bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 GENETIC BASES OF APOSEMATIC TRAITS: INSIGHTS FROM THE SKIN

2 TRANSCRIPTIONAL PROFILES OF Oophaga POISON FROGS

3

4

5 Andrés Posso-Terranova1-2*, José Andres1-3.

6

7

8 1. Department of Biology. University of Saskatchewan. 112 Science Pl. Saskatoon. SK.

9 Canada.

10 2. Universidad Nacional de Colombia sede Palmira. A.A. 237. Palmira. Colombia.

11 3. Department of Ecology and Evolution. Cornell University. Corson Hall. Ithaca. NY.

12 14853.

13

14 *Corresponding author

15 Email: [email protected]

16

17 All authors contributed equally to this work. bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

18 ABSTRACT

19 Aposematic organisms advertise their defensive toxins to predators using a variety

20 of warning signals, including bright coloration. While most Neotropical poison frogs

21 (Dendrobatidae) rely on crypsis to avoid predators, Oophaga poison frogs from South

22 America advertise their chemical defenses, a complex mix of diet-derived alkaloids, by

23 using conspicuous hues. The present study aimed to characterize the skin transcriptomic

24 profiles of the South American clade of Oophaga poison frogs (O. anchicayensis, O.

25 solanensis, O. lehmanni and O. sylvatica). Our analyses showed very similar

26 transcriptomic profiles for these closely related species in terms of functional annotation

27 and relative abundance of gene ontology terms expressed. Analyses of expression profiles

28 of Oophaga and available skin transcriptomes of cryptic anurans allowed us to propose

29 possible mechanisms for the active sequestration of alkaloid-based chemical defenses and

30 to highlight some genes that may be potentially involved in resistance mechanisms to

31 avoid self-intoxication and skin coloration. In doing so, we provide an important

32 molecular resource for the study of warning signals that will facilitate the assembly and

33 annotation of future poison frog genomes.

34

35 KEYWORDS

36 Dendrobatids, RNA sequencing, transcriptomes, gene ontology, candidate

37 genes. bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

38 INTRODUCTION

39 Aposematic organisms advertise their defensive toxins to predators using a variety

40 of warning signals [1-3]. Among aposematic species, the poison frogs (Dendrobatidae)

41 from the tropical rain forests of Central and South America represent one of the most

42 spectacular examples of warning coloration. While the majority of dendrobatids rely on

43 crypsis to avoid predators, some members of this family are both brightly colored and

44 chemically defended [4]. From a genetic point of view, these aposematic defenses can be

45 defined as a complex phenotype resulting from the integration (i.e. covariation) of

46 different genetic elements related to conspicuousness, bold behavior, unpalatability, diet

47 specialization, etc. While a long history of research has been devoted to understand the

48 genetics of warning coloration in arthropods, particularly in Heliconius butterflies [5, 6],

49 the molecular underpinnings of aposematism in vertebrates, particularly the mechanisms

50 whereby individuals become toxic (or distasteful) remain mostly unknown. In this study,

51 our primary aim was to characterize the skin transcriptomic profiles of Oophaga species

52 as a first attempt to shed light on the molecular genetic bases of aposematic components

53 in poison frogs: the ability to sequester alkaloid-based chemical defenses, warning

54 coloration, and the resistance mechanisms to avoid self-intoxication.

55 The frogs of this complex inhabit the lowland Pacific rainforests of the

56 Colombian and Ecuadorian Chocó. Preliminary molecular data from nuclear and

57 mitochondrial markers showed a similar genetic background among lineages in contrast

58 to an extraordinary diversity of morphotypes [7]. Individuals from different allopatric

59 lineages can be relatively homogeneous, striped, or spotted, and their colors range from

60 bright red, to orange and yellow (Fig 1) [8]. These polymorphic coloration patterns serve bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

61 as a warning signal of their chemical defenses [9], a complex mix of diet-derived

62 alkaloids secreted by the dermal glands [10, 11]. Although these chemicals may involve

63 metabolism and transport through other tissues, the final accumulation of toxins as well

64 as the production of pigmentary cells is performed in the skin tissue of these organisms

65 even during the adult stage [12-14]. Thus, we expected that the genes, pathways, and/ or

66 gene networks potentially associated with coloration, alkaloid metabolism, transport and

67 storage, to be expressed in the skin of these lineages.

68 Although the lineages of the Oophaga studied here display a wide variety of

69 warning signals, most of them share a black background coloration (Fig 1). In adult frogs,

70 this melanistic coloration is controlled by the lack of iridophores and xanthophores in the

71 dermal chromatophore unit, as well as the size and dispersion pattern of the melanin-

72 containing organelles (melanosomes) within the dermal melanophores [15]. Thus, we

73 also hypothesized that in adult individuals, genes involved in the amount, size and

74 distribution of the melanosomes should be differentially expressed in the skin. Similarly,

75 because the secreted alkaloids of chemically-defended dendrobatids are known to disrupt

76 the normal ion-channel activity and to alter the neurotransmitter-receptor binding

77 capacity of nerve and muscle cells [16-18], we hypothesized that the fraction of genes

78 differentially expressed in the skin of poison frogs (when compared to non-aposematic

79 species) is also enriched in genes related to alkaloid sequestration, transport and

80 autotoxicity avoidance. bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

81 Fig 1. Hierarchical clustering (bootstrap=999) and heatmap contrasting the 82 differentially expressed unigenes from skin tissue between Oophaga and cryptic species. 83 Color within the heatmap represent the log2 fold change values. Dark blue indicates 84 upregulation while a lighter coloration indicates downregulation. Rows of the heatmap 85 correspond to the differentially expressed unigenes (n=1,931) detected in this study. 86 bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

87 To test these hypotheses, we first assembled the skin transcriptomes of five

88 different Harlequin poison frogs species: O. histrionica [19], O. lehmanni [20], O.

89 sylvatica, [21] and the recently described O. anchicayensis and O. solanensis [22]. Then,

90 we compared the transcription profiles of these aposematic species with that of a diverse

91 group of cryptic anurans. Functional annotation of unigenes showing a phylogenetic

92 signal (i.e. highly represented and differentially expressed in Oophaga poison frogs)

93 allowed us to propose plausible metabolic pathways (and candidate genes) related with

94 toxicity and color. Overall, this study provides an important molecular resource for the

95 study of aposematism within poison frogs and will facilitate the future assembling and

96 annotation of the complex dendrobatid genomes [23].

97

98 METHODS

99 Library preparation and sequencing

100 Due the conservation status of these frogs (CR, EN, VU; IUCN) and the

101 restrictions imposed by the Ethics Committee, we were allowed to euthanize a very

102 limited number of samples per species. Seven individuals (O. lehmanni and O.

103 anchicayensis, n=2 per each species; O. histrionica, O. sylvatica, and O. solanensis, n=1;

104 each species) were euthanized in the field with benzocaine gel at 5% by direct application

105 into the mouth cavity. Individual skin samples were taken and stored at -80 °C in

106 RNAlater® (Life Technologies, Carlbad, CA). RNA extractions were performed using

107 Tri-Reagent (Molecular Research Center, Cincinnati, OH). After quality check (Agilent

108 Bioanalyzer Agilent Technologies, Wilmington, DE) samples were used to generate

109 RNA-seq libraries using the Illumina Truseq RNA Sample Prep protocol (Illumina, San bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

110 Diego, CA). Libraries were cleaned using AMPure XP and sequenced on a single

111 Illumina HiSeq2000 lane (TruSeq SBS v. 3) as follows: four libraries (O. anchicayensis,

112 O. histrionica, O. lehmanni and O. sylvatica) were sequenced using single-end (150-bp)

113 while the remaining three libraries (O. anchicayensis, O. solanensis and O. lehmanni)

114 were run in a single lane of a paired-end module (100-bp, x 2). All animal procedures

115 were approved by the Ethics Committee of Universidad Nacional de Colombia (Acta

116 No.03, July 22nd, 2015) and were conducted based on the NIH Guide for the Principles of

117 Animal Care. Sampling was conducted according to the research permits granted by

118 Autoridad Nacional de Licencias Ambientales ANLA, Resolución 0255 del 12 de Marzo

119 de 2014 and Resolución 1482 del 20 de Noviembre de 2015. Sequencing was carried out

120 by the Cornell University’s BioResource Center. Datasets including raw RNA sequence

121 reads are available from the authors upon request.

122 Transcriptome assemblies and functional annotation

123 Initial read quality trimming, filtering and removal of adapters was performed

124 using FLEXBAR v.2.5 [24]. All retained reads were ≥ 50bp with an average quality of ≥

125 30 and less than two uncalled (ambiguous) bases (S1 Table). For each library, a de novo

126 transcriptome was assembled using TRINITY v.2.1.0 [25] with default settings (kmer=25,

127 minimum contig length=48) and keeping only the longest transcript per cluster for

128 subsequent analyses. Varying these parameters (i.e. default settings) did not result in

129 assemblies with longer N50 values. To account for differences in the quality and

130 sequencing strategy among individual transcriptomes, we constructed a composite de

131 novo reference transcriptome. Briefly, we followed [26] and combined 20% randomly

132 selected reads (>33 million in total) from each of the four single-end libraries (O. bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

133 anchicayensis, O. histrionica, O. lehmanni and O. sylvatica). Then, a TRINITY assembly

134 was performed using a minimum contig length of 350 bp. To filter out highly similar

135 contigs that may potentially represent alternatively spliced transcripts, we implemented

136 the error correction module of iAssembler v1.3.2 [27] with default parameters (maximum

137 length of end clips=30 bp, minimum overlap length=40 bp, minimum percent sequence

138 identity=95%).

139 Gene annotation was conducted using a sequential BLASTX search to both, the

140 available Xenopus transcriptomes and the NCBI non-redundant (nr) database. The

141 composite transcripts were first compared with that of the Xenopus databases retaining

142 annotations with E-values ≤ 10-5. Unannotated contigs were then submitted for BLASTX

143 to the nr protein database for possible identification. Gene ontology (GO) annotation and

144 term mapping was performed using Blast2Go v 5.2.0 with default significance cutoffs

145 [28].

146 To estimate the completeness of each transcriptome, we implemented the

147 expected gene content of Benchmarking Universal Single-Copy Orthologs (BUSCO v3)

148 [29] as implemented in gVolante v1.1.0 [30]. This widely used metrics for transcriptome

149 assembly [31] assesses the quality of any given transcriptome by estimating the

150 completeness of core vertebrate genes predicted to be ubiquitous in eukaryotes. The pre-

151 processing of the reads and all BLASTX analyses were run in the Bugaboo Dell Xeon

152 cluster of the western Canada's WestGrid computer facilities (www.westgrid.ca).

153 Transcriptome profiles

154 Analyses on the distribution of reads and the relative abundance of different

155 transcripts within each individual sample (i.e. lineage) were carried out by mapping bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

156 RNA-seq reads back to the composite de novo assembly. In order to identify highly

157 represented unigenes, we first implemented eXpress (probabilistic assignment of

158 ambiguously mapping sequenced fragments) [32] to estimate the effective number of

159 reads (ER) that mapped to the contigs in the reference transcriptome after adjusting for

160 read number and length biases. Then, to compare the proportion of reads that mapped to a

161 transcript in each Oophaga RNA-library, we estimated the number of transcripts per

162 million (TPM), a measure of RNA abundance that allows the comparison between

163 samples (sum of all TPMs in each sample are the same) [33]. We visually explored for

164 highly represented unigenes in each RNA-library by using dispersion plots of the ER

165 values and percentile plots of the TPM distribution. Additionally, we performed a

166 Principal Component Analysis (PCA) of the reference contigs dataset and using each

167 library TPMs values as independent variables and selected the overall top expressed

168 unigenes (2%).

169 To further characterize the skin transcriptome profile of Oophaga poison frogs,

170 we compared the transcription levels of annotated unigenes in this clade with those

171 observed in several cryptic (i.e. non-aposematic) anurans, including a toad (Bufo

172 gargarizans), and five frog species (Pelophylax nigromaculatus, Polypedates

173 megacephalus, Rana catesbeiana, Rana sylvatica, and Xenophrys sangzhiensis, S2 Table)

174 [34, 35]. RNA-seq data from these species was obtained from the Sequence Read Archive

175 platform (SRA) and represented the only skin-based RNA-seq available to us for

176 comparison purposes. Although transcriptomic datasets are available for

177 phylogenetically closer and cryptic species (i.e Colostethus) [36], these datasets were not

178 included in our analysis because they are not derived from skin tissue and may bias our bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

179 analyses. Read quality, contamination screening, duplicate removal, and quality trimming

180 steps were performed in FLEXBAR v.2.5 as previously described. The filtered raw reads

181 from individual libraries (Oophaga, n=7; cryptic species n=6) were mapped to our

182 composite de novo reference transcriptome using Bowtie2 v2.3.4.1 [37]. To accommodate

183 for sequence divergence among the taxa included in our study (≈75–98 similarity), we

184 followed [38] and ran the alignment algorithm allowing for soft clipping (--local) and

185 with a maximum penalty value of three (--mp 3) . Then, we implemented eXpress [32] to

186 estimate the effective number of reads (ER) that mapped to the contigs in the reference

187 transcriptome after adjusting for read number and length biases. Only unigenes with an

188 average of >10 ER/library were included in the differential expression analyses [39].

189 To identify genes showing a phylogenetic signal in their expression levels and to

190 estimate fold changes in expression levels between aposematic (i.e. Oophaga) and cryptic

191 species, we followed [40] and implemented the R package DEseq [41] to select unigenes

192 with an adjusted p-value of <0.05. To visualize patterns of expression, we constructed a

193 multidimensional scaling plot (Euclidean distances) of the ER data using Past v. 3.18

194 [42]. A heatmap of the expression differences between Oophaga and cryptic anuran

195 lineages (ngenes=1,931) was generated using the ‘heatmap.2’ function of the ‘gplots’

196 package, with Euclidean distances and complete linkage for clustering (bootstrap=999)

197 [43]. BLAST analysis and GO term mapping for the 1,931 unigenes included in this

198 analysis was performed as described above.

199

200 RESULTS

201 Transcriptome assemblies and functional annotation bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

202 Illumina sequencing produced an average number of reads per sample of 118.3

203 million for paired-end and 37.7 million for single-end libraries. After a stringent read

204 trimming involving the removal of low-quality sequences, duplicated reads and reads

205 containing adapter/primer sequences, we retained over 81% of the initial sequencing data.

206 Paired-end libraries produced a fairly consistent number of reads (S1 Table).

207 After the removal/merging of highly similar contigs (paired-end:7,5% - 9,6%;

208 single-end: 6.0% - 13.4%), the 150-bp based transcriptomes recovered a large number of

209 contigs ranging from 35,287 (O. anchicayensis) to 107,381 (O. sylvatica). Paired-reads

210 libraries (100-bp; 2X) generated transcriptomes with a relatively similar number of

211 contigs (40,398 for O. solanensis and 60,494 for O. lehmanni). N50 values and average

212 transcript length (AL) were lower for paired-end libraries (N50= 498-561bp; AL=

213 441.78±379 - 475.93±436bp) than for assemblies produced with single-end libraries

214 (N50=667-1579bp; AL=538.9±1001bp - 668.6±819 bp) (Table 1). Assembled and

215 annotated transcriptomes are available from the authors upon request. bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

216 Table 1. Summary statistics and quality assessment estimators of the eight de novo assembled transcriptomes generated in this study 217 (Oophaga species n=7; composite reference n=1). 218 219 Paired-end libraries Single-end libraries

O. solanensis O. anchicayensis O. lehmanni O. sylvatica O. lehmanni O. anchicayensis O. histrionica Reference

22.23 32.06 35.7 93.9 77.3 23.16 85.97 31.6 File size (Mbytes) 43,690 63,520 66,937 123,916 117,082 37,552 111,823 31,498 # contigs # contigs after filtration 40,398 57,516 60,494 107,381 104,523 35,287 96,892 31,498 (removal/merging of highly similar contigs) 19,853,219 28,061,593 31,856,993 81,266,530 66,367,430 20,238,091 74,768,159 31,968,628 # bases 17,164,733 23,756,585 26,287,312 58,224,462 50,845,151 15,278,290 53,582,406 31,968,628 # bases after filtration 7.5% 9.5% 9.6% 13.3% 10.7% 6.0% 13.4% 0.0% Merged contigs Average transcript 454.41± 405 441.78± 379 475.93 ± 436 655.8 ± 799 566.8± 648 538.9 ± 1579 668.6 ± 819 1,014.91 ± 948.43 length (AL) 6,115 7,915 8,400 15,102 12,906 54,265 15,136 16,041 Maximum length 528 498 561 1079 809 667 1,101 1,316 N50 46.5% 47.7% 50.8% 42.1% 41.6% 53.4% 44.5% 62.6% Contigs with BLAST hits 220 bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

221 Comparative BLAST analysis indicated that our transcriptomes recovered a

222 significant proportion of the X. laevis and X. tropicalis transcriptomes (50,592 and 41,042

223 unigenes respectively). Paired-end transcriptomes showed a lower number of significant

224 BLAST hits (18,263 – 29,670) than single-end libraries (18,526 – 40,651) (S1 Fig). In

225 general, individual de-novo transcriptomes were of good quality and recovered 51-79%

226 of the core eukaryotic universal single-copy orthologs [29] (S3 Table). The final de novo

227 composite skin transcriptome reconstructed from all the RNA reads of the Oophaga

228 species yielded 31,498 contigs greater than 350 bp. The overall assembly incorporated

229 94% of all initial reads and the level of fragmentation was low with half of all base pairs

230 clustered into contigs of 1,316 bp in length or greater. The maximum contig length was

231 16,041 bp and the AL was 1014.91 ± 948.43 (S2A Fig). Nucleotide-based BLAST

232 analyses (BLASTX) revealed that ~63% of the contigs (n=19,732) show significant

233 similarities with either annotated gene products and/or known protein domains (E-value ≤

234 10−5) (S2B Fig) and only a small fraction of unigenes (3.4%) showed significant

235 homology to the same annotated transcript. The highest BLAST scores were found with X.

236 tropicalis (74%), X. laevis (2%) and the green sea turtle Chelonia mydas (0.5%). The

237 composite skin transcriptome was of higher quality when compared with individual

238 assemblies, having 88.54% of the ortholog count of BUSCO [29] (S3 Table).

239 Gene Ontology (GO) assignments were used to classify the functions of the

240 predicted unigenes based on contigs with significant BLASTX (E-value ≤ 10−5). Based on

241 GO level II, unigenes were assigned to 27 biological processes (BP), 22 cell components

242 (CC) and 15 molecular functions (MF) (S2C Fig). Some unigenes were associated with

243 multiple GO annotations because a single sequence may be annotated in any or all bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

244 categories, giving more GO annotations than sequences annotated [44]. Within BP, ~47%

245 of the annotations were assigned to basic cellular, metabolic processes and biological

246 regulation. The remaining unigenes were involved in a broad range of BP such as

247 response to stimulus (7%), response to stress (7%), localization (6%), developmental

248 process (5%), signal transduction (4%), biogenesis (4%), immune response (2%),

249 reproductive process (1.7%) and cellular adhesion (1%). Within the CC category, other

250 than the essential cell constituents (44%), the membrane components were highly

251 represented in the transcriptome (19%). Within MF, most of the unigenes were assigned

252 to binding and catalytic activities (72%) followed by transporter activity (9%), molecular

253 transducers (7%) and molecular function regulators (4%) (S2C Fig).

254 Transcriptome profiles

255 Dispersion plots of ER values in Oophaga RNA libraries showed a common

256 pattern of over-represented unigenes across RNA-libraries. That is, the same group of

257 unigenes showed similar patterns of over-representation no matter RNA library’s origin

258 (i.e. different Oophaga species) or composition (paired vs. single-read libraries) (S3 Fig).

259 The TPM represents a measure of RNA abundance and hence, it provides a general

260 overview of gene expression levels in a particular sample. Interestingly, TPM

261 distribution plots (percentiles) showed a distinct spike in expression levels towards

262 percentile 98% for all our RNA-libraries (S4A Fig). After the conservative filtration of

263 the 2% top expressed unigenes, we selected a subset of 1,437 unigenes with particular

264 higher levels of expression (higher TPM values and PCA outliers, S4B Fig) in Oophaga

265 skin tissue. Homology BLAST analysis of these unigenes revealed that 76% (n=1,092)

266 show significant similarities with annotated gene products and/or known protein domains bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

267 distributed mainly across amphibian (frogs) species (X. tropicalis=30%, X. laevis=14%

268 and Rana catesbeiana=4%).

269 After adjusting for library size and length bias, the maximum effective number of

270 reads (ER) in Oophaga species ranged from 2,461 (O. anchicayensis single-end) to 5,813

271 (O. sylvatica – single-end) while in the cryptic species varied from 82,509 (Rana

272 catesbeiana) to 452,018 (Bufo gargarizans) (higher coverage in cryptic species, S2

273 Table). This indicated that after the optimization of our alignment parameters, a

274 considerable amount of reads from each RNA-seq library actually mapped to our

275 composite de novo transcriptome. After the removal of contigs with low expression

276 profiles (<10 read counts), our RNA-seq dataset was composed of reads mapping to

277 20,771 genes. Multidimensional scaling plots (stress<0.05) revealed a clear separation by

278 groups (Oophaga vs. cryptic species, p<0.001 Bonferroni corrected) with higher variation

279 among the cryptic species, as expected given the broad taxonomic range of the members

280 of this group (Fig 2).

281 About 9% of the highly expressed genes (n= 1,931) also showed a phylogenetic

282 signal (i.e. significant fold changes in expression levels between Oophaga and all other

283 anurans). Most of these genes (n=1,656; 85.8%) show higher levels of expression in

284 Oophaga. The clustering analysis (Fig 1) revealed that the toad (B. gargarizans) has the

285 closest transcription profile to Oophaga species. One possible explanation is the

286 presence of alkaloid-based toxins in its skin glands [45].

287 bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

288 Fig 2. Multidimensional scaling (MDS) plot of 13 RNA-sequencing samples 289 from skin tissue of Oophaga (n=7, red dots) and cryptic species (n=6, blue dots). RNA- 290 seq data shown here represent the effective read counts (ER) for 31,498 skin-specific 291 contigs. Color ellipses represent a 95% confidence interval. bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

292 Homology BLAST analyses revealed that 65% (n=1,252) show significant

293 similarities with annotated gene products and/or known protein domains (S4 Table; S5A

294 Fig) distributed mainly across amphibian (frogs) species (X. tropicalis=32%, X.

295 laevis=19% and Rana catesbeiana=4%) (S5B Fig). Based on GO mapping level II, these

296 unigenes were assigned to 24 BP, 20 CC and 13 MF (S5C Fig). Multilevel GO term

297 classification assigned the highly expressed unigenes to 16 BP, 5 CC and 9 MF (S5C

298 Fig). Within BP, 13% of unigenes were associated with oxidation-reduction processes,

299 followed by response to stimulus (10%) and regulation of macromolecule metabolic

300 processes (7.3%). Within CC, the integral components of the cell membrane were highly

301 represented (39%), followed by protein complex (27%) and nuclear components (20%).

302 Within MF, most of the differentially expressed unigenes were assigned to metal ion

303 binding activity (21%), followed by protein binding activity and transmembrane

304 transporters (17%), oxidoreductase activity (12%) and structural molecule activity (10%)

305 (S5C Fig). The full functional annotation of these genes allowed us to propose plausible

306 mechanisms that may be involved in the ability to sequester alkaloid-based chemical

307 defenses, warning coloration and the auto-resistance mechanisms to avoid self-

308 intoxication (S5 Table).

309

310 DISCUSSION

311 Studies focusing on highly represented and differential gene expression in related

312 lineages have provided valuable insights into the molecular underpinnings of phenotypic

313 variation [46, 47]. These analyses rely on the premises that lineages show contrasting

314 phenotypes of interest, and that most differentially expressed genes relate to the trait of bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

315 interest rather than the overall of genomic differentiation between lineages. Here, we

316 examined shared patterns of gene expression in a small clade of closely related

317 aposematic frogs (Oophaga species). Because all members of this monophyletic group

318 show contrasting hues and accumulate alkaloid-derived toxins in their dermal glands [48,

319 49], we assumed that the genes related with these traits are likely to 1) be highly

320 expressed in the skin of Oophaga poison frogs and 2) showed contrasting expression

321 patterns between aposematic and cryptic and less (or not) poisonous species. Using

322 expression levels in a multivariate framework, we first identified ortholog genes that are

323 highly represented in Oophaga skin tissue and then, we compared with those

324 differentially expressed in the skin of contrasting groups and that show similar patterns of

325 expression among Oophaga species. We finally used a functional annotation analyses to

326 highlight a series of genes that are possibly associated with a series of aposematic traits.

327 The characterization of our skin transcriptomic profiles allowed us to identify

328 genes that are potentially associated with alkaloid transportation and resistance to

329 autotoxicity in poison frogs (Dendrobatidae). Because the alkaloids secreted by these

330 frogs are structurally very similar to those present in plants [18, 50-52], it is reasonable to

331 assume that the transportation and accumulation of alkaloids in these frogs may be

332 carried out by similar systems to those described in plants in which alkaloids are

333 transferred from source to sink organs [53-55]. If so, at least, two alternative membrane

334 mechanisms may help to explain the transport and storage of unmodified diet-derived

335 alkaloid by the specialized cells of skin secretory glands. First, alkaloids in a lipophilic

336 state may freely pass through the cell membranes by simple diffusion and accumulate in

337 acidic secretory lysosomes if they become protonated to form hydrophilic cations. This bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

338 ion-trap mechanism is not energy-dependent and does not necessarily require the

339 expression of any transporters. Alternatively, the transportation of alkaloids may be

340 managed by proton-antiport carrier systems in an energy-requiring manner [56-58].

341 Under this alternative model, diet derived alkaloids may be taken up by ABC transporters

342 [59, 60] and may accumulate in the secretory lysosomes of the skin gland cells by a

343 cation exchanger antiporter (CAX) system, dependent on the pH gradient generated by

344 “vacuolar” type ATPases (V-H+-ATPases) and/or pyrophosphatases (V-H+-PPases). To

345 find some support for these two potential models that might explain the accumulation and

346 posterior secretion of dietary alkaloids, we investigated the common transcriptional

347 profile across Oophaga species. For each of these species, our differential expression and

348 transcript abundance analysis revealed that at least 15 homologs of different type II CAX

349 are highly expressed in skin tissue (S5 Table). A result suggesting the existence of an

350 active membrane transportation-accumulation mechanism of alkaloids in harlequin

351 poison frogs. Consistent with this hypothesis, we also found putative ABC transporters

352 and at least one V-H+-ATPase on the skin transcriptome. All together, these results

353 suggest that active lysosomal exocytosis may play a key role in the secretion of alkaloids

354 in these frogs.

355 The accumulation of toxic compounds implies that organisms must avoid self-

356 intoxication (auto-resistance). While the membrane transportation mechanisms described

357 above reduce auto-toxicity by compartmentalizing the sequestered alkaloids, other non-

358 alternative excluding mechanisms are likely to contribute to auto-toxicity resistance. In

359 harlequin poison frogs, the major toxic components present in skin tissue are

360 histrionicoxins (HTX) [51]. These alkaloids are known to cause temporary paralysis or bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

361 even death by inactivating or blocking voltage-gate ion channels [61]. Resistance to this

362 type of cytotoxic compounds usually arises through either, an increased expression level

363 of P450 enzymes (CYPs) that metabolize the toxin, or through target insensitivity via

364 mutations that reduce the toxin's ability to bind the ion channel itself [62]. Our analyses

365 revealed that five CYP homologs are amongst the most highly expressed genes in the

366 skin of harlequin poison frogs (S5 Table). This result suggests that the oxidative

367 biotransformation of lipophilic alkaloids to hydrophilic compounds [63] is an important

368 auto resistance mechanism in these frogs. Although the overproduction of the CYP-

369 enzymes has been interpreted as evidence for metabolic detoxification [64], there is an

370 alternative explanation for the high expression levels of CYP proteins in skin of harlequin

371 poison frogs. While many of the alkaloids sequestered in the wild are accumulated

372 unchanged in the dermal glands, at least two species of dendrobatids, stereo-selectively

373 hydroxylate the toxic pumiliotoxin PTX-(+)-251D to convert it into a much more potent

374 toxin, the allopumiliotoxin aPTX-(+)-267A [17]. Both PTX-(+)-251D and aPTX-(+)-

375 267A have been isolated from the skin of O. histrionica, and CYPs are known to be

376 involved in the stereo-selective hydroxylation of alkaloids [65]. Thus, it is also possible

377 that the CYPs expressed in the skin of harlequin poison frogs may enhance the

378 antipredator potency of ingested PTXs.

379 Despite the major role that detoxification enzymes may have in the resistance to

380 diet acquired toxins, mutations conferring constitutive resistance to alkaloids are also

381 likely to be involved in auto-resistance in chemically-defended dendrobatids. In insects,

382 single amino-acid substitution in the voltage-gated sodium channels (Na+K+-ATPases)

383 are responsible for resistance to host-plant phytochemicals [66-68]. In amphibians, the bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

384 only documented example of this this type of target insensitivity is from distantly-related

385 lineages of alkaloid-defended frogs, 24 species of Neotropical dendrobatids and the

386 aposematic Madagascar frog Mantella aurantiaca [69]. In all of these cases, they carried

387 different amino acid replacements in the inner pore of the voltage-gated sodium channel

388 Nav1.4 that are not found in other frog [70]. Our analyses revealed eight genes encoding

389 voltage-gated ion channel proteins (VGIC), one of which (8966), encodes the gamma-1

390 subunit of a sodium channel.

391 Aposematism in harlequin frogs is a complex phenotype that results from the

392 integration of different elements including unpalatability and conspicuousness. Thus,

393 another of our goals was to identify genes potentially related with warning coloration.

394 Many aposematic organisms, including the Oophaga poison frogs studied here exhibit

395 color patterns that show strong color and/or luminance contrast such between black and

396 red, orange or yellow [15]. It has been proposed that such patterning increases aversion

397 learning as well as conspicuousness and distinctiveness from palatable prey [71-76]. In

398 anurans, coloration relates to both the structure and pigment composition of the dermal

399 chromatophore units in which three types of pigment cells (xanthophores, iridophores and

400 melanophores) are laid one on another [77]. In dark/black skin areas of harlequin poison

401 frogs, no xanthophores or iridophores are present and the distal fingers of the

402 melanophores are filled with melanin granules (melanosomes) obscuring the dermis [15].

403 Thus, genes involved in the amount, size and distribution of the melanosomes are likely

404 to play a significant role in the coloration pattern of these frogs.

405 In one of the few detailed studies of coloration in anurans [78], authors suggest a

406 common origin for of all the pigment granules found in the cells of the chromatophore: a bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

407 primordial organelle (vesicle) derived from the rough endoplasmic reticulum (RER).

408 According to this model, in the formation of melanosomes, the pre-melanosomes are

409 derived from cisternae of the RER, which then fuse with vesicles derived from the Golgi

410 complex containing tyrosinase enzymes [12, 78-81]. A highly expressed gene identified

411 here (tyrosinase regulator, 25062) may contribute to regulate this mechanism, which in

412 turn might translate into hue differences. Interestingly, among Oophaga species, we have

413 found populations that are characterized by light-brown background coloration as oppose

414 to black [15, 22]. One tantalizing possibility is that this phenotypic difference is indeed

415 associated with the expression differences of these tyrosinase enzymes.

416 Obviously, there are other molecular and cellular mechanisms that might be

417 associated to the difference between light and dark background coloration. In fishes and

418 amphibians, dark hues are known to be produced by the interaction between high levels

419 of melanocyte-stimulating hormone (α-MSH) and several variants of its transmembrane

420 receptor (MC1R) through the dispersion of melanosomes within the melanophore (by

421 increasing cAMP intracellular levels) [82, 83]. Thus, it is possible that structural or

422 expression differences in the MC1R might contribute to dark phenotypes in Oophaga

423 frogs. While there is a strong evidence that different mutations at MC1R cause either

424 light or dark phenotypes in many mammals, birds and reptiles [84-88], the only two

425 studies conducted in frogs are inconclusive [89, 90]. A detailed inspection of the coding

426 sequences recovered for this gene revealed the presence of different length isoforms,

427 making MC1R a promising candidate gene candidate to explain the differences in

428 background coloration in poison frogs [15]. Finally, our study revealed another two

429 highly and differentially expressed genes (14447-Melanoregulin-; 2038-Melanocortin bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

430 phosphoprotein), which may also contribute to dark hues. In this case, the predicted

431 products of these genes are key proteins that mediate the melanosome transport and

432 distribution in epidermal cells through the formation of a tripartite protein complex [91].

433 The disruption of the transport protein complex results in pigmentary dilution and lighter

434 phenotypes by the clustering of melanosomes around the nucleus [92].

435 Overall, this study demonstrates the utility of using RNA-sequencing with non-

436 model organisms to identify loci that may be of adaptive importance. Altogether, these

437 data enabled us to provide a first global study of Oophaga poison frogs transcriptomes

438 and propose potential mechanisms for alkaloid sequestration, auto-resistance to toxic

439 compounds, and variation in coloration and patterns. It is important to recognized that

440 due the sampling restrictions imposed to us (see methods), we are most likely missing

441 some of the genes responsible for these traits. It is also probable that the genes associated

442 with coloration, alkaloid metabolism, transport and storage are differentially expressed

443 not only in skin, but other tissues in the organism. Despite the potential caveats, other

444 molecular studies have shown that some of the genes identified in this study are indeed

445 potentially related with aposematic traits in dendrobatids [15, 70, 93].

446 Despite the critical impact of genetic basis of coloration in the evolution and

447 diversification of aposematic phenotypes, the genetic architecture of coloration in poison

448 frogs (Dendrobatidae) remains virtually unexplored. Here, we report probably the first

449 RNA-seq study in Oophaga poison frogs, a model system for understanding the

450 relationship between toxicity, diet and coloration. The skin-expressed genes that we have

451 identified here provide initial working hypotheses to further unravel the molecular

452 genetics mechanisms to sequester alkaloid-based chemical defenses, warning coloration bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

453 and the auto-resistance mechanisms to avoid self-intoxication. Hence, further analysis

454 aiming to compare the amino acid substitutions in the expressed cDNAs will provide

455 insights about the structure and function of each of these genes. Finally, our comparative

456 transcriptome data provide an important new resource to better understand the evolution

457 of warning signals in nature.

458

459 Acknowledgements

460 We would like to thank the former members of Rick Harrison’s lab (requiescat in

461 pace) at Cornell University for their valuable discussions and comments on early versions

462 of the manuscript, which was greatly improved by the thoughtful comments of Dr.

463 Andrew Crawford at Universidad de los Andes (Colombia). This study was funded by

464 COLCIENCIAS (Departamento Administrativo de Ciencia, Tecnología e Innovación,

465 Colombia, 529-2011) grant to A.P.-T. and an NSERC-Discovery grant to J.A.A. bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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745 SUPPORTING INFORMATION

746 SUPPORTING TABLES

747 S1 Table. Number of reads and library type (i.e paired vs. single-end) for each 748 individual RNA-seq experiment. 749

Raw number of Reads after Remaining % Library Individual library reads filtering of reads type O. solanensis 128,599,724 105,179,065 81% Paired O. anchicayensis 124,232,804 110,217,128 88% reads* O. lehmanni 102,225,680 91,493,848 89%

O. sylvatica 48,929,329 45,147,654 92%

O. lehmanni 43,633,080 41,086,379 94% Single reads O. anchicayensis) 7,998,636 7,173,285 89%

O. histrionica 50,204,617 47,376,912 94%

Reference transcriptome N/A 33,895,474 100% Single reads

*. Number of reads correspond to the combined value of both ends libraries. 750 bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

751 S2 Table. RNA-sequence datasets from cryptic species used for the differential 752 expression analysis of this work. 753 Species Experiment (SRA*) RNA-seq library Total bases Date available Bufo gargarizans SRX2640691 Paired-end (150 b) 19.2 Gbp 2017-03-15 Pelophylax nigromaculatus SRX2640690 Paired-end (150 b) 18.0 Gbp 2017-03-15 Polypedates megacephalus SRX1720193 Paired-end (100 b) 6.3 Gbp 2016-04-25 Rana catesbeiana SRX2988982 Paired-end (100 b) 9.2 Gbp 2017-07-07 Rana sylvatica SRX2989023 Paired-end (100 b) 8.9 Gbp 2017-07-07 Xenophrys sangzhiensis SRX1720191 Paired-end (100 b) 6.8 Gbp 2016-04-25 * Sequence Read Archive accession number from Genbank. 754 755 756 S3 Table. Quality assessment report for each individual and the composite reference 757 transcriptome based on the protein count of the core eukaryotic genes (BUSCO, see 758 methods in the main article) 759

Busco v.2 mapping Transcriptome Library Complete Partial Complete + Partial O. solanensis Paired 20.60% 30.47% 51.07% O. anchicayensis Paired 24.46% 37.77% 62.23% O. lehmanni Paired 29.61% 40.77% 70.38% O. sylvatica Single 45.92% 33.48% 79.40% O. lehmanni Single 33.91% 39.91% 73.82% O. anchicayensis Single 9.87% 30.47% 40.34% O. histrionica Single 39.06% 37.77% 76.83% Reference assembly Single 45.92% 42.62% 88.54% 760

761 S4 Table. Summary statistics of the differentially expressed unigenes (see methods) and 762 their BLAST annotations.

Differentially With Without expressed unigenes annotation annotation

Up-regulated 1656 (85.6%) 996 (51.6%) 660 (34.2%) Down-regulated 275 (14.2%) 256 (13.3%) 19 (1%) Total 1931 (100%) 1252 (64.9%) 679 (35.2%) 763 bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

764 S5 Table. Transcripts with potential important functions in the alkaloid sequestration system, auto-resistance to toxic compounds and 765 variation in coloration in harlequin poison frogs. The corresponding composite Oophaga transcriptome is available from the authors 766 upon request. 767 Length Transcript ID Fold change padj BLAST description GO Names list (bp) C:cell; P:gonad development; P:thyroid gland development; P:luteinizing hormone secretion; P:thyroid hormone generation; P:cellular response to hormone 14256 779.11 0.00 Transmembrane protein 41B 1253 stimulus; P:follicle-stimulating hormone secretion; P:negative

regulation of organ growth; C:extracellular region; F:hormone activity 26699 517.76 0.00 Transmembrane protein 181 1079 C:extracellular space 14644 297.83 0.00 Transmembrane protein 116-like 1743 14399 192.77 0.01 Transmembrane protein 26 1039 6476 174.07 0.00 Transmembrane protein 1690 F:structural constituent of eye lens F:chondroitin sulfate binding; C:nucleus; F:identical protein binding; F:calcium-dependent protein binding; C:cytosol; C:chromaffin granule membrane; 15428 105.09 0.02 Transmembrane protein 220 697 P:regulation of transcription,

DNA-templated; F:heparin transportation Alkaloid binding; P:negative regulation of cellular process; F:carbohydrate binding; C:apical plasma membrane 8646 100.29 0.01 Transmembrane protein 69 936 20904 66.89 0.04 Transmembrane protein 186-like 615 bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

P:'de novo' pyrimidine nucleobase biosynthetic process; F:orotidine- 19857 53.30 0.05 Transmembrane protein 70, mitochondrial 532 5'-phosphate decarboxylase activity; P:'de novo' UMP biosynthetic process 24171 45.71 0.01 Transmembrane protein 125-like 4098 Keratinocyte-associated Transmembrane protein 2- 19808 37.86 0.04 1043 like P:negative regulation of potassium ion transmembrane transport; P:positive regulation of myofibroblast contraction; P:response to salt; P:negative regulation of platelet aggregation; P:response to progesterone; 16724 27.39 0.02 Transmembrane protein 206 1589 P:positive regulation of vasoconstriction; P:negative regulation of gastrin-induced gastric acid secretion; C:neuronal cell body; P:negative regulation of insulin secretion; F:prostaglandin …. 24522 25.75 0.00 Transmembrane protease serine 13-like 2292 F:oxidoreductase activity P:response to dehydroepiandrosterone; F:gamma-catenin binding; C:extracellular space; P:response 25621 7.42 0.05 Cation-transporting ATPase 13A4 469 to 11-deoxycorticosterone; C:membrane; P:metabolic process; P:response to progesterone; P:response to estradiol; C:desmosome; F:hydrolase activity F:calcium ion binding; 9463 -6.16 0.04 Lysosomal-trafficking regulator 850 P:keratinization 768 bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Length Transcript ID Fold change padj BLAST description GO Names list (bp) F:calcium ion binding; 15350 175.29 0.00 EF-hand calcium-binding domain-containing 10-like 436 C:cellular_component P:single-organism cellular localization; P:cell cycle process; F:binding; P:organelle organization; C:intracellular organelle part; P:single-organism transport; P:establishment of protein localization; C:endosome; C:membrane-bounded vesicle; 16951 95.46 0.01 S100 calcium binding protein Z 2161 P:vesicle-mediated transport; site insensitivity site

P:regulation of cellular process; - C:membrane; C:cytoplasmic vesicle; P:establishment of localization in cell; P:cellular protein localization; P:establishment of organelle localization C:extracellular space; F:carboxypeptidase activity; 1216 63.08 0.03 Extracellular calcium-sensing receptor-like 1214 P:bradykinin catabolic process; P:proteolysis; P:response to

glucocorticoid target resistance: - P:ion transport; P:regulation of membrane potential; C:integral component of membrane; F:extracellular ligand-gated ion Potassium Voltage-gated channel subfamily E channel activity; P:synaptic 18107 4239.03 0.00 1864 member 3-like transmission; C:synapse; F:binding; P:regulation of biological process; P:neurological

system process; P:response to auto Alkaloid stimulus; C:plasma membrane part bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

F:high voltage-gated calcium channel activity; F:protein C- terminus binding; C:voltage-gated calcium channel complex; 16614 9.90 0.04 MGC114646 protein 1514 P:calcium ion transmembrane transport; C:cytosol; P:positive regulation of cytosolic calcium ion concentration 907 -6.46 0.01 Voltage-dependent anion channel 2 990 376 -10.41 0.00 Voltage-dependent anion channel 388 C:sarcoplasmic reticulum; C:voltage-gated calcium channel complex; F:calcium channel regulator activity; F:voltage-gated calcium channel activity; F:mating pheromone activity; P:pheromone- dependent signal transduction Sodium channel, voltage-dependent, gamma subunit 8966 102.76 0.01 640 involved in conjugation with 1 cellular fusion; C:T-tubule; C:extracellular exosome; F:metal ion binding; P:mating; P:regulation of ion transmembrane transport; P:calcium ion transmembrane transport; P:regulation of calcium ion transport 769 bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Fold change padj BLAST description Length (bp) GO Names list

420.10 0.04 Cytochrome P450-like TBP 408 36.47 0.01 Cytochrome P450, family 4, subfamily 458 F:oxygen binding; P:oxygen transport; C:voltage-gated F, polypeptide 22 potassium channel complex; F:delayed rectifier potassium channel activity; F:A-type (transient

outward) potassium channel activity; P:protein homooligomerization; C:neuronal cell body; P:membrane repolarization; F:oxygen transporter activity; F:ion channel binding; F:heme binding; C:hemoglobin complex; C:dendrite; C:caveola; C:perinuclear endoplasmic reticulum; F:iron ion

binding; P:cellular response to BMP stimulus; C:sarcolemma; P:regulation of ion transmembrane transport; P:potassium ion export

30.72 0.02 Cytochrome P450 2F2-like 1201 metabolic resistance: - 17.97 0.00 Cytochrome P450 2D26-like 1851 P:sucrose biosynthetic process; C:chromosome;

F:binding detoxification 6.93 0.04 Cytochrome P450 2K1-like 429 C:nucleus; F:DNA binding; P:cytoskeleton organization; F:protein heterodimerization activity; F:calcium ion binding; C:nucleosome; F:calcium- dependent phospholipid binding

auto Alkaloid

bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Fold change Padj BLAST description Length (bp) GO Names list 273.95 0.00 Melanoregulin-like protein 1131 25.44 0.03 Melanocortin receptor binding NECAP 2070 P:regulation of appetite; C:secretory granule; Coloration endocytosis associated 2 C:peroxisomal matrix; P:positive regulation of transcription from RNA polymerase II promoter; F:hormone activity; P:regulation of blood pressure; C:extracellular space; P:neuropeptide signaling pathway; P:glucose homeostasis; F:type 1 melanocortin receptor binding; P:cellular pigmentation; P:regulation of corticosterone secretion; F:type 3 melanocortin receptor binding; F:type 4 melanocortin receptor binding; P:regulation of glycogen metabolic process; P:negative regulation of tumor necrosis factor production 12.71 0.05 Melanocortin m-phase phosphoprotein 2732 P:regulation of nucleobase-containing compound 8 metabolic process; P:G-protein coupled receptor signaling pathway; P:single-multicellular organism process; F:peptide binding; F:melanocortin receptor activity; F:protein binding; P:cellular macromolecule metabolic process; F:carbohydrate derivative binding; C:intracellular membrane-bounded organelle; P:homeostatic process; C:membrane; P:regulation of gene expression; C:macromolecular complex 13.52 0.00 Tyrosine kinase, non-receptor 2 1981 F:protein binding; C:proton-transporting ATP synthase complex; P:single-organism cellular process; P:nucleobase-containing compound metabolic process; P:transport; C:mitochondrial inner membrane; F:hydrolase activity

770 bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

771 SUPPORTING FIGURES

772 S1 Fig. Pie charts representing the number and proportion of contigs with significant 773 BLAST hits (E<1.0E-5) for each individual transcriptome from Oophaga species. 774 Transcriptomes obtained using paired-ends RNA-seq libraries are represented in (A) and 775 those obtained with single-end libraries in (A). bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

776 S2 Fig. A) Contig length distribution of the de-novo composite reference transcriptome 777 of Oophaga species from skin tissue. B) Pie chart representing the number and proportion 778 of contigs with significant BLAST hits (E<1.0E-5) in the reference Oophaga 779 transcriptome. C) Gene ontology (GO) categories distribution (level II) for the annotated 780 unigenes in the Oophaga reference transcriptome. bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

781 S3 Fig. Distribution plots of the effective number of reads (ER) from Oophaga RNA-seq 782 experiments that mapped to the composite reference transcriptome contigs (n=31,498). bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

783 S4 Fig. a) Percentile plot of the estimated transcripts per million (TPM) in the composite 784 reference transcriptome as calculated based on raw RNA reads from individual libraries. 785 Color names are equivalent to those in tendency lines. b) Principal component analyses 786 (PCA) plot of the reference contigs dataset using TPMs values as independent variables. 787 Red dots represent the selected highly represented unigenes (2% of the total contigs; n= 788 1,437) while dark dots represent the remaining ones (n=30,061) bioRxiv preprint doi: https://doi.org/10.1101/706655; this version posted July 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

789 S5 Fig. A) Pie chart representing the total number of differentially expressed unigenes 790 and the proportion with significant BLAST hits (E<1.0E-5). B) BLAST hit species 791 distribution of highly expressed unigenes. C) Gene ontology (GO) categories distribution 792 (multilevel) for the annotated highly expressed unigenes.