The Round Goby Genome

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1 Evolved for success in novel environments: The round goby genome

3 Authors

4 Irene Adrian-Kalchhauser1, Anders Blomberg2*, Tomas Larsson3*, Zuzana Musilova4*, Claire R Peart5*,

5 Martin Pippel6*, Monica Hongroe Solbakken7*, Jaanus Suurväli8*, Jean-Claude Walser9*, Joanna

6 Yvonne Wilson10*, Magnus Alm Rosenblad2,11§, Demian Burguera4§, Silvia Gutnik12§, Nico Michiels13§,

7 Mats Töpel2§, Kirill Pankov10§, Siegfried Schloissnig14§, Sylke Winkler6§

9 Author contributions

10 * equal contribution, section lead authors, listed alphabetically

11 § co-authors with equal contribution, listed alphabetically

13 Affiliations

14 1 Program Man-Society-Environment, Department of Environmental Sciences, University of Basel,

15 Vesalgasse 1, 4051 Basel, Switzerland

16 2 Department of Chemistry and Molecular Biology, University of Gothenburg, Medicinaregatan 9C,

17 41390 Gothenburg, Sweden

18 3 Department of Marine Sciences, University of Gothenburg, Medicinaregatan 9C, 41390 Gothenburg,

19 Sweden

20 4 Department of Zoology, Charles University, Vinicna 7, CZ-128 44 Prague, Czech Republic

21 5 Division of Evolutionary Biology, Faculty of Biology, Ludwig-Maximilians-Universität München,

22 Grosshaderner Strasse 2, 82152 Planegg-Martinsried, Germany

23 6 Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307

24 Dresden, Germany

25 7 Centre for Ecological and Evolutionary Synthesis, University of Oslo, Blindernveien 31, 0371 Oslo,

26 Norway

27 8 Institute for Genetics, University of Cologne, Zülpicher Strasse 47a, D-50674 Köln, Germany

28 9 Genetic Diversity Centre, ETH, Universitätsstrasse 16, 8092 Zurich, Switzerland

29 10 Department of Biology, McMaster University, 1280 Main Street West, Hamilton, ON, Canada

30 11 NBIS Bioinformatics Infrastructure for Life Sciences, University of Gothenburg, Medicinaregatan 9C,

31 41390 Gothenburg, Sweden

32 12 Biocenter, University of Basel, Klingelbergstrasse 50/70, 4056 Basel, Switzerland

33 13 Institute of Evolution and Ecology, University of Tuebingen, Auf der Morgenstelle 28, 72076

34 Tübingen, Germany

35 14 Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC), 1030 Vienna, Austria.

37 Corresponding author

38 Irene Adrian-Kalchhauser

39 email: [email protected]

40 mail: University of Basel, Vesalgasse 1, 4051 Basel

41 phone: +41 61 2070410

43 Keywords

44 PacBio, Neogobius melanostomus, invasive species, fish, genomics, evolution, adaptation, gene

45 duplication, vision, olfaction, innate immunity, detoxification, osmoregulation, epigenetics

46 Abstract

48 Since the beginning of global trade, hundreds of species have colonized territories outside of their

49 native range. Some of these species proliferate at the expense of native ecosystems, i.e., have

50 become invasive. Invasive species constitute powerful in situ experimental systems to study fast

51 adaptation and directional selection on short ecological timescales. They also present promising case

52 studies for ecological and evolutionary success in novel environments.

54 We seize this unique opportunity to study genomic substrates for ecological success and adaptability

55 to novel environments in a vertebrate. We report a highly contiguous long-read based genome

56 assembly for the most successful temperate invasive fish, the benthic round goby (Neogobius

57 melanostomus), and analyse gene families that may promote its impressive ecological success.

59 Our approach provides novel insights from the large evolutionary scale to the small species-specific

60 scale. We describe expansions in specific cytochrome P450 enzymes, a remarkably diverse innate

61 immune system, an ancient duplication in red light vision accompanied by red skin fluorescence,

62 evolutionary patterns in epigenetic regulators, and the presence of genes that may have contributed to

63 the round goby’s capacity to invade cold and salty waters.

65 A recurring theme across all analyzed gene families are gene expansions. This suggests that gene

66 duplications may promote ecological flexibility, superior performance in novel environments, and

67 underlie the impressive colonization success of the round goby. Gobiidae generally feature fascinating

68 adaptations and are excellent colonizers. Further long-read genome approaches across the goby

69 family may reveal whether the ability to conquer new habitats relates more generally to gene copy

70 number expansions.

71 Introduction

73 Since the beginning of global trade and the colonial period, hundreds of species have colonized

74 territories outside their native range. A fraction of those species proliferates at the expense of native

75 species and ecosystems, i.e., they are invasive. While invasive species present challenges for

76 biodiversity and ecosystem conservation, they also constitute exciting eco-evolutionary models for

77 adaptation and ecological success in novel or changing environments (1–4).

79 The benthic round goby Neogobius melanostomus (Figure 1A) is one of the most widespread invasive

80 fish species. Since 1990, round gobies have been detected in over 20 countries outside their native

81 Ponto-Caspian range. In some regions of Europe and North America, they have become the most

82 common fish species (5–7) (Figure 1B). Lasting impacts on biodiversity and on ecosystems have

83 been observed (see (8) for a summary of the impacts). In recent years, the round goby has therefore

84 become a novel model for ecology, behavior and evolution (Figure 1C).

86 The round goby effortlessly outcompetes native species with similar ecology, and is therefore a

87 promising candidate to study fundamental questions on long-term ecological and evolutionary

88 success. Round goby sequence data are presently restricted to a handful of phylogenetic markers (9–

89 15). However, genome analyses have previously provided significant insights into fish ecology and

90 evolution. Examples are genome compaction (16), the transition from fin to limb (17), loss of major

91 parts of adaptive immunity (18), or effects of genome duplication (19). We therefore expect relevant

92 insights into round goby biology and into success in novel environments from the round goby genome

93 sequence.

94 Figure 1

95 The round goby, a benthic invasive fish species. A Wild-caught round goby in aquaria. Individuals

96 are usually brightly colored or spotted with a characteristic black dot on the first dorsal fin. During the

97 reproductive season, territorial males develop a black body color (first panel). B The growing scientific

98 relevance of the species is reflected by records in Web of Science (orange) when compared to a non-

99 invasive fish with similar ecology (European bullhead, Cottus gobio; grey). C Current distribution of

100 round goby. The round goby has spread from its native region (green) to many European rivers and

101 lakes, the Baltic Sea, the Great Lakes and their tributaries (orange). D Phylogenetic position of the

102 round goby among fishes.

103 The survival of an individual in a novel environment depends on how well it can perceive, react to, and

104 accommodate to its new surroundings. In this study, we therefore explore a high quality and

105 contiguous genome assembly of the round goby for genes related to three categories: environmental

106 perception, reaction to environmental conditions, and long-term accommodation to novel

107 environments. We focus on gene families that have been hypothesized to play a role in the

108 colonization of novel environments and on gene families that may relate to round goby invasion

109 ecology.

110

111 For environmental perception, we investigated genes responsible for sensory perception in fishes. We

112 specifically focused on the opsin genes for visual perception, as well as on the olfactory receptors for

113 odor perception. Vision in fishes is often specifically adapted to environmental conditions, such as

114 darkness in deep water (20), modified color spectrum in turbid water (21, 22), habitat color (23), or

115 specific light regimes or light compositions (24–26). The overall spectral sensitivity range of teleost

116 fishes exceeds the human visual range and, in many cases, includes the UV (23) and far-red (27)

117 spectrum. Similarly, olfaction is an essential chemoreception sense for fish, allowing for fast responses

118 to predators and alarm cues as well as for intra-species communication. Pheromones have play an

119 important role in the round goby (28–30), and males attract females into their nests by releasing them

120 (31). A particularly specialized sense of smell therefore may provide an advantage during initial

121 population establishment in novel environments.

122

123 We further investigated genes that may mediate responses to novel environments, namely genes

124 involved in detoxification, ion transport and the immune system. The round goby occurs even in

125 chemically contaminated harbors (32–34) and appears to tolerate xenobiotic compounds well. This

126 suggests that the round goby may be particularly well equipped to degrade and eliminate chemical

127 pollutants. We therefore analyze the cytochrome P450 gene superfamily, which is a particularly

128 important and conserved part of the xenobiotic response (35). The round goby also tolerates a wide

129 range of salinities (0 to 25 PSU / ‰) and temperatures (0°C-30°C) and occurs at latitudes ranging

130 from <40° N in the Ponto-Caspian region to >60° N in the Baltic Sea. Most fishes tolerate only a

131 narrow range of salinities (36); the round goby however belongs to a specialized group, the euryhaline

132 fish species, which thrive in fresh and brackish environments and includes estuarine species and

133 migratory species such as salmon. We study the genetic basis of osmoregulation and osmolyte

134 production in round goby to gain insights into the evolution of salinity and cold tolerance, and to

135 possibly predict future range expansions. Invasive species encounter an array of previously unknown

136 pathogens when they colonize a habitat, and invasion success may be related to a species’ ability to

137 tackle novel immune challenges (37). Intriguingly, the round goby displays a low parasite load at the

138 invasion front (38). We therefore characterize key factors of the innate and the adaptive immune

139 system.

140

141 We also investigated conserved gene regulators because such genes might be involved in long-term

142 adaptation to a novel environment. Mechanisms such as DNA methylation and histone modifications

143 promote long- and short-term gene expression regulation and therefore mediate adaptations to altered

144 conditions at the cellular level (39), but also regulate genome-scale evolutionary processes such as

145 the distribution of meiotic recombination events (40) or transposon activity (41), and provide stochastic

146 variability as basis for selection (42). Epigenetic variants have been proposed to cause fitness-relevant

147 differences in gene expression and phenotype (43, 44). The ecological flexibility of the round goby has

148 been linked to enhanced gene expression plasticity in response to environmental stimuli (45) and to

149 their ability to pass information on water temperature to their offspring through maternal RNA (46). To

150 understand the features of core epigenetic regulators in the round goby, we focused on two widely

151 conserved and well characterized parts of the epigenetic machinery: the histone-methylating PRC2

152 complex and the DNA methylases. Both mechanisms are thought to restrict developmental plasticity,

153 downregulate gene expression (at least in mammals), and have been linked to plastic responses,

154 behavioral changes, and environmental memory (47–51).

155

156 Finally, we take advantage of the high genome contiguity to investigate sex determination using RAD

157 sequencing data. Fish display a wide variety of sex determination mechanisms, ranging from sex

158 chromosomes to multilocus genetic sex determination to environmental sex determination (52), and

159 sex determination in the round goby has not previously been investigated.

160 Results

161

162 1. The round goby genome

163

164 The round goby genome assembly (“RGoby_Basel_V2”, BioProject accession PRJNA549924,

165 BioSample SAMN12099445, GenBank genome accession VHKM00000000, release date July 22

166 2019) consists of 1364 contigs with a total length of 1.00 Gb (1’003’738’563 bp), which is within the

167 expected size range (53–55). It is assembled to high contiguity (NG50 at 1’660’458 bp and N50 at

168 2’817’412 bp). GC content is 41.60%. An automated Maker gene annotation predicts a total of 38,773

169 genes and 39,166 proteins, of which 30,698 are longer than 100 amino acids (Table 1; annotation

170 track available as Supplemental_Material_S1). The genome does not appear to contain a sex

171 chromosome or a large sex determining region, since a RAD-tag dataset from 40 females and 40

172 males with an estimated resolution of 25,000 – 45,000 bp does not contain any sex-specific loci.

173

174 Approximately 47% of the genome assembly is masked as various types of repetitive sequences by

175 RepeatMasker in the Maker annotation pipeline. The genome consists of approximately 9% predicted

176 interspersed repeats (Supplemental_Table_S1), which is much lower than for zebrafish (Danio rerio,

177 total genome size 1427.3Mb, 46% predicted as interspersed repeats) but higher than for the more

178 closely related three-spined stickleback (Gasterosteus aculeatus, total genome size 446.6Mb, 3.2%

179 predicted as interspersed repeats). Among interspersed repeats, the long terminal repeat (LTR)

180 retrotransposon family is the most common in many species including fish (Repbase,

181 https://www.girinst.org/repbase/). RepeatMasker identifies 0.9% LTR retrotransposons in the round

182 goby genome, but separately run de novo predictions with LTRfinder and LTRharvest

183 (Supplemental_Table_S1) indicate an underestimation of LTR retrotransposons and interspersed

184 repeats by this approach. The latter approaches estimate that the proportion of LTR retrotransposons

185 in the round goby genome is 11.2% (3.8% LTRs with target-site-repeats; LTRfinder) or 4.9%

186 (LTRharvest), respectively.

187

188 In addition to the genome sequence, we provide raw short read sequencing data from various

189 published and ongoing projects. They include RNA sequencing data from early cleavage embryos

190 (46), DNA methylation capture data from adult male brains (47), as well as RAD tags from two local

191 Swiss populations and ATAC seq reads from brain and liver (unpublished; Table 1).

192

193 Table 1. Round goby genome assembly and annotation statistics.

Assembly Number of contigs 1364 Total genome length (bp) 1,003,738,563 Longest contig (bp) 19,396,355 Smallest contig (bp) 21,178 N50 contig length (bp) 2,817,412 Annotation Number of genes 38,773 Genomic repeat content (%) 47 G + C (%) 41.60 LTR retrotransposons (%) 4.9 - 11.2 NCBI BioProject PRJNA549924 Accession Accession VHKM00000000 Additional sequencing data Embryonic transcriptome (1-32 cell stages) from 16 clutches RNA (Adrian-Kalchhauser 2018) NCBI BioProject PRJNA547711 NCBI SRA SRR9317352 - SRR9317366 Brain DNA methylation data from 15 males DNAme (Somerville 2019) NCBI BioProject PRJNA515617 NCBI SRA SRR8450505 - SRR8450528 RAD Seq data from 120 individuals RADseq (unpublished) NCBI BioProject PRJNA547536 NCBI SRA SRR9214152 - SRR9214154 ATAC Seq data of liver and brain from 50 individuals ATACseq (unpublished) NCBI BioProject PRJNA551348 NCBI SRA SRR9714857 - SRR9714901 194

195 2. Sensory perception genes: Vision

196

197 Vertebrates perceive color with cone cells expressing one of four types of opsin proteins (usually

198 sensitive to the red, green, blue, and ultraviolet part of the spectrum) and dim light with rod cells

199 expressing the rod opsin. The UV and blue light is detected by the short-wavelength sensitive SWS1

200 and SWS2 opsins, the green part of the spectrum is perceived mostly by the rhodopsin-like RH2

201 opsins, and the red color by the long-wavelength sensitive LWS opsins. Rod cells are active in the

202 dim-light conditions and contain the rod opsin RH1 (56). Gene duplications and losses of the opsin

203 genes during fish evolution correlate to certain extent with adaptations to specific environments (20,

204 57).

205

206 We identified two cone opsin gene duplications in the round goby genome. Firstly, the genome

207 features a recent duplication of the green-sensitive RH2 gene. RH2 duplications are a common

208 phenomenon in fish (Figure 2). Secondly, the genome features an ancient duplication of the long-

209 wave red-sensitive LWS gene. The event can be traced most likely to the ancestor of all teleosts, or

210 possibly even to the ancestor of Neopterygii (Figure 2; see Supplemental_Fig_S1 for full tree). As

211 expected, the round goby genome further contains one dim-light rod opsin (RH1) gene, two blue-

212 sensitive SWS2 genes (57), and as previously reported for gobies, lacks the UV/violet-sensitive SWS1

213 gene (20, 25, 57).

214

215 The proposed ancestral position of the red opsin gene duplication is supported by three lines of

216 evidence. First, the monophyly of all other teleost + gar LWS genes is strongly supported by the

217 Bayesian analysis (Bayesian posterior probability value = 1). Second, the distant phylogenetic position

218 is supported by trees based on individual exons, which indicate a low probability of a compromised

219 phylogenetic signal, e.g. due to the partial gene conversion. Three of four exons cluster at the same

220 position as the whole gene, while the fourth exon (exon 4) cluster with the genes resulting from a more

221 recent teleost-specific LWS duplication specific to Astyanax and Scleropages (58)

222 (Supplemental_Fig_S2). Third, the choice of outgroup (parietopsin or pinopsin) does not affect the

223 position of the LWS2 gene. Together, these analyses suggest either (1) the presence of an ancient

224 gene duplication event of the LWS gene in the ancestor of teleost and holostean fishes (i.e.

225 Neopterygii) which was retained only in the goby family, or (2) a teleost-specific event, possibly

226 identical to that reported for characins and bony tongues (58), with a subsequent concerted goby-

227 specific sequence diversification in exons 2, 3 and 5.

228

229 The spectral sensitivity of photopigments, i.e. their excitation wavelength can be modified by

230 substitutions in certain key amino acids (59). We find that round goby LWS1 and LWS2 differ in the

231 key spectral tuning site at amino acid 277 (position 261 of bovine rhodopsin, Table 2) suggesting a

232 sensitivity shift of 10 nm.

233

234 Figure 2

235 Phylogenetic tree of vertebrate opsin gene sequences. Maximum-likelihood phylogenetic tree

236 based on the cone and rod visual opsins and using VA opsins and pinopsins as outgroup. The round

237 goby genome contains two LWS gene copies, which seem to be the results of an ancient gene

238 duplication event, and two more recently duplicated RH2 gene copies. Round goby is indicated in

239 orange. Red opsin branches are indicated in red. Green opsin branches are indicated in green. Non-

240 teleost species and the outgroup (VA opsins and pinopsins) are indicated in grey. Grey boxes highlight

241 Gobiidae.

242

243 To find a possible link to the ecological significance of the red opsin duplication, we checked for the

244 presence of red skin fluorescence in the round goby. Interestingly, round goby individuals of both

245 sexes and of all sizes (n=10) feature weakly red fluorescent crescents above the eyes (Figure 3).

246 Whether such pattern has any relevance for the putatively enhanced vision in the red spectrum

247 remains elusive.

248

249 Figure 3

250 Red fluorescence in the round goby. Round gobies exhibit red fluorescence above the eyes when

251 exposed to green light.

252

253 Table 2. Amino acid analysis of key tuning sites in Gobiidae red opsins proteins.

max. spectral key tuning amino acid site in sensitivity reference species ecology gene round goby (wavelength) 180 197 277 285 308 bovine rhodopsin equivalent site: 164 181 261 269 292

Boleophthalmus terrestrial LWS1 A H Y T A 553 nm pectinirostris mudskipper LWS2 A H F A A 531 nm (25) Periophthalmus terrestrial LWS1 S H Y T A 560 nm magnuspinnatus mudskipper LWS2 A H F T A 546 nm freshwater LWS1 S H Y T A 560 nm this study Neogobius temperate melanostomus rivers and lakes LWS2 S H F T A 550 nm* this study * = predicted by the key tuning sites, and Y261F shift of 10 nm; Yokoyama, 2008.

254 3. Sensory perception genes: Olfaction

255

256 Olfactory receptors (OR) in vertebrates are 7-transmembrane-domain G-protein coupled

257 transmembrane proteins. They are expressed in neurons embedded in membranes of the olfactory

258 lamellae. Mammals usually have several hundred OR genes that cluster in two major types (~400 in

259 human, and ~1000 genes in mouse) (60). Teleost fishes possess fewer OR genes but feature a higher

260 diversity (5 kinds of type 2 ORs in teleosts as compared to 2 kinds of type 2 ORs in mammals) (61).

261 The binding properties of individual ORs, especially in fishes, are virtually unexplored.

262

263 We identified 112 putative olfactory receptor genes in the round goby genome. To put this result into

264 evolutionary context, all analyses were carried out in comparison with two Gobiidae species (blue-

265 spotted mudskipper and giant mudskipper) and two percomorph species (three-spined stickleback,

266 Gasterosteus aculeatus and Nile tilapia, Oreochromis niloticus; Figure 4A). The round goby presented

267 similar number of ORs (112) to the giant mudskipper (106) and stickleback (115), notably less than the

268 blue-spotted mudskipper (154) and near half the amount compared to Nile tilapia (214). We find that

269 all ORs belong to one of two transmembrane domain subtypes according to the Pfam database (7tm4

270 or 7tm1; Figure 4B; Supplemental_Fig_S3). This matches a previous large-scale phylogenetic

271 analysis which identified two main types of olfactory receptor genes in vertebrates (61). The functional

272 differences between the domain subtypes are unclear, but their different consensus sequences may

273 confer distinct biochemical properties.

274

275 Our analyses identify several cases of clade-specific gene expansions. Certain OR genes are

276 expanded in parallel in several lineages (Figure 4C). Likely, those expansion events are the result of

277 clade-restricted gene duplications, although a secondary role for gene conversion after species

278 divergence cannot be ruled out. While the Nile tilapia features the greatest overall amount of

279 expansions, the round goby presents the highest number of genes and expansions within the 7tm1

280 subfamily, a trend that is consistent in the other Gobiidae species (Figure 4D).

281

282

283 Figure 4

284 Phylogenetic tree of percomorph olfactory receptor protein sequences. A Phylogenetic

285 relationship among five analyzed percomorph species, i.e. three gobiids, one cichlid and one

286 stickleback. B Maximum-likelihood phylogenetic tree constructed with adrenergic receptors as

287 outgroup. Sequences were identified de novo except for nile tilapia (blue). Branches magnified in

288 panels C and D are highlighted with grey boxes. C Branch of the 7tm4 family featuring large

289 independent expansions in all species analyzed. D Branch of the 7tm1 family featuring several

290 expansions in Gobiidae (red, orange) that are not paralleled in other percomorph species (blue).

291

292 4. Response to the environment: Detoxification

293

294 The CYP gene superfamily is an essential part of the defensome, a collection of genes that provide

295 protection against harmful chemicals (35). Vertebrate genomes contain between 50-100 CYP genes.

296 The genomes of fugu and zebrafish, for example, encode 54 (62) and 94 (63) CYP genes respectively.

297 Expansions of individual CYP families occur in both mammals and fish. For example, zebrafish have

298 three times as many CYP2 family members (40) as most other vertebrate species (13-15), and similar

299 expansions of CYP2 genes have been observed in mice and rats (64).

300

301 We find that the round goby genome contains few CYP genes. We identify 25 complete or partial CYP

302 genes, as well as 21 gene fragments. Pseudogenes are common for CYP genes (62, 63, 65), which is

303 why strict annotation criteria are applied first before smaller fragments are considered. In total, the

304 genome contains approximately 50 CYP genes (Supplemental_Table_S2).

305

306 When including gene fragments, all expected CYP families are present in the round goby, and the

307 phylogenetic analyses show the expected relationships between gene families and between

308 vertebrates (Figure 5). Fish and most vertebrates have CYP genes from 17 families (CYP 1-5, 7, 8,

309 11, 17, 19, 20, 21, 24, 26, 27, 46 and 51) (62), while the CYP39 family occurs in humans and

310 zebrafish, but not in fugu (62, 63). In the round goby, the complete or partial genes could be assigned

311 to 9 CYP families (CYP 1- 4, 8, 19, 26, 27 and 51). The families CYP7, CYP11, CYP17 and CYP21

312 were present among the sequence fragments.

313

314 Contrary to expectations, the classical detoxification families CYP1-3 were not expanded (Figure 5).

315 CYP1, 2, 3 and to a lesser extent CYP4 proteins are responsible for the oxidative metabolism of

316 xenobiotic compounds (pollutants, drugs, etc.). In rodents and humans, the CYP1 family metabolizes

317 planar cyclic aromatic hydrocarbon compounds (reviewed in (66)), the CYP2 family metabolizes

318 structurally diverse drugs, steroids and carcinogens, the CYP4 family catalyzes the ω-hydroxylation of

319 the terminal carbon of fatty acids and xenobiotics, and CYP3 genes metabolize a range of structurally

320 different compounds in the liver and intestines. Over 50% of all pharmaceutical compounds are

321 metabolized by CYP3A genes in human. The goby genome contains three or four CYP1 genes: one

322 CYP1B gene, two CYP1C genes, and one CYP1A fragment. The latter lacks two main characteristics

323 (I- and K-helix) and could therefore be a pseudogene. As expected for a vertebrate (64), the genome

324 contains many CYP2 genes. The most important fish CYP2 families were represented, including

325 CYP2J, CYP2N, CYP2Y and CYP2AD. Finally, the round goby had a single CYP3A gene and a

326 potential CYP3A fragment. This is somewhat unusual because fish often feature species-specific

327 CYP3 subfamilies in addition to CYP3A. For example, medaka also contains CYP3B genes, zebrafish

328 CYP3C genes, and Acanthopterygii fish CYP3D genes (67).

329

330 We find that the round goby genome contains six CYP8 genes, which is more than expected based on

331 observations from the other gobies. The closely related large blue-spotted mudskipper has only two

332 CYP8 genes (XM_020924471 and XM_020919000.1; about 73-85% identity); no sequences were

333 found in other mudskipper species. Accordingly, we assume that the CYP8B genes have undergone

334 species-specific tandem duplications in the round goby, as is also known for the subfamilies CYP2AA,

335 CYP2X and CYP2K in zebrafish (64). Five round goby CYP8 genes locate to the same contig with

336 high sequence similarity (~90%), which is similar to zebrafish CYP8B1-3 that also colocalize on the

337 same chromosome (63). Misidentification of closely related CYP7, CYP8, and CYP39 genes as CYP8

338 is unlikely given the colocalization and high sequence similarity. The function of the expansion is

339 presently unclear, although expression patterns in zebrafish suggest a role in the early embryo (63). In

340 humans, CYP8 genes act as prostacyclin synthases that mediate steroid metabolic pathways in bile

341 acid production or prostaglandin synthesis (68). Based on structural similarities with yeast proteins,

342 CYP8 genes might also have E3 ubiquitin ligase activity. The almost identical crystal structures of

343 zebrafish and human CYP8A1 suggest similar functions in fish and mammals (69).

344

345 Figure 5.

346 Phylogenetic tree of vertebrate CYP protein sequences. Maximum likelihood phylogenetic tree

347 with 100 bootstraps, rooted with the CYP51 family. Detoxification genes CYP1-3 do not feature

348 unusual expansions, while the CYP8 family is expanded to six members (grey boxes). Non-fish

349 vertebrates are printed in grey. Fragments too short for tree building but attributable to a certain family

350 are indicated by orange half circles next to the root of the respective family.

351

352

353 5. Response to the environment: Osmoregulation

354

355 Osmotic homeostasis depends on passive ion and water uptake through cell membranes and the

356 intercellular space, on the active uptake or excretion of ions, and on the production and accumulation

357 of osmolytes. To understand the ability of round goby to tolerate a wide range of salinities, we

358 therefore compared the round goby repertoire of osmoregulatory genes to those of a stenohaline

359 freshwater species (zebrafish) and of euryhaline species (Nile tilapia, blue-spotted mudskipper and

360 three-spine stickleback).

361

362 Passive ion and water transport across membranes (transcellular permeability) depends on the

363 superfamily of aquaporin proteins. Aquaporins transport water (classical aquaporins), water and

364 glycerol (aquaglyceroporins), ammonia (aquaammoniaporins), or additional undescribed molecules

365 (unorthodox aquaporins; Figure 6). Primary sequences are only moderately conserved between the

366 classes (approximately 30% identity), but all aquaporins share six membrane-spanning segments and

367 five connecting loops. We find 15 aquaporin genes in the round goby, which compares to the number

368 in human (n=13) or zebrafish (n=20) and is lower than in the euryhaline Atlantic salmon (n=42) (70,

369 71). With 5 classical water aquaporins, 6 aquaglyceroporins, 2 aquaammonioporins, and 2 unorthodox

370 aquaporins, the round goby features the same types of aquaporins as freshwater stenohaline fish

371 (e.g., zebrafish) and highly euryhaline fish (e.g., tilapia; Figure 6).

372

373 Ion and water flow between cells in epithelia (paracellular permeability) is regulated by tight junctions,

374 of which claudin and occludin proteins are the most important components. Mammalian genomes

375 contain ~ 20 claudin genes, invertebrates such as Caenorhabditis elegans or Drosophila melanogaster

376 contain 4-5 genes, and fish often feature large expansions. For example, the fugu genome contains 56

377 claudins, of which some occur in clusters of > 10 genes (72). The round goby genome features 40

378 claudin paralogues, which is in line with numbers known from other fish. All human claudin genes were

379 represented as homologues (Supplemental_Fig_S4), and the round goby genome contains one

380 occludin gene in each of the two known subclades of the protein family (Supplemental_Fig_S5).

381

382

383

384 Figure 6

385 Phylogenetic tree of fish aquaporin proteins. Maximum-likelihood tree with 100 bootstraps of round

386 goby (Neogobius melanostomus, orange) in relation to cyprinid zebrafish (Danio rerio) and

387 percomorph three spine stickleback (Gasterosteus aculeatus), nile tilapia (Oreochromis niloticus), and

388 great blue-spotted mudskipper (Boleophthalmus pectinirostris). Zebrafish was used as outgroup in

389 each aquaporin subfamiliy. The main classes of aquaporins are labeled with human genes names.

390

391 In the kidney, intestine and gills, fish use active ion transport (mostly sodium transporters) to maintain

392 osmotic balance. Mechanisms mediating sodium uptake include electroneutral Na+/H+ exchange via

393 the NHE3b protein, Na+/Cl- cotransport via the NCC protein, and coupling of Na+ absorption with H+

394 secretion by a V-type H+-ATPase (73). We find 12 Na+/H+ exchanger genes, 5 Na+-K+-ATPase

395 catalytic alpha subunits and 6 Na+-K+-ATPase regulatory beta subunits in the round goby genome.

396 The round goby thus contains the same types of genes, but less copies, than either zebrafish or tilapia

397 (Supplemental_Fig_S6). We find that round goby, and also mudskippers, feature an interesting

398 distribution of Na+/Cl- co-transporters to subgroups; while most zebrafish and tilapia Na+/Cl- co-

399 transporters belong to the NKCC1 subgroup, Gobiidae feature more genes in the NKCC2 subgroup

400 (Figure 7).

401

402 Figure 7

403 Phylogenetic tree of human and fish sodium/potassium/chloride co-transporter proteins

404 (NKCC). Maximum likelihood tree with 100 bootstraps of round goby (Neogobius melanostomus,

405 orange), zebrafish (Danio rerio), three spine stickleback (Gasterosteus aculeatus), nile tilapia

406 (Oreochromis niloticus), great blue-spotted mudskipper (Boleophthalmus pectinirostris) and as

407 contrast humans (Homo sapiens, grey). Potassium/chloride co-transporters (KCC) are used as

408 outgroup.

409

410 Finally, fish produce osmolytes to actively take up and retain water. In particular, the cyclic polyol myo-

411 inositol is used by euryhaline teleosts to acclimate to high salinity. Two enzymes are required for its

412 production: myo-D inositol 3-phosphate synthase (MIPS) and inositol monophosphatase (IMPA). In

413 addition, some fish actively accumulate myo-inositol with a sodium/myo-inositol cotransporter (SMIT)

414 (74, 75). This transporter is of particular importance for marine fish exposed to high salt concentrations

415 (76, 77), while freshwater fish lack a SMIT gene (e.g. the freshwater stenohaline zebrafish lacks the

416 SMIT gene). The presence of SMIT has therefore been proposed to be a critical prerequisite for high

417 salinity tolerance in fish (78). We find that the round goby genome contains MIPS and IMPA, and

418 importantly, also a SMIT gene (Supplemental_Fig_S7).

419

420 6. Response to the environment: Immune System

421

422 It has been speculated that invasion success may relate to the ability to fight novel immune challenges

423 (37). We therefore characterized key genes related to the immune system, focusing on genes that

424 span both the innate and adaptive immune system such as pattern recognition receptors, selected

425 cytokines and chemokines, antigen presentation, T-cell surface receptors and antibodies

426 (Supplemental_Table_S3; Supplemental_Table_S4).

427

428 We find that the round goby genome features a classical adaptive immunity setup (Table 3).

429 Vertebrate adaptive immunity is characterized by the Major Histocompatibility Complex (MHC) class I

430 and MHC class II proteins and their regulators. MHCI presents antigens derived from a cell’s

431 intracellular environment, while MHCII presents antigens derived from material engulfed by

432 macrophages, B-cells or dendritic cells (79). We find 26 full length MHCI sequences from the classic

433 U-lineage and one sequence from the teleost-specific Z-lineage (80) (Supplemental_Table_S5).

434 MHCII is represented by 8 alpha (2 fragments) and 9 beta copies (Supplemental_Table_S6). The

435 uneven numbers may be attributed to assembly issues, but also, additional small fragments were not

436 further investigated (data not shown). We also identify the key MHC-supporting peptides Beta-2-

437 Microglobulin, CD74, TAP1/2 and tapasin. Beta-2-Microglobulin (B2M) is present in two copies, one of

438 which contains several indels, a diverged region, and no stop codon and thus may be a pseudogene.

439 The round goby has two copies of TAP2, which promotes the delivery of peptides to MHCI (annotated

440 as TAP2 and TAP2T; Supplemental_Table_S4; Supplemental_Fig_S8). Two TAP2 genes have also

441 been described in zebrafish, and our results thus suggest this is conserved feature among teleosts

442 (81). In addition, we identify the MHC transcriptional regulators CIITA and NLRC5

443 (Supplemental_Table_S3). The presence of the thymus transcription factor AIRE and the T-cell

444 receptors CD4 and CD8 confirms the presence of helper T cells and cytotoxic T cells in the round

445 goby.

446

447 Table 3. Overview of manually annotated key adaptive immune genes

NEME Contig Gene Start End Strand annotation annotation CIITA NEME_493 Contig_2585 3 985 719 3 993 128 Antisense AICDA NEME_58 Contig_447 597 424 599 014 sense AIRE NEME_9 Contig_79 14 106 230 14 113 573 antisense B2M NEME_421 Contig_2242 363 050 363 352 antisense B2M_pseudo NEME_421 Contig_2242 368 352 368 721 antisense CD4 NEME_213 Contig_1334 340 445 348 248 sense CD74 NEME_71 Contig_593 791 743 796 652 antisense CD8a NEME_729 Contig_3231 634 222 648 487 antisense CD8b NEME_729 Contig_3231 656 030 660 462 antisense RAG1 NEME_106 Contig_787 4 690 414 4 695 142 sense RAG2 NEME_106 Contig_787 4 699 042 4 700 651 antisense TAP1 NEME_582 Contig_2864 694 776 722 339 sense TAP2 NEME_387 Contig_2107 2 987 106 2 993 287 antisense TAP2T NEME_299 Contig_1786 3 697 645 3 704 089 sense Tapasin NEME_387 Contig_2107 3 111 989 3 119 308 sense 448

449 Similarly, the humoral adaptive immune response (also termed the B-cell mediated immune response)

450 is intact in the round goby. Humoral immunity in fish is characterized by three antibody isotypes

451 consisting of immunoglobulin heavy chains delta (IgD), mu (IgM), and tau (IgT). We identify a contig-

452 spanning immunoglobulin heavy chain locus (Supplemental_Fig_S9) containing 8 delta constant

453 domains, and 4 constant mu domains, as well as genes responsible for heavy chain recombination

454 and immunoglobulin hypermutation (RAG1/2 and AID(AICDA); Table 3; Supplemental_Table_S3).

455 There is no evidence for the presence of immunoglobulin tau constant domains, which are commonly

456 found in carps and salmonids (82).

457

458 While round goby adaptive immunity conforms to vertebrate standards, its innate immune repertoire

459 displays remarkable and unusual features. We find that all components of the inflammasome (a

460 signaling pathway involved in inflammatory responses; Figure 8) are expanded. Inflammasome

461 assembly is activated through pathogen pattern recognition receptors (83), and ultimately triggers a

462 local or systemic acute phase response by producing IL-1 family cytokines (83, 84) and/or promotes

463 cell death via pyroptosis (84). In the round goby genome, components of the entire cascade (pattern

464 recognition receptors, ASC adaptor proteins, IL-1, and acute phase proteins) are present in

465 unexpectedly large numbers (Figure 8; Supplemental_Table_S8). In the following, our findings are

466 described step-by-step from the cell surface down to the acute phase response.

467

468

469 Figure 8.

470 The inflammasome pathway. Several components of the pathway are expanded in the round goby

471 (gene numbers in round goby, or novel groups for NLRs, are indicated in orange). Pathogen-

472 associated patterns are recognized by pattern recognition receptors such as Toll-like receptors at the

473 cell surface, or NLRs in the cytoplasm. This interaction triggers the transcription of cytokine precursors

474 via NFkB, and the activation and assembly of inflammasome components (NLRs, Pro-Caspase-1, and

475 ASC). Inflammasome-activated Caspase-1 then initiates the maturation of cytokines and an acute

476 phase inflammatory response (CRP, APCS proteins), and / or pyroptosis through gasdermin.

477

478 Perhaps the best studied pattern recognition receptors are the Toll-like Receptors (TLRs), pathogen-

479 recognizing molecules that are generally expressed either at the plasma membrane or on the

480 endosomal membranes. Sixteen TLR types with slightly differing ligand binding activities are

481 conserved across vertebrates, and most vertebrate genomes contain one to three copies of each type.

482 As expected for a teleost, the round goby genome does not contain the LPS-detecting TLR4 genes.

483 However, in total we find 56 TLRs, of which 40 appear to originate from an expansion of Toll-Like

484 Receptor 23-like genes (Figure 9). Small expansions of specific TLRs are somewhat common in fish

485 (85), and indeed, we find minor TLR22 and TLR23 expansions to 6-13 copies in the genomes of other

486 Gobiidae. However, the extent of the expansion of TLR23 exceeds even what is observed for TLR22

487 in the relatives of cod (Gadiformes) (86). Phylogenetically, the identified TLR23 sequences form three

488 clades, of which two are specific to Gobiidae, while the third contains TLR23 sequences from other

489 teleosts as well (Supplemental_Fig_S10). In terms of genomic location, round goby TLRs 22 and 23

490 were distributed across several contigs with some copies arranged in tandem, which suggests several

491 independent duplication events.

492

493 Figure 9

494 Phylogenetic tree of teleost Toll Like Receptor protein sequences. A maximum likelihood

495 phylogenetic tree run with the JTT substitution model and 500 bootstrap replicates on the

496 transmembrane, linker and TIR domain of all TLRs found in a selected set of teleosts in the Ensembl

497 database, the Atlantic cod genome version 2, and all manually investigated Gobiiformes. A TLR

498 sequence from the lancelet Branchiostoma belcheri was used as an outgroup and the root was placed

499 upon its corresponding branch. Green triangles, Atlantic cod. Orange circles, round goby. Grey box,

500 TLR22 and TLR23.

501

502 For intracellular pathogen recognition receptors of the NACHT domain and Leucine-rich Repeat

503 containing receptor (NLR) family, we identify two new, previously undescribed families (Group 5 and 6)

504 present in the round goby and also in the mudskipper B. pectinirostris (Figure 10).

505

506 Figure 10

507 Phylogenetic tree of the NLR nucleotide-binding domain sequences in round goby, great blue

508 dotted mudskipper, zebrafish and human. Maximum Likelihood phylogenetic tree with 500 bootstraps

509 rooted at the split between NB-ARC (found in APAF) and NACHT domains (present in all the other

510 NLRs). NB-ARC domains from APAF1 orthologs were used as an outgroup. Bootstrap values are

511 shown for nodes that determine an entire cluster. The tree resolves all three major classes of

512 vertebrate NLRs (NLR-A, NLR-B, NLR-C). NLR-A genes were conserved in all analyzed species, no

513 NLR-B genes were found from the gobies. Six groups of NLR-C genes were identified, four of which

514 are exclusive to zebrafish (Group 1-4) and two contain only sequences from gobies (Groups 5 and 6).

515 Within the goby-specific groups, lineage-specific expansions can be seen for both round goby

516 (orange) and mudskipper (brown).

517

518 NLRs have diverse roles from direct pathogen recognition to transcriptional regulation of the MHC

519 (NLRs CIITA and NLRC5) and contribute to inflammasome activation. Mammalian genomes display

520 20-40 NLRs in families NLR-A and NLR-B, while fish also feature a fish-specific subfamily (NLR-C)

521 (87) and a much expanded NLR repertoire (e.g. 405 NLR-C genes in zebrafish) (88, 89). The round

522 goby genome contains at least 353 NLRs (Supplemental_Table_S8), which include 9 highly

523 conserved vertebrate NLRs (NOD1, NOD2, NLRC3, NLRC5, NLRX1, NWD1, NWD2, APAF1, CIITA)

524 as well as 344 NLR-C genes. Fish NLRs cluster into 6 groups of which 2 represent novel NLR-C

525 clades (groups 5 and 6, Figure 10). The novel groups are supported by phylogenetic analyses as well

526 as motif presence/absence (Table 4). NLR-C groups are characterized by highly conserved versions

527 of the sequence motif Walker A. The most common sequence for Walker A observed in both goby

528 NLR-C groups, GVAGVGKT, is not associated with any of the four major NLR-C groups in zebrafish

529 (88). Also, NLR subtypes often carry group-specific combinations of the protein-protein interaction

530 domain PYD and/or B30.2 domain. This holds true for Gobiidae NLR-C groups, since only group 5

531 NLRs can carry an N-terminal PYD domain and/or a C-terminal B30.2 domain (88), similar to the

532 zebrafish Group 1 and 2 NLRs (Table 4). In contrast, some group 6 NLRs have C-terminal CARD

533 domains, which in both human and zebrafish are attached to specific inflammasome-associated NLR-

534 B genes (90). The round goby C-terminal CARD-containing NLRs are found on the same few scaffolds

535 and share a high degree of sequence similarity, indicative of a recent expansion. This expansion is

536 absent from mudskipper and thus restricted to the round goby lineage. Many other Group 6 NLRs are

537 fragmented, with large insertions in the middle of their conserved 2 kb exon

538 (Supplemental_Table_S8).

539

540

541 Table 4. Key features of each of the six NLR-C subgroups. x denotes a variable amino acid.

Identified Group in this Walker A Last residues of the largest exon PYD? B30.2? study 1 GIAGVGKT L(I/M)PVVKNT(T/R)RA + + 2 GVAGIGKS LSAVIKTSKRA + + 3 GIAGIGKT L(IP/TA)AV(R/S)NC(RK/TR/RR)A - + 4 GVAGIGKT LPV(I/V)xxxx(A/V)x - - 5 x GVAG(V/A/I)GKT (L/M)PV(V/I)KASxK(A/V) + + 6 x GVAG(V/A)GKT L(I/V)P(A/V)VRNCRKA - - 542

543 Once activated, some NLRs (including those with a C-terminal CARD) can oligomerize and form an

544 inflammasome in order to activate specific caspases (usually Caspase 1, Figure 8). The interaction

545 between NLRs and the caspase are mediated by the adaptor protein ASC (also known as PYCARD),

546 which itself oligomerizes into large structures known as “specks” (91). Vertebrates have 1-2 copies of

547 ASC, which are characterized by a characteristic combination of a single PYD and CARD domain. In

548 the round goby genome, we find 20 cases of this domain combination. Since the genomes of other

549 gobies contain 1-2 PYD-ASC combinations, the expansion appears to be specific to the round goby

550 (Figure 11A). The effector protein Caspase 1 is present as one gene in humans and as two genes in

551 zebrafish. We find that the round goby genome features an expansion to 18 copies. Interestingly,

552 some of those genes appear to contain a CARD domain (as seen in mammals and several species of

553 fish) while others have PYD (as seen in zebrafish). This suggests that a caspase with both domains

554 may have existed in the common ancestor of fish and tetrapods, with most lineages having retained

555 only one of the two. However, phylogenetic analyses reveal that all round goby Caspase 1 genes are

556 the result of a single expansion event specific to this species (Figure 11B). An alternative explanation

557 for the presence of both PYD- and CARD-caspases 1 genes would be a recurrent acquisition of PYD

558 in different lineages. In any case, in addition to Caspase 1 genes, caspase 3 (a key component of

559 apoptosis which may be activated by Caspase 1) is also expanded to 5 copies. Caspase 4 and 5, on

560 the other hand, appear to be absent.

561

562 Finally, we find that genes encoding for two peptides produced in the course of inflammation, the

563 acute phase reactants C-reactive protein (CRP) and serum amyloid component P (APCS), are

564 expanded to a total of 25 copies (compared to 2-7 in fish, and 5-19 in the other Gobiidae). In fish, CRP

565 and APCS are closely related and cannot be distinguished based on BLAST scores or phylogeny. As

566 seen in other fish species, all investigated CRP/APCS sequences resolve into two major phylogenetic

567 clades, with the mammalian sequences in a third (Supplemental_Figure_S11).

568

569

570 Figure 11

571 A Phylogenetic tree of gnathostome ASC protein sequences. Maximum Likelihood phylogenetic

572 tree with 500 bootstraps rooted at the split between tetrapods and ray-finned fish. Tetrapods were

573 used as outgroup. Round goby is indicated in orange. Gobiidae are highlighted with a grey box. The

574 goby sequences form a clear separate cluster (marked with the box), with a large expansion apparent

575 in the round goby. B Phylogenetic tree of gnathostome Caspase 1 protein sequences The

576 Caspase 1 tree comprises all protein sequences annotated as CASP1 in the investigated Gobiiformes

577 genomes aligned together with reference sequences from Ensembl and GenBank. The root was

578 placed on the branch containing the mammalian sequences.

579

580

581 7. Adaptation to novel environments: Epigenetic regulators

582

583 The PRC2 complex establishes and maintains gene repression (92) and thus represents a plasticity-

584 restricting mechanism. The complex mediates di- and trimethylation of lysine 27 on histone H3 and

585 contains four proteins: a catalytic subunit (either enhancer of zeste EZH1 or EZH2), suppressor of

586 zeste SUZ12, embryonic ectoderm development EED and RB Binding Protein 4 RBBP4 (50). In

587 mammals, the alternative catalytic subunits EZH1 and EZH2 have partially complementary roles (93,

588 94), and requirements for the two alternative catalytic subunits differ between species – in contrast to

589 mammals, zebrafish develop in the absence of either catalytic subunit (95, 96). We find that the round

590 goby genome contains the usual complement of PRC2 components: two copies of SUZ12 (of which

591 one appears quite diverged), one copy of EED, one copy of RBBP4, and two copies of EZH (with

592 multiple isoforms determined by RACE experiments). For SUZ12, EED, and RBBP4, sequence-based

593 identification was straightforward, and phylogenetic analyses followed the known phylogenetic

594 relationships of fish, mammals, and other vertebrates (Supplemental_Fig_S12). The catalytically

595 active subunits EZH1 and EZH2 do cluster with the closest species in the phylogeny, the mudskipper

596 B. pectinirostris (Figure 12), but the deeper relationships within EZH2 are poorly supported and may

597 suggest a complex evolutionary history.

598

599

600 Figure 12

601 Phylogenetic tree of vertebrate EZH proteins. Midpoint-rooted Bayesian phylogenetic tree. Note the

602 position of the Australian ghost shark (potential outgroup) within the poorly supported EZH2 branch.

603 When rooting with Australian ghost shark, teleost EZH2 genes cluster with EZH1 (data not shown).

604 Round goby is indicated in orange.

605

606 Methylation marks similarly regulate gene expression and are deposited by conserved enzymes called

607 DNA methyltransferases (DNMTs). Mammals feature two types of DNMTs, DNMT3 (three genes A, B,

608 and L) and DNMT1 (one gene). The two types perform both de novo and maintenance methylation,

609 respectively, in a dynamic division of labor (97). Interestingly, fish feature a variable repertoire of

610 DNMT3 genes. Medaka, fugu, zebrafish, and carp have three, five, six, and twelve DNMT3 genes,

611 respectively (98). We find that the round goby genome features one DNMT1 that follows the expected

612 phylogenies, and five DNMT3 genes, of which two cluster with vertebrate DNMT3A sequences, and

613 three with vertebrate DNMT3B sequences (Figure 13). The number of DNMT3 genes in round goby

614 corresponds to that seen in in stickleback, fugu and tilapia (99). In general, the DNMT3 phylogeny is

615 not well supported, which limits conclusions about the evolution of specific DNMT3 genes.

616

617

618

619 Figure 13

620 Phylogenetic tree of vertebrate DNMT3 proteins. Midpoint-rooted Bayesian phylogenetic tree. The

621 Australian ghost shark (potential outgroup) is positioned among DNMT3A genes. Round goby is

622 indicated in orange. Zebrafish, the only other fish with well-annotated DNMT3 genes, is indicated in

623 green.

624

625 Discussion

626

627 General observations

628

629 Our analyses depict a genome that, in many respects, is similar to other teleost genomes. There is no

630 evidence for recent genome duplications, and genome size, gene content and GC content are within

631 the ordinary range. Transposable elements can create genetic variation and mediate ecological

632 success (100), but repeat analyses do not reveal unusual transposon activities in the round goby.

633 Small genome size has been proposed to foster invasiveness (101), but the round goby genome is not

634 particularly small. Phylogenetic analyses reveal that many of the analyzed gene families conform to

635 expectations. For example, green opsin gene duplications and the loss of the UV opsin are observed

636 in many fish lineages (20). Similarly, the expected gene families and overall gene complements are

637 found for olfactory receptors, cytochrome P450, and osmoregulatory proteins, for adaptive immunity

638 and epigenetic regulators. Multilocus sex determination has previously been suggested for freshwater

639 gobies (102), and indeed our data suggest a multigenic and/or environmental sex determination

640 system for the round goby, rather than a large sex-determining region or a sex chromosome. Overall,

641 these findings support the validity of the sequencing and assembly approach, and suggest that

642 selected findings of interest are not artefacts. In addition, the round goby genome sequence also

643 reveals several novel and interesting findings of which some pertain to teleost genomes in general,

644 some to Gobiidae, and some to specific gene families, with possible implications for invasive potential.

645

646 Environmental perception

647

648 We find that the visual system of Gobiidae may be more efficient in the red parts of the light spectrum.

649 This is intriguing considering the benthic life style of gobies and their occurrence in turbid areas. In

650 clear waters, red light from the sun is the least abundant part of the spectrum (and virtually absent

651 below 15m of depth) because red light penetrates least through water, but many organisms convert

652 the deeply penetrating green and blue wavelengths into red. Indeed, the eyes of gammarids, a

653 common prey of round goby, strongly reflect red light (103). An enhanced red perception through an

654 additional red opsin gene may thus be relevant for round goby predation success. In turbid waters, red

655 is the most common part of the light spectrum because long wavelengths experience least scattering

656 (21). Round gobies readily establish populations in turbid environments. The retention of two red opsin

657 genes may thus possibly relate to the remarkable ability of the round goby to colonize turbid habitats.

658 Our predictions based on the key amino-acid substitution suggest that LWS1 is expected to be most

659 sensitive at 560 nm (same as one of the mudskipper gobies) (25), while LWS2 is expected to be most

660 sensitive at 550 nm (59). Similar small differences in the sensitivity maximum can indeed result in

661 functionally different spectral tuning palettes (e.g. during development or in different environmental

662 conditions) (104).

663

664 The presence of red fluorescence on top of the eye in round goby is the first unequivocal description

665 of fluorescence in a freshwater fish and might be interpreted as being associated with the ability to

666 discriminate different shades of red colors. However, the fluorescence in the specimens investigated

667 was quite weak. Unless fluorescence expression is stronger under natural conditions or in the

668 ancestral population from which the invading populations stem, a visual function of the weak

669 fluorescence observed here seems unlikely (see warnings by (105)). Fluorescence is, however,

670 widespread and stronger among several marine gobies (106). Although the fluorescent "eyebrows" of

671 the round goby show a striking similarity to those of some marine gobies, their function will remain

672 unclear until properly tested. Social functions are possible – for example, in sand gobies, dark eyes

673 indicate female readiness to spawn (107). Alternatively, they may simply provide camouflage for

674 individuals buried in bright sand up to the eyes. Functional hypotheses for fluorescence, such as

675 communication, camouflage and improved prey detection have been extensively reviewed by (108).

676 The genetic tools now available for the round goby may allow for experimental manipulation of

677 fluorescence expression, once the actual fluorophores that produce the fluorescent signal have been

678 identified.

679

680 Response to the environment

681

682 With respect to ecological and physiological aspects of success and invasiveness, some findings on

683 CYP genes, on osmoregulation, and on innate immunity call for further attention. The mostly minimal

684 complement of cytochrome P450 proteins present in the round goby is unexpected considering the

685 occurrence of round goby in polluted areas (109, 110). The CYP1-3 gene complement for xenobiotic

686 metabolism is similar to other teleost genomes, and the ability of the round goby to survive in

687 contaminated environments must therefore have other reasons. Round goby may cope with

688 contaminations at the level of gene expression, either through higher basal expression values or by a

689 particularly rapid response to exposure (45). Alternatively, this species may have peculiarities in other,

690 not yet analyzed areas of the defensome (e.g. transporters). Analyses of the tissue expression of CYP

691 families 1, 2 and 3, and also the study of other defensome gene families, including the nuclear

692 receptors regulating CYP gene expression, transporters and conjugating enzyme families, may be

693 useful in this respect.

694

695 Another potentially relevant finding is the ability of the round goby to not only produce, but also

696 accumulate osmolytes. Species distribution constraints often arise from physiological limitations. The

697 round goby is one of the most geographically wide-ranging invasive fish species in Europe and North

698 America, and the ability to accumulate osmolytes may impact its range expansion in three ways.

699 Firstly, 0-25 PSU (common for coastal waters, but lower than the ocean) is the species' current limit

700 for unperturbed osmoregulation (111). However, the round goby’s repertoire of key-genes in myo-

701 inositol production and accumulation might bestow the species with the potential to eventually tolerate

702 higher salinities, for example through the evolution of altered gene regulation patterns, and colonize

703 higher PSUs. Secondly, osmolytes improve water retention and thus desiccation tolerance. In this

704 context, myo-inositol accumulation may have contributed to overland dispersal. Overland dispersal of

705 eggs or larvae with boats or fishing gear involves air exposure, and indeed, round goby eggs

706 withstand desiccation for up to 48 hours (112). Finally, osmolytes essentially act as anti-freeze agents

707 and molecular chaperones, and contribute to cryoprotection in diverse organisms from bacteria (113)

708 to flies (114). Osmolytes may thus enable the round goby to combat a number of environmental

709 conditions and to colonize new areas. The surprising and unexpected ability of the round goby to

710 colonize cold areas well below its temperature optimum of 22°C, such as the Northern Baltic Sea, may

711 be linked to osmolyte production.

712

713 Lastly, the “strike fast, strike hard” innate immune system and the impressively large inflammation

714 machinery of the round goby may enhance the species’ colonization potential. Fish immunity appears

715 to be quite plastic. For example, cod have disposed of some core adaptive immunity components (18),

716 yellow croaker feature an expanded TNF repertoire (115), and channel catfish retain a high number of

717 recent duplications and SNPs in immune genes (116). Meanwhile, in salmonids, genes specifically

718 retained after the 4th whole genome duplication are not immune genes (117).

719

720 We find that the round goby genome contains multiple copies of genes for inflammasome assembly,

721 activation, and function. This is interesting because the fish inflammasome complex is much more

722 poorly characterized than that of mammals. Maturation of IL-1 by inflammasome-activated Caspase 1

723 cleavage in fish is a matter of debate, since teleost IL-1 proteins lack the conserved caspase cleavage

724 site present in mammalian IL-1b and IL-18 (118). However, as has been shown for zebrafish, Caspase

725 1 can also utilize an alternative site to cleave and mature IL-1 (90, 119). In any case, the caspases

726 also mediate cell death via pyroptosis and the presence of other components such as ASC, caspases

727 and pro-IL1 and pro-IL18 supports a role for inflammasomes in fish. Zebrafish ASC oligomerize and

728 form “specks” as seen in mammals (90). The molecular dynamics of inflammasome activation

729 therefore represent a potential future research avenue in the round goby.

730

731 In terms of ecological success, the round goby’s expanded repertoire of pathogen recognition

732 receptors may broaden the scope of its immune response and increase the range of detectable

733 ligands and pathogens. The expanded acute phase repertoire may also contribute to a fast response,

734 or inversely, may limit excessive cell damage. In humans, the acute phase protein CRP contains

735 inflammation as part of a negative feedback loop (120). Thus, the round goby may re-enter

736 homeostasis faster compared to other fish species with smaller CRP/APCS repertoires. The larger

737 acute phase repertoire may also function to limit the cellular damage caused by the potentially large

738 amount of inflammasome combinations the round goby can generate. In this context, we suggest

739 systematic investigations into a potential relation between inflammasome expansions and

740 invasiveness in Gobiidae, in combination with immune challenge experiments.

741

742 Long-term adaptation

743

744 We identify a potentially interesting evolutionary history for the conserved PRC2 component EZH in

745 fish, and add to the previous observation that the conserved de novo DNA methylation machinery

746 features a surprising diversity in fish. These results underscore the need for in-depth investigations

747 into the role and relevance of epigenetic regulation and transgenerational inheritance in teleosts. Our

748 findings support the emerging idea that epigenetic regulation in fish follows somewhat different rules

749 than in mammals. For the histone methylating complex PRC2, our results suggest interesting

750 phylogenetic relationships of EZH proteins in fish. EZH proteins act in tissue specific complexes

751 comprised of core SUZ12, EED, and RBBP4, but also AEBP2, PCL proteins and JARID2. These

752 proteins enhance PRC2 efficiency, contribute to recruitment to target sites, or inhibit the complex (50,

753 92). Small sequence changes can have strong effects on the entire complex, since the precise

754 interactions among the components and with other gene regulators impact its function and localization

755 (121–124). For example, species-specific insertions (125) are thought to regulate PRC2 recruitment

756 and/or exclusion from target genes (126). We suggest that the future incorporation of more sequences

757 of both EZH1 and EZH2 from a greater range of taxa and the inclusion of currently unannotated

758 versions of the genes associated with both the teleost specific whole genome duplication and lineage

759 specific duplications (96) would aid understanding of the evolutionary history of the entire complex. We

760 expect that studying PRC2 in non-mammalian vertebrates may reveal ancestral or less abundant

761 interactions, functions or also complex associations of PRC2.

762

763 Similarly, our results warrant an in-depth exploration of DNA methylation in fish. Originally, DNA

764 methylation evolved to distinguish own (methylated) DNA from foreign (non-methylated) DNA such as

765 introduced by viruses. Therefore, cytosines in CG base contexts are by default methylated. In

766 mammals, DNA methylation in CG dense regions (CG islands) is associated with gene repression.

767 However, DNA methylation also features species- and taxon-specific differences, even among

768 vertebrates, which are still greatly underappreciated. For example, non-methylated genome regions in

769 fish are unexpectedly CG-poor (127), fish differ from mammals with respect to the distribution of

770 methylated CpGs in the genome (128), algorithms developed on mammals fail to identify CpG islands

771 in fish (129), genome-wide CpG island predictions in cold-blooded animals consist primarily of false

772 positives (130), and fish CG methylation occurs mainly in coding regions, where it correlates positively

773 with gene expression levels (131). These curious differences are further enhanced by the seemingly

774 random copy number variation in the de novo DNA methyltransferase DNMT3 in teleosts, which do not

775 reflect genome duplication events in teleosts (99). DNMT3 genes display highly spatiotemporal

776 expression patterns particularly during development (132–135), and an in-depth and species-aware

777 exploration of the role of DNA methylation in fish is clearly warranted.

778

779 Gene expansions

780

781 A general theme across several of the analyzed gene families is gene expansions. Gene expansions

782 have been linked to invasive potential before (136) and are recurrent in fish genomes, both within

783 (117, 137) and outside (116, 138, 139) the context of whole genome duplications. Many duplicated

784 genes are known to experience rapid neofunctionalization rather than subfunctionalization (137), and

785 have the potential to compensate against mutation even after divergence (140). The round goby and

786 its relatives are definitely strong candidates for further and systematic investigation of a link between

787 gene expansions and colonization or invasion potential. The Benthophilinae group is recently

788 diversified crowd of fish with many members inexplicably on the move (141), and Gobiidae in general

789 share a remarkable colonization potential (10, 142). Importantly, recent gene expansions can be

790 difficult to resolve with short reads, and genomes based on long read sequencing (as presented here)

791 will be instrumental in this regard.

792

793 Among the receptor families analyzed, the NLRs, TLRs, and olfactory receptors, we identify a couple

794 of particularly beautiful case studies for recent expansions and repeated radiations. Our identification

795 of two previously undescribed NLR-C gene families (88), here termed group 5 and group 6, indicates

796 substantial diversification of NLRs in fish. Different teleost lineages appear to feature different NLR-C

797 subfamilies with large lineage-specific expansions reminiscent of olfactory receptor repertoires.

798 Similarly, we identify interesting cases of parallel expansions across families, and also family-specific

799 expansions, among olfactory receptors. Both cases warrant investigations into the evolution of ligand

800 binding repertoires. For example, 7tm1 subfamily members may be involved in the detection of

801 distinctive types of odors relevant for round goby, and possibly, Gobiidae ecology (28–30). Which

802 types of odorants are detected by parallel expanded ORs, and whether these expansions serve to

803 detect similar or different types of odorant molecules in different species, remains to be studied.

804 Finally, the massively expanded TLR22 and TLR23 families warrant an exploration of their ligand

805 binding properties. TLR22 and TLR23 have been suggested to recognize nucleic acid ligands (85), but

806 some also react to protein or lipid pathogen-associated patterns (143–145), and their role in fish is

807 currently unclear.

808

809 In summary, this work provides a solid basis for future research on the genomic, genetic, and

810 epigenetic basis of ecological success. Clearly, many more gene families or pathways may contribute

811 to the round goby’s invasion success. For example, the presented analyses barely scratch the surface

812 of epigenetic regulation, innate immunity and transporters (e.g. of toxins). We did not investigate

813 endocrine pathways (which govern growth and reproductive success) nor antimicrobial peptides

814 (which contribute to innate immune defense), areas which may yield fruitful information of the success

815 of this invader. We welcome future research using this novel genomic resource, and encourage

816 experts on those pathways to contribute their knowledge.

817 Methods

818

819 A relevant note upfront is that this manuscript is the product of a long-standing collaboration of leading

820 experts in their respective fields. The gene families analyzed differ widely with regard to sequence

821 conservation, the number and similarity of genes within and between species, the scope of questions

822 in the field, etc. Compare, for example, the de novo identification of hundreds of virtually identical NLR

823 receptors with the manual annotation of a handful of extremely conserved DNA methyltransferases, or

824 the phylogenetic analysis of the conserved vertebrate CYP gene family with a fish-centered

825 comparison of osmotic balance regulators. Accordingly, each collaborator applied methods that were

826 suited for the respective situation. As a common theme, genes were identified by blast, sequences

827 were extracted and aligned with other fish and/or other vertebrates, trees were constructed with either

828 bayesian or maximum-likelihood methods, and findings were always verified against the mudskipper

829 genomes.

830

831 Genomic DNA library preparation and PacBio sequencing

832

833 Genomic DNA was extracted from the liver of one male individual of round goby caught in Basel,

834 Switzerland (47° 35′ 18″ N, 7° 35′ 26″ E). At the Genome Center Dresden, Germany, 300 mg of liver

835 tissue were ground by mortar and pestle in liquid nitrogen and lysed in Qiagen G2 lysis buffer with

836 Proteinase K. RNA was digested by RNase A treatment. Proteins and fat were removed with two

837 cycles of phenol-chloroform extraction and two cycles of chloroform extraction. Then, DNA was

838 precipitated in 100% ice cold ethanol, spooled onto a glass hook, eluted in 1x TE buffer, and stored at

839 4 °C. 10 μg of DNA was cleaned using AMPure beads. From this DNA, five long insert libraries were

840 prepared for PacBio sequencing according to the manufacturer’s protocols. Genomic DNA was

841 sheared to 30-40 kb using the Megaruptor device. The PacBio libraries were size selected for

842 fragments larger than 15-17.5 kb using the BluePippin device. PacBio SMRT sequencing was

843 performed with the P6/C4 chemistry using 240 min sequencing runs. Average read length was 11-12

844 kb. In total, 86 SMRT cells were sequenced on the PacBio RSII instrument resulting in 46 gigabases

845 (Gb; an estimated 46x coverage for a putative ~1 Gb genome) polymerase reads.

846

847

848 Assembly of the round goby genome

849

850 The round goby genome was assembled at the Heidelberg Institute for Theoretical Studies HITS

851 gGmbH. Raw PacBio reads were assembled using the Marvel (146, 147) assembler with default

852 parameters unless mentioned otherwise. Marvel consisted of three major steps, namely the setup

853 phase, patch phase and the assembly phase. In the setup phase, reads were filtered by choosing only

854 the best read of each Zero-Mode Waveguide as defined by the H5dextract tool (146) and requiring

855 subsequently a minimum read length of 4k. The resulting 3.2 million reads were stored in an internal

856 Marvel database. The patch phase detected and fixed read artefacts including missed adapters,

857 polymerase strand jumps, chimeric reads and long low-quality segments that were the primary

858 impediments to long contiguous assemblies (146). To better resolve those artefacts only low

859 complexity regions were masked (DBdust) and no further repeat masking was done. The resulting

860 patched reads longer than 3k (41x coverage) were then used for the final assembly phase. The

861 assembly phase stitched short alignment artefacts from bad sequencing segments within overlapping

862 read pairs. This step was followed by repeat annotation and the generation of the overlap graph,

863 which was subsequently toured in order to generate the final contigs. By using an alignment-based

864 approach, the final contigs were separated into a primary set and an alternative set containing bubbles

865 and spurs in an overlap graph. To correct base errors, we first used the correction module of Marvel,

866 which made use of the final overlap graph and corrected only the reads that were used to build the

867 contigs. After tracking the raw reads to contigs, PacBio’s Quiver (148) algorithm was applied twice to

868 further polish contigs as previously described (146).

869

870 Automated annotation of the round goby genome

871

872 The round goby genome assembly was annotated using Maker v2.31.8 (149, 150). Two

873 iterations were run with assembled transcripts from round goby embryonic tissue (46) and data

874 from eleven other actinopterygian species available in the ENSEMBL database (downloaded

875 the 15th February 2016, http://www.ensembl.org, see Table 5) as well as the SwissProt protein

876 set from the uniprot database as evidence (downloaded March 2, 2016;

877 https://www.uniprot.org/downloads). In addition, an initial set of reference sequences obtained

878 from a closely related species, the sand goby Pomatoschistus minutus, sequenced by the

879 CeMEB consortium at University of Gothenburg, Sweden (https://cemeb.science.gu.se), was

880 included. The second maker iteration was run after first training the gene modeler SNAP

881 version 2006-07-28 (151) based on the results from the first run. Augustus v3.2.2 (152) was

882 run with initial parameter settings from Zebrafish. Repeat regions in the genome were masked

883 using RepeatMasker known elements (153) and repeat libraries from Repbase (154) as well as

884 de novo identified repeats from the round goby genome assembly obtained from a

885 RepeatModeler analysis (153).

886

887 Table 5. Summary of reference data from Ensembl used for the annotation.

Reference species Number of protein Assembly version from ENSEMBL sequences (downloaded 15th Feb 2016) Astyanax mexicanus 23698 AstMex102 Danio rerio 44487 GRCz10 Gadus morhua 22100 gadMor1 Gasterosteus aculeatus 27576 BROADS1 Lepisosteus oculatus 22483 LepOcu1 Oreochromis niloticus 26763 Orenil1.0 Oryzias latipes 24674 MEDAKA1 Poecilia formosa 30898 PoeFor_5.1.2 Takifugu rubripes 47841 FUGU4 Tetraodon nigroviridis 23118 TETRAODON8 Xiphophorus maculatus 20454 Xipmac4.4.2 888

889 In order to ensure the completeness and quality of the current assembly and the associated gene

890 models, the assembly and the predicted protein sequences were run against reference sets at two

891 different taxonomical levels (303 eukaryotic and 4584 actinopterygian single copy orthologues) using

892 the BUSCO pipeline v2.0 (155, 156).

893

894 The maker annotation results were used to generate a database for JBrowse/Webapollo using the

895 script “maker2jbrowse” included with JBrowse (157, 158). Predicted protein and transcript sequences

896 were used to query the uniprot database, using blastp and blastn respectively, and the best hit

897 descriptions were transferred to the fasta headers with scripts bundled with Maker as described in

898 (150). The annotated genome is currently hosted on a WebApollo genome browser and Blast server at

899 the University of Gothenburg, Sweden at http://albiorix.bioenv.gu.se/.

900 Our analyses reveal that some degree of care is warranted regarding gene models. De novo

901 annotation without transcriptome data tends to be biased towards known and conserved genes,

902 homopolymer sequencing errors may cause annotation errors, and fish proteins have diverged faster

903 than mammalian homologs (159). For example, 25% of human genes cannot be identified in the

904 pufferfish (16). Even in the well-characterized zebrafish, targeted approaches have the potential to

905 reveal additional novel genes (160). We therefore encourage researchers to consider genome-wide

906 blast searches in addition to a consultation of round goby gene models, and hope that extensive RNA

907 sequencing data can be generated in the future to improve the predictions.

908

909 Sex determining regions

910

911 To investigate whether the round goby genome features large sex determining regions, we analyzed

912 own available RAD sequencing data. We prepared restriction site–associated DNA (RAD) (161)

913 libraries following the protocol used by (162, 163), which is largely based on (166). In short, we used

914 the Sbf1 enzyme on DNA extracted from 57 females, 56 males, and 5 juveniles caught in Basel,

915 Switzerland, and pooled 39-40 individuals per library for SR 100bp sequencing with Illumina. 45

916 females and 47 males retained sufficient numbers of reads (>150000) per sample after cleaning and

917 demultiplexing, were processed with the Stacks pipeline using the genome independent approach

918 (164), and were analyzed for sex-specific loci present exclusively in males or females. Considering a

919 genome size of ~1GB, the presence of 23 chromosomes (165), and a calling success of 21877 loci in

920 95 or 96 individuals (49220 loci in at least 40 individuals), we expected an average density of one

921 RAD locus every 45710 (20316) bp and an average number of 951 (2140) markers for an average

922 sized chromosome. The presence of a sex chromosome should thus be indicated by hundreds of sex-

923 specific RAD loci, while a contiguous sex determining region larger than 45000 bp would be indicated

924 by one or more sex specific RAD loci. Read numbers per locus for each sample were extracted from

925 the *.matches.tsv file output from Stacks and analyzed for sex-specific loci with standard R table

926 manipulations.

927

928 Vision

929

930 Opsin genes were extracted from the genome assembly using the Geneious software

931 (http://www.geneious.com) (167) by mapping the genomic scaffolds (Medium Sensitivity, 70% identity

932 threshold) against individual opsin exons of Nile tilapia (Oreochromis niloticus; GenBank Acc. no.:

933 MKQE00000000.1). This led to capturing of all scaffolds containing any visual opsin. The genes were

934 then annotated by mapping back of the single exons of tilapia against each scaffold separately (High

935 Sensitivity; 50% identity threshold) combined with the Live Annotate & Predict function as

936 implemented in Geneious, based on the Nile tilapia and mudskipper (25) opsin gene annotation. All

937 regions upstream and downstream from every opsin gene, as well as the intergenic regions were

938 separately tested for presence of any further opsin gene or its fragment (pseudogene). The annotated

939 genes were checked for the reading frame and the putative protein product was predicted.

940

941 We next performed phylogenetic analysis on the visual opsin genes (i.e. SWS1, SWS2, RH2, RH1 and

942 LWS opsins) across vertebrates, with focus on selected model species of teleost fishes. We further

943 specifically focused on the LWS genes from the fish species or lineages known to possess multiple

944 LWS copies, such as livebearers and pupfishes (Cyprinodontiformes) (168), zebrafish (Danio rerio)

945 (169), salmon (Salmo salar) (170), common carp (Cyprinus carpio) (170), cavefish (Astyanax

946 mexicanus) (171), Northern pike (Esox lucius) (170), labyrinth fishes (Anabas testudineus) (20), Asian

947 arowana (Scleropages formosus) (170) as well as other gobies, such as mudskippers (25) and reef

948 gobies (20). The opsin gene sequences from round goby and other fish species, including outgroup of

949 non-visual opsins (pinopsin, parietopsin, vertebrate-ancestral opsins and opn3 opsin;

950 Supplemental_Material_S2) were aligned using the MAFFT (172) plugin (v1.3.5) under the L-ins-i

951 algorithm as implemented in Geneious. Exon 5 (exon 6 in case of LWS) and part of exon 1 (or entire

952 exon 1 in case of LWS), which provided ambiguous alignment due to their higher variability, were

953 discarded. We estimated the model parameters by jModeltest 2.1.6 (173, 174), and subsequently used

954 the bayesian inference to calculate single-gene phylogeny using the MrBayes 3.2.6 (175) software as

955 implemented on the CIPRES Science gateway (176).

956

957 Olfaction

958

959 Olfactory receptor (OR) peptide sequences to be used as a reference were extracted from a publicly

960 available Oreochromis niloticus protein dataset (177). Those references were blasted (tblastn) against

961 the genomes of the round goby (Neogobius melanostomus), the blue-spotted mudskipper

962 (Boleophthalmus pectinirostris) (25), the giant mudskipper (Periophthalmodon magnuspinatus) (25)

963 and the three-spined stickleback (Gasterosteus aculeatus) (178), using an e-value threshold of 10e-50.

964 Only the hit with highest bit-score for each genomic position with more than one alignment was

965 employed in subsequent steps. Mapped hits belonging to contiguous positions of the protein

966 (maximum overlap of 15 aminoacids) and with a genomic distance smaller than 10kb were joined as

967 exons of the same CDS-gene model. Obtained sequences were translated to proteins using

968 TransDecoder (http://transdecoder.github.io), filtering all models that produce peptides smaller than

969 250 aminoacids. While many ORs are usually around 300 aminoacids long in total, 250 is close to the

970 average size of their main transmembrane domain, which is centrally located in the protein and more

971 suitable to interspecific alignment compared to N-terminal and C-terminal ends. We acknowledge that

972 this method might introduce a reduced proportion of recent pseudogenes that could lead to a small

973 overestimation of OR genes with coding capacity, although all species should be affected equivalently.

974

975 Next, an hmmscan (http://hmmer.org/) was produced against Pfam database to identify the domain

976 with highest score for each obtained protein sequence. We also filtered against false positive detection

977 using blast against confident OR and non-OR protein datasets. For phylogenetic analysis, sequences

978 (Supplemental_Material_S3) were aligned with MAFFT (https://mafft.cbrc.jp/alignment/server/) and a

979 Maximum Likelihood methodology was employed to build the tree using W-IQ-TREE software (179)

980 with standard parameters and Ultrafast bootstrap (180). Four adrenergic receptor sequences from

981 Oreochromis niloticus were used as an outgroup. Monophyletic groups formed by five or more genes

982 of the same species were considered as lineage-specific gene expansions. Because of the

983 phylogenetic proximity of the two mudskippers and the differences in their genome assembly statistics,

984 only B. pectinirostris was considered and P. magnuspinatus sequences were allowed to be included in

985 their lineage-specific expansion groups.

986

987 Detoxification

988

989 The Basic Local Alignment Search Tool (BLAST, v. 2.2.31) (181) was used to identify local alignments

990 between the round goby genome and a query including all annotated CYPs in humans and zebrafish

991 (vertebrate) and the most dissimilar invertebrate CYPs from Drosophila melanogaster (arthropod),

992 Caenorhabditis elegans (nematode) and Capitella teleta (annelid; Supplemental_Material_S4). Only

993 BLAST high scoring pairs with Expect values of 1.0 x 10-10 or smaller were considered significant.

994

995 The JBrowse genome viewer (v1.12.1) was used to manually annotate the significant regions of each

996 genome from the BLAST search, identifying start (ATG) and stop (TGA/TAA/TAG) codons, exon

997 number, and splice site signals (GT/AG) at intron-exon boundaries. The lengths of the potential CYPs

998 were identified and considered full length at ~500 amino acid residues long. Potential genes were

999 matched to the well-curated cytochrome P450 HMM in the Pfam protein family database (182) to

1000 confirm identity. The ScanProsite tool (183) was used to verify the presence of four largely conserved

1001 CYP motifs: the I-helix, K-helix, meander coil and heme loop. Each gene was classified as ‘complete’

1002 (proper length with start and stop codon, all motifs present, and match to the HMM) or ‘partial’

1003 (presence of at least the entire ~120 amino acid region that contains all motifs but clearly less than full

1004 length). Any potential CYP that was missing at least one of the motifs was considered a gene

1005 ‘fragment’ (Supplemental_Table_S9).

1006

1007 All of the ‘complete’ and ‘partial’ round goby CYPs (Supplemental_Table_S9) were included in further

1008 analyses. Clustal Omega (v1.2.4) (184) was used to generate a multiple sequence alignment of the

1009 round goby sequences and a variety of well-known vertebrate CYPs from humans, Danio rerio, Mus

1010 musculus, Xenopus laevis, Gallus gallus, and Rattus norvegicus (125 sequences in total;

1011 Supplemental_Material_S5). Mesquite (v3.10) (185) was utilized to trim the alignment, especially at

1012 the termini of the protein sequences where significant variation is typically observed, leaving only the

1013 portion of the alignment representative of the homology of the sequences. The final ‘masked’

1014 alignment (Supplemental_Material_S6) was used as input for the Randomized Axelerated Maximum

1015 Likelihood program (RAxML v8.2.10) (186). 100 bootstrap trees were generated with the rapid

1016 generation algorithm (-x) and a gamma distribution. The JTT substitution matrix with empirical

1017 frequencies was implemented in tree generation. The final maximum likelihood phylogenetic tree was

1018 visualized with Figtree (v1.4.3) (187) and rooted with the CYP51 family of enzymes.

1019

1020 Osmoregulation

1021

1022 Protein sequences for aquaporins, tight junction proteins, ion transporters, and enzymes in osmolyte

1023 production pathways were retrieved from the round goby genome by BLASTing well-characterized

1024 proteins from zebrafish, downloaded from Uniprot (March 2018), against the round goby gene

1025 models/proteins. Only round goby gene-models/proteins for which the predicted protein covered at

1026 least 70%, with a sequence identity of at least 40% and with E-value < 10-20 of the corresponding

1027 protein in zebrafish were used for the phylogenetic analyses. Well-established paralogues belonging

1028 to different subclasses of the respective protein family, based on either literature search or from initial

1029 phylogenetic analysis of that particular protein family, were used as additional query sequences to

1030 minimize the risk of missing relevant round goby sequences. Osmoregulatory genes from human and

1031 zebrafish were used for overall classification of clades in the respective protein family. Some

1032 modifications were made to the retrieved round goby sequences before analysis: i) For NHE ion

1033 transporters, a 780 aa long non-homologous N-terminus from one of the Neogobius sequences was

1034 removed before the phylogenetic analysis. ii) Some of the claudin genes were subjected to manual

1035 curation of Maker predicted proteins. The claudin genes in fish consist of several tandem arrays, which

1036 in some cases results in merging of 2-4 claudin genes by the Maker software. Claudins have a typical

1037 trans-membrane (TM) pattern with four distinct TM domains. All manually curated claudin genes from

1038 round goby were examined to have the expected four TM domains by TMHMM searches. Round goby

1039 protein sequences after manual curation are available in the supplement

1040 (Supplemental_Material_S7).

1041

1042 No myo-inositol phosphate synthase (MIPS) and sodium/inositol cotransporter (SMIT) proteins from

1043 zebrafish was found in Uniprot. To confirm that there are truly no MIPS and SMIT genes in zebrafish,

1044 the zebrafish genome at NCBI was also searched for homologies using blastp and tblastn using as

1045 query the MIPS and SMIT protein sequences from tilapia as query, and no hits were found. Thus, in

1046 the case of MIPS and SMIT, tilapia sequences were used for searching for round goby homologues.

1047 For the phylogenetic analyses, protein sequences from zebrafish (Danio rerio), three spine stickleback

1048 (Gasterosteus aculeatus), tilapia (Oreochromis niloticus), mudskipper (Boleophthalmus pectinirostris)

1049 and Homo sapiens (exception for human NKA-beta) were used in comparison to round goby, and

1050 were obtained from Uniprot (zebrafish, stickleback, tilapia, human) or RefSeq (mudskipper;

1051 Supplemental_Material_S7). Phylogenetic analyses of osmoregulatory proteins in round goby were

1052 performed using maximum likelihood with PhyML v3.0 with 100 bootstraps and using Gblocks to

1053 eliminate poorly aligned positions and highly divergent regions. PhyML analyses were performed at

1054 the Phylogeny.fr website (http://www.phylogeny.fr) using default settings.

1055

1056 Immune system

1057

1058 To perform an overall characterization of key genes related to the immune system, protein queries

1059 representing core components of innate and acquired immunity from several fish species as well as

1060 mammalian reference sequences were downloaded from UniProt and Ensembl. The protein queries

1061 were aligned prior to usage to ensure sequence homology. We also added previously extracted

1062 protein sequences from the Toll-like receptor family, reported by (85), and MHCI sequences reported

1063 by (80). All queries are listed in Supplemental_Table_S4. To enable comparative analyses between

1064 sequenced Gobiiformes, the genomes of Periophthalmodon schlosseri (GCA_000787095.1),

1065 Periophthalmus magnuspinatus (GCA_000787105.1), Scartelaos histophorus (GCA_000787155.1)

1066 and Boleophthalmus pectinirostris (GCA_000788275.1) were additionally downloaded from NCBI.

1067

1068 All protein queries were used in a tblastn (blast+ v. 2.6.0) towards the round goby genome assembly

1069 using default parameters and a e-value cutoff of 1e-10 (188). Some queries (caspase-1, TLRs, IL1

1070 and IL8) were also used in an identical tblastn towards the other Gobiiformes genomes. Genomic hit

1071 regions were extracted using BEDtools (v. 2.17.0) extending both up- and downstream as needed to

1072 obtain full length gene sequences (189). The extracted genomic regions were imported into MEGA7,

1073 the reading frame was adjusted for each exon and aligned as proteins to the corresponding translated

1074 coding sequence of queries using MUSCLE with default parameters. Intronic sequences were

1075 removed leaving an in-frame coding sequence (190, 191). All alignments were subjected to manual

1076 evaluation before subsequent analysis.

1077

1078 To generate phylogenetic trees, protein alignments were made and model tested using the ProtTest3

1079 server (http://darwin.uvigo.es/software/prottest_server.html) specifying BIC and no tree optimization

1080 (server has been disabled but ProtTest is available for download from GitHub) (192). All alignments

1081 reported the JTT model as best hit. Maximum likelihood trees were produced by using RAxML-

1082 PTHREADS (v 8.0.26), PROTCATJTT, rapid bootstrap and 500 bootstrap replicates (193). The final

1083 trees were imported into FigTree (http://tree.bio.ed.ac.uk/software/figtree/), and subsequently Adobe

1084 Illustrator, for presentation purposes.

1085

1086 In order to identify members of the large multigenic family of fish-specific NACHT and Leucine-Rich

1087 Repeats containing genes (NLRs; the fish-specific subset is also known as NLR-C) (87), an alignment

1088 of 368 zebrafish NLR-C proteins was obtained from (88). A combination of tblastn, HMMER3

1089 searches (194) and alignments with MAFFT v7.310 (195) was used to generate first an initial list of

1090 “candidate regions” potentially containing an NLR (Supplemental_Material_S8) and then an

1091 annotation of the characteristic domains in round goby NLR-C family members (see

1092 Supplemental_Material_S9) and Supplemental_Table_S8 for details), consisting of 25 PYRIN , 1 N-

1093 terminal CARD, 12 C-terminal CARD, 343 FISNA-NACHT and 178 B30.2 domains. Custom HMM

1094 models for major NLR exons (FISNA-NACHT, and PRY-SPRY/B30.2) were generated and utilized

1095 during this process (Supplementary Methods, Supplemental_Material_S10). The majority of

1096 identified FISNA-NACHT exons contained frameshifts or a large insertion, indicating either

1097 pseudogenization, acquisition of new introns, problems with the assembly, or a combination of the

1098 three (196). In any case, for the subsequent phylogenetic analysis, only the 61 clearly intact NLRs

1099 were used. These were aligned with NLRs from human, zebrafish and the mudskipper goby using

1100 MAFFT (Supplemental_Material_S9; Supplemental_Material_S11); Maximum Likelihood trees were

1101 produced with RAxML-PTHREADS, PROTCATJTT, rapid bootstrap and 500 bootstrap replicates

1102 (193). The final trees were imported into FigTree (http://tree.bio.ed.ac.uk/software/figtree/), and

1103 subsequently Adobe Illustrator. The alignments were inspected manually for presence of the

1104 conserved Walker A motifs and sequence logos for these were generated with WebLogo (197).

1105 Finally, we performed a survey of the PYD domains, Peptidase_C14 domains (Caspases) and CARD.

1106 All cases of a PYD domain followed by an adjacent CARD in the round goby (putative apoptosis-

1107 associated speck-like protein containing a CARD (ASC), also known as PYD-CARD or PYCARD)

1108 were identified from the HMMER3 dataset. The open reading frames containing these were translated,

1109 concatenated, and aligned with similarly structured proteins from human, mouse, lizard, frog and all

1110 the fish in Ensembl, and with PYD-CARDs identified from the other available goby assemblies

1111 (Supplemental_Material_S12). A phylogenetic tree was generated as described above. The

1112 annotation for NLR-C genes consists of predicted positions for all of the major conserved NLR-

1113 associated domains (PYD, CARD, FISNA-NACHT-helices, LRRs, B30.2; Supplemental_Table_S8).

1114

1115 Epigenetic regulators

1116

1117 We focused on two gene expression regulators which are conserved among all eukaryotes: the

1118 Polycomb Repressive Complex 2 (PRC2), which deposits repressive histone methylation marks, and

1119 the DNA methylases, which methylate cytosine in CpG contexts. The presence of both marks is

1120 commonly associated with a downregulation of gene expression. The protein sequences of zebrafish

1121 orthologues of PRC2 components RBBP2, EED, EZH1-2, and SUZ12 (50) and of DNA methylases

1122 DNMT1 and DNMT3 (198) were blasted against the round goby genome using default parameters of

1123 the Albiorix Blast server. The protein sequence of predicted proteins at the hit site was extracted

1124 manually in the round goby genome browser and aligned with mouse, human, and zebrafish protein

1125 sequences. When the first and/or last exon sequences as predicted in the round goby genome differed

1126 significantly from the mouse, human, and zebrafish sequences, we attempted confirmation by 3’ and

1127 5’RACE on RNA extracted from whole juvenile animals (see Supplemental_Material_S13 for primer

1128 sequences and PCR conditions). A putative CDS was combined from automated annotation and

1129 RACE results, and aligned to sequences extracted from a variety of fish taxa, shark, chicken, frog,

1130 lizard, and human (Supplemental_Material_S14). Given the high conservation of these proteins in

1131 eukaryotes, and the absence of major unexpected differences between round goby and other

1132 vertebrates, additional Gobiidae were not included in the analyses. In order to perform codon aware

1133 alignment MACSE (199) was used. The model and partitioning scheme used for each phylogenetic

1134 analysis was estimated using PartitionFinder2 (200) using PhyML (201) with corrected AIC scores

1135 (AICc) used for model selection. Phylogenetic analyses were performed using MrBayes 3.2.6 (175,

1136 202) with three independent runs for each gene. Analyses were run for 2,000,000 generations or until

1137 the standard deviation of split frequencies was below 0.01 up to a maximum of 20,000,000

1138 generations. In order to aid convergence in the EZH analyses the temperature parameter was set to

1139 0.05.

1140

1141 Transposable elements

1142

1143 A number of different applications were used for the repeat annotation of the genome. They are

1144 described in the repeat annotation report (Supplemental_Material_S15). In summary, in addition to

1145 the identification of repeats with RepeatModeler (as described above), we used TRF (203) to predict

1146 tandem repeats. RepeatMasker (153), a homology-based approach was used to produce a genome-

1147 wide overview of interspersed repeats. LTR Finder (204) and LTRharvest (205) in combination with

1148 LTRdigest (206), both de novo approaches, were used to predict LTRs.

1149

1150 Availability of data and materials

1151

1152 The Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession

1153 VHKM00000000. The version described in this paper is version VHKM01000000.

1154 Various Illumina reads are available under the accessions indicated in Table 1.

1155 Other datasets supporting the conclusions of this article are included within the article and its

1156 additional files.

1157

1158 Additional files

1159

1160 Supplemental Figure S1: .pdf; full opsin phylogenetic tree

1161 Supplemental Figure S2: .pdf; opsin tree constructed with individual exons

1162 Supplemental Figure S3: .pdf; full olfactory receptor phylogenetic tree

1163 Supplemental Figure S4: .pdf; claudin phylogenetic tree

1164 Supplemental Figure S5: .pdf; occludin phylogenetic tree

1165 Supplemental Figure S6: .pdf; sodium transporter phylogenetic trees

1166 Supplemental Figure S7: .pdf; myo-inositol production and accumulation gene phylogenetic trees

1167 Supplemental Figure S8: .pdf; TAP gene phylogenetic tree

1168 Supplemental Figure S9: .pdf; Ig locus schematic

1169 Supplemental Figure S10: .pdf; TLR phylogenetic tree of Gobiidae

1170 Supplemental Figure S11: .pdf; CRP / APCS phylogenetic tree

1171 Supplemental Figure S12: .pdf; SUZ12, EED, and RBBP4 phylogenetic trees

1172 Supplemental Material S1: .gz; annotation tracks for genome

1173 Supplemental Material S2: .txt; opsin sequences used for tree building

1174 Supplemental Material S3: .txt; olfactory receptor sequences used for tree building

1175 Supplemental Material S4: .txt; CYP sequences used as query

1176 Supplemental Material S5: .txt; CYP sequences used for tree building

1177 Supplemental Material S6: .phy; CYP alignment

1178 Supplemental Material S7: .doc; sequences for osmoregulatory proteins

1179 Supplemental Material S8: .fas; NLR candidate regions

1180 Supplemental Material S9: .doc; detailed methods for NLR annotation

1181 Supplemental Material S10: zip of .hmm; hmm models used to identify NLRs

1182 Supplemental Material S11: .fas; NLR sequences used for vertebrate tree building

1183 Supplemental Material S12: .fas; NLR sequences used for Gobiidae tree building

1184 Supplemental Material S13: .doc; detailed methods for RACE

1185 Supplemental Material S14: zip of .nex; dnmt1, dnmt3, eed, ezh, rbbp4, and suz12 alignments

1186 Supplemental Material S15: .txt; detailed methods for repeat annotation

1187 Supplemental Table S1: .xls; overview and results for repeat sequences analysed

1188 Supplemental Table S2: .xls; results for CYP genes (location, sequence, length)

1189 Supplemental Table S3: .doc; overview table of immune genes analysed

1190 Supplemental Table S4: .xls; immune gene sequences used as query

1191 Supplemental Table S5: .doc; results for MHCI

1192 Supplemental Table S6: .doc; results for MHCII

1193 Supplemental Table S7: .txt; results for other immune genes analysed

1194 Supplemental Table S8: .xls; results for NLRs

1195 Supplemental Table S9: .xls; results for CYPs

1196

1197 Funding

1198

1199 ZM was funded by the Czech Science Foundation (16-09784Y) and the Swiss National Science

1200 Foundation (PROMYS - 166550). DB was funded by CZ.02.2.69/0.0/0.0/16_027/0008495 -

1201 International Mobility of Researchers at Charles University. Genome sequencing was funded with a

1202 contribution of the Freiwillige Akademische Gesellschaft Basel to IAK. KP was funded by an

1203 Undergraduate Student Research Award and a Discovery grant RGPIN5767-16 (to JYW) from the

1204 Natural Sciences and Engineering Research Council of Canada. MT, TL and MAR were funded by the

1205 Center for Marine Evolutionary Biology. JS was funded by grants LE 546/9-1 and WI 3081/5-1 within

1206 the Deutsche Forschungsgemeinschaft (DFG) - funded Priority Programme SPP1819. MHS was

1207 funded by the Norwegian Research Council (grant numbers 199806/S40 and 222378/F20). AB was

1208 funded by the Swedish Research Council (VR; #2017-04559).

1209

1210 Acknowledgements

1211

1212 We are grateful to Prof. Patricia Burkhardt-Holm for her continuous support and encouragement. We

1213 thank Bernd Egger, Astrid Böhne, Philipp Hirsch and Patricia Burkhardt-Holm for critically reading the

1214 manuscript. We thank Fabio Cortesi for his insightful comments and the Center for Marine

1215 Evolutionary Biology for hosting a Blast server and a genome browser. Computational resources were

1216 provided by the CESNET LM2015042 and the CERIT Scientific Cloud LM2015085, provided under the

1217 programme "Projects of Large Research, Development, and Innovations Infrastructures". We thank

1218 Maria Leptin for her helpful comments and advice during annotation of the inflammasome

1219 components.

1220

1221 Author contributions

1222

1223 Sylke Winkler isolated DNA and generated PacBio reads, Martin Pippel and Siegfried Schloissnig

1224 assembled the genome sequence, and Tomas Larsson, Mats Tölpel and Magnus Alm Rosenblad

1225 performed automated annotation and provided the genome browser and Blast server.

1226

1227 Jean-Claude Walser provided transposable element analyses, Silvia Gutnik, Claire Peart, and Irene

1228 Adrian-Kalchhauser provided DNA methyltransferase and PRC2 analyses, Anders Blomberg provided

1229 osmoregulation analyses, Monica Hongroe Solbakken and Jaanus Suurväli provided immune gene

1230 analyses, Zuzana Musilova and Demian Burguera provided vision and olfaction analyses, Joanna

1231 Yvonne Wilson and Kirill Pankov provided CYP gene analyses, Nico Michiels investigated red

1232 fluorescence.

1233

1234 Irene Adrian-Kalchhauser initiated, designed, and supervised the project, acquired the necessary

1235 funding, coordinated annotation efforts, compiled the manuscript and handled the submission process.

1236

1237 Permissions

1238

1239 Fish used in this work were caught in accordance with permission 2-3-6-4-1 from the Cantonal Office

1240 for Environment and Energy, Basel Stadt.

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Danio rerio opn3 NM001111164 outgroup Oryzias latipes pinopsin XM011483381 Supplemental_Fig_S1 1 Anolis carolinensis pinopsin XM016997727 0.628 Oreochromis niloticus pinopsin XM005467442 Xenopus tropicalis parietopsin DQ284780 0.938 Periophthalmodon schlosseri LWS2 ex345 pseudogen VA opsins and pinopsin The round goby genome 1 Xenopus tropicalis parapinopsin NM 213665 0.959 Cyprinus carpio VAopsin AF233520 1 Danio rerio VA1 NM131586 coding 1 Danio rerio VA2 NM001110280 (outgroup) 0.653 Oryzias latipes VAopsin NM001136515 0.957 0.988 Salmo salar VAopsin SSAF001499 Gallus gallus pinopsin U15762 Callorhinchus milii LWS1 EF565165 1 Callorhinchus milii LWS2 EF565166 0.691 0.996 Gallus gallus LWS NM205440 Geotria australis LWS AY366491 1 0.657 Homo sapiens LWS NM020061 LWS (red) 1 Homo sapiens MWS NM000513 0.823 Neogobius melanostomus Contig 2403 LWS02 ex 1 Periophthalmus magnuspinnatus LWS2 1 Boleophthalmus pectinirostris LWS2 0.645 1 Scartelaos histophorus LWS 02 ex34 Lepisosteus oculatus LWS Scleropages formosus LWS 01 1 Astyanax mexicanus LWS 02 1 1 Astyanax mexicanus LWS 03 Astyanax mexicanus LWS 01 0.974 Cyprinus carpio LWS 01 pseudogen 0.990 0.999 Cyprinus carpio LWS 02 1 1 Danio rerio LWS A 0.906 Danio rerio LWS B 0.905 Pimephales promelas LWS A 1 Pimephales promelas LWS B Scleropages formosus LWS 05 ex12345 Clupea harengus LWS ex123 0.625 Xenopus laevis LWS NM001090645 Percopsis transmontana LWS 0.654 Salmo salar LWS 04 0.901 1 1 Esox lucius LWS C 1 Esox lucius LWS D 0.655 1 Esox lucius LWS A 0.998 Esox lucius LWS B 0.853 Salmo salar LWS 03 1 Salmo salar LWS 01 Salmo salar LWS 02 0.830 Neoniphon sammara LWS 1 Cetomimus sp LWS 1 Rondeletia loricata LWS Oreochromis niloticus AF247128 LWS Oryzias latipes LWS A 1 Oryzias latipes LWS B 0.638 Neogobius melanostomus Contig 623 LWS01 0.925 0.989 Boleophthalmus pectinirostris LWS1 1 Scartelaos histophorus LWS 01 Periophthalmodon schlosseri LWS 1 0.997 Periophthalmus magnuspinnatus LWS1 1 Anabas testudineus LWS 01 0.959 0.936 Anabas testudineus LWS 02 1 1 Helostoma temminckii LWS A 0.969 Helostoma temminckii LWS B Takifugu rubripes LWS 0.518 Gasterosteus aculeatus LWS 0.1 0.687 Perca fluviatilis LWS ex123 04 cDNA part 1 Perca fluviatilis LWS ex123 01 cDNA part 1 Perca fluviatilis LWS ex123 03 cDNA part 0.509Perca fluviatilis LWS ex123 02 cDNA part 1 Cyprinodon variegatus LWS A Cyprinodon variegatus LWS C 0.952 Cyprinodon variegatus LWS B 1 Cyprinodon variegatus LWS retrotransposed 1 Anableps anableps FJ711155 S180r 0.999 Poecilia latipinna JF823554 LWS R 0.897 Anableps anableps FJ711157 S180 gamma 1 0.682Anableps anableps FJ711154 S180 alpha 0.889 Anableps anableps FJ711158 S180 beta Poecilia latipinna JF823552 LWS 2 1 1 Poecilia latipinna JF823551 LWS 1 Poecilia latipinna JF823553 LWS 3 Geotria australis SWS1 AY366495 1 Xenopus laevis SWS1 NM001085652 Anolis carolinensis SWS1 AF134192 3 4 0.846 0.996 Gallus gallus SWS1 M90239 1 Homo sapiens SWS1 NM001708 SWS1 (UV) 0.809 Lepistosteus oculatus SWS1 A 1 Lepisosteus oculatus SWS1 B missing ex4 0.948 Scleropages formosus SWS1 Cyprinus carpio SWS1 1 1 Pimephales promelas SWS1 1 Danio rerio SWS1 AF109373 coding 1 Esox lucius SWS1 1 1 Salmo salar SWS1 01 1 Salmo salar SWS1 02 pseudo Gasterosteus aculeatus SWS1 1 Oreochromis niloticus SWS1 BAC 0.997 Oryzias latipes SWS1 0.986 Poecilia latipinna SWS1 KX768645 1 Anableps anableps FJ711153 0.527 Cyprinodon nevadensis SWS1 2 0.940 Cyprinodon nevadensis SWS1 1 Geotria australis SWS2 AY366492 Gallus gallus SWS2 NM205517 1 0.991 0.853 Anolis carolinensis SWS2 AF133907 coding Xenopus laevis SWS2 green BC080123 1 Lepisosteus oculatus SWS2 SWS2 (violet/blue) 0.999 Scleropages formosus SWS2 0.593 Salmo salar SWS2 NM001123706 0.609 1 Astyanax mexicanus SWS2 1 Astyanax fasciatus SWS2 AH007939 1 Cyprinus carpio SWS2 gen01 1 Cyprinus carpio SWS2 gen02 1 Pimephales promelas SWS2 0.999 Danio rerio SWS2 AF109372 coding Neogobius melanostomus Contig 623 SWS2A second cDNA 1 0.976 Periophthalmus magnuspinnatus SWS2B 1 Boleophthalmus pectinirostris SWS2B ex1234 0.654 1 Scartelaos histophorus SWS2B Takifugu rubripes SWS2B AY598947 1 1 Anabas testudineus SWS2B KP004284 0.972 Helostoma temminckii SWS2B KP004249 Pseudochromis fuscus SWS2B coding 0.865 Oreochromis niloticus AF247120 SWS2B 1 0.951 Oryzias latipes SWS2B cutFromGenome 1 Cyprinodon variegatus SWS2B 1 Anableps anableps SWS2B HM627004 0.999 Poecilia reticulata SWS2B Oryzias latipes AB223056 SWS2A Poecilia reticulata SWS2Aalpha 1 1 0.734 Anableps anableps SWS2A FJ711152 0.691 Cyprinodon variegatus SWS2Aalpha 1 Oreochromis niloticus AF247116 SWS2A 1 Anabas testudineus SWS2Aalpha KP004284 0.803 Helostoma temminckii SWS2Aalpha KP004249 Pseudochromis fuscus SWS2Aalpha complete coding 1 Pseudochromis fuscus SWS2Abeta coding 1 Gasterosteus aculeatus BT027452 SWS2A 1 Neogobius melanostomus Contig 623 SWS2B first cDNA 1 Boleophthalmus pectinirostris SWS2Abeta 1 Scartelaos histophorus SWS2Abeta 0.661 Periophthalmus magnuspinnatus SWS2Abeta Geotria australis RHA AY366493 0.590 Gallus gallus RH1 NM 001030606 0.999 Homo sapiens RH1 NM000539 1 1 Anolis carolinensis NM 001291387 0.650 Xenopus laevis RH1 NM001087048 Callorhinchus milii RH1 EF565167 RH1 (dim vision) 0.993 Lepisosteus oculatus RH1 Scleropages formosa RH1 0.523 Astyanax mexicanus RH1 01 1 Pimephales promelas RH1 02 1 0.998 1 Danio rerio RH1 02 0.677 Cyprinus carpio RH1 01 like pseud Cyprinus carpio RH1 02 like 0.960 Astyanax mexicanus RH1 02 1 Pimephales promelas RH1 01 0.665 Danio rerio RH1 01 1 1 Cyprinus carpio RH1 03 Cyprinus carpio RH1 04 1 0.998 Salmo salar RH1a pseudo 1 1 Esox lucius RH1 XM010902101 Salmo salar RH1c Neogobius melanostomus Contig 305 RH1 0.998 1 0.940 Boleophthalmus pectinirostris RH1 1 Scartelaos histophorus RH1 1 Periophthalmodon schlosseri RH1 1 Periophthalmus magnuspinnatus RH1 Gasterosteus aculeatus RH1 0.647 Takifugu rubripes RH1 0.998 1 Anabas testudineus RH1 Helostoma temminckii RH1 1 Oryzias latipes RH1 0.756 1 Cyprinodon variegatus RH1 0.802 Anableps anableps FJ711156 Poecilia latipinna RH1 XM015047258 Geotria australis RHB AY366494 1 Anolis carolinensis RH2 AH007735 coding 0.986 Gallus gallus RH2 green coding M92038 0.885 Lepisosteus oculatus RH2 1 Callorhinchus milii RH2 EF565168 RH2 (green) Astyanax mexicanus RH2 1 Danio rerio RH2 gen01 0.799 Danio rerio RH2 gen02 0.999 Pimephales promelas RH2 gen01 0.999 1 Danio rerio RH2 gen03 Danio rerio RH2 gen04 1 1 Pimephales promelas RH2 gen02 1 Pimephales promelas RH2 gen03 ex2345 0.997 Cyprinus carpio RH2 gen01 1 0.605Cyprinus carpio RH2 gen03 transcriptome Cyprinus carpio RH2 gen02 Salmo salar RH2 gen01 0.832 1 Salmo salar RH2 gen04 1 1 Salmo salar RH2 gen02 Salmo salar RH2 gen03 1 1 Esox lucius RH2 gen01 1 Esox lucius RH2 gen02 1 Esox lucius RH2 gen03 Esox lucius RH2 gen04 0.923 Takifugu rubripes RH2 gen01 ex2345 Oreochromis niloticus RH2B BAC 1 0.995 Oryzias latipes RH2 gen01 1 1 Cyprinodon variegatus RH2 gen01 0.991 Poecilia reticulata RH2 gen01 1 Boleophthalmus pectinirostris RH2 A 0.608 Periophthalmus magnuspinnatus RH2 gen01 0.667 Periophthalmodon schlosseri RH2 ex45 pse 0.999 Periophthalmodon schlosseri RH2 0.791 Periophthalmus magnuspinnatus RH2 ex45 gen03 pse Boleophthalmus pectinirostris RH2 B ex1345 1 Gasterosteus aculeatus RH2 gen01 1 Gasterosteus aculeatus RH2 gen02 0.994 Takifugu rubripes RH2 gen02 0.999 Anabas testudineus RH2 gen01 1 1 Helostoma temminckii RH2 02 ex123 0.914 Anabas testudineus RH2 02 ex123 1 Helostoma temminckii RH2 01 ex123 1 Boleophthalmus pectinirostris RH2 C Periophthalmus magnuspinnatus RH2 gen02 1 Neogobius melanostomus Contig 1118 RH2 1 1 Neogobius melanostomus Contig 4222 RH2 2 0.769 1 Oreochromis niloticus RH2Aalpha BAC Oreochromis niloticus RH2Abeta BAC 0.994 1 Oryzias latipes RH2 gen02 Oryzias latipes RH2 gen03 0.779 Poecilia reticulata RH2 gen02 1 1 Cyprinodon variegatus RH2 gen02 pse Cyprinodon variegatus RH2 gen03

Phylogenetic tree of vertebrate opsin protein sequences. Maximum-likelihood phylogenetic tree with VA opsins and pinopbsins as outgroup. Round goby is indicated in orange. Non-teleost species and the outgroup (VA opsins and pinopsins) are indicated in grey. bioRxiv preprint doi: https://doi.org/10.1101/708974; this version posted August 6, 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.

Xenopus tropicalis parietopsin DQ284780 Oryzias latipes pinopsin XM011483381 1 Anolis carolinensis pinopsin XM016997727 Supplemental_Fig_S2 0.959 Oreochromis niloticus pinopsin XM005467442 Xenopus tropicalis parapinopsin NM 213665 VA opsins and pinopsin Cyprinus carpio VAopsin AF233520 1 Danio rerio VA1 NM131586 coding The round goby genome 1 Danio rerio VA2 NM001110280 0.53 Oryzias latipes VAopsin NM001136515 (outgroup) 0.998 Salmo salar VAopsin SSAF001499 Gallus gallus pinopsin U15762 Callorhinchus milii LWS1 EF565165 1 Callorhinchus milii LWS2 EF565166 0.687 Gallus gallus LWS NM205440 0.98 Geotria australis LWS AY366491 Homo sapiens LWS NM020061 1 1 Homo sapiens MWS NM000513 LWS (red) Boleophthalmus pectinirostris LWS2 ex2 Boleophthalmus pectinirostris LWS2 ex5 1 Neogobius melanostomus Contig 2403 LWS02 ex2 0.921 Neogobius melanostomus Contig 2403 LWS02 ex3 0.60Neogobius9 melanostomus Contig 2403 LWS02 ex5 Periophthalmus magnuspinnatus LWS2 ex2 Periophthalmus magnuspinnatus LWS2 ex5 Boleophthalmus pectinirostris LWS2 ex3 0.5Per4 iophthalmus magnuspinnatus LWS2 ex3 Lepisosteus oculatus LWS Scleropages formosus LWS 05 ex12345 Clupea harengus LWS ex123 Xenopus laevis LWS NM001090645 0.621 Scleropages formosus LWS 01 0.576 1 Astyanax mexicanus LWS 02 0.97 Astyanax mexicanus LWS 03 Neogobius melanostomus Contig 2403 LWS02 ex4 0.983 0.993 0.599 Boleophthalmus pectinirostris LWS2 ex4 0.987 Periophthalmus magnuspinnatus LWS2 ex4 Astyanax mexicanus LWS 01 Cyprinus carpio LWS 01 pseudogen 0.941 1 Cyprinus carpio LWS 02 1 Danio rerio LWS A 1 Danio rerio LWS B 0.906Pimephales promelas LWS A 1 Pimephales promelas LWS B Percopsis transmontana LWS Salmo salar LWS 04 0.686 1 Esox lucius LWS C 1 Esox lucius LWS D 0.736 Esox lucius LWS A 0.99Esox4 lucius LWS B 0.73Salmo5 salar LWS 03 0.88Salmo2 salar LWS 01 1Salmo salar LWS 02 0.602 Neoniphon sammara LWS 1 Cetomimus sp LWS 1 Rondeletia loricata LWS Oreochromis niloticus AF247128 LWS Oryzias latipes LWS A 1Oryzias latipes LWS B 0.920.518 7 Neogobius melanostomus Contig 623 LWS01 1 1 Boleophthalmus pectinirostris LWS1 1 Scartelaos histophorus LWS 01 0.99Periophthalmodon9 schlosseri LWS 0.999 Periophthalmus magnuspinnatus LWS1 0.938 Takifugu rubripes LWS 1Anabas testudineus LWS 01 0.950.994 7Anabas testudineus LWS 02 1 Helostoma temminckii LWS A 0.564 Helostoma temminckii LWS B Gasterosteus aculeatus LWS 0.61 perca LWS ex123 04 cDNA part 1 perca LWS ex123 01 cDNA part 1perca LWS ex123 03 cDNA part 0.83perca5 LWS ex123 02 cDNA part Cyprinodon variegatus LWS A 1Cyprinodon variegatus LWS C 0.94C7yprinodon variegatus LWS B 1 Cyprinodon variegatus LWS retrotransposed 1 Anableps anableps FJ711155 S180r 0.999Poecilia latipinna JF823554 LWS R 0.95Anableps5 anableps FJ711157 S180 gamma 1 0.70Anableps6 anableps FJ711154 S180 alpha 0.84Anableps2 anableps FJ711158 S180 beta Poecilia latipinna JF823552 LWS 2 1 Poecilia latipinna JF823551 LWS 1 1Poecilia latipinna JF823553 LWS 3 Geotria australis SWS1 AY366495 Xenopus laevis SWS1 NM001085652 1 Anolis carolinensis SWS1 AF134192 3 4 0.778 0.998 Gallus gallus SWS1 M90239 SWS1 (UV) 1 Homo sapiens SWS1 NM001708 0.753 Lepisosteus oculatus SWS1 A 1 Lepisosteus oculatus SWS1 B missing ex4 0.897 Scleropages formosus SWS1 Cyprinus carpio SWS1 1 1 Pimephales promelas SWS1 1 Danio rerio SWS1 AF109373 coding 0.999 Esox lucius SWS1 1 1 Salmo salar SWS1 01 1 Salmo salar SWS1 02 pseudo Gasterosteus aculeatus SWS1 1 Oreochromis niloticus SWS1 BAC 0.999 Oryzias latipes SWS1 0.993 Anableps anableps FJ711153 0.652 Poecilia latipinna SWS1 KX768645 1 Cyprinodon nevadensis SWS1 2 0.892 Cyprinodon nevadensis SWS1 1 Geotria australis SWS2 AY366492 Gallus gallus SWS2 NM205517 1 0.993 Anolis carolinensis SWS2 AF133907 coding 0.852 Xenopus laevis SWS2 green BC080123 1 Lepisosteus oculatus SWS2 SWS2 (violet/blue) 1 Scleropages formosus SWS2 0.932 Salmo salar SWS2 NM001123706 Astyanax mexicanus SWS2 1 1Astyanax fasciatus SWS2 AH007939 Cyprinus carpio SWS2 gen01 1 1Cyprinus carpio SWS2 gen02 1 Pimephales promelas SWS2 1 Danio rerio SWS2 AF109372 coding Neogobius melanostomus Contig 623 SWS2A second cDNA 1 Periophthalmus magnuspinnatus SWS2B 0.916 1 Boleophthalmus pectinirostris SWS2B ex1234 0.507 1 Scartelaos histophorus SWS2B Takifugu rubripes SWS2B AY598947 1 Anabas testudineus SWS2B KP004284 1 Helostoma temminckii SWS2B KP004249 0.983 Pseudochromis fuscus SWS2B coding 0.823 Oreochromis niloticus AF247120 SWS2B 1 0.937 Oryzias latipes SWS2B cutFromGenome 1 Cyprinodon variegatus SWS2B 1 Anableps anableps SWS2B HM627004 0.99Poecilia6 reticulata SWS2B Oryzias latipes AB223056 SWS2A Poecilia reticulata SWS2Aalpha 1 1 Anableps anableps SWS2A FJ711152 0.674Cyprinodon variegatus SWS2Aalpha 0.749 1 Oreochromis niloticus AF247116 SWS2A Anabas testudineus SWS2Aalpha KP004284 0.7331 Helostoma temminckii SWS2Aalpha KP004249 Pseudochromis fuscus SWS2Aalpha complete coding 1 Pseudochromis fuscus SWS2Abeta coding 0.999 Gasterosteus aculeatus BT027452 SWS2A 1 Neogobius melanostomus Contig 623 SWS2B first cDNA 1 Boleophthalmus pectinirostris SWS2Abeta 1 Scartelaos histophorus SWS2Abeta 0.50Perioph7 thalmus magnuspinnatus SWS2Abeta Geotria australis RHA AY366493 0.998 Gallus gallus RH1 NM 001030606 Homo sapiens RH1 NM000539 1 Anolis carolinensis NM 001291387 1 Xenopus laevis RH1 NM001087048 RH1 (dim vision) 0.817 Callorhinchus milii RH1 EF565167 Lepisosteus oculatus RH1 0.998 Scleropages formosa RH1 0.9 Astyanax mexicanus RH1 01 1 Pimephales promelas RH1 02 0.995 1 Danio rerio RH1 02 1 Cyprinus carpio RH1 01 like pseud 0.86Cyprinus7 carpio RH1 02 like 0.994 Astyanax mexicanus RH1 02 0.734 Pimephales promelas RH1 01 1 Danio rerio RH1 01 1 Cyprinus carpio RH1 03 1Cyprinus carpio RH1 04 1 Salmo salar RH1a pseudo 1 1 Esox lucius RH1 XM010902101 1 Salmo salar RH1c 1 Gasterosteus aculeatus RH1 Neogobius melanostomus Contig 305 RH1 1 Boleophthalmus pectinirostris RH1 1 0.91Scartelaos1 histophorus RH1 1 Periophthalmodon schlosseri RH1 0.673 1 Periophthalmus magnuspinnatus RH1 Takifugu rubripes RH1 Oryzias latipes RH1 0.9990.549 1 Anabas testudineus RH1 1 Helostoma temminckii RH1 Cyprinodon variegatus RH1 1 Anableps anableps FJ711156 0.828Poecilia latipinna RH1 XM015047258 Geotria australis RHB AY366494 1 Anolis carolinensis RH2 AH007735 coding 0.988 Gallus gallus RH2 green coding M92038 Lepisosteus oculatus RH2 0.794 RH2 (green) 1 Callorhinchus milii RH2 EF565168 Astyanax mexicanus RH2 Danio rerio RH2 gen01 1 0.805 Danio rerio RH2 gen02 0.997 Pimephales promelas RH2 gen01 1 Danio rerio RH2 gen03 1 Danio rerio RH2 gen04 1 1 Pimephales promelas RH2 gen02 1 Pimephales promelas RH2 gen03 ex2345 0.995Cyprinus carpio RH2 gen02 1 Cyprinus carpio RH2 gen01 0.99Cyprinus5 carpio RH2 gen03 transcriptome Salmo salar RH2 gen01 0.897 1 Salmo salar RH2 gen04 Salmo salar RH2 gen02 1 1 Salmo salar RH2 gen03 1 Esox lucius RH2 gen01 0.998Esox lucius RH2 gen02 1 Esox lucius RH2 gen03 1 Esox lucius RH2 gen04 Periophthalmodon schlosseri RH2 1 Periophthalmus magnuspinnatus RH2 ex45 gen03 pse 1 0.75Boleophthalmus4 pectinirostris RH2 B ex1345 0.995 Boleophthalmus pectinirostris RH2 A 1 0.71Per9 iophthalmus magnuspinnatus RH2 gen01 0.68 Periophthalmodon schlosseri RH2 ex45 pse Takifugu rubripes RH2 gen01 ex2345 0.96 Oreochromis niloticus RH2B BAC 0.935 Oryzias latipes RH2 gen01 1 1 Cyprinodon variegatus RH2 gen01 1 Poecilia reticulata RH2 gen01 Gasterosteus aculeatus RH2 gen01 1Gasterosteus aculeatus RH2 gen02 0.995 Takifugu rubripes RH2 gen02 0.999Anabas testudineus RH2 gen01 1 Helostoma temminckii RH2 02 ex123 1 0.84Anabas7 testudineus RH2 02 ex123 1 Helostoma temminckii RH2 01 ex123 Boleophthalmus pectinirostris RH2 C 1 Periophthalmus magnuspinnatus RH2 gen02 1 Neogobius melanostomus Contig 1118 RH2 1 1 Neogobius melanostomus Contig 4222 RH2 2 0.538 Oreochromis niloticus RH2Aalpha BAC 1 Oreochromis niloticus RH2Abeta BAC 0.999 Oryzias latipes RH2 gen02 1 Oryzias latipes RH2 gen03 0.2 0.955 Poecilia reticulata RH2 gen02 1 Cyprinodon variegatus RH2 gen02 pse 1 Cyprinodon variegatus RH2 gen03

Phylogenetic tree of vertebrate opsin protein sequences, using single exons. Maximum-likelihood phylogenetic tree with VA opsins and pinopbsins as outgroup. Round goby is indicated in orange. Non-teleost species and the outgroup (VA opsins and pinopsins) are indicated in grey. bioRxiv preprint doi: https://doi.org/10.1101/708974; this version posted August 6, 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.

ADRENERGIC3.Oreochromis niloticus (Nile tilapia) ADRENERGIC5.Oreochromis niloticus (Nile tilapia) ADRENERGIC1.Oreochromis niloticus (Nile tilapia) ADRENERGIC2.Oreochromis niloticus (Nile tilapia) XP_003445365.1-LOC_360.1.OR.7tm_4.exons1.NW_018345971.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003445365.1-LOC_200.1.OR.7tm_4.exons1.KN461151.1.Periophthalmus magnuspinnatus (mudskipper) XP_013126850.1-LOC_341.1.OR.7tm_4.exons1.NW_018353850.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_013126850.1-LOC_360.1.OR.7tm_4.exons1.Contig_363.Neogobius melanostomus (round goby) Supplemental_Fig_S3 XP_003445438.1-LOC_344.1.OR.7tm_4.exons1.NW_018353850.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_013126850.1-LOC_173.1.OR.7tm_4.exons1.KN465618.1.Periophthalmus magnuspinnatus (mudskipper) XP_013126850.1-LOC_353.1.OR.7tm_4.exons1.Contig_363.Neogobius melanostomus (round goby) XP_003445438.1-LOC_358.1.OR.7tm_4.exons1.Contig_363.Neogobius melanostomus (round goby) XP_013126850.1-LOC_345.1.OR.7tm_4.exons1.NW_018353850.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_013126850.1-LOC_174.1.OR.7tm_4.exons1.KN465618.1.Periophthalmus magnuspinnatus (mudskipper) XP_013126850.1-LOC_347.1.OR.7tm_4.exons1.NW_018353850.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003445438.1-LOC_159.1.OR.7tm_4.exons1.KN460619.1.Periophthalmus magnuspinnatus (mudskipper) XP_003445438.1-LOC_148.1.OR.7tm_4.exons1.JACL01048658.1.Periophthalmus magnuspinnatus (mudskipper) XP_003445435.1.Oreochromis niloticus (Nile tilapia) XP_013126849.2.Oreochromis niloticus (Nile tilapia) XP_019207996.1.Oreochromis niloticus (Nile tilapia) The round goby genome XP_003445437.1.Oreochromis niloticus (Nile tilapia) XP_003445438.1.Oreochromis niloticus (Nile tilapia) XP_019207990.1.Oreochromis niloticus (Nile tilapia) XP_019207991.1.Oreochromis niloticus (Nile tilapia) XP_019207992.1.Oreochromis niloticus (Nile tilapia) XP_019207993.1.Oreochromis niloticus (Nile tilapia) XP_013126850.1.Oreochromis niloticus (Nile tilapia) XP_019207995.1.Oreochromis niloticus (Nile tilapia) XP_019207994.1.Oreochromis niloticus (Nile tilapia) XP_003445438.1-LOC_181.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_013126850.1-LOC_267.2.OR.7tm_4.exons1.NW_018358927.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_013126850.1-LOC_352.1.OR.7tm_4.exons1.Contig_3474.Neogobius melanostomus (round goby) XP_013126851.2-LOC_342.1.OR.7tm_4.exons1.NW_018353850.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_013126851.2-LOC_211.1.OR.7tm_4.exons1.KN462114.1.Periophthalmus magnuspinnatus (mudskipper) XP_003445445.1-LOC_343.1.OR.7tm_4.exons1.NW_018353850.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003445445.1-LOC_212.1.OR.7tm_4.exons1.KN462114.1.Periophthalmus magnuspinnatus (mudskipper) XP_013126851.2-LOC_359.1.OR.7tm_4.exons1.Contig_363.Neogobius melanostomus (round goby) XP_003445445.1-LOC_346.1.OR.7tm_4.exons1.NW_018353850.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_013126853.1-LOC_202.1.OR.7tm_4.exons1.JACL01048658.1.Periophthalmus magnuspinnatus (mudskipper) XP_003445365.1.Oreochromis niloticus (Nile tilapia) XP_019208363.1.Oreochromis niloticus (Nile tilapia) XP_003445366.1.Oreochromis niloticus (Nile tilapia) XP_013126851.2.Oreochromis niloticus (Nile tilapia) XP_013126852.2.Oreochromis niloticus (Nile tilapia) XP_003445443.2.Oreochromis niloticus (Nile tilapia) XP_013126853.1.Oreochromis niloticus (Nile tilapia) XP_005452049.2.Oreochromis niloticus (Nile tilapia) XP_003445445.1.Oreochromis niloticus (Nile tilapia) XP_013126851.2-LOC_180.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_013126851.2-LOC_196.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_013126852.2-LOC_194.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_013126851.2-LOC_182.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_013126851.2-LOC_184.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_013126851.2-LOC_191.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_013126851.2-LOC_188.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_013126851.2-LOC_193.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_013126851.2-LOC_192.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_013126851.2-LOC_195.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_013126851.2-LOC_197.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_003456642.1.Oreochromis niloticus (Nile tilapia) XP_003443339.2.Oreochromis niloticus (Nile tilapia) XP_019208022.1.Oreochromis niloticus (Nile tilapia) XP_013125661.1.Oreochromis niloticus (Nile tilapia) XP_019208023.1.Oreochromis niloticus (Nile tilapia) XP_019208026.1.Oreochromis niloticus (Nile tilapia) XP_019208024.1.Oreochromis niloticus (Nile tilapia) XP_019208028.1.Oreochromis niloticus (Nile tilapia) XP_019208029.1.Oreochromis niloticus (Nile tilapia) XP_019208025.1.Oreochromis niloticus (Nile tilapia) XP_019208030.1.Oreochromis niloticus (Nile tilapia) XP_013125661.1-LOC_307.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_013125661.1-LOC_308.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_013125661.1-LOC_309.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_003443339.2-LOC_312.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_003443339.2-LOC_317.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_003443339.2-LOC_325.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_003443339.2-LOC_319.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_003443339.2-LOC_320.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_013125661.1-LOC_311.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_019207989.1.Oreochromis niloticus (Nile tilapia) XP_013125660.1-LOC_337.1.OR.7tm_4.exons1.NW_018353850.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_013125660.1-LOC_208.1.OR.7tm_4.exons1.KN462114.1.Periophthalmus magnuspinnatus (mudskipper) XP_013125660.1-LOC_362.1.OR.7tm_4.exons2.Contig_363.Neogobius melanostomus (round goby) XP_003460622.1-LOC_340.1.OR.7tm_4.exons1.NW_018353850.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003460622.1-LOC_210.1.OR.7tm_4.exons1.KN462114.1.Periophthalmus magnuspinnatus (mudskipper) XP_013125660.1-LOC_361.1.OR.7tm_4.exons1.Contig_363.Neogobius melanostomus (round goby) XP_003460622.1-LOC_339.1.OR.7tm_4.exons1.NW_018353850.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003460622.1-LOC_209.1.OR.7tm_4.exons1.KN462114.1.Periophthalmus magnuspinnatus (mudskipper) XP_003460622.1.Oreochromis niloticus (Nile tilapia) XP_019208295.1.Oreochromis niloticus (Nile tilapia) XP_019208286.1.Oreochromis niloticus (Nile tilapia) XP_013125660.1-LOC_178.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_013125660.1.Oreochromis niloticus (Nile tilapia) XP_013125660.1-LOC_306.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_013125660.1-LOC_310.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_013125660.1-LOC_318.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_013125660.1-LOC_400.1.OR.7tm_4.exons1.NW_018355507.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_013125660.1-LOC_224.1.OR.7tm_4.exons1.JACL01048922.1.Periophthalmus magnuspinnatus (mudskipper) XP_013125660.1-LOC_401.1.OR.7tm_4.exons1.NW_018355507.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_013125660.1-LOC_404.1.OR.7tm_4.exons1.NW_018355507.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_013125660.1-LOC_169.1.OR.7tm_4.exons1.KN464856.1.Periophthalmus magnuspinnatus (mudskipper) XP_013125660.1-LOC_170.1.OR.7tm_4.exons1.KN464856.1.Periophthalmus magnuspinnatus (mudskipper) XP_013125660.1-LOC_250.1.OR.7tm_4.exons1.KN463219.1.Periophthalmus magnuspinnatus (mudskipper) XP_013125660.1-LOC_397.1.OR.7tm_4.exons1.Contig_3994.Neogobius melanostomus (round goby) XP_013125660.1-LOC_399.1.OR.7tm_4.exons1.Contig_3994.Neogobius melanostomus (round goby) XP_013125660.1-LOC_398.1.OR.7tm_4.exons1.Contig_3994.Neogobius melanostomus (round goby) XP_013125660.1-LOC_401.1.OR.7tm_4.exons1.Contig_3994.Neogobius melanostomus (round goby) XP_013125660.1-LOC_400.1.OR.7tm_4.exons1.Contig_3994.Neogobius melanostomus (round goby) XP_013125660.1-LOC_402.1.OR.7tm_4.exons1.NW_018355507.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_013125660.1-LOC_406.1.OR.7tm_4.exons1.NW_018355507.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_013125660.1-LOC_251.1.OR.7tm_4.exons1.KN463219.1.Periophthalmus magnuspinnatus (mudskipper) XP_013125660.1-LOC_405.1.OR.7tm_4.exons1.NW_018355507.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_013125660.1-LOC_252.1.OR.7tm_4.exons1.KN463219.1.Periophthalmus magnuspinnatus (mudskipper) XP_013125660.1-LOC_407.1.OR.7tm_4.exons1.NW_018355507.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_013125660.1-LOC_408.1.OR.7tm_4.exons1.NW_018355507.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_005462779.1-LOC_371.2.OR.7tm_4.exons1.NW_018345971.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_005462779.1-LOC_313.1.OR.7tm_4.exons1.NW_018360113.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_005462779.1-LOC_216.1.OR.7tm_4.exons1.KN462165.1.Periophthalmus magnuspinnatus (mudskipper) XP_005462779.1-LOC_312.1.OR.7tm_4.exons1.NW_018360113.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_005462779.1-LOC_347.1.OR.7tm_4.exons1.Contig_3355.Neogobius melanostomus (round goby) XP_005462779.1-LOC_349.1.OR.7tm_4.exons1.Contig_3355.Neogobius melanostomus (round goby) XP_005462779.1-LOC_223.1.OR.7tm_4.exons1.KN462546.1.Periophthalmus magnuspinnatus (mudskipper) XP_005462779.1-LOC_225.1.OR.7tm_4.exons1.KN462546.1.Periophthalmus magnuspinnatus (mudskipper) XP_005462779.1-LOC_252.1.OR.7tm_4.exons1.chrI.Gasterosteus aculeatus (three-spined stickleback) XP_005462779.1-LOC_262.1.OR.7tm_4.exons1.chrI.Gasterosteus aculeatus (three-spined stickleback) XP_003457863.1.Oreochromis niloticus (Nile tilapia) XP_005462779.1.Oreochromis niloticus (Nile tilapia) XP_003457893.1.Oreochromis niloticus (Nile tilapia) XP_003457891.3.Oreochromis niloticus (Nile tilapia) XP_003457892.2.Oreochromis niloticus (Nile tilapia) XP_003460046.2-LOC_252.1.OR.7tm_4.exons1.NW_018357150.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003457890.1-LOC_240.1.OR.7tm_4.exons1.KN463138.1.Periophthalmus magnuspinnatus (mudskipper) XP_003460046.2-LOC_340.1.OR.7tm_4.exons1.Contig_3355.Neogobius melanostomus (round goby) XP_003460044.3.Oreochromis niloticus (Nile tilapia) XP_003457889.1.Oreochromis niloticus (Nile tilapia) XP_003457890.1.Oreochromis niloticus (Nile tilapia) XP_003460045.2.Oreochromis niloticus (Nile tilapia) XP_003460046.2.Oreochromis niloticus (Nile tilapia) XP_003460047.2.Oreochromis niloticus (Nile tilapia) XP_003457854.2.Oreochromis niloticus (Nile tilapia) XP_019223090.1.Oreochromis niloticus (Nile tilapia) XP_003460049.3.Oreochromis niloticus (Nile tilapia) XP_019222742.1.Oreochromis niloticus (Nile tilapia) XP_003457890.1-LOC_315.1.OR.7tm_4.exons1.chrI.Gasterosteus aculeatus (three-spined stickleback) XP_003460049.3-LOC_282.1.OR.7tm_4.exons1.chrI.Gasterosteus aculeatus (three-spined stickleback) XP_013121252.1.Oreochromis niloticus (Nile tilapia) XP_013121252.1-LOC_167.1.OR.7tm_4.exons1.chrI.Gasterosteus aculeatus (three-spined stickleback) XP_019222741.1.Oreochromis niloticus (Nile tilapia) XP_003457866.1.Oreochromis niloticus (Nile tilapia) XP_003457866.1-LOC_241.1.OR.7tm_4.exons1.chrI.Gasterosteus aculeatus (three-spined stickleback) XP_003457866.1-LOC_293.1.OR.7tm_4.exons1.chrI.Gasterosteus aculeatus (three-spined stickleback) XP_003457866.1-LOC_304.1.OR.7tm_4.exons1.chrI.Gasterosteus aculeatus (three-spined stickleback) XP_003457866.1-LOC_271.1.OR.7tm_4.exons1.chrI.Gasterosteus aculeatus (three-spined stickleback) XP_003457887.3.Oreochromis niloticus (Nile tilapia) XP_005462815.2.Oreochromis niloticus (Nile tilapia) XP_003460046.2-LOC_223.1.OR.7tm_4.exons1.NW_018347324.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003460046.2-LOC_232.1.OR.7tm_4.exons1.NW_018347324.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003457891.3-LOC_352.1.OR.7tm_4.exons1.NW_018354195.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003460046.2-LOC_258.1.OR.7tm_4.exons1.NW_018357150.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019222741.1-LOC_226.1.OR.7tm_4.exons1.KN462546.1.Periophthalmus magnuspinnatus (mudskipper) XP_003457890.1-LOC_345.1.OR.7tm_4.exons1.Contig_3355.Neogobius melanostomus (round goby) XP_003460044.3-LOC_348.1.OR.7tm_4.exons1.Contig_3355.Neogobius melanostomus (round goby) XP_003460044.3-LOC_257.1.OR.7tm_4.exons1.NW_018357150.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003457889.1-LOC_227.1.OR.7tm_4.exons1.KN462546.1.Periophthalmus magnuspinnatus (mudskipper) XP_019222741.1-LOC_343.1.OR.7tm_4.exons1.Contig_3355.Neogobius melanostomus (round goby) XP_003457889.1-LOC_256.1.OR.7tm_4.exons1.NW_018357150.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003457889.1-LOC_228.1.OR.7tm_4.exons1.KN462546.1.Periophthalmus magnuspinnatus (mudskipper) XP_003457889.1-LOC_242.1.OR.7tm_4.exons1.NW_018357150.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003457889.1-LOC_243.1.OR.7tm_4.exons1.NW_018357150.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003457889.1-LOC_244.1.OR.7tm_4.exons1.NW_018357150.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003457889.1-LOC_232.1.OR.7tm_4.exons1.KN463138.1.Periophthalmus magnuspinnatus (mudskipper) XP_003457889.1-LOC_215.1.OR.7tm_4.exons1.KN462165.1.Periophthalmus magnuspinnatus (mudskipper) XP_003457889.1-LOC_245.1.OR.7tm_4.exons1.NW_018357150.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003457889.1-LOC_246.1.OR.7tm_4.exons1.NW_018357150.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003457889.1-LOC_248.1.OR.7tm_4.exons1.NW_018357150.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003457889.1-LOC_249.1.OR.7tm_4.exons1.NW_018357150.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003457889.1-LOC_247.1.OR.7tm_4.exons1.NW_018357150.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003457889.1-LOC_236.1.OR.7tm_4.exons1.KN463138.1.Periophthalmus magnuspinnatus (mudskipper) XP_003457889.1-LOC_233.1.OR.7tm_4.exons1.KN463138.1.Periophthalmus magnuspinnatus (mudskipper) XP_003457889.1-LOC_234.1.OR.7tm_4.exons1.KN463138.1.Periophthalmus magnuspinnatus (mudskipper) XP_003457889.1-LOC_237.1.OR.7tm_4.exons1.KN463138.1.Periophthalmus magnuspinnatus (mudskipper) XP_003457889.1-LOC_253.1.OR.7tm_4.exons1.NW_018357150.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003457889.1-LOC_241.1.OR.7tm_4.exons1.KN463138.1.Periophthalmus magnuspinnatus (mudskipper) XP_003457889.1-LOC_341.1.OR.7tm_4.exons1.Contig_3355.Neogobius melanostomus (round goby) XP_013121252.1-LOC_250.1.OR.7tm_4.exons1.NW_018357150.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_013121252.1-LOC_239.1.OR.7tm_4.exons1.KN463138.1.Periophthalmus magnuspinnatus (mudskipper) XP_003457887.3-LOC_240.1.OR.7tm_4.exons1.NW_018347324.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003460046.2-LOC_251.1.OR.7tm_4.exons1.NW_018347324.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003457866.1-LOC_261.1.OR.7tm_4.exons1.NW_018347324.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003457887.3-LOC_387.1.OR.7tm_4.exons1.Contig_3772.Neogobius melanostomus (round goby) XP_003457887.3-LOC_388.1.OR.7tm_4.exons1.Contig_3772.Neogobius melanostomus (round goby) XP_003457887.3-LOC_389.1.OR.7tm_4.exons1.Contig_3772.Neogobius melanostomus (round goby) XP_003457887.3-LOC_390.1.OR.7tm_4.exons1.Contig_3772.Neogobius melanostomus (round goby) XP_003457887.3-LOC_391.1.OR.7tm_4.exons1.Contig_3772.Neogobius melanostomus (round goby) XP_003457866.1-LOC_254.1.OR.7tm_4.exons1.NW_018357150.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003457887.3-LOC_231.1.OR.7tm_4.exons1.KN462546.1.Periophthalmus magnuspinnatus (mudskipper) XP_003460044.3-LOC_342.1.OR.7tm_4.exons1.Contig_3355.Neogobius melanostomus (round goby) XP_003460047.2-LOC_238.1.OR.7tm_4.exons1.NW_018357150.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003460047.2-LOC_239.1.OR.7tm_4.exons1.NW_018357150.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003460047.2-LOC_265.1.OR.7tm_4.exons1.KN463418.1.Periophthalmus magnuspinnatus (mudskipper) XP_003460047.2-LOC_267.1.OR.7tm_4.exons1.KN463418.1.Periophthalmus magnuspinnatus (mudskipper) XP_003460047.2-LOC_339.1.OR.7tm_4.exons1.Contig_3355.Neogobius melanostomus (round goby) XP_003457866.1-LOC_403.1.OR.7tm_4.exons1.NW_018347324.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003460049.3-LOC_257.1.OR.7tm_4.exons1.JACL01051979.1.Periophthalmus magnuspinnatus (mudskipper) XP_003460049.3-LOC_300.1.OR.7tm_4.exons1.Contig_2825.Neogobius melanostomus (round goby) XP_003460049.3-LOC_301.1.OR.7tm_4.exons1.Contig_2825.Neogobius melanostomus (round goby) XP_003460049.3-LOC_392.1.OR.7tm_4.exons1.Contig_3772.Neogobius melanostomus (round goby) XP_003460049.3-LOC_396.1.OR.7tm_4.exons1.Contig_3772.Neogobius melanostomus (round goby) XP_003460047.2-LOC_269.1.OR.7tm_4.exons1.KN463418.1.Periophthalmus magnuspinnatus (mudskipper) XP_003460045.2-LOC_241.1.OR.7tm_4.exons1.NW_018357150.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003460046.2-LOC_268.1.OR.7tm_4.exons1.KN463418.1.Periophthalmus magnuspinnatus (mudskipper) XP_003460044.3-LOC_255.1.OR.7tm_4.exons1.NW_018357150.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003460049.3-LOC_230.1.OR.7tm_4.exons1.KN462546.1.Periophthalmus magnuspinnatus (mudskipper) XP_003460049.3-LOC_382.1.OR.7tm_4.exons1.NW_018345971.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003460049.3-LOC_199.1.OR.7tm_4.exons1.KN461151.1.Periophthalmus magnuspinnatus (mudskipper) XP_003457866.1-LOC_314.1.OR.7tm_4.exons1.NW_018360113.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019222742.1-LOC_283.2.OR.7tm_4.exons1.NW_018349577.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019222742.1-LOC_294.2.OR.7tm_4.exons1.NW_018349577.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003460044.3-LOC_236.1.OR.7tm_4.exons1.NW_018355915.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_013121252.1-LOC_214.1.OR.7tm_4.exons1.KN462165.1.Periophthalmus magnuspinnatus (mudskipper) XP_003457866.1-LOC_315.2.OR.7tm_4.exons1.NW_018360113.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003457866.1-LOC_197.2.OR.7tm_4.exons1.KN461151.1.Periophthalmus magnuspinnatus (mudskipper) XP_003457866.1-LOC_419.1.OR.7tm_4.exons1.Contig_4528.Neogobius melanostomus (round goby) XP_003457887.3-LOC_393.1.OR.7tm_4.exons1.NW_018345971.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003457889.1-LOC_198.1.OR.7tm_4.exons1.KN461151.1.Periophthalmus magnuspinnatus (mudskipper) XP_003445425.1.Oreochromis niloticus (Nile tilapia) XP_019207986.1.Oreochromis niloticus (Nile tilapia) XP_019207987.1.Oreochromis niloticus (Nile tilapia) XP_019208766.1.Oreochromis niloticus (Nile tilapia) XP_003445426.1.Oreochromis niloticus (Nile tilapia) XP_019207988.1.Oreochromis niloticus (Nile tilapia) XP_019208310.1.Oreochromis niloticus (Nile tilapia) XP_019208343.1.Oreochromis niloticus (Nile tilapia) XP_005452071.2.Oreochromis niloticus (Nile tilapia) XP_013126862.2.Oreochromis niloticus (Nile tilapia) XP_013126847.2.Oreochromis niloticus (Nile tilapia) XP_019208337.1.Oreochromis niloticus (Nile tilapia) XP_013126865.1.Oreochromis niloticus (Nile tilapia) XP_013126866.1.Oreochromis niloticus (Nile tilapia) XP_013126866.1-LOC_254.1.OR.7tm_4.exons1.chrUn.Gasterosteus aculeatus (three-spined stickleback) XP_013126866.1-LOC_176.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_013126866.1-LOC_255.1.OR.7tm_4.exons1.chrUn.Gasterosteus aculeatus (three-spined stickleback) XP_013126866.1-LOC_175.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_013126866.1-LOC_170.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_013126866.1-LOC_172.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_013126866.1-LOC_173.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_013126866.1-LOC_174.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_013126866.1-LOC_171.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_013126866.1-LOC_169.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_003457854.2-LOC_260.1.OR.7tm_4.exons1.NW_018357571.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003457854.2-LOC_263.1.OR.7tm_4.exons1.NW_018357571.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003460044.3-LOC_185.1.OR.7tm_4.exons1.KN467007.1.Periophthalmus magnuspinnatus (mudskipper) XP_003457866.1-LOC_429.1.OR.7tm_4.exons1.Contig_1398.Neogobius melanostomus (round goby) XP_003460049.3-LOC_262.1.OR.7tm_4.exons1.NW_018357571.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003460049.3-LOC_264.1.OR.7tm_4.exons1.NW_018357571.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003460049.3-LOC_186.1.OR.7tm_4.exons1.KN467007.1.Periophthalmus magnuspinnatus (mudskipper) XP_003460049.3-LOC_430.1.OR.7tm_4.exons1.Contig_1398.Neogobius melanostomus (round goby) XP_003443300.3-LOC_350.1.OR.7tm_4.exons1.NW_018353960.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003443300.3-LOC_203.1.OR.7tm_4.exons1.KN461324.1.Periophthalmus magnuspinnatus (mudskipper) XP_019208020.1-LOC_363.1.OR.7tm_4.exons1.NW_018354295.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003443300.3-LOC_150.1.OR.7tm_4.exons1.KN463805.1.Periophthalmus magnuspinnatus (mudskipper) XP_019208020.1-LOC_364.1.OR.7tm_4.exons1.NW_018354295.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003443300.3-LOC_151.1.OR.7tm_4.exons1.KN463805.1.Periophthalmus magnuspinnatus (mudskipper) XP_003443300.3-LOC_263.1.OR.7tm_4.exons2.Contig_1319.Neogobius melanostomus (round goby) XP_003443300.3-LOC_463.1.OR.7tm_4.exons1.Contig_1319.Neogobius melanostomus (round goby) XP_003443300.3.Oreochromis niloticus (Nile tilapia) XP_019208020.1.Oreochromis niloticus (Nile tilapia) XP_019207943.1-LOC_213.1.OR.7tm_4.exons1.NW_018355507.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003449579.1-LOC_245.1.OR.7tm_4.exons1.KN463219.1.Periophthalmus magnuspinnatus (mudskipper) XP_019209300.1-LOC_220.1.OR.7tm_4.exons1.NW_018355507.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003456639.1-LOC_221.1.OR.7tm_4.exons1.NW_018355507.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003456639.1-LOC_222.1.OR.7tm_4.exons1.NW_018355507.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003449564.1-LOC_268.1.OR.7tm_4.exons1.Contig_2711.Neogobius melanostomus (round goby) XP_003449564.1-LOC_269.1.OR.7tm_4.exons1.Contig_2711.Neogobius melanostomus (round goby) XP_003449564.1-LOC_290.1.OR.7tm_4.exons1.Contig_2711.Neogobius melanostomus (round goby) XP_003449564.1-LOC_276.1.OR.7tm_4.exons1.Contig_2711.Neogobius melanostomus (round goby) XP_003449582.1-LOC_279.1.OR.7tm_4.exons1.Contig_2711.Neogobius melanostomus (round goby) XP_003449564.1-LOC_280.1.OR.7tm_4.exons1.Contig_2711.Neogobius melanostomus (round goby) XP_019211167.1-LOC_283.1.OR.7tm_4.exons1.Contig_2711.Neogobius melanostomus (round goby) XP_003449564.1-LOC_291.1.OR.7tm_4.exons1.Contig_2711.Neogobius melanostomus (round goby) XP_003449564.1-LOC_292.1.OR.7tm_4.exons1.Contig_2711.Neogobius melanostomus (round goby) XP_019211166.1-LOC_293.1.OR.7tm_4.exons1.Contig_2711.Neogobius melanostomus (round goby) XP_019211165.1-LOC_294.1.OR.7tm_4.exons1.Contig_2711.Neogobius melanostomus (round goby) XP_003449579.1-LOC_410.1.OR.7tm_4.exons1.NW_018355507.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003449579.1-LOC_248.1.OR.7tm_4.exons1.KN463219.1.Periophthalmus magnuspinnatus (mudskipper) XP_019211167.1-LOC_412.1.OR.7tm_4.exons1.NW_018355507.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019211167.1-LOC_247.1.OR.7tm_4.exons1.KN463219.1.Periophthalmus magnuspinnatus (mudskipper) XP_003449564.1-LOC_248.1.OR.7tm_4.exons1.chrUn.Gasterosteus aculeatus (three-spined stickleback) XP_003449564.1-LOC_205.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_003449564.1-LOC_199.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_003449564.1-LOC_209.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_003449564.1-LOC_213.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_003449564.1-LOC_227.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_003449564.1-LOC_217.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_003449564.1-LOC_219.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_003449564.1-LOC_222.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_003449564.1-LOC_250.1.OR.7tm_4.exons1.chrUn.Gasterosteus aculeatus (three-spined stickleback) XP_003449564.1-LOC_203.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_003449564.1-LOC_204.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_003449564.1-LOC_218.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_003449564.1-LOC_202.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_003449564.1-LOC_208.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_003449564.1-LOC_228.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_003449564.1-LOC_210.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_003449564.1-LOC_211.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_003449564.1-LOC_230.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_003449564.1-LOC_253.1.OR.7tm_4.exons1.chrUn.Gasterosteus aculeatus (three-spined stickleback) XP_003449564.1-LOC_200.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_003449564.1-LOC_198.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_003449564.1-LOC_303.1.OR.7tm_4.exons1.chrXII.Gasterosteus aculeatus (three-spined stickleback) XP_003449564.1-LOC_226.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_003449579.1-LOC_214.1.OR.7tm_4.exons1.NW_018355507.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003449564.1-LOC_215.1.OR.7tm_4.exons1.NW_018355507.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003449579.1-LOC_244.1.OR.7tm_4.exons1.KN463219.1.Periophthalmus magnuspinnatus (mudskipper) XP_003449582.1-LOC_249.1.OR.7tm_4.exons1.KN463219.1.Periophthalmus magnuspinnatus (mudskipper) XP_003449579.1-LOC_216.1.OR.7tm_4.exons1.NW_018355507.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019207942.1-LOC_217.1.OR.7tm_4.exons1.NW_018355507.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003449569.1-LOC_218.1.OR.7tm_4.exons1.NW_018355507.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003449569.1-LOC_219.1.OR.7tm_4.exons1.NW_018355507.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003449579.1-LOC_242.1.OR.7tm_4.exons1.KN463219.1.Periophthalmus magnuspinnatus (mudskipper) XP_003449579.1-LOC_243.1.OR.7tm_4.exons1.KN463219.1.Periophthalmus magnuspinnatus (mudskipper) XP_019211167.1-LOC_232.1.OR.7tm_4.exons1.chrXVI.Gasterosteus aculeatus (three-spined stickleback) XP_003449569.1.Oreochromis niloticus (Nile tilapia) XP_003456640.1.Oreochromis niloticus (Nile tilapia) XP_003449582.1.Oreochromis niloticus (Nile tilapia) XP_003449583.1.Oreochromis niloticus (Nile tilapia) XP_019207940.1.Oreochromis niloticus (Nile tilapia) XP_003449542.2.Oreochromis niloticus (Nile tilapia) XP_019207941.1.Oreochromis niloticus (Nile tilapia) XP_019207942.1.Oreochromis niloticus (Nile tilapia) XP_003449581.1.Oreochromis niloticus (Nile tilapia) XP_003456639.1.Oreochromis niloticus (Nile tilapia) XP_019211167.1.Oreochromis niloticus (Nile tilapia) XP_019211168.1.Oreochromis niloticus (Nile tilapia) XP_003460472.2.Oreochromis niloticus (Nile tilapia) XP_019207955.1.Oreochromis niloticus (Nile tilapia) XP_003449541.2.Oreochromis niloticus (Nile tilapia) XP_003449564.1.Oreochromis niloticus (Nile tilapia) XP_003449568.2.Oreochromis niloticus (Nile tilapia) XP_003460559.2.Oreochromis niloticus (Nile tilapia) XP_019211169.1.Oreochromis niloticus (Nile tilapia) XP_019211170.1.Oreochromis niloticus (Nile tilapia) XP_003449570.2.Oreochromis niloticus (Nile tilapia) XP_019211166.1.Oreochromis niloticus (Nile tilapia) XP_003449571.1.Oreochromis niloticus (Nile tilapia) XP_003460345.2.Oreochromis niloticus (Nile tilapia) XP_019207952.1.Oreochromis niloticus (Nile tilapia) XP_019207953.1.Oreochromis niloticus (Nile tilapia) XP_019207956.1.Oreochromis niloticus (Nile tilapia) XP_019212193.1.Oreochromis niloticus (Nile tilapia) XP_003449565.2.Oreochromis niloticus (Nile tilapia) XP_003449566.2.Oreochromis niloticus (Nile tilapia) XP_019211165.1.Oreochromis niloticus (Nile tilapia) XP_019211164.1.Oreochromis niloticus (Nile tilapia) XP_003449574.2.Oreochromis niloticus (Nile tilapia) XP_003449575.2.Oreochromis niloticus (Nile tilapia) XP_003449576.2.Oreochromis niloticus (Nile tilapia) XP_019207944.1.Oreochromis niloticus (Nile tilapia) XP_013123517.2.Oreochromis niloticus (Nile tilapia) XP_003449578.1.Oreochromis niloticus (Nile tilapia) XP_019207943.1.Oreochromis niloticus (Nile tilapia) XP_003449579.1.Oreochromis niloticus (Nile tilapia) XP_013129445.2.Oreochromis niloticus (Nile tilapia) XP_019207939.1.Oreochromis niloticus (Nile tilapia) XP_019209300.1.Oreochromis niloticus (Nile tilapia) XP_019209546.1.Oreochromis niloticus (Nile tilapia) XP_019211809.1.Oreochromis niloticus (Nile tilapia) XP_003460030.1-LOC_398.1.OR.7tm_4.exons2.NW_018354704.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003460030.1-LOC_259.1.OR.7tm_4.exons2.NW_018357150.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003460030.1-LOC_263.1.OR.7tm_4.exons2.KN463418.1.Periophthalmus magnuspinnatus (mudskipper) XP_003460030.1-LOC_337.1.OR.7tm_4.exons2.Contig_3355.Neogobius melanostomus (round goby) XP_003460030.1-LOC_168.1.OR.7tm_4.exons1.chrI.Gasterosteus aculeatus (three-spined stickleback) XP_003460030.1.Oreochromis niloticus (Nile tilapia) XP_013123033.1-LOC_237.1.OR.7tm_4.exons1.NW_018357150.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_013123033.1-LOC_264.1.OR.7tm_4.exons1.KN463418.1.Periophthalmus magnuspinnatus (mudskipper) XP_013123033.1-LOC_335.1.OR.7tm_4.exons1.Contig_3355.Neogobius melanostomus (round goby) XP_013123033.1-LOC_338.1.OR.7tm_4.exons1.Contig_3355.Neogobius melanostomus (round goby) XP_003460027.2.Oreochromis niloticus (Nile tilapia) XP_019222743.1.Oreochromis niloticus (Nile tilapia) XP_003460042.1.Oreochromis niloticus (Nile tilapia) XP_013123022.2.Oreochromis niloticus (Nile tilapia) XP_019223008.1.Oreochromis niloticus (Nile tilapia) XP_013123033.1.Oreochromis niloticus (Nile tilapia) XP_019222760.1-LOC_322.1.OR.7tm_4.exons1.NW_018361464.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019222760.1-LOC_160.1.OR.7tm_4.exons1.KN464118.1.Periophthalmus magnuspinnatus (mudskipper) XP_019222760.1-LOC_167.1.OR.7tm_4.exons1.KN464599.1.Periophthalmus magnuspinnatus (mudskipper) XP_019222760.1-LOC_166.1.OR.7tm_4.exons1.KN464599.1.Periophthalmus magnuspinnatus (mudskipper) XP_019222760.1-LOC_306.1.OR.7tm_4.exons1.Contig_2833.Neogobius melanostomus (round goby) XP_019222713.1.Oreochromis niloticus (Nile tilapia) XP_019222760.1.Oreochromis niloticus (Nile tilapia) XP_019222760.1-LOC_247.1.OR.7tm_4.exons1.chrI.Gasterosteus aculeatus (three-spined stickleback) XP_005455158.2-LOC_323.1.OR.7tm_4.exons1.NW_018361464.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_005455158.2-LOC_282.1.OR.7tm_4.exons1.KN463719.1.Periophthalmus magnuspinnatus (mudskipper) XP_005455158.2-LOC_324.1.OR.7tm_4.exons1.NW_018361464.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_005455158.2-LOC_325.1.OR.7tm_4.exons1.NW_018361464.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_005455158.2-LOC_283.1.OR.7tm_4.exons1.KN463719.1.Periophthalmus magnuspinnatus (mudskipper) XP_005455158.2-LOC_421.1.OR.7tm_4.exons1.Contig_788.Neogobius melanostomus (round goby) XP_019222760.1-LOC_304.1.OR.7tm_4.exons1.Contig_2833.Neogobius melanostomus (round goby) XP_005455158.2-LOC_302.1.OR.7tm_4.exons1.Contig_2833.Neogobius melanostomus (round goby) XP_005455158.2-LOC_326.1.OR.7tm_4.exons1.NW_018361464.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_005455158.2-LOC_327.1.OR.7tm_4.exons1.NW_018361464.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_005455158.2-LOC_284.1.OR.7tm_4.exons1.KN463719.1.Periophthalmus magnuspinnatus (mudskipper) XP_005455158.2-LOC_422.1.OR.7tm_4.exons1.Contig_788.Neogobius melanostomus (round goby) XP_005455158.2.Oreochromis niloticus (Nile tilapia) XP_019222759.1.Oreochromis niloticus (Nile tilapia) XP_003450214.2.Oreochromis niloticus (Nile tilapia) XP_019222761.1.Oreochromis niloticus (Nile tilapia) XP_019222762.1.Oreochromis niloticus (Nile tilapia) XP_019222763.1.Oreochromis niloticus (Nile tilapia) XP_019222764.1.Oreochromis niloticus (Nile tilapia) XP_019222765.1.Oreochromis niloticus (Nile tilapia) XP_019222761.1-LOC_244.1.OR.7tm_4.exons1.chrI.Gasterosteus aculeatus (three-spined stickleback) XP_019222761.1-LOC_245.1.OR.7tm_4.exons1.chrI.Gasterosteus aculeatus (three-spined stickleback) XP_019222761.1-LOC_246.1.OR.7tm_4.exons1.chrI.Gasterosteus aculeatus (three-spined stickleback) XP_019222768.1.Oreochromis niloticus (Nile tilapia) XP_019222769.1.Oreochromis niloticus (Nile tilapia) XP_019222775.1.Oreochromis niloticus (Nile tilapia) XP_003457602.1-LOC_334.1.OR.7tm_4.exons1.NW_018353795.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003457602.1-LOC_181.1.OR.7tm_4.exons1.KN466353.1.Periophthalmus magnuspinnatus (mudskipper) XP_003457602.1.Oreochromis niloticus (Nile tilapia) XP_003457602.1-LOC_433.1.OR.7tm_4.exons1.Contig_1986.Neogobius melanostomus (round goby) XP_003457602.1-LOC_278.1.OR.7tm_4.exons1.chrVII.Gasterosteus aculeatus (three-spined stickleback) XP_003457602.1-LOC_280.1.OR.7tm_4.exons1.chrVII.Gasterosteus aculeatus (three-spined stickleback) XP_019219095.1-LOC_279.1.OR.7tm_4.exons1.chrVII.Gasterosteus aculeatus (three-spined stickleback) XP_019219095.1-LOC_285.1.OR.7tm_4.exons1.chrVII.Gasterosteus aculeatus (three-spined stickleback) XP_019219095.1-LOC_295.1.OR.7tm_4.exons1.chrVII.Gasterosteus aculeatus (three-spined stickleback) XP_019219198.1-LOC_287.1.OR.7tm_4.exons1.chrVII.Gasterosteus aculeatus (three-spined stickleback) XP_019219091.1-LOC_288.1.OR.7tm_4.exons1.chrVII.Gasterosteus aculeatus (three-spined stickleback) XP_019219095.1-LOC_291.1.OR.7tm_4.exons1.chrVII.Gasterosteus aculeatus (three-spined stickleback) XP_019219095.1-LOC_292.1.OR.7tm_4.exons1.chrVII.Gasterosteus aculeatus (three-spined stickleback) XP_019219198.1-LOC_294.1.OR.7tm_4.exons1.chrVII.Gasterosteus aculeatus (three-spined stickleback) XP_019219198.1-LOC_296.1.OR.7tm_4.exons1.chrVII.Gasterosteus aculeatus (three-spined stickleback) XP_019219095.1-LOC_289.1.OR.7tm_4.exons1.chrVII.Gasterosteus aculeatus (three-spined stickleback) 7tm4 XP_019219198.1-LOC_286.1.OR.7tm_4.exons1.chrVII.Gasterosteus aculeatus (three-spined stickleback) XP_019219091.1.Oreochromis niloticus (Nile tilapia) XP_019219098.1.Oreochromis niloticus (Nile tilapia) XP_019219198.1.Oreochromis niloticus (Nile tilapia) XP_019219095.1.Oreochromis niloticus (Nile tilapia) XP_019219198.1-LOC_335.1.OR.7tm_4.exons1.NW_018353795.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019219091.1-LOC_305.1.OR.7tm_4.exons1.Contig_1395.Neogobius melanostomus (round goby) XP_003457603.1.Oreochromis niloticus (Nile tilapia) XP_003458326.1.Oreochromis niloticus (Nile tilapia) XP_019219102.1.Oreochromis niloticus (Nile tilapia) XP_019219100.1.Oreochromis niloticus (Nile tilapia) XP_005462404.3.Oreochromis niloticus (Nile tilapia) XP_019219093.1.Oreochromis niloticus (Nile tilapia) XP_019219094.1.Oreochromis niloticus (Nile tilapia) XP_005462403.2.Oreochromis niloticus (Nile tilapia) XP_019219223.1.Oreochromis niloticus (Nile tilapia) XP_019219100.1-LOC_266.1.OR.7tm_4.exons1.NW_018358439.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019219100.1-LOC_192.1.OR.7tm_4.exons1.KN467396.1.Periophthalmus magnuspinnatus (mudskipper) XP_019219095.1-LOC_366.1.OR.7tm_4.exons1.Contig_1395.Neogobius melanostomus (round goby) XP_019219100.1-LOC_266.1.OR.7tm_4.exons1.JACL01054066.1.Periophthalmus magnuspinnatus (mudskipper) XP_003457595.3-LOC_353.1.OR.7tm_4.exons1.NW_018354260.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003457601.1-LOC_355.1.OR.7tm_4.exons1.NW_018354260.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003457601.1-LOC_217.1.OR.7tm_4.exons1.KN462446.1.Periophthalmus magnuspinnatus (mudskipper) XP_003457601.1-LOC_358.1.OR.7tm_4.exons1.NW_018354260.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003457601.1-LOC_220.1.OR.7tm_4.exons1.KN462446.1.Periophthalmus magnuspinnatus (mudskipper) XP_003457601.1-LOC_356.1.OR.7tm_4.exons1.NW_018354260.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003457601.1-LOC_218.1.OR.7tm_4.exons1.KN462446.1.Periophthalmus magnuspinnatus (mudskipper) XP_003457601.1-LOC_404.1.OR.7tm_4.exons1.Contig_1395.Neogobius melanostomus (round goby) XP_003457601.1-LOC_357.1.OR.7tm_4.exons1.NW_018354260.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003457595.3-LOC_219.1.OR.7tm_4.exons1.KN462446.1.Periophthalmus magnuspinnatus (mudskipper) XP_003457595.3.Oreochromis niloticus (Nile tilapia) XP_019219086.1.Oreochromis niloticus (Nile tilapia) XP_003457598.2.Oreochromis niloticus (Nile tilapia) XP_019219088.1.Oreochromis niloticus (Nile tilapia) XP_013121051.1.Oreochromis niloticus (Nile tilapia) XP_003457601.1.Oreochromis niloticus (Nile tilapia) XP_019219089.1.Oreochromis niloticus (Nile tilapia) XP_003457601.1-LOC_277.1.OR.7tm_4.exons1.chrVII.Gasterosteus aculeatus (three-spined stickleback) XP_019219085.1-LOC_359.1.OR.7tm_4.exons1.NW_018354260.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019219085.1-LOC_221.1.OR.7tm_4.exons1.KN462446.1.Periophthalmus magnuspinnatus (mudskipper) XP_019219085.1-LOC_426.1.OR.7tm_4.exons1.Contig_1395.Neogobius melanostomus (round goby) XP_019219084.1.Oreochromis niloticus (Nile tilapia) XP_019219085.1.Oreochromis niloticus (Nile tilapia) XP_019219085.1-LOC_274.1.OR.7tm_4.exons1.chrVII.Gasterosteus aculeatus (three-spined stickleback) XP_019219085.1-LOC_276.1.OR.7tm_4.exons1.chrVII.Gasterosteus aculeatus (three-spined stickleback) XP_019219085.1-LOC_275.1.OR.7tm_4.exons1.chrVII.Gasterosteus aculeatus (three-spined stickleback) XP_019219085.1-LOC_361.1.OR.7tm_4.exons1.NW_018354260.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019219085.1-LOC_222.1.OR.7tm_4.exons1.KN462446.1.Periophthalmus magnuspinnatus (mudskipper) XP_019219085.1-LOC_427.1.OR.7tm_4.exons1.Contig_1395.Neogobius melanostomus (round goby) XP_019219085.1-LOC_428.1.OR.7tm_4.exons1.Contig_1395.Neogobius melanostomus (round goby) XP_019219085.1-LOC_273.1.OR.7tm_4.exons1.chrVII.Gasterosteus aculeatus (three-spined stickleback) XP_005462572.1.Oreochromis niloticus (Nile tilapia) XP_019219008.1.Oreochromis niloticus (Nile tilapia) XP_019219008.1-LOC_263.1.OR.7tm_4.exons2.chrV.Gasterosteus aculeatus (three-spined stickleback) XP_019219008.1-LOC_267.1.OR.7tm_4.exons2.chrV.Gasterosteus aculeatus (three-spined stickleback) XP_019219064.1-LOC_365.1.OR.7tm_4.exons1.NW_018354490.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019219064.1-LOC_372.1.OR.7tm_4.exons1.NW_018354571.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019219064.1-LOC_153.1.OR.7tm_4.exons1.KN463989.1.Periophthalmus magnuspinnatus (mudskipper) XP_019219063.1-LOC_154.1.OR.7tm_4.exons1.KN463989.1.Periophthalmus magnuspinnatus (mudskipper) XP_019219063.1-LOC_371.1.OR.7tm_4.exons1.Contig_3683.Neogobius melanostomus (round goby) XP_019219064.1-LOC_375.1.OR.7tm_4.exons1.Contig_3683.Neogobius melanostomus (round goby) XP_019219063.1-LOC_381.1.OR.7tm_4.exons1.NW_018354571.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019219063.1-LOC_383.1.OR.7tm_4.exons1.NW_018354571.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019219063.1-LOC_388.1.OR.7tm_4.exons1.NW_018354571.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019219063.1-LOC_389.1.OR.7tm_4.exons1.NW_018354571.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019219063.1-LOC_384.1.OR.7tm_4.exons1.NW_018354571.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019219063.1-LOC_387.1.OR.7tm_4.exons1.NW_018354571.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019219063.1-LOC_385.1.OR.7tm_4.exons1.NW_018354571.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019219063.1-LOC_386.1.OR.7tm_4.exons1.NW_018354571.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019219063.1-LOC_390.1.OR.7tm_4.exons1.NW_018354571.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019219063.1-LOC_183.1.OR.7tm_4.exons1.KN466677.1.Periophthalmus magnuspinnatus (mudskipper) XP_019219063.1-LOC_373.1.OR.7tm_4.exons1.NW_018354571.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019219063.1-LOC_155.1.OR.7tm_4.exons1.KN463989.1.Periophthalmus magnuspinnatus (mudskipper) XP_003457730.1-LOC_382.1.OR.7tm_4.exons1.Contig_3685.Neogobius melanostomus (round goby) XP_003457730.1-LOC_385.1.OR.7tm_4.exons1.Contig_3713.Neogobius melanostomus (round goby) XP_005462570.1-LOC_368.1.OR.7tm_4.exons1.NW_018354571.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019219064.1-LOC_370.1.OR.7tm_4.exons1.NW_018354571.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_005462570.1-LOC_369.1.OR.7tm_4.exons1.NW_018354571.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_005462570.1-LOC_280.1.OR.7tm_4.exons1.KN463700.1.Periophthalmus magnuspinnatus (mudskipper) XP_005462570.1-LOC_152.1.OR.7tm_4.exons1.KN463989.1.Periophthalmus magnuspinnatus (mudskipper) XP_005462570.1-LOC_281.1.OR.7tm_4.exons1.KN463700.1.Periophthalmus magnuspinnatus (mudskipper) XP_019219064.1-LOC_363.1.OR.7tm_4.exons1.Contig_3682.Neogobius melanostomus (round goby) XP_003457730.1-LOC_391.1.OR.7tm_4.exons1.NW_018354571.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003457730.1-LOC_392.1.OR.7tm_4.exons1.NW_018354571.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003457730.1-LOC_394.1.OR.7tm_4.exons1.NW_018354571.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019219064.1-LOC_184.1.OR.7tm_4.exons1.KN466677.1.Periophthalmus magnuspinnatus (mudskipper) XP_019219063.1-LOC_367.1.OR.7tm_4.exons3.Contig_3683.Neogobius melanostomus (round goby) XP_003457730.1-LOC_378.1.OR.7tm_4.exons2.Contig_3685.Neogobius melanostomus (round goby) XP_003457730.1-LOC_370.1.OR.7tm_4.exons2.Contig_3683.Neogobius melanostomus (round goby) XP_003457730.1-LOC_386.1.OR.7tm_4.exons2.Contig_3713.Neogobius melanostomus (round goby) XP_003457730.1.Oreochromis niloticus (Nile tilapia) XP_019219063.1.Oreochromis niloticus (Nile tilapia) XP_019219064.1.Oreochromis niloticus (Nile tilapia) XP_005462570.1.Oreochromis niloticus (Nile tilapia) XP_005462570.1-LOC_299.1.OR.7tm_4.exons1.chrVII.Gasterosteus aculeatus (three-spined stickleback) XP_005462570.1-LOC_301.1.OR.7tm_4.exons1.chrVII.Gasterosteus aculeatus (three-spined stickleback) XP_003457729.1.Oreochromis niloticus (Nile tilapia) XP_019219065.1-LOC_269.1.OR.7tm_4.exons1.chrV.Gasterosteus aculeatus (three-spined stickleback) XP_019219065.1.Oreochromis niloticus (Nile tilapia) XP_019219065.1-LOC_268.1.OR.7tm_4.exons1.chrV.Gasterosteus aculeatus (three-spined stickleback) XP_003455983.1-LOC_318.1.OR.7tm_4.exons1.NW_018361464.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003455983.1-LOC_190.1.OR.7tm_4.exons1.KN461045.1.Periophthalmus magnuspinnatus (mudskipper) XP_003455983.1-LOC_309.1.OR.7tm_4.exons1.Contig_2833.Neogobius melanostomus (round goby) XP_003455983.1.Oreochromis niloticus (Nile tilapia) XP_003455983.1-LOC_238.1.OR.7tm_4.exons1.chrI.Gasterosteus aculeatus (three-spined stickleback) XP_003455988.1-LOC_319.1.OR.7tm_4.exons1.NW_018361464.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003455988.1-LOC_179.1.OR.7tm_4.exons1.KN461045.1.Periophthalmus magnuspinnatus (mudskipper) XP_003455988.1-LOC_308.1.OR.7tm_4.exons1.Contig_2833.Neogobius melanostomus (round goby) XP_003455988.1.Oreochromis niloticus (Nile tilapia) XP_003455988.1-LOC_239.1.OR.7tm_4.exons1.chrI.Gasterosteus aculeatus (three-spined stickleback) XP_019200981.1-LOC_320.1.OR.7tm_4.exons1.NW_018361464.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019200981.1-LOC_168.1.OR.7tm_4.exons1.KN461045.1.Periophthalmus magnuspinnatus (mudskipper) XP_019200981.1-LOC_240.1.OR.7tm_4.exons1.chrI.Gasterosteus aculeatus (three-spined stickleback) XP_019200981.1-LOC_321.1.OR.7tm_4.exons1.NW_018361464.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019200981.1-LOC_161.1.OR.7tm_4.exons1.KN464118.1.Periophthalmus magnuspinnatus (mudskipper) XP_019200981.1-LOC_307.1.OR.7tm_4.exons1.Contig_2833.Neogobius melanostomus (round goby) XP_019200981.1.Oreochromis niloticus (Nile tilapia) XP_019200982.1.Oreochromis niloticus (Nile tilapia) XP_019200983.1.Oreochromis niloticus (Nile tilapia) XP_019200981.1-LOC_242.1.OR.7tm_4.exons1.chrI.Gasterosteus aculeatus (three-spined stickleback) XP_019200981.1-LOC_243.1.OR.7tm_4.exons1.chrI.Gasterosteus aculeatus (three-spined stickleback) XP_005462566.1.Oreochromis niloticus (Nile tilapia) XP_005462567.1.Oreochromis niloticus (Nile tilapia) XP_005462568.1.Oreochromis niloticus (Nile tilapia) XP_019219061.1.Oreochromis niloticus (Nile tilapia) XP_019219062.1.Oreochromis niloticus (Nile tilapia) XP_013120661.1.Oreochromis niloticus (Nile tilapia) XP_013120661.1-LOC_262.1.OR.7tm_1.exons1.KN463369.1.Periophthalmus magnuspinnatus (mudskipper) XP_013120661.1-LOC_305.1.OR.7tm_1.exons1.chrXII.Gasterosteus aculeatus (three-spined stickleback) XP_003451939.1-LOC_272.1.OR.7tm_4.exons1.NW_018349355.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003451939.1-LOC_307.1.OR.7tm_1.exons1.NW_018359103.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019214035.1-LOC_317.1.OR.7tm_4.exons1.NW_018351983.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003451939.1-LOC_330.1.OR.7tm_1.exons1.NW_018351983.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003451939.1-LOC_332.1.OR.7tm_1.exons1.NW_018351983.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003451939.1-LOC_274.1.OR.7tm_4.exons1.KN463566.1.Periophthalmus magnuspinnatus (mudskipper) XP_003451939.1-LOC_437.1.OR.7tm_1.exons1.Contig_2522.Neogobius melanostomus (round goby) XP_003451939.1-LOC_487.1.OR.7tm_1.exons1.Contig_2528.Neogobius melanostomus (round goby) XP_003451939.1-LOC_439.1.OR.7tm_1.exons1.Contig_2522.Neogobius melanostomus (round goby) XP_003451939.1-LOC_484.1.OR.7tm_1.exons1.Contig_2528.Neogobius melanostomus (round goby) XP_003451939.1-LOC_304.1.OR.7tm_4.exons1.NW_018359103.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019214035.1-LOC_462.1.OR.7tm_1.exons2.Contig_2523.Neogobius melanostomus (round goby) XP_003451939.1-LOC_444.1.OR.7tm_1.exons1.Contig_2522.Neogobius melanostomus (round goby) XP_003451939.1-LOC_445.1.OR.7tm_1.exons1.Contig_2522.Neogobius melanostomus (round goby) XP_003451939.1-LOC_453.1.OR.7tm_1.exons1.Contig_2522.Neogobius melanostomus (round goby) XP_003451939.1-LOC_461.1.OR.7tm_1.exons1.Contig_2523.Neogobius melanostomus (round goby) XP_019214035.1-LOC_451.1.OR.7tm_4.exons1.Contig_2522.Neogobius melanostomus (round goby) XP_019214035.1-LOC_454.1.OR.7tm_4.exons2.Contig_2523.Neogobius melanostomus (round goby) XP_003451939.1-LOC_452.1.OR.7tm_1.exons1.Contig_2522.Neogobius melanostomus (round goby) XP_019214035.1-LOC_224.1.OR.7tm_1.exons1.NW_018355659.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003451939.1-LOC_226.1.OR.7tm_1.exons1.NW_018355659.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019214035.1-LOC_225.1.OR.7tm_4.exons2.NW_018355659.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019214035.1-LOC_227.1.OR.7tm_4.exons2.NW_018355659.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003451939.1-LOC_229.1.OR.7tm_4.exons1.NW_018355659.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019214035.1-LOC_176.1.OR.7tm_1.exons1.KN466150.1.Periophthalmus magnuspinnatus (mudskipper) XP_019214035.1-LOC_438.1.OR.7tm_4.exons1.Contig_2522.Neogobius melanostomus (round goby) XP_019214035.1-LOC_486.1.OR.7tm_4.exons1.Contig_2528.Neogobius melanostomus (round goby) XP_019214035.1-LOC_410.1.OR.7tm_1.exons1.Contig_4011.Neogobius melanostomus (round goby) XP_019214035.1-LOC_235.1.OR.7tm_4.exons1.JACL01049760.1.Periophthalmus magnuspinnatus (mudskipper) XP_003451939.1-LOC_231.1.OR.7tm_4.exons1.NW_018355659.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003451939.1-LOC_194.1.OR.7tm_4.exons1.KN467957.1.Periophthalmus magnuspinnatus (mudskipper) XP_003451939.1-LOC_408.1.OR.7tm_1.exons1.Contig_4011.Neogobius melanostomus (round goby) XP_019214035.1.Oreochromis niloticus (Nile tilapia) XP_019214062.1.Oreochromis niloticus (Nile tilapia) XP_019214037.1-LOC_268.1.OR.7tm_1.exons1.NW_018359070.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019214037.1-LOC_269.2.OR.7tm_1.exons1.NW_018359070.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019214037.1-LOC_271.1.OR.7tm_1.exons1.NW_018359070.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019214037.1-LOC_273.2.OR.7tm_1.exons1.NW_018359070.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019214037.1-LOC_253.1.OR.7tm_1.exons1.KN463335.1.Periophthalmus magnuspinnatus (mudskipper) XP_019214037.1-LOC_256.1.OR.7tm_1.exons1.KN463335.1.Periophthalmus magnuspinnatus (mudskipper) XP_019214036.1-LOC_258.1.OR.7tm_1.exons2.KN463335.1.Periophthalmus magnuspinnatus (mudskipper) XP_003451937.1-LOC_317.2.OR.7tm_1.exons1.Contig_3034.Neogobius melanostomus (round goby) XP_003451937.1-LOC_321.1.OR.7tm_1.exons1.Contig_3279.Neogobius melanostomus (round goby) XP_003451937.1-LOC_330.1.OR.7tm_1.exons1.Contig_3280.Neogobius melanostomus (round goby) XP_003451937.1-LOC_323.1.OR.7tm_1.exons1.Contig_3279.Neogobius melanostomus (round goby) XP_003451937.1-LOC_327.1.OR.7tm_1.exons1.Contig_3279.Neogobius melanostomus (round goby) XP_003451937.1-LOC_332.1.OR.7tm_1.exons1.Contig_3280.Neogobius melanostomus (round goby) XP_003451937.1-LOC_328.1.OR.7tm_1.exons4.Contig_3279.Neogobius melanostomus (round goby) XP_003451939.1-LOC_282.1.OR.7tm_1.exons1.NW_018359103.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003451939.1-LOC_261.1.OR.7tm_4.exons1.KN463363.1.Periophthalmus magnuspinnatus (mudskipper) XP_019214036.1-LOC_465.1.OR.7tm_4.exons2.Contig_2523.Neogobius melanostomus (round goby) XP_003451939.1-LOC_467.1.OR.7tm_1.exons1.Contig_2523.Neogobius melanostomus (round goby) XP_003451939.1-LOC_469.1.OR.7tm_1.exons1.Contig_2523.Neogobius melanostomus (round goby) XP_019214036.1-LOC_473.1.OR.7tm_4.exons2.Contig_2523.Neogobius melanostomus (round goby) XP_019214036.1-LOC_466.1.OR.7tm_4.exons2.Contig_2523.Neogobius melanostomus (round goby) XP_019214037.1-LOC_285.1.OR.7tm_1.exons1.NW_018359103.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019214037.1-LOC_287.1.OR.7tm_1.exons1.NW_018359103.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019214037.1-LOC_290.1.OR.7tm_1.exons1.NW_018359103.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019214037.1-LOC_292.1.OR.7tm_1.exons1.NW_018359103.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019214037.1-LOC_293.1.OR.7tm_1.exons1.NW_018359103.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019214037.1-LOC_295.1.OR.7tm_1.exons1.NW_018359103.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019214037.1-LOC_298.1.OR.7tm_1.exons1.NW_018359103.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019214037.1-LOC_297.1.OR.7tm_1.exons1.NW_018359103.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019214037.1-LOC_162.1.OR.7tm_1.exons1.KN464236.1.Periophthalmus magnuspinnatus (mudskipper) XP_019214037.1-LOC_165.1.OR.7tm_1.exons1.KN464236.1.Periophthalmus magnuspinnatus (mudskipper) XP_019214036.1-LOC_163.1.OR.7tm_1.exons2.KN464236.1.Periophthalmus magnuspinnatus (mudskipper) XP_019214037.1-LOC_300.1.OR.7tm_1.exons1.NW_018359103.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019214037.1-LOC_302.1.OR.7tm_1.exons1.NW_018359103.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003451939.1-LOC_271.1.OR.7tm_1.exons1.KN463566.1.Periophthalmus magnuspinnatus (mudskipper) XP_003451939.1-LOC_464.1.OR.7tm_1.exons1.Contig_2523.Neogobius melanostomus (round goby) XP_019214036.1.Oreochromis niloticus (Nile tilapia) XP_003451939.1-LOC_275.1.OR.7tm_1.exons1.NW_018359103.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) 7tm1 XP_003451939.1-LOC_279.1.OR.7tm_1.exons1.NW_018359103.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003451940.2-LOC_277.1.OR.7tm_1.exons1.NW_018359103.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003451939.1-LOC_259.1.OR.7tm_1.exons1.KN463363.1.Periophthalmus magnuspinnatus (mudskipper) XP_003451939.1.Oreochromis niloticus (Nile tilapia) XP_003451940.2.Oreochromis niloticus (Nile tilapia) XP_019214037.1.Oreochromis niloticus (Nile tilapia) XP_003451937.1.Oreochromis niloticus (Nile tilapia) XP_003451937.1-LOC_235.1.OR.7tm_1.exons2.chrXVII.Gasterosteus aculeatus (three-spined stickleback) XP_003451940.2-LOC_236.1.OR.7tm_1.exons1.chrXVII.Gasterosteus aculeatus (three-spined stickleback) XP_003451940.2-LOC_281.1.OR.7tm_1.exons1.NW_018359103.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003451930.1.Oreochromis niloticus (Nile tilapia) XP_013130921.2-LOC_233.1.OR.7tm_1.exons1.NW_018355659.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_013130921.2-LOC_195.1.OR.7tm_1.exons2.KN467957.1.Periophthalmus magnuspinnatus (mudskipper) XP_013130921.2-LOC_405.1.OR.7tm_1.exons2.Contig_4011.Neogobius melanostomus (round goby) XP_013130921.2.Oreochromis niloticus (Nile tilapia) XP_003451931.1-LOC_234.1.OR.7tm_4.exons4.NW_018355659.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003451931.1-LOC_403.1.OR.7tm_1.exons4.Contig_4011.Neogobius melanostomus (round goby) XP_003451931.1.Oreochromis niloticus (Nile tilapia) XP_003451931.1-LOC_234.1.OR.7TM_GPCR_Srx.exons3.chrXVII.Gasterosteus aculeatus (three-spined stickleback) XP_003440337.2-LOC_235.1.OR.7tm_1.exons1.NW_018355755.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003440337.2-LOC_205.1.OR.7tm_1.exons1.KN461928.1.Periophthalmus magnuspinnatus (mudskipper) XP_003440336.2.Oreochromis niloticus (Nile tilapia) XP_003440337.2.Oreochromis niloticus (Nile tilapia) XP_003440337.2-LOC_179.1.OR.7tm_1.exons1.chrI.Gasterosteus aculeatus (three-spined stickleback) XP_003455968.2-LOC_348.1.OR.7tm_1.exons1.NW_018353872.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003455968.2.Oreochromis niloticus (Nile tilapia) XP_003455969.1.Oreochromis niloticus (Nile tilapia) XP_013119980.2.Oreochromis niloticus (Nile tilapia) XP_003455969.1-LOC_190.1.OR.7tm_1.exons1.chrI.Gasterosteus aculeatus (three-spined stickleback) XP_003455969.1-LOC_201.1.OR.7tm_1.exons1.chrI.Gasterosteus aculeatus (three-spined stickleback) Maximum-likelihood phylogenetic tree of XP_003455969.1-LOC_212.1.OR.7tm_1.exons1.chrI.Gasterosteus aculeatus (three-spined stickleback) XP_003455969.1-LOC_223.1.OR.7tm_1.exons1.chrI.Gasterosteus aculeatus (three-spined stickleback) XP_003455969.1-LOC_233.1.OR.7tm_1.exons1.chrI.Gasterosteus aculeatus (three-spined stickleback) XP_003455990.2.Oreochromis niloticus (Nile tilapia) XP_019222780.1.Oreochromis niloticus (Nile tilapia) XP_005474851.1.Oreochromis niloticus (Nile tilapia) XP_019219040.1-LOC_336.1.OR.7tm_1.exons1.NW_018353795.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_019219054.1-LOC_204.1.OR.7tm_1.exons1.KN461589.1.Periophthalmus magnuspinnatus (mudskipper) XP_003452603.2.Oreochromis niloticus (Nile tilapia) XP_003452606.1.Oreochromis niloticus (Nile tilapia) XP_019219040.1.Oreochromis niloticus (Nile tilapia) percomorph olfactory receptor protein XP_003452604.1.Oreochromis niloticus (Nile tilapia) XP_003452607.2.Oreochromis niloticus (Nile tilapia) XP_003452609.2.Oreochromis niloticus (Nile tilapia) XP_019219055.1.Oreochromis niloticus (Nile tilapia) XP_019219056.1.Oreochromis niloticus (Nile tilapia) XP_019219057.1.Oreochromis niloticus (Nile tilapia) XP_003452611.1.Oreochromis niloticus (Nile tilapia) XP_003452610.1.Oreochromis niloticus (Nile tilapia) XP_019219053.1.Oreochromis niloticus (Nile tilapia) XP_019219054.1.Oreochromis niloticus (Nile tilapia) XP_005457412.2.Oreochromis niloticus (Nile tilapia) XP_005457413.2.Oreochromis niloticus (Nile tilapia) sequences. XP_019219040.1-LOC_302.2.OR.7tm_4.exons1.chrVII.Gasterosteus aculeatus (three-spined stickleback) XP_019219053.1-LOC_367.1.OR.7tm_1.exons1.NW_018354571.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_005457412.2-LOC_298.1.OR.7tm_1.exons1.Contig_2789.Neogobius melanostomus (round goby) XP_005457413.2-LOC_299.1.OR.7tm_1.exons2.Contig_2789.Neogobius melanostomus (round goby) XP_005457413.2-LOC_312.1.OR.7tm_1.exons1.Contig_2861.Neogobius melanostomus (round goby) XP_019219054.1-LOC_313.1.OR.7tm_1.exons1.Contig_2861.Neogobius melanostomus (round goby) XP_005457413.2-LOC_417.1.OR.7tm_1.exons2.Contig_4414.Neogobius melanostomus (round goby) 0.5 XP_019219040.1-LOC_310.1.OR.7tm_1.exons1.Contig_2861.Neogobius melanostomus (round goby) XP_019219040.1-LOC_311.1.OR.7tm_1.exons1.Contig_2861.Neogobius melanostomus (round goby) XP_019219054.1-LOC_206.1.OR.7tm_1.exons1.KN461942.1.Periophthalmus magnuspinnatus (mudskipper) XP_019219054.1-LOC_171.1.OR.7tm_1.exons1.KN465204.1.Periophthalmus magnuspinnatus (mudskipper) XP_019219054.1-LOC_172.1.OR.7tm_1.exons1.KN465315.1.Periophthalmus magnuspinnatus (mudskipper) XP_019219054.1-LOC_193.1.OR.7tm_1.exons1.KN467561.1.Periophthalmus magnuspinnatus (mudskipper) XP_003458926.1-LOC_351.1.OR.7tm_1.exons1.NW_018354082.1.Boleophthalmus pectinirostris (great blue spotted mudskipper) XP_003458926.1-LOC_207.1.OR.7tm_1.exons1.KN462097.1.Periophthalmus magnuspinnatus (mudskipper) XP_003458926.1-LOC_333.1.OR.7tm_1.exons1.Contig_331.Neogobius melanostomus (round goby) XP_003458926.1.Oreochromis niloticus (Nile tilapia) XP_003458926.1-LOC_270.1.OR.7tm_1.exons1.chrVII.Gasterosteus aculeatus (three-spined stickleback) bioRxiv preprint doi: https://doi.org/10.1101/708974; this version posted August 6, 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.

Supplemental_Fig_S4 The round goby genome

0 Boleophthalmus pectinirostris XP 020776079.1 claudin-14-like Neogobius melanostomus 00014068 Boleophthalmus pectinirostris XP 020778469.1 claudin-4-like 0.730.044 Homo sapiens sp P57739 CLD2 Claudin-2 0.87 0.95 Neogobius melanostomus 00008797 2 Boleophthalmus pectinirostris XP 020778519.1 claudin-4-like Neogobius melanostomus 00008798 Homo sapiens sp P56750 CLD17 Claudin-17 0.520.93 0.28 Homo sapiens sp P56748 CLD8 Claudin-8 Neogobius melanostomus 00031809 0.81 Boleophthalmus pectinirostris XP 020778480.1 claudin-like 0 0 Neogobius melanostomus 00008796 0.88 Neogobius melanostomus 00013771 5 0.72 Homo sapiens sp Q96B33 CLD23 Claudin-23 0.94 Boleophthalmus pectinirostris XP 020788752.1 claudin-4-like 0 Neogobius melanostomus 00013768 2 0 Boleophthalmus pectinirostris XP 020784215.1 claudin-4-like Neogobius melanostomus 00013771 2 0 0.8 Homo sapiens sp Q8N7P3 CLD22 Claudin-22 0 Neogobius melanostomus 00013768 3 0.88 Neogobius melanostomus 00024587 0.86 Boleophthalmus pectinirostris XP 020796789.1 claudin-14-like Neogobius melanostomus 00010016 1 Boleophthalmus pectinirostris XP 020772860.1 claudin-10-like Neogobius melanostomus 000054002 0.960 Neogobius melanostomus 00026018 1 Boleophthalmus pectinirostris XP 020778788.1 claudin-15-like 0.68 0.8 Neogobius melanostomus 00004176 0.75 Boleophthalmus pectinirostris XP 020792553.1 claudin-19 0.9 Boleophthalmus pectinirostris XP 020793507.1 claudin-15 0 0 Neogobius melanostomus 00002234 Homo sapiens sp Q9NY35 CLDN1 Claudin domain-containing protein 0.36 0.86 Boleophthalmus pectinirostris XP 020786823.1 claudin-15-like 0.81 Boleophthalmus pectinirostris XP 020785871.1 claudin-7-B-like 0.83 0.69 Boleophthalmus pectinirostris XP 020796827.1 claudin-8-like 0 0.45 Neogobius melanostomus 00034332 Neogobius melanostomus 00010201 0.89 Homo sapiens sp O75508 CLD11 Claudin-11 0.93 Homo sapiens sp P56856 CLD18 Claudin-18 0.64 Boleophthalmus pectinirostris XP 020774615.1 claudin-18-like Boleophthalmus pectinirostris XP 020794856.1 claudin-10-like 0.76 0.55 Homo sapiens sp P78369 CLD10 Claudin-10 0.91 Boleophthalmus pectinirostris XP 020794860.1 claudin-10-like 0 0 0.83 Boleophthalmus pectinirostris XP 020772853.1 claudin-10-like 0.76 Neogobius melanostomus 00005400 1 0.65 Boleophthalmus pectinirostris XP 020795461.1 claudin-19-like partial 0.78 Homo sapiens sp Q8N6F1 CLD19 Claudin-19 Homo sapiens sp P56746 CLD15 Claudin-15 Homo sapiens sp O95500 CLD14 Claudin-14 0.99 Boleophthalmus pectinirostris XP 020778485.1 claudin-4-like 0.81 Neogobius melanostomus 00008800 0.75 Homo sapiens sp O95471 CLD7 Claudin-7 0 Neogobius melanostomus 00027152 0 0.8 Boleophthalmus pectinirostris XP 020772920.1 claudin-1-like Neogobius melanostomus 00029461 0.88 Boleophthalmus pectinirostris XP 020784214.1 claudin-4-like Neogobius melanostomus 00013768 1 0 Neogobius melanostomus 00013533 0.93 0.46 Boleophthalmus pectinirostris XP 020784320.1 claudin-4-like 0 1 Boleophthalmus pectinirostris XP 020778488.1 claudin-3-like Neogobius melanostomus 00008801 1 Neogobius melanostomus 00008799 2 0.61 Boleophthalmus pectinirostris XP 020796873.1 claudin-8-like 0.96 0.86 Neogobius melanostomus 00026112 0.8 0.9 Boleophthalmus pectinirostris XP 020796819.1 claudin-7-A-like 0.82 Neogobius melanostomus 00026117 2 0.99 Boleophthalmus pectinirostris XP 020784149.1 claudin-4-like 0.78 Neogobius melanostomus 00013771 3 Homo sapiens sp O95484 CLD9 Claudin-9 0.89 Boleophthalmus pectinirostris XP 020784254.1 claudin-4-like 0.82 0.9 Neogobius melanostomus 00013772 Homo sapiens sp O95832 CLD1 Claudin-1 0 Boleophthalmus pectinirostris XP 020796858.1 claudin-8-like 0.9 Boleophthalmus pectinirostris XP 020796849.1 claudin-8-like 0.72 0.77 Neogobius melanostomus 00026117A 1 0.76 1 Boleophthalmus pectinirostris XP 020784148.1 claudin-4-like 0 Neogobius melanostomus 00013771 6 Homo sapiens sp O15551 CLD3 Claudin-3 0 Boleophthalmus pectinirostris XP 020772846.1 claudin-10-like 0.99 Boleophthalmus pectinirostris XP 020786984.1 claudin-9-like 0.81 Neogobius melanostomus 00027886 Homo sapiens sp O14493 CLD4 Claudin-4 0.88 Boleophthalmus pectinirostris XP 020784216.1 claudin-4-like Neogobius melanostomus 00013771 1 0.8 Neogobius melanostomus 00008799 1 0 Boleophthalmus pectinirostris XP 020797042.1 claudin-5 isoform X1 0 Neogobius melanostomus 00012075 0.77 0.99 Boleophthalmus pectinirostris XP 020787613.1 claudin-5-like 0.78 0.81 Neogobius melanostomus 00005100 0.79 Homo sapiens sp O00501 CLD5 Claudin-5 0.73 Neogobius melanostomus 00008801 2 Neogobius melanostomus 00013771 4 0.89 Homo sapiens sp P56747 CLD6 Claudin-6 0 Boleophthalmus pectinirostris XP 020784179.1 claudin-4-like 0 0 Boleophthalmus pectinirostris XP 020778465.1 claudin-4-like 0.8 Homo sapiens sp H7C241 CLD34 Claudin-34 0 Neogobius melanostomus 00003787 Boleophthalmus pectinirostris XP 020778510.1 claudin-3-like 0.7 Boleophthalmus pectinirostris XP 020778518.1 claudin-4-like Neogobius melanostomus 00008797 1 Homo sapiens sp P56880 CLD20 Claudin-20 0.36 0.67 Homo sapiens sp C9JDP6 CLD25 Putative claudin-25 0.73 Boleophthalmus pectinirostris XP 020794871.1 claudin-10-like 0.83 Homo sapiens sp P56749 CLD12 Claudin-12 0 Homo sapiens sp Q9Y5I7 CLD16 Claudin-16 Boleophthalmus pectinirostris XP 020780034.1 claudin-11-like

Phylogenetic tree of vertebrate claudin genes. Maximum-likelihood tree with 100 bootstraps of round goby (Neogobius melanostomus, orange) in relation to great blue-spotted mudskipper (Boleophthalmus pectinirostris) and human (Homo sapiens, grey). bioRxiv preprint doi: https://doi.org/10.1101/708974; this version posted August 6, 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.

Supplemental_Fig_S5 The round goby genome

1 Lo Homo sapiens tr A1BQX2 A1BQX2 0.82 Homo sapiens tr D6RA09 D6RA09 1 1 Boleophthalmus pectinirostris XP 020793838.1 Neogobius melanostomus 00012039 Boleophthalmus pectinirostris XP 020796584.1 Danio rerio tr Q08BB8 Q08BB8 0.98 Danio rerio tr E7F2I5 E7F2I5 0.72 0.85 Boleophthalmus pectinirostris XP 020789066.1 1 Neogobius melanostomus 00018861 1 0.96 Gasterosteus aculeatus tr G3NY69 G3NY69 Oreochromis niloticus tr I3JV07 I3JV07 Homo sapiens sp Q16625 OCLN Danio rerio tr Q4KM13 Q4KM13 0.74 0.96 0.97 Gasterosteus aculeatus tr G3N596 G3N596 0.83 Boleophthalmus pectinirostris XP 020793842.1 1 Neogobius melanostomus 00012037 0.5 Danio rerio tr F1QFR8 F1QFR8 1 0.93 Gasterosteus aculeatus tr G3NI06 G3NI06 0.59 Oreochromis niloticus tr I3JW93 I3JW93 Boleophthalmus pectinirostris XP 020789581.1

Phylogenetic tree of vertebrate occludin genes. Maximum-likelihood tree with 100 bootstraps of round goby (Neogobius melanostomus, orange) in relation to great blue-spotted mudskipper (Boleophthalmus pectinirostris), stickleback (Gasterosteus aculeatus), nile tilapia (Oreochromis niloticus), zebrafish (Danio rerio), and human (Homo sapiens, grey). bioRxiv preprint doi: https://doi.org/10.1101/708974; this version posted August 6, 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.

Supplemental_Fig_S6 The round goby genome

Na+/H+ exchanger Na+-K+-ATPase alpha

0 tr_Q5PR36_Q5PR36 Danio rerio 0 tr_A0A0R4IT04_A0A0R4IT04 Danio rerio 0 tr_A9XPA5_A9XPA5 Danio rerio sp_P54707_AT12A Homo sapiens 0.8 tr_F1QHS0_F1QHS0 Danio rerio 0.87 tr_G3P2F2_G3P2F2 Gasterosteus aculeatus 0.850 tr_G3NDF8_G3NDF8 Gasterosteus aculeatus 0.93 tr_I3JF83_I3JF83 Oreochromis niloticus tr_G3NDF5_G3NDF5 Gasterosteus aculeatus 0.97 0.9 0 00002724 Neogobius melanostomus tr_I3KLH0_I3KLH0 Oroechromis niloticus XP_020792751.1 Boleophthalmus pectinirostris XP_020785393.1 Boleophthalmus pectinirostris 0.97 tr_Q9DGL5_Q9DGL5 Danio rerio sp_Q8IVB4_SL9A9 Homo sapiens tr_B0S5Q5_B0S5Q5 Danio rerio 0.73 0.075 0 XP_020774408.1 Boleophthalmus pectinirostris tr_Q90X34_Q90X34 Danio rerio 0 0.73 XP_020774407.1 Boleophthalmus pectinirostris tr_G3Q0H8_G3Q0H8 Gasterosteus aculeatus 0 0.8 0 00003765 Neogobius melanostomus 0.89 tr_I3KD16_I3KD16 Oreochromis niloticus 0 tr_I3JA90_I3JA90 Oroechromis niloticus 0 XP_020796901.1 Boleophthalmus pectinirostris tr_F1QI95_F1QI95 Danio rerio 0.87 sp_Q13733_AT1A4 Homo sapiens 0 tr_F1Q610_F1Q610 Danio rerio 0 sp_P13637_AT1A3 Homo sapiens 0.88 tr_A9XPA4_A9XPA4 Danio rerio 0 0.96 tr_G3NB58_G3NB58 Gasterosteus aculeatus 0 tr_Q4V9A5_Q4V9A5 Danio rerio tr_G3NB56_G3NB56 Gasterosteus aculeatus 0.78 0.91 0 tr_A0A0R4IZF2_A0A0R4IZF2 Danio rerio 0.84 00012929 Neogobius melanostomus sp_Q92581_SL9A6 Homo sapiens NHE6 0 XP_020793662.1 Boleophthalmus pectinirostris sp_Q96T83_SL9A7 Homo sapiens NHE7 tr_I3JBJ7_I3JBJ7 Oreochromis niloticus XP_020782715.1 Boleophthalmus pectinirostris 0 0.92 0 tr_G3P4Q7_G3P4Q7 Gasterosteus aculeatus 0 tr_A9XPA3_A9XPA3 Danio rerio 0.85 0.74 tr_G3P4S9_G3P4S9 Gasterosteus aculeatus 0.84 0 tr_F1R354_F1R354 Danio rerio 0.98 0.92 tr_G3P4R4_G3P4R4 Gasterosteus aculeatus 0 tr_F8W586_F8W586 Danio rerio 0.88 tr_I3K5Q8_I3K5Q8 Oreochromis niloticus 0.88 0 00008284 Neogobius melanostomus XP_020796248.1 Boleophthalmus pectinirostris tr_I3JPC6_I3JPC6 Oroechromis niloticus 0 tr_Q6P271_Q6P271 Danio rerio 0.92 tr_I3JPC7_I3JPC7 Oroechromis niloticus 0 0.84 tr_Q9DEY2_Q9DEY2 Danio rerio tr_G3QAV3_G3QAV3 Gasterosteus aculeatus 0 0 tr_Q9DEU2_Q9DEU2 Danio rerio tr_G3PYY9_G3PYY9 Gasterosteus aculeatus tr_Q4KMK4_Q4KMK4 Danio rerio 0 0 tr_A0A0R4IX40_A0A0R4IX40 Danio rerio sp_P50993_AT1A2 Homo sapiens 0 tr_F1QSJ6_F1QSJ6 Danio rerio 0 sp_P05023_AT1A1 Homo sapiens 0.85 tr_A4QP93_A4QP93 Danio rerio 0.96 tr_Q9DEU1_Q9DEU1 Danio rerio 0 0.079 00008682 Neogobius melanostomus tr_B0R068_B0R068 Danio rerio 0 XP_020779128.1 Boleophthalmus pectinirostris 0 0.81 tr_B8JKT0_B8JKT0 Danio rerio XP_020779124.1 Boleophthalmus pectinirostris 0.9 tr_Q7T2D6_Q7T2D6 Danio rerio 0 tr_I3J3A8_I3J3A8 Oroechromis niloticus 0 0.81 tr_Q9DEY3_Q9DEY3 Danio rerio tr_I3J3A7_I3J3A7 Oroechromis niloticus 0.99 tr_B3DFZ2_B3DFZ2 Danio rerio tr_I3JEY2_I3JEY2 Oroechromis niloticus tr_Q8JIV1_Q8JIV1 Danio rerio sp_Q6AI14_SL9A4 Homo sapiens NHE4 0025176 Neogobius melanostomus tr_B7ZV95_B7ZV95 Danio rerio 0 0.89 0 XP_020792263.1 Boleophthalmus pectinirostris 0 tr_B0S734_B0S734 Danio rerio 0.8 0 tr_I3K3B9_I3K3B9 Oreochromis niloticus 0 tr_A0A0R4IF22_A0A0R4IF22 Danio rerio 0.88 tr_I3K3G3_I3K3G3 Oreochromis niloticus 0.69 tr_A9XPA0_A9XPA0 Danio rerio 0.74 0.99 tr_R9WR11_R9WR11_9CICH tr_G3PQ84_G3PQ84 Gasterosteus aculeatus 0.76 0.76 tr_Q9DEU0_Q9DEU0 Danio rerio XP_020797456.1 Boleophthalmus pectinirostris 0 0 0.92 tr_B8JKS7_B8JKS7 Danio rerio 0.91 00035431 Neogobius melanostomus 0 tr_Q90X33_Q90X33 Danio rerio XP_020794800.1 Boleophthalmus pectinirostris 0 0.75 tr_Q9DGL4_Q9DGL4 Danio rerio 0.86 tr_I3K9T8_I3K9T8 Oroechromis niloticus 0 0 0.89 tr_B8JKS9_B8JKS9 Danio rerio tr_G3NEX4_G3NEX4 Gasterosteus aculeatus 0.99 tr_Q503J4_Q503J4 Danio rerio sp_Q9UBY0_SL9A2 Homo sapiens NHE2 tr_A0A0R4IJ10_A0A0R4IJ10 Danio rerio 00015753 Neogobius melanostomus 0 tr_I3K395_I3K395 Oreochromis niloticus 0 XP_020773382.1 Boleophthalmus pectinirostris 0.94 tr_I3K372_I3K372 Oreochromis niloticus 0.96 tr_G3Q3R6_G3Q3R6 Gasterosteus aculeatus 0 tr_G3PMS8_G3PMS8 Gasterosteus aculeatus tr_I3KMB0_I3KMB0 Oroechromis niloticus 0 0.820.89 tr_G3PMS4_G3PMS4 Gasterosteus aculeatus 0.98 00014717 Neogobius melanostomus 0.84 tr_G3PMT3_G3PMT3 Gasterosteus aculeatus 00032425 Neogobius melanostomus 0 0.84 tr_G3PMU0_G3PMU0 Gasterosteus aculeatus tr_I3KHH2_I3KHH2 Oroechromis niloticus 0 tr_A0A0R4IRT1_A0A0R4IRT1 Danio rerio 0 tr_I3K8I6_I3K8I6 Oroechromis niloticus 0 tr_I3K3J4_I3K3J4 Oreochromis niloticus 0.92 sp_P19634_SL9A1 Homo sapiens NHE1 0 00036611 Neogobius melanostomus 0.99 tr_F1QR72_F1QR72 Danio rerio 0 0 00025177 Neogobius melanostomus tr_A0A0G2KZI8_A0A0G2KZI8 Danio rerio 0 0 XP_020792240.1 Boleophthalmus pectinirostris 0.76 0.73 0 00001997 Neogobius melanostomus 0 tr_A9XP99_A9XP99 Danio rerio 0.93 tr_Q9DGL6_Q9DGL6 Danio rerio 0 XP_020790001.1 Boleophthalmus pectinirostris tr_Q7ZU25_Q7ZU25 Danio rerio tr_G3NC67_G3NC67 Gasterosteus aculeatus 1 XP_020796885.1 Boleophthalmus pectinirostris 0.26 sp_P98194_AT2C1 Homo sapiens 0 tr_I3KML0_I3KML0 Oroechromis niloticus 00025762 Neogobius melanostomus 0 0 tr_G3PK51_G3PK51 Gasterosteus aculeatus 0.95 0 XP_020795955.1 Boleophthalmus pectinirostris sp_O14983_AT2A1 Homo sapiens 00017961 Neogobius melanostomus sp_P20020_AT2B1 Homo sapiens 0 tr_F1R610_F1R610 Danio rerio 0 tr_A9XPA2_A9XPA2 Danio rerio 0 00018498 Neogobius melanostomus 0.7 0 00018498_N-trunk Neogobius melanostomus 0 tr_G3PZW9_G3PZW9 Gasterosteus aculeatus 0.87 XP_020780200.1 Boleophthalmus pectinirostris tr_I3J5H3_I3J5H3 Oroechromis niloticus 0.98 sp_Q14940_SL9A5 Homo sapiens NHE5

0 0.82 XP_020789995.1 Boleophthalmus pectinirostris 00002007 Neogobius melanostomus 0.95 tr_G3NCZ3_G3NCZ3 Gasterosteus aculeatus tr_A9XP97_A9XP97 Danio rerio 0 0 tr_A3KPJ8_A3KPJ8 Danio rerio 0 tr_I3K858_I3K858 Oroechromis niloticus 0 tr_I3K859_I3K859 Oroechromis niloticus 0 tr_Q9DGI4_Q9DGI4 Danio rerio 0.4 0 tr_I3K857_I3K857 Oroechromis niloticus 0 tr_Q8QHI1_Q8QHI1 Danio rerio tr_I3K860_I3K860 Oroechromis niloticus 0.38 1 0 tr_F1QYB9_F1QYB9 Danio rerio tr_A9XPA1_A9XPA1 Danio rerio 0.99 0.95 tr_Q90266_Q90266 Danio rerio tr_B7ZVJ6_B7ZVJ6 Danio rerio 0.76 tr_A0A0R4IE14_A0A0R4IE14 Danio rerio tr_I3JWW2_I3JWW2 Oroechromis niloticus 1 1 0.36 tr_G3PT60_G3PT60 Gasterosteus aculeatus 0.99 Neogobius melanostomus 00029449-RA XP_020772934.1 Bolephthalmus pectinirostris 0.84 XP_020796651.1 Bolephthalmus pectinirostris 0.89 tr_I3KC45_I3KC45 Oroechromis niloticus tr_Q90Z34_Q90Z34 Danio rerio XP_020796323.1 Bolephthalmus pectinirostris 0.98 0.75 tr_G3N891_G3N891 Gasterosteus aculeatus tr_I3JHR1_I3JHR1 Oroechromis niloticus 0.99 0.99 1 tr_I3JHR0_I3JHR0 Oroechromis niloticus

1 tr_A0A0R4IAE6_A0A0R4IAE6 Danio rerio 0 tr_A0A0R4IEL3_A0A0R4IEL3 Danio rerio 0.6 tr_Q9DGL2_Q9DGL2 Danio rerio 0.36 1 tr_G3QAN7_G3QAN7 Gasterosteus aculeatus 0.18 tr_G3QAN6_G3QAN6 Gasterosteus aculeatus 0.85 XP_020778771.1 Bolephthalmus pectinirostris 0.99 Neogobius melanostomus 00026002-RA 1 tr_I3J8Y6_I3J8Y6 Oroechromis niloticus Na+-K+-ATPase beta tr_I3J8Y5_I3J8Y5 Oroechromis niloticus 1 tr_G3PRI4_G3PRI4 Gasterosteus aculeatus 0.95 tr_G3PRI3_G3PRI3 Gasterosteus aculeatus 0.26 tr_I3J546_I3J546 Oroechromis niloticus 1 XP_020794724.1 Bolephthalmus pectinirostris Neogobius melanostomus 00020908-RA tr_E7F9R1_E7F9R1 Danio rerio 1 1 tr_I3J2V6_I3J2V6 Oroechromis niloticus 0.91 tr_I3J2V5_I3J2V5 Oroechromis niloticus 1 XP_020779213.1 Boleophthalmus pectinirostris 0.36 Neogobius melanostomus 00028573-RA tr_I3KT86_I3KT86 Oroechromis niloticus 1 0.91 Neogobius melanostomus 00003737-RA XP_020774501.1 Bolephthalmus pectinirostris 0.86 tr_G3PKB5_G3PKB5 Gasterosteus aculeatus tr_Q6NYH5_Q6NYH5 Danio rerio 0.89 1 0.27 tr_Q9DGL3_Q9DGL3 Danio rerio tr_Q7ZVX2_Q7ZVX2 Danio rerio 0.83 1 tr_F1Q6G8_F1Q6G8 Danio rerio tr_Q9DEY4_Q9DEY4 Danio rerio 0.71 0.96 XP_020797469.1 Bolephthalmus pectinirostris 0.66 Neogobius melanostomus 00022721-RA 0.93 tr_I3J761_I3J761 Oroechromis niloticus 0.6 1 tr_G3PRG7_G3PRG7 Gasterosteus aculeatus tr_G3PRF6_G3PRF6 Gasterosteus aculeatus

Phylogenetic tree of vertebrate ion transporters. Maximum-likelihood tree with 100 bootstraps of round goby (Neogobius melanostomus, orange) in relation to great blue-spotted mudskipper (Boleophthalmus pectinirostris), stickleback (Gasterosteus aculeatus), nile tilapia (Oreochromis niloticus), zebrafish (Danio rerio), and human (Homo sapiens, grey) bioRxiv preprint doi: https://doi.org/10.1101/708974; this version posted August 6, 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.

Supplemental_Fig_S7 The round goby genome

MIPS 0.83 Neogobius melanostomus 00015537 Bolephthalmus pectinirostris XP_020775377.1 0.94 Gasterosteus aculeatus tr_G3P635 Oreochromis niloticus tr_I3IXX3 Homo sapiens sp_Q9NPH2_INO1

0.1

IMPA 1 Neogobius melanostomus 00006008 0.9 Bolephthalmus pectinirostris XP_020786419.1

1 0.95 Gasterosteus aculeatus tr_G3NMA2 Gasterosteus aculeatus tr_G3NMA6 Oreochromis niloticus tr_I3JGL5 0.91 0.97 Gasterosteus aculeatus tr_G3ND95 0.63 Oreochromis niloticus tr_I3K906 0.082 Bolephthalmus pectinirostris XP_020796829.1 0.98 Danio rerio tr_Q6DGB2 Danio rerio tr_B0S5J9 0.95 Bolephthalmus pectinirostris XP_020786424.1 0.44 Neogobius melanostomus 00006010 1 Oreochromis niloticus tr_I3JGM2 0.27 Gasterosteus aculeatus tr_G3NMF7 0.91 Homo sapiens tr_A0A140VJL8 Danio rerio tr_Q503S9_

0.3

SMIT

1 Bolephthalmus pectinirostris XP_020773420.1 0.97 Bolephthalmus pectinirostris XP_020795862.1 0.65 Bolephthalmus pectinirostris XP_020783330.1 1 Bolephthalmus pectinirostris XP_020788626.1 0.96 Bolephthalmus pectinirostris XP_020788177.1 Homo sapiens sp_Q8WWX8_SC5AB Danio rerio tr_B8JHU7 Homo sapiens sp_P53794_SC5A3 1 0.72 Danio rerio tr_E9QDC2 0.91 0.91 Bolephthalmus pectinirostris XP_020787565.1 0.97 0.71 augustus_masked-Neogobius melanostomus 48-processed-gene-8.6-mRNA-1 0.29 Oreochromis niloticus tr_I3KXY0 Gasterosteus aculeatus 0.2 tr_G3NHQ8 0.93 Oreochromis niloticus tr_I3KDP7 1 Bolephthalmus pectinirostris XP_020783839.1 Neogobius melanostomus ORF_194840_L_2061_GC_48.7

Phylogenetic tree of vertebrate genes promoting osmolyte production. Maximum-likelihood tree with 100 bootstraps of round goby (Neogobius melanostomus, orange) in relation to great blue-spotted mudskipper (Boleophthalmus pectinirostris), stickleback (Gasterosteus aculeatus), nile tilapia (Oreochromis niloticus), zebrafish (Danio rerio), and human (Homo sapiens, grey) bioRxiv preprint doi: https://doi.org/10.1101/708974; this version posted August 6, 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.

Supplemental_Fig_S8 The round goby genome

Lepisosteus oculatus ABCB9 ENSLOCG00000005562 ENSLOCT00000006724 Poecilia formosa TAP2T ENSPFOG00000018394 ENSPFOT00000018542 Xiphophorus maculatus TAP2T ENSXMAG00000014741 ENSXMAT00000014798 100 100 TAP2T Takifugu rubripes TAP2T ENSTRUG00000010603 ENSTRUT00000026877 100 Gasterosteus aculeatus TAP2T ENSGACG00000004139 ENSGACT00000005455 100 Neogobius melanostomus TAP2T Contig 2864 GOBY 693836-723297 75 Tetraodon nigroviridis TAP2T ENSTNIG00000011842 ENSTNIT00000014998 48 Oreochromis niloticus TAP2T ENSONIG00000008663 ENSONIT00000010899 47 Danio rerio TAP2T ENSDARG00000033446 ENSDART00000153401 100 Latimeria chalumnae TAP2T ENSLACG00000013948 ENSLACT00000015949

Latimeria chalumnae TAP1 ENSLACG00000014125 ENSLACT00000016151 Poecilia formosa TAP1 ENSPFOG00000007480 ENSPFOT00000007589 TAP1 100 Neogobius melanostomus TAP1 Contig 1786 GOBY 3696645-3705080 100 Astyanax mexicanus TAP1 ENSAMXG00000017074 ENSAMXT00000017573 100 86 Oryzias latipes TAP1 ENSORLG00000016158 ENSORLT00000020221 48 Oreochromis niloticus TAP1 ENSONIG00000018168 ENSONIT00000022909 100 78 Danio rerio TAP1 ENSDARG00000079766 ENSDART00000150156 97 Gasterosteus aculeatus TAP1 ENSGACG00000013054 ENSGACT00000017303 Xiphophorus maculatus TAP1 ENSXMAG00000018614 ENSXMAT00000018691 100 Neogobius melanostomus TAP2 rev Contig 2107 GOBY 2986109-2994269 Oreochromis niloticus TAP2 ENSONIG00000020042 ENSONIT00000025265 10048 Danio rerio TAP2 ENSDARG00000036787 ENSDART00000031380 TAP2 0.3 100 82 Gasterosteus aculeatus TAP2 ENSGACG00000000135 ENSGACT00000000178 Gasterosteus aculeatus TAP2 ENSGACG00000000128 ENSGACT00000000169 34 Poecilia formosa TAP2 ENSPFOG00000003049 ENSPFOT00000003025 100 Takifugu rubripes TAP2 ENSTRUG00000003771 ENSTRUT00000008905

Phylogenetic tree of fish TAP genes. Maximum-likelihood tree with 500 bootstraps. Round goby (Neogobius melanostomus) is labeled orange. Outgroup: Lepistosteus oculatus ABC transporter. bioRxiv preprint doi: https://doi.org/10.1101/708974; this version posted August 6, 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.

Supplemental_Fig_S9 The round goby genome

Partial sequence overlap between contigs Contig_3340 Contig_4064 antisense_1-28711 bp antisense_368544-387339 bp end of contig end of contig 24354-28711 1-18725 368544-387339

IgM/D IgM 1 IgM 2 IgM 3 IgD 1 IgD 2 IgD 3 IgD 4 IgD 5 IgD 6 IgD 7 IgD 8

The Ig locus spans two contigs and contains IgM and IgD domains. bioRxiv preprint doi: https://doi.org/10.1101/708974; this version posted August 6, 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.

Supplemental_Fig_S10 The round goby genome

Periophthalmodon schlosseri JACM01076057.1 5267-8338 endscf

Neogobius melanostomus NEME 589 34823-39929 Periophthalmus magnuspinnatus KN461375.1 42951-46379 rev Boleophthalmus pectinirostris KN525449.1 9045-15080 noTIR

Periophthalmodon schlosseri KN485528.1 72662-76041 rev

Boleophthalmus pectinirostris KN523599.1 423653-427329 rev

Neogobius melanostomus NEME 589 26151-32607 Neogobius melanostomus NEME Neogobius905 214042-220426 melanostomus NEMEmissing 219 TIR7749-13602 rev Neogobius melanostomus NEMENeogobius 219 53016-58870 melanostomus NEME rev 591 58000-66017 Scartelaos histophorus KN511955.1 1-2535 rev Neogobius melanostomussuibogoeN NEME 904 120879-130318Neogobius missing melanostomussumotsonalem TIR NEME 591EMEN 75143-81627 572 286256-328846

Neogobius melanostomus NEME 218 88776-102838

Neogobius melanostomus NEME 421 1324405-1328761 missingTIR

Neogobius melanostomus NEME 425 83723-91189 rev

Neogobius melanostomus NEME 220 90224-96678 rev Neogobius melanostomus NEME 220 100000-106983 rev Neogobius melanostomus NEME 421 1143202-1149101 missing small piece Neogobius melanostomus NEME 420 599030-604249

Neogobius melanostomus NEME 422 54330-59545 Scartelaos histophorus KN517762.1 4170-9588

Boleophthalmus pectinirostris KN522985.1 775076-779804 0 7 9 8 5-9 8 8 3 5 1 9 5 E M E N su m otson ale m suib og oe N Neogobius melanostomus NEME 62 9292-19167 indels Neogobius melanostomus NEME 275 652829-662120 Boleophthalmus pectinirostris KN522985.1 779913-784431

100

98 100 100 Neogobius melanostomus NEME 591 121397-129072

100 99 NeogobiusBoleophthalmus melanostomus pectinirostris NEME 592 KN524665.1 43710-51365 291182-294509 rev

99 Neogobius melanostomus NEME 428 560000-568859 100 Periophthalmus magnuspinnatus KN465702.1 13179-16828 rev

100 100 Boleophthalmus pectinirostris KN523599.1 415005-418430 rev Periophthalmodon schlosseri JACM01014076.1 1-2876 rev 100 100 92 100 Scartelaos histophorus KN518146.1 1-2270 rev endscf

100 49 Periophthalmodon schlosseri JACM01014076.1 6988-10909 rev Scartelaos histophorus KN510011.1 1-4558 rev endscf 100 86 66 100 Neogobius melanostomus NEME 591 43284-48607 99 63 100 48 Neogobius melanostomus NEME 589 48061-53303 missing small piece 67 Periophthalmus magnuspinnatus KN464753.1 23376-26151 100 30 100 Periophthalmus magnuspinnatus KN461375.1 35364-38560 Periophthalmodon schlosseri JACM01007922.1 1-6953 rev endscf notTIR 71 84 Scartelaos histophorus JACN01062416.1 1-3963 rev 100 54 99 Periophthalmodon schlosseri KN469087.1 14886-18682 Boleophthalmus pectinirostris KN521858.1 3700-8604 rev noTIR 100 100 100 Boleophthalmus pectinirostris KN525449.1 16565-20363 noTIR 99 Periophthalmus magnuspinnatus KN464753.1 6708-11934 rev 99 100 Neogobius melanostomus NEME 591 68100-73292 79 45 Neogobius melanostomus NEME 589 10285-15324 100 95 78 Neogobius melanostomus NEME 591 113421-118500 Periophthalmodon schlosseri KN469087.1 1-4944 rev 100 100 100 Neogobius melanostomus NEME 592 35362-40507

Boleophthalmus pectinirostris KN521980.1 1638-4365 rev noTIR 100 Neogobius melanostomus NEME 592 27556-32696 100 Neogobius melanostomus NEME 592 13327-17777 98 91 0.799 98 Boleophthalmus pectinirostris KN521980.1 18744-23406 Neogobius melanostomus NEME 591 87605-95849 90 100 40 99 Neogobius melanostomus NEME 592 5439-10472 100 93 Periophthalmodon schlosseri KN472561.1 5104-10861 rev endscf Neogobius melanostomus NEME 591 83518-88605 100 91 Neogobius melanostomus NEME 589 19142-24248 56 Periophthalmus magnuspinnatus KN465791.1 18038-24309 rev 70 Neogobius melanostomus NEME 591 95849-110708 100 46 100 Neogobius melanostomus NEME 592 16777-25791 52 99 Boleophthalmus pectinirostris KN524893.1 3242813-3246424 100 Danio rerio TLR26b 93 100 48 Danio rerio TLR26a 88 100 Scartelaos histophorus KN513524.1 1-3086 rev endscf 100 Astyanax mexicanus TLR26 99 TLR26 100 100 80 Latimeria chalumnaeGasterosteus TLR21 baculeatus TLR21 b 100 45 97 Periophthalmus magnuspinnatus TLR21a Oreochromis niloticus TLR23 b 100 Neogobius melanostomus NEME 387 3297687-3303353 59 100 100 Periophthalmus magnuspinnatus TLR21b Poecilia formosa TLR23 c LatimeriaPeriophthalmus chalumnae TTLR21 magnuspinnatus a TLR21c

100 Astyanax mexicanus TLR21 Poecilia formosa TLR23 b 100 100 100 45 100 Danio rerio TLR21 71 100 Poecilia formosa TLR23 a 100 44 32 99 28 100 21 Neogobius melanostomus NEME 33 3513131-3517961 rev 100 Neogobius melanostomus NEME 36 81864-86305 rev 32 Xiphophorus maculatus TLR23 93 100 Takifugu rubripes TLR21 100 Takifugu rubripes TLR23 54 52 Tetraodon nigroviridis TLR21 Gasterosteus aculeatus TLR21 a 001 100 Oreochromis niloticus TLR21

55 39 100 Gasterosteus aculeatus TLR22 Poecilia formosa TLR21 Tetraodon nigroviridis TLR23 95 100 100 Takifugu rubripes TLR22 TLR21

100 Tetraodon nigroviridis TLR22 100 100 Oryzias latipes TLR22 Oryzias latipes TLR21 100 Oreochromis niloticus TLR22

100 100 100 PoeciliaXiphophorus formosa TLR22 maculatus TLR22a 87 100 Xiphophorus maculatus TLR22b 100 100 Neogobius melanostomus NEME 34 2141440-2150656 rev 100

100

100 Neogobius melanostomus NEME 34 2112375-2123097 rev Neogobius melanostomus NEME 34 2125314-2132370 rev

Periophthalmodon schlosseri KN483411.1 44254-47119

3716921-1050921 1.148125NK sirtsorinitcep sumlahthpoeloB sirtsorinitcep 1.148125NK 3716921-1050921 Periophthalmus magnuspinnatus KN467472.1 46754-53789 rev Boleophthalmus pectinirostris KN523573.1 415711-427643 rev Astyanax mexicanus TLR23 c AstyanaxLepisosteus mexicanus oculatus TLR23 TLR23b Periophthalmodon schlosseri JACM01029142.1 1-3778 endscf Periophthalmus magnuspinnatus KN464467.1 18015-20835 Astyanax mexicanus TLR23 a

s o aletra c S Boleophthalmus pectinirostris KN525645.1 49077-54885 TLR23

Neogobius melanostomus NEME 310 3033417-3041179 rev

Neogobius melanostomus NEME 310 3023003-3028748 rev

0 3 0 1 0 N C A J suro h p otsih

51 1.429

Boleophthalmus pectinirostris KN525591.1 94353-101283 rev 1

Scartelaos Periophthalmodonhistophorus JACN01102642.1 schlosseri JACM01015485.1 1946-5376 endscf 1-5695 rev Scartelaos histophorus KN506245.1 4744-9235

81-0

Periophthalmus magnuspinnatus KN466808.1 129592-134594 130

Neogobius melanostomus NEME 1348 29641-50380 rev Boleophthalmus pectinirostris KN522700.1 840192-844087 rev Periophthalmodon schlosseri KN469728.1 2068-6190 rev

Boleophthalmus pectinirostris KN522700.1 834783-838999 rev Boleophthalmus pectinirostris KN522735.1Periophthalmus 585-4410 magnuspinnatus smallindel KN461469.1 42612-46730 Periophthalmodon schlosseri KN478351.1 1709-5643 endscf

Periophthalmus magnuspinnatus KN465255.1 493311-498074

Neogobius melanostomus NEME 1348 17940-23154 rev missing last exon TLR22

0.3

Phylogenetic tree of Gobiidae Toll Like Receptor protein sequences. A maximum likelihood phylogenetic tree run with the JTT substitution model and 500 bootstrap replicates on the transmembrane, linker and TIR domain of all TLRs found in Gobiiformes (Neogobius melanostomus, orange; other Gobiidae, black) and selected other fish (grey) for context. bioRxiv preprint doi: https://doi.org/10.1101/708974; this version posted August 6, 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.

Supplemental_Fig_S11 The round goby genome

100 CRP_Homo sapiens (human) CRP_Mus musculus (mouse) 79 100 APCS_Mus musculus (mouse) 61 APCS_Homo sapiens (human) 100 MTPX2_pentaxin_Mus musculus (mouse) MTPX1_pentaxin_Mus musculus (mouse) Latimeria chalumnae (West Indian ocean coelacanth) 100 Latimeria chalumnae (West Indian ocean coelacanth) Latimeria chalumnae (West Indian ocean coelacanth) Latimeria chalumnae (West Indian ocean coelacanth) 60 Tetraodon nigroviridis (pufferfish) 100 Oroechromis niloticus (Nile tilapia) 79 Latimeria chalumnae (West Indian ocean coelacanth) 78 Lepisosteus oculatus (spotted gar) Gadus morhua (Atlantic cod) 57 99 Neogobius melanostomus (round goby) 100 Neogobius melanostomus (round goby) 83 Oroechromis niloticus (Nile tilapia) 100 37 66 76 Oroechromis niloticus (Nile tilapia) 38 Oroechromis niloticus (Nile tilapia) Poecilia formosa (Amazon molly) 45 Tetraodon nigroviridis (pufferfish) 52 Tetraodon nigroviridis (pufferfish) Tetraodon100 nigroviridis (pufferfish) 36 68 Tagifugu rubripes (Japanese pufferfish) 85 Tagifugu rubripes (Japanese pufferfish) Danio rerio (zebrafish) 100 Danio rerio (zebrafish) 92 Danio rerio (zebrafish) 78 Danio rerio (zebrafish) 23 44 Danio rerio (zebrafish) 64 Astyanax mexicanus (blind cave fish) 100 Astyanax mexicanus (blind cave fish) 32 Astyanax mexicanus (blind cave fish) 96 Danio rerio (zebrafish) 93 Neogobius melanostomus (round goby) Tetraodon nigroviridis (pufferfish) 10052Tagifugu rubripes (Japanese pufferfish) 18 Gadus morhua (Atlantic cod) 54 Orizias latipes (medaka) 23 Oroechromis niloticus (Nile tilapia) 10 Gasterosteus aculeatus (three-spined stickleback) 52 Poecilia formosa (Amazon molly) 98Xiphophorus maculatus (Southern platyfish) Lepisosteus oculatus (spotted gar) 100 Lepisosteus oculatus (spotted gar) 60 Oroechromis niloticus (Nile tilapia) Danio rerio (zebrafish) 84 Tetraodon nigroviridis (pufferfish) 100 Tagifugu rubripes (Japanese pufferfish) 100 49 Tagifugu rubripes (Japanese pufferfish) Tagifugu72 rubripes (Japanese pufferfish) Gadus morhua (Atlantic cod) Gadus morhua (Atlantic cod) 81 99 Gadus morhua (Atlantic cod) 100Gadus morhua (Atlantic cod) Oroechromis niloticus (Nile tilapia) 21 25 Oroechromis niloticus (Nile tilapia) 44 Poecilia formosa (Amazon molly) 65 95 Xiphophorus maculatus (Southern platyfish) 96 65 Xiphophorus maculatus (Southern platyfish) 24 Poecilia formosa (Amazon molly) Neogobius melanostomus (round goby) 91 Neogobius melanostomus (round goby) 49 Neogobius melanostomus (round goby) 31 94 Neogobius melanostomus (round goby) 35 Neogobius melanostomus (round goby) 24 Neogobius melanostomus (round goby) 32 Neogobius melanostomus (round goby) 100Neogobius melanostomus (round goby) Poecilia formosa (Amazon molly) 100Xiphophorus maculatus (Southern platyfish) Gasterosteus aculeatus (three-spined stickleback) 100Gasterosteus aculeatus (three-spined stickleback) 70 60 Orizias latipes (medaka) 100 Orizias latipes (medaka) 100 Orizias latipes (medaka) 46 Oroechromis niloticus (Nile tilapia) 100Oroechromis niloticus (Nile tilapia) 28 Poecilia formosa (Amazon molly) 100Xiphophorus maculatus (Platyfish) 10 Tetraodon nigroviridis (pufferfish) 99 Tagifugu rubripes (Japanese pufferfish) Neogobius melanostomus (round goby) 50 89 Neogobius melanostomus (round goby) 98 Neogobius melanostomus (round goby) 70 Neogobius melanostomus (round goby) 99 Neogobius melanostomus (round goby) 83 Neogobius melanostomus (round goby) 100 Neogobius melanostomus (round goby) 87 Neogobius melanostomus (round goby) 99 Neogobius melanostomus (round goby) 96 Neogobius melanostomus (round goby) 67Neogobius melanostomus (round goby) 100 Neogobius melanostomus (round goby) 98 95Neogobius melanostomus (round goby) 0.3 Neogobius melanostomus (round goby)

Phylogenetic tree of fish CRP/APCS sequences. Maximum Likelihood phylogenetic tree with 500 bootstraps rooted at the split between tetrapods and ray-finned fish. Tetrapods were used as outgroup and are indicated in grey. Round goby is indicated in orange. bioRxiv preprint doi: https://doi.org/10.1101/708974; this version posted August 6, 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.

Supplemental_Fig_S12 The round goby genome

EED C_milii_lcl_NW_006890057_1_cds_XP_007900557_1_1961__gene_eed_ A_carolinensis_lcl_NC_014778_1_cds_XP_003219406_1_11052__gene_eed_ 0.91 G_gallus_lcl_NC_006088_4_cds_NP_001026547_1_5392__db_xref_GeneID_426381_ X_laevis_lcl_NC_030726_1_cds_NP_001089517_1_11030__gene_eed_L_ 1 1 X_laevis_lcl_NC_030727_1_cds_NP_001082354_1_14041__gene_eed_S_ 0.69 X_tropicalis_lcl_NC_030678_1_cds_NP_001017325_1_9358__gene_eed_ 0.9 I_punctatus_lcl_NC_030441_1_cds_XP_017313699_1_42921__gene_eed_ 0.98 A_mexicanus_lcl_NC_035908_1_cds_XP_007254895_1_14566__gene_eed_ 1 C_carpio_lcl_NW_017545551_1_cds_XP_018958640_1_63569__gene_eed_ D_rerio_lcl_NC_007112_7_cds_NP_001017766_2_7__gene_eed_ 0.96 L_oculatus_lcl_NC_023195_1_cds_XP_006639249_1_32158__db_xref_GeneID_102685474_ 0.99 S_formosus_lcl_NW_017371699_1_cds_XP_018603581_1_20192__gene_eed_ 0.99 1 S_formosus_lcl_NW_017371699_1_cds_XP_018603582_1_20193__gene_eed_ 0.95 C_harengus_lcl_NW_012220521_1_cds_XP_012678797_1_11521__gene_eed_ E_lucius_lcl_NC_025989_3_cds_XP_010884408_1_44258__gene_eed_ 1 0.2 0.55 S_salar_lcl_NC_027315_1_cds_XP_014006483_1_61375__db_xref_GeneID_106574878_ 0.94 B_pectinirostris_lcl_NW_018348300_1_cds_XP_020797464_1_3071__gene_eed_ 1 EED_mRNA_20171128_putative_after_RACE 1 T_rubripes_lcl_NC_018897_1_cds_XP_003966763_1_9370__db_xref_GeneID_101076119_ O_latipes_lcl_NC_019860_1_cds_XP_011479497_1_1654__gene_eed_ 1 0.94 O_latipes_lcl_NC_019860_1_cds_NP_001098326_1_1653__gene_eed_ O_niloticus_lcl_NC_031986_1_cds_XP_003459967_1_50744__gene_eed_ 0.92 0.8 N_coriiceps_lcl_NW_011339380_1_cds_XP_010767513_1_6357__gene_eed_ 0.82 C_variegatus_lcl_NW_015150606_1_cds_XP_015232873_1_10717__gene_eed_ 1 P_latipinna_lcl_NW_015114037_1_cds_XP_014912452_1_31495__gene_eed_ 1 P_reticulata_lcl_NC_024332_1_cds_XP_008422393_1_2796__gene_eed_ SUZ12 C_milii_lcl_NW_006890063.1_cds_XP_007886606.1_4406__gene_suz12_ 1C_milii_lcl_NW_006890063.1_cds_XP_007886605.1_4405__gene_suz12_ G_gallus_lcl_NC_006105.4_cds_XP_004946226.1_33525__db_xref_GeneID_417406_ 1 A_carolinensis_lcl_NW_003338745.1_cds_XP_008112590.1_19460__gene_suz12_ 1 X_tropicalis_lcl_NW_016683713.1_cds_NP_001072292.1_37989__gene_suz12_ 1X_tropicalis_lcl_NW_016683713.1_cds_XP_012822185.1_37990__gene_suz12_ 1 X_laevis_lcl_NC_030741.1_cds_NP_001124416.1_51559__gene_suz12.S_ 1X_laevis_lcl_NC_030741.1_cds_XP_018092618.1_51558__gene_suz12.S_ 0.63X_laevis_lcl_NC_030741.1_cds_XP_018092619.1_51557__gene_suz12.S_ 1 L_oculatus_lcl_NC_023188.1_cds_XP_015211984.1_23445__db_xref_GeneID_102688802_ SUZ12_mRNA_20171128_putative_after_RACE_NEME386 0.55 C_carpio_lcl_NW_017538041.1_cds_XP_018931038.1_38788__gene_LOC109058280_ 1 D_rerio_lcl_NC_007114.7_cds_NP_001003529.1_6083__gene_suz12a_ 1 1D_rerio_lcl_NC_007114.7_cds_XP_021327339.1_6082__gene_suz12a_ C_harengus_lcl_NW_012219739.1_cds_XP_012674006.1_7192__gene_suz12_ A_mexicanus_lcl_NC_035909.1_cds_XP_022530939.1_14975__gene_suz12_ 1 1 I_punctatus_lcl_NC_030427.1_cds_XP_017337919.1_21781__gene_suz12_ 0.98 1I_punctatus_lcl_NC_030427.1_cds_XP_017337918.1_21780__gene_suz12_ 1 C_carpio_lcl_NC_031708.1_cds_XP_018961624.1_8633__gene_LOC109092321_ D_rerio_lcl_NC_007117.7_cds_NP_001076293.2_12801__gene_suz12b_ 1 D_rerio_lcl_NC_007117.7_cds_XP_021332567.1_12802__gene_suz12b_ 1D_rerio_lcl_NC_007117.7_cds_XP_021332565.1_12799__gene_suz12b_ 1 D_rerio_lcl_NC_007117.7_cds_XP_021332566.1_12800__gene_suz12b_ D_rerio_lcl_NW_018395252.1_cds_NP_001076293.2_55853__gene_suz12b_ 1D_rerio_lcl_NW_018394637.1_cds_NP_001076293.2_50281__gene_suz12b_ E_lucius_lcl_NC_025976.3_cds_XP_010871257.1_16917__gene_suz12_ 1 0.2 E_lucius_lcl_NC_025976.3_cds_XP_010871260.1_16919__gene_suz12_ B_pectinirostris_lcl_NW_018360135.1_cds_XP_020794126.1_22728__gene_suz12_ 1 1 SUZ12_mRNA_20171206_after_RACE_RT_NEME4 O_niloticus_lcl_NC_031971.1_cds_XP_005470002.1_13223__gene_suz12_ 1 1 O_niloticus_lcl_NC_031971.1_cds_XP_005470003.1_13224__gene_suz12_ O_latipes_lcl_NC_019859.1_cds_XP_004065843.1_456__gene_suz12_ 0.86 O_latipes_lcl_NC_019859.1_cds_XP_020556824.1_453__gene_suz12_ 1O_latipes_lcl_NC_019859.1_cds_XP_011472328.1_454__gene_suz12_ O_latipes_lcl_NC_019859.1_cds_XP_011472323.1_455__gene_suz12_ 0.61 T_rubripes_lcl_NC_018906.1_cds_XP_003972026.2_17938__db_xref_GeneID_101078630_ N_coriiceps_lcl_NW_011346291.1_cds_XP_010772407.1_10753__gene_suz12_ 0.95 N_coriiceps_lcl_NW_011346291.1_cds_XP_010772408.1_10751__gene_suz12_ 1N_coriiceps_lcl_NW_011346291.1_cds_XP_010772405.1_10750__gene_suz12_ N_coriiceps_lcl_NW_011346291.1_cds_XP_010772410.1_10755__gene_suz12_ N_coriiceps_lcl_NW_011346291.1_cds_XP_010772409.1_10754__gene_suz12_ 1N_coriiceps_lcl_NW_011346291.1_cds_XP_010772406.1_10752__gene_suz12_

RBBP4 C_milii_lcl_NW_006890174.1_cds_XP_007902488.1_18542__gene_rbbp4_ X_laevis_lcl_NC_030726.1_cds_NP_001083811.1_9338__gene_rbbp4.L_ 0.69X_laevis_lcl_NW_016694858.1_cds_NP_001085185.1_55546__gene_rbbp4.S_ 1 X_tropicalis_lcl_NC_030678.1_cds_XP_017946704.1_7318__gene_rbbp4_ 1X_tropicalis_lcl_NC_030678.1_cds_NP_001011394.2_7317__gene_rbbp4_ 1 G_gallus_lcl_NC_006110.4_cds_NP_990183.1_37554__db_xref_GeneID_395658_ 1G_gallus_lcl_NC_006110.4_cds_XP_015153087.1_37555__db_xref_GeneID_395658_ 1 A_carolinensis_lcl_NW_003339848.1_cds_XP_016854465.1_32886__gene_rbbp4_ 1A_carolinensis_lcl_NW_003339848.1_cds_XP_003230003.3_32885__gene_rbbp4_ A_carolinensis_lcl_NW_003339848.1_cds_XP_016854466.1_32887__gene_rbbp4_ 1 O_niloticus_lcl_NC_031985.1_cds_XP_003458547.1_47457__gene_LOC100706337_ 1 N_coriiceps_lcl_NW_011353176.1_cds_XP_010777891.1_15687__gene_LOC104952724_ E_lucius_lcl_NC_025977.3_cds_XP_012990906.1_19465__gene_LOC105012361_ E_lucius_lcl_NC_025977.3_cds_XP_012990905.1_19466__gene_LOC105012361_ 1E_lucius_lcl_NC_025977.3_cds_XP_012990907.1_19464__gene_LOC105012361_ 1 E_lucius_lcl_NC_025977.3_cds_XP_019906053.1_19467__gene_LOC105012361_ S_formosus_lcl_NW_017372081.1_cds_XP_018615456.1_30816__gene_rbbp4_ 1S_formosus_lcl_NW_017372081.1_cds_XP_018615457.1_30817__gene_rbbp4_ 0.97 C_harengus_lcl_NW_012223807.1_cds_XP_012695418.1_26555__gene_LOC105911176_ 0.69 0.96 1L_oculatus_lcl_NC_023184.1_cds_XP_006631293.1_13898__db_xref_GeneID_102682231_ 0.87 L_oculatus_lcl_NC_023184.1_cds_XP_006631294.1_13897__db_xref_GeneID_102682231_ I_punctatus_lcl_NC_030439.1_cds_XP_017310551.1_40377__gene_rbbp4_ 1 A_mexicanus_lcl_NC_035919.1_cds_XP_007249347.1_27940__gene_rbbp4_ 1 D_rerio_lcl_NC_007130.7_cds_NP_997760.1_37139__gene_rbbp4_ 1 C_carpio_lcl_NW_017538293.1_cds_XP_018938592.1_45599__gene_LOC109066061_ 0.52 1 0.07 C_carpio_lcl_NW_017538293.1_cds_XP_018938593.1_45600__gene_LOC109066061_ E_lucius_lcl_NC_025982.3_cds_XP_010877212.2_30351__gene_LOC105015627_ 1 S_salar_lcl_NC_027308.1_cds_NP_001133542.1_28960__db_xref_GeneID_100195041_ RBBP4_mRNA_20171128_putative_after_RACE_long3UTR 0.63 1 B_pectinirostris_lcl_NW_018353624.1_cds_XP_020775066.1_5642__gene_LOC110155313_ N_coriiceps_lcl_NW_011369099.1_cds_XP_010790592.1_27110__gene_LOC104963675_ 1 1N_coriiceps_lcl_NW_011369099.1_cds_XP_010790594.1_27112__gene_LOC104963675_ T_rubripes_lcl_NC_018891.1_cds_XP_003962472.1_2764__db_xref_GeneID_101078368_ 1 0.64 T_rubripes_lcl_NC_018891.1_cds_XP_011614536.1_2763__db_xref_GeneID_101078368_ O_niloticus_lcl_NC_031983.1_cds_XP_003447806.1_43853__gene_LOC100711479_ O_latipes_lcl_NC_019880.1_cds_XP_004082417.1_31998__gene_rbbp4_ 1 0.84 C_variegatus_lcl_NW_015150933.1_cds_XP_015245400.1_21991__gene_rbbp4_ 1 P_reticulata_lcl_NC_024352.1_cds_XP_008397708.1_39670__gene_rbbp4_ 1 P_latipinna_lcl_NW_015113120.1_cds_XP_014895791.1_16552__gene_LOC106952289_

Phylogenetic tree of vertebrate PRC2 components EED, SUZ12, and RBBP4. Bayesian phylogenetic tree rooted with Australian ghostshark (C. milii). Round goby is indicated in orange.