Jason R. Gallant

Genomic basis for the convergent evolution of electric organs

Only one way to make an electric organ? Electric fish have independently evolved electric organs that help them to communicate, navigate, hunt, and defend themselves. Gallant et al. analyzed the genome of the electric eel and the genes expressed in two other distantly related electric fish. The same genes were recruited within the different species to make evolutionarily new structures that function similarly. Science, this issue p. 1522 Multiple divergent fish lineages have used the same evolutionary toolkit to produce electric organs. Little is known about the genetic basis of convergent traits that originate repeatedly over broad taxonomic scales. The myogenic electric organ has evolved six times in fishes to produce electric fields used in communication, navigation, predation, or defense. We have examined the genomic basis of the convergent anatomical and physiological origins of these organs by assembling the genome of the electric eel (Electrophorus electricus) and sequencing electric organ and skeletal muscle transcriptomes from three lineages that have independently evolved electric organs. Our results indicate that, despite millions of years of evolution and large differences in the morphology of electric organ cells, independent lineages have leveraged similar transcription factors and developmental and cellular pathways in the evolution of electric organs.

Ancient homology underlies adaptive mimetic diversity across butterflies

Convergent evolution provides a rare, natural experiment with which to test the predictability of adaptation at the molecular level. Little is known about the molecular basis of convergence over macro-evolutionary timescales. Here we use a combination of positional cloning, population genomic resequencing, association mapping and developmental data to demonstrate that positionally orthologous nucleotide variants in the upstream region of the same gene, WntA, are responsible for parallel mimetic variation in two butterfly lineages that diverged >65 million years ago. Furthermore, characterization of spatial patterns of WntA expression during development suggests that alternative regulatory mechanisms underlie wing pattern variation in each system. Taken together, our results reveal a strikingly predictable molecular basis for phenotypic convergence over deep evolutionary time.

Signal variation and its morphological correlates in Paramormyrops kingsleyae provide insight into the evolution of electrogenic signal diversity in mormyrid electric fish

We describe patterns of geographic variation in electric signal waveforms among populations of the mormyrid electric fish species Paramormyrops kingsleyae. This analysis includes study of electric organs and electric organ discharge (EOD) signals from 553 specimens collected from 12 localities in Gabon, West-Central Africa from 1998 to 2009. We measured time, slope, and voltage values from nine defined EOD “landmarks” and determined peak spectral frequencies from each waveform; these data were subjected to principal components analysis. The majority of variation in EODs is explained by two factors: the first related to EOD duration, the second related to the magnitude of the weak head-negative pre-potential, P0. Both factors varied clinally across Gabon. EODs are shorter in eastern Gabon and longer in western Gabon. Peak P0 is slightly larger in northern Gabon and smaller in southern Gabon. P0 in the EOD is due to the presence of penetrating-stalked (Pa) electrocytes in the electric organ while absence is due to the presence of non-penetrating stalked electrocytes (NPp). Across Gabon, the majority of P. kingsleyae populations surveyed have only individuals with P0-present EODs and Pa electrocytes. We discovered two geographically distinct populations, isolated from others by barriers to migration, where all individuals have P0-absent EODs with NPp electrocytes. At two sites along a boundary between P0-absent and P0-present populations, P0-absent and P0-present individuals were found in sympatry; specimens collected there had electric organs of intermediate morphology. This pattern of geographic variation in EODs is considered in the context of current phylogenetic work. Multiple independent paedomorphic losses of penetrating stalked electrocytes have occurred within five Paramormyrops species and seven genera of mormyrids. We suggest that this key anatomical feature in EOD signal evolution may be under a simple mechanism of genetic control, and may be easily influenced by selection or drift throughout the evolutionary history of mormyrids.

Differential expression of genes and proteins between electric organ and skeletal muscle in the mormyrid electric fish Brienomyrus brachyistius

SUMMARY Electric organs (EOs) have evolved independently in vertebrates six times from skeletal muscle (SM). The transcriptional changes accompanying this developmental transformation are not presently well understood. Mormyrids and gymnotiforms are two highly convergent groups of weakly electric fish that have independently evolved EOs: while much is known about development and gene expression in gymnotiforms, very little is known about development and gene expression in mormyrids. This lack of data limits prospects for comparative work. We report here on the characterization of 28 differentially expressed genes between SM and EO tissues in the mormyrid Brienomyrus brachyistius, which were identified using suppressive subtractive hybridization (SSH). Forward and reverse SSH was performed on tissue samples of EO and SM resulting in one cDNA library enriched with mRNAs expressed in EO, and a second library representing mRNAs unique to SM. Nineteen expressed sequence tags (ESTs) were identified in EO and nine were identified in SM using BLAST searching of Danio rerio sequences available in NCBI databases. We confirmed differential expression of all 28 ESTs using RT-PCR. In EO, these ESTs represent four classes of proteins: (1) ion pumps, including the α- and β-subunits of Na+/K+-ATPase, and a plasma membrane Ca2+-ATPase; (2) Ca2+-binding protein S100, several parvalbumin paralogs, calcyclin-binding protein and neurogranin; (3) sarcomeric proteins troponin I, myosin heavy chain and actin-related protein complex subunit 3 (Arcp3); and (4) the transcription factors enhancer of rudimentary homolog (ERH) and myocyte enhancer factor 2A (MEF2A). Immunohistochemistry and western blotting were used to demonstrate the translation of seven proteins (myosin heavy chain, Na+/K+-ATPase, plasma membrane Ca2+-ATPase, MEF2, troponin and parvalbumin) and their cellular localization in EO and SM. Our findings suggest that mormyrids express several paralogs of muscle-specific genes and the proteins they encode in EOs, unlike gymnotiforms, which may post-transcriptionally repress several sarcomeric proteins. In spite of the similarity in the physiology and function of EOs in mormyrids and gymnotiforms, this study indicates that the mechanisms of development in the two groups may be considerably different.

From Sequence to Spike to Spark: Evo-devo-neuroethology of Electric Communication in Mormyrid Fishes

Abstract Mormyrid fishes communicate using pulses of electricity, conveying information about their identity, behavioral state, and location. They have long been used as neuroethological model systems because they are uniquely suited to identifying cellular mechanisms for behavior. They are also remarkably diverse, and they have recently emerged as a model system for studying how communication systems may influence the process of speciation. These two lines of inquiry have now converged, generating insights into the neural basis of evolutionary change in behavior, as well as the influence of sensory and motor systems on behavioral diversification and speciation. Here, we review the mechanisms of electric signal generation, reception, and analysis and relate these to our current understanding of the evolution and development of electromotor and electrosensory systems. We highlight the enormous potential of mormyrids for studying evolutionary developmental mechanisms of behavioral diversification, and make the case for developing genomic and transcriptomic resources. A complete mormyrid genome sequence would enable studies that extend our understanding of mormyrid behavior to the molecular level by linking morphological and physiological mechanisms to their genetic basis. Applied in a comparative framework, genomic resources would facilitate analysis of evolutionary processes underlying mormyrid diversification, reveal the genetic basis of species differences in behavior, and illuminate the origins of a novel vertebrate sensory and motor system. Genomic approaches to studying the evo-devo-neuroethology of mormyrid communication represent a deeply integrative approach to understanding the evolution, function, development, and mechanisms of behavior.

Unique patterns of transcript and miRNA expression in the South American strong voltage electric eel (Electrophorus electricus)

BackgroundWith its unique ability to produce high-voltage electric discharges in excess of 600 volts, the South American strong voltage electric eel (Electrophorus electricus) has played an important role in the history of science. Remarkably little is understood about the molecular nature of its electric organs.ResultsWe present an in-depth analysis of the genome of E. electricus, including the transcriptomes of eight mature tissues: brain, spinal cord, kidney, heart, skeletal muscle, Sachs’ electric organ, main electric organ, and Hunter’s electric organ. A gene set enrichment analysis based on gene ontology reveals enriched functions in all three electric organs related to transmembrane transport, androgen binding, and signaling. This study also represents the first analysis of miRNA in electric fish. It identified a number of miRNAs displaying electric organ-specific expression patterns, including one novel miRNA highly over-expressed in all three electric organs of E. electricus. All three electric organ tissues also express three conserved miRNAs that have been reported to inhibit muscle development in mammals, suggesting that miRNA-dependent regulation of gene expression might play an important role in specifying an electric organ identity from its muscle precursor. These miRNA data were supported using another complete miRNA profile from muscle and electric organ tissues of a second gymnotiform species.ConclusionsOur work on the E. electricus genome and eight tissue-specific gene expression profiles will greatly facilitate future research on determining the coding and regulatory sequences that specify the function, development, and evolution of electric organs. Moreover, these data and future studies will be informed by the first comprehensive analysis of miRNA expression in an electric fish presented here.

Electric fish genomics: Progress, prospects, and new tools for neuroethology

Electric fish have served as a model system in biology since the 18th century, providing deep insight into the nature of bioelectrogenesis, the molecular structure of the synapse, and brain circuitry underlying complex behavior. Neuroethologists have collected extensive phenotypic data that span biological levels of analysis from molecules to ecosystems. This phenotypic data, together with genomic resources obtained over the past decades, have motivated new and exciting hypotheses that position the weakly electric fish model to address fundamental 21st century biological questions. This review article considers the molecular data collected for weakly electric fish over the past three decades, and the insights that data of this nature has motivated. For readers relatively new to molecular genetics techniques, we also provide a table of terminology aimed at clarifying the numerous acronyms and techniques that accompany this field. Next, we pose a research agenda for expanding genomic resources for electric fish research over the next 10years. We conclude by considering some of the exciting research prospects for neuroethology that electric fish genomics may offer over the coming decades, if the electric fish community is successful in these endeavors.

Electrostatic Tuning of a Potassium Channel in Electric Fish

Molecular variation contributes to the evolution of adaptive phenotypes, though it is often difficult to understand precisely how. The adaptively significant electric organ discharge behavior of weakly electric fish is the direct result of biophysical membrane properties set by ion channels. Here, we describe a voltage-gated potassium-channel gene in African electric fishes that is under positive selection and highly expressed in the electric organ. The channel produced by this gene shortens electric organ action potentials by activating quickly and at hyperpolarized membrane potentials. The source of these properties is a derived patch of negatively charged amino acids in an extracellular loop near the voltage sensor. We demonstrate that this negative patch acts by contributing to the global surface charge rather than by local interactions with specific amino acids in the channels extracellular face. We suggest a more widespread role for this loop in the evolutionary tuning of voltage-dependent channels.

The genome and adult somatic transcriptome of the mormyrid electric fish Paramormyrops kingsleyae

Abstract Several studies have begun to elucidate the genetic and developmental processes underlying major vertebrate traits. Few of these traits have evolved repeatedly in vertebrates, preventing the analysis of molecular mechanisms underlying these traits comparatively. Electric organs have evolved multiple times among vertebrates, presenting a unique opportunity to understand the degree of constraint and repeatability of the evolutionary processes underlying novel vertebrate traits. As there is now a completed genome sequence representing South American electric eels, we were motivated to obtain genomic sequence from a linage that independently evolved electric organs to facilitate future comparative analyses of the evolution and development of electric organs. We report here the sequencing and de novo assembly of the genome of the mormyrid Paramormyrops kingsleyae using short-read sequencing. In addition, we have completed a somatic transcriptome from 11 tissues to construct a gene expression atlas of predicted genes from this assembly, enabling us to identify candidate housekeeping genes as well as genes differentially expressed in the major somatic tissues of the mormyrid electric fish. We anticipate that this resource will greatly facilitate comparative studies on the evolution and development of electric organs and electroreceptors.

Microevolutionary processes underlying macroevolutionary patterns of electric signal diversity in mormyrid fish

This study examines evolutionary causes underlying the maintenance of diversity in electric courtship signals (EODs) emitted by the mormyrid electric fish species Paramormyrops kingsleyae. P. kingsleyae are polymorphic for an EOD feature which characterizes interspecific signal diversity among the rapidly diverged Paramormyrops genus. We collected 338 specimens and recorded EOD signals from 9 populations distributed throughout Gabon, west central Africa, collected in 1999-2009. First, we demonstrate using microsatellite genotyping a significant signature of isolation by distance between populations. Second, utilizing principal components analysis of 21 landmarks measured from EOD waveforms, we find that EOD duration and the magnitude of a small head negative pre-potential (P0) are highly correlated with patterns of spatial and genetic structure. Finally, utilizing a behavioral assay, we demonstrate that P. kingsleyae individuals can discriminate between P0-absent and P0-present EOD waveforms, although genetic and morphological analysis indicate no assortative mating between signal types. Together, these results support the hypothesis that patterns of signal variation in P. kingsleyae have resulted from genetic drift acting upon isolated populations. As P. kingsleyae represents a microcosm of signal diversity among mormyrids, these findings illustrate a potential mechanism by which interspecific patterns of EOD diversity may originate at the population level.The mormyrid fish species Paramormyrops kingsleyae emits an electric organ discharge (EOD) with a dual role in communication and electrolocation. Populations of P. kingsleyae have either biphasic or triphasic EODs, a feature which characterizes interspecific signal diversity among the Paramormyrops genus. We quantified variation in EODs of 327 P. kingsleyae from 9 populations throughout Gabon and compared it to genetic variation estimated from 5 neutral microsatellite loci. We found no correlation between electric signal and genetic distances, suggesting that EOD divergence between populations of P. kingsleyae cannot be explained by drift alone. An alternative hypothesis is that EOD differences are a cue for assortative mating, which would require P. kingsleyae be capable of differentiating between divergent EOD waveforms. Using a habituation-dishabituation assay, we found that P. kingsleyae can discriminate between triphasic and biphasic EOD types. Nonetheless, patterns of genetic and electric organ morphology divergence provide evidence for hybridization between signal types. Although reproductive isolation with respect to signal type is not absolute, our results suggest that EOD variation in P. kingsleaye has the potential to serve as a cue for assortative mating and point to selective forces rather than drift as important drivers of signal evolution.