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1 2 How the Central American Seaway and

3 an ancient northern passage affected

4 diversification

5 6 Lisa Byrne1, François Chapleau1, and Stéphane Aris-Brosou*,1,2 7 8 1Department of Biology, University of Ottawa, Ottawa, ON, CANADA 9 2Department of Mathematics & Statistics, University of Ottawa, Ottawa, ON, CANADA 10 11 *Corresponding author: E-mail: [email protected] 12

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13 Abstract 14 While the natural history of flatfish has been debated 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 trans-Arctic northern route, a hypothesis 26 fully compatible with paleogeographic reconstructions. 27 28 Keywords: Pleuronectiformes, Bayesian dating, trans-Arctic migration, vicariance, 29 Central 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 (Chanet 1997; Friedman 2012), and 51 placed with confidence on the flatfish evolutionary tree (Parham et al. 2012; Campbell et 52 al. 2014a; Harrington et al. 2016). As a result, calibrating a molecular clock becomes 53 challenging. On the other hand, a dense species sampling may enable us to take 54 advantage of a singular feature of flatfishes: some extant species are found both in the 55 Pacific and Atlantic oceans. Furthermore, the existence of geminate species pairs of 56 flatfishes, where sister taxa have one member in each ocean, suggests a speciation event 57 pre-dating the formation of the Isthmus of Panama, which occurred approximately 12 to 3 58 million years ago [MYA] (Haug and Tiedemann 1998; O'Dea et al. 2016). Also possible 59 is the role played by the opening of the Bering Strait around 5.5-5.4 MYA (Gladenkov et 60 al. 2002), which would have permitted the trans-Arctic migration of ancestral populations

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61 from one ocean to the other, as documented in marine invertebrates (e.g., Durham and 62 MacNeil 1967; Reid 1990) or vertebrates (e.g., Grant 1986), but to date, never in flatfish. 63 Our driving hypothesis is then that the sole information relative to the formation of the 64 Isthmus of Panama can be used to calibrate molecular clocks, and allows us to unravel 65 the timing of flatfish evolution, as how rapidly they diversified remains an unsolved 66 question. We show here that the diversification of flatfish in the seas surrounding the 67 Americas followed a complex pattern, related to not only the closure of the Isthmus of 68 Panama, but also to a warming event that opened up a trans-Arctic northern route.

69 Results 70 To test how the evolutionary dynamics of flatfish were affected by a major geological 71 event, the closure of the Central American Seaway (CAS), we reconstructed dated 72 Bayesian phylogenetic trees from a large data matrix under four relaxed molecular clock 73 models, each one of them being based on a different calibration scheme (Fig. 1). Under 74 the first model, no calibration priors were placed on internal nodes. This initial tree, with 75 the rogue sequences removed (data on GitHub: see Methods), was used to identify pairs 76 of sister taxa that are split between the two oceans, with one species in the Atlantic and 77 the other in the Pacific. This led us to single out twelve pairs of such species. These sister 78 species happened not to be evenly distributed on the estimated phylogeny (Fig. 2, top), 79 but they are the only geminate species included in GenBank (as of August 2016). On 80 each pair, we placed calibration date priors corresponding to the closure of the CAS (the 81 ‘ALL’ model). An examination of the posterior distributions of their divergence times 82 suggests that some species have a very narrow speciation window, where all the mass of 83 the posterior distribution is between 5-3 MYA, while others have a wider distribution 84 (Fig. 3A). Closer inspection of these distributions further reveals that most of the species 85 with narrow posterior distributions have a northern range (Fig. 2, and 3A, in blue), while 86 those with the wider posterior distributions have a “southern” distribution, closer to the 87 Isthmus of Panama (Fig. 2, and 3A, in red). To further assess this observation, we first 88 went back to the original clock model, removed all priors initially placed on sister taxa 89 (the ‘NONE’ model), and were able to validate that even in this case: northern and 90 southern species showed, to one exception each (Hippoglossus hippoglossus and H.

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91 stenolepis in the north, and Poecilopsetta natalensis and P. hawaiiensis in the south; Fig. 92 2), shifted posterior distributions (Fig. 3B). The former pair was actually not estimated as 93 being sister species in any of the four clock models (Fig. 1), while P. natalensis and P. 94 hawaiiensis, although inhabiting the Western Indian and Eastern Pacific oceans, occupy 95 ranges that do not extend to the coasts of the Americas as with the other identified sister 96 taxa. Models with priors placed only on northern (the ‘NORTH’ model: Fig. 3C) or 97 southern (the ‘SOUTH’ model: Fig. 3D) species also showed a similar temporal shift. 98 This shift suggested that southern species diverged early, before the complete closing of 99 the CAS, while northern species diverged later, at or possibly after the isthmus was 100 completed. Averaging these posterior distributions for the northern and southern species, 101 to the exception of the two outliers noted above, showed these results more clearly (Fig. 102 3E-H). To assess the robustness of this result to the inclusion of the P. natalensis and P. 103 hawaiiensis sister species, we conducted an additional set of analyses removing all prior 104 information from this pair. The ALL and SOUTH models, which are the only ones where 105 this prior occurs, show that the general pattern that we found above holds: southern 106 species diverged early, before the complete closing of the CAS, and northern species 107 diverged later, at or possibly after the isthmus was completed (Fig. S1). 108 All these results were obtained assuming that Psettodidae is an appropriate outgroup 109 for these analyses. However, the position of Psettodidae in flatfishes is still debated 110 (Betancur-R. and Orti 2014; Campbell et al. 2014a; Campbell et al. 2014b; Harrington et 111 al. 2016), so that their inclusion might affect our results. To assess this possibility, we 112 reran all four models without Psettodidae, letting the molecular clock root the tree (Aris- 113 Brosou and Rodrigue 2012). Figure S2 shows the exact same results as above (Fig. 3), so 114 that our dating results are also robust to the inclusion, or not, of Psettodidae. Our dating 115 results are also robust to the inclusion of internal calibration priors as used in Harrington 116 et al. (2016), and based on the Scophthalmus rhombus / Bothus pantherinus and the 117 Crossorhombus kobensis / Bothus pantherinus divergences, dated at 29.62 118 (Vandenberghe et al. 2012) and 11.06 MYA (Carnevale et al. 2006), respectively (Fig. 119 S3). 120 In an attempt to tease out these models (including Psettodidae and without the two 121 internal calibration priors) and their predictions about the exact timing of divergence

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122 between northern and southern species, we assessed model fit by means of the modified 123 Akaike’s Information Criterion (AICM; Baele et al. 2013), which accounts for 124 uncertainty in the MCMC sampling (Raftery et al. 2007). Even if model ranking based on 125 this measure is known to be unstable (Baele et al. 2013), it is clear that the models with 126 priors only on the northern or on the southern species perform significantly (> 200 AIC 127 units) worse than the two other models, which may be difficult to tease apart (Table 1). 128 The predictions of the best models suggest that H. hippoglossus and H. stenolepis, both 129 northern species, consistently diverged before the complete closure of the seaway, in 130 tandem with the average southern species, and that the average northern species diverged 131 in tandem with P. natalensis and P. hawaiiensis (Fig. 3E-F). 132 One intriguing result from these analyses is that the northern sister species seem to 133 come from a single clade (Fig. 2), which would suggest that it is possible to identify the 134 direction of the trans-Arctic migration. For this, we retrieved the current species range of 135 all flatfish analyzed here (Fig. S4), and indicated their average latitude on the 136 phylogenetic trees (Fig. S5). This northern clade, highly supported (posterior probability 137 > 0.9; see asterisk in Fig. S5) even in the analyses including the two additional internal 138 priors (Fig. S6), is mostly comprised of species currently found in the Pacific (e.g., 139 Reinhardtius evermanni), suggesting that the northern sister species that we identified 140 originated from the Pacific, and that their trans-Arctic migration was from the Pacific into 141 the Atlantic in a northern direction.

142 Discussion 143 Our results show that flatfish underwent a first speciation at the time when the CAS 144 closed, which led to the species that, today, have an equatorial range (Fig. 2), as the 145 formation of the Isthmus of Panama resulted in a barrier to gene flow leading to their 146 speciation. Our results also show that species that today have a northern range (Fig. 2) 147 either emerged at the closure of the seaway, or after its closure. These timing estimates 148 imply that this second bout of speciation was not caused by gene flow impeded by the 149 closure of the seaway, but demand an interpretation involving trans-Arctic gene flow, 150 through a northern route, before being interrupted, most likely this time by a climatic 151 event. Critically, these conclusions are robust to the inclusion of outgroup species

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152 (Psettodidae), of outliers species (P. natalensis and P. hawaiiensis), and are consistently 153 found across the four clock models implemented here, including the one (NONE) relying 154 on no other fossil information than the 47.8-42.1 MYA ancestor to this order (Chanet 155 1997; Friedman 2012) – hereby indicating that our data are highly informative with 156 respect to the timing of the divergence of these sister species (and rejecting our initial 157 hypothesis) following their trans-CAS and trans-Arctic migrations. 158 Trans-Arctic migrations from the Pacific to the Atlantic are not new, and have been 159 described in both invertebrates (e.g., Durham and MacNeil 1967) and vertebrates (e.g., 160 Grant 1986), but never using a relaxed molecular clock relying on as little information as 161 a single fossil and a large multigene data set covering an entire order (Pleuronectiformes). 162 While seminal work was usually based on paleontological (Durham and MacNeil 1967), 163 or on morphological evidence (Reid 1990), subsequent work started using allozymes and 164 other proteins (Grant 1986; Zaslavskaya et al. 1992), followed by genetic evidence, 165 mostly at the population level using mitochondrial DNA (Rawson and Harper 2009). 166 However, most of the recent work that relies on molecular clocks either makes strong 167 assumptions on substitution rates (Rawson and Harper 2009; Liu et al. 2011), uses 168 divergences that are vicariance-motivated but not supported by any fossil information 169 (McCusker and Bentzen 2010), or posit in which direction the trans-Arctic migration 170 took place (Dodson et al. 2007) to infer trans-Arctic migrations. Our work is the first to 171 use sister species to demonstrate the existence and elucidate the timing of both the trans- 172 CAS and the trans-Arctic events, while also inferring in which direction the latter took 173 place, without making too many assumptions. 174 Strikingly, the geological evidence is directly in line with our date estimates. The 175 fossil record suggests that the first aquatic connection between the Pacific and Arctic 176 (and Atlantic) oceans through the Bering Strait occurred approximately 5.5-5.4 MYA 177 (Gladenkov et al. 2002) due to a rise in sea levels, linked to tectonic activity 178 (Marincovich and Gladenkov 2001). This would have permitted the trans-Arctic 179 migration of populations ancestral to today’s northern species from one ocean to the other 180 through this ancient “northern passage.” This global warming event, between the late 181 Miocene to early Pleiocene, was then followed by a significant period of cooling during 182 the Pleiocene into the Pleistocene (Zachos et al. 2001), leading to periods of repeated

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183 glaciations and a subsequent ice age. These cold events would have resulted in the 184 closure of this ancient “northern passage,” hereby stopping the trans-Arctic gene flow 185 between the two oceans, and leading to the more recent speciation of the northern taxa. 186 For approximately a million years after the first opening of the Bering Strait, water 187 flowed through the strait in a southern direction, from the Arctic to the Pacific ocean, 188 until the formation of the Isthmus of Panama occurred close to the equator (Berta 2012). 189 With the formation of the Isthmus, and hence the closing of the CAS, the ocean currents 190 reversed due to a change in global ocean circulation (Haug and Tiedemann 1998; De 191 Schepper et al. 2015), and have since flowed in a northern direction through the Bering 192 Strait, from the Pacific to the Arctic (Marincovich 2000). This again is absolutely 193 consistent with our results (Fig. S4), with dispersal or migration from the Pacific into the 194 Atlantic in a northern direction through this strait, which is known as the trans-Arctic 195 interchange (Vermeij 1991). Fossil data also show that the Bering land bridge has been 196 exposed and submerged on multiple occasions since the Pleistocene (Gladenkov and 197 Gladenkov 2004). These openings and closings of the Bering Strait could have provided a 198 mechanism for divergence and the evolution of sister taxa (Taylor and Dodson 1994; 199 Väinölä 2003). 200 Our results also have implication at the family level of flatfish. Further significant 201 global cooling during the Pleistocene resulted in major glaciation events (Zachos et al. 202 2008) that could be responsible for creating barriers that isolated populations. All of the 203 remaining sister taxa in our analysis, who have divergence estimates of less than 2 MYA 204 in our study belong to the family (sensu Cooper and Chapleau 1998). The 205 Pleuronectidae are the predominant flatfish family found in cold and temperate seas of 206 the northern hemisphere (Norman 1934; Cooper and Chapleau 1998). There are far more 207 Pleuronectidae species in the Pacific Ocean, most of them endemic to the north Pacific 208 Ocean off of North America and Asia in the region extending from the Bering Strait to 209 the gulf of California (Norman 1934). None of the arctic species are restricted solely to 210 arctic waters (Munroe 2015). Munroe also noted that Cooper (in an unpublished 211 manuscript) identified areas of endemism among the current distribution of the 212 Pleuronectidae. It has been shown that during the trans-Arctic interchange, there was a 213 far higher number of species (up to eight times higher) migrating to the Atlantic than to

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214 the Pacific (Vermeij 1991). Fossil and phylogenetic evidence suggest the Pacific Ocean 215 as the origin for diversification of the Pleuronectidae (Munroe 2015), and our 216 phylogenetic results are highly congruent with this hypothesis. 217 It is possible that the outliers, the Atlantic and Pacific halibut (H. hippoglossus and H. 218 stenolepis, respectively), diverged during one of the first openings of the Bering Strait. 219 The estimated dates from the molecular clock analysis are approximately 5.5 MYA, in 220 accordance with the hypothesized dates for the first aquatic connection (Marincovich and 221 Gladenkov 2001). The remaining sister taxa have a younger age estimate of ~1-2 MYA, 222 corresponding with global cooling during the Pleistocene and repeated glaciations 223 (Zachos et al. 2001), which likely formed more barriers to genetic flow. In the second 224 pair of outliers, P. hawaiiensis inhabits waters of the Eastern Central Pacific to the 225 Hawaiian Islands, while P. natalensis inhabits coastal waters of Eastern Africa (Fig. 2). 226 Due to the far reaching range of P. natalensis, and the relatively younger age estimates 227 for divergence (1-2 MYA), these results beg for future research into other vicariant 228 hypotheses, or dispersal routes, as these two members of Poecilopsetta diverged long 229 after the Isthmus had closed. One candidate would be the Indo-Pacific barrier, which 230 formed an almost continuous land bridge between Australia and Asia, and could have 231 hence limited dispersal between the Pacific and the Indian oceans, hereby promoting 232 speciation (Gaither et al. 2011). This barrier formed during the Pleistocene glacial cycles 233 (2.58-0.01 MYA; Voris 2000), which is, again, fully consistent with our date estimates 234 (Fig. 3, S1). 235 Based on the most extensive multigene sequence alignment available to date across 236 all flatfish species, we have showed here that the evolutionary dynamics of sister species 237 that are distributed across the two oceans strongly supported the existence of two bouts of 238 speciation: one triggered by the closure of the CAS 12-3 MYA, and a second one due to 239 the closure of an ancient northern passage 5-0.01 MYA. Our work adds to a growing 240 body of evidence pointing not only to the role of geological processes in shaping biotic 241 turnovers (Bacon et al. 2015), but also on the consequences of climate change that can 242 either trigger speciation events (Reid 1990), or promote faunal interchanges whose 243 ecological and economic impacts are difficult to predict (Wisz et al. 2015).

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244 Materials and Methods

245 Data retrieval and alignment 246 Prior to retrieving sequence data, GenBank was surveyed to identify all the genes 247 belonging to species of Pleuronectiform families (as of August 2016), based on 248 GenBank’s browser. DNA sequences for a total of 332 flatfish species (out of 249 over 800 species in the order) were identified and downloaded for five nuclear genes 250 (KIAA1239, MYH6, RIPK4, RAG1, SH3PX3), and four mitochondrial genes (12S, 16S, 251 COX1 and CYTB). These represented all the taxa having at least one of these six gene 252 sequences in GenBank; see Table S1 for the corresponding accession numbers. Diversity 253 was richly sampled as species from 13 of the 14 families in the order Pleuronectiformes 254 were included in our catchment. In line with current consensus in flatfish systematics, the 255 family Psettodidae was chosen as the outgroup to all other taxa (Chapleau 1993). 256 These sequences were aligned using MUSCLE ver. 3.8.31 (Edgar 2004) on a gene- 257 by-gene basis. Each alignment was visually inspected with AliView ver. 1.18 (Larsson 258 2014), and was manually edited where necessary. In particular, large indels (> 10 bp) 259 were removed prior to all phylogenetic analyses. The 5’ and 3’ ends of sequences were 260 also trimmed. The aligned sequences were then concatenated into a single alignment 261 using a custom R script.

262 Data pre-processing 263 To gauge the phylogenetic content of our data set, we performed a first series of 264 molecular clock analyses on the concatenated data matrix, with all the nine gene 265 sequences obtained above (12S, 16S, COI, Cyt-b, KIAA1239, MYH6, RIPK4, SH3PX3 266 and RAG1), and all the 332 taxa representing all families of flatfish. To account for rate 267 heterogeneity along this alignment, partitions were first identified with PartitionFinder 2 268 (Lanfear 2012), selecting the best-fit model of nucleotide substitution based on Akaike’s 269 Information Criterion (AIC). This optimal partition scheme was then employed in a first 270 Bayesian phylogenetic analysis, conducted with BEAST ver. 1.8.3 (Drummond and 271 Rambaut 2007). This program implements a Markov chain Monte Carlo (MCMC) 272 sampler, which co-estimates both tree topology and divergence times. As determined by 273 PartionFinder, a GTR+I+Γ model was applied to each data partition. These models of

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274 evolution across partitions were assumed to be independent (unlinked, in BEAST 275 parlance), while both clock model and tree model partitions were shared (linked) by all 276 partitions. An uncorrelated relaxed clock was employed with a lognormal distribution 277 prior on rates, and a Yule speciation prior (Drummond et al. 2006). Due to the paucity of 278 reliably placed fossils on the flatfish tree, a unique calibration point was placed on the 279 most recent common ancestor (MRCA) of the ingroup, as a mean-one exponential prior, 280 with an offset of 40 million years reflecting the age of 47.8-42.1 MYA (Chanet 1997; 281 Friedman 2012). To stabilize the estimate, a lognormal ln(0, 1.5) prior with a 40 MYA 282 offset was also placed on the root of the tree (root height). Two separate MCMC samplers 283 were run, each for 10,000,000 generations. Trees were sampled every 5,000 generations, 284 and convergence was checked visually using Tracer ver. 1.6 (available at: 285 http://tree.bio.ed.ac.uk/software/ tracer/). Tree log files from each run were combined in 286 LogCombiner, after conservatively removing 10% of each run as burn-in. The resulting 287 maximum a posteriori (MAP) tree was generated with TreeAnnotator (Drummond and 288 Rambaut 2007). 289 As the topology of this resulting MAP tree was unconventional, we suspected the 290 presence of rogue taxa, i.e. species evolving either much faster or much slower than the 291 majority, which can contribute negatively to consensus tree support (Aberer et al. 2013). 292 Rogue taxa were identified using the RogueNaRok (Aberer et al. 2013) webserver 293 (http://rnr.h-its.org). The consensus trees from the preliminary analysis using 10,000 294 iterations were used as the tree set for rogue taxon analysis. A total of 22 rogues were 295 identified and pruned from the analysis, leaving 310 species.

296 Molecular Dating 297 To assess the impact of the closure of the CAS on flatfish evolutionary dynamics, a 298 second set of partitioned relaxed molecular clock analyses was performed (without the 299 rogue sequences). The timing of the closure of the CAS is estimated to have occurred 300 between 12 and 3 MYA (Duque-Caro 1990; Coates et al. 1992; Haug and Tiedemann 301 1998; O'Dea et al. 2016), and we used this time window as a prior to inform the relaxed 302 molecular clock-based phylogenetic reconstructions. The analyses were performed on the 303 same concatenated data set, with the same single fossil calibration, but we also placed a 304 lognormal prior ln(3,1.5), that has most of its mass on the 12-3 MYA window, on the

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305 MRCA of each pair of sister taxa (the ‘ALL’ model). From the initial BEAST analyses, 306 twelve pairs of taxa were selected based on two criteria: (i) being sister species on that 307 initial tree, (ii) with one species distributed in the Pacific and one in the Atlantic ocean 308 (Fig. 2). Again, two independent MCMC samplers were run each for 100 million 309 iterations, with samples taken every 5000 step. 310 Because these pairs of sister species show a contrasted geographic distribution, 311 having either a southern (equatorial) or a northern range (Fig. 2), two additional sets of 312 analyses were performed. In a first set, calibration priors (ln(3,1.5), as per above) were 313 placed only sister taxa that had a geographic range in the southern hemisphere (the 314 ‘SOUTH’ model), while in a second set, identical calibration priors were placed only on 315 sister species with a northern range (the ‘NORTH’ model). Finally, a set of analyses was 316 performed using no sister taxa calibrations at all (the ‘NONE’ model). For each analysis, 317 results from the two MCMC runs were combined using LogCombiner after removing an 318 even more conservative burn-in of 50%. The final MAP tree was generated with 319 TreeAnnotator. 320 In an attempt to rank these different models (priors on all sister taxa; only on southern 321 taxa; only on southern taxa; no “CAS” priors), the AICM was computed for each model 322 (Baele et al. 2013). These computations were performed in Tracer for each of the four 323 different calibration models, based on 100 replicates.

324 Species distribution data 325 In order to map the distribution of each species of flatfish, we resorted to the Global 326 Biodiversity Information Facility (GBIF: https://www.gbif.org/) database, accessed with 327 the R library rgbif (Chamberlain 2017). Up to 1,000 records were retrieved for each 328 species, and plotted on a geographic map based on GBIF observational records. Where 329 needed, species locations were summarized by mean latitude and longitude (Fig. S4). 330 Approximate attribution to a particular ocean was based on species-specific mean 331 longitudes (Atlantic: between 20˚ and 120˚; Indian: 120˚ and -100˚; Pacific otherwise).

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332 Availability of Computer Code and Data 333 Accession numbers, sequence alignments, BEAST models (as xml files), estimated 334 phylogenetic trees (maximum a posteriori trees from BEAST), and the R scripts used to 335 plot the figures in this study are available from https://github.com/sarisbro. 336

337 Acknowledgments 338 This work was supported by the University of Ottawa (LB), and the Natural Sciences 339 Research Council of Canada (FC, SAB). We are grateful to Jonathan Dench for 340 discussions and comments, to two anonymous reviewers for very constructive comments, 341 and to Compute Canada and Ontario’s Center for Advanced Computing for providing us 342 access to their resource. This work was completed while SAB was hosted by Yutaka 343 Watanuki, at the University of Hokkaido in Hakodate, thanks to an Invitational 344 Fellowship from the Japanese Society for the Promotion of Science.

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487 Table 1. AICM values for the phylogenetic analyses using four different calibration 488 schemes. Models are ranked from the best AICM model (top) to the worst model. SE: 489 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 +/-3.789 490 491

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492 Figure captions 493 494 FIG. 1. Phylogenetic trees reconstructed based on relaxed clock models. Four models 495 were employed, representing different specifications of prior distributions set on sister 496 taxa. (A) No priors were set on sister taxa. (B) Priors were set on all pairs of taxa. (C) 497 Priors were set only on sister taxa with a current northern range. (D) Priors were set only 498 on sister taxa with a current southern range. Horizontal scale is in million years ago 499 (MYA). The closure of the CAS (12-3 MYA) is shown between vertical gray broken 500 lines. 501

502 FIG. 2. Distribution of the geminate species used in this study. Geographic distribution of 503 the twelve pairs of sister species of flatfish used to calibrate the relaxed molecular clock 504 models. Original data come from GBIF (www.gbif.org; accessed Nov 9, 2017). The top 505 panel shows the phylogenetic distribution of these species based on the clock model 506 including prior ages on all twelve pairs of species; scale bar: 5 MYA. 507

508 FIG. 3. Posterior densities of divergence times for sister taxa used as calibration points in 509 the relaxed molecular clock analyses, under four different calibration schemes. NONE: 510 no priors were placed on sister taxa; ALL: priors were placed on all pairs of sister taxa; 511 NORTH: priors were placed only on pairs of sister taxa with a northern distribution; 512 SOUTH: priors were placed only on pairs of sister taxa with a southern distribution. Top 513 row (A-D) shows all 17 distributions, while bottom row (E-H) shows range-averaged 514 distributions (solid) to the exception of outlier pairs (dashed lines). Densities are color- 515 coded for species with northern (blue) and southern (red) range. Dashed vertical gray 516 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 0.95 0.97 1.00 0.99 0.99 Soleidae Cynoglossidae 0.99 0.90 Soleidae 0.94 Soleidae 1.00 0.98 1.00 0.95 0.94 1.00 Poecilopsettidae 1 1.00 0.99 0.94 Poecilopsettidae 1 1.00 Poecilopsettidae 1 0.97 0.99 Poecilopsettidae 1 1.00 Samaridae 1.00 1.00 1.00 Samaridae Samaridae Samaridae 1.00 1.00 Achiridae Achiridae-Soleidae 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 1.00 0.71 0.70 Achiropsettidae Poecilopsettidae 2 1.00 1.00 0.73 Poecilopsettidae 2 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 1.00 1.00 0.80 Achiropsettidae Scophthalmidae 0.71 1.00 "Cyclopseta" gr. Bothidae 1.00 1.00 1.00 1.00 Citharidae "Cyclopsetta" gr. Scopthalmidae 1.00 1.00 1.00 Psettodidae Psettodidae 1.00 Psettodidae Psettodidae

40 30 20 MYA 10 0 40 30 20 MYA 10 0 40 30 20 MYA 10 0 40 30 20 MYA 10 0 C y noglo ss u s _pun ct i c ep s Cynoglossus_lingu a Cynoglossus_lid a ●● Paraplagusia_bilineat a Cynoglossus_sinicu s Cynoglossus_acaudatu s C y noglo ss u s _ cy noglo s su s ● Cynoglossus_broadhurst i ● Cynoglossus_semilaevi s C y noglo ss u s _ r oule i Cynoglossus_purpureomaculatu s C ynog l ossus_abbreviatu s Cynoglossus_carpenter i Cynoglossus_marley i Cynoglossus_nigropinnatu s Cynoglossus_light i Cynoglossus_joyner i C y noglo ss u s _oligolepi s C y noglo ss u s _ r obu st u s Cynoglossus_are l Cynoglossus_attenuatu s Cynoglo ss u s _macrolepido t u s Cynoglossus_elongatu s Cynoglossus_ochiai i ●● C y noglo ss u s _ s inu s arabic i C y noglo ss u s _in t e rr up t u s

. Paraplagusia_bloch i ● Cynoglossus_brown i ● ● ● ●

Paraplagusia_japonic a ● Cynoglossus_zanzibarensi s 2:P. natalensis Cynoglossus_monod i Cynoglossus_senegalensi s C ynog l ossus_dub i u s The copyright holder for this preprint (which was not The copyright holder for this preprint (which Symphurus_plagius a Symphurus_civitatiu m Symphurus_plagusi a Symphurus_ginsburg i Sym phu r u s _dio m edeanu s ● ● ●●

Symphurus_n i grescen s ● ● ●● ● Symphurus_arawa k ● Symphurus_tessellatu s ● ●

Symphurus_atricaudu s ● ●● ● Symphurus_ommaspilu s ● ●●●●●●●● ●● ●● ● Symphurus_rafinesqu e ● Symphurus_orien t ali s Sym phu r u s _leu c o c hilu s Symphurus_thermophilu s ● Symphurus_strictu s Sym phu r u s _ m ega s o m u s

Symphurus_hondoensi s ●● Symphurus_microrhynchu s

Sym phu r u s _longi r o str i s ● Solea_senegalensi s ●

Solea_aegyptiac a ● S olea_ s ole a Solea_vulgari s ●● ●●●●●● ●● Solea_klein i ●● Solea_kleini i ●

Synapturichthys_kleini i ● Pegusa_impa r

Solea_lascari s ● S olea_i m pa r Pegusa_cadenat i ● Dicologlossa_cuneat a Microchirus_boscanio n Michrochirus_boscanio n Michrochirus_ocelatu s Dicologlossa_hexophthalm a M i cr o c hi r u s _a z e v i a Michrochirus_azevi a ●

Microchirus_variegatu s ●● ● ● ● ● ●

Monochirus_Hispidu s ●● ● ●

Monochirus_hispidu s ● ● ● ●

Microchirus_ocellatu s ● ● Bathysolea_profundicol a ●

Buglossidium_lu t eu m ● ●● ● ●● ● ● ● ●● ● ●

Pegusa_nasut a ● ● ● ● ● ● ● Solea_ovat a ●●● ● ● ● ●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●● ●●●●●●●●●●● ●●●●●●●●● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ●●●● ● ● ● ●● ●● ●● ●● ●● ●● ●● ● ● ●● ●

Aesopia_cornut a ●● ● ●●●●● ● ● ● ● ● ● ● ● ● ● ● ● ●●●●●●●●●●● ●●●● ●●● ●● ● ● ● ●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ● ● Zebrias_scalari s ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ●● ●● ●●●●●●● ●●●●●●● ● ●●●● ● ● ●●●●●●●●●●●● ●●●●●●●● ● ●●●●●●●●●●●●●●●●●● ●●●● ● ●●●● ● ●● ● ●●● ●● ●●● ●● ●●● ● ● ● ● ● ● ● ● ● ●● ●● ● ● ●● ● ●● ● ● ●● ● ●● ●●

Zebrias_zebr a ●●●●●●●●●● ●● ● ● ● ● ●● ● ● ● ●●●●●●●●●●●● ●●●● ● ● ● ● ●●●●●●●●●●●●●●●● ● ●● ● ● ● ● ●●● ● ● ● ●● ● ● ● ● ●●●●● ● ● ●●● ● ● Zebrias_synapturoide s ● ● ●● ● ● ● ●● ●● ● ● ● ● ● ●

Brachirus_annulari s ●● ● ● ●● ●● ● ●● ● ●● ● ●●●●● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ●●● ●●●

Pseudaesopia_japonic a ● ● ● ● ●● ●● ● ● ● ●

Soleich t hys_he t erorhino s ● ● ●●● ● ● ●●●● Heteromycteris_matsubara i ● ● ●●● ●● ● ●●●● ● ● ● ● ●●●●●●●●●●●●● ●● ● ● ● ● ●● ●● Heteromycteris_japonicu s ● ●● ● ● ● ● ● ● ● ●●●●● ● ●●● ● ● ● ● ● ●●● Aseraggodes_kaianu s ●● ● ● ● ● ● ●●●●● ● ●●●●● ●● ● ●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●● ● Pardachirus_morrow i ● ● ● ●

As e r aggode s _hee mstr a i ● ● ●●●●●● ●● ●● ●●●●●● ●●● ●●● ●●●●●●●● ●●●●● ●●●●● ●● ●● ●●●●● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●● ●●●●●●● ● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● Aseraggodes_melanostictu s ●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ●

Aseraggodes_kobensi s ● ● ● ● ● ● ●●●● ● ● ●● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●●● ● ●● ● ● ● ●●●●●●●●●●● ●●●●● ●●●●●●● ● ● ●● ● ●●● ●●● ●●●●●●●●●● ● ●● ●●● ● ●

P a r da c hi r u s _ m a rm o r a t u s ●● ●●●●●●●●●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●

P a r da c hi r u s _pa v oninu s ● ● ● ●● ● ●●● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ● ●

Dage t ich t hys_commersonni i ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ●● ●●● ● Sy nap t ura_margina t a ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ●● ● ● ● ● ● ● ●

Dagetichthys_lusitanic a ● ● ● ●● ● ●● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ●● ●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●● ● ● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ● ●● ● ● ● ● ●● ● ● ● ●● ●●●● ● ● ● ●● ● ● ● ● ● ● ● ● ● S y nap t ura_lusi t ani c a ● ● ● ● ● ● ● ●●● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ●● ● ● ● ●

Austroglossus_microlepi s ● ● ●● ●●●●●●●●●●●●●●●●●●●● ●● ● ● ● ●● ● ● ● ●● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ●● ● ● ● ● ●● ●● ●●● ● ● ● ● ● ● ● ● ● ●● ● ● Austrog l ossus_pectora li s ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●●● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● Zebrias_zebrinu s ● ●● ●● ●● ●● ●● ● ● ● ● ● ● ● ● ● ●

Zebrias_fasciatu s ● Zebrias_quagg a ● ● ● ●● ●● ● Zebrias_regan i ● ● ●● ●● ● Poecilopsetta_plinthu s ● ● ● ● ● ● ● ● ● ●

Ci t ha r oide s _ m a cr olepido t u s ● Poecilopsetta_beani i Poecilopsetta_bean i Samariscus_japonicu s Samariscus_latu s Samariscus_longimanu s Samariscus_xenicu s Plagiopsetta_gloss a Samaris_cristatu s Samariscus_triocellatu s Achirus_declivi s Achirus_achiru s

Achirus_linea t u s 7:P. platessa H y po c line m u s _ m en t ali s Soleonasus_fini s Apionichthys_dumeril i Trinectes_microphthalmu s

T rinec t es_macula t u s 9:L. limanda Tr ine ct e s _pauli st anu s T rinec t es_inscrip t u s this version posted April 24, 2018. G ymnachirus_ t exa e G ymnachirus_mela s Gymnachirus_nudu s ●

; Catathyridium_jenynsi i Catathyridium_jenyns i Ci t ha r oide s _ m a cr olepi s Citharus_linguatul a CC-BY-NC-ND 4.0 International license Rhombosolea_ t apirin a Rhombosolea_plebei a R homboso l ea_ l epor i n a a P el t o r ha m phu s _no v ae z eelandia e Pelotretis_flavilatu s Ammotretis_rostratu s Achiropsetta_tricholepi s Neoachiropsetta_milford i ● M an c op s e tt a_ m a c ula t a ● Oncopterus_darwini i Poecilopsetta_hawaiiensis

P oec ilopse tta_nat alensis 2 Poecilopsetta_praelong a ● Marleyella_bicolorat a Zeugopterus_punctatu s Phrynorhombus_norvegicu s Lepidorhombus_whiffiagoni s Lepidorhombus_bosci i 12:G. cynoglossus ● Scophtha l mus_max i mu s ● Psetta_maeotic a ● ●

Psetta_maxim a ●● Scoph t halmus_rhombu s ●

Platichthys_stellatu s ● ● ● ● Platichthys_flesu s 11:H. hippoglossus Liopsetta_pinnifasciat a ● ● ● ● ●●●● ● ● ● ●

Kare i us_b i co l oratu s ● 10:H. platessoides ● 7 Pleuronectes_platessa ● Pleuronectes_quadrituberculatus ● ●● ●

Limanda_punctatissim a ●● ●●● ● ●●● ●● ● ●● ● ●● ● ●●● ● ●● ● ●● ●● ● ●●● ● ●●● ●● ●● ●●● ●● ● ●● ●● ● ●● ●●● ●●● ●●● ●● ● ●●● ●●●● ●●● ● ●●● ●●● ● ●● ●● ● ● ●● ●● ●●● ●● ●● ●● ●● ●● ● ● ● ●●● ● ●●● ●● ● ●● ●●● ●●● ●● ● ● ● ●● ●● ●●● ●●● ●●● ●●● ●●● ●● ●● ●●● ●●● ●● ●●● ● ● ● ●●● ● ●● ●● ● ●● ●● ●●●● ●●● ●●● ●● ●● ●● ●● ●●● ●●● ●● Limanda_proboscidea ●● ●●● ●● ●● ● ● ●●● ●● ●● ●● ● ●● ●●● ●● ● ●●●● ●●● ●●● ● ●● ● ●● ●● ●●● ●● ● ●● ●●● ●● ●●●● ●●● ●●● ● ● ● ●● ●●● ● ●● ●● ● ● ● ●●● ●●● ●●● ● ●●● ●● ●●●● ●● ●●● ●● ●● ● ●●● ● ●● ●●● ●● ●● ●● ●●● ●● ●●● ● ● ●● ●●● ● ●● ●● ● ● ●● ● ●● ● ●● ●● 8 ● ● ●●● ●● ●● ●●● ●●● ● ● ● ●● ● ●●● ● ●● ●● ● ● ●●● ●● ● ● ● ●● ●● ●●● ●● ●●● ● ●●● ● ●●● ● ●● ● ●● ● ● ●●● ●● ● ●●● ● ●● ●● ●●●● ●●● ●● ●● ●● ●● ● ●● ●

Limanda_ferruginea ●● ●● ● ●● ●●● ● ● ●● ●● ● ● ●●● ●● ●● ● ● ● ●● ●● ●● ●●● ● ●●● ●● ● ● ● ● ● ●●● ●●● ●● ●● ●● ● ● ● ●●● ● ● ● ●● ● ●● ●● ●● ●●● ● ● ●● ● ●●● ●●● ●● ● ●● ● ●● ● ●●● ●● ● ●● ● ●● ●● ●●●●● ●● ●● ● ●●● ●● ● ●● ●● ●●●● ●●● ●● ● ●●● ●●●● ●● ● ● ● ●● ● ● ● ● ●● ●●● ● ●● ●●●●● ●●●● ●● ●● ●●●● ● ● ● ●● ●●● ●●● ● ●● ●● ●●● ● ●●● ●● ●● ● ● ●● ●● ● ●● ● ● ●● ●● ●● ●● ●● ●● ●●● ●● ●● ●● ●●●● ● ●●● ●● ● ●● ●● ● ●● ●● ●● ● 4:C. minutus ●● ●●● ●● ●● ●●●● ●● ● ● ● ● ●●●● ●● ●● ●● ●● ● ●● ●●● ● ● ●●●● ●●● ●● ● ● ●● ●●● ●●● ●● ● ● ● ●● ● ●●● ●●● ● ●●● ●● ●● ●● ● ●●● ●● ●● ● ●● ● ●●● ●● ●● ● ●● ●●● ● ● ●● ●●●● ●● ●● ●● ●● ●● ●●●● ●●● ●●● ●●● ● ●●● ●● ● ●●●

Psettichthys_melanostictu s ● ● ● ●● ●● ●● ●● ● ● ●● ●● ● ● ● ●● ●●● ● ●● ●● ● ● ●● ● ● ● ●● ●● ●● ●●● ●● ● ●●● ● ● ●● ●● ● ● ●● ● ● ● ● ●● ● ● ●● ●● ● ● ● ● ●● ● ● ●● ●● ●●● ●● ●●● ● ●● ●● ●● ● ● ●● ● ● ● ●●● ●● ● ●● ●● ●● ● ●● ● ●● ●● ● ●●● ●●● ●●●● ●● ● ● ●●●● ●● ●● ● ●●● ●●●●●● ●● ● ●● ●● ● ● ●● ●● ●● ●● ● ● ●● ● ● ● ●● ●●● ● ● ●● ●●●● ●● ●● ●●● ●●● ● ●● ● ●●● ● ●●●● ●● ●● ● ●●●● ● ●● ●● ●● ●●● ● ●●● ●●● ●● ●● ●● ● 1:H. oblonga ●● ● ● ●●●●●● ● ● ●● ●● ● ●● ● ● ● ●● ● ●● ●● ●● ● ● ●●●● ● ● ●● ●● ● ●●● ● ●●● ●●● ●● ● ● ●● ●● ●● ●● ●● ●● ● ●● ● ●● ●● ●● ●● ● ● ●●●● ● Parophrys_ve t ul a ● ●● ●● ●● ● ●● ●● ● ● ●● ●● ● ●● ● ● ●● ●●●● ● ●● ● ● ● ● ●● ● ●● ● ●● ●● ●● ●● ● ●● ●● ● ● ● ●● ● ●●● ●● ● ● ●● ●●● ● ● ● ●●●● ●● ●● ●●●●●● ● ● ●● ●●●● ●● ● ● ● ●● ● ●●● ● ● ● ● ●● ●● ●● ●● ● ●● ●● ● ●● ● ●●● ● ● ●●● ●● ● ●● ● ●● ●● ●● ● ●● ●● ●● ● ●● ●● ● ●● ●●●● ●● ●● ●● ●● ● ● ● ●● ●● ●● ●●● ●●● ●● ● ●●● ●● ● ●● ● ● ●● ●● ●● ●●● ●● ●●●● ●● ●● ●●●●●●●● ●● ●● ●● ●●● ●●●● ●● ●● ●●●● ●● ●●● ● ●● ●● ●● ●● ●● ● ●● ● ●●● ●● ● ●● ●● ●●●● ● ●●● ●● ●●●● ● ●● ●●● ●● ●●● ●●● ●● ●●●● ●●● ●● ● ●● ●● ●● ●●● ● ●● ●● ●● ●● ●●●● ● ●●● ● ●●● ● ●● ● ●● ●● ● ●● ●● ● ●●●● ●● ●●

Lepidopsetta_bilineat a ●● ● ●● ●●●● ●● ●●● ●● ●●● ●● ●●● ● ● ●● ●● ● ●● ●● ●● ●●● ●● ●● ●● ●● ● ● ● ●●● ●● ● ● ● ●● ●●● ● ●● ●●●● ●● ●●● ●● ●● ●● ●● ●●● ● ● ● ● ●● ●●●● ●●● ●●●● ●● ●● ● ● ●● ●● ●● ●● ●●● ●● ● ●●● ●● ●●●● ●● ●● ●● ●● ● ●●● ●●●●● ●● ●● ●● ● ● ● ●●● ●● ●●● ● ●●●●●● ●● ●● ●●●● ● ● ●●●●●●●● ●● ●● ● ●●● ●● ●● ●●● ●●●● ●●● ●●● ● ●● ●● ●● ●● ●● ● ●● ● ●●● ● ●● ●●●● ●●● ●● ●●●● ●● ●● ●●●● ●●●● ●● ● ● ●●●● ●● ●● ●● ●● ●●● ●●● ●● ●●●● ●●●● ●●● ●● ●● ●● ● ●● ●● ● ● ●● ● ●● ●● ● ●● ●● ●●●●● ● ● ●● ●●● ●●●● ●● ●●●● ●● ●●● ● ●●●● ●● ●●● ●● ●● ● ●● ●● ●● ●● ●● ●● ●● ●● ●●● ●●● ● ●●● ●● ●●●● ●● ●● ● ●● ●● ●●●● ●● ●● ● ●●●● ● ● ●● ●● ●●●● ●●

Is op s e tt a_i s olepi s ● ●● ● ●● ●●● ●●●●● ●● ●● ●● ● ●● ● ●● ●● ●● ● ●● ●● ● ●● ●●●●● ● ●● ●● ●● ●● ●● ●● ●●● ●●●● ●● ●●● ●● ●●●● ●● ●● ●● ●● ●●● ●● ● ●● ● ●●●●● ●● ●●● ●● ● ●● ●●● ● ●● ● ●●● ●●●● ●● ●●●● ●● ●● ●● ● ● ●●●● ● ●● ●●●● ●●●● ●●●● ● ●● ●● ● ●● ●●● ●●● ●● ●● ● ●●●●● ●● ● ●● ●● ●● ●● ●●●●● ●● ●● ●●● ●● ●● ●●● ●● ●● ● ● ●● ●●● ●●● ● ●● ● ●● ●● ●● ●● ●● ●● ●● ● ●● ●● ●● ●● ●● ● ●● ●● ●● ●● ● ●●●● ●● ●● ●● ● ●● ●●● ●●●● ●● ●● ● ●● ●● ● ●● ●● ●● ●●●●●● ●● ●● ●● ●● ●● ●● ● ●● ●● ●●●● ●● ● ●● ●● ●● ● ●● ●●●● ●●● ●● ●●● ● ●● ●● ● ●● ●● ● ● ●● ● ●●● ● ●● ●●● ●● ●● ●●● ●● ● ●● ●● ●●● ●● ●● ● ●● ●●●● ●● ● ●●● ●● ●● ●● ●● ●● ●●● ●● ●● ●●● ●● ●●●● ●● ● ●● ● ●● ●● ●● ●● ●● Lepidopsetta_polyxystr a ● ●●●●● ●●● ●● ●● ●● ●● ●● ●● ●● ● ●● ●●●● ●●●● ●● ●●● ●● ●● ●●●● ●● ●● ●● ●● ● ● ● ●● ●● ●● ● ●● ●●● ● ●● ●● ●● ●● ●● ●● ● ● ●● ● ●●●● ●● ●●●● ●● ●● ●● ●● ●● ●● ●● ●● ●● ●● ●● ●● ●● ● ●● ●●●● ● ●● ●● ●●● ● ●● ●●●● ●● ●● ●●● ●● ●● ●● ● ● ●● ●● ●● ●● ●● ●● ● ●● ●● ●● ●● ●● ●● ● ●●● ●●● ●● ● ● ●● ● ●●● ●●● ● ● ●● ●●● ● ●● ●● ● ● ● ●●● ●● ●● ●● ●●● ●● ●●● ●● ●● ● ●● ●●● ● ●● ●● ●● ●

Lepidopsetta_mochigare i ●● ●● ●● ● ● ● ●●● ●● ●● ● ● ● ●● ●● ●● ● ● ● ● ●● ●● ● ● ●● ●● ● ● ● ●● ● ●●● ● ●● ●●● ●● ●●● ●● ● ● ● ●● ●● ● ●●● ● ●●● ●● ●● ●● ●● ●●● ●●● ●● ● ●●● ●● ●●● ●● ● ● ●● ●● ●● ● ●● ●● ● ●●●●● ●● ●● ● ●● ● ●● ●●●●● ●●●● ● ●● ● ● ●● ●● ● ● ●●●● ● ● ●● ● ● ●●● ●●● ● ●●

Parophrys_ v e t ulu s ● ● ●● ●● ● ●●●● ●●●●● ●●● ●● ●● ●● ●● ●●● ●● ● ●●●● ● ●● ● ●● ●●● ●● ●●● ●● ● ●● ●● ● ● ●● ●● ● ●●●● ●● ● ●● ●● ● ● ●● ●● ●● ●● ●●● ●● ● ●● ●● ● ●● ●● ●●●● ● ● ●●● ●● ●● ● ●● ●● ●● ●● ●● ●● ●● ●● ●● ● ● ●● ●●● ● ● ●●● ●● ●●● ●● ●● ●● ●● ● ●● ●● ●●● ●●● ●● ●● ● ● ● ● ● ●● ●● ● ●● ●● ●●● ●● ●● ●● ●● ●● ● ● ●● ●● ● ●● ● ●● ●● ● ●● ●● ●● ●●● ●● ●●●● ● ●● ●● ●●●●● ● ●● ● ● ●●● ●●● ●●

Pseudopleuronectes_schrenk i ● ●● ●● ● ●● ● ●● ●●● ●● ● ●● ●●● ●● ●● ●●● ●●● ●●● ● ●● ● ●● ● ●● ● ●●● ●●● ●● ● ●●● ●● ●●●● ● ●● ●●● ● ● ●● ●● ● ● ●● ● ● ●● ●● ●● ● ●● ● ●● ● ●● ● ● ●● ● ●● ● ●● ●● ● ●●● ●● ●● ●● ● ●● ●●●● ●●●● ●● ● ● ● ●● ●●●● ● ● ● ●● ●● ●● ●● ●● ●● ●●● ●● ● ●● ●● ● ● ●● ●●● ●●●● ●● ●● ●● ●● ● ●● ● ● ● ●● ●● ●● ● ● ●● ●● ● ●●●● ●●● ●●● ●● ●● ●● ●●●● ●● ● ●● ● ●● ● ●●● ●●●● ●● ●● ● ●●● ● ●●●● ●●● ● ● ● ●●● ●● ●● ●● ●●●● ●● ●● ●●● ●● ●● ●● ●● ●● ● ●●●● ● ●● ●● ● ● ● ●● ●● ●●●● ● ● ●● ●● ●●●● ● ●● ●● ●● ●● ●●●● ●● ●●●● ●●●● ● ● ● ● ●● ● ● ● ●● ●● ●● ●● ● ●● ●● ● ● ● ● ●● ● ●●● ● ● ● ● ● ●● ● ● ● ●● ● ● ●● ●●● ● ● ●● ● ● ●● ● ● ●● ●● ●● ●● ●● ●●●● ●●●● ● ●●●● ● ●●● ●● ●●●● ●● ● ● ●●● ●● ● ●● ●●●● ●●●●●●● ●●● ● ● ●●● ●● ●● ●● ●● ●● ●● ●● ●● ●● ● ●●● ●● ● ●● ●● ●● ●● ● ●● ●● ●● ●● ●● ●● ● ●●● ●● ●● ● ● ●● ●● ●● ●● ● ●●●● ●● ●● ● ●● ●●●● ●● ● ●● ●● ●● ● ●● ●● ●● ●● ●● ●● ●● ● ● ●●● ●● ●●● ●● ●● ●● ●●●● ●● ● ●● ●● ●● ●● ● ●● ● ●● ●●●● ●● ● ●● ● ●● ●● ● ●● ●●● ● ●● ●● ●● ●● ●● ●●● ● ●● ●● ●● ●● ●● ● ● ● ●●●● ●● ●● ●● ●● ● ●● ● ●● ●● ●●● ●● ●● ● ●● ●● ● ●● ●●●● ●●●● ●● ● ●● ●●● ●● ●● ●●●● ●● ●● ●● ●●●● ● ●● ●● ●● ● ● ●●●● ● ●● ●●● ●● ● ●● ● ●● ●● ●● ●● ●●●●●● ●● ●● ● ●● ●● ●● ● ●● ●● ● ● ●● ● ●●● ● ●● ●● ●● ●● ● ●● ●●●● ●● ●● ●● ●●●● ●● ● ●●● ●● ●● ● ● ●● ●●●● ● ●● ● ●● ●● ●●●

Pseudopleuronec t es_yokohama e ● ● ●● ● ●● ●● ●●●● ● ●● ● ●● ● ●●● ●● ● ●●●● ● ●● ●● ●● ●● ●● ●● ●● ●● ●● ●● ●● ●● ● ●● ●● ●● ●●● ●● ●● ●● ●● ●● ●● ●● ●● ●●● ●● ● ● ●● ●●●● ●● ● ●● ●●● ● ●● ●● ●●● ●● ●● ●●●● ●● ● ●● ●● ●● ● ●● ●● ●● ●●● ●● ●● ● ●● ●● ●● ● ●● ●●● ● ●● ●● ●● ● ●● ●● ● ● ●●●● ●●●● ●● ●●●● ● ●● ● ●●● ●●● ●● ●● ●● ● ●● ● ●● ●● ●●● ●● ● ●● ●● ● ● ●● ●● ●● ● ●●● ●●●● ● ●●● ●● ● ●● ●● ●●● ●●● ●● ●● ● ●● ●●●● ●●●● ●● ●● ●● ●● ●●● ● ●● ●●● ●● ●● ●● ●●●●●● ●●●● ● ●● ● ● ● ●● ● ●● ●● ●● ●● ●● ●● ● ●●● ●● ●● ● ●● ● ● ● ●● ●● ●● ● ●● ●● ●●● ● ●●● ●● ●● ● ●●● ●● ● ●● ●●●● ●● ●● ● ●● ● ●● ●● ●●● ●● ●● ●● ● ●● ●● ●● ●● ● ●● ●● ● ● ●● ●● ● ● ● ●● ●● ●●●● ●● ● ●●● ● ●● ●● ●● ●●● ●● ●● ●● ●● ●● ● ● ● ●● ● ●●●● ●● ●● ●● ● ●● ● ●● ●● ● ● ●● ●● ● ●● ●● ●● ● ●● ●● ●● ●●● ●● ● ●● ●●●● ●● ● ●● ●● ● ●● ●●●● ●● ● ● ● ●●●● ● ●● ●●●● ●● ●● ● ● ●● ● ● ●●● ●●●● ●●● ● ● ●● ●●● ● ●● ● ● ●●● ● ●● ●● ●●● ●●●● ● ●● ●●●●● ●●●● ●● ●● ● ● ● ●● ● ●● ●● ● ●● ●● ●●● ● ●● ● ● ● ● ●●●● ●●● ●● ● ● ● ● ●●●● ● ●● ●● ●● ● ●● ●●●● ● ●● ●● ●● ●● ●●●● ●● ●● ●● ● ●● ● ●● ●● ● ● ●● ● ●●● ● ●● ● ●●●● ●●●● ●●●● ●● ●● ● ●● ● ● ●●●● ● ● ●●●● ●● ● ●● ● ● ●● ● ●● ●● ●● ●● ● ●●● ● ●●● ● ●●●● ●●●● ●● ● ● ● ●●● ●●● ● ●●●● ●●●● ● ●●● ●●●● 8:L. ferruginea ●● ●● ●●●●●● ●● ●● ● ● ● ● ● ●●●● ●● ●● ● ●● ● ●● ●● ● ● ● ●●

Pseudopleuronectes_obscuru s ●● ●●●● ● ●● ●● ● ●● ● ●● ●● ●●●● ●●●● ●● ● ● ● ● ●● ● ● ●● ●● ● ● ● ● ●● ● ● ● ● ●●● ● ● ●●● ● ● ●● ● ●● ● ● ● ● ●●● ● ● ● ●● ●● ● ●● ●● ● ● ● ●● ●● ● ●● ●● ●● ● ●● ●●●● ● ●● ●● ● ●●● ●● ●● ●●● ●● ●● ● ● ● ●● ● ●●●● ●●●● ●●●● ●● ●●●● ●●●● ●● ● ●● ●● ●● ●● ●● ●●●● ● ●● ●●●● ●● ● ● ●●●● ●● ●● ●●● ●● ●● ●● ● ●●●● ●● ●● ●● ●● ● ●●● ●●● ● ●● ●●●● ●● ● ●● ●●● ● ●● ●● ● ●● ● ●● ●● ●● ●●●● ●●●● ●● ●● ●● ●● ●●●● ● ●●●● ●● ●● ●● ●●● ● ● ● ●● ●● ●●●● ● ●●●● ●● ●● ●● ●● ●● ●●● ●

Pseudopleuronectes_herzenstein i ●●●● ●● ●● ● ●● ●●●● ● ●●● ●● ●● ●● ●● ●● ●●●● ●●●● ●● ● ● ● ● ●● ●●● ●● ●● ● ●● ●● ●● ●● ● ● ● ● ●● ● ●● ● ● ●●●● ●●●● ● ●● ● ●●●● ●● ●● ● ● ●● ● ●● ● ●● ●●● ●● ●●● ●● ●● ● ●● ●● ● ● ●●●● ●●●● ●● ●●●● ●● ● ●● ●● ●●●● ●● ● ● ● ● ●● ● ●● ●● ●● ●●●● ●● ●● ●● ●●●● ● ●● ● ● ● ● ●● ● ●● ●● ●●

Pseudopleuronec t es_americanu s ● ● ●● ● ●● ●● ●● ● ●● ●● ● ●● ●● ● ●● ●● ●●●● ●● ●● ●● ●● ●●●● ●● ●● ●● ●● ● ●● ●● ●● ●●●● 6:C. chittendeni ●●● ●●● ●● ● ●● ● ●● ● ●● ● ●● ●●●● ●● ●● ●● ● ● ●● ●●●● ●● ●● ●● ●●●● ●● ●● ●● ●● ●● ●●●● ● ●●●● ● ●●●● ●●● ● ●● ● ●●●● Hippoglossoides_robus t u s ● ● ● ● ● ●● ●●●● ●● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ● ●

Hi ppog l osso i des_e l assodo n ● ● ● ● ● ●● ●● ●● ●● ● ● ● ●● ●● ● ● ●● Hippoglossoides_platessoides ● ●● ●● ● ● ● ● ● ●● ● ● ● ●● ●● ● ● ●● ●●

Hippoglossoides_dubius ● ● ●● 3:C. arctifrons ● ● ● ● ● ● ● ● ● ●● ●● ● ● ● ●● ● ● 10 ●● ● ● ●● ● ●● Cleis t henes_pine t oru m ●●● ● ● ●● ● ●● ●● ● ● ● ● ● ●● ● ● ●● ●● ● 5:S. micrurum ● ● ● ● ●● ● ● ● ●●● ● ●

Cleisthenes_herzenstein i ●● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ●●● ●● ● ● ● ● ● ●● ● ● ● ● ● ●● ●● Dexistes_rikuzeniu s ● ● ● ●●● ●● ●● ● ● ● ● ● ● ● ● ● ● ● ●

Limanda_limanda ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ●● 9 ●● ● Limanda_aspera ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●

Li m anda_ s a k halinen s i s ● ● ● ● ● ● ●●● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● Embassich t hys_ba t hybiu s ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●●● ● ●●

Microstomus_bathybiu s ● ● ●● ● ● ●●● ● ●● ● ● ●● ● ●● ● ● ●● ● ● ● ● ●●●● ● ● ● ● ● ●● ●

Micros t omus_ki t t ● ● ● ●● ●● ● ● ● ● ● ● ● ● ●●●●● ● Microstomus_achn e ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●

Micros t omus_paci f icu s ● ● ● ● ● ● ● ●● ● ● ● Glyptocephalus_cynoglossus ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●●● ●●● ●●●●● ●● ●● ● ●● ● ●● ●● ●● ●● ● ●●●●● ●● ●●●● ●● ●●●●●● ●●●● ●●●●● ●● ●● ● ●●●●● ●● ● ●●● ●● ●●●● ●●●● ●● ●● ●● ●● ●● ●● ●● ●● ●●●●●●●● ●●●●●●●● ●● ● ●● ●●●●●●●● ●●●● ●● ●●●●●● ●●●● ●● ●● ●● ● ● ●● ●● ●● ●● ●● ●● ●● ●●● ●●●● ●● ●● ●● ●●● ●●●● ●● ●● ●● ●● ● ●● ●● ●●●● ●● ●●●● ●● ●● ●● ●● ●● ●●●● ● ●●●● ●●●● ●●●● ● ●●●● ● ●● ●● ●● ●●●●●● ●●●●●●●● ●●● ●● ●●● ● ●●●● ●● ●●●● ●●●●●● ● ●● ●● ●● ●● ●●●● ●● ●●●●●●●●●● ●●●●●● ● ● ●● ●● ●● ● ●●● ● ●●●● ●● ●● ●●●● ●● ●● ●● ● ●● ●● ●● ●●●● ●● ●● ●● ●● ●●●● ●● ●● ●● ●● ●● ●●●● ●● ● ●●●● ●● ●● ●● ●● ●●●●●● ●●●●●●●● ●● ●● ●● ● ●● ●● ●● ● ● ●● ●●●● ●● ●● ●● ●● ●● ●●●● ● ●●●●● ●● ●● ● ●●●● ●● ● ●●●● ●●● ●●●●●●● ●●●●●● ●● ●●●●● ●● ● ●● ●●●●●●● ●●●● ●● ●● ●● ●● ● ● ●● ● ●●●● ●● ●● ●● ●● ● ●● ●●● ● ●● ●● ●●●● ●●●●● ● ●● ●● ●● ●● ●● ● ●● ●● ●● ●● ●● ●● ●● ●● ●● ●●● ●●●● ●●●● ● ●● ●●●●●● ●● ●● ●●●●●● ●●● ● ●●●● ● ●●● ●● ● ● ●●●● ●●●●●●●● ●● ●●●● ●● ●●●● ●● ●● ●● ●● ●● ●●●●●●●● ●● ●●●● ● ●● ●● ● ● ● ●● ●● ●● ●● ●●● ●● ● ●●● ●● ●● ● ●● ●●●● ●● ●●● ●●●● ●●●●● ●● ●●●●● ● ●●●● ●● ●● ●● ●● ●● ●● ●● ●●●● ●●●●●●●● ●● ●● ●● ●●●● ●● ●● ● ●● ●● ●●●●●● ●● ●● ●● ●●●● ●●●●● ● ●●●● ●● ●● ●●● ●● ●● ● ●●●●●●●● ● ●● ●●●● ● ●●●●● ●●●●●● ● ●● ●● ● ● ●●● ● ● ●●●● ●● ● ●● ●●● ●● ●● ● ● ● ●● ●● ●● ● ●● ●●●● ● ● ●●●● ●● ●● ● ●● ●● ●● ● ● ● ●●● ●● ●● ●● ●● ●● ●● ● ●● ● ●● ●●●● ● ●●●● ●● ●● ●●●●●● ●● ●● ●● ●● ●●●● ●●●● ●● ●● ●● ●● ●●●● ●●●●●●●●● ●●● ●●●●●● ●●●●●● ●●● ●● ● ● G lypt ocephalus_st elleri ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ●

G lyp t o c ephalu s _zachiru s ● ●● 12 ● ●● ● ●● ● ● ● ● ● ● ● ● ● ●

Tanakius_kitahara e ● ● ● ● V e r a s pe r _ m o s e r i ● ● ● ● ●●●●●●● ● ● ● ●

Verasper_variega t u s ● ●● ● ● ●

Eopsetta_grigorjew i ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ●●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ●● ●● ● ●● ●● ● ● ● ● ● ●● ● ● Eopsetta_jordan i ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ●●● ● ● ●

Clidoderma_asperrimu m ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●● ●

Reinhardtius_hippoglossoide s ● ● ● ●●● ● ●● Hippoglossus_hippoglossus ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ●● ● Hippoglossus_stenolepis ● ● ● ●● ●● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● Southern ● ● ● ● ● ● ● ● ● ●●●●●●●●●● ● ●● ● ● ● ● ● ● ● ● ● ●

Lyopse tt a_exili s ●● ●● ● ●● 11 ● ● ● ● ● ● ● ●● ● ●● ● ● Northern ● ● ● ● ● ● ●● ● ●●● ●● ●● ●● ● ● ●● ●● ●● ●● ●●

Pleuronich t hys_ver t icali s ●● ● ● ● ●● ●● ●● ● ● ●● ● ● ● ● ● ● ●● ●●● ● ●● ●● ● ● ● ●● ●● ● ●● ●● ● ●

Pleuronichthys_coenosu s ● ● ●● ●● ● ●● ●● ● ● ●● ●● ● ● ● ● ●● ● ●● ● ● ●● ●● ●● ●● ●● ● ● ● ● ●● ● ●● ● ● ● ●● ● ●●●● ●● ● ● ● ●●●●●● ●● ● ● ● ●● ● ● ● ●● ● ● ● ●● ● ●● ● ● ●

Pleuronichthys_ritter i ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● P l euron i chthys_decurren s ●● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● Pleuroni c h t hys_cornu t u s ●●● ●● ●● ● ● ● ● ● ●● ●● ●● ●● ●●●●● ●●●●● ●● ●● ● ● ●● ●● ●● ●● ●● ● ● ● ●● ●● ●● ●● Pleuronichthys_japonicu s ●●

Pleuronichthys_guttulatu s ● https://doi.org/10.1101/247304 Reinhardtius_evermann i ● ●

A t heres t hes_evermann i ● ● ● ● Atheresthes_stomia s ● Pseudorhombus_oligodo n ●● ●● ●●

Pseudorhombus_malayanu s ● Pseudorhombus_elevatu s ● ● ●● Ps eudo r ho m bu s _jen y n s i i ● ●● ● ● ● ● ● ● ●● ●● ● ● ●●● ●● ● ● ● ● ● ●●●● ●● ●● ●● ● ● ● ● ● ●

Pseudorhombus_dup li c i oce ll atu s ● ● ● ● ● ●●●● ●●●● ● 4:C. darwini ●● ● ● ●● ●● ●● ● ● ● ● ●●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ●● ● ● ● ● ●

Pseudorhombus_arsiu s ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ●● ●● ● ●● ●●● ● ●●● ● ● ● Pseudorhombus_natalensi s ● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ●● ● ● ●

Pseudorhombus_pentophthalmu s ● 6:C. querna ● ●●●● ●●●● ● ● doi: Pse tt ina_hainanensi s ● ●● ● ● ● ● ●● ● ● ● ● ● ● ● ● Tephrinectes_sinensi s ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●

Tarphops_oligolepi s ● ● ● ●● ●● ● ● ● ● ● ● ● ●● ● ● ● T arphops_elegan s ● ● ●● ● ● ● ●● ●● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

P seudorhombus_le v isquami s ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● 1

Hippoglossina_oblonga ● ●● ● ●●● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●●● ●● ● ● ● ● ●

Paralichthys_oblongu s ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● P a r ali c h t h ys _pa t agoni c u s ● ● ● ● ● Paralichthys_adspersu s ● ● ●● ● ● ●● ● ● ● ●●● ● ●●●● ● ●● ● ●● ● ● ● ● ● ● ● ● ●●●●●●●●● ● ●●●● ● ●● ● ● ● ●●● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ●

Paralich t hys_cali f ornicu s ● ● ● ● ● ●●●● ●●●●●●● ●●●●●●●●●● ●●●●●●●●● ● ● ●● ●● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ●●●● ● ●●●●●●●●●●●●●● ●●●●●●●●●●●● ●●●● ● ●●●●●●●● ●● ● ●●●● ● ● ● ● ● ● Paralichthys_squamilentu s ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●●●●● ● ●● ● ● ●● ●● ●● ●●●● ●●●●●●●●●●●●●●● ● ● ● ●●●●●●●●●●●●●●●●●●● ● ● ● ● ● ● ● ● ● ●●●●●●●●●●●●●●● ● ● ● ●● ● ● ● ● ●● ● ● ● ●

Paralichthys_albigutt a ● ●●● ●●●●●●● ●●●●●●●●●●●●●● ●●●●●● ●●●●●●●●●●●●●●●●●●●● ●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●●●●

Paralichthys_olivaceu s ● ● ● ● 3:C. platophrys ● ●● ●●● ●

Paralichthys_isoscele s ● ● Xystr eu rys _ r a s il e ● Xystreurys_liolepi s ● ● Scoph t halmus_aquosu s ● Arnoglossus_capensi s Arnoglossus_imperiali s ● Arnoglossus_ t hor i Arnoglossus_latern a ● Crossorhombus_kanekoni s ● Arnoglossus_tapeinosom a ● Crossorhombus_valderos t ra t u s ● Crossorhombus_kobensi s Crossorhombus_azureu s Ar noglo ss u s _ m a cr olophu s Hippoglossina_stArnoglossus_aspiloomata s ● 5:S. maculiferum Parabothus_chlorospilu s ● Ps e tt ina_iiji m a e ● ●●● Psettina_gigante a ● ● ● ● ● Psettina_tosan a ● ● ●

Engyprosopon_macro l epi s ● ● A rnoglo ss us_pol ys pilu s ● ● Lophonectes_gallu s 1:H. stomata

Engyprosopon_xenandru s ● ● Laeop s _nig r o m a c ula t u s ●

Arnoglossus_scaph a ● ● ●

Laeops_pectorali s ● ●

E ng y pro s opon_maldivensi s ● Laeops_macroph t halmu s

E ng y p r o s opon_ m ul t i s qua m a ●

Kamoharaia_megastom a ● ● bioRxiv preprint Chascanopse tt a_lugubri s ● ●

Chascanopsetta_proriger a ● ● Grammatobothus_polyophthalmu s ● ● ● ● ● 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 hu s _leopardinu s ● ● ●

Engyophrys_sent a 8:L.proboscidea ● ●● ● ● ● ● ●

Trichopsetta_ventrali s ● ● ● ●● ● ●● ● ● ●● ● ● M onolene_ s e ss ili c aud a ● ●●● ●● ●●● ● ● ●● ● ● ● ● ● Engyprosopon_grandisquam a ● ●● ● ● ●●●● ●●●●●●●●●●●●● ●●●●●●●●● ● ● ● ● ●●●● ●●●● ● ● ● ● ● ● ● ● ● ● ● ● ● Bothus_ocellatu s ● ●● ● ●● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ●

Bo t hus_maculi f eru s ● ● ● ● ● ● ● ●

Bothus_poda s ● ● ● ● ● ●● ● ●● ●● ● ● ● ● ● ● ●● Bothus_robins i ● ● ● ● ●● ● ● ● ● ● ● ● ●● ●

Bothus_lunatu s ● ●● ● ● ● ● ● ● ● ● ● ●● ● ●● ●● ● ● ● ●● ●● ● ● ● ●● ● ● ●● ● ●● ● ● ●● ● ● ● ●● ● ● ● ●● ● ● ● Bo t hus_mancu s ● ●● ●●●● ● ● ● ● ● ● ●●●● ● ● 11:H. stenolepis Bothus_pantherinu s ● ● ● ● ●● ● ●● Bothus_myriaste r ● ● ● ●● ● ● ● ●●●●● ●●●● ●● ●●● ●●● ●●● ●● ● ● ● ● ● ● Japonolaeops_dentatu s ● ●● ●●● ● ● ●●●●●●●●●● ● ● ● ● ● ● ● ● ●● ●

Laeop s _ k i t aha r a e ● ● ● ● ● ●

Arnoglossus_yamanaka i ●● ● ● ●●● ● ●● ●● ● ● ●●● ● ● ● ●●●● ●●●●●●● ●●● ● Neolaeops_microphthalmu s ● ●● ●● ●●● ●● ●●●●●●●●● ●●●●● ● ● ● ●● ● ● ●●● ●●●●●● ●●●●●● ●●●●●●●●●●● ●●●●●●●●●● ●●●●●●●●●● ● ●● ●● ●●● ● ●● Arnoglossus_tenui s ●●●●●● ●●●●●● ●● ● ● ●● ●● ●●●● ●●● ●● ●●● ●● ●●●● ●●●●●●●● ●●●●●●●●●●●●●● ●●●●●●●●●●● ●●●●●●●●● ● ● ● ● ● ●●●●●●●●● ● ●●●●●●●●● ●●●● ● ●● ● ● ●●●●●●●●●●●●● ●●●●●●●●●●● ●●●●●● ●●●●●● ●●●●●● ●●●●● ●●● ● ●● ● ●●●●● ●●●●● ● ●●●●●● ●●●●● ●●●●●●●●●●● ●●●●●●●●●●● ●●●●●●●●●●●●●● ●●● ● ● ● ● ● ●●●●●●●●●●● ●●●●●●●●●●● ●●●●●●●●● ●●●●● ● ● ● ● ●●●●●● ●●●●●●●●● ●●●●●●●●●●●●●●● ●●●●●●●●●●●●●● ●●●●●●● ●●●●● ●●●●● ●●●●● ●●● ● ●●● ●●●● ● ●● ●●●●● ●●●●●●● ●●●●●●●●●●●● ●●●●●●●●●●● ● ● ●●●●●●●●● ●●●●●●●● ●●●●●●●● ●●●●●●●●● ● ● Citharichthys_arctifrons ●●●●● ●●●●●●●●●●●●● ●●●●●●●●●●●●●● ●●●●●●●●●●●●● ●●●●●●●●●●●●●● ●●●●●●●● ●●●●●● ●●●● ●● ●●● ●●● ●●●● ● ●● ●● ●●●●●● ●●●●●● ●●●●●● ●●●●● ●●●●●●●●● ● ● ● ●●●●●●●● ●●●●●●● ●●●●●●●●● ●●●●●●●●●●●●● ●●●●●●●● ●● ● ●● ●●● ●● ● ●●●●●●●● ●●●●●●●●●● ●●●●●●●●●●●●● ●●●●●●●●● ●●●●●● ●●● ●●●●●● ●●●●●● ●●● ● ●● ●●● ●●●● ●●●● ●●●●● ●●●● ●● ●● ●●●●●●● ●●●●●● ●●● ●●●● ● ●● ● ● ● 3 ●●●●●● ●●●●●● ●●●●● ●●●●●●●●●●● ●●●●●●●●●●● ●●●●●●●●●●●●● ●● ●● ● ● ●●●● ●●●●● ●●●●●●●● ●●●●●● ●●●●●●●●● ●●●●● ●●● ● ●● ●●●●● ●●●● ●●●● ●●●●● ●●●● ●●●●●●● ●●●● ●●●● ●●●●●●● ●●●●● ●●● ●●●●●●● ●●●●●●● ●● ● ● ● ●● ●●●● ● ● ● ●●● ●●●●●● ●●●●●●● ●●●●●●●●●●●● ●●●●●●●●●●● ●●●●●●●●● ●●● ● ● ● ●●●●● ● ●●● ● ● ● ●●● ●●●●● ●●●●● ●●●●●● ●●●● ●●●● ●●●●●●● ●●● ●●●●●● ●●●●●●● ●●●●●● ●●●●●● ●●●● ●●●●● ●●● ● ● ● ● ●● ● ● ● ● ● ●●●●● ●●●●●●●● ●●● ●●●●● ●●●●●● ● ●●●● ●●●● ●●●●●● ●●●●● ●●●●● ●●●● ●●● ●●● ●●●●● ●●●●●●● ●●●●●●●● ●●●● ●● ●●●● ●●●● ●●●●●●● ●●●●● ●●● ●●●●●●●●● ●●● ● Citharichthys_xanthostigm a ● ● ●●●●● ●●●●● ●●●●●● ●●● ●●●● ●●●●● ●●●●● ●●●●●●●●● ●●●●● ●●●●●●● ●●●●●● ●●●●●●● ●● ● ● ●● ●● ● ● ● ● ● ● ●● ● ● ●●● ●●● ● ● ●● ● ● ●● ● ● ●● ●●● ●●●●● ●●●● ●●●● ●●● ●●●● ●●●●●●●●● ●●●●●●●● ●●●●●●●● ●●●●●●●●●●● ●●●●●●●●● ●●● ● ● ●●●● ● ●● ●●● ●● ● ● ● ●● ● ●● ●●●● ● ●●●●● ●●●●●● ●●●●● ●●●●● ●●●● ●●●●● ●●●●● ●●●●●●●●●●● ●●●●●●● ●●●●●●●● ●●●●●●●● ●●●●● ●●● ●● ● ●

Citharichthys_sordidu s ● ●● ● ● ●● ● ● ● ● ●●● ●●● ●●●● ●●●● ●●● ●●●●● ●●● ●●●●● ●●●●●●●●● ●●●●●●●●●●●●●●● ●●●●●●●●●● ●●● ● ● ●●● ● ● ●● ● ●● ● ● ● ●●● ● ●● ● ● ● ●●● ●● ● ● ● ● ●● ● ● ●● ● ● ● ● ●●●●●● ●●●●● ●●●● ●●●●● ● ●● ●●●● ●●●●●●●● ●●●●●●●●● ●●●●●●●●●● ●●●●●● ●● ● ● ● ●● ● ● ● ● ● ● ●● ● ● Etropus_microstomu s ● ●●● ●●●● ●●●●● ●●●●● ●● ●●●●● ●●●●● ●●●●●●●● ●●●●●●●●● ●●●●●●●●● ●● ● ●● ● ● ● ● ●● ● 2:P. hawaiiensis ● ● ●● ● ● ●● ●● ●●●● ●●●● ●●● ●● ● ●●●● ●●●●●● ●●●●●●●●●●● ●●●●●●●●●●● ●●●●●● ●●●● ●● ●● ●●● ● ●● ● ●● ● ● ● ●●●●●●●●● ● ● ● ●●● ● ● ● ●●● ●●●● ●●●● ●● ●● ●●●●● ●●● ●●●●●●●●●●●● ●●●●●●●● ●●●●●●●●● ●●●● ● ●● ●● ●● ●● Ci t ha r i c h t h ys _a r ena c eu s ●●●● ●●●● ● ● ●● ●●●●● ● ● ● ●● ●●● ●●●●●●●● ●●●●●●●●●● ●●●●●● ●● ●● ● ●● ●● ● ●● ● ● ●●●● ●● ●●●● ●●●●●●●● ●●●●●●● ●●● ●● ● ●●● ●● ● ● Citharichthys_gilbert i ●●●●● ● ● ●● ●● ●●● ●● ● ● ● ●●● ● ●●● ● ●●● ●●● ● ● ● ●● ●● ● ● Citharichthys_spilopteru s ●● ●●● ●●●●● ●●●● ●●●● ●● ● ●●●● ●●● ●● ●●● ● ●●● ●●● ●●● ●●●● ●●● ●●●● ●● ●●●●● ●●●●●●●● ●●●●●●●●● ●●● ●● ●● ● ●●● ●● ●●● ●●● ●● ●● ●●●●●●●●●●● ●●●●●●●● ●●●●●●●● ● ●●● ●● ●●●● ●●● ●●● ●●● ●●● ●●● ● ●●●●●●●● ●●●●●●●● ●●●●●●●●● ●●●●●●● ●●● ●● ●●

Ci t harich t hys_macrop s ●●●●●●●●●● ● ● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●● ●●● ● ●●●●● ●●● ●●●●● ●●●●●● ●●●●●●●●●● ●●●●●● ●●●●●●●●●● ●●●●●●●●●●● ●●●●●●● ●●●●● ●●●● ●● ● ● ● ●●● ●●●●● ●●●●● ●●●●●● ●●●●●●● ●●●●●●●●● ●●●●●● ●●●●●●●● ●●●●● ●●●● ●●●●● ●●●● ●●● ●● Citharichthys_darwini ● ● ● ● ● ●● ●●●● ●●●●● ●●●●●● ●●●●●● ●●●●●●●●● ●●●● ●●●● ●●● ●●● ●● ●●●●●● ●●●●● ●● ● ●●● ●●● ● ●● ●● ●●●● ●●●●● ●●●● ●●●● ●● ●● ●● ●● ●● ●● ●● ●● ● ●●● 4 ● ● ●●●●● ● ● ● ● ● ●● ●● Cit haricht hys_minutus ● ●● ●● ●●● ●●●●●● ●● ● ●●●●● ●●●● ●● ●● ●●● ●●●● ● ● ● ●● ●● ● ● ●●●● ●●● ● ●●● ●●●●● ● ●●● ●●● ●●●●● ●●●● ● ●● ● ●● ● ● ●● ● ● ●● ●● ●●● ●●● ●●● ●●●● ●●●●●● ●● ● ●●● ●● ●● ● ●● ● ●●● ● ● Ci t harich t hys_s t igmaeu s ●● ●● ● ●● ● ●●●●● ●●●●● ●●●● ●●●●●●● ●●

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

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

Et ropu s _ c ro ss o t u s ● ● ● ● ● ● ●● ●●●●● ● ●●

●● ● 5 . 0 ●●● ● ●● ●●●●● ●●● ●●● ● ● ● ● ● Etropus_longimanu s ●●●● ●●●● ●●●● ●●● ● ●● 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 Poecilopsetta hawaiiensis/Poecilopsetta 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_papillosu m ● ●● ●● ●● ● ● ● ● ● ● ●

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● ● ● ● ● ● ● ● ● ● ● ●

●● ● ● ● ● ● ● ● Cyclopsetta_chittendeni ●

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● ● ● ● ●

● 6

● ● Cyclopsetta_panamensi s 7:P. quadrituberculatus Paralichthys_dentatu s

Citharichthys_platophrys

Cyclopsetta_querna bioRxiv preprint doi: https://doi.org/10.1101/247304; this version posted April 24, 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