Expansion of Voltage-dependent Na1 Channel Gene Familyin Early Tetrapods Coincided with the Emergence ofTerrestriality and Increased Brain Complexity

Harold H. Zakon,*,1,2 Manda C. Jost,3 and Ying Lu1

1Section of Neurobiology, The University of Texas2Bay Paul Center for Comparative and Molecular Biology, The Marine Biological Laboratory, Woods Hole, MA3Department of Natural Sciences, Western New Mexico University

*Corresponding author: E-mail: h.zakon@mail.utexas.edu.

Associate editor: David Irwin

Abstract

Mammals have ten voltage-dependent sodium (Nav) channel genes. Nav channels are expressed in different cell types withdifferent subcellular distributions and are critical for many aspects of neuronal processing. The last common ancestor ofteleosts and tetrapods had four Nav channel genes, presumably on four different chromosomes. In the lineage leading tomammals, a series of tandem duplications on two of these chromosomes more than doubled the number of Nav channelgenes. It is unknown when these duplications occurred and whether they occurred against a backdrop of duplication offlanking genes on their chromosomes or as an expansion of ion channel genes in general. We estimated key dates of theNav channel gene family expansion by phylogenetic analysis using teleost, elasmobranch, lungfish, amphibian, avian, lizard,and mammalian Nav channel sequences, as well as chromosomal synteny for tetrapod genes. We tested, and exclude, thenull hypothesis that Nav channel genes reside in regions of chromosomes prone to duplication by demonstrating the lackof duplication or duplicate retention of surrounding genes. We also find no comparable expansion in other voltage-dependent ion channel gene families of tetrapods following the teleost–tetrapod divergence. We posit a specific expansionof the Nav channel gene family in the Devonian and Carboniferous periods when tetrapods evolved, diversified, andinvaded the terrestrial habitat. During this time, the amniote forebrain evolved greater anatomical complexity and noveltactile sensory receptors appeared. The duplication of Nav channel genes allowed for greater regional specialization in Navchannel expression, variation in subcellular localization, and enhanced processing of somatosensory input.

Key words: sodium channel, tetrapods, amniotes, terrestriality, gene duplication, brain.

IntroductionVoltage-dependent sodium (Nav) channels are critical forelectrical excitability and neuronal computation. Mammalshave ten Nav channels with distinct biophysical properties,types of modulation by neurotransmitters, and tissue andsubcellular distributions (Angelino and Brenner 2007). Forexample, a distinct Nav channel (Nav1.4) is predominantlyexpressed in skeletal muscle and another Nav channel(Nav1.5) predominantly in cardiac muscle. Different Navchannels are expressed in unmyelinated axons (Nav1.2)and at the nodes of Ranvier in myelinated axons(Nav1.6) (Westenbroek et al. 1989; Caldwell et al. 2000).Specific Nav channels (Nav1.7, 1.8, and 1.9) are highly ex-pressed in nociceptors (Akopian et al. 1996; Cummins et al.1999; Dib-Hajj et al. 2002) or may be upregulated specifi-cally in neurons in the nociceptive pathway following injury(Nav1.3) (Hains et al. 2003). Some cell types, such as fast-firing parvalbumin-positive inhibitory neurons, mainly ex-press one type of Nav channel (Nav1.1), whereas anotherNav channel is expressed in neighboring pyramidal neurons(Nav1.6) (Ogiwara et al. 2007; Lorincz and Nusser 2010).Different Nav channels may even be expressed in different

subcellular domains in neurons: distinct Nav channels areresponsible for initiating the action potential at the axoninitial segment (Nav1.6) and for backpropagation of theaction potential into the soma (Nav1.2), a critical functionfor activity-dependent synaptic plasticity (Hu et al. 2009).

Recent studies have clarified the evolutionary relation-ships among and timing of the origin of vertebrate Navchannels (Okamura et al. 1994; Plummer and Meisler1999; Lopreato et al. 2001; Goldin 2002; Piontkivska andHughes 2003; Novak et al. 2006; Hill et al. 2008). Early in ver-tebrate evolution, a single Nav channel gene of early chor-dates (Okamura et al. 1994) duplicated twice, presumablyduring two consecutive whole genome duplication(WGD) events, giving rise to four Nav channel genes, eachpresumed on a different chromosome (Plummer andMeisler 1999; Lopreato et al. 2001; Novak et al. 2006). In tele-osts, this number jumped to eight Nav channel genes via athird teleost-specificWGD (Lopreato et al. 2001; Novak et al.2006), whereas a series of tandem duplications on two ofthese chromosomes at unknown times in the lineage leadingto mammals resulted in a total of ten Nav channel genes inrodents and humans and presumably other mammals(Plummer and Meisler 1999). A major goal of this study

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Mol. Biol. Evol. 28(4):1415–1424. 2011 doi:10.1093/molbev/msq325 Advance Access publication December 9, 2010 1415

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was to determine the timing and significance of these tan-dem duplications for tetrapod evolution. Additionally, wewished to investigate whether the duplication and retentionwas unique to Nav channel genes and, therefore, possiblyadaptive or merely the result of passive factors such as chro-mosomal ‘‘hotspots’’ for duplication. Finally, we askedwhether the expansion of the Nav channel gene familywas part of a general expansion of other ion channel genefamilies or a unique event.

Materials and Methods

Genomic SequencesWe obtained the whole complement of Nav channel aminoacid sequences from human (Homo sapiens) and rat(Rattus norvegicus) from GenBank. Using a BLAT searchwith human and rodent Nav channel genes, we derivedand translated nucleotide sequences from the Ensemblegenome databases for western clawed frog (Xenopus tropi-calis, v4.1, August 2005), green anole lizard (Anoliscarolinensis, AnoCar1, assembly 2007), platypus (Ornitho-rhynchus anatinus, v5.0, assembly January 2007), grayshort-tailed opossum (Monodelphis domestica; MonDom5,October 2006), chicken (Gallus gallus, v2.1, May 2006), andelephant shark (Callorhinchus milii; v1.0, 2007). About halfof the Nav channel genes from these species had alreadybeen deposited in GenBank, but the other half had notyet been annotated. Additional sequences from lamprey(Petromyzon marinus), newt (Cynops pyrrhogaster), and theAfrican clawed frog (Xenopus laevis) were also used for phy-logeny estimation (supplementary table I, SupplementaryMaterial online).

Because Xenopus is extensively used as a developmentalbiological model, very good Xenopus expressed sequencetag (EST) databases are available from the TIGR database(http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/Blast/in-dex.cgi). We utilized this EST database to confirm Xenopusgenomic sequences (supplementary fig. 1, SupplementaryMaterial online).

Nav channels comprise four repeating domains (DI–DIV),interconnecting extra- and intracellular loops and N and Ctermini. Sequences from all mammals, Xenopus, Anolis, andGallus, were mostly full length (N to C termini) with theoccasional small region of missing sequence due to geneassembly problems. Due to the low coverage (1.4�) ofthe Callorhinchus genome, contigs were short and typicallycontained only one or a few exons.

Sequences Derived by Reverse transcriptionPolymerase Chain ReactionWe cloned additional Nav channel sequences by Reverse tran-scription (RT) polymerase chain reaction (PCR) from varioustissues of a few key species for which sequenced genomeswere unavailable. Lungfish (Dipnoi) and coelacanths arethe most basal living sarcopterygii and are basal to the tetra-pods, so we cloned Nav channel transcripts from heart, mus-cle, brain, and spinal cord of the South American lungfish(Lepidosiren paradoxa). Additionally, we cloned Nav channel

genes from genomic DNA of lungfish to attempt to captureany genes that might be expressed in low levels or not ex-pressed in the tissues from which we extracted RNA.

The Chondrichthyes (e.g., sharks, rays, skates, and chime-ras) diverged from the lineage leading to tetrapods;525Main the mid-Cambrian period (Hedges 2009), presumably fol-lowing the second vertebrate WGD (Kuraku 2008). We wereable to obtain muscle and heart from a horn shark (Hetero-dontus francisci) and the brain of an Atlantic stingray (Da-syatis sabina) and clonedNav channel transcripts from themto estimate the number of Nav channel genes in verte-brates well before the emergence of tetrapods.

We used nested primer sets developed in our laboratoryfor cloning Nav channel genes from a range of vertebratespecies. These primarily targeted domains I, II, and/or IIIand variable interconnecting intracellular loops. The RT re-action either utilized the lower primer of step one toattempt to target Nav channel genes or a polyTTT primerto ensure that we had not missed any transcripts. Forlungfish, the primers were as follows: first PCR (upper:CCRTGGAAYTGKCTKGATTT and lower: RTRAARRA-DGABCCRAADRTGATG) and second PCR (upper:ATGRCGTAYVYYACVGAGTT and lower: TACATDATNYC-CATCCANCCTTT). We used the following primer setsfor: —1) shark heart: first PCR (upper: TGYGGYGARTGGA-TYGARAC and lower: RTRAARRADGABCCRAADRTGATG)and second PCR (upper: ATGTGGGAYTGYATGGARGT andlower: TACATDATNYCCATCCANCCTTT) and 2) sharkmuscle: first PCR (upper: TCYMGAGGBTTCTGYDTTGGand lower: RTRAARRADGABCCRAADRTGATG) and sec-ond PCR (upper: CCRTGGAAYTGKCTKGATTT and lower:TACATDATNYCCATCCANCCTTT). For skate brain, weused a similar primer set as in lungfish muscle. Temperaturesand times were 53 �C for annealing and 94 �C for denaturingsteps (30–45 s), and 74 �C for extension steps (extensiontime dependent on the length of predicted PCRproducts, 1 min/1,000 bp) for a total of 35 cycles.

Nav Channel PhylogenyNav channel amino acid sequences were aligned in ClustalXusing default parameters, and poorly aligned regions,mainly long intracellular loops, were removed. Final align-ments were output into NEXUS files. To reconstruct Navchannel gene phylogeny, two independent Bayesian anal-yses (Mr Bayes, version 3) were conducted, each with fiveMCMC chains and run for 200,000 generations, assumingsix substitution types and a gamma distribution of ratevariation among sites. For each analysis, posterior probabil-ities were calculated using majority-rule consensus of alltrees saved after the log-likelihood asymptote (burn-in).Lamprey Nav channel gene sequences were specified forrooting.

Analysis of Non-Nav Channel GenesWe wished to determine whether duplications of non-Navchannel genes occurred within the early tetrapod lineagefollowing the teleost–tetrapod divergence. Human nucle-otide sequences were initially used to search the National

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Center for Biotechnology Information (NCBI) (nr/nt) data-base for orthologous or paralogous sequences fromchicken, frog, and teleosts (supplementary table II, Supple-mentary Material online). Then, all the above nucleotidesequences were used for BLAT searches of Ensemblegenome databases of zebrafish and chicken to assure thatno unannotated genes were missed. No attempt was madeto reconstruct the history of these genes within teleosts, soextensive searches were not made of teleost genomes.However, if no teleostean ortholog was initially recoveredfrom GenBank or from the zebrafish genome, furthersearches were made in the genomes of stickleback (Gaster-osteus aculeatus), medaka (Oryzias latipes), and pufferfish(Takifugu rubripes and Tetraodon nigroviridis) in case a pu-tative teleostean ortholog might have been lost in zebrafishbut retained in other teleost lineages. Finally, the teleosteansequences were used for BLAT searches of the NCBI andEnsemble databases to ensure that all tetrapod orthologshad been uncovered. If a gene had duplicated in tetrapods,each duplicate would be equally likely to be identified by itsteleostean ortholog. Analysis of teleostean and tetrapodnon-Nav channel amino acid trees was done using theNeighbor-Joining algorithm with default values in ClustalXwith 1,000 bootstrap replications.

Determination of SyntenyChromosomal location of human and chicken Nav channeland flanking genes was available from NCBI. Synteny wasdetermined for lizard and frog Nav and flanking genes bymanually assembling genes from scaffolds from Ensemble,determining gene identity by BLAT with the NCBI databaseand by their locations in comparison with human andchicken chromosomes.

Results

Description of DataFrom BLAT searches on archived genomes, we recovered sixNav channel genes from Xenopus, nine from lizard, six fromplatypus (plus two smaller fragments that were not usedfor analysis), eight from opossum, and nine from chicken.Where possible, predicted X. tropicalis gene sequences wereconfirmed by ESTs, and in two cases (xt236 and xt464b),incomplete genomic sequences were filled in by overlapwith ESTs (supplementary fig. 1, Supplementary Materialonline).

We derived pieces of Nav channel genes from elephantshark. Due to the low coverage, each contig had one or atmost a few exons, and contigs could not be unambiguouslyconnected. However, we identified 3–4 distinct pieces cor-responding to most exons of the Nav channel gene thatsuggested a total of four Nav channel genes. We recoveredfour contigs with all or part of the most 3# exon (the lon-gest exon in all vertebrate Nav channel genes); one contigincluded two other exons. This gave us sequences of from;200 to 500 amino acids.

By RT-PCR, we cloned three Nav channel genes from lung-fish, one each from brain and spinal cord, muscle, and heart

tissue; two from horn shark, one from muscle and one fromheart; and one from skate brain. These were not completesequences but covered domains II–III (lungfish muscleand skate brain) or domains I–III (shark and lungfish heart,lungfish brain and lungfish muscle). Given the limitations ofour tissue samples and the RT-PCR method, we do not claimthat this provides the full complement of Nav channel genesof these species.

As we show below, we have a strong case for homologiz-ing the amniote Nav channel genes; therefore, we use themammalian gene nomenclature (SCNxA, where x5 a num-ber) for Nav channel genes frommammals, birds, and lizards(protein designation 5 Nav; gene designation 5 SCN). Butbecause not all the nonamniote genes are orthologs of am-niote genes, we did not use the mammalian nomenclaturefor these.

Expansion of Nav Channel Genes Occurred in theDevonian and Carboniferous PeriodsWe generated phylogenetic trees without (align-ment1: fig. 1) and with (alignment2: supplementary fig.2, Supplementary Material online) the four elephant sharksequences. Resulting tree topologies of alignment1 wereidentical for the two runs with minor variation in the pos-terior probabilities for a few branches. Alignment2 was runonly once due to the low support values in the parts of thetree including the elephant shark sequences (supplemen-tary fig. 2, Supplementary Material online). We will focuson alignment1.

SCN8A has a simple history with no duplications tracingback to an ancestral gene that is also represented inelasmobranchs (skatebrain), lungfish (lungfishbrain), andamphibians (xt67) (fig. 1: shaded in light blue). There aretwo duplicates in zebrafish due to a teleost-specificWGD. As in humans, SCN8A orthologs of frog, chicken,and lizard reside alone on a chromosome (supplementarytable I, Supplementary Material online).

SCN4A shows a similar history with amniote orthologsgrouping with genes from frog (xt43), lungfish (lungfish-muscle), and shark (sharkmuscle) and a pair of duplicategenes in teleosts (fig. 1: shaded in yellow). The orthologsof SCN4A also reside singly on a chromosome (supplementarytable I, Supplementary Material online).

Orthologs of the three Nav channel genes on humanchromosome 3 (SCN5A, SCN10A, and SCN11A) are foundin other mammals, chicken, and lizard, and these threegenes have shared synteny (fig. 1: shaded in light red). Theyderive from a single gene represented in our sample by or-thologs in shark (sharkheart), zebrafish (SCN5Laa andSCN5Lab), lungfish (lungfishheart), and frog (xt28). The sin-gle frog gene is syntenically related to the amniote genes(fig. 2). The presence of a single gene at the amphibian–amniote split and of three genes before the synapsid (mam-mals)–diapsid (reptiles and birds) divergence means thattwo duplications of the ancestral gene occurred in a 30-My window at the origin of amniotes in the lower tomid-Carboniferous periods.

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The history of the Nav channel genes on human chro-mosome 2 is more complex. These genes derive froma fourth ancestral gene (Novak et al. 2006), although wewere unable to detect an ortholog of this gene in lungfishtissues or skate brain despite extensive attempts to amplifyit from RNA and genomic DNA. As expected, there are twogene duplicates in zebrafish (SCN1Laa and SCN1Lab). Thegene tree suggests that a single ancestral gene underwentindependent duplications in amphibians and amniotes(fig. 1: shaded in green). In this scenario, multiple duplica-tions of a putative ancestral Nav channel gene would have

generated SCN1A, SCN2A, SCN3A, and SCN7A/SCN9A (theprecursor to separate SCNA7a and SCN9A genes). Becausethese all have orthologs in mammals, lizards, and chicken,these duplications would have occurred within the same30-My window as the triplicated genes on human chromo-some 3 (fig. 1). The final duplication of SCN7A/SCN9A intoSCN7A and SCN9A likely occurred after the divergence ofmonotreme and therian mammals (220 Ma) precedingthe marsupial–placental split (175 Ma). However, giventhe low values of the posterior probabilities in our trees, thereis some uncertainty about the timing of this duplication.

FIG. 1. Phylogenetic tree for amino acid translation of tetrapod Nav channel genes determined by Bayesian analysis. Amniote Nav channelgenes are homologized with and named according to human gene nomenclature. Genes from the frog, Xenopus tropicalis, are named after theirscaffold location and genes from lungfish, skate, and shark according to the tissue from which they were amplified. Names of the mammalianNav channel proteins are given on the right side of the figure. Human Nav channel genes that are on the same chromosome and theirhomologs from other species are within a block of color.

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On the other hand, the most parsimonious interpreta-tion of the synteny (fig. 3) is that two duplications had al-ready occurred in the common ancestor of amphibians andamniotes. This is because the Nav channel and other genesin this region of the amphibian and amniote chromosomeshave the same syntenic relationships. Additionally, oneNav channel gene in each lineage and in the same relativechromosomal position (amphibian xt464b and amnioteSCN2A) is oppositely oriented on the chromosome to allthe other Nav channel genes (arrows, fig. 3). This suggeststhat most of the duplications occurred at a slower rate over130 My.

What might account for the discrepancy in interpreta-tion between the gene tree and synteny data? It is unlikelyto be due to Xenopus Nav channel genes that were missedor misassembled in genome sequencing. First, all the ESTsthat we uncovered uniquely matched a specific gene (inmost cases multiple ESTs were mapped to each gene),and all Xenopus Nav channel genes were represented inthe EST database (supplementary fig. 1, SupplementaryMaterial online). Second, the few additional amphibianNav channel genes available from GenBank of sufficientlength to align (e.g., newt and xlaev1.2) appeared to be or-thologs of genes that we had already uncovered in X. tro-picalis. Finally, the Xenopus scaffolds were assembled fromoverlapping reads of shotgun sequence de novo so that theapparent synteny is not an artifact (Hellsten et al. 2010). Itis possible, but seems unlikely, that independent duplica-tions in amphibians and amniotes could have resulted inidentical patterns of synteny. If the duplications had oc-curred in the common ancestor of amphibians and am-niotes as suggested by synteny, then the nonoverlappingclustering of amphibian and amniote genes in the treemight be explained by some amount of gene conversion

within the amniote and/or amphibian lineages, as some-times occurs following gene duplications (Kellis et al. 2004).

Alignment2 included the fragments from elephant shark(supplementary fig. 2, Supplementary Material online). Asexpected, the inclusion of these short sequences resulted inlow posterior probabilities for them and their neighboringbranches; these were too low to trust their exact position-ing in the tree. However, each of the 4 sequences groupedwith 1 of the 4 clades of Nav channel genes with extremelystrong (posterior probabilities 5 100) support. Further-more, a BLAT search of GenBank with the nucleotide se-quences of each of the four 3# exons had top matches withsequences in one specific clade. The inclusion of these se-quences supports our contention (Lopreato et al. 2001;Novak et al. 2006) that the ancestor of teleosts and tetra-pods had four Nav channel genes and gives an indication ofthe ‘‘missing’’ ancestor of the fourth Nav channel gene clade.

In sum, we suggest that after the second WGD (esti-mated at ;550 Ma, Meyer and Schartl 1999; Dehal andBoore 2005; Panopoulou and Poustka 2005; Blommeet al. 2006) brought the number of Nav channel genesto four, there was a period of stasis. Then a series of tandemNav channel gene duplications occurred in a 30- to 130-Myperiod during early tetrapod evolution, following which theNav channel gene family remained largely stable for another300 My (fig. 5).

Genes Flanking the Nav Channel Genes Did NotDuplicate or Were Not RetainedWe posit that the retention of the Nav channel gene paral-ogs was due to selection. The null hypothesis is that the Navchannel gene expansion was simply a consequence of insta-bility in the regions of these two chromosomes in which theNav channel genes reside that led to duplication and

FIG. 2. Synteny for tetrapod Nav channel genes referenced to Navchannel genes on human chromosome 3. Black boxes represent Navchannel genes, and gray boxes represent other genes.

FIG. 3. Synteny for tetrapod Nav channel genes referenced to Navchannel genes on human chromosome 2. Black boxes represent Navchannel genes, and gray boxes represent other genes. Arrowsrepresent Nav channel gene whose chromosomal orientation isopposite to all the other Nav channel genes.

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retention of all the genes in this region. We tested the nullhypothesis by examining whether flanking genes show a sim-ilar history of duplication and retention. We sampled onlythose flanking genes that were located on chromosomes orscaffolds of the species for which we had synteny informa-tion. In our sample (figs. 2 and 3) 14 of 15 genes showed notandemduplications on these chromosomes, with SLC22A13and SLC22A14 being a duplicate pair. Given that the Navchannel genes on these two chromosomes have both dupli-cated (2 out of 2: this analysis ignores the fact that the Navchannel genes underwent multiple duplications), this is sig-nificantly greater than expected given the number of dupli-cations of the flanking non-Nav channel genes on these twochromosomes (1 out of 14) (P , 0.0001, two-tailed chi-square).

However, it is also possible that these flanking genes du-plicated but were then dispersed throughout the genome,whereas the Nav channel genes were retained where theyduplicated. We derived sequences for the 16 flanking genesin figures 2 and 3 from GenBank or searches from genomedatabases and constructed trees for them. We found thatmost showed indications of the initial two rounds of WGD

but no duplications in the 450 My following the actino-pterygian–sarcopterygian split (fig. 4A). The only excep-tion, mentioned above, was the duplicate pair of SLC22Amembers13 and 14. This gene family has a history of exten-sive duplication (.25 members) with the ‘‘center of grav-ity’’ of duplication elsewhere in the genome.

We conclude that the genes flanking the Nav channelgenes either did not duplicate or, if they did so, were notretained. This supports the hypothesis that the Nav channelgene duplications were retained as a result of selection.

Other Ion Channel Gene Families Did NotDuplicate or Were Not RetainedVoltage-dependent ion channels are fundamental to theelectrical activity of the brain. We next asked whether therewas a general expansion of other six transmembrane (6TM)voltage-dependent ion channel gene families during tetra-pod evolution or whether this expansion was specific to theNav channel gene family. We addressed this question usingboth published literature (Saito and Shingai 2006; Hoeggand Meyer 2007; Jackson et al. 2007) and gene trees that

FIG. 4. Representative non-Nav channel gene trees. (A) CSNRP, a gene flanking the Nav channel genes, which was studied to test for duplicationand retention of neighboring non-Nav genes. (B) CACNA1, the calcium channel gene family, an example of another ion channel family. Noattempt was made to systematically sample teleostean orthologs; therefore, these trees do not represent detailed phylogenies of teleosteangenes. Teleost orthologs were only used to establish the number of duplications that occurred in tetrapods following the teleost–tetrapoddivergence. Asterisks, bootstrap values of 100.

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we constructed with sequences from teleost, human, frog,and chicken databases. The channel that we investigatedincluded the major depolarizing (Ca2þ, transient receptorpotential [TRP], and hyperpolarization-activated cyclic nu-cleotide-gated ion channel [HCN]) and hyperpolarizing(Kv, ether-a-go-go related [ERG], and slo) channels. Wesampled all 17 members of the voltage-dependent Kþ

channel (Kv), 4 members of the HCN), 7 members ofthe canonical TRP, 6 members of the TRP, vanilloid sensi-tive (TRPV), 4 members of the TRP, melastatin (TRPM), 1member of the TRP ankyrin repeat, 8 members of the ERG,3 members of the large-conductance calcium-activated Kþ

family (slo), and 10 members of the voltage-dependentcalcium (CACNA1 or Cav) gene families (fig. 4B).

Most (54 out of 60) of the ion channel genes in our sam-ple showed no duplications following the teleost–tetrapoddivergence. There is a mammalian-specific duplication in theslo gene family (slo3). A number of TRP channels do nothave teleost orthologs (TRPV1/TRPV2, TRPV3, TRPV5/TRPV6, TRPM6, and TRPM8). However, the absence ofTRPM8 and TRPV3 in teleosts is likely due to losses in tele-osts rather than duplications in amniotes (Saito and Shingai2006). TRPV5/TRPV6 are a pair that clearly duplicated in am-niotes (Saito and Shingai 2006). The timing of the TRPV1/TRPV2 duplication is not resolvable (Saito and Shingai 2006),but it may represent a tetrapod-specific duplication. Evenassuming that all apparent duplications in tetrapods are realrather than reflecting losses of teleost genes, Nav channelgenes duplicated (2 out of 4: again, this analysis ignores re-peated duplications of each ancestral paralog) significantlymore than other ion channel genes (6 out of 60) (P, 0.007,two-tailed chi-square).

Discussion

Relationship to Previous Nav Channel PhylogeniesBased on chromosomal location and phylogeny of the Navchannel family in mammals, Plummer and Meisler (1999)proposed that the ten mammalian Nav channels resultedfrom a single ancestral chordate Nav channel gene that un-derwent two rounds of genome duplication early in verte-brate evolution. These duplications ended in a single Navchannel gene on each of four chromosomes followed bya series of tandem duplications on two of those chromo-somes. Inclusion of teleosten Nav channel sequences insubsequent analyses supported this idea and further dem-onstrated that the initial two rounds of duplications pre-ceded the teleost–tetrapod divergence, teleosts have eightNav channel genes likely as the result of a WGD, and theduplications in teleosts and tetrapods occurred indepen-dently (Lopreato et al. 2001; Novak et al. 2006). Novaket al. (2006), which also included chicken sequences, didnot attempt to resolve the timing of the tandem duplica-tions within tetrapods. The trees from that study wouldsuggest that many of the Nav channel genes from chickenand mammals duplicated independently. However, the se-quences then available from the chicken were likely misas-sembled (and some have been discontinued by NCBI),

thereby confusing the relationship of mammalian andavian genes. In the current study, we addressed this issueby determining the chicken sequences manually froma newer Ensemble release of the chicken genome (version2.1). Furthermore, we added sequence from key taxa suchas amphibians, lizard, and monotreme and marsupialmammals as well as including sequences cloned in our lab-oratory from lungfish and elasmobranchs. A strong conclu-sion from the current study is that, with the exception ofa single mammalian-specific duplication, all mammalianNav channel genes were present in an early amniote ances-tor (fig. 5).

In addition, the current phylogeny supports our previ-ous conclusion that four Nav channel genes existed in thecommon ancestor of teleosts and tetrapods. A novel resultin the current study is that we identified orthologs of thesefour genes in lungfish and elasmobranchs. Orthologs to 3 ofthese 4 were easily cloned from lungfish and elasmobranchtissues. Despite numerous attempts, we were unable to am-plify the fourth gene, the ancestor of the SCNA1, SCN2A,etc. complex (which are mainly neural-expressing genes),from lungfish brain, spinal cord, skate brain, or lungfish ge-nomic DNA. However, we identified a likely ortholog in theelephant shark genome. We suggest that this gene is notexpressed, or at least not at high levels, in the central ner-vous system of elasmobranchs and lungfish.

Nav Channel Gene Expression and TetrapodEvolutionThe Nav channel gene family expansion occurred in twochromosomal regions. It was accompanied neither by du-plication and/or retention of flanking genes nor by wide-spread expansion of other ion channel genes; it was specificand presumably advantageous. The Nav channel gene tan-dem duplications were concurrent with the origin of tetra-pods and their invasion of the terrestrial habitat. It largelypreceded the diversification of amniotes into synapsid(mammal) and diapsid (reptiles/birds) lineages (Hedges2009; Shedlock and Edwards 2009).

As tetrapods took to land, they evolved new modes oflocomotion, coped with loss of buoyancy, confronteda novel sensory environment, and exploited new food re-sources (Glenner et al. 2006; Clack 2007). Meeting thesechallenges was facilitated by the evolution of new sensoryreceptors in their skin and muscles. For example, early tet-rapods evolved muscle spindles (Maeda et al. 1983; Rosset al. 2007), and different lineages of tetrapods laterevolved other kinds of somatosensory receptors (e.g., la-mellated Pacinian/Herbst corpuscles in amniotes, domepressure receptors in crocodilians, etc.) (Hunt 1961;Proske 1969; Dorward and Macintyre 1971; von Duringand Miller 1979; Soares 2002). This was accompaniedby greater anatomical and physiological complexity in thedorsal root ganglion system (Sneddon 2002; Sneddonet al. 2003). Little is known about the expression ofNav channel genes in the dorsal root ganglia of nonmam-malian tetrapods. In mammals, a number of different Navchannels are expressed in dorsal root ganglion neurons

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and axons—Nav1.6, Nav1.3, Nav1.8, Nav1.9, andNav1.7—and these contribute to the systematic variationin conduction velocity and other biophysical properties ofdifferent classes of dorsal root ganglion neurons (Herzoget al. 2003). Of these channels, all but Nav1.6 derive fromthe tetrapod-specific duplications. Thus, the duplicationsof Nav channel genes enabled the diversity of Nav channeltypes in dorsal root ganglion cells.

Besides the obvious advantages of possessing a repertoireof Nav channels that can be called upon for performingvarious coding ‘‘jobs,’’ matching Nav channel biophysicalproperties with signaling requirements also results in met-abolic efficiency (Hasenstaub et al. 2010; Schmidt-Hieberand Bischofberger 2010). A more expansive repertoire ofNav channel genes might also have been selected on thebasis of energy savings.

The Nav channel gene duplications also occurred ata time when the amniote brain, especially the forebrain,was robustly expanding and adding new anatomical re-gions (Northcutt 2002). We do not believe that the increasein number of Nav channel genes was causal to the increasein forebrain complexity. Rather, these likely happened inparallel, both as manifestations of increasing brain com-plexity. Also, because we have only examined 6TM ionchannel gene families, we cannot comment on the possiblerole of other brain-expressing genes, such as neurotrans-mitters and their receptors, etc. in the evolution of theamniote brain.

Parallel Expansion of Nav Channel Gene Family inTeleostsGlobal cataclysmic events in the late Devonian initiateda period of mass extinction for most vertebrate lineages(Clack 2007; Sallan and Coates 2010). The main survivorsof this event were the tetrapods (the other sarcopterygiantaxa mostly went extinct and are represented today solelyby lungfishes and coelacanths), on land, and the actino-pterygii (and chondrichthyes), in water. Similar to the evo-lutionary success of tetrapods, one group of actinopterygii,the teleosts, eventually came to dominate the marine andaquatic habitats.

Interestingly, the Nav channel gene family indepen-dently expanded in teleosts to almost the same numberof genes (eight Nav channel genes) as in amniotes (Lopreatoet al. 2001; Novak et al. 2006). In contrast to tetrapods,this duplication arose suddenly as a result of a third teleost-specific WGD (Meyer and Schartl 1999; Hurley et al. 2007)and no tandem duplications occurred over the next;250–300 My. As in tetrapods, all the Nav channel geneduplicates have been retained. It will be interesting to re-construct the detailed histories of other ion channel genefamilies in teleosts and determine if these are all retained asthe Nav channel genes have been or whether there hasbeen greater loss of other ion channel genes back to a ‘‘base-line’’ pre-WGD number. In other words, is there a relativeincrease in Nav over other ion channel genes in teleostsas in tetrapods? In a further parallel to the amniotes,

0 MYA

100 MYA

200 MYA

400 MYA

600 MYA

300 MYA

500 MYA

Synapsid-Diapsid

Amphibia-Amniota

Actinopterygii-Sarcopterygii

Chondrichthyes-Osteichthyes

FIG. 5. Schematic timeline for Nav channel gene duplications. Each set of boxes represents the lineage of four ancestral genes. Timing of theduplication of the orthologs of human chromosomes 2 (the darkest boxes) is conservatively estimated according to the most parsimoniousinterpretation offered by synteny. Four Nav channel genes were present in the last common ancestor of teleosts and tetrapods(actinopterygian–sarcopterygian divergence, ;450 Ma) and likely also in the common ancestor of chondrichthyes and osteichthyes (;525Ma). The vertical dotted lines imply that these four genes resulted from the second of two vertebrate WGD events estimated at ;550 Ma.Divergence times from Hedges (2009), Madsen (2009), and Shedlock and Edwards (2009).

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the teleostean forebrain also increased in complexity com-pared with that of nonteleost actinopterygian fishes (e.g.,bowfin, gar, and sturgeon) (Northcutt 2002).

Recent Nav Channel Gene Duplicates AreDifferentially ExpressedIt has been proposed that recently duplicated genes showmore restricted expression or greatest sequence divergencethan those that duplicated in the distant past (Farre andAlba 2009; Milinkovitch et al. 2010). SCN4A, with no historyof duplication since the last WGD, is predominantly ex-pressed in mammalian muscle; in lungfish, its orthologwas only expressed in muscle. SCN8A, also with no historyof duplication since the WGD, is expressed in brain (andnot heart or muscle) in lungfish and is expressed at uniformlevels throughout the mammalian brain (Whitaker et al.2000, 2001) (no data from reptiles and birds; althoughSCN8A is also expressed in mammalian heart, Maieret al. 2004). On the other hand, two of the complex of trip-licated genes represented by human chromosome 3 andone from the complex localized to human chromosome2 are expressed in neurons of the peripheral somatosensorysystem, and some have ‘‘unusual’’ biophysical properties(Akopian et al. 1996; Cummins et al. 1999, 2001; Dib-Hajjet al. 2002).

The complex of genes that show the greatest duplica-tion (human chromosome 2) are mainly expressed inbrain and show the greatest variation in regional pat-terns of expression (telencephalon vs. brain stem) orin subcellular distribution (axons vs. somata) in themammalian brain (Westenbroek et al. 1989; Akopianet al. 1996; Cummins et al. 1999, 2001; Caldwell et al.2000; Whitaker et al. 2000, 2001; Dib-Hajj et al. 2002;Jarnot and Corbett 2006; Ogiwara et al. 2007; van Wartet al. 2007; Duflocq et al. 2008; Hu et al. 2009). The finalgene duplication that occurred before the origin of ther-ian mammals gave rise to a unique Nav channel with ahighly derived sequence that has lost its voltage sensitiv-ity but is still permeable to Naþ ions (Nax) (Watanabeet al. 2006). This channel is more involved in Naþ ionregulation than neural computation.

ConclusionThe Nav channel gene family of tetrapods underwent a se-ries of duplications 300–450 Ma largely during their earlyevolution (fig. 5). This wave of duplications did not involvethe duplication or retention of flanking genes or other ionchannel genes. We speculate that the rapid expansion ofthe Nav channel gene family accommodated greater com-plexity in neural processing and was a seminal event in theevolution of the amniote brain.

Supplementary MaterialSupplementary figures 1 and 2 and tables I and II are avail-able at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).

AcknowledgmentsThe authors gratefully acknowledge Marianna Grenadierfor artwork, Dr Robert Dores, University of Denver, for sup-plying tissues, and Dr Hans Hofmann for commenting onthe manuscript.National Science Foundation (IBN 0236147 to H.H.Z. andM.C.J.); National Institutes of Health (R01GM084879 toH.H.Z.).

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