Does autoresistance co-evolve with in poison-dart ?

Supplementary material Supplementary Tables

Table S1. Specimen information and accession numbers of the sequences used in ancestral sequence reconstructions. For sequences obtained from publicly available genomes or transcriptomes where sequences do not have individual accession numbers (i.e. Nanorana parkeri, Rhinella marina, Rana pipiens) we provide the name of the contig or gene annotation.

Species Voucher Locality DI DII DIII DIV Source Observations Allobates femoralis- Lumbaquí, KT989177 - - KT989148 Tarvin et al. 2016 Allobates talamancae - Tundaloma, Ecuador KT989178 - - KT989149 Tarvin et al. 2016 - Río Pastaza, Ecuador KT989179 - - KT989150 Tarvin et al. 2016 bilinguis- Venecia, Ecuador KT989180 KT989205 KT989220 KT989151 Tarvin et al. 2016 - Canelos, Ecuador KT989181 - - KT989152 Tarvin et al. 2016 Ameerega parvula- Gualaquiza, Ecuador KT989182 KT989206 - KT989153 Tarvin et al. 2016 Panguana Biological Ameerega petersi GECOH1407 Station, Puerto Inca, Perú TBA - - TBA This Study Panguana Biological Ameerega picta GECOH1406 Station, Puerto Inca, Perú TBA - - TBA This Study Leticia, Amazonas, TNHC-FS466 TBA - - TBA This Study La Celia, Risaralda, Andinobates bombetes GECOH1495 Colombia TBA - - TBA This Study Yotoco, Valle del Cauca, Andinobates bombetes GECOH297 Colombia TBA - - TBA This Study Andinobates fulguritus GECOH1314 Lloró, Chocó, Colombia TBA - - TBA This Study Cantón de San Pablo, Andinobates fulguritus GECOH1615 Chocó, Colombia TBA - - TBA This Study Fort Sherman, Colón, panamansis TNHC-FS4889 Panama KT989193 - - KT989164 Tarvin et al. 2016 auratus ANW12085 - LT984935 - - - Yuan & Wang 2018 Dendrobates auratus ANW12086 - LT984936 - - - Yuan & Wang 2018 Dendrobates auratus ANW12087 - LT984939 - - - Yuan & Wang 2018 Dendrobates auratus ANW12088 - LT984937 - - - Yuan & Wang 2018 Dendrobates auratus ANW12164 - LT984938 - - - Yuan & Wang 2018 Dendrobates auratus ANW12221 - LT984933 - - - Yuan & Wang 2018 Dendrobates auratus ANW12222 - LT984932 - - - Yuan & Wang 2018 Lago Bayano, Panama, Dendrobates auratus GECOH1824 Panama TBA - - TBA This Study Dendrobates auratus GECOH1844 Veraguas, Panama TBA - - TBA This Study Dendrobates auratus IW163 - LT984940 - - - Yuan & Wang 2018 Dendrobates auratus IW175 - LT984934 - - - Yuan & Wang 2018 Dendrobates auratus IW177 - LT984926 - - - Yuan & Wang 2018 Dendrobates auratus IW931 - LT984927 - - - Yuan & Wang 2018 Dendrobates auratus IW933 - LT984928 - - - Yuan & Wang 2018 Dendrobates auratus IW934 - LT984929 - - - Yuan & Wang 2018 Dendrobates auratus IW936 - LT984930 - - - Yuan & Wang 2018 Dendrobates auratus IW937 - LT984931 - - - Yuan & Wang 2018 Captive bred, Understory Dendrobates tinctorius- Enterprises KT989189 KT989210 KT989224 KT989160 Tarvin et al. 2016 Dendrobates truncatus GECOH1589 Nuquí, Chocó, Colombia TBA - - TBA This Study Dendrobates truncatus GECOH2035 Lérida, Tolima, Colombia TBA - - TBA This Study anthonyi - El Progreso, Ecuador KT989195 - KT989166 Tarvin et al. 2016 Epipedobates boulengeri - Arasha, Ecuador KT989196 KT989212 KT989227 KT989167 Tarvin et al. 2016 Epipedobates boulengeri Buenaventura, Valle del north GECOH594 Cauca, Colombia TBA - - TBA This Study Epipedobates boulengeri Buenaventura, Valle del north GECOH640 Cauca, Colombia TBA - - TBA This Study Epipedobates boulengeri Guapi, Cauca, Colombia north GECOH691 (mainland) TBA - - TBA This Study Epipedobates darwinwallacei- Mindo, Ecuador KT989197 - - KT989168 Tarvin et al. 2016 - Manta Real, Ecuador KT989198 - - KT989169 Tarvin et al. 2016 Epipedobates tricolor - Chazo Juan, Ecuador KT989199 KT989213 KT989229 KT989170 Tarvin et al. 2016 Espadarana callistomma - Alto Tambo, Ecuador KT989175 KT989203 KT989217 KT989144 Tarvin et al. 2016 Gastrotheca litonedis- Ñamarín, Ecuador KT989173 KT989202 KT989216 KT989143 Tarvin et al. 2016 italoi- Canelos, Ecuador KT989184 KT989207 KT989221 KT989155 Tarvin et al. 2016 Hyloxalus nexipus- Río Negro, Ecuador KT989185 KT989208 KT989222 KT989156 Tarvin et al. 2016 Hypsiboas picturatus El Placer, Ecuador - KT989204 KT989218 KT989146 Tarvin et al. 2016 Incilius nebulifer TNHC-FS6877 Austin, Texas, USA KT989172 KT989201 KT989215 KT989142 Tarvin et al. 2016 Méndez, Morona Lithodytes lineatus QCAZ16621 Santiago, Ecuador KT989174 - - KT989145 Tarvin et al. 2016 Captive bred, Understory Mantella aurantiaca- Enterprises KT989171 KT989200 KT989214 KT989141 Tarvin et al. 2016 Obtained from Npar_R017212 Nanorana parkeri- Sun et al. 2015 genome granulifera IW1005 Rio Baru LT984951 - - - Yuan & Wang 2018 Oophaga granulifera IW1006 Rio Baru LT984950 - - - Yuan & Wang 2018 Oophaga granulifera IW1007 Rio Baru LT984949 - - - Yuan & Wang 2018 Oophaga granulifera IW1008 Rio Baru LT984948 - - - Yuan & Wang 2018 Oophaga granulifera IW1010 - LT984947 - - - Yuan & Wang 2018 Oophaga granulifera IW1012 Drake Bay LT984946 - - - Yuan & Wang 2018 Oophaga granulifera IW1013 Drake Bay LT984945 - - - Yuan & Wang 2018 Oophaga granulifera IW1014 Drake Bay LT984944 - - - Yuan & Wang 2018 Oophaga granulifera IW926 Fila Chonta LT984943 - - - Yuan & Wang 2018 Oophaga granulifera IW928 Fila Chonta LT984942 - - - Yuan & Wang 2018 Oophaga granulifera IW939 Rio Saverge LT984941 - - - Yuan & Wang 2018 Buenaventura, Valle del Oophaga histrionica GECOH1002 Cauca, Colombia TBA - - TBA This Study Buenaventura, Valle del Oophaga histrionica GECOH1008 Cauca, Colombia TBA - - TBA This Study Oophaga pumilio GECOH2007 Kilambé, Nicaragua TBA - - TBA This Study Oophaga pumilio IW001 - LT984967 - - - Yuan & Wang 2018 Oophaga pumilio IW002 - LT984968 - - - Yuan & Wang 2018 Oophaga pumilio IW003 - LT984969 - - - Yuan & Wang 2018 Oophaga pumilio IW004 - LT984970 - - - Yuan & Wang 2018 Oophaga pumilio IW016 - LT984955 - - - Yuan & Wang 2018 Oophaga pumilio IW018 - LT984956 - - - Yuan & Wang 2018 Oophaga pumilio IW019 - LT984971 - - - Yuan & Wang 2018 Oophaga pumilio IW020 - LT984957 - - - Yuan & Wang 2018 Oophaga pumilio IW021 - LT984958 - - - Yuan & Wang 2018 Oophaga pumilio IW047 - LT984965 - - - Yuan & Wang 2018 Oophaga pumilio IW048 - LT984974 - - - Yuan & Wang 2018 Oophaga pumilio IW050 - LT984972 - - - Yuan & Wang 2018 Oophaga pumilio IW052 - LT984973 - - - Yuan & Wang 2018 Oophaga pumilio IW053 - LT984959 - - - Yuan & Wang 2018 Oophaga pumilio IW055 - LT984966 - - - Yuan & Wang 2018 Oophaga pumilio IW092 - LT984960 - - - Yuan & Wang 2018 Oophaga pumilio IW093 - LT984961 - - - Yuan & Wang 2018 Oophaga pumilio IW094 - LT984975 - - - Yuan & Wang 2018 Oophaga pumilio IW095 - LT984976 - - - Yuan & Wang 2018 Oophaga pumilio IW096 - LT984962 - - - Yuan & Wang 2018 Oophaga pumilio IW142 - LT984977 - - - Yuan & Wang 2018 Oophaga pumilio IW143 - LT984978 - - - Yuan & Wang 2018 Oophaga pumilio IW169 - LT984963 - - - Yuan & Wang 2018 Oophaga pumilio IW171 - LT984952 - - - Yuan & Wang 2018 Oophaga pumilio IW180 - LT984954 - - - Yuan & Wang 2018 Oophaga pumilio IW181 - LT984964 - - - Yuan & Wang 2018 Oophaga pumilio IW182 - LT984953 - - - Yuan & Wang 2018 Oophaga pumilio IW189 - LT984979 - - - Yuan & Wang 2018 Oophaga pumilio IW193 - LT984986 - - - Yuan & Wang 2018 Oophaga pumilio IW194 - LT984980 - - - Yuan & Wang 2018 Oophaga pumilio IW196 - LT984981 - - - Yuan & Wang 2018 Oophaga pumilio IW197 - LT984982 - - - Yuan & Wang 2018 Oophaga pumilio IW199 - LT984983 - - - Yuan & Wang 2018 Oophaga pumilio IW200 - LT984984 - - - Yuan & Wang 2018 Oophaga pumilio IW201 - LT984985 - - - Yuan & Wang 2018 north GECOH1145 Nuquí, Chocó, Colombia TBA - - TBA This Study Phyllobates aurotaenia north GECOH1155 Nuquí, Chocó, Colombia TBA - - TBA This Study Phyllobates aurotaenia north GECOH1307 Lloró, Chocó, Colombia TBA - - TBA This Study Phyllobates aurotaenia north GECOH1484 Tadó, Chocó, Colombia TBA - - TBA This Study Phyllobates aurotaenia north GECOH705 Tadó, Chocó, Colombia TBA - - TBA This Study Phyllobates aurotaenia north GECOH706 Tadó, Chocó, Colombia TBA - - TBA This Study Phyllobates aurotaenia Buenaventura, Valle del south GECOH1170 Cauca, Colombia TBA - - TBA This Study Phyllobates aurotaenia Buenaventura, Valle del south GECOH1190 Cauca, Colombia TBA - - TBA This Study Phyllobates aurotaenia Buenaventura, Valle del south GECOH1194 Cauca, Colombia TBA - - TBA This Study Captive bred, Parents from Pueblo Rico, TBA Obtained from - Risaralda, Colombia This Study transcriptome San José del Palmar, Phyllobates bicolor GECOH1039 Chocó, Colombia TBA - - TBA This Study Pueblo Rico, Risaralda, Phyllobates bicolor GECOH1174 Colombia TBA - - TBA This Study Pueblo Rico, Risaralda, Phyllobates bicolor GECOH1176 Colombia TBA - - TBA This Study Pueblo Rico, Risaralda, Phyllobates bicolor GECOH1485 Colombia TBA - - TBA This Study Pueblo Rico, Risaralda, Phyllobates bicolor GECOH523 Colombia TBA - - TBA This Study Isla Colón, Panamá, Phyllobates lugubris GECOH1865 Panamá TBA - - TBA This Study Isla Veraguas, Panamá, Phyllobates lugubris GECOH1866 Panamá TBA - - TBA This Study Puerto Viejo de talamanca, Limón, Costa Phyllobates lugubris GECOH963 Rica TBA - - TBA This Study Puerto Viejo de talamanca, Limón, Costa Phyllobates lugubris GECOH964 Rica TBA - - TBA This Study Puerto Viejo de talamanca, Limón, Costa Phyllobates lugubris GECOH966 Rica TBA - - TBA This Study Captive bred, Black Phyllobates terribilis- Jungle Terrarium Supply KT989191 KT989211 - KT989162 Tarvin et al. 2016 Buenaventura, Valle del Phyllobates terribilis GECOH1165 Cauca, Colombia TBA - TBA TBA This Study Timbiquí, Cauca, Phyllobates terribilis GECOH1183 Colombia TBA - TBA TBA This Study Buenaventura, Valle del Phyllobates terribilis GECOH646 Cauca, Colombia TBA - TBA TBA This Study Captive bred, Black Jungle Terrarium Supply Phyllobates terribilis RMPC025 and Josh’s Frogs TBA - TBA TBA This Study Mint morhp Phyllobates terribilis RMPC026 Captive bred, Black TBA - TBA TBA This Study Mint morhp Jungle Terrarium Supply and Josh’s Frogs Captive bred, Black Jungle Terrarium Supply Phyllobates terribilis RMPC027 and Josh’s Frogs TBA - TBA TBA This Study Mint morhp Captive bred, Black Jungle Terrarium Supply Phyllobates terribilis RMPC038 and Josh’s Frogs TBA - TBA TBA This Study Captive bred, Black Jungle Terrarium Supply Phyllobates terribilis RMPC039 and Josh’s Frogs TBA - TBA TBA This Study Captive bred, Black Jungle Terrarium Supply Phyllobates terribilis RMPC040 and Josh’s Frogs TBA - TBA TBA This Study Captive bred, Black Jungle Terrarium Supply Phyllobates terribilis RMPC041 and Josh’s Frogs TBA - TBA TBA This Study Captive bred, Black Jungle Terrarium Supply Phyllobates terribilis RMPC042 and Josh’s Frogs TBA - TBA TBA This Study Captive bred, Black Jungle Terrarium Supply Phyllobates terribilis RMPC043 and Josh’s Frogs TBA - TBA TBA This Study Captive bred, Black Jungle Terrarium Supply Phyllobates terribilis RMPC044 and Josh’s Frogs TBA - TBA TBA This Study Golfo Dulce, Puntarenas, Phyllobates vittatus GECOH976 Costa Rica TBA - - TBA This Study Golfo Dulce, Puntarenas, Phyllobates vittatus GECOH979 Costa Rica TBA - - TBA This Study Captive bred, Science Kit Obtained from Transcript 366577 Rana pipiens- & Boreal Labs Christenson et al. 2014 transcriptome toraro GECOH1698 Leticia, Amazonas, TBA - - TBA This Study Colombia Leticia, Amazonas, Ranitomeya toraro GECOH1702 Colombia TBA - - TBA This Study Ranitomeya Leticia, Amazonas, ventrimaculata GECOH1699 Colombia TBA - - TBA This Study Ubaque, Cundinamarca, Rheobates palmatus GECOH2050 Colombia TBA - - TBA This Study Ubaque, Cundinamarca, Rheobates palmatus GECOH2051 Colombia TBA - - TBA This Study Gene_27569_Rm102599d1524284t1_g_27569_m_27 Obtained from Rhinella marina - Australia (multiple sites) 569 Richardson et al. 2017 transcriptome Acandí, Chocó, nubicola GECOH2034 Colombia TBA - - TBA This Study Buenaventura, Valle del Silverstoneia cf. erasmios GECOH2039 Cauca, Colombia TBA - - TBA This Study Buenaventura, Valle del Silverstoneia cf. erasmios GECOH2040 Cauca, Colombia TBA - - TBA This Study Annotated ENSXETP00000031166 Xenopus tropicalis - - ENSEMBL gene Annotated ENSLACP00000010984 Latimeria chalumnae*- - ENSEMBL gene Annotated ENSRNOP00000016841 Rattus norvegicus*- - ENSEMBL gene Annotated ENSPSIP00000008805 Pelodiscus sinensis*- - ENSEMBL gene Annotated ENSACAP00000012689 Anolis carolinensis*- - ENSEMBL gene * Not used in analyses. Only included in figures for comparison. Table S2. Mutations of NaV channels known to confer resistance, at least partially to BTX. hNaV stands for human sodium channels, rNaV for rat sodium channels, and BgNaV for the cockroach sodium channel. Site numbers follow the rat SCN4A sequence.

Domain Position Substitution NaV Paralog Reference

429 S → K hNaV1.5 (Wang, Tikhonov, Zhorov, et al. 2007)

429 S → R hNaV1.5 (Wang, Tikhonov, Zhorov, et al. 2007)

1 429 S → W hNaV1.5 (Wang, Tikhonov, Mitchell, et al. 2007) DI 433 I → K rNaV1.4 (Wang and Wang 1998)

434 N → K rNaV1.4 (Wang and Wang 1998)

437 L → K rNaV1.4 (Wang and Wang 1998)

784 N → K rNaV1.4, hNaV1.5 (Wang et al. 2001; Wang, Tikhonov, DII Mitchell, et al. 2007)

788 L → K rNaV1.4 (Wang et al. 2001)

1236 F → K hNaV1.5 (Wang et al. 2006)

1236 F → R hNaV1.5 (Wang et al. 2006)

1 1275 G → A BgNaV1-1 (Du et al. 2011)

DIII 1276 S → K rNaV1.4, BgNaV1-1 (Wang et al. 2000; Du et al. 2009)

1277 F → K BgNaV1-1 (Du et al. 2011)

1 1277 F → A BgNaV1-1 (Du et al. 2011)

1280 L → K rNaV1.4, BgNaV1-1 (Wang et al. 2000; Du et al. 2009)

1579 F → K rNaV1.4 (Wang and Wang 1999)

2 1579 F → A rNaV1.2, rNaV1.3 (Linford et al. 1998; Li et al. 2002)

1579 F → I rNaV1.3 (Li et al. 2002)

1583 V → C rNaV1.4 (Vendantham and Cannon 2000) DIV 1584 N → T rNaV1.4 (Wang and Wang 2017)

1584 N → K rNaV1.4 (Wang and Wang 1999)

1Produces partial resistance to BTX. 2 Mutant F1579A was shown to be resistant to BTX in rNaV1.2 and 1.3, but this same mutant was readily modified by BTX in rNaV1.4 (Wang et al. 1999). Table S3. Sequences excluded from analyses due to possible contamination. See the Results section and Figure S1 of Yuan and Wang (2018) for further information.

Species Voucher Locality DI DII DIII DIV Adelphobates galactonotus TNHC-FS4889 Captive bred KT989187 - - KT989158 Ameerega trivittata TNHC-GDC17589 - KT989183 - - KT989154 San Félix, Táchira, Aromobates saltuensis TNHC-FS5544 KT989176 - - KT989147 Colostethus fugax- Canelos, Ecuador KT989192 - KT989225 KT989163 captivus - Zumbi, Ecuador KT989186 KT989209 KT989223 KT989157 LSUMZH-15103 Isla de Diamante, Oophaga pumilio (OMNH-33297) Nicaragua KT989188 - - KT989159 Quibdó, Chocó, Phyllobates aurotaenia TNHC-FS4990 Colombia KT989190 - - KT989161

Table S4. Primers designed for the amplification of SCN4A gene regions, their locations in the gene, and the annealing temperatures and elongation times used in PCR cycles. All reactions used the following cycling protocol: Initial 2 min at 95°C, followed by 35 cycles of 30s at 95°C, 40 sec at T A, tE at 72°C, and a final extension step of 5 min at 72 °C.

Name Location Sequence (3’ - 5’) TA tE E9F DI S6-S6 Linker ACCCTAAGAGCTGCAGGTAA 50°C 0:20 E9R DI-DII Linker TGTTTCTTTAGCTGTTCTAGCATGT 50°C 0:20 E21F DIII S6-S5 Linker ATTGGGGATCAACCTGKMTAYG 50°C 1:00 E22R DIII-DIV Linker CGGTCTAGGAATTGGTTTCACCGGC 50°C 1:00 I21R1 Intron 21 AATATGATCRTTCTGCCCYSATA 48°C 1:00 I21R2 Intron 21 GTTTRCTATGAATTGTRAATSACC 48°C 1:00 I21R3 Intron 21 GWAATWCCCCACAGACTATCA 48°C 1:00 E24F DIV S5 TGCCYTGATGATGTCWCTCCCWGC 58°C 1:00 E24R C-terminal Intracellular loop TCCAAYCACCATYGGGAGRTCCA 58°C 1:00 Fig. S1: SCN4A Gene genealogies ! We used a phylogenetic approach to look for potential contamination or other artifacts in our dataset. Sequences for each species were aligned to extract unique haplotypes, which were then aligned among themselves, in both cases using MUSCLE (Edgar 2004). These alignments were then used to infer maximum likelihood trees in PhyML (Guindon and Gascuel 2003) with a single data partition and the best model of DNA evolution, chosen under the AICc criterion in jModelTest2 (Darriba et al. 2012). Tolopogy searches were done with nearest-neighbor interchange (NNI) moves, starting from a BioNJ tree, and nodal support was evaluated with approximate Bayes support values (aBayes; Guindon et al. 2010). In all cases we recovered trees with no conspicuous deviations from current phylogenetic hypotheses, suggesting that no contamination or artifacts were present in our data. Below we present each genealogy along with the best model. Numbers on branches or in front of nodes correspond to aBayes support. Support values of 0 were removed to improve visualization. ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! DI

1 Rana_pipiens Nanorana parkeri 0.65 Mantella aurantiaca Gastrotheca litonedis 0.52 Espadarana callistomma Lithodytes lineatus 1 0.65 Rhinella marina Incilius nebulifer 0.86 0.47 Allobates talamancae Allobates femoralis 1 Allobates zaparo Rheobates palmatus 0.68 Hyloxalus nexipus 1 Hyloxalus italoi Oophaga granulifera A1 0.68 Dendrobates tinctorius 1 Dendrobates truncatus 1 Denrobates auratus A1 0.97 Dendrobates auratus A2 Oophaga pumilio A4 0.99 Oophaga pumilio A1 Oophaga pumilio A2 Ranitomeya toraro A2 Oophgaga histrionica Oophaga pumilio A3 Ranitomeya toraro A1 0.59 Ranitomeya ventrimaculata 0.96 Andinobates fulguritus 0.67 0.84 Andinobates bombetes Phylobates vittatus Phylobates aurotaenia South 1 Phylobates lugubris Phylobates terribilis Phylobates bicolor Phylobates aurotaenia North Ameerega bilinguis 0.96 Ameererga petersi 0.92 Ameerega parvula Ameerega trivittata A2 271 nucleotide sites 0.87 Ameerega hahneli Best model: HKY+G 0.96 Ameerega picta Colostethus panamansis 0.91 1 Silverstoneia cf. erasmios Silverstoneia nubicola Epipedobates boulengeri Epipedobates boulengeri North A2 Epipedobates anthonyi 1 Epipedobates boulengeri North A1 0.05 Epipedobates tricolor Epipedobates machalilla Epipedobates darwinwallacei ENSXETT00000031166 DII

Mantella aurantiaca

Nanorana parkeri

Rana pipiens

Gastrotheca lithonedis 0.82 Epipedobates boulengeri 0.95

Epipedobates tricolor 0.64 0.6 Dendrobates tinctorius

0.88 Phyllobates bicolor 0.65 0.99 1 Phyllobates terribilis

Ameerega bilinguis 1 1

Ameerega parvula

Hyloxalus italoi 0.37 0.53 Hyloxalus nexipus 249 nucleotide sites Best model: TPM1uf+G Rhinella marina 1

0.4 Incilius nebulifer

Espadarana callistomma

0.85 0.04 Hypsiboas picturatus

Xenopus tropicalis ! ! DIII

Mantella aurantiaca

Rana pipiens

0.8 Nanorana parkeri

Espadarana callistoma

Gastrotheca litonedis

1 Hypsiboas picturatus

Incilius nebulifer 1 0.97 Rhinella marina

Hyloxalus nexipus 0.53

Hyloxalus italoi 0.86 0.58 Phyllobates bicolor 0.99 0.82 Phyllobates terribilis

252 nucleotide sites 1 Dendrobates tinctorius Best model: HKY+I Ameerega bilinguis

0.76 Epipedobates tricolor 1

Epipedobates boulengeri 0.03 Xenopus tropicalis ! DIV

1 Rana pipiens Nanorana parkeri 0.58 Mantella aurantiaca 0.92 Espadarana callistomma Lithodytes lineatus 0.42 0.43 1 R.marina Incilius nebulifer Gastrotheca litonedis 0.92 Hypsiboas picturatus 1 Rheobates palmatus 1 Allobates talamancae Allobates zaparo 0.39 Allobates femoralis 1 1 Dendrobates tinctorius Dendrobates auratus 0.86 Dendrobates truncatus A1 Dendrobates truncatus A2 0.99 Andinobates fulguritus 1 0.99 Andinobates bombetes A2 Andinobates bombetes A1 1 Ranitomeya toraro A1 1 Ranitomeya ventrimaculata 0.99 Ranitomeya toraro A2 1 Oophaga histrionica Oophaga pumilio 0.55 Hyloxalus italoi 0.45 Hyloxalus nexipus 1 Epipedobates tricolor Epipedobates darwinwallacei Epipedobates boulengeri 0.98 Epipedobates anthonyi 0.98 Epipedobates boulengeri 1 Epipedobates machalilla E.boulengeri north A1 E.boulengeri north A2 Silverstoneia cf. erasmios 0.45 0.96 Silverstoneia nubicola 0.99 Colostethus panamansis 0.98 Ameerega picta Ameerega hahneli 0.84 1 Ameerega petersi Ameerega trivittata Ameerega bilinguis 0.95 Ameerega parvula 1 Phyllobates terribilis A1 Phyllobates bicolor 548 nucleotide sites Phyllobates aurotaenia North A2 Best model: TPM3uf+G Phyllobates aurotaenia North A1 Phyllobates aurotaenia South Phyllobates vittatus Phyllobates terribilis A2 Phyllobates lugubris A1 1 Phyllobates lugubris A2 0.05 Phyllobates lugubris A3 Xenopus tropicalis Fig. S2: Additional P-loop and S6 alignments ! Below we present alignments for regions in the DIII P-loop and S6 segments, and the DIV P- loop of NaV1.4 proximal to BTX binding sites (Wang et al. 2000; Wang et al. 2006). No mutations potentially associated with resistance to BTX or other alkaloids were found in these regions. Full alignments of all the sequenced sites are available in phylip format in the Dryad repository (http://dx.doi.org/xxxx/xxxx). As in Figures 2-3, sites where directed mutagenesis has caused BTX resistance in vitro are shaded in gray.

DIV-Ploop

FNFETFGNSI ICLFQITTSAGWDGLL Latimeria chalumnae ...... Rattus norvegicus ...... E ...... Pelodiscus sinensis ...... Anolis carolinensis ...... M...... Xenopus tropicalis ...... Nanorana parkeri ...... M....E...... Mantella aurantiaca --...... Rana pipiens ...... Gastrotheca litonedis --...... E...... Hypsiboas picturatus ------Espadarana callistomma --...... E...... Lithodytes lineatus --...... G..M...... Incilius nebulifer --...... Rhinella marina ...... Rheobates palmatus ...... Allobates zaparo --...... Allobates femoralis --...... Allobates talamancae --...... Hyloxalus italoi --...... Hyloxalus nexipus --...... Phyllobates vitattus ...... Phyllobates lugubris ...... Phyllobates bicolor ...... Phyllobates aurotaenia North ...... Phyllobates aurotaenia South ...... Phyllobates terribilis A1 ...... Phyllobates terribilis A2 --...... Ranitomeya ventrimaculata ...... Ranitomeya toraro ...... Andinobates bombetes ...... S...... Andinobates fulguritus ...... Oophaga granulifera ------Oophaga pumilio ...... Oophaga histrionica ...... Dendrobates truncatus ...... Dendrobates auratus ...... Dendrobates tinctorius --.....S...... Colostethus panamansis - - ...... Ameerega bilinguis --...... Ameerega picta ...... Ameerega trivittata ...... Ameerega petersi ...... Ameerega hahneli --...... Ameerega parvula ------Silverstoneia cf. erasmios ...... Silverstoneia nubicola ...... Epipedobates boulengeri North ...... Epipedobates boulengeri --...... Epipedobates tricolor --...... Epipedobates machalilla --...... Epipedobates anthonyi --...... Epipedobates darwinwallacei --...... DIII-Ploop DIII-S6

SLLQVATFKGWMD IM IFI IFGAFFTLNLF IG Latimeria chalumnae ...... E...... Rattus norvegicus ...... S...... Anolis carolinensis ...... V...... Pelodiscus sinensis ...LGTGAP.TLAAL ...... Xenopus tropicalis ...... Nanorana parkeri .....------Mantella aurantiaca ...... Rana pipiens ...... Gastrotheca litonedis ...... S...... Hypsiboas picturatus ...... Espadarana callistomma ...... Incilius nebulifer ...... Rhinella marina ...... Hyloxalus italoi ...... Hyloxalus nexipus ...... Phyllobates terribilis ------...... Phyllobates bicolor ...... Dendrobates tinctorius ...... Ameerega parvula ...... Ameerega bilinguis ...... Epipedobates boulengeri ......

! ! Fig S3: Orthology to SCN4A ! After obtaining protein sequences for each species (see Methods section in the main text), we built a gene genealogy of all members of the SCNA gene family from several tetrapods to confirm that all our sequences were orthologous to SCN4A. We first aligned the full SCNA protein sequences from Rattus norvegicus (brown rat), Latimeria chalumnae (coelacanth) Xenopus tropicalis, Nanorana parkeri, Rhinella marina, and Rana pipiens using MUSCLE. These sequences were either obtained from ENSEMBL, or by blasting the transcriptomes/ genomes of each species as indicated in the main text. Subsequently, we used this alignment as a scaffold to which each of the alignments performed for ancestral sequence reconstruction were merged with the “Profile Align” function in Geneious (Drummond et al. 2010). Finally, we used ProtTest (Darriba et al. 2011) to chose the best model of protein substitution (JTT+I+G+F), and built a gene tree with PhyML as detailed in Fig. S1. Our sequences formed a clade with all other SCN4A sequences (shown below in red), confirming their orthology to this gene. Accession numbers for sequences derived from a single individual are indicated in taxon labels. Branch labels correspond to aBayes support values; support below 0.5 was not annotated for visualization purposes. Coelacanth - ENSLACP00000003463 - SCN10A 1 1 Rat - ENSRNOP00000069875 - SCN5A 1 Rat - ENSRNOP00000047944 - SCN10A 0.8 Rat - ENSRNOP00000033224 - SCN 11A 1 X.tropicalis - ENSXETP00000009252 - SCN5A 1 R.marina - Gene.848+74846+96082 R.pipiens - Transcript_397926 - SCN5A 1 0.94 N.parkeri - Npar_R004585 - SCN5A 1 Rat - ENSRNOP00000008160 - SCN8A Coelacanth - ENSLACP00000021496 - SCN8A X.tropicalis - ENSXETP00000015904 - SCN8A 0.63 1 1 R.marina - Gene.124028 - SCN8A 1 1 R.pipiens - Transcript_267747 - SCN8A N.parkeri - Npar_R004585 - SCN8A Coelacanth - ENSLACP00000008553 - Unnamed 1 Rat - ENSRNOP00000068852 - SCN9A 1 1 Rat - ENSRNOP00000044385 - SCN7A 1 Rat - ENSRNOP00000072091 - SCN3A 1 Rat - ENSRNOP00000074819 - SCN1A 1 Rat - ENSRNOP00000007069 - SCN2A 1 X.tropicalis - ENSXETP00000019653 - SCN3A 1 R.pipiens - Transcript_152834 R.marina - Gene.94782 1 0.88 N.parkeri - Npar_R004590 1 X.tropicalis - ENSXETP00000045061 - SCN1A 1 R.marina - Gene.124028 1 N.parkeri - Npar_R004585 1 X.tropicalis - ENSXETP00000019636 -SCN2A 1 R.marina - Gene.124028 1 R.pipiens - Transcript_416622+152876 N.parkeri - Npar_R004589 Coelacanth - ENSLACP00000010984 - SCN4A 1 0.99 D.tinctorius_D2 - KT989210 1 Rat - ENSRNOP00000016841 - SCN4A G.litonedis_D3 - KT989216 0.93 G.litonedis_D1 - KT989173 X.tropicalis - ENSXETP00000031166 - SCN4A 1 N.parkeri - Npar_R004585 M.aurantiaca_D4 - KT989141 G.litonedis_D2 - KT989202 M.aurantiaca_D3 - KT989214 1 R.pipiens - Transcript_366577 0.98 Hyp.picturatus_D2 - KT989204 Es.callistoma_D2 - KT989203 Hyp.picturatus_D4 - KT989146 M.aurantiaca_D2 - KT989200 0.78 Am.bilinguis_D3 - KT989220 L.lineatus_D1 - KT989174 R.palmatus_D4 Al.femoralis_D1 - KT989177 Al.talamancae_D1 - KT989178 Al.talamancae_D4 - KT989149 Es.callistoma_D4 - KT989144 0.62 Hyl.italoi_D3 - KT989221 R.marina - Gene.27569 I.nebulifer_D4 - KT989142 L.lineatus_D4 - KT989145 I.nebulifer_D1 - KT989172 I.nebulifer_D2 - KT989201 Ep.boulengeri_D3 - KT989227 Es.callistoma_D1 - KT989175 Hyl.italoi_D4 - KT989155 C.panamansis_D4 - KT989164 Hyl.italoi_D2 - KT989207 A.picta_D1 A.trivittata_A2_D1 S.flotator_D1 Am.hahneli_D1 - KT989181 Hyl.italoi_D1 - KT989184 Am.bilinguis_D1 - KT989180 Hyl.nexipus_D3 - KT989222 Ep.tricolor_D3 - KT989229 Hyp.picturatus_D3 - KT989218 D.tinctorius_D3 - KT989224 S.nubicola_D1 Am.bilinguis_D2 - KT989205 Am.parvula_D2 - KT989206 0.79 E.boulengeri_North_D4 Hyl.nexipus_D1 - KT989185 1 Ep.tricolor_D4 - KT989170 E.boulengeri_D4 Ep.darwinwallacei_D4 - KT989168 Ep.anthonyi_D4 - KT989166 Ep.machalilla_D4 - KT989169 1 S.nubicola_D4 S.flotator_D4 I.nebulifer_D3 - KT989215 Es.callistoma_D3 - KT989217 C.panamansis_D1 - KT989193 R.palmatus_D1 A.petersi_D1 Hyl.nexipus_D2 - KT989208 Hyl.nexipus_D4 - KT989156 O.pumilio_D1 0.74 A.fulguritus_D1 Ep.anthonyi_D1 - KT989195 O.histrionica_D1 A.bombetes_D1 R.ventrimaculata_D1 R.toraro_D1 0.94 A.trivittata_D4 Am.bilinguis_D4 - KT989151 0.99 Am.parvula_D4 - KT989153 A.picta_D4 Am.hahneli_D4 - KT989152 A.petersi_D4 Al.talamancae_D4 - KT989149 G.litonedis_D4 - KT989143 0.99 0.95 Al.talamancae_D4 - KT989149 Al.femoralis_D4 - KT989148 E.boulengeri_D1 O.granulifera_A1_D1 E.boulengeri_North_D1 0.98 D.truncatus_D1 D.auratus_A1_D1 Am.parvula_D1 - KT989182 Ep.tricolor_D1 - KT989199 Ep.darwinwallacei_D1 - KT989197 Ep.machalilla_D1 - KT989198 M.aurantiaca_D1 - KT989171 Ep.tricolor_D2 - KT989213 D.tinctorius_D1 - KT989189 Ep.boulengeri_D2 - KT989212 P.terribilis_D2 - KT989211 0.5 P.lugubris_D4 P.aurotaenia_South_D4 P.bicolor_D4 P.terribilisA1_D4 P.vittatus_D4 P.auro_North_D4 P.bicolor_Transcript P.terribilisA1_D1 P.terribilisA2_D1 P.aurotaenia_South_D1 P.vittatus_D1 P.aurotaenia_North_D1 P.lugubris_D1 P.terribilisA2_D4 P.bicolor_D1 P.terribilis_D3 O.pumilio_D4 O.histrionica_D4 1 D.tinctorius_D4 - KT989160 D.truncatus_D4 D.auratus_D4 1 0.56 A.bombetes_D4 A.bombetes_A1_D4 0.3 0.98 A.fulguritus R.toraro R.ventrimaculata