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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 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 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, 111 et 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. 115 In an attempt to tease out these models and their predictions about the exact timing of 116 divergence between northern and southern species, we assessed model fit by means of the 117 modified Akaike’s Information Criterion (AICM; Baele et al. 2013), which accounts for 118 uncertainty in the MCMC sampling (Raftery et al. 2007). Even if model ranking based on 119 this measure is known to be unstable (Baele et al. 2013), it is clear that the models with 120 priors only on the northern or on the southern species perform significantly (> 200 AIC 121 units) more poorly than the two other models, which may be difficult to tease apart

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122 (Table 1). The predictions of the best models suggest that H. hippoglossus and H. 123 stenolepis, both northern species, consistently diverged before the complete closure of the 124 seaway, in tandem with the average southern species, and that the average northern 125 species diverged in tandem with P. natalensis and P. hawaiiensis (Fig. 3E-F). 126 One intriguing result from these analyses is that the northern sister species seem to 127 come from a single clade (Fig. 2), which would suggest that it is possible to identify the 128 direction of the trans-Arctic migration. For this, we retrieved the current species range of 129 all flatfish analyzed here (Fig. S3), and indicated their average latitude on the 130 phylogenetic trees (Fig. S4). This northern clade, highly supported (posterior probability 131 > 0.9; see asterisk in Fig. S4), is mostly comprised of species currently found in the 132 Pacific (e.g., Reinhardtius evermanni), suggesting that the northern sister species that we 133 identified originated from the Pacific, and that their trans-Arctic migration was from the 134 Pacific into the Atlantic in a northern direction.

135 Discussion 136 Our results show that flatfish underwent a first speciation at the time when the CAS 137 closed, which led to the species that, today, have an equatorial range (Fig. 2), as the 138 formation of the Isthmus of Panama resulted in a barrier to gene flow leading to their 139 speciation. Our results also show that species that today have a northern range (Fig. 2) 140 either emerged at the closure of the seaway, or after its closure. These timing estimates 141 imply that this second bout of speciation was not caused by gene flow impeded by the 142 closure of the seaway, but demand an interpretation involving trans-Arctic gene flow, 143 through a northern route, before being interrupted, most likely this time by a climatic 144 event. Critically, these conclusions are robust to the inclusion of outgroup species 145 (Psettodidae), of outliers species (P. natalensis and P. hawaiiensis), and are consistently 146 found across the four clock models implemented here, including the one (NONE) relying 147 on no other fossil information than the 47.8-42.1 MYA ancestor to this order (Chanet 148 1997; Friedman 2012) – hereby indicating that our data are highly informative with 149 respect to the timing of the divergence of these sister species (and rejecting our initial 150 hypothesis) following their trans-CAS and tans-Arctic migrations.

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151 Trans-Arctic migrations from the Pacific to the Atlantic are not new, and have been 152 described in both invertebrates (e.g., Durham and MacNeil 1967) and vertebrates (e.g., 153 Grant 1986), but never using a relaxed molecular clock relying on as little information as 154 a single fossil and a large multigene data set covering an entire order (Pleuronectiformes). 155 While seminal work was usually based on paleontological (Durham and MacNeil 1967), 156 or on morphological evidence (Reid 1990), subsequent work started using allozymes and 157 other proteins (Grant 1986; Zaslavskaya et al. 1992), followed by genetic evidence, 158 mostly at the population level using mitochondrial DNA (Rawson and Harper 2009). 159 However, most of the recent work that relies on molecular clocks either makes strong 160 assumptions on substitution rates (Rawson and Harper 2009; Liu et al. 2011), uses 161 divergences that are vicariance-motivated but not supported by any fossil information 162 (McCusker and Bentzen 2010), or posit in which direction the trans-Arctic migration 163 took place (Dodson et al. 2007) to infer trans-Arctic migrations. Our work is the first to 164 use sister species to demonstrate the existence and elucidate the timing of both the trans- 165 CAS and the trans-Arctic events, while also infering in which direction the latter took 166 place, without making too many assumptions. 167 Strikingly, the geological evidence is directly in line with our date estimates. The 168 fossil record suggests that the first aquatic connection between the Pacific and Arctic 169 (and Atlantic) oceans through the Bering Strait occurred approximately 5.5-5.4 MYA 170 (Gladenkov et al. 2002) due to a rise in sea levels, linked to tectonic activity 171 (Marincovich and Gladenkov 2001). This would have permitted the trans-Arctic 172 migration of populations ancestral to today’s northern species from one ocean to the other 173 through this ancient “northern passage.” This global warming event, between the late 174 Miocene to early Pleiocene, was then followed by a significant period of cooling during 175 the Pleiocene into the Pleistocene (Zachos et al. 2001), leading to periods of repeated 176 glaciations and a subsequent ice age. These cold events would have resulted in the 177 closure of this ancient “northern passage,” hereby stopping the trans-Arctic gene flow 178 between the two oceans, and leading to the more recent speciation of the northern taxa. 179 For approximately a million years after the first opening of the Bering Strait, water 180 flowed through the strait in a southern direction, from the Arctic to the Pacific ocean, 181 until the formation of the Isthmus of Panama occurred close to the equator (Berta 2012).

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182 With the formation of the Isthmus, and hence the closing of the CAS, the ocean currents 183 reversed due to a change in global ocean circulation (Haug and Tiedemann 1998; De 184 Schepper et al. 2015), and have since flowed in a northern direction through the Bering 185 Strait, from the Pacific to the Arctic (Marincovich 2000). This again is absolutely 186 consistent with our results (Fig. S4), with dispersal or migration from the Pacific into the 187 Atlantic in a northern direction through this strait, which is known as the trans-Arctic 188 interchange (Vermeij 1991). Fossil data also show that the Bering land bridge has been 189 exposed and submerged on multiple occasions since the Pleistocene (Gladenkov and 190 Gladenkov 2004). These openings and closings of the Bering Strait could have provided a 191 mechanism for divergence and the evolution of sister taxa (Taylor and Dodson 1994; 192 Väinölä 2003). 193 Our results also have implication at the family level of flatfish. Further significant 194 global cooling during the Pleistocene resulted in major glaciation events (Zachos et al. 195 2008) that could be responsible for creating barriers that isolated populations. All of the 196 remaining sister taxa in our analysis, who have divergence estimates of less than 2 MYA 197 in our study belong to the family (sensu Cooper and Chapleau 1998). The 198 Pleuronectidae are the predominant flatfish family found in cold and temperate seas of 199 the northern hemisphere (Norman 1934; Cooper and Chapleau 1998). There are far more 200 Pleuronectidae species in the Pacific Ocean, most of them endemic to the north Pacific 201 Ocean off of North America and Asia in the region extending from the Bering Strait to 202 the gulf of California (Norman 1934). None of the arctic species are restricted solely to 203 arctic waters (Munroe 2015). Munroe also noted that Cooper (in an unpublished 204 manuscript) identified areas of endemism among the current distribution of the 205 Pleuronectidae. It has been shown that during the trans-Arctic interchange, there was a 206 far higher number of species (up to eight times higher) migrating to the Atlantic than to 207 the Pacific (Vermeij 1991). Fossil and phylogenetic evidence suggest the Pacific Ocean 208 as the origin for diversification of the Pleuronectidae (Munroe 2015), and our 209 phylogenetic results are highly congruent with this hypothesis. 210 It is possible that the outliers, the Atlantic and Pacific halibut (H. hippoglossus and H. 211 stenolepis, respectively), diverged during one of the first openings of the Bering Strait. 212 The estimated dates from the molecular clock analysis are approximately 5.5 MYA, in

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

237 Materials and Methods

238 Data retrieval and alignment 239 Prior to retrieving sequence data, GenBank was surveyed to identify all the genes 240 belonging to species of Pleuronectiform families (as of August 2016), based on 241 GenBank’s taxonomy browser. DNA sequences for a total of 332 flatfish species (out of

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242 over 800 species in the order) were identified and downloaded for five nuclear genes 243 (KIAA1239, MYH6, RIPK4, RAG1, SH3PX3), and four mitochondrial genes (12S, 16S, 244 COX1 and CYTB). These represented all the taxa having at least one of these six gene 245 sequences in GenBank; see Table S1 for the corresponding accession numbers. Diversity 246 was richly sampled as species from 13 of the 14 families in the order Pleuronectiformes 247 were included in our catchment. In line with current consensus in flatfish systematics, the 248 family Psettodidae was chosen as the outgroup to all other taxa (Chapleau 1993). 249 These sequences were aligned using MUSCLE ver. 3.8.31 (Edgar 2004) on a gene- 250 by-gene basis. Each alignment was visually inspected with AliView ver. 1.18 (Larsson 251 2014), and was manually edited where necessary. In particular, large indels (> 10 bp) 252 were removed prior to all phylogenetic analyses. The 5’ and 3’ ends of sequences were 253 also trimmed. The aligned sequences were then concatenated into a single alignment 254 using a custom R script.

255 Data pre-processing 256 To gauge the phylogenetic content of our data set, we performed a first series of 257 molecular clock analyses on the concatenated data matrix, with all the nine gene 258 sequences obtained above (12S, 16S, COI, Cyt-b, KIAA1239, MYH6, RIPK4, SH3PX3 259 and RAG1), and all the 332 taxa representing all families of flatfish. To account for rate 260 heterogeneity along this alignment, partitions were first identified with PartitionFinder 2 261 (Lanfear 2012), selecting the best-fit model of nucleotide substitution based on Akaike’s 262 Information Criterion (AIC). This optimal partition scheme was then employed in a first 263 Bayesian phylogenetic analysis, conducted with BEAST ver. 1.8.3 (Drummond and 264 Rambaut 2007). This program implements a Markov chain Monte Carlo (MCMC) 265 sampler, which co-estimates both tree topology and divergence times. As determined by 266 PartionFinder, a GTR+I+Γ model was applied to each data partition. These models of 267 evolution across partitions were assumed to be independent (unlinked, in BEAST 268 parlance), while both clock model and tree model partitions were shared (linked) by all 269 partitions. An uncorrelated relaxed clock was employed with a lognormal distribution 270 prior on rates, and a Yule speciation prior (Drummond et al. 2006). Due to the paucity of 271 reliably placed fossils on the flatfish tree, a unique calibration point was placed on the 272 most recent common ancestor (MRCA) of the ingroup, as a mean-one exponential prior,

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273 with an offset of 40 million years reflecting the age of 47.8-42.1 MYA (Chanet 1997; 274 Friedman 2012). To stabilize the estimate, a lognormal ln(0, 1.5) prior with a 40 MYA 275 offset was also placed on the root of the tree (root height). Two separate MCMC samplers 276 were run, each for 10,000,000 generations. Trees were sampled every 5,000 generations, 277 and convergence was checked visually using Tracer ver. 1.6 (available at: 278 http://tree.bio.ed.ac.uk/software/ tracer/). Tree log files from each run were combined in 279 LogCombiner, after conservatively removing 10% of each run as burn-in. The resulting 280 maximum a posteriori (MAP) tree was generated with TreeAnnotator (Drummond and 281 Rambaut 2007). 282 As the topology of this resulting MAP tree was unconventional, we suspected the 283 presence of rogue taxa, i.e. species evolving either much faster or much slower than the 284 majority, which can contribute negatively to consensus tree support (Aberer et al. 2013). 285 Rogue taxa were identified using the RogueNaRok (Aberer et al. 2013) webserver 286 (http://rnr.h-its.org). The consensus trees from the preliminary analysis using 10,000 287 iterations were used as the tree set for rogue taxon analysis. A total of 22 rogues were 288 identified and pruned from the analysis, leaving 310 species.

289 Molecular Dating 290 To assess the impact of the closure of the CAS on flatfish evolutionary dynamics, a 291 second set of partitioned relaxed molecular clock analyses was performed (without the 292 rogue sequences). The timing of the closure of the CAS is estimated to have occurred 293 between 12 and 3 MYA (Duque-Caro 1990; Coates et al. 1992; Haug and Tiedemann 294 1998; O'Dea et al. 2016), and we used this time window as a prior to inform the relaxed 295 molecular clock-based phylogenetic reconstructions. The analyses were performed on the 296 same concatenated data set, with the same single fossil calibration, but we also placed a 297 lognormal prior ln(3,1.5), that has most of its mass on the 12-3 MYA window, on the 298 MRCA of each pair of sister taxa (the ‘ALL’ model). From the initial BEAST analyses, 299 twelve pairs of taxa were selected based on two criteria: (i) being sister species on that 300 initial tree, (ii) with one species distributed in the Pacific and one in the Atlantic ocean 301 (Fig. 2). Again, two independent MCMC samplers were run each for 100 million 302 iterations, with samples taken every 5000 step.

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303 Because these pairs of sister species show a contrasted geographic distribution, 304 having either a southern (equatorial) or a northern range (Fig. 2), two additional sets of 305 analyses were performed. In a first set, calibration priors (ln(3,1.5), as per above) were 306 placed only sister taxa that had a geographic range in the southern hemisphere (the 307 ‘SOUTH’ model), while in a second set, identical calibration priors were placed only on 308 sister species with a northern range (the ‘NORTH’ model). Finally, a set of analyses was 309 performed using no sister taxa calibrations at all (the ‘NONE’ model). For each analysis, 310 results from the two MCMC runs were combined using LogCombiner after removing an 311 even more conservative burn-in of 50%. The final MAP tree was generated with 312 TreeAnnotator. 313 In an attempt to rank these different models (priors on all sister taxa; only on southern 314 taxa; only on southern taxa; no “CAS” priors), the AICM was computed for each model 315 (Baele et al. 2013). These computations were performed in Tracer for each of the four 316 different calibration models, based on 100 replicates.

317 Species distribution data 318 In order to map the distribution of each species of flatfish, we resorted to the Global 319 Biodiversity Information Facility (GBIF: https://www.gbif.org/) database, accessed with 320 the R library rgbif (Chamberlain 2017). Up to 1,000 records were retrieved for each 321 species, and plotted on a geographic map based on GBIF observational records. Where 322 needed, species locations were summarized by mean latitude and longitude (Fig. S4). 323 Approximate attribution to a particular ocean was based on species-specific mean 324 longitudes (Atlantic: between 20˚ and 120˚; Indian: 120˚ and -100˚; Pacific otherwise).

325 Availability of Computer Code and Data 326 Accession numbers, sequence alignments, BEAST models (as xml files), estimated 327 phylogenetic trees (maximum a posteriori trees from BEAST), and the R scripts used to 328 plot the figures in this study are available from https://github.com/sarisbro. 329

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330 Acknowledgments 331 This work was supported by the University of Ottawa (LB), and the Natural Sciences 332 Research Council of Canada (FC, SAB). We are grateful to Jonathan Dench for 333 discussions and comments, and to Compute Canada and Ontario’s Center for Advanced 334 Computing for providing us access to their resource.

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470 Table 1. AICM values for the phylogenetic analyses using four different calibration 471 schemes. Models are ranked from the best AICM model (top) to the worst model. SE: 472 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 +/-3.789 473 474

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475 Figure captions 476 477 FIG. 1. Phylogenetic trees reconstructed based on relaxed clock models. Four models 478 were employed, representing different specifications of prior distributions set on sister 479 taxa. (A) No priors were set on sister taxa. (B) Priors were set on all pairs of taxa. (C) 480 Priors were set only on sister taxa with a current northern range. (D) Priors were set only 481 on sister taxa with a current southern range. Horizontal scale is in million years ago 482 (MYA). The closure of the CAS (12-3 MYA) is shown between vertical gray broken 483 lines. 484

485 FIG. 2. Distribution of the geminate species used in this study. Geographic distribution of 486 the twelve pairs of sister species of flatfish used to calibrate the relaxed molecular clock 487 models. Original data come from GBIF (www.gbif.org; accessed Nov 9, 2017). The top 488 panel shows the phylogenetic distribution of these species based on the clock model 489 including prior ages on all twelve pairs of species; scale bar: 5 MYA. 490

491 FIG. 3. Posterior densities of divergence times for sister taxa used as calibration points in 492 the relaxed molecular clock analyses, under four different calibration schemes. NONE: 493 no priors were placed on sister taxa; ALL: priors were placed on all pairs of sister taxa; 494 NORTH: priors were placed only on pairs of sister taxa with a northern distribution; 495 SOUTH: priors were placed only on pairs of sister taxa with a southern distribution. Top 496 row (A-D) shows all 17 distributions, while bottom row (E-H) shows range-averaged 497 distributions (solid) to the exception of outlier pairs (dashed lines). Densities are color- 498 coded for species with northern (blue) and southern (red) range. Dashed vertical gray 499 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 The copyright holder for this preprint (which was not The copyright holder for this preprint (which Cynoglossus_senegalensi s C ynog l ossus_dub i u s 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 G ymnachirus_ t exa e G ymnachirus_mela s ●

this version posted March 26, 2018. 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 ● Reinhardtius_evermann i ● ●

A t heres t hes_evermann i ● ● ● https://doi.org/10.1101/247304 ● 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 ● ●●●● ●●●● ● ●

Pse tt ina_hainanensi s ● ●● ● ● ● ● ●● ● ● ● ● ● ● ● ● Tephrinectes_sinensi s ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ●

Tarphops_oligolepi s ● ● ● doi: ●● ●● ● ● ● ● ● ● ● ●● ● ● ● 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 ● ●

Chascanopse tt a_lugubri s ● ●

Chascanopsetta_proriger a ● ● bioRxiv preprint 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 ●●●● ●●●● ●●●● ●●● ● ●● Citharichthys 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 March 26, 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