track. They slow their approach, become disoriented and drift away from the target within seconds of the field being switched off. This result suggests that the fish are unable to construct a cognitive map of a dipole source from brief exposure to local electrosensory stimuli. The second set of trials shows that Brachyhypopomus diazi and Gymnotus carapo bend their body to track electric vectors which are suddenly jumped to a new direction. The latency of the bend response is 0.5 s after the jump. Bending initiates a turn that reduces to zero the error between the fish's direction and the electric field vector and helps keep the fish aligned with the local electric field vector. Together, these experiments suggest that passive electrolocation is stimulus-bound and that these fish find the electrical sources simply by tracking instantaneous local electric current vectors.

The dorsal filament of the weakly electric Apteronotidae (Gymnotiformes; Teleostei) is specialized for electroreception.

The Apteronotidae, a family of weakly electric fish from South America (Gymnotiformes), possess a structure called the dorsal filament with an unknown function and evolutionary origin. This study compared the gross anatomy of the dorsal filament of 13 species of apteronotids and used light microscopy to examine the filaments of Adontosternarchus balaenops, Apteronotus albifrons, and Apteronotus leptorhynchus. The dorsal filament is an unscaled, thin, tapering structure attached to a mid-dorsal groove on the posterior half of the fish's back. The interior of the filament is a gelatinous mucopolysaccharide matrix (connective tissue) containing blood vessels and a bilateral nerve in which nearly all the afferents are large (8-10 mu m) and heavily myelinated. The location of the anterior origin of the filament varies from 0.48 to 0.66 of the body length, posterior to the snout, in 13 species. The filament is covered with hundreds of large-type tuberous electroreceptors and some ampullary receptors, at approximately the same density and ratio as those on the nearby back. The morphology of the large-type tuberous receptors and their afferents suggests that they are phase-coding T-units. A double layer of epithelial cells separates the ventral side of the filament from the groove in the trunk of the fish, except at the anterior origin where the interior of the filament is continuous with the body. This specialized double epithelium could provide a high resistance barrier to electrical current. This study was unable to distinguish between two hypotheses: that the dorsal filament is a modified adipose fin (as suggested previously), retained only in this family of Gymnotiformes; or that it is a uniquely derived character of the Apteronotidae.

Convergent designs for electrogenesis and electroreception.

New- and old-world tropical electric fish lack a common electrical ancestor, suggesting that the mechanisms of signal generation and recognition evolved independently in the two groups. Recent research on convergent designs for electrogenesis and electroreception has focused on the structure of electric organs, the neural circuitry controlling the pacemaker driving the electric organ, and the neural circuitry underlying time coding of electric waveforms.

Directional characteristics of tuberous electroreceptors in the weakly electric fish, Hypopomus (Gymnotiformes).

This paper is an electrophysiological study of the directionality of the tuberous electroreceptors of weakly electric fish. We recorded from two classes of tuberous electroreceptors known for pulse gymnotiforms: Burst Duration Coders (BDCs), and Pulse Markers (PMs). Both code for stimulus amplitude, although the dynamic range for BDCs is greater, and both exhibit strong directional preferences. Polar plots of spike number (for BDCs) or spike threshold (for PMs) versus electric field azimuth, are figure-8 shaped with two asymmetrical, elliptical lobes separated by 180 degrees. The best azimuth of these two types of receptors from a given body region correlate with each other and with measures of best azimuth for transepidermal current flow. The shape and asymmetry of the directionality profiles appear to be caused by filter dynamics of the receptors. Pulse Markers are located on the anterior part of the body surface while Burst Duration Coders are located all over. The best directions of receptors in the anterior third of the body vary systematically with location from 0 degrees to 180 degrees. This region is probably critical for determining the direction of local electric fields. Together these receptors provide the CNS with sufficient information to construct a map of horizontal plane electric field directions.

Directional sensitivity of tuberous electroreceptors: polarity preferences and frequency tuning.

This paper examines the directionality of tuberous electroreceptor responses and relates them to a polarity bias seen for passive electrolocation by electric fish (Hypopomus). We recorded from Burst Duration Coders (BDCs) while stimulating with 1 kHz single period sine waves with electric fields oriented horizontally in different directions. Electroreceptors have figure-8 directional sensitivity profiles with two, usually unequal lobes of sensitivity separated by 180 degrees. For most units the larger lobe points inward, while for a few, the lobes are symmetrical or the larger lobe points outward. The differences correlate with differences in frequency tuning of the receptors. We can alter, and even reverse, the directional asymmetry of a single unit by changing the frequency of the stimulus. Two general response profiles results, with two corresponding classes of tuning curves. The degree of asymmetries and the effects of stimulus frequency and of tuning can be modeled with a linear/non-linear/linear cascade filter. The behavioral preference for approaching the head end (+) of an electrode is difficult to understand in light of the asymmetry of responses we report for amplitude-coding BDCs but can be understood by reference to the time-coding Pulse Marker (PM) receptors.

Functional analysis of sexual dimorphism in an electric fish, Hypopomus pinnicaudatus, order Gymnotiformes.

Hypopomus pinnicaudatus, an electric fish, has a marked sexual dimorphism in its tail filament. Sexually mature males have long, 'feathered' tails as compared with females. The sexual dimorphism emerges when a fish reaches about 110 mm total length. Mature males have larger electrocytes which are more widely spaced and more numerous than those in mature females. The biphasic electric organ discharge (EOD) is longer in males than in females. The peak-to-peak amplitude of the male's EOD is weaker than a female's of the same total length. The weaker discharge is unexpected given the increase in size and number of electrocytes. It is suggested that the reduction in EOD amplitude is a consequence of the increase in EOD duration among males. Further, female choice probably played a role in the evolution of long duration EODs among males, and males may have secondarily grown long tails to compensate for the loss in active space that would otherwise accompany a weaker EOD.

Electric fish approach stationary signal sources by following electric current lines.

African electric fish of a pulse species, Brienomyrus brachyistius (Mormyridae), housed singly in a large, circular arena, were presented with electrical stimuli which mimicked a conspecific intruder. Stimuli were produced with either dipolar or bipolar electrodes in three different geometries. We tracked the unconditioned approach response paths taken by the fish and compared tracks for each of the geometries. The results suggest that B. brachyistius can determine neither the distance nor the direction of an electric dipole from afar, but that they do manage to find the source by maintaining a precise alignment of their body axis parallel to the direction of the local electric field vector (parallel to current lines) while swimming. This behaviour ultimately leads to the current source. We propose that this behaviour may be a simple mechanism mediating the approach response of one electric fish to another.

Acoustic communication in an electric fish, Pollimyrus isidori (Mormyridae).

It has been known since von Frisch's work in the 1930's that mormyrid electric fishes are quite sensitive to sound. We now describe a repertoire of natural sounds produced by the mormyrid, Pollimyrus isidori, during breeding and aggression; reception of communication sounds is probably a major function for mormyrid audition. In aquaria, Pollimyrus isidori produce 'grunts', 'moans', 'growls', 'pops' and 'hoots' at various phases during nesting, courtship, and territory defense. All five sounds are produced primarily at night. Territorial males produce grunts, moans and growls during courtship. Vocalizing is stimulated by the presence of a gravid female on the male's territory and decreases with the onset of spawning. Hoots and pops are given during agonistic behavior. Grunts are bursts of acoustic pulses, stereotyped for an individual, with the potential as individual signatures. The electric organ is silent during grunts and moans and is discharged at a reduced rate during growls. The courtship and spawning of Pollimyrus isidori is described.

Temporal structure of non-propagated electric communication signals.

Acoustic and electric communication differ in one important respect: while acoustic communication signals propagate through air or water as a wave, electric signals do not propagate, but exist instead as electrostatic fields. As a result of propagation, acoustic signals are distorted during transmission in a largely unpredictable way. Sound receivers, therefore, may not be able to recognize fine details in the waveform of an acoustic signal, but may have to rely instead upon time intervals between repetitions of a waveform, on the frequency of a signal, or on frequency modulations. By contrast, non-propagating electric communication signals are immune to many sources of signal distortion that affect sounds. Consequently, electric signal receivers may reliably use waveform cues to recognize a sender's identity and discriminate between signals. As examples, mormyrid electric fish encode species and sex differences in the fine structure of the electric organ discharge waveform and sense the differences using temporal cues. Gymnotiform pulse-discharging electric fish may employ scan-sampling for waveform analysis: a specialized mechanism analogous to a digital sampling oscilloscope for slowly scanning a repetitive waveform.

Time domain processing of electric organ discharge waveforms by pulse-type electric fish.

We explored coastal streams, rivers, and swamps in the Guianas of South America and found eleven species of gymnotiform fishes with pulse discharges. Each species has a characteristic electric organ discharge (EOD) waveform of 0.5-5 ms duration; at least two species appear to have a natural sex difference in their EODs which is apparent when comparing large adult males and females. Three sensory coding mechanisms are proposed to explain how electric fish might be able to determine species and sex identity from such short electrical pulses. Spectral Coding: electroreceptors tuned to different frequencies encode the spectrum of the EOD as a cross-fiber stimulation pattern. Temporal Coding: EODs are encoded as a volley of nerve spikes patterned in the time domain. Scan Sampling: a receiver detects a signaler's EOD as an amplitude modulation or 'beat' set up by the combination of its own discharge with the signaler's. The receiver uses the modulation envelope to assess the signaler's EOD waveform. To distinguish between these three coding mechanisms, we tested the ability of one pulse gymnotiform, Hypopomus beebei, to discriminate one electric waveform from another by comparing the acceleration of the discharge rate to different stimuli. Stimuli are presented under two conditions: when the stimulus pulse train is free-running compared to the fish's pulse train, and when the stimulus train is phase-locked to the fish's discharge pulse train. Under the former condition scan sampling may be used; under the latter it will be impossible. Hypopomus discriminates the polarity of a single period sinusoidal stimulus under scanning conditions but does not discriminate under clamped conditions. Hypopomus gives the strongest response to single period sine waves of 670 Hz and weaker responses to sinusoids of lower and higher frequencies. Free-running and phase-locked stimuli evoke similar responses. Under free-running conditions, Hypopomus discriminates a series of EOD-like stimuli that have been phase-shifted by varying amounts, but under phase-locked conditions does not. Scan sampling is presented as a possible waveform recognition mechanism for pulse-discharging gymnotiform fishes.

Hormonal control of sexual differentiation: changes in electric organ discharge waveform.

Males and females of some mormyrid electric fishes generate electrical pulses that differ in waveform and duration. For one such species, testosterone or dihydrotestosterone induces females and immature males to produce the mature male electric organ discharge which is two times the duration of the female or immature discharge. Estradiol has only a weak effect. For a second species where males and females have similar electric organ discharges, testosterone produces no effect. The data suggest that androgens affect the electric organ itself.