bioRxiv preprint doi: https://doi.org/10.1101/247304; this version posted January 12, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 How the Central American Seaway and

2 an ancient northern passage affected 3 diversification

4 1 1 1,2,* 5 LISA BYRNE , FRANÇOIS CHAPLEAU , AND STÉPHANE ARIS-BROSOU 6 7 1Department of Biology, University of Ottawa, Ottawa, ON, CANADA; 2Department of 8 Mathematics & Statistics, University of Ottawa, Ottawa, ON, CANADA 9 10 *Correspondence to be sent to: Department of Biology, University of Ottawa, 30 Marie 11 Curie Pvt., Ottawa, ON, CANADA; Email: [email protected] 12

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13 Abstract 14 While the natural history of flatfish has been contentious for decades, the mode of 15 diversification of this biologically and economically important group has never been 16 elucidated. To address this question, we assembled the largest molecular data set to date, 17 covering > 300 species (out of ca. 800 extant), from 13 of the 14 known families over 18 nine genes, and employed relaxed molecular clocks to uncover their patterns of 19 diversification. As the fossil record of flatfish is contentious, we used sister species 20 distributed on both sides of the American continent to calibrate clock models based on 21 the closure of the Central American Seaway (CAS), and on their current species range. 22 We show that flatfish diversified in two bouts, as species that are today distributed 23 around the Equator diverged during the closure of CAS, while those with a northern 24 range diverged after this, hereby suggesting the existence of a post-CAS closure dispersal 25 for these northern species, most likely along a northern route, a hypothesis fully 26 compatible with paleogeographic reconstructions. 27 28 Keywords: Pleuronectiformes, molecular clock, Bayesian dating, vicariance, Central 29 American Seaway, Isthmus of Panama

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30 The Pleuronectiformes, or , are a speciose group of ray-finned fish, containing

31 14 families and over 800 known species (Munroe 2015). Flatfish begin life in the pelagic

32 zone, but undergo a larval metamorphosis in which one eye, either left or right,

33 depending on the species, migrates to the other side of the cranium. The adult fish then

34 adopts a benthic lifestyle. Flatfish have asymmetric, laterally-compressed bodies, and

35 have lost their swim bladders during transformation. With eyes facing upwards, flatfish

36 are also capable of protruding them. This singular morphology long puzzled taxonomists

37 (Norman 1934), and the phylogeny of this group remains poorly resolved.

38 At the highest taxonomic level, flatfishes are generally considered to be monophyletic,

39 based on both morphological (Chapleau 1993), and molecular evidence (Berendzen et al.

40 2002; Pardo et al. 2005; Azevedo et al. 2008; Betancur-R et al. 2013; Harrington et al.

41 2016). All these studies also support the monophyletic status of most families within the

42 order, to the exception of the . As all molecular studies to date have

43 essentially focused on the monophyletic status of the order, they were based on as many

44 representative species of each order. As a result, intra-ordinal relationships, among

45 genera and even families, are still debated. It can therefore be expected that taking

46 advantage of both species- and gene-rich evidence, while incorporating paleontological

47 and/or geological data in the framework of molecular clocks, should help clarify not only

48 the phylogenetic status of this family (dos Reis et al. 2015), but also – and more critically

49 here – their evolutionary dynamics.

50 However, very few flatfish fossils are known, and placed with confidence on the

51 flatfish evolutionary tree (Parham et al. 2012). As a result, calibrating a molecular clock

52 becomes challenging. On the other hand, a dense species sampling may enable us to take

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53 advantage of a singular feature of flatfishes: some extant species are found both in the

54 Pacific and Atlantic oceans. Furthermore, the existence of geminate species pairs of

55 flatfishes, where sister taxa have one member in each ocean, suggests a speciation event

56 pre-dating the closure of the Isthmus of Panama, which occurred approximately 12 to 3

57 million years ago [MYA] (Haug and Tiedemann 1998; ODea et al. 2016). Our driving

58 hypothesis is then that this information can be used to calibrate molecular clocks, and

59 allow us to unravel the timing of flatfish evolution, as how rapidly they diversified

60 remains an unsolved question. We show here that the diversification of flatfish in the sea

61 surrounding the Americas followed a complex pattern, related to not only the closure of

62 the Isthmus of Panama, but also to a warming event that opened up a northern route.

63 Results

64 To test how the evolutionary dynamics of flatfish were affected by a major geological

65 event, the closure of the CAS, we reconstructed dated Bayesian phylogenetic trees from a

66 large data matrix under four relaxed molecular clock models, each one of them being

67 based on a different calibration scheme (Fig. 1). Under the first model, no calibration

68 priors were placed on internal nodes. The initial data survey tree, with the rogue

69 sequences removed (data on GitHub) was used to identify pairs of sister taxa that are split

70 between the two oceans, with one species in the Atlantic and the other in the Pacific. This

71 led us to single out twelve pairs of such species. These sister species happened not to be

72 evenly distributed on the estimated phylogeny (Fig. 2, top), but they are the only

73 geminate species included in GenBank (as of August 2016). On each pair, we placed

74 calibration date priors corresponding to the closure of the CAS. An examination of the

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75 posterior distributions of their divergence times suggests that some species had a very

76 narrow speciation window, where all the mass of the posterior distribution is between 5-3

77 MYA, while others have a wider distribution (Fig. 3A). Closer inspection of these

78 distributions further reveals that most of the species with narrow posterior distributions

79 have a northern range (Fig. 2, and 3A, in blue), while those with the wider posterior

80 distributions have a “southern” distribution, closer to the Isthmus of Panama (Fig. 2, and

81 3A, in red). To further assess this observation, we first went back to the original clock

82 model, with no priors on sister taxa, and were able to validate that even in this case,

83 northern and southern species showed, to one exception each (Hippoglossus hippoglossus

84 and H. stenolepis in the north, and Poecilopsetta natalensis and P. hawaiiensis in the

85 south; Fig. 2), shifted posterior distributions (Fig. 3B). The former pair was actually not

86 estimated as being sister species in any of the four clock models (Fig. 1), while P.

87 natalensis and P. hawaiiensis, although inhabiting the Western Atlantic and Eastern

88 Pacific oceans, occupy ranges that do not extend to the coasts of the Americas as with the

89 other identified sister taxa. Models with priors placed only on northern (Fig. 3C) or

90 southern (Fig. 3D) species also showed a similar temporal shift. This shift suggested that

91 southern species diverged early, before the complete closing of the CAS, while northern

92 species diverged later, at or possibly after the isthmus was completed. Averaging these

93 posterior distributions for the northern and southern species, to the exception of the two

94 outliers noted above, showed these results more clearly (Fig. 3E-H).

95 In an attempt to tease out these models and their predictions about the exact timing of

96 divergence between northern and southern species, we assessed model fit by means of

97 AICM. Even if model ranking based on this measure is known to be unstable (Baele et al.

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98 2012), it is clear that the models with priors only on the northern or on the southern

99 species perform significantly (> 200 AIC units) more poorly than the two other models,

100 which may be difficult to tease apart (Table 1). Their predictions suggest that H.

101 hippoglossus and H. stenolepis, both northern species, consistently diverged before the

102 complete closure of the seaway, in tandem with the average southern species, and that the

103 average northern species diverged in tandem with P. natalensis and P. hawaiiensis (Fig.

104 3E-F).

105 Discussion

106 Our results show that flatfish underwent a first speciation at the time when the CAS

107 closed, which led to the species that, today, have an equatorial range (Fig. 2), as the

108 formation of the Isthmus of Panama resulted in a barrier to gene flow leading to their

109 speciation. Our results also show that species that today have a northern range (Fig. 2)

110 either emerged at the closure of the seaway, or after its closure. These timing estimates

111 imply that this second bout of speciation was not caused by gene flow impeded by the

112 closure of the seaway, but demand an interpretation involving gene flow through a

113 different route, a northern route, before being interrupted, most likely this time by a

114 climatic event. Strikingly, the geological evidence is directly in line with our date

115 estimates. The fossil record suggests that the first aquatic connection between the Pacific

116 and Arctic (and Atlantic) oceans through the Bering Strait occurred approximately 5.5-

117 5.4 MYA (Gladenkov et al. 2002) due to a rise in sea levels, linked to tectonic activity

118 (Marincovich and Gladenkov 2001). This would have permitted the migration of

119 populations ancestral to today’s northern species from one ocean to the other through this

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120 ancient “northern passage.” This global warming event, between the late Miocene to

121 early Pleiocene, was then followed by a significant period of cooling during the Pleiocene

122 into the Pleistocene (Zachos 2001), leading to periods of repeated glaciations and a

123 subsequent ice age. These cold events would have resulted in the closure of this ancient

124 “northern passage,” hereby stopping gene flow between the two oceans, and leading to

125 the more recent speciation of the northern taxa.

126 For approximately a million years after the first opening of the Bering Strait, water

127 flowed in a southern direction, until the formation of the Isthmus of Panama occurred

128 close to the equator (Berta 2012). With the formation of the Isthmus, and the closing of

129 the CAS, the ocean currents reversed due to a change in global ocean circulation (Haug

130 and Tiedemann 1998; De Schepper et al. 2015), and have since flowed from the Pacific

131 to the Arctic (Marincovich 2000). Dispersal or migration from the Pacific into the

132 Atlantic in a northern direction through this strait is known as the trans-Arctic

133 interchange (Vermeij 1991). Fossil data also show that the Bering land bridge has been

134 exposed and submerged on multiple occasions since the Pleistocene (Gladenkov and

135 Gladenkov 2004). These openings and closings of the Bering Strait could have provided a

136 mechanism for divergence and the evolution of sister taxa (Taylor and Dodson 1994;

137 Väinölä 2003).

138 Our results also have implication at the family level of flatfish. Further significant

139 global cooling during the Pleistocene resulted in major glaciation events (Zachos et al.

140 2008) that could be responsible for creating barriers that isolated populations. All of the

141 remaining sister taxa in our analysis, who have divergence estimates of less than 2 MYA

142 in our study belong to the family (sensu Cooper and Chapleau 1998). The

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143 Pleuronectidae are the predominant flatfish family found in cold and temperate seas of

144 the northern hemisphere (Norman 1934; Cooper and Chapleau 1998). There are far more

145 Pleuronectidae species in the Pacific Ocean, most of them endemic to the north Pacific

146 Ocean off of north America and Asia in the region extending from the Bering Strait to the

147 gulf of California (Norman 1934). None of the arctic species are restricted solely to arctic

148 waters (Munroe 2015). Munroe also noted that Cooper (in an unpublished manuscript)

149 identified areas of endemism among the current distribution of the Pleuronectidae. It has

150 been shown that during the trans-Arctic interchange, there was a far higher number of

151 species (up to eight times higher) migrating to the Atlantic than to the Pacific (Vermeij

152 1991). Fossil and phylogenetic evidence suggest the Pacific Ocean as the origin for

153 diversification of the Pleuronectidae (Munroe 2015) and our phylogenetic results are

154 highly congruent with this hypothesis.

155 It is possible that the outliers, the Atlantic and Pacific halibut (H. hippoglossus and H.

156 stenolepis, respectively), diverged during one of the first openings of the Bering Strait.

157 The estimated dates from the molecular clock analysis are approximately 5.5 MYA, in

158 accordance with the hypothesized dates for the first aquatic connection (Marincovich and

159 Gladenkov 2001). The remaining sister taxa have a younger age estimate of ~1-2 MYA,

160 corresponding with global cooling during the Pleistocene and repeated glaciations

161 (Zachos 2001) which likely formed more barriers to genetic flow. In the second pair of

162 outliers, P. hawaiiensis inhabits waters of the Eastern Central Pacific to the Hawaiian

163 Islands, while P. natalensis inhabits coastal waters of Eastern Africa (Fig. 2). Due to the

164 far reaching range of P. natalensis, and the relatively younger age estimates for

165 divergence (1-2 MYA), these results beg for future research into other vicariant

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166 hypotheses, or dispersal routes, as these two members of Poecilopsetta diverged long

167 after the Isthmus had closed.

168 Based on the most extensive multigene sequence alignment available to date across

169 all flatfish species, we have showed here that the evolutionary dynamics of sister species

170 that are distributed across the two oceans strongly supported the existence of two bouts of

171 speciation: one triggered by the closure of the CAS 12-3 MYA, and a second one due to

172 the closure of an ancient northern passage 5-0.01 MYA. The most intriguing implication

173 of our results is that other sea critters than the flatfish should have been affected by the

174 two same geological processes, the closure of the CAS (Bacon et al. 2015) and, most

175 intriguingly, of the ancient northern passage, so that future studies should be able to

176 extend our findings to other living forms inhabiting these two oceans.

177 Methods and Materials

178 Data retrieval and alignment

179 Prior to retrieving sequence data, GenBank was surveyed to identify all the genes

180 belonging to species of Pleuronectiform families (as of August 2016), based on

181 GenBank’s browser. DNA sequences for a total of 332 flatfish species (out of

182 over 800 species in the order) were identified and downloaded for five nuclear genes

183 (KIAA1239, MYH6, RIPK4, RAG1, SH3PX3), and four mitochondrial genes (12S, 16S,

184 COX1 and CYTB). These represented all the taxa having at least one of these six gene

185 sequences in GenBank; see Table S1 for the corresponding accession numbers. Diversity

186 was richly sampled as species from 13 of the 14 families in the order Pleuronectiformes

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187 were included in our catchment. In line with current consensus in flatfish systematics, the

188 family Psettodidae was chosen as the outgroup to all other taxa (Chapleau 1993).

189 These sequences were aligned using MUSCLE ver. 3.8.31 (Edgar 2004) on a gene-

190 by-gene basis. Each alignment was visually inspected with AliView ver. 1.18 (Larsson

191 2014), and was manually edited where necessary. In particular, large indels (> 10 bp)

192 were removed prior to all phylogenetic analyses. The 5’ and 3’ ends of sequences were

193 also trimmed. The aligned sequences were then concatenated into a single alignment

194 using a custom R script.

195 Data pre-processing

196 To gauge the phylogenetic content of our data set, we performed a first series of

197 molecular clock analyses on the concatenated data matrix, with all the nine gene

198 sequences obtained above (12S, 16S, COI, Cyt-b, KIAA1239, MYH6, RIPK4, SH3PX3

199 and RAG1), and all the 332 taxa representing all families of flatfish. To account for rate

200 heterogeneity along this alignment, partitions were first identified with PartitionFinder 2

201 (Lanfear 2012), selecting the best-fit model of nucleotide substitution based on Akaike’s

202 Information Criterion (AIC). This optimal partition scheme was then employed in a first

203 Bayesian phylogenetic analysis, conducted with BEAST ver. 1.8.3 (Drummond and

204 Rambaut 2007). This program implements a Markov chain Monte Carlo (MCMC)

205 sampler, which co-estimates both tree topology and divergence times. As determined by

206 PartionFinder, a GTR+I+Γ model was applied to each data partition. These models of

207 evolution across partitions were assumed to be independent (unlinked, in BEAST

208 parlance), while both clock model and tree model partitions were shared (linked) by all

209 partitions. An uncorrelated relaxed clock was employed with a lognormal distribution

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210 prior on rates, and a Yule speciation prior (Drummond et al. 2006). Due to the paucity of

211 reliably placed fossils on the flatfish tree, a unique calibration point was placed on the

212 most recent common ancestor (MRCA) of the ingroup, as a mean-one exponential prior,

213 with an offset of 40 million years reflecting the age of 47.8-42.1 MYA (Chanet 1997). To

214 stabilize the estimate, a lognormal ln(0, 1.5) prior with a 40 MYA offset was also placed

215 on the root of the tree (root height). Two separate MCMC samplers were run, each for

216 10,000,000 generations. Trees were sampled every 5,000 generations, and convergence

217 was checked visually using Tracer ver. 1.6. (Rambaut et al. 2014). Tree log files from

218 each run were combined in LogCombiner, after conservatively removing 10% of each

219 run as burn-in. The resulting maximum a posteriori (MAP) tree was generated with

220 TreeAnnotator (Drummond and Rambaut 2007).

221 As the topology of this resulting MAP tree was unconventional, we suspected the

222 presence of rogue taxa, i.e. species evolving either much faster or much slower than the

223 majority, which can contribute negatively to consensus tree support (Aberer et al. 2013).

224 Rogue taxa were identified using the RogueNaRok (Aberer et al. 2013) webserver

225 (http://rnr.h-its.org). The consensus trees from the preliminary analysis using 10,000

226 iterations were used as the tree set for rogue taxon analysis. A total of 22 rogues were

227 identified and pruned from the analysis, leaving 310 species.

228 Molecular Dating

229 To assess the impact of the closure of the Central American Seaway (CAS; forming the

230 Isthmus of Panama) on flatfish evolutionary dynamics, a second set of partitioned relaxed

231 molecular clock analyses was performed (without the rogue sequences). The timing of

232 the closure of the CAS is estimated to have occurred between 12 and 3 MYA (Duque-

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233 Caro 1990; Coates et al. 1992; Haug and Tiedemann 1998; ODea et al. 2016), and we

234 used this time window as a prior to inform the relaxed molecular clock-based

235 phylogenetic reconstructions. The analyses were performed on the same concatenated

236 data set, with the same single fossil calibration, but we also placed a lognormal prior

237 ln(3,1.5), that has most of its mass on the 12-3 MYA window, on the MRCA of each pair

238 of sister taxa. From the initial BEAST analyses, twelve pairs of taxa were selected based

239 on two criteria: (i) being sister species on that initial tree, (ii) with one species distributed

240 in the Pacific and one in the Atlantic ocean (Fig. 2). Again, two independent MCMC

241 samplers were run each for 100 million iterations, with samples taken every 5000 step.

242 Because these pairs of sister species show a contrasted geographic distribution,

243 having either a southern (equatorial) or a northern range (Fig. 2), two additional sets of

244 analyses were performed. In a first set, calibration priors (ln(3,1.5), as per above) were

245 placed only sister taxa that had a geographic range in the southern hemisphere, while in a

246 second set, identical calibration priors were placed only on sister species with a northern

247 range. Finally, a set of analyses was performed using no sister taxa calibrations at all. For

248 each analysis, results from the two MCMC runs were combined using LogCombiner after

249 removing an even more conservative burn-in of 50%. The final MAP tree was generated

250 with TreeAnnotator.

251 In an attempt to rank these different models (priors on all sister taxa; only on southern

252 taxa; only on southern taxa; no “CAS” priors), a modified Akaike’s Information Criterion

253 (AICM) that accounts for uncertainly in the MCMC sampling (Raftery et al. 2007) was

254 computed for each model (Baele et al. 2013). These computations were performed in

255 Tracer for each of the four different calibration models, based on 100 replicates.

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256 Availability of Computer Code and Data

257 Accession numbers, sequence alignments, and BEAST models (as xml files) used in this

258 study are available from https://github.com/sarisbro.

259

260 Acknowledgments

261 This work was supported by the University of Ottawa (LB), and the Natural Sciences

262 Research Council of Canada (FC, SAB). We are grateful to Jonathan Dench for

263 discussions and comments, as well as to Compute Canada and Ontario’s Center for

264 Advanced Computing for providing us access to their resource.

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312 asymmetry arose in a blink of the evolutionary eye. BMC Evol. Biol. 16:224. 313 Haug G.H., Tiedemann R. 1998. Effect of the formation of the Isthmus of Panama on 314 Atlantic Ocean thermohaline circulation. Nature. 393:673–676. 315 Larsson A. 2014. AliView: A fast and lightweight alignment viewer and editor for large 316 datasets. Bioinformatics. 30:3276–3278. 317 Marincovich L., Gladenkov A.Y. 2001. New evidence for the age of Bering Strait. Quat. 318 Sci. Rev. 20:329–335. 319 Marincovich L.J. 2000. Central American paleogeography controlled Pliocene Arctic 320 Ocean molluscan migrations. Geology. 28:551–554. 321 Munroe T.A. 2015. Systematic Diversity of the Flatfishes. In: Robin Gibson, Richard 322 Nash, Audrey Geffen H.V. der V., editor. Flatfishes: biology and exploitation. Wiley. 323 p. 13–44. 324 Norman J.R. 1934. A systematic monograph of the flatfishes (Heterosomata). By J.R. 325 Norman, London. 326 ODea A., Lessios H.A., Coates A.G., Eytan R.I., Restrepo-Moreno S.A., Cione A.L., 327 Collins L.S., de Queiroz A., Farris D.W., Norris R.D., Stallard R.F., Woodburne 328 M.O., Aguilera O., Aubry M.-P., Berggren W.A., Budd A.F., Cozzuol M.A., 329 Coppard S.E., Duque-Caro H., Finnegan S., Gasparini G.M., Grossman E.L., 330 Johnson K.G., Keigwin L.D., Knowlton N., Leigh E.G., Leonard-Pingel J.S., Marko 331 P.B., Pyenson N.D., Rachello-Dolmen P.G., Soibelzon E., Soibelzon L., Todd J.A., 332 Vermeij G.J., Jackson J.B.C. 2016. Formation of the Isthmus of Panama. Sci. Adv. 333 2:e1600883–e1600883. 334 Pardo B.G., Machordom A., Foresti F., Porto-Foresti F., Azevedo M.F.C., Bañon R., 335 Sánchez L., Martínez P. 2005. Phylogenetic analysis of flatfish (Order 336 Pleuronectiformes) based on mitochondrial 16S rDNA sequences. Sci. Mar. 69:531– 337 543. 338 Parham J.F., Donoghue P.C.J., Bell C.J., Calway T.D., Head J.J., Holroyd P.A., Inoue 339 J.G., Irmis R.B., Joyce W.G., Ksepka D.T., Patané J.S.L., Smith N.D., Tarver J.E., 340 Van Tuinen M., Yang Z., Angielczyk K.D., Greenwood J.M., Hipsley C.A., Jacobs 341 L., Makovicky P.J., Müller J., Smith K.T., Theodor J.M., Warnock R.C.M., Benton 342 M.J. 2012. Best practices for justifying fossil calibrations. Syst. Biol. 61:346–359.

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343 Raftery A.E., Newton M.A., Satagopan J.M., Krivitsky P.N. 2007. Estimating the 344 integrated likelihood via posterior simulation using the Harmonic Mean Identity. 345 Bayesian Stat.:1–45. 346 Rambaut A., Suchard M.A., Xie D., Drummond A.J. 2014. Tracer v1.6. 347 dos Reis M., Donoghue P.C.J., Yang Z. 2015. Bayesian molecular clock dating of species 348 divergences in the genomics era. Nat. Rev. Genet. 17:71–80. 349 De Schepper S., Schreck M., Beck K.M., Matthiessen J., Fahl K., Mangerud G. 2015. 350 Early Pliocene onset of modern Nordic Seas circulation related to ocean gateway 351 changes. Nat. Commun. 6:8659. 352 Taylor E.B., Dodson J.J. 1994. A molecular analysis biogeography within (genus 353 Osmevus) of relationships and a species complex of Holarctic fish. Mol. Ecol. 354 3:235–248. 355 Väinölä R. 2003. Repeated trans-Arctic invasions in littoral bivalves: Molecular 356 zoogeography of the Macoma balthica complex. Mar. Biol. 143:935–946. 357 Vermeij G.J. 1991. Anatomy of an invasion: the trans-Arctic interchange. Paleobiology. 358 17:281–307. 359 Zachos J. 2001. Trends, rhythms, and aberrations in global climate 65 Ma to present. 360 Science (80-. ). 292:686–693. 361 Zachos J.C., Dickens G.R., Zeebe R.E. 2008. An early Cenozoic perspective on 362 greenhouse warming and carbon-cycle dynamics. Nature. 451:279–283. 363 364

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365 Table 1. AICM values for the phylogenetic analyses using four different calibration 366 schemes. SE: standard error.

Calibration Model AICM value SE All 374,082.199 +/-1.579 None 374,110.488 +/-3.789 South 374,367.749 +/-0.696 North 374,746.628 +/-3.789 367 368

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369 Figure captions 370 371 FIG. 1. Phylogenetic trees reconstructed based on relaxed clock models. Four models 372 were employed, representing different specifications of prior distributions set on sister 373 taxa. (A) No priors were set on sister taxa. (B) Priors were set on all pairs of taxa. (C) 374 Priors were set only on sister taxa with a current northern range. (D) Priors were set only 375 on sister taxa with a current southern range. Horizontal scale is in million years ago 376 (MYA). The closure of the CAS (12-3 MYA) is shown between vertical gray broken 377 lines. 378

379 FIG. 2. Distribution of the geminate species used in this study. Geographic distribution of 380 the twelve pairs of sister species of flatfish used to calibrate the relaxed molecular clock 381 models. Original data come from GBIF (www.gbif.org; accessed Nov 9, 2017). The top 382 panel shows the phylogenetic distribution of these species based on the clock model 383 including prior ages on all twelve pairs of species; scale bar: 5 MYA. 384

385 FIG. 3. Posterior densities of divergence times for sister taxa used as calibration points in 386 the relaxed molecular clock analyses, under four different calibration schemes. NONE: 387 no priors were placed on sister taxa; ALL: priors were placed on all pairs of sister taxa; 388 NORTH: priors were placed on only pairs of sister taxa with a northern distribution; 389 SOUTH: priors were placed on only pairs of sister taxa with a southern distribution. Top 390 row (A-D) shows all 17 distributions, while bottom row (E-H) shows range-averaged 391 distributions (solid) to the exception of outlier pairs (dashed lines). Densities are color- 392 coded for species with northern (blue) and southern (red) range. Dashed vertical gray 393 lines indicate the closure of the CAS (21-3 MYA).

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A NONE 0.95 B ALL 0.96 C NORTH 0.98 D SOUTH 1.00 Cynoglossidae Cynoglossidae Cynoglossidae 0.99 0.95 0.97 1.00 0.99 Soleidae Soleidae Cynoglossidae 0.99 0.90 Soleidae 0.94 1.00 0.98 1.00 0.95 0.94 1.00 Poecilopsettidae 1 1.00 0.99 Poecilopsettidae 1 0.94 Poecilopsettidae 1 1.00 Poecilopsettidae 1 0.97 0.99 1.00 Samaridae 1.00 Samaridae 1.00 Samaridae 1.00 Samaridae Achiridae 1.00 Achiridae-Soleidae 1.00 Achiridae 1.00 1.00 Achiridae Citharidae 1.00 Citharidae 0.99 1.00 0.73 1.00 Pleuronectidae Citharidae 0.99 0.99 Rhombosoleidae Pleuronectidae 0.99 0.99 0.94 Paralichthyidae Achiropsettidae Rhombosoleidae Paralichthyidae 1.00 1.00 0.94 1.00 Achiropsettidae 1.00 0.71 Poecilopsettidae 2 1.00 0.70 1.00 Poecilopsettidae 2 0.73 0.77 Poecilopsettidae 2 1.00 1.00 1.00 Scopthlamidae 1.00 Bothidae "Cyclopsetta" gr. 1.00 1.00 Scopthalmidae 0.99 1.00 1.00 0.99 Pleuronectidae 0.99 "Cyclopsetta" gr. 1.00 Rhombosoleidae 0.708 Pleuronectidae 1.00 1.00 Paralichthyidae 1.00 0.71 1.00 0.85 Achiropsettidae Paralichthyidae 0.95 0.76 Rhombosoleidae Poecilopsettidae 2 0.79 0.71 0.97 0.94 1.00 Bothidae 1.00 1.00 Achiropsettidae 1.00 1.00 0.80 Scophthalmidae 0.71 1.00 "Cyclopseta" gr. Bothidae 1.00 1.00 1.00 Citharidae 1.00 "Cyclopsetta" gr. Scopthalmidae 1.00 1.00 1.00 Psettodidae Psettodidae 1.00 Psettodidae Psettodidae

-50 4 0 3 0 2 0 1 0 0 4 0 3 0 2 0 MYA 1 0 0 MYA -50 4 0 3 0 2 0 MYA 1 0 0 -50 4 0 3 0 2 0 MYA 1 0 0 C y noglossu s _puncti c eps

Cynoglossus_lingua ●● Cynoglossus_lida Paraplagusia_bilineata Cynoglossus_sinicus Cynoglossus_acaudatus C y noglossu s _ cynoglos sus ● Cynoglossus_broadhursti ● Cynoglossus_semilaevis C y noglossu s _ r oulei Cynoglossus_purpureomaculatus C ynogl ossus_abbreviatus Cynoglossus_carpenteri Cynoglossus_marleyi Cynoglossus_nigropinnatus Cynoglossus_lighti Cynoglossus_joyneri C y noglossu s _oligolepis C y noglossu s _ r obustu s Cynoglossus_arel Cynoglossus_attenuatus Cynoglossu s _macrolepidot u s Cynoglossus_elongatus natalensis Cynoglossus_ochiaii ●● C y noglossu s _ s inus arabici C y noglossu s _int e rrupt u s

. Paraplagusia_blochi ● Cynoglossus_browni ● ● The copyright holder for this preprint (which was not The copyright holder for this preprint (which ● ● Paraplagusia_japonica ● Cynoglossus_zanzibarensis 2:P. Cynoglossus_monodi Cynoglossus_senegalensis C ynogl ossus_dubi u s Symphurus_plagiusa Symphurus_civitatium Symphurus_plagusia

Symphurus_ginsburgi ● ● Symphur u s _diom edeanus ●● ●

Symphurus_ni grescens ● ●● ● ●

Symphurus_arawak ● ● Symphurus_tessellatus

Symphurus_atricaudus ● ●● ● ● ●●●●●●●●

Symphurus_ommaspilus ●● ●● ● Symphurus_rafinesque ● Symphurus_orient alis

Symphur u s _leuc o c hilus ● Symphurus_thermophilus Symphurus_strictus Symphur u s _ m egas o m u s Symphurus_hondoensis ●● Symphurus_microrhynchus

Symphur u s _longir o stri s ● Solea_senegalensis ●

Solea_aegyptiaca ● S olea_s olea Solea_vulgaris ●● ●●●●●● ●● Solea_kleini ●●

Solea_kleinii ●

Synapturichthys_kleinii ● Pegusa_impar Solea_lascaris ●

S olea_im par ● Pegusa_cadenati Dicologlossa_cuneata Microchirus_boscanion Michrochirus_boscanion Michrochirus_ocelatus Dicologlossa_hexophthalma

M i cro c hir u s _az e v i a ● Michrochirus_azevia Microchirus_variegatus ●● ● ● ● ● ● Monochirus_Hispidus ●● ● ●

Monochirus_hispidus ● ● ● ● ● Microchirus_ocellatus ● Bathysolea_profundicola ● ● Buglossidium_lut eum ●● ● ●● ● ● ● ●● ● ● ● ● ● Pegusa_nasuta ● ● ● ● Solea_ovata ●●● ● ● ● ●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●● ●●●●●●●●●●● ●●●●●●●●● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ●●●● ● ● ● ●● ●● ●● ●● ●● ●● ●● ● ● ●● ● ●● ● ●●●●● ● ● ● ● ● ● ● Aesopia_cornuta ● ● ● ● ● ●●●●●●●●●●● ●●●● ●●● ●● ● ● ● ●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● Zebrias_scalaris ● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ●● ●● ●●●●●●● ●●●●●●● ● ●●●● ● ● ●●●●●●●●●●●● ●●●●●●●● ● ●●●●●●●●●●●●●●●●●● ●●●● ● ●●●● ● ●● ● ●●● ●● ●●● ●● ●●● ● ● ● ● ● ● ● ● ● ●● ●● ● ● ●● ● ●● ● ● ●● ● ●● ●● ●●●●●●●●●● ●● ● ●

Zebrias_zebra ● ● ●● ● ● ● ●●●●●●●●●●●● ●●●● ● ● ● ● ●●●●●●●●●●●●●●●● ● ●● ● ● ● ● ●●● ● ● ● ●● ● ● ● ● ●●●●● ● ● ●●● ● ● ● ● ●● ●

Zebrias_synapturoides ● ● ●● ●● ● ● ● ● ● ● ●●

Brachirus_annularis ● ● ●● ●● ● ●● ● ●● ● ●●●●● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ●●● ●●● ● ● ●

Pseudaesopia_japonica ● ●● ●● ● ● ● ● ● ●

Soleicht hys_het erorhinos ●●● ● ● ●●●● ● ● ●●● ●● ● ●●●● ● ● ●

Heteromycteris_matsubarai ● ●●●●●●●●●●●●● ●● ● ● ● ● ●● ●● ● ●● ● ● ● ● Heteromycteris_japonicus ● ● ● ●●●●● ● ●●● ● ● ● ● ● ●●● ●● ● ● ● ● ● ●●●●●

Aseraggodes_kaianus ● ●●●●● ●● ● ●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●● ● Pardachirus_morrowi ● ● ● ● ● ● ●●●●●● ●● Ase r aggodes _heemstra i ●● ●●●●●● ●●● ●●● ●●●●●●●● ●●●●● ●●●●● ●● ●● ●●●●● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●● ●●●●●●● ● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●

Aseraggodes_melanostictus ● ● ● ● ● ● Aseraggodes_kobensis ● ● ●●●● ● ● ●● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●●● ● ●● ● ● ● ●●●●●●●●●●● ●●●●● ●●●●●●● ● ● ●● ● ●●● ●●● ●●●●●●●●●● ● ●● ●●● ● ● P a r dac hir u s _ m a rmo r a t u s ●● ●●●●●●●●●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●●● ● ● ● ● ● ●● ● ● P a r dac hir u s _pav oninus ●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●

Daget icht hys_commersonnii ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ●● ●●● ● ● ● ● ● ● ●● ● ● ● ● ● Synapt ura_marginat a ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ●

Dagetichthys_lusitanica ●● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ●● ●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●● ● ● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ● ●● ● ● ● ● ●● ● ● ● ●● ●●●● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ●

S y napt ura_lusit anic a ●● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ●● ●●●●●●●●●●●●●●●●●●●● ●● ● ●

Austroglossus_microlepis ● ●● ● ● ● ●● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ●● ● ● ● ● ●● ●● ●●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ● Austrogl ossus_pectoralis ● ● ●● ● ●●● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ●● ●● ●● ●● ●● ● ● ●

Zebrias_zebrinus ● ● ● ● ● ● ●

Zebrias_fasciatus ● Zebrias_quagga ● ● ● ●● ●● ● Zebrias_regani ● ● ●● ●● ● ● ●

Poecilopsetta_plinthus ● ● ● ● ● ● ● ● Cit har oides _ m a crolepidot u s ● Poecilopsetta_beanii Poecilopsetta_beani Samariscus_japonicus Samariscus_latus Samariscus_longimanus Samariscus_xenicus Plagiopsetta_glossa Samaris_cristatus Samariscus_triocellatus Achirus_declivis Achirus_achirus

Achirus_lineat u s 7:P. platessa H y poc linem u s _ m ent alis Soleonasus_finis Apionichthys_dumerili Trinectes_microphthalmus

T rinect es_maculat u s 9:L. limanda Trinecte s _paulistanus T rinect es_inscript u s G ymnachirus_t exae G ymnachirus_melas Gymnachirus_nudus ● Catathyridium_jenynsii Catathyridium_jenynsi this version posted January 12, 2018. Cit har oides _ m a crolepis

CC-BY-NC-ND 4.0 International license Citharus_linguatula Rhombosolea_t apirina ; Rhombosolea_plebeia R hombosol ea_l epori n a a P elt o r ham phus _nov aez eelandiae Pelotretis_flavilatus Ammotretis_rostratus Achiropsetta_tricholepis Neoachiropsetta_milfordi ● M anc ops e tta_m a c ulat a ● Oncopterus_darwinii Poecilopsetta_hawaiiensis

P oec ilopse tta_nat alensis 2 Poecilopsetta_praelonga ● Marleyella_bicolorata Zeugopterus_punctatus Phrynorhombus_norvegicus Lepidorhombus_whiffiagonis Lepidorhombus_boscii 12:G. cynoglossus ● Scophthal mus_maxi mus ● Psetta_maeotica ● ● Psetta_maxima ●● Scopht halmus_rhombus ● Platichthys_stellatus ● ● ● ● Platichthys_flesus 11:H. hippoglossus ●

Liopsetta_pinnifasciata ● ● ● ●●●● ● ● ● ● Karei us_bi col oratus ● 10:H. platessoides ● 7 Pleuronectes_platessa ● Pleuronectes_quadrituberculatus ● ●● ● ●● ●●● ● ●●●

Limanda_punctatissima ●● ● ●● ● ●● ● ●●● ● ●● ● ●● ●● ● ●●● ● ●●● ●● ●● ●●● ●● ● ●● ●● ● ●● ●●● ●●● ●●● ●● ● ●●● ●●●● ●●● ● ●●● ●●● ● ●● ●● ● ● ●● ●● ●●● ●● ●● ●● ●● ●● ● ● ● ●●● ● ●●● ●● ● ●● ●●● ●●● ●● ● ● ● ●● ●● ●●● ●●● ●●● ●●● ●●● ●● ●● ●●● ●●● ●● ●●● ● ● ● ●●● ● ●● ●● ● ●● ●● ●●●● ●●● ●●● ●● ●● ●● ●● ●●● ●●● ●● ●● ●●● ●● ●● ● ● ●●● ●● ●● ●● ● ●● ●●● ●● ● ●●●● ●●● ●●● ● ●● ●

Limanda_proboscidea ●● ●● ●●● ●● ● ●● ●●● ●● ●●●● ●●● ●●● ● ● ● ●● ●●● ● ●● ●● ● ● ● ●●● ●●● ●●● ● ●●● ●● ●●●● ●● ●●● ●● ●● ● ●●● ● ●● ●●● ●● ●● ●● ●●● ●● ●●● ● ● ●● ●●● ● ●● ●● ● ● ●● ● ●● ● ●● ●● 8 ● ● ●●● ●● ●● ●●● ●●● ● ● ● ●● ● ●●● ● ●● ●● ● ● ●●● ●● ● ● ● ●● ●● ●●● ●● ●●● ● ●●● ● ●●● ● ●● ● ●● ● ● ●●● ●● ● ●●● ● ●● ●● ●●●● ●●● ●● ●● ●● ●● ● ●● ● ●● ●● ● ●● ●●● ● ● ●● ●● ● ● ●●● ●● ●● ● ● ● ●● ●● ●● ●●● ● ●●● ●● ● ● ● ● ● ●●● ●●● ●● ●● ●● ● ●

Limanda_ferruginea ● ●●● ● ● ● ●● ● ●● ●● ●● ●●● ● ● ●● ● ●●● ●●● ●● ● ●● ● ●● ● ●●● ●● ● ●● ● ●● ●● ●●●●● ●● ●● ● ●●● ●● ● ●● ●● ●●●● ●●● ●● ● ●●● ●●●● ●● ● ● ● ●● ● ● ● ● ●● ●●● ● ●● ●●●●● ●●●● ●● ●● ●●●● ● ● ● ●● ●●● ●●● ● ●● ●● ●●● ● ●●● ●● ●● ● ● ●● ●● ● ●● ● ● ●● ●● ●● ●● ●● ●● ●●● ●● ●● ●● ●●●● ● ●●● ●● ● ●● ●● ● ●● ●● ●● ● 4:C. minutus ●● ●●● ●● ●● ●●●● ●● ● ● ● ● ●●●● ●● ●● ●● ●● ● ●● ●●● ● ● ●●●● ●●● ●● ● ● ●● ●●● ●●● ●● ● ● ● ●● ● ●●● ●●● ● ●●● ●● ●● ●● ● ●●● ●● ●● ● ●● ● ●●● ●● ●● ● ●● ●●● ● ● ●● ●●●● ●● ●● ●● ●● ●● ●●●● ●●● ●●● ●●● ● ●●● ●● ● ●●● ● ● ● ●● ●● ●● ●● ● ● ●● ●● ● ● ● ●● ●●● ● ●● ●● ● ● ●● ● ● ● ●● ●● ●● ●●● ●● ● ●●● ● Psettichthys_melanostictus ● ●● ●● ● ● ●● ● ● ● ● ●● ● ● ●● ●● ● ● ● ● ●● ● ● ●● ●● ●●● ●● ●●● ● ●● ●● ●● ● ● ●● ● ● ● ●●● ●● ● ●● ●● ●● ● ●● ● ●● ●● ● ●●● ●●● ●●●● ●● ● ● ●●●● ●● ●● ● ●●● ●●●●●● ●● ● ●● ●● ● ● ●● ●● ●● ●● ● ● ●● ● ● ● ●● ●●● ● ● ●● ●●●● ●● ●● ●●● ●●● ● ●● ● ●●● ● ●●●● ●● ●● ● ●●●● ● ●● ●● ●● ●●● ● ●●● ●●● ●● ●● ●● ● 1:H. oblonga ●● ● ● ●●●●●● ● ● ●● ●● ● ●● ● ● ● ●● ● ●● ●● ●● ● ● ●●●● ● ● ●● ●● ● ●●● ● ●●● ●●● ●● ● ● ●● ●● ●● ●● ●● ●● ● ●● ● ●● ●● ●● ●● ● ● ●●●● ● ● ●● ●● ●● ● ●● ●● ● ● ●● ●● ● ●● ● ● ●● ●●●● ● ●● ● ● ● ● ●● ● ●● ● ●●

Parophrys_vet ula ●● ●● ●● ● ●● ●● ● ● ● ●● ● ●●● ●● ● ● ●● ●●● ● ● ● ●●●● ●● ●● ●●●●●● ● ● ●● ●●●● ●● ● ● ● ●● ● ●●● ● ● ● ● ●● ●● ●● ●● ● ●● ●● ● ●● ● ●●● ● ● ●●● ●● ● ●● ● ●● ●● ●● ● ●● ●● ●● ● ●● ●● ● ●● ●●●● ●● ●● ●● ●● ● ● ● ●● ●● ●● ●●● ●●● ●● ● ●●● ●● ● ●● ● ● ●● ●● ●● ●●● ●● ●●●● ●● ●● ●●●●●●●● ●● ●● ●● ●●● ●●●● ●● ●● ●●●● ●● ●●● ● ●● ●● ●● ●● ●● ● ●● ● ●●● ●● ● ●● ●● ●●●● ● ●●● ●● ●●●● ● ●● ●●● ●● ●●● ●●● ●● ●●●● ●●● ●● ● ●● ●● ●● ●●● ● ●● ●● ●● ●● ●●●● ● ●●● ● ●●● ● ●● ● ●● ●● ● ●● ●● ● ●●●● ●● ●● ●● ● ●● ●●●● ●● ●●● ●● ●●● ●● ●●● ● ● ●● ●● ● ●● ●● ●● ●●● ●● ●● ●● ●● ● ● ● ●●● ●● ● ●

Lepidopsetta_bilineata ● ●● ●●● ● ●● ●●●● ●● ●●● ●● ●● ●● ●● ●●● ● ● ● ● ●● ●●●● ●●● ●●●● ●● ●● ● ● ●● ●● ●● ●● ●●● ●● ● ●●● ●● ●●●● ●● ●● ●● ●● ● ●●● ●●●●● ●● ●● ●● ● ● ● ●●● ●● ●●● ● ●●●●●● ●● ●● ●●●● ● ● ●●●●●●●● ●● ●● ● ●●● ●● ●● ●●● ●●●● ●●● ●●● ● ●● ●● ●● ●● ●● ● ●● ● ●●● ● ●● ●●●● ●●● ●● ●●●● ●● ●● ●●●● ●●●● ●● ● ● ●●●● ●● ●● ●● ●● ●●● ●●● ●● ●●●● ●●●● ●●● ●● ●● ●● ● ●● ●● ● ● ●● ● ●● ●● ● ●● ●● ●●●●● ● ● ●● ●●● ●●●● ●● ●●●● ●● ●●● ● ●●●● ●● ●●● ●● ●● ● ●● ●● ●● ●● ●● ●● ●● ●● ●●● ●●● ● ●●● ●● ●●●● ●● ●● ● ●● ●● ●●●● ●● ●● ● ●●●● ● ● ●● ●● ●●●● ●● ● ●● ● ●● ●●● ●●●●● ●● ●● ●● ● ●● ● ●● ●● ●● ● ●● ●● ● ●● ●●●●● ● ●● ●● ●● ●● ●● ●● ●●● ●●●● ●● ●●● ●● ●●●● ●● ●● ●● ●● ●●● ●● ● ●● ● ●●●●● ●● ●●● ●● ●

Isops e tta_is olepis ●● ●●● ● ●● ● ●●● ●●●● ●● ●●●● ●● ●● ●● ● ● ●●●● ● ●● ●●●● ●●●● ●●●● ● ●● ●● ● ●● ●●● ●●● ●● ●● ● ●●●●● ●● ● ●● ●● ●● ●● ●●●●● ●● ●● ●●● ●● ●● ●●● ●● ●● ● ● ●● ●●● ●●● ● ●● ● ●● ●● ●● ●● ●● ●● ●● ● ●● ●● ●● ●● ●● ● ●● ●● ●● ●● ● ●●●● ●● ●● ●● ● ●● ●●● ●●●● ●● ●● ● ●● ●● ● ●● ●● ●● ●●●●●● ●● ●● ●● ●● ●● ●● ● ●● ●● ●●●● ●● ● ●● ●● ●● ● ●● ●●●● ●●● ●● ●●● ● ●● ●● ● ●● ●● ● ● ●● ● ●●● ● ●● ●●● ●● ●● ●●● ●● ● ●● ●● ●●● ●● ●● ● ●● ●●●● ●● ● ●●● ●● ●● ●● ●● ●● ●●● ●● ●● ●●● ●● ●●●● ●● ● ●● ● ●● ●● ●● ●● ●● ● ●●●●● ●●● ●● ●● ●● ●● ●● ●● ●● ● ●● ●●●● ●●●● ●● ●●● ●● ●● ●●●● ●● ●● ●● ●● ● ● ● ●● ●● ●● ● ●● ●●● ●

Lepidopsetta_polyxystra ●● ●● ●● ●● ●● ●● ● ● ●● ● ●●●● ●● ●●●● ●● ●● ●● ●● ●● ●● ●● ●● ●● ●● ●● ●● ●● ● ●● ●●●● ● ●● ●● ●●● ● ●● ●●●● ●● ●● ●●● ●● ●● ●● ● ● ●● ●● ●● ●● ●● ●● ● ●● ●● ●● ●● ●● ●● ● ●●● ●●● ●● ● ● ●● ● ●●● ●●● ● ● ●● ●●● ● ●● ●● ● ● ● ●●● ●● ●● ●● ●●● ●● ●●● ●● ●● ● ●● ●●● ● ●● ●● ●● ● ●● ●● ●● ● ● ● ●●● ●● ●● ●

Lepidopsetta_mochigarei ● ● ●● ●● ●● ● ● ● ● ●● ●● ● ● ●● ●● ● ● ● ●● ● ●●● ● ●● ●●● ●● ●●● ●● ● ● ● ●● ●● ● ●●● ● ●●● ●● ●● ●● ●● ●●● ●●● ●● ● ●●● ●● ●●● ●● ● ● ●● ●● ●● ● ●● ●● ● ●●●●● ●● ●● ● ●● ● ●● ●●●●● ●●●● ● ●● ● ● ●● ●● ● ● ●●●● ● ● ●● ● ● ●●● ●●● ● ●● ● ● ●● ●● ● ●●●● ●●●●● ●●● ●● ●● ●● ●● ●●● ●● ● ●●●● ● ●● ● ●● ●●● ●● ●●● ●● ● ●● ●● ● ● ●● ●● ● ●●●●

Parophrys_v e t ulus ●● ● ●● ●● ● ● ●● ●● ●● ●● ●●● ●● ● ●● ●● ● ●● ●● ●●●● ● ● ●●● ●● ●● ● ●● ●● ●● ●● ●● ●● ●● ●● ●● ● ● ●● ●●● ● ● ●●● ●● ●●● ●● ●● ●● ●● ● ●● ●● ●●● ●●● ●● ●● ● ● ● ● ● ●● ●● ● ●● ●● ●●● ●● ●● ●● ●● ●● ● ● ●● ●● ● ●● ● ●● ●● ● ●● ●● ●● ●●● ●● ●●●● ● ●● ●● ●●●●● ● ●● ● ● ●●● ●●● ●● ● ●● ●● ● ●● ● ●● ●●● ●● ● ●● ●●● ●● ●● ●●● ●●● ●●● ● ●● ● ●● ● ●● ● ●●● ●●● ●● ● ●●● ●● ●●●● ●

Pseudopleuronectes_schrenki ●● ●●● ● ● ●● ●● ● ● ●● ● ● ●● ●● ●● ● ●● ● ●● ● ●● ● ● ●● ● ●● ● ●● ●● ● ●●● ●● ●● ●● ● ●● ●●●● ●●●● ●● ● ● ● ●● ●●●● ● ● ● ●● ●● ●● ●● ●● ●● ●●● ●● ● ●● ●● ● ● ●● ●●● ●●●● ●● ●● ●● ●● ● ●● ● ● ● ●● ●● ●● ● ● ●● ●● ● ●●●● ●●● ●●● ●● ●● ●● ●●●● ●● ● ●● ● ●● ● ●●● ●●●● ●● ●● ● ●●● ● ●●●● ●●● ● ● ● ●●● ●● ●● ●● ●●●● ●● ●● ●●● ●● ●● ●● ●● ●● ● ●●●● ● ●● ●● ● ● ● ●● ●● ●●●● ● ● ●● ●● ●●●● ● ●● ●● ●● ●● ●●●● ●● ●●●● ●●●● ● ● ● ● ●● ● ● ● ●● ●● ●● ●● ● ●● ●● ● ● ● ● ●● ● ●●● ● ● ● ● ● ●● ● ● ● ●● ● ● ●● ●●● ● ● ●● ● ● ●● ● ● ●● ●● ●● ●● ●● ●●●● ●●●● ● ●●●● ● ●●● ●● ●●●● ●● ● ● ●●● ●● ● ●● ●●●● ●●●●●●● ●●● ● ● ●●● ●● ●● ●● ●● ●● ●● ●● ●● ●● ● ●●● ●● ● ●● ●● ●● ●● ● ●● ●● ●● ●● ●● ●● ● ●●● ●● ●● ● ● ●● ●● ●● ●● ● ●●●● ●● ●● ● ●● ●●●● ●● ● ●● ●● ●● ● ●● ●● ●● ●● ●● ●● ●● ● ● ●●● ●● ●●● ●● ●● ●● ●●●● ●● ● ●● ●● ●● ●● ● ●● ● ●● ●●●● ●● ● ●● ● ●● ●● ● ●● ●●● ● ●● ●● ●● ●● ●● ●●● ● ●● ●● ●● ●● ●● ● ● ● ●●●● ●● ●● ●● ●● ● ●● ● ●● ●● ●●● ●● ●● ● ●● ●● ● ●● ●●●● ●●●● ●● ● ●● ●●● ●● ●● ●●●● ●● ●● ●● ●●●● ● ●● ●● ●● ● ● ●●●● ● ●● ●●● ●● ● ●● ● ●● ●● ●● ●● ●●●●●● ●● ●● ● ●● ●● ●● ● ●● ●● ● ● ●● ● ●●● ● ●● ●● ●● ●● ● ●● ●●●● ●● ●● ●● ●●●● ●● ● ●●● ●● ●● ● ● ●● ●●●● ● ●● ● ●● ●● ●●● ● ● ●● ● ●● ●● ●●●● ● ●● ● ●● ● ●●● ●● ● ●●●● ● ●● ●● ●● ●● ●● ●● ●● ●● ●● ●● ●● ●● ● ●● ●● ●● ●●● ●● ●● ●● ●● ●● ●● ●● ●● ●●● ●● ● ● ●● ●●●● ●● ● ●● ●●● ● ●● ●● ●●● ●● ●● ●●●● ●● ● ●● ●● ●● ● ●● ●● ●● ●●● ●● ●● ● ●● ●● ●● ● ●● ●●● ● ●● ●● ●● ● ●● ●● ● ● ●●●● ●●●● ●● ●●●● ● ●● ● ●●● ●●●

Pseudopleuronect es_yokohamae ●● ●● ●● ● ●● ● ●● ●● ●●● ●● ● ●● ●● ● ● ●● ●● ●● ● ●●● ●●●● ● ●●● ●● ● ●● ●● ●●● ●●● ●● ●● ● ●● ●●●● ●●●● ●● ●● ●● ●● ●●● ● ●● ●●● ●● ●● ●● ●●●●●● ●●●● ● ●● ● ● ● ●● ● ●● ●● ●● ●● ●● ●● ● ●●● ●● ●● ● ●● ● ● ● ●● ●● ●● ● ●● ●● ●●● ● ●●● ●● ●● ● ●●● ●● ● ●● ●●●● ●● ●● ● ●● ● ●● ●● ●●● ●● ●● ●● ● ●● ●● ●● ●● ● ●● ●● ● ● ●● ●● ● ● ● ●● ●● ●●●● ●● ● ●●● ● ●● ●● ●● ●●● ●● ●● ●● ●● ●● ● ● ● ●● ● ●●●● ●● ●● ●● ● ●● ● ●● ●● ● ● ●● ●● ● ●● ●● ●● ● ●● ●● ●● ●●● ●● ● ●● ●●●● ●● ● ●● ●● ● ●● ●●●● ●● ● ● ● ●●●● ● ●● ●●●● ●● ●● ● ● ●● ● ● ●●● ●●●● ●●● ● ● ●● ●●● ● ●● ● ● ●●● ● ●● ●● ●●● ●●●● ● ●● ●●●●● ●●●● ●● ●● ● ● ● ●● ● ●● ●● ● ●● ●● ●●● ● ●● ● ● ● ● ●●●● ●●● ●● ● ● ● ● ●●●● ● ●● ●● ●● ● ●● ●●●● ● ●● ●● ●● ●● ●●●● ●● ●● ●● ● ●● ● ●● ●● ● ● ●● ● ●●● ● ●● ● ●●●● ●●●● ●●●● ●● ●● ● ●● ● ● ●●●● ● ● ●●●● ●● ● ●● ● ● ●● ● ●● ●● ●● ●● ● ●●● ● ●●● ● ●●●● ●●●● ●● ● ● ● ●●● ●●● ● ●●●● ●●●● ● ●●● ●●●● 8:L. ferruginea ●● ●● ●●●●●● ●● ●● ● ● ● ● ● ●●●● ●● ●● ● ●● ● ●● ●● ● ● ● ●● ●● ●●●● ● ●● ●● ● ●● ● ●● ●● ●●●● ●●●● ●● ● ● ● ● ●● ● ● ●● ●● ● ● ● ● ●● ● ● ● ● ●●● ● ● ●●● ● ● ●● ● ●● ● ● ● ● ●●● ● ● ● ●● ●●

Pseudopleuronectes_obscurus ● ●● ●● ● ● ● ●● ●● ● ●● ●● ●● ● ●● ●●●● ● ●● ●● ● ●●● ●● ●● ●●● ●● ●● ● ● ● ●● ● ●●●● ●●●● ●●●● ●● ●●●● ●●●● ●● ● ●● ●● ●● ●● ●● ●●●● ● ●● ●●●● ●● ● ● ●●●● ●● ●● ●●● ●● ●● ●● ● ●●●● ●● ●● ●● ●● ● ●●● ●●● ● ●● ●●●● ●● ● ●● ●●● ● ●● ●● ● ●● ● ●● ●● ●● ●●●● ●●●● ●● ●● ●● ●● ●●●● ● ●●●● ●● ●● ●● ●●● ● ● ● ●● ●● ●●●● ● ●●●● ●● ●● ●● ●● ●● ●●● ● ●●●● ●● ●● ● ●● ●●●● ● ●●● ●● ●● ●● ●● ●● ●●●● ●●●● ●● ●

Pseudopleuronectes_herzensteini ● ● ● ●● ●●● ●● ●● ● ●● ●● ●● ●● ● ● ● ● ●● ● ●● ● ● ●●●● ●●●● ● ●● ● ●●●● ●● ●● ● ● ●● ● ●● ● ●● ●●● ●● ●●● ●● ●● ● ●● ●● ● ● ●●●● ●●●● ●● ●●●● ●● ● ●● ●● ●●●● ●● ● ● ● ● ●● ● ●● ●● ●● ●●●● ●● ●● ●● ●●●● ● ●● ● ● ● ● ●● ● ●● ●● ●● ● ● ●● ● ●● ●● ●● ● ●● ●● ● ●● ●● Pseudopleuronect es_americanus ● ●● ●● ●●●● ●● ●● ●● ●● ●●●● ●● ●● ●● ●● ● ●● ●● ●● ●●●● 6:C. chittendeni ●●● ●●● ●● ● ●● ● ●● ● ●● ● ●● ●●●● ●● ●● ●● ● ● ●● ●●●● ●● ●● ●● ●●●● ●● ●● ●● ●● ●● ●●●● ● ●●●● ● ●●●● ●●● ● ●● ● ●●●● ● ● ● ● ● ●● ●●●● ●●

Hippoglossoides_robust u s ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● Hippogl ossoi des_el assodon ● ● ●● ●● ●● ●● ● ● ● ●● ●● ● ● ●● ● ●● ●● Hippoglossoides_platessoides ● ● ● ● ● ●● ● ● ● ●● ●● ● ● ●● ●● ● ● ●● 3:C. arctifrons ● Hippoglossoides_dubius ● ● ● ● ● ● ● ● ●● ●● ● ● ● ●● ● ● 10 ●● ● ● ●● ● ●● ●●● ● ●

Cleist henes_pinet orum ●● ● ●● ●● ● ● ● ● ● ●● ● ● ●● ●● ● 5:S. micrurum ● ● ● ● ●● ● ● ● ●●● ● ● ●● ●

Cleisthenes_herzensteini ● ●● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ●●● ●● ● ● ● ● ● ●● ● ● ● ● ● ●● ●● ● ● ● ●●●

Dexistes_rikuzenius ●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

Limanda_limanda ● ● ● ● ●● ● ● ● ● ●● ● ●● 9 ●● ● ● ●

Limanda_aspera ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● Lim anda_s a k halinens i s ● ● ●●● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● Embassicht hys_bat hybius ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●●● ● ●● ● ● ●● ● Microstomus_bathybius ● ●●● ● ●● ● ● ●● ● ●● ● ● ●● ● ● ● ● ●●●● ● ● ● ● ● ●● ● ● ● ●

Microst omus_kit t ●● ●● ● ● ● ● ● ● ● ● ●●●●● ● ● ● ● ● ●

Microstomus_achne ● ● ● ● ● ● ● ● ●● ● Microst omus_pacif icus ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

Glyptocephalus_cynoglossus ● ● ● ● ● ● ●● ●●● ●●● ●●●●● ●● ●● ● ●● ● ●● ●● ●● ●● ● ●●●●● ●● ●●●● ●● ●●●●●● ●●●● ●●●●● ●● ●● ● ●●●●● ●● ● ●●● ●● ●●●● ●●●● ●● ●● ●● ●● ●● ●● ●● ●● ●●●●●●●● ●●●●●●●● ●● ● ●● ●●●●●●●● ●●●● ●● ●●●●●● ●●●● ●● ●● ●● ● ● ●● ●● ●● ●● ●● ●● ●● ●●● ●●●● ●● ●● ●● ●●● ●●●● ●● ●● ●● ●● ● ●● ●● ●●●● ●● ●●●● ●● ●● ●● ●● ●● ●●●● ● ●●●● ●●●● ●●●● ● ●●●● ● ●● ●● ●● ●●●●●● ●●●●●●●● ●●● ●● ●●● ● ●●●● ●● ●●●● ●●●●●● ● ●● ●● ●● ●● ●●●● ●● ●●●●●●●●●● ●●●●●● ● ● ●● ●● ●● ● ●●● ● ●●●● ●● ●● ●●●● ●● ●● ●● ● ●● ●● ●● ●●●● ●● ●● ●● ●● ●●●● ●● ●● ●● ●● ●● ●●●● ●● ● ●●●● ●● ●● ●● ●● ●●●●●● ●●●●●●●● ●● ●● ●● ● ●● ●● ●● ● ● ●● ●●●● ●● ●● ●● ●● ●● ●●●● ● ●●●●● ●● ●● ● ●●●● ●● ● ●●●● ●●● ●●●●●●● ●●●●●● ●● ●●●●● ●● ● ●● ●●●●●●● ●●●● ●● ●● ●● ●● ● ● ●● ● ●●●● ●● ●● ●● ●● ● ●● ●●● ● ●● ●● ●●●● ●●●●● ● ●● ●● ●● ●● ●● ● ●● ●● ●● ●● ●● ●● ●● ●● ●● ●●● ●●●● ●●●● ● ●● ●●●●●● ●● ●● ●●●●●● ●●● ● ●●●● ● ●●● ●● ● ● ●●●● ●●●●●●●● ●● ●●●● ●● ●●●● ●● ●● ●● ●● ●● ●●●●●●●● ●● ●●●● ● ●● ●● ● ● ● ●● ●● ●● ●● ●●● ●● ● ●●● ●● ●● ● ●● ●●●● ●● ●●● ●●●● ●●●●● ●● ●●●●● ● ●●●● ●● ●● ●● ●● ●● ●● ●● ●●●● ●●●●●●●● ●● ●● ●● ●●●● ●● ●● ● ●● ●● ●●●●●● ●● ●● ●● ●●●● ●●●●● ● ●●●● ●● ●● ●●● ●● ●● ● ●●●●●●●● ● ●● ●●●● ● ●●●●● ●●●●●● ● ●● ●● ● ● ●●● ● ● ●●●● ●● ● ●● ●●● ●● ●● ● ● ● ●● ●● ●● ● ●● ●●●● ● ● ●●●● ●● ●● ● ●● ●● ●● ● ● ● ●●● ●● ●● ●● ●● ●● ●● ● ●● ● ●● ●●●● ● ●●●● ●● ●● ●●●●●● ●● ●● ●● ●● ●●●● ●●●● ●● ●● ●● ●● ●●●● ●●●●●●●●● ●●● ●●●●●● ●●●●●● ●●● ●● ● ● ● ● ● ●

G lypt ocephalus_st elleri ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● G lypt o c ephalus _zachirus ● ●● 12 ● ●● ● ●● ● ● ● ● ● ● ● ● ● ● Tanakius_kitaharae ● ● ● ● ● ●

V e r a s per _ m o s e r i ● ● ●●●●●●● ● ● ● ● ●

Verasper_variegat u s ●● ● ● ● ● ● ● ● ● ● Eopsetta_grigorjewi ● ● ● ● ● ● ●● ● ●● ●●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ●● ●● ● ●● ●● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ●

Eopsetta_jordani ● ● ●● ● ● ● ● ● ●●● ● ● ● ● ● ●●

Clidoderma_asperrimum ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ●●● Reinhardtius_hippoglossoides ● ●● ● ●

Hippoglossus_hippoglossus ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ●● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ●

Hippoglossus_stenolepis ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● Southern ● ● ● ● ● ● ● ● ● ●●●●●●●●●● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ●●

Lyopsetta_exilis 11 ● ● ● ● ● ● ● ●● ● ●● ● ● Northern ● ● ● ● ● ● ●● ● ●●● ●● ●● ●● ● ● ●● ●● ●● ●● ●● ●● ● ● ● ●●

Pleuronicht hys_vert icalis ●● ●● ● ● ●● ● ● ● ● ● ● ●● ●●● ● ●● ●● ● ● ● ●● ●● ● ●● ●● ● ● ● ● ●● ●● ● ●● ●● ● ● ●● ●● Pleuronichthys_coenosus ● ● ● ● ●● ● ●● ● ● ●● ●● ●● ●● ●● ● ● ● ● ●● ● ●● ● ● ● ●● ● ●●●● ●● ● ● ● ●●●●●● ●● ● ● ● ●● ● ● ● ●● ● ● ● ●● ● ●● ● ● ● ● ● ● ● Pleuronichthys_ritteri ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●●

P l euroni chthys_decurrens ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ●●● ●● ●● ● ● ● ● ● ●● ●● ●● ●● ●●●●●

Pleuronic h t hys_cornut u s ●●●●● ●● ●● ● ● ●● ●● ●● ●● ●● ● ● ● ●● ●● ●● ●● Pleuronichthys_japonicus ●●

Pleuronichthys_guttulatus ● Reinhardtius_evermanni ● ● A t herest hes_evermanni ● ● ● ● Atheresthes_stomias ● Pseudorhombus_oligodon ●● ●● ●● ● ● https://doi.org/10.1101/247304 Pseudorhombus_malayanus Pseudorhombus_elevatus ● ●● ●

Pseudor hom bus _jeny n s i i ●● ● ● ● ● ● ● ●● ●● ● ● ●●● ●● ● ● ● ● ● ●●●● ●● ●● ●● ● ● ● ● ● ● ●

Pseudorhombus_duplic i ocellatus ● ● ● ● ●●●● ●●●● ● 4:C. darwini ●● ● ● ●● ●● ●● ● ● ● ● ●●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● Pseudorhombus_arsius ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ●● ●● ● ●● ●●● ● ●●● ● ● ● ● ●

Pseudorhombus_natalensis ● ● ● ● ●● ● ● ● ● ●● ● ● ●● ● ● ● ● 6:C. querna Pseudorhombus_pentophthalmus ● ●●●● ●●●● ● ● ●

Psettina_hainanensis ●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●

Tephrinectes_sinensis ●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ●●

Tarphops_oligolepis ● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ● T arphops_elegans ● ● ●● ●● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

P seudorhombus_lev isquamis ● ● ●● ● doi: ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● 1 ● ●● ●

Hippoglossina_oblonga ●●● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●●● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● Paralichthys_oblongus ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● P a r alic h t h ys_pat agonic u s ● ● ● ● ● ● ● ●● Paralichthys_adspersus ● ● ●● ● ● ● ●●● ● ●●●● ● ●● ● ●● ● ● ● ● ● ● ● ● ●●●●●●●●● ● ●●●● ● ●● ● ● ● ●●● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●

Paralicht hys_calif ornicus ●●●● ●●●●●●● ●●●●●●●●●● ●●●●●●●●● ● ● ●● ●● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ●●●● ● ●●●●●●●●●●●●●● ●●●●●●●●●●●● ●●●● ● ●●●●●●●● ●● ● ●●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●●●●●

Paralichthys_squamilentus ● ●● ● ● ●● ●● ●● ●●●● ●●●●●●●●●●●●●●● ● ● ● ●●●●●●●●●●●●●●●●●●● ● ● ● ● ● ● ● ● ● ●●●●●●●●●●●●●●● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ●●● ●●●●●●● ●●●●●●●●●●●●●● ●●●●●● ●●●●●●●●●●●●●●●●●●●● Paralichthys_albigutta ●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●●●● ● ● ● Paralichthys_olivaceus ● 3:C. platophrys ● ●● ●●● ● ●

Paralichthys_isosceles ● Xystreurys_ r a s ile ● Xystreurys_liolepis ● ● Scopht halmus_aquosus ●

Arnoglossus_capensis ● Arnoglossus_imperialis

Arnoglossus_t hori ●

Arnoglossus_laterna ● Crossorhombus_kanekonis Arnoglossus_tapeinosoma ● Crossorhombus_valderost rat u s ● Crossorhombus_kobensis Crossorhombus_azureus Arnoglossu s _ m a crolophus Hippoglossina_stomata ● Arnoglossus_aspilos 5:S. maculiferum Parabothus_chlorospilus ● Pse ttina_iijim a e ● ●●● ●

Psettina_gigantea ● ● ● ● ●

Psettina_tosana ● ●

Engyprosopon_macrol epis ● ● A rnoglossus_polyspilus ● ● Lophonectes_gallus 1:H. stomata

Engyprosopon_xenandrus ● ● Laeops _nigr o m a c ulat u s ●

Arnoglossus_scapha ● ● ● Laeops_pectoralis ● ●

E ngy pros opon_maldivensis ● Laeops_macropht halmus

E ngy p r o s opon_m ult i s quam a ● Kamoharaia_megastoma ● ● Chascanopsetta_lugubris ● ●

Chascanopsetta_prorigera ● ● ●

Grammatobothus_polyophthalmus ● ● ● ● 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 who has granted bioRxiv a license to display the preprint in perpetuity. It is made available certified by peer review) is the author/funder, B o t hus _leopardinus ● ● ● bioRxiv preprint 8:L.proboscidea

Engyophrys_senta ● ●● ● ● ● ● ●

Trichopsetta_ventralis ● ● ● ●● ● ●● ● ● ●● ● ● ●

M onolene_s e ssilic auda ●●● ●● ●●● ● ● ●● ● ● ● ● ● ● ●● ● ● ●●●● ●●●●●●●●●●●●● ●●●●●●●●● ●

Engyprosopon_grandisquama ● ● ● ●●●● ●●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●

Bothus_ocellatus ●● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ● Bot hus_maculif erus ● ● ● ● ● ● ● ● ● Bothus_podas ● ● ● ● ●● ● ●● ●● ● ● ● ● ● ● ●● Bothus_robinsi ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ●● ●

Bothus_lunatus ● ● ● ● ● ● ● ● ●● ● ●● ●● ● ● ● ●● ●● ● ● ● ●● ● ● ●● ● ●● ● ● ●● ● ● ● ●● ● ● ● ●● ● ● ● ● ●● ●●●● ● ● ● Bot hus_mancus ● ● ● ●●●● ● ● 11:H. stenolepis ●

Bothus_pantherinus ● ● ● ●● ● ●● ● ● Bothus_myriaster ● ●● ● ● ● ●●●●● ●●●● ●● ●●● ●●● ●●● ●● ● ● ● ● ● ● ● ●● ●●● ● ●

Japonolaeops_dentatus ●●●●●●●●●● ● ● ● ● ● ● ● ● ●● ● ●

Laeops _ k i t ahar a e ● ● ● ● ● ●● ● ●

Arnoglossus_yamanakai ●●● ● ●● ●● hawaiiensis ● ● ●●● ● ● ● ●●●● ●●●●●●● ●●● ● ● ●●

Neolaeops_microphthalmus ●● ●●● ●● ●●●●●●●●● ●●●●● ● ● ● ●● ● ● ●●● ●●●●●● ●●●●●● ●●●●●●●●●●● ●●●●●●●●●● ●●●●●●●●●● ● ●● ●● ●●● ● ●● ●●●●●● ●●●●●● ●● ● ● ●● ●● ●●●● ●●● ●● ●●● ●● ●●●● ●●●●●●●● ●●●●●●●●●●●●●● ●●●●●●●●●●● ●●●●●●●●● ● ● ● Arnoglossus_tenuis ● ● ●●●●●●●●● ● ●●●●●●●●● ●●●● ● ●● ● ● ●●●●●●●●●●●●● ●●●●●●●●●●● ●●●●●● ●●●●●● ●●●●●● ●●●●● ●●● ● ●● ● ●●●●● ●●●●● ● ●●●●●● ●●●●● ●●●●●●●●●●● ●●●●●●●●●●● ●●●●●●●●●●●●●● ●●● ● ● ● ● ● ●●●●●●●●●●● ●●●●●●●●●●● ●●●●●●●●● ●●●●● ● ● ● ● ●●●●●● ●●●●●●●●● ●●●●●●●●●●●●●●● ●●●●●●●●●●●●●● ●●●●●●● ●●●●● ●●●●● ●●●●● ●●● ● ●●● ●●●● ● ●● ●●●●● ●●●●●●● ●●●●●●●●●●●● ●●●●●●●●●●● ● ● ●●●●●●●●● ●●●●●●●● ●●●●●●●● ●●●●●●●●● ● ● ●●●●● ●●●●●●●●●●●●● ●●●●●●●●●●●●●● ●●●●●●●●●●●●● ●●●●●●●●●●●●●● ●●●●●●●● ●●●●●● ●●●● ●● ●●● ●●● ●●●● ● ●● ●● ●●●●●● ●●●●●● ●●●●●● ●●●●● ●●●●●●●●●

Citharichthys_arctifrons ● ● ● ●●●●●●●● ●●●●●●● ●●●●●●●●● ●●●●●●●●●●●●● ●●●●●●●● ●● ● ●● ●●● ●● ● ●●●●●●●● ●●●●●●●●●● ●●●●●●●●●●●●● ●●●●●●●●● ●●●●●● ●●● ●●●●●● ●●●●●● ●●● ● ●● ●●● ●●●● ●●●● ●●●●● ●●●● ●● ●● ●●●●●●● ●●●●●● ●●● ●●●● ● ●● ● ● ● 3 ●●●●●● ●●●●●● ●●●●● ●●●●●●●●●●● ●●●●●●●●●●● ●●●●●●●●●●●●● ●● ●● ● ● ●●●● ●●●●● ●●●●●●●● ●●●●●● ●●●●●●●●● ●●●●● ●●● ● ●● ●●●●● ●●●● ●●●● ●●●●● ●●●● ●●●●●●● ●●●● ●●●● ●●●●●●● ●●●●● ●●● ●●●●●●● ●●●●●●● ●● ● ● ● ●● ●●●● ● ● ● ●●● ●●●●●● ●●●●●●● ●●●●●●●●●●●● ●●●●●●●●●●● ●●●●●●●●● ●●● ● ● ● ●●●●● ● ●●● ● ● ● ●●● ●●●●● ●●●●● ●●●●●● ●●●● ●●●● ●●●●●●● ●●● ●●●●●● ●●●●●●● ●●●●●● ●●●●●● ●●●● ●●●●● ●●● ● ● ● ● ●● ● ● ● ● ● ●●●●● ●●●●●●●● ●●● ●●●●● ●●●●●● ● ●●●● ●●●● ●●●●●● ●●●●● ●●●●● ●●●● ●●● ●●● ●●●●● ●●●●●●● ●●●●●●●● ●●●● ●● ●●●● ●●●● ●●●●●●● ●●●●● ●●● ●●●●●●●●● ●●● ● ● ● ●●●●● ●●●●● ●●●●●● ●●● ●●●● ●●●●● ●●●●● ●●●●●●●●● ●●●●● ●●●●●●● ●●●●●● ●●●●●●● ●● ● ● ●● ●● ● ● ● ● Citharichthys_xanthostigma ● ● ●● ● ● ●●● ●●● ● ● ●● ● ● ●● ● ● ●● ●●● ●●●●● ●●●● ●●●● ●●● ●●●● ●●●●●●●●● ●●●●●●●● ●●●●●●●● ●●●●●●●●●●● ●●●●●●●●● ●●● ● ● ●●●● ● ●● ●●● ●● ● ● ● ●● ● ●● ●●●● ● ●●●●● ●●●●●● ●●●●● ●●●●● ●●●● ●●●●● ●●●●● ●●●●●●●●●●● ●●●●●●● ●●●●●●●● ●●●●●●●● ●●●●● ●●● ●● ● ● ● ●● ● ● ●● ● ● ● ● Citharichthys_sordidus ●●● ●●● ●●●● ●●●● ●●● ●●●●● ●●● ●●●●● ●●●●●●●●● ●●●●●●●●●●●●●●● ●●●●●●●●●● ●●● ● ● ●●● ● ● ●● ● ●● ● ● ● ●●● ● ●● ● ● ● ●●● ●● ● ● ● ● ●● ● ● ●● ● ● ● ● ●●●●●● ●●●●● ●●●● ●●●●● ● ●● ●●●● ●●●●●●●● ●●●●●●●●● ●●●●●●●●●● ●●●●●● ●● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ●●● ●●●● ●●●●● ●●●●● ●● ●●●●● ●●●●● ●●●●●●●● ●●●●●●●●● ●●●●●●●●● ●● ● ●● ● ● ● ● ●●

Etropus_microstomus ● 2:P. ● ● ●● ● ● ●● ●● ●●●● ●●●● ●●● ●● ● ●●●● ●●●●●● ●●●●●●●●●●● ●●●●●●●●●●● ●●●●●● ●●●● ●● ●● ●●● ● ●● ● ●● ● ● ● ●●●●●●●●● ● ● ● ●●● ● ● ● ●●● ●●●● ●●●● ●● ●● ●●●●● ●●● ●●●●●●●●●●●● ●●●●●●●● ●●●●●●●●● ●●●● ● ●● ●● ●● ●● ●●●● ●●●● ● Cit har i c h t h ys_ar enac eus ● ●● ●●●●● ● ● ● ●● ●●● ●●●●●●●● ●●●●●●●●●● ●●●●●● ●● ●● ● ●● ●● ● ●● ● ● ●●●● ●● ●●●● ●●●●●●●● ●●●●●●● ●●● ●● ● ●●● ●● ● ● Citharichthys_gilberti ●●●●● ● ● ●● ●● ●●● ●● ● ● ● ●●● ● ●●● ● ●●● ●●● ● ● ● ●● ●● ● ● ●● ●●● ●●●●● ●●●● ●●●● ●● ● ●●●● ●●● ●● ●●● ● Citharichthys_spilopterus ●●● ●●● ●●● ●●●● ●●● ●●●● ●● ●●●●● ●●●●●●●● ●●●●●●●●● ●●● ●● ●● ● ●●● ●● ●●● ●●● ●● ●● ●●●●●●●●●●● ●●●●●●●● ●●●●●●●● ● ●●● ●● ●●●● ●●● ●●● ●●● ●●● ●●● ● ●●●●●●●● ●●●●●●●● ●●●●●●●●● ●●●●●●● ●●● ●● ●● ●●●●●●●●●● ● ● ● Cit haricht hys_macrops ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●● ●●● ● ●●●●● ●●● ●●●●● ●●●●●● ●●●●●●●●●● ●●●●●● ●●●●●●●●●● ●●●●●●●●●●● ●●●●●●● ●●●●● ●●●● ●● ● ● ● ●●● ●●●●● ●●●●● ●●●●●● ●●●●●●● ●●●●●●●●● ●●●●●● ●●●●●●●● ●●●●● ●●●● ●●●●● ●●●● ●●● ●● ● ● ● Citharichthys_darwini ● ● ●● ●●●● ●●●●● ●●●●●● ●●●●●● ●●●●●●●●● ●●●● ●●●● ●●● ●●● ●● ●●●●●● ●●●●● ●● ● ●●● ●●● ● ●● ●● ●●●● ●●●●● ●●●● ●●●● ●● ●● ●● ●● ●● ●● ●● ●● ● ●●● 4 ● ● ●●●●● ● ● ● ● ● ●● ●● Cit haricht hys_minutus ● ●● ●● ●●● ●●●●●● ●● ● ●●●●● ●●●● ●● ●● ●●● ●●●● ● ● ● ●● ●● ● ● ●●●● ●●● ● ●●● ●●●●● ● ●●● ●●● ●●●●● ●●●● ● ●● ● ●● ● ● ●● ● ● ●● ●● ●●● ●●● ●●● ●●●● ●●●●●● ●● ● ●●● ●● ●● ● ●● ● ●●● ● ●

Cit haricht hys_st igmaeus ●● ●● ● ●● ● ●●●●● ●●●●● ●●●● ●●●●●●● ●● ●● ●● ● ● ● ● ● ● ● ●● ●●● ●●● ●● ●● ●●● ●●● ●● ●●●● ●●● ●●● ● ●●

●● ●● ● ● ● ● ● ●● ●● ●●●●● ●● ●●●● ●●●●●● ●●● ●● 9:L. aspera ●

● Etropus _ c rosso t u s ● ● ● ● ●● ●●●●● ● ●●

●● ● ●●● ● ●● ●●●●● ●●● ●●● ● ● ● ● ● 5 . 0 ●●●● ●●●● ●●●● ●●● ● ●● darwini/Citharichthys minutus darwini/Citharichthys 4:Citharichthys Syacium maculiferum/Syacium micrurum maculiferum/Syacium 5:Syacium 6:Cyclopsetta chittendeni/Cyclopsetta querna 7:Pleuronectes platessa/Pleuronectes quadrituberculatus Limanda ferruginea/Limanda proboscidea 8:Limanda ferruginea/Limanda Limanda aspera/Limanda limanda 9:Limanda aspera/Limanda Hipoglossoides dubius/Hippoglossoides platessoides 10:Hipoglossoides dubius/Hippoglossoides Hippoglossus stenolepis/Hippoglossus hippoglossus 11:Hippoglossus stenolepis/Hippoglossus 12:Glyptocephalus stelleri/Glyptocephalus cynoglossus Citharichthys arctifrons/Citharichthys platophrys arctifrons/Citharichthys 3:Citharichthys Hippoglossina oblonga/Hipoglossina stomata 1:Hippoglossina oblonga/Hipoglossina

Etropus_longimanus natalensis hawaiiensis/Poecilopsetta 2:Poecilopsetta

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●● ● ● ● ● ●● ●● ●●●●●● ●●● ●●●●● ●●● ●● ●● ●

● ● ●● Syacium_micrurum ● ●● ●● ● ●●● ● ●● ●

5

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S yac ium_mac ulif erum ● ● ●● ● ●● ● ●

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●

●● ● ●●● ●● ● ● ● ● ● ●

●● ●● ●● ● ● ● ● ● ●

Syacium_papillosum ●

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● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ●

Cyclopsetta_chittendeni ●

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● 6

● ● Cyclopsetta_panamensis 7:P. quadrituberculatus Paralichthys_dentatus

Citharichthys_platophrys

Cyclopsetta_querna bioRxiv preprint doi: https://doi.org/10.1101/247304; this version posted January 12, 2018. 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.

A NONE B ALL C NORTH D SOUTH Northern sp. Southern sp. Density 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 E F G H Density 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0

0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 MYA MYA MYA MYA