Old gene duplication facilitates origin and diversification of an innovative communication system--twice

Abstract

The genetic basis of parallel innovation remains poorly understood due to the rarity of independent origins of the same complex trait among model organisms. We focus on two groups of teleost fishes that independently gained myogenic electric organs underlying electrical communication. Earlier work suggested that a voltage-gated sodium channel gene (Scn4aa), which arose by whole-genome duplication, was neofunctionalized for expression in electric organ and subsequently experienced strong positive selection. However, it was not possible to determine if these changes were temporally linked to the independent origins of myogenic electric organs in both lineages. Here, we test predictions of such a relationship. We show that Scn4aa co-option and rapid sequence evolution were tightly coupled to the two origins of electric organ, providing strong evidence that Scn4aa contributed to parallel innovations underlying the evolutionary diversification of each electric fish group. Independent evolution of electric organs and Scn4aa co-option occurred more than 100 million years following the origin of Scn4aa by duplication. During subsequent diversification of the electrical communication channels, amino acid substitutions in both groups occurred in the same regions of the sodium channel that likely contribute to electric signal variation. Thus, the phenotypic similarities between independent electric fish groups are also associated with striking parallelism at genetic and molecular levels. Our results show that gene duplication can contribute to remarkably similar innovations in repeatable ways even after long waiting periods between gene duplication and the origins of novelty.

Fig. 1.

Voltage-gated Na⁺ channel α-subunit. (Upper) Transmembrane folding diagram. Each domain contains six segments (yellow) with intracellular (black) and extracellular (gray) loops. Green triangles bound the investigated sequence. S4 of each domain contains positively charged residues that serve as voltage sensors. P-loops between S5 and S6 form the outer pore. An inactivation gate in the linker between domains III and IV mediates fast inactivation via a conserved binding particle (IFM across many species). (Lower) Representation of the α-subunit in the cell membrane.

Fig. 2.

Selection on Na⁺ channel paralogs along branches of the teleost phylogeny. Shown are optimal categorizations of branches into selective categories (tick marks along heat scale). Branches assigned to a given dN/dS category share the same color. Deeper blue indicates strongly constrained lineages, whereas hotter colors indicate lineages with elevated dN/dS. Following branch categorization, dN/dS category values and branch lengths were re-estimated in HyPhy allowing dN/dS variation across sites. Branch lengths are drawn proportional to expected numbers of nonsynonymous substitutions per codon. A single tree topology inferred from the dataset Concat was used for both genes, but bootstrap support values (%) are indicated in only one tree (asterisks: 100%). Trees were rooted for display purposes according to accepted fish relationships, with length of the branch on which the root falls split equally on each side. Arrows indicate the most parsimonious branches in which Scn4aa was co-opted for exclusive expression in EO_myo.

Fig. 3.

Scn4aa and Scn4ab expression in EO_myo and SM of electric fish compared with nonelectrogenic relatives. (Upper) Three gymnotiform species with EO_myo (Right) compared with one gymnotiform lacking EO_myo in adults and two nonelectrogenic ostariophysan fishes (Left). (Lower) Three mormyroid species compared with two nonelectrogenic osteoglossomorphs and a more distantly related percomorph. Note the loss of Scn4aa expression (i.e., lack of bands) in SM and the gain of expression (presence of bands) in EO_myo for all electric fish possessing adult EO_myo.

Fig. 4.

Pattern of selection across Scn4aa. Per-window dN/dS estimates (window size: 10 codons) for Scn4aa: red, gymnotiforms with adult EO_myo; blue, mormyroids, all of which have adult EO_myo; black, nonelectric fish (smaller values plotted in front such that all values are visible). Black bars are mirrored because the same set of nonelectric fish species was compared with each group of electric fish. Color scheme of middle axis matches channel schematic in Fig. 1. Lower axis provides standard residue numbers from electric eel (34). The breakpoint spans excluded hyper-variable sequence. dN/dS estimates in pink circles are significantly different from nonelectric fish; asterisks indicate windows that are significantly different in both electric fish groups. The clipped dN/dS estimate for mormyroids in the second window after the breakpoint is 2.56 (nonsignificant).

Fig. 5.

Functionally important regions of Na_v1.4a exhibiting amino acid substitutions in electric fish at otherwise conserved sites. Positions conserved across most vertebrates but variable in electric fish are indicated by black triangles below the sequences. Variable residues at conserved sites are shown by differently colored shadings for each amino acid. Deletions are shown as red hyphens. Colored asterisks and lines next to the human and Xenopus sequences indicate residues with critical roles in channel gating, human channelopathies, or neurotoxin function (Inset).