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1 LETTER doi: /nature13451 The evolution of the placenta drives a shift in sexual selection in livebearing fish B. J. A. Pollux 1,2, R. W. Meredith 1,3, M. S. Springer 1, T. Garland 1 & D. N. Reznick 1 The evolution of the placenta from a non-placental ancestor causes a shift of maternal investment from pre- to post-fertilization, creating a venue for parent offspring conflicts during pregnancy 1 4. Theory predicts that the rise of these conflicts should drive a shift from a reliance on pre-copulatory female mate choice to polyandry in conjunction with post-zygotic mechanisms of sexual selection 2. This hypothesis has not yet been empirically tested. Here we apply comparative methods to test a key prediction of this hypothesis, which is that the evolution of placentation is associated with reduced pre-copulatory female mate choice. We exploit a unique quality of the livebearing fish family : placentas have repeatedly evolved or been lost, creating diversity among closely related lineages in the presence or absence of placentation 5,6. We show that post-zygotic maternal provisioning by means of a placenta is associated with the absence of bright coloration, courtship behaviour and exaggerated ornamental display traits in males. Furthermore, we found that males of placental species have smaller bodies and longer genitalia, which facilitate sneak or coercive mating and, hence, circumvents female choice. Moreover, we demonstrate that post-zygotic maternal provisioning correlates with superfetation, a female reproductive adaptation that may result in polyandry through the formation of temporally overlapping, mixed-paternity litters. Our results suggest that the emergence of prenatal conflict during the evolution of the placenta correlates with a suite of phenotypic and behavioural male traits that is associated with a reduced reliance on pre-copulatory female mate choice. Viviparity creates a venuefor parent offspring conflictsinutero 7 caused by a fundamental discord between mothers and developing embryos over the level of maternal investment during pregnancy. Females are selected to maximize their lifetime reproductive success by optimizing the allocation to each offspring while individual offspring are selected to demand a greater investment from the mother than is optimal for her to provide 1 4. The ensuing evolutionary dynamics of perpetual adaptation and counter-adaptation between mother and developing embryo are hypothesized to be the driving force behind a rapid divergence in the genomic, developmental and physiological details of the placenta 1,3,6. A central tenet of the parent offspring conflict theory is that offspring must be able to manipulate the transfer of resources 1,7. However, not all viviparous taxa have this capability 4 6. Lecithotrophic viviparous species lack placentas and allocate all resources necessary for embryo development to the eggs before fertilization. This limits the potential for viviparitydriven conflict, because maternal investment pre-dates the expression of the paternal genome 1,2,4,6. The evolution of the placenta from a nonplacental lecithotrophic ancestor causes a shift in maternal investment from pre- to post-fertilization 5,6, offering embryos the opportunity to influence maternal investment throughout gestation 6,8,9. This creates the potential for genomic conflicts, the magnitude of which depend on the extent of post-zygotic investment 1 4,6. Theory predicts that the emergence of genomic conflicts, early in the evolution of the placenta, should drive a shift from a reliance on precopulatory mate choice to increasing levels of polyandry in conjunction with post-zygotic mechanisms of sexual selection 2. Lecithotrophic species produce large, costly (that is, fully provisioned) eggs 5,6, gaining most reproductive benefits by carefully selecting suitable mates based on phenotype or behaviour 2. These females, however, run the risk of mating with genetically inferior (for example, closely related or dishonestly signalling) males, because genetically incompatible males are generally not discernable at the phenotypic level 10. Placental females may reduce these risks by producing tiny, inexpensive eggs and creating large mixedpaternity litters by mating with multiple males. They may then rely on the expression of the paternal genomes to induce differential patterns of post-zygotic maternal investment among the embryos and, in extreme cases, divert resources from genetically defective (incompatible) to viable embryos 1 4,6,11. Here we apply comparative methods to examine potential conflictdriven shifts in sexual selection associated with the evolution of postfertilization maternal provisioning within the livebearing fish family (order Cyprinodontiformes). This family presents a unique opportunity, because (1) it contains closely related species that differ markedly in the degree and timing of maternal provisioning, ranging from strict pre-zygotic yolk provisioning to extreme levels of post-zygotic investment associated with integrated maternal and fetal tissues specialized for nutrient transfer (that is, placentas 5,6 ); (2) placentas were lost or evolved multiple times independently 5,6 ; and (3) there is great interspecific variation in reproductive traits associated with pre-copulatory sexual selection, including caudal swords, enlarged dorsal fins and bright coloration in males Furthermore, a number of lineages have evolved the ability to carry multiple, temporally overlapping litters that are fertilized at different points in time (that is, superfetation 6,13,14 ). In mammals, superfetation facilitates the formation of mixed-paternity litters (polyandry) Finally, molecular and experimental studies suggest that prenatal genomic conflicts occur in this family 18 and can result in differential patterns of post-zygotic maternal investment between developing embryos 19. If substantial post-fertilization maternal provisioning intensifies fetal maternal conflict 1,3 causing reduced female reliance on pre-copulatory cues in mate choice 2, then males of species withextensive post-fertilization maternal provisioning should display less developed, or the absence of, traits that facilitate female mate choice before copulation. Such traits include sexual dichromatism, courtship behaviour or ornaments. Moreover, if superfetation facilitates multiple paternity 15 17, then species with relatively high levels of post-fertilization provisioning should also have a higher probability of having superfetation 20,21. Finally, copulation is known to incur costs to the females (for example, physical injury, reduced feeding opportunity, increased risk of predation and/or sexually transmitted diseases) 13,14. If substantial post-fertilization maternal provisioning coincides with an increase in the frequency of polyandry, the ensuing sexual conflict should drive the evolution of female (resistance) traits that reduce the costs associated with superfluous mating attempts and, at the same time, male traits that enhance male mating success in the face of female resistance 14. In, a smaller male size relative to female size and an increase in gonopodium length (the male copulatory organ) increases the reproductive success of males 1 Department of Biology, University of California, Riverside, California 92521, USA. 2 Experimental Zoology Group, Wageningen University, 6708 WD Wageningen, the Netherlands. 3 Department of Biology and Molecular Biology, Montclair State University, Montclair, New Jersey 07043, USA. 1 1 S E P T E M B E R V O L N A T U R E Macmillan Publishers Limited. All rights reserved

2 LETTER during sneaky or coercive copulation, which enables males to circumvent female choice 14, We thus predict that males of species with a relatively higher post-zygotic maternal investment should display relatively smaller body sizes and longer gonopodia. To test these hypotheses, we first quantified the degree of postfertilization maternal provisioning for each species with the matrotrophy index, which is the estimated dry mass of the offspring at birth divided by the dry mass of the egg at fertilization. The matrotrophy index provides an objective, dimensionless measure of the degree of post-fertilization maternal provisioning that presents a proxy for the level of placentation 5,6. Lecithotrophic species have matrotrophy index values of less than 1, because embryos lose dry mass during gestation 5,6. Placentotrophic species have matrotrophy index values greater than 1, because post-fertilization maternal provisioning causes growth during development 5,6. We employed a well-resolved phylogeny to test for predicted evolutionary shifts in sexual selection with Bayesian tests Phalloptychus januarius wrayi manni vittata hurtadoi atrora affinis holbrooki geiseri Belonesox belizanus Heterandria bimaculata couchianus gordoni xiphidium variatus maculatus alvarezi hellerii clemenciae monticolus multilineatus nigrensis cortezi nezahualcoyotl pygmaeus Priapella compressa? Priapella intermedia Scolichthys greenwayi Carlhubbsia stuarti Carlhubbsia kidderi Neoheterandria tridentiger Neoheterandria elegans occidentalis lucida prolifica infans monacha? viriosa presidionis turneri gracilis turrubarensis scarlii latidens? fasciata? baenschi retropinna elongata paucimaculata? Priapichthys darienensis Heterandria formosa Xenophallus umbratilis? Alfaro hubberi Alfaro cultratus? terrabensis roseni rhabdophora? holdridgei? Phallichthys tico Phallichthys amates parismina cascajalensis Priapichthys annectens Girardinus metallicus heterandria melanogaster zonata perugiae? tridens nigrofasciata dominicensis? caymanensis vittata hollandi? minor? scalpridens sphenops mexicana? gracilis gilli orri latipinna velifera petenensis caucana vivipara wingei reticulata? branneri parae picta Cnesterodon decemmaculatus Phalloceros caudimaculatus Xenodexia ctenolepis Ln(matrotrophy index) Figure 1 Phylogenetic tree showing relationships among 94 species of the fish family. Boxes at the terminal ends of the branches are coded according to the male sexual selection index: black 5 3, dark grey 5 2, light grey 5 1 and white 5 0; the boxed question mark indicates incomplete information (Supplementary Table 1). Branch colours depict a maximum likelihood reconstruction of maternal provisioning for log-transformed matrotrophy indices. The ancestral reconstruction was performed with phytools 30 and a Brownian motion model of trait evolution. The arrow on the scale bar corresponds to a matrotrophy index value of 1.0, which indicates the division between lecithotrophic and placentotrophic species. In agreement with previous analyses 5,6, the ancestral reconstruction suggests a complex history for the evolution of placentotrophy. The current analysis suggests that the common ancestor of the family has a placenta and that there were multiple losses and gains of placentation within the family. A caveat is that the single egglayer within (Tomeurus gracilis) was excluded from the analysis. The inclusion of this taxon, along with outgroups that contain both livebearers and egg-layers, may yield different results for the evolutionary history of placentotrophy within N A T U R E V O L S E P T E M B E R Macmillan Publishers Limited. All rights reserved 2014

3 LETTER of correlated trait evolution and phylogenetic linear and logistic regressions that explain the variation in sexually selected traits as a function of matrotrophy index (Fig. 1). Bayesian tests show that matrotrophy index, and sexually dimorphic coloration (dichromatism) and courtship behaviour, respectively, have evolved in a correlated fashion (log(bayes factor) and 3.438, respectively). Phylogenetic logistic regressions further show that both traits are negatively correlated to matrotrophy index (b , P and b , P , respectively), indicating that these are significantly less likely to be found in placental lineages (Fig. 2a, b). The presence of exaggerated male display traits (that is, enlarged dorsal fins and filamentous extensions on upper maxillae in the genus or extension of ventral part of the caudal fin to form a sword in ) is also negatively correlated with matrotrophy index, but this trend is not significant after correcting for phylogeny (b , P ; log(bayes factor) ; Fig. 2c). The sexual selection index, defined as the summed presence of these three male traits (dichromatism, courtship behaviour and ornamental display traits), decreases significantly with increasing matrotrophy index (phylogenetic generalized least-squares regression: F , P ; log(bayes factor) ), indicating that lecithotrophic males have significantly more traits to facilitate female choice before copulation than highly placental species (Fig. 2d). Superfetation is strongly correlated with matrotrophy index (phylogenetic logistic regression: b , P, 0.001; log(bayes factor) ; Fig. 2e), indicating that placental species are more likely to have it. The relative length of the gonopodium is positively correlated with matrotrophy index (phylogenetic generalized least-squares regression: F , P ; log(bayes factor) ; Fig. 2f), demonstrating a strong association between longer genitalia and the presence of post-fertilization maternal provisioning. The size dimorphism index is also positively correlated with matrotrophy index, both for body weight (F , P, 0.001; log(bayes factor) ; Fig. 2g) and standard length (F , P, 0.001; log(bayes factor) ; Fig. 2h), indicating that the difference in body size between males and females is larger in lineages with higher levels of post-zygotic maternal investment. This increase is caused by a decrease in male size (log 10 (male wet mass): F , P , log(bayes factor) ; log 10 (male standard length): F , P , log(bayes factor) ; Fig. 2i, j blue lines) in association with increasing matrotrophy index, rather than an increase in female size (log 10 (female wet mass): F , P log(bayes factor) ; log 10 (female standard length): F , P , log(bayes factor) ; Fig. 2i, j red lines). Our findings yield three important insights. First, male traits that facilitate pre-copulatory female mate choice are less well developed in placental lineages. This is supported by patterns within individual clades. In the northern clade of the genus 5, males from lecithotrophic species are melanic whereas males from derived placental species have the same coloration as females. In the subgenus Micropoecilia of, males belonging to the lecithotrophic clade are far more intensely coloured than the males in the derived placental clade, suggesting that here too sexually dimorphic coloration is disappearing. Extreme male ornamental display traits used during courtship are only found in lecithotrophic clades ( and subgenus Mollienesia of ) and are notably absent in placental species. Since sexual selection in is influenced by pre-copulatory cues 12 14, these results suggest that phenotype- or behaviourally based female mate choice is of greater importance in lecithotrophic species than in species with substantial post-zygotic maternal provisioning. Second, male traits that help circumvent female mate choice during sneak or coercive mating are more developed in placental species. These findings concur with the theory that sexual conflict can result in the evolution of sexual dimorphism 25,26 and rapid phenotypic divergence in genitalia 27,28. In poeciliids, large males and short genitals are associated with courtship behaviour aimed at attracting cooperative females Smaller males and longer genitalia are associated with sneak copulation, a Dichromatism c Ornamentation e Superfetation g SDI body weight i Log 10 (body weight) the small size allowing males to approach females from behind without being detected and enabling them to manoeuvre more easily when inserting the gonopodium into the gonoduct of uncooperative females, while longer gonopodia enable a more efficient sperm transfer during unsolicited matings 12 14, Third, the degree of post-zygotic maternal provisioning is strongly correlated with superfetation, which is found in all placental lineages save for Phalloceros caudimaculatus and the subgenus Pamphorichthys of. This reproductive adaptation is thought to diminish the b Courtship behaviour d Sexual selection index Gonopodium length h SDI standard length Log 10 (standard length) j Ln(matrotrophy index) f Figure 2 Phylogenetic logistic and linear regressions. The regressions evaluate the effect of the natural-log-transformed matrotrophy index on (a) dichromatism (n 5 94 taxa), (b) courtship behaviour (n 5 79), (c) ornamental male display traits (n 5 94), (d) sexual selection index (defined as the total number of male traits present ranging from 0 (none of the three traits present) to maximum 3 (all three traits present); n 5 79), (e) superfetation (n 5 92), (f) relative gonopodium length (n taxa), (g) size dimorphism index (SDI) for body weight (n 5 87), (h) size dimorphism index for standard length (n 5 ), (i) log 10 -transformed male (blue dots and line, n 5 99) and female (red dots and line, n 5 87) body weight, and (j) log 10 -transformed male (blue dots and line, n 5 107) and female (red dots and line, n 5 ) standard length. 1 1 S E P T E M B E R V O L N A T U R E Macmillan Publishers Limited. All rights reserved

4 LETTER probability of a single male monopolizing an entire litter by fertilizing all embryos. Instead, by dividing offspring into multiple, smaller temporally overlapping litters, each fertilized at different points in time, and by using sperm derived from the most recent mating event ( last male sperm precedence 13,14 ), superfetation increases a female s likelihood of creating multiple-paternity litters Prior research has shown that male coloration, courtship behaviour and ornamental display traits play an important role in pre-copulatory female mate choice 13 14, that small male size and long genitalia facilitate sneak copulation (a strategy that circumvents female choice) 13,14,22 24 and that superfetation facilitates the formation of mixed-paternity litters (polyandry) The correlation of these traits with the level of post-zygotic maternal provisioning provides support for an association between the placenta and weaker pre-copulatory mate choice. What remains to be shown is that this relationship is causal and is associated with an increase in multiple paternities. Our study provides the first empirical evidence concurring with the hypothesis 2 that the rise of parent offspring conflicts during the evolutionary transition from pre- to post-zygotic maternal provisioning correlates with a shift in sexual selection. This study will help to understand the elusive consequences of viviparity-driven conflict and may advance our knowledge about the evolution of reproductive traits in other viviparous lineages that evolved placentas, since all share the same potential for genomic conflict. METHODS SUMMARY The maximum likelihood phylogeny was constructed using RAxML (ref. 29). Different phylogenetic comparative approaches were used to test for correlated trait evolution. Online Content Methods, along with any additional Extended Data display items andsourcedata, areavailable intheonlineversionofthepaper; references unique to these sections appear only in the online paper. Received 12 August 2013; accepted 2 May Published online 9 July; corrected online 10 September 2014 (see full-text HTML version for details). 1. Haig, D. Genetic conflicts in human pregnancy. Q. Rev. Biol. 68, (1993). 2. Zeh, D. W. & Zeh, J. A. Reproductive mode and speciation: the viviparity-driven conflict hypothesis. Bioessays 22, (2000). 3. Wilkins, J. R. & Haig, D. What good is genomic imprinting: the function of parentspecific gene expression. Nature Rev. Genet. 4, (2003). 4. Crespi, B. & Semeniuk, C. Parent-offspring conflict in the evolution of vertebrate reproductive mode. Am. Nat. 163, (2004). 5. Reznick, D. N., Mateos, M. & Springer, M. S. Independent origins and rapid evolution of the placenta in the fish genus. Science 298, (2002). 6. Pollux, B. J. A., Pires, M. N., Banet, A. I. & Reznick, D. N. The evolution of placentas in the fish family an empirical study of macroevolution. Annu. Rev. Ecol. Evol. Syst. 40, (2009). 7. Trivers, R. L. Parent-offspring conflict. Am. Zool. 14, (1974). 8. Banet, A. I., Au, A. G. & Reznick, D. N. Is mom in charge? Implications of resource provisioning on the evolution of the placenta. Evolution 64, (2010). 9. Pollux, B. J. A. & Reznick, D. N. Matrotrophy limits a female s ability to adaptively adjust offspring size and fecundity in fluctuating environments. Funct. Ecol. 25, (2011). 10. Zeh, J. A. & Zeh, D. W. Toward a new sexual selection paradigm: polyandry, conflict and incompatibility. Ethology 109, (2003). 11. Haig, D. Brood reduction and optimal parental investment when offspring differ in quality. Am. Nat. 136, (1990). 12. Bisazza, A. Male competition, female mate choice and sexual size dimorphism in poeciliid fishes. Mar. Behav. Physiol. 23, (1993). 13. Meffe, G. K. & Snelson, F. F. Jr. (eds). Ecology and Evolution of Livebearing Fishes () (Prentice Hall, 1989). 14. Evans, J. P., Pilastro, A. & Schlupp, I. (eds) Ecology and Evolution of Poeciliid Fishes (Univ. Chicago Press, 2011). 15. Shackelford, R. M. Superfetation in the ranch mink. Am. Nat. 86, (1952). 16. Yamaguchi, N., Dugdale, H. L. & MacDonald, D. W. Female receptivity, embryonic diapause, and superfetation in the European badger (Meles meles): implications for the reproductive tactics of males and females. Q. Rev. Biol. 81, (2006). 17. Dugdale, H. L., MacDonald, D. W., Pope, L. C. & Burke, T. Polygynandry, extra-group paternity and multiple-paternity litters in European badger (Meles meles) social groups. Mol. Ecol. 16, (2007). 18. O Neill, M. J. et al. Ancient and continuing Darwinian selection on insulin-like growth factor II in placental fishes. Proc. Natl Acad. Sci. USA 104, (2007). 19. Schrader, M. & Travis, J. Testing the viviparity-driven-conflict hypothesis: parentoffspring conflict and the evolution of reproductive isolation in a poeciliid fish. Am. Nat. 172, (2008). 20. Coleman, S. W., Harlin-Cognato, A. & Jones, A. G. Reproductive isolation, reproductive mode, and sexual selection: Empirical tests of the viviparity-driven conflict hypothesis. Am. Nat. 173, (2009). 21. Schrader, M. & Travis, J. Variation in offspring sizewith birthorder in placental fish: A role for asymmetric sibling competition? Evolution 66, (2012). 22. Bisazza, A. & Marin, G. Sexual selection and sexual size dimorphism in the eastern mosquitofish holbrooki (Pisces ). Ethol. Ecol. Evol. 7, (1995). 23. Pilastro, A., Giacomello, E. & Bisazza, A. Sexual selection for small size in male mosquitofish ( holbrooki). Proc. R. Soc. Lond. B 264, (1997). 24. Evans, J. P. et al. Intraspecific evidence from guppies for correlated patterns of male and female genital trait diversification. Proc. R. Soc. B 278, (2011). 25. Arnqvist, G. & Rowe, L. Antagonistic coevolution between the sexes in a group of insects. Nature 415, (2002). 26. Bonduriansky, R. & Chenoweth, S. F. Intralocus sexual conflict. Trends Ecol. Evol. 24, (2009). 27. Arnqvist, G. Comparative evidence for theevolution of genitalia by sexualselection. Nature 393, (1998). 28. Hosken, D. J. & Stockley, P. Sexual selection and genital evolution. Trends Ecol. Evol. 19, (2004). 29. Stamatakis, A. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22, (2006). 30. Revell, L. J. phytools: an R package for phylogenetic comparative biology (and other things). Methods Ecol. Evol. 3, (2012). Supplementary Information is available in the online version of the paper. Acknowledgements We thank all individuals and institutions who provided samples for this study (Rehoboth Aquatics, H. Bart Jr, R. Davis, D. Fromm, J. de Greef, H. Hieronimus, B. Hobbs, T. Hrbek, J. Johnson, B. Kohler, J. Langenhammer, C. Li, J. Lundberg, M. Mateos, A. Meyer, D. Nelson, L. Page, L. Parenti, M. Sabaj Pérez, R. Robins, R. de Ruiter, S. Schaefer, M. Schartl, J. Sparks, M. Stiassny, J. Travis, J. Trexler and J. Williams); L. Rowe and A. Furness for discussions and reading the manuscript; and C. Oufiero and M. Banet for help in collecting part of the data. This study was supported by Rubicon grant of the Netherlands Organisation for Scientific Research and Marie Curie IIF grant of the European Union to B.J.A.P. and grant DEB of the US National Science Foundation to D.N.R. and M.S.S. Author Contributions B.J.A.P. and D.N.R. designed the study, D.N.R. quantified the matrotrophy indices, R.W.M. and M.S.S. constructed the molecular phylogeny, T.G. taught B.J.A.P. how to do phylogenetic regression and aided in the preliminary analysis of the data, and B.J.A.P. measured the morphological traits, performed the final analyses of the data and wrote the paper. All authors discussed the results and commented on the manuscript. Author Information Reprints and permissions information is available at The authors declare no competing financial interests. Readers are welcome to comment on the online version of the paper. Correspondence and requests for materials should be addressed to B.J.A.P. ([email protected]; [email protected]) or D.N.R. ([email protected]) N A T U R E V O L S E P T E M B E R Macmillan Publishers Limited. All rights reserved 2014

5 LETTER METHODS Matrotrophy index. The degree of pre- versus post-fertilization maternal provisioning was estimated for 110 species of the family by calculating the matrotrophy index, which is defined as the ratio of the estimated dry mass of the offspring at birth divided by the dry mass of the egg at fertilization. The stage of embryo development was assigned on the basis of morphological criteria 5 and converted into a numerical score that ranged from 0 (yolked egg, no development) to 45 (fully developed embryo, ready to be born), with stage 50 representing a newborn young. The numerator and denominator were estimated from a regression line fitted to the log-transformed dry masses of embryos (y axis) and their stage of development (x axis). The matrotrophy index provides an objective dimensionless measure of the degree of post-fertilization maternal provisioning that can be used as a proxy for the level of placentation 5. Lecithotrophic species have matrotrophy index values ranging from 0.5 to 0.75, meaning that the embryos lose 25 50% of their dry mass during gestation due to metabolic costs associated with development; similar mass losses are observed in egg-laying species of fish 5. Placentotrophic species have matrotrophy index values that range from near 1, implying some post-fertilization provisioning to offset the costs of development, to more than, indicating extensive post-fertilization maternal provisioning. Sexually selected traits and superfetation. We obtained information on superfetation and the presence of sexually dimorphic coloration (dichromatism), courtship behaviour and exaggerated display traits in males from the literature and personal observations (all data are provided in Supplementary Table 1). Observed species were monitored twice for 20 min at room temperature in 37.8 l stock tanks containing gravel, aquatic plants and artificial lighting. We calculated a pre-copulatory sexual selection index for each species by assigning a value of 1 to each of the three male traits if they were present and then taking the sum. The sexual selection index indicated the total number of these traits present in the males of each species and ranged from 0 (indicating an absence of these male traits) to maximum 3 (indicating the presence of all three traits). Morphological measurements. Information on sexual size dimorphism and genital length was obtained from preserved specimens (all data are provided in Supplementary Table 2). Standard length of preserved specimens was measured (to the nearest 0.01 mm) from the tip of the upper jaw to the outer margin of the hypural plate, using digital callipers. Gonopodium length was taken as the distance between the base of the gonopodium and its distal tip. The gonopodium is the male s copulatory organ derived from a metamorphosed anal fin and used to transfer sperm bundles (spermatozeugmata) into the female s urogenital opening. Relative gonopodium length was calculated as the ratio of gonopodium length to male standard length. The wet mass was measured to the nearest 0.01 mg on a Mettler AE 163 Microbalance (Mettler Instruments) after removal of excess liquid. Sexual size dimorphism (for body weight and standard length) was quantified as (1) the log of size ratio between females and males (sexual size dimorphism 5 log(female size/male size)) and (2) the size dimorphism index, which takes the ratio of the larger to the smaller sex and then subtracts 1 (size dimorphism index 5 (larger sex/smaller sex) 2 1). This latter value is made negative if males are the larger sex and positive if females are the larger sex 31,32. For all statistical analyses, body weight and standard length were log 10 transformed. Molecular sequences and phylogeny reconstruction. We assembled a data set that consisted of segments from 28 different genes (20 nuclear, 8 mitochondrial). Sequences were extractedfrom GenBank and supplemented with 920 new sequences for seven nuclear genes (ENC1, Glyt, SH3PX3, MYH6, Rag1, Rh, X-src) and two mitochondrial genes (Cytb, ND2). The data set comprised sequences for 288 species (293 terminals) of clupeocephalans of which 177 were poeciliids. Taxon sampling and GenBank accession numbers are provided in Supplementary Table 3. Tissue/ D samples for this project were provided to D.N.R. by different individuals/ institutions (see Acknowledgements) and are now housed at the University of California Riverside. Genomic D was extracted from either whole fish or fin clips using AccuPrep Genomic D Extraction Kits (Bioneer). Some samples proved difficult to amplify. In these cases we used Illustra GenomiPhi V2 D Amplification Kits (GE Healthcare) to amplify whole genomic D before PCR. Two mitochondrial and seven nuclear gene regions were targeted in PCR reactions. Mitochondrial D amplicons were as follows: (1) 39 end of tr Glu, complete cytochrome b (cytb), and 59 end of tr Thr ; and (2) 39 end of tr Gln, complete tr Met, complete DH dehydrogenase subunit 2 (ND2), complete tr Trp, complete tr Ala, and 59 end of tr Asn. Nuclear amplicons were as follows: (1) two partial exons (8 and 10), all of exon 9, and two introns (8 and 9) of the tyrosine kinase gene (X-src); (2) exon 1 of myosin, heavy polypeptide 6 (MYH6); (3) exon 2 of ectodermal-neural cortex 1 like protein (ENC1); (4) exon 2 of glycosyltransferase (Glyt); (5) exon 1 of SH3 and PX domain containing 3 (SH3PX3); (6) a portion of the 7 transmembrane receptor region of rhodopsin (Rh); and (7) exon 3 of recombination activating gene-1 (Rag1). The majority of primers used have previously been described: X-src (refs 33 35); MYH6, ENC1, Glyt, SH3PX3 (refs 34 36); Rh (refs 34, 35, 37); Rag1, ND2 (refs 34, 35, 38 41); cytb (refs 34, 35, 42). Additional primers new to this study were designed as necessary. All primers used in this study can be found in Supplementary Table 4. PCR reactions, including nested PCRs, were performed following protocols outlined in refs 34, 35. All PCR products were run out on 1% agarose gels and the product of interest was excised and cleaned using AccuPrep Gel Purification kits (Bioneer). Cleaned PCR products were sequenced in both directions using an automated D sequencer (ABI 3730xl) at the University of California Riverside s Core Genetics Institute. Sequencher 4.8 was used to assemble contigs. All sequences were manually aligned in Se-Al 43. Alignment ambiguous regions, including several transfer R (trs) (Gln, Met, Trp, Ala, Asn), were visually identified and removed before performing phylogenetic analyses. The final D alignment for the 28 gene segments without alignment ambiguous regions was 20,828 base pairs. We divided the concatenated data set into 22 partitions. The mitochondrial gene regions were partitioned into proteincoding genes and Rs (tr, ribosomal R) and each nuclear gene region was given its own partition. jmodeltest was used to determine the best-fit models of D substitution 44,45 for each partition as suggested by the Akaike information criterion (Extended Data Table 1). Given that the C distribution accounts for rate heterogeneity, we did not include a proportion of invariant sites as recommended in ref. 46. If the model suggested by jmodeltest was not implemented in RAxML (ref. 29), we used the next most complex model. The 22-partition maximum likelihood analysis used RAxML (ref. 29) (Extended DataTable1; Supplementary Figs 1 and 2). RAxML analyses started from randomized maximum parsimony starting trees and employed the GTRCAT model with 500 bootstrap pseudoreplicates, and the fast hill-climbing algorithm. All other free parameters were estimated. Bootstrapping and a search for the maximum likelihood tree were performed in the same analysis (TreeBase submission number 15653). Bayesian inference of correlated evolution. BayesTraits version 2.0 (software available from was used to test for correlated evolution between the natural-log-transformed matrotrophy index and all other traits. The BayesTraits-Continuous module was used to compare two models of trait evolution: a dependent model in which two traits are assumed to evolve in a correlated fashion on the phylogeny, and an independent model in which the correlation between traits is set to zero using the testcorrel command. Three different sets of analyses were performed: the first set of analyses was run using the maximum likelihood phylogram from RAxML (Extended Data Table 2); the second was performed using ten trees sampled at regular intervals of 50 from the RAxML bootstrap analysis to control for phylogenetic uncertainty 48 (Extended Data Table 3); and the third was conducted using the maximum likelihood phylogram from RAxML and an imputed data set for all 177 poeciliid taxa in our phylogeny to evaluate the potential influence of missing values (Extended Data Table 4). The imputed data set was obtained using the phylogenetic data imputation technique implemented in PhyloPars 49, a web-based program that uses a maximum likelihood based method for estimating missing values assuming a Brownian motion model of trait evolution. The BayesTraits-Continuous module treats binary traits as continuous variables (BayesTraits does not allow mixture of continuous and binary variables, but see phylogenetic logistic regression below). If a binary trait is significantly correlated with the natural-log-transformed matrotrophy index (a quantitative variable), then the mean matrotrophy index for the group coded as zero differs significantly from the mean of the group coded as 1 (ref. 48). Average trait values were calculated for species with data from multiple populations and used in the analyses. Markov chain Monte Carlo analyses were run for 5,050,000 generations sampled every 1,000th iteration, with a burn-in of 50,000. The acceptance values of the transition rate parameter (ratedev) were within the required 20 40% for all analyses, ensuring adequate mixing among chains 48. The scaling parameter lambda (l) was simultaneously estimated to assess the contribution of the phylogeny to trait evolution (l 5 0 indicates phylogenetic independence and l 5 1 indicates phylogenetic dependence 47,48 ). Best-fit models of trait evolution were selected by appraisal of the log(bayes factors) 48,50, calculated as follows: log(bayes factor) 5 2(log[harmonic mean(dependent model)] 2 log[harmonic mean(independent model)]). On the log-scale, negative log(bayes factors) argue in favour of the independent model of evolution; positive values support a dependent model of evolution, with log(bayes factors) greater than 2 offering positive evidence and values greater than 5 strong evidence in favour of the dependent model 48. Phylogenetic logistic regression. Phylogenetic logistic regressions (with Firth correction) were performed using PLogReg.m version 18Aug10 (ref. 51) in Matlab (Mathworks). The phylogeny was first exported from Mesquite version 2.75 (ref. 52) as a PDI file and converted to a phylogenetic variance covariance matrix using the DOS program PDDIST 53 (available from the PDAP package in Mesquite). Sexual dimorphic coloration (dichromatism), courtship behaviour, exaggerated display traits and superfetation are binary traits. Their presence within each species was categorized as 1 and their absence as 0. The independent variable (the natural-logtransformed matrotrophy index) was standardized to have mean equal to 0 and 2014 Macmillan Publishers Limited. All rights reserved

6 LETTER standard deviation equal to 1, so that the regression coefficients represented effect sizes of the independent variable whose magnitudes reflected the size of effect of the variable 51. A bootstrapping procedure with 2,000 simulations was used to generate the confidence intervals and test for statistical significance of the slope of the regression model. Convergence of model parameters was achieved in all cases (Extended Data Table 5). Phylogenetic generalized least-squares regression. Regressionv2.m version 16Mar11 (ref. 54) was used to assess linear relationships between the naturallog-transformed matrotrophy index and the sexual selection index, relative gonopodium length, sexual size dimorphism and log 10 -transformed male and female standard lengths and body weights. Mesquite version 2.75 (ref. 52) was employed to add populations as soft polytomies to the phylogeny. Population branch lengths were set at based on the mean of all population branch lengths in the original maximum likelihood phylogram, with the constraint that the tips of the population branches had to be lined up with the tip of the original species branch in the phylogeny. The MODEL procedure in SAS version 9.2 (SAS Institute) was used before all regressions to confirm a constant variance of residuals (homoscedasticity) by means of White s general and Breusch Pagan tests (Extended Data Table 6; SAS/ETS 12.1 User s Guide 55 ). Three regression models were then computed: (1) ordinary linear least-squares regressions assuming a star phylogeny; (2) phylogenetic generalized least-squares regressions assuming a Brownian motion process of trait evolution; and (3) phylogenetic generalized least-squares regressions assuming an Ornstein Uhlenbeck process of trait evolution, which incorporates stabilizing selection towards an optimum value with different possible means for different groups. Ornstein Uhlenbeck regression allows branch lengths to vary, estimating the optimal Ornstein Uhlenbeck transformation parameter, d, with a value of 0 indicating that a star phylogeny (ordinary linear least-squares model) best fits the data, a value of 1 that the original input tree (phylogenetic generalized least-squares model) best fits the data and a value between 0 and 1 that intermediate branch lengths provide the best fit 54. For all statistical analyses, body weight and standard length were log 10 transformed. To allow for soft polytomies, the degrees of freedom was corrected, using an adjusted degrees of freedom when comparing test statistics with critical F values of the total number of branches (N) (z 1 2), where z is the number of branches that are set to a length of zero 56,57. Two approaches were used to select the best-fit linear regression model. First, we assessed differences in the Akaike information criterion (AIC): AIC 5 (22 3 ln(ml likelihood)) 1 (2 3 number of parameters) between models, expressed as D i 5 AIC i 2 AIC min (where AIC min is the value of the model with the lowest AIC value and AIC i is the value for the alternative model i). D i provides a heuristic measure of the fit of alternative model i relative to the fit of the best model: D i, 2 suggests that there is substantial support for alternative model i relative to the best model; 4, D i, 7 indicates that the alternative model has considerably less support; and D i. 10 signifies that the alternative model is highly unlikely 58. Second, where one model was a nested subset of the other (compared with the ordinary linear least-squares and phylogenetic generalized least-squares models, the Ornstein Uhlenbeck model contains one more estimated parameter), we compared them by maximum likelihood ratio tests, where twice the difference in natural-log likelihoods between models (D 5 22(maximum likelihood for best model 2 maximum likelihood for alternative model i)) is assumed to be distributed asymptotically as a x 2 distribution with degrees of freedom equal to the difference in the number of parameters in the two models. Following ref. 54, we also used likelihood ratio tests to compare the phylogenetic generalized least-squares and ordinary linear least-squares models. Although the degrees of freedom in these comparisons is zero (both models have the same number of parameters), a difference in likelihoods greater than (which is the ninety-fifth percentile of the distribution of x 2 with one degree of freedom) is often taken to indicate a significant difference (P. 0.05) in the fit of the two models 54,59 (Supplementary Table 6). 31. Lovich, J. E. & Gibbons, J. W. A review of techniques for quantifying sexual size dimorphism. Growth Dev. Aging 56, (1992). 32. Fairbairn, D. J., Blanckenhorn, W. U. & Székely, T. (eds) Sex, Size and Gender Roles: Evolutionary Studies of Sexual Size Dimorphism (Oxford Univ. Press, 2007). 33. Meyer, A. & Lydeard, C. The evolution of copulatory organs, internal fertilization, placentae and viviparity in killifishes (Cyprinodontiformes) inferred from a D phylogeny of the tyrosine kinase gene X-src. Proc. R. Soc. Lond. B 254, (1993). 34. Meredith, R. W., Pires, M. N., Reznick, D. N. & Springer, M. S. Molecular phylogenetic relationships and the evolution of the placenta in (Micropoecilia) (: Cyprinodontiformes). Mol. Phylogenet. Evol. 55, (2010). 35. Meredith, R. W., Pires, M. N., Reznick, D. N. & Springer, M. S. Molecular phylogenetic relationships and the coevolution of placentotrophy and superfetation in (: Cyprinodontiformes). Mol. Phylogenet. Evol. 59, (2011). 36. Li, C., Ortí, G., Zhang, G. & Lu, G. A practical approach to phylogenomics: the phylogeny of ray-finned fish (Actinopterygii) as a case study. BMC Evol. Biol. 7, 44 (2007). 37. Chen, W. J., Bonillo, C. & Lecointre, G. Repeatability of clades as a criterion of reliability: a case study for molecular phylogeny of Acanthomorpha (Teleostei) with larger number of taxa. Mol. Phylogenet. Evol. 26, (2003). 38. Hrbek, T., Seckinger, J. & Meyer, A. A phylogenetic and biogeographic perspective on the evolution of poeciliid fishes. Mol. Phylogenet. Evol. 43, (2007). 39. Kocher, T. D., Conroy, J. A., McKaye, K. R., Stauffer, J. R. & Lockwood, S. F. Evolution of DH dehydrogenase subunit 2 in East African cichlid fish. Mol. Phylogenet. Evol. 4, (1995). 40. Ptacek, M. B. & Breden, F. Phylogenetic relationships among the mollies (: : Mollienesia group) based on mitochondrial D sequences. J. Fish Biol. 53 (Suppl. A), (1998). 41. Breden, F., Ptacek, M. B., Rashed, M., Taphorn, D. & Figueiredo, C. A. Molecular phylogeny of the live-bearing fish genus (Cyprinodontiformes: ). Mol. Phylogenet. Evol. 12, (1999). 42. Schmidt, T. R., Bielawski, J. P. & Gold, J. R. Molecularphylogenetics andevolution of the cytochrome b gene in the cyprinid genus Lythrurus. Copeia 1998, (1998). 43. Rambaut, A. Se-Al: Sequence Alignment Editor v.2.0a11 ( software/sea1, 1996). 44. Guindon, S. & Gascuel, O. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52, (2003). 45. Posada, D. jmodeltest: phylogenetic model averaging. Mol. Biol. Evol. 25, (2008). 46. Yang, Z. Computational Molecular Evolution (Oxford Univ. Press, 2006). 47. Pagel, M. Inferring the historical patterns of biological evolution. Nature 401, (1999). 48. Pagel, M. & Meade, A. User s Manual for BayesTraits V2 ( ). 49. Bruggeman, J., Heringa, J. & Brandt, B. W. PhyloPars: estimation of missing parameter values using phylogeny. Nucleic Acids Res. 37, W179 W184 (2009). 50. Kass, R. E. & Raftery, A. E. Bayes factors. J. Am. Stat. Assoc. 90, (1995). 51. Ives, A. R. & Garland, T. Jr. Phylogenetic logistic regression for binary dependent variables. Syst. Biol. 59, 9 26 (2010). 52. Maddison, W. P. & Maddison, D. R. Mesquite: A Modular System forevolutionary Analysis v.2.75 ( 2011). 53. Garland, T. Jr, Dickerman, A. W., Janis, C. M. & Jones, J. A. Phylogenetic analysis of covariance by computer simulation. Syst. Biol. 42, (1993). 54. Lavin, S. R., Karasov, W. H., Ives, A. R., Middleton, K. M. & Garland, T. Jr. Morphometrics of the avian small intestine compared with that of nonflying mammals: a phylogenetic approach. Physiol. Biochem. Zool. 81, (2008). 55. SAS Institute. SAS/ETS 12.1 User s Guide (SAS Institute, 2012). 56. Purvis, A. & Garland, T. Jr. Polytomies in comparative analyses of continuous characters. Syst. Biol. 42, (1993). 57. Garland, T. Jr & Díaz-Uriarte, R. Polytomies and phylogenetically independent contrasts: an examination of the boundeddegrees of freedom approach. Syst. Biol. 48, (1999). 58. Burnham, K. P. & Anderson, D. R. Model Selection and Multimodel Inference: A Practical Information-Theoretic Approach 2nd edn, (Springer, 2002). 59. Felsenstein, J. Inferring Phylogenies (Sinauer, 2004) Macmillan Publishers Limited. All rights reserved

7 LETTER Extended Data Table 1 Results of models of molecular evolution chosen by jmodelltest 2014 Macmillan Publishers Limited. All rights reserved

8 LETTER Extended Data Table 2 Bayesian inference of correlated evolution between the natural-log-transformed matrotrophy index and other lifehistory traits within the family 2014 Macmillan Publishers Limited. All rights reserved

9 LETTER Extended Data Table 3 Bayesian inference of correlated evolution between the natural-log-transformed matrotrophy index and other lifehistory traits within the family using a subset of ten trees to control for phylogenetic uncertainty 2014 Macmillan Publishers Limited. All rights reserved

10 LETTER Extended Data Table 4 Bayesian inference of correlated evolution between the natural-log-transformed matrotrophy index and other lifehistory traits after data imputation with PhyloPars 2014 Macmillan Publishers Limited. All rights reserved

11 LETTER Extended Data Table 5 Ordinary and phylogenetic logistic regression parameter estimates for the effect of the natural-log-transformed matrotrophy index on dichromatism, courtship behaviour, ornamental male display traits and superfetation within the family 2014 Macmillan Publishers Limited. All rights reserved

12 LETTER Extended Data Table 6 White s general and Breusch Pagan tests for homoscedasticity 2014 Macmillan Publishers Limited. All rights reserved

13 doi: /nature Supplementary Table 1 Taxa, natural log-transformed matrotrophy index (LNMI), dichromatism (DICHROM: 1 = dichromatic, 0 = monochromatic), courtship behavior (COURT: 1 = courtship present, 0 = courtship absent), ornamental display traits (ORM: 1 = traits present, 0 = traits absent), sexual selection index (SSI) and superfetation (SUPER: 1 = superfetation present, 0 = superfetation absent). SPECIES LMNI DICHROM COURT ORM SSI SUPER References Alfaro cultratus ,12,13,60 Alfaro hubberi ,60 Belonesox belizanus ,12,13,65,78 cascajalensis ,13,60 holdridgei ,60 parismina ,13,60 rhabdophora ,63,64,65 roseni ,60,65 terrabensis ,13 Carlhubbsia kidderi ,13,60 Carlhubbsia stuarti ,12,13 Cnesterodon decemmaculatus ,12,13 affinis ,12,13,66,67 atrora ,13 geiseri ,60 holbrooki ,14,23 hurtadoi ,60 manni ,13,60 vittata ,12,13 wrayi ,12 Girardinus metallicus ,60 Heterandria bimaculata ,12,13 Heterandria formosa ,12,13,60 Neoheterandria elegans ,12 Neoheterandria tridentiger ,13,70 Phallichthys amates ,12,13,80 Phallichthys tico ,13,80 Phalloceros caudimaculatus ,12,13,60 Phalloptychus januarius ,9,13,60 (Limia) caymanensis (Limia) dominicensis ,13,35,60,61,68,69 (Limia) heterandria ,35,60 (Limia) melanogaster ,13, 35,60,61,68,69 (Limia) nigrofasciata ,13,60,61,68,69 (Limia) perugiae ,13,60,61,68,69 (Limia) tridens ,13,60,68,69 (Limia) vittata ,13,60,61,68,69 (Limia) zonata ,13,60,68,69 (Micropoecilia) branneri ,60 (Micropoecilia) parae ,12,13,35,60,61 (Micropoecilia) picta ,12,13,14,35,60,61 (Micropoecilia) reticulata ,12,13,14,35,60,61 (Micropoecilia) wingei ,35,60 (Mollienesia) caucana ,35,60,71 (Mollienesia) gilli (Mollienesia) gracilis (Mollienesia) latipinna * ,13,60,71 (Mollienesia) mexicana ,13,60,71 (Mollienesia) orri ,71 1

14 (Mollienesia) petenensis * ,60,71 (Mollienesia) sphenops ,13,14,60,72 (Mollienesia) velifera * 3-60,71 (Mollienesia) vivipara ,60 (Pamphorichthys) hollandi ,13,60 (Pamphorichthys) minor ,35,60 (Pamphorichthys) scalpridens ,35 baenschi ,13,60 elongata ,13,60,73 fasciata ,13,60 gracilis ,12,13,60 infans ,13,60 latidens ,13,60 lucida ,12,13,60 monacha ,12,13,60 occidentalis ,12,13,60,74 paucimaculata ,73 presidionis ,13,60 prolifica ,13,60 retropinna ,13,60,73 scarlii ,13,60 turneri ,13,60 turrubarensis ,13,60,73 viriosa ,13,60 Priapella compressa ,12,60 Priapella intermedia ,13 Priapichthys annectens ,12,13 Priapichthys darienensis ,60 Scolichthys greenwayi ,12,60 Xenodexia ctenolepis ,12,13,60 Xenophallus umbratilis , 60 alvarezi ,13,60,75 clemenciae ,13,60,75 cortezi ,13,60,75 couchianus ,60 gordoni ,13,60 hellerii ,13,60,75 maculatus ,13,60 monticolus ,75,76 multilineatus ,75,77,79 nezahualcoyotl nigrensis ,13,60,62,75 pygmaeus ,13,60,75,76 variatus ,13,60 xiphidium ,75 Type of male ornament: * Enlarged dorsal fin ( sailfin ); filamentous extensions on upper maxilla ( moustache ); extension of ventral part of the caudal fin ( sword ). Additional references: 60 this study; 61 Wischnath (1993) Altas of livebearers of the World, T.F.H. Publications, Inc., USA; 62 Crapon de Caprona & Ryan (1990) Anim. Behav. 39: ; 63 Basolo (2004) Anim. Behav. 68:75-82; 64 Johnson & Belk (2001) Oecologia 126: ; 65 Bussing (1988) Rev. Biol. Trop. 36:81-87; 66 Hughes (1985) Behav. Ecol. Sociobiol. 17: ; 67 Itzkowitz (1971) Chesapeake Science 12: ; 68 Farr (1984) Z. Tierpsychol. 65: ; 69 Hamilton (2001) Mol. Phyl. Evol. 19: ; 70 McPhail (1978) Behav. 64: ; 71 Ptacek (2005) Viviparous fishes, New Life Publications, Florida; 72 Schlupp (2010) Behav. Ecol. Sociobiol. 64: ; 73 BJA Pollux, AI Furness & C Garita-Alvarado, field observations; 74 Constanz (1975) Ecology 56: ; 75 Marcus & McCune (1999) Syst. Biol. 48: ; 76 Franck (1964) Zool. Jb. Physiol. 71: ; 77 Rios-Cardenas et al. (2007) Anim. Behav. 74: ; 78 Horth (2004) Florida Scientist 67: ; 79 Rios-Cardenas et al. (2007) Anim. Behav. 74: ; 80 Regus et al. (2013) J. Fish Biol. 83:

15 Supplementary Table 2 Taxa, natural log-transformed matrotrophy index (LNMI), number of males (NMAL), number of females (NFEM), relative gonopodium length (PROPGL), log 10 transformed male standard length (LG10MSL), log 10 transformed male body weight (LG10MBW), log 10 transformed female standard length (LG10FSL), log 10 transformed female body weight (LG10FBW), size dimorphism index body weight (SDIBW), sexual size dimorphism body weight (SSDBW), size dimorphism index standard length (SDISL) and sexual size dimorphism standard length (SSDSL). SPECIES (POPULATION) LNMI NMAL NFEM PROPGL LG10MSL LG10MBW LG10FSL LG10FBW SDIBW SSDBW SDISL SSDSL Alfaro cultratus Belonesox belizanus cascajalensis episcopi holdridgei parismina rhabdophora roseni terrabensis Carlhubbsia stuarti Cnesterodon decemmaculatus Cnesterodon hypselurus affinis holbrooki manni punctata Girardinus metallicus Heterandria bimaculata Heterandria formosa (DNR1) Heterandria formosa (DNR2) Neoheterandria cana Neoheterandria elegans Neoheterandria tridentiger Phallichthys amates Phallichthys tico Phalloceros caudimaculatus Phalloptychus januarius (Limia) caymanensis (Limia) heterandria (Limia) melanogaster (Limia) nigrofasciata (Limia) sulphurophila (Limia) tridens (Limia) vittata (Micropoecilia) bifurca (Micropoecilia) branneri (Micropoecilia) obscura (Micropoecilia) parae (Micropoecilia) picta (FB) (Micropoecilia) picta (Mausica pool) (Micropoecilia) picta (Rio la Ceiba) (Micropoecilia) reticulata (Micropoecilia) sarrafae (Micropoecilia) wingei (Mollienesia) caucana (Mollienesia) gilli (Mollienesia) gracilis (Mollienesia) latipinna (Mollienesia) mexicana (Mollienesia) salvatoris (Mollienesia) sphenops (Mollienesia) velifera (Mollienesia) vivipara (Pamphorichthys) araguaiensis (Pamphorichthys) hasemani (Pamphorichthys) hollandi (Magu River)

16 (Pamphorichthys) hollandi (Utinga River) (Pamphorichthys) minor baenschi elongata (DNR) elongata (Panam Vieja) fasciata gracilis infans latidens (El Palillo) latidens (Rio Moccorito) lucida monacha occidentalis (Aguajito Canyon) occidentalis (DNR) paucimaculata presidionis (DNR) presidionis (Rio Moccorito) prolifica (Rio Mocorito) prolifica (Rio Presidio) retropinna (DNR) retropinna (Rio Ceibo) scarlii (Rio Tomatlon) scarlii (Rio San Blas) turneri turrubarensis (Rio Abrojo) turrubarensis (Rio la Plata) viriosa Priapella chamulae Priapella compressa (DNR) Priapella compressa (Rio Tacotalpa) Priapella compressa (Rio Usumacinta) Priapichthys annectens Priapichthys darienensis Pseudopoecilia festae Scolichthys greenwayi Scolichthys iota Xenodexia ctenolepis Xenophallus umbratilis alvarezi clemenciae cortezi couchianus hellerii maculatus monticolus multilineatus nezahualcoyotl nigrensis pygmaeus variatus xiphidium

17 Supplementary Table 3 Taxon sampling and Genbank accession numbers. Table **: Taxon sampling and GenBank accession numbers. X = New to this study (KJ KJ697673); = Not present. GenBank taxonomy does not follow the taxonomy of the genus as used in this paper. Specifically, Limia, Micropoecilia, and Pamphorichthys are recognized as full genera in GenBank, whereas we recognize each of these taxa as subgenera of 42. Thus, GenBank nomenclature for (Limia) is Limia; (Micropoecilia) is Micropoecilia; (Pamphorichthys) is Pamphorichthys. Rs Higher Level Species 12S tr Val 16S 5 prime end 16S 3 End tr Leu Anablepidae Anableps anableps Anablepidae Anableps dowei Anablepidae Jenynsia lineata Jenynsia Anablepidae multidentata Anablepidae Oxyzygonectes dovii Aphredoderidae Aphredoderus sayanus NC NC NC NC NC Aplocheilidae Fenerbahce formosus Aplocheilidae Aphyoplatys duboisi Aphyosemion Aplocheilidae bitaeniatum Aplocheilidae Aplocheilus lineatus Aplocheilidae Epiplatys annulatus Aplocheilidae Fundulopanchax Argentiniformes Argentina AY Argentina sialis NC Menidia menidia Atheriniformes Atheriniformes NC Menidia menidia NC Menidia menidia Aulopiformes Batrachoidiformes Synodus Porichthys NC Synodus variegatus NC_ Porichthys myriaster NC Synodus variegatus NC_ Porichthys myriaster NC Synodus variegatus NC_ Porichthys myriaster NC Synodus variegatus NC_ Porichthys myriaster NC Synodus variegatus NC_ Porichthys myriaster Beloniformes Oryzias latipes AP AP AP AP AP AP AP AP AP AP Myripristis Myripristis berndti Myripristis Myripristis Myripristis berndti berndti berndti Beryciformes Beryciformes berndti Characiformes Cichlidae Cichlidae Clupeidae Characiformes Cichlasomatinae NC Pygocentrus nattereri NC Hypselecara temporalis GU Oreochromis niloticus NC Pygocentrus nattereri NC Hypselecara temporalis GU Oreochromis niloticus NC Pygocentrus nattereri NC Hypselecara temporalis GU Oreochromis niloticus NC Pygocentrus nattereri NC Hypselecara temporalis GU Oreochromis niloticus NC Pygocentrus nattereri NC Hypselecara temporalis GU Oreochromis niloticus Oreochromis Dorosoma cepedianum DQ DQ DQ DQ DQ Cypriniformes Danio rerio NC NC NC NC NC Cypriniformes Notemigonus crysoleucas AB AB AB AB AB AF AF AF AF Semotilus Semotilus Semotilus Semotilus Semotilus atromaculatus atromaculatus atromaculatus Cypriniformes atromaculatus atromaculatus Cubanichthys Cyprinodontidae cubensis Cyprinodontidae Cubanichthys pengelleyi 5

18 Cyprinodontidae Cyprinodon variegatus Cyprinodontidae Floridichthys carpio Cyprinodontidae Jordanella floridae Cyprinodontidae Orestias Esociformes Esox lucius NC NC NC NC NC Fundulidae Fundulus cingulatus Fundulidae Fundulus lineolatus Fundulidae Lucania goodei Fundulidae Lucania parva Gadidae Gadus morhua NC NC NC NC NC Gasterosteus Gasterosteiformes aculeatus NC NC NC NC NC Gonorynchiformes Chanos chanos NC NC NC NC NC Allodontichthys hubbsi Allodontichthys polylepis Allodontichthys tamazulae Allodontichthys zonistius Alloophorus robustus Allotoca catarinae Allotoca diazi Allotoca dugesii Allotoca goslinei Allotoca maculata Allotoca meeki Allotoca regalis Allotoca sp. MNCN3676 Allotoca zacapuensis Ameca splendens Ataeniobius toweri Chapalichthys encaustus Chapalichthys pardalis Characodon audax Characodon lateralis Crenichthys baileyi Crenichthys nevadae Empetrichthys latos Girardinichthys multiradiatus Girardinichthys viviparus Goodea atripinnis Goodea gracilis Hubbsina turneri 6

19 Ilyodon amecae Ilyodon furcidens Ilyodon whitei Ilyodon xantusi Neotoca bilineata Profundulus guatemalensis Profundulus labialis Profundulus punctatus Skiffia bilineatus Skiffia francesae Skiffia lermae Skiffia multipunctata Xenoophorus captivus Xenotaenia resolanae Xenotoca eiseni NC NC NC NC NC Xenotoca melanosoma Xenotoca variatus Zoogoneticus quitzeoensis Zoogoneticus tequila AB AB AB Apteronotus Apteronotus albifrons Apteronotus albifrons albifrons Gymnotiformes Lampriformes Lophiiformes Lutjanidae Macrouridae Moronidae Mugiliformes Myctophiformes Ophidiiformes Osmeriformes Pleuronectiformes Gymnotiformes Lampriformes Lophius Lutjanus Macrouridae Morone Mugil Myctophiformes Ophidiiformes Osmeriformes Pleuronectiformes AB Apteronotus albifrons NC Stylephorus chordatus AP Lophius americanus NC Lutjanus sebae AP Bathygadus antrodes NC Morone saxatilis NC Mugil cephalus AP Neoscopelus microchir NC Sirembo imberbis AP Alepocephalus agassizii GQ Cynoglossus abbreviatus NC Stylephorus chordatus AP Lophius americanus NC Lutjanus sebae AP Bathygadus antrodes NC Morone saxatilis NC Mugil cephalus AP Neoscopelus microchir NC Sirembo imberbis AP Alepocephalus agassizii GQ Cynoglossus abbreviatus NC Stylephorus chordatus AP Lophius americanus NC Lutjanus sebae AP Bathygadus antrodes NC Morone saxatilis NC Mugil cephalus AP Neoscopelus microchir NC Sirembo imberbis AP Alepocephalus agassizii GQ Cynoglossus abbreviatus NC Stylephorus chordatus AP Lophius americanus NC Lutjanus sebae AP Bathygadus antrodes NC Morone saxatilis NC Mugil cephalus AP Neoscopelus microchir NC Sirembo imberbis AP Alepocephalus agassizii GQ Cynoglossus abbreviatus AB Apteronotus albifrons NC Stylephorus chordatus AP Lophius americanus NC Lutjanus sebae NC Morone saxatilis NC Mugil cephalus AP Neoscopelus microchir NC Sirembo imberbis AP Alepocephalus agassizii GQ Cynoglossus abbreviatus Alfaro cultratus EF EF EF EF EF Alfaro hubberi 7

20 Aplocheilichthys normani Aplocheilichthys spilauchen Belonesox belizanus EF EF EF cascajalensis hartwegi EF EF EF EF EF holdridgei parismina rhabdophora EF EF EF EF EF roseni terrabensis EF EF EF EF EF Carlhubbsia kidderi Carlhubbsia stuarti EF EF EF EF EF Cnesterodon decemmaculatus EF EF EF EF EF Cnesterodon hypselurus Cnesterodon septentrionalis Dactylophallus denticulatus Dactylophallus ramsdeni Dactylophallus sp. DDEN10 Fluviphylax pygmaeus EF EF EF EF EF Fluviphylax simplex affinis AP AP AP AP EF atrora EF EF caymanensis eurystoma geiseri heterochir hispaniolae holbrooki hubbsi hurtadoi luma manni marshi melapleura nicaraguensis oligosticta panuco punctata puncticulata rhizophorae 8

21 sexradiata sp. LLSTC4571 vittata EF EF EF EF EF wrayi EF EF EF EF EF yucatana zarskei Girardinus creolus EF EF EF EF EF Girardinus metallicus EF EF EF EF EF Girardinus microdactylus Girardinus rivasi Girardinus sp. GMIC19 Glaridichthys falcatus Glaridichthys uninotatus Heterandria bimaculata EF EF EF EF EF Heterandria formosa EF EF EF EF EF Heterandria jonesi EF EF EF EF EF Heterophallus milleri EF EF EF EF EF Heterophallus rachovii Limia caymanensis Limia dominicensis EF EF EF EF EF Limia garnieri Limia grossidens Pseudolimia heterandria Limia melanogaster EF EF EF EF EF Limia melanonotata Limia nigrofasciata Limia pauciradiata Limia perugiae Limia rivasi Limia sulfurophila Limia tridens EF EF EF EF EF Limia versicolor Limia vittata Limia zonata Micropoecilia bifurca Micropoecilia branneri Acanthophacelus obscura Micropoecilia parae (French Guiana) 9

22 Micropoecilia parae (Suriname) Micropoecilia picta Micropoecilia picta (Trinidad and Tobago) Acanthophacelus reticulata Micropoecilia sarrafae Acanthophacelus wingei Acanthophacelus wingei (Venezuela) Neoheterandria cana Neoheterandria elegans EF EF EF Neoheterandria tridentiger EF EF EF EF EF Pamphorichthys araguaiensis Pamphorichthys hasemani Pamphorichthys hollandi Pamphorichthys minor Pamphorichthys scalpridens Phallichthys amates EF EF EF EF EF Phallichthys pittieri Phallichthys quadripunctatus DQ Phallichthys tico EF EF EF EF EF Phalloceros caudimaculatus EF EF EF EF EF Phalloptychus januarius EF EF EF Mollienesia butleri Mollienesia catemaconis Mollienesia caucana EF EF EF EF EF Mollienesia chica Mollienesia gilli Mollienesia gracilis Mollienesia latipinna Mollienesia latipunctata EF EF EF EF EF Mollienesia mexicana limanturi Mollienesia mexicana mexicana Mollienesia orri Mollienesia petenensis 10

23 Campeche Mollienesia petenensis MP523 Mollienesia salvatoris Mollienesia sphenops Mollienesia sulphuraria Mollienesia velifera vivipara vivipara (Trinidad and Tobago) baenschi balsas catemaco elongata fasciata EF EF EF EF EF gracilis hnilickai EF EF EF EF EF infans latidens lucida monacha occidentalis paucimaculata pleurospilus presidionis prolifica retropinna scarlii sonoriensis turneri turrubarensis viriosa Priapella chamulae Priapella compressa EF EF EF Priapella intermedia EF EF EF EF EF Priapella olmecae EF EF EF EF EF Priapichthys annectens EF EF EF EF EF Priapichthys darienensis Priapichthys panamensis DQ Priapichthys puetzi DQ Pseudopoecilia festae EF EF EF EF EF

24 Quintana atrizona EF EF EF EF EF Scolichthys greenwayi EF EF EF EF EF Scolichthys iota Tomeurus gracilis EF EF EF EF EF Xenodexia ctenolepis EF EF EF EF EF Xenophallus umbratilis EF EF EF EF EF alvarezi andersi birchmanni clemenciae continens cortezi couchianus evelynae EF EF EF EF EF gordoni hellerii FJ FJ FJ FJ EF maculatus AP AP AP AP AP malinche mayae meyeri milleri mixei montezumae monticolus multilineatus nezahualcoyotl nigrensis pygmaeus signum variatus xiphidium EF EF EF EF EF NC NC NC NC NC Polymixia Polymixia japonica Polymixia Polymixia Polymixia japonica japonica japonica Polymixiidae Polymixia japonica Pristigasteridae Chirocentrus dorab NC NC NC NC NC Pristigasteridae Pellona NC Pellona flavipinnis NC Pellona flavipinnis NC Pellona flavipinnis NC Pellona flavipinnis NC Pellona flavipinnis Rivulidae Rivulus hartii Oncorhynchus Salmoniformes mykiss NC NC NC NC NC Scorpaeniformes Scorpaeniformes EU Sebastes ruberrimus EU Sebastes ruberrimus EU Sebastes ruberrimus 12

25 Siluriformes Ictalurus punctatus NC NC NC NC NC Stomiiformes Stomiiformes AP Chauliodus sloani AP Chauliodus sloani AP Chauliodus sloani AP Chauliodus sloani AP Chauliodus sloani Synbranchiformes Monopterus albus NC NC NC NC NC Tetraodontidae Takifugu rubripes NC NC NC NC NC Tetraodon Tetraodontidae nigroviridis NC NC NC NC NC Valenciidae Valencia hispanica Zeidae Zeus faber NC NC NC NC NC Zoarcoidea Lycodes NC Lycodes toyamensis NC Lycodes toyamensis NC Lycodes toyamensis NC Lycodes toyamensis NC Lycodes toyamensis Mitochondrial Protein Coding Genes Family Species COI Cytb ND1 ND2 Anablepidae Anableps anableps X EF Anablepidae Anableps dowei X Anablepidae Jenynsia lineata X X Jenynsia Anablepidae multidentata X X Anablepidae Oxyzygonectes dovii EF EF Aphredoderidae Aphredoderus sayanus NC NC NC NC Aplocheilidae Fenerbahce formosus X Aplocheilidae Aphyoplatys duboisi X X Aphyosemion Aplocheilidae bitaeniatum X X Aplocheilidae Aplocheilus lineatus X X Aplocheilidae Epiplatys annulatus X X DQ Fundulopanchax sjostedti Aplocheilidae Fundulopanchax FJ Argentiniformes Argentina Argentina striata NC Atheriniformes Atheriniformes Menidia menidia NC Synodus Aulopiformes Synodus variegatus NC_ Porichthys Batrachoidiformes Porichthys myriaster X Fundulopanchax gardneri FJ Argentina striata NC NC Menidia NC Menidia menidia menidia Menidia menidia NC NC Synodus NC Synodus variegatus Synodus variegatus variegatus NC_ Porichthys myriaster NC_ Porichthys myriaster NC_ Porichthys myriaster Beloniformes Oryzias latipes AP AP AP AP Beryciformes Beryciformes AP Myripristis berndti AP Myripristis berndti AP Myripristis berndti AP Myripristis berndti Characiformes Characiformes NC Pygocentrus nattereri NC Pygocentrus nattereri NC Pygocentrus nattereri NC Pygocentrus nattereri Cichlidae Cichlidae Clupeidae Cichlasomatinae NC Hypselecara temporalis GU Oreochromis niloticus NC Hypselecara temporalis GU Oreochromis niloticus NC Hypselecara temporalis GU Oreochromis niloticus NC Hypselecara temporalis GU Oreochromis niloticus Oreochromis Dorosoma cepedianum DQ DQ DQ DQ Cypriniformes Danio rerio NC NC NC NC Notemigonus Cypriniformes crysoleucas AB AB AB AB

26 Cypriniformes Cyprinodontidae Cyprinodontidae Cyprinodontidae Semotilus atromaculatus JN HQ Cubanichthys cubensis X X Cubanichthys pengelleyi X X Cyprinodon variegatus X AF Cyprinodontidae Floridichthys carpio X X Cyprinodontidae Jordanella floridae AY X AY X Cyprinodontidae Orestias Orestias silustani Orestias agassizii Esociformes Esox lucius NC NC NC NC Fundulidae Fundulus cingulatus X X Fundulidae Fundulus lineolatus X X Fundulidae Lucania goodei X X Fundulidae Lucania parva X Gadidae Gadus morhua NC NC NC NC Gasterosteus Gasterosteiformes aculeatus NC NC NC NC Gonorynchiformes Chanos chanos NC NC NC NC Allodontichthys hubbsi AY AF Allodontichthys polylepis AY AF Allodontichthys tamazulae AY AF Allodontichthys zonistius AY AF Alloophorus robustus AY AF Allotoca catarinae AY AF Allotoca diazi AY AF Allotoca dugesii AY AF Allotoca goslinei AY AF Allotoca maculata AY AF Allotoca meeki AF Allotoca regalis AY AF Allotoca sp. MNCN3676 AF Allotoca zacapuensis AF Ameca splendens AY X X Ataeniobius toweri AY X X Chapalichthys encaustus AY AF Chapalichthys pardalis AY X X Characodon audax AY AF Characodon lateralis AY AF Crenichthys baileyi AY AF Crenichthys nevadae X X Empetrichthys latos AY ELU09108 Girardinichthys multiradiatus AY AF Girardinichthys viviparus AY X X 14

27 Goodea atripinnis AY AF Goodea gracilis X X Hubbsina turneri AY AF Ilyodon amecae AF Ilyodon furcidens AY X X Ilyodon whitei AY AF Ilyodon xantusi AF Neotoca bilineata AF Profundulus guatemalensis AY Profundulus labialis AY Profundulus punctatus AY Skiffia bilineatus AY Skiffia francesae AY AF Skiffia lermae AY AF Skiffia multipunctata AY AF X Xenoophorus captivus AY X X Xenotaenia resolanae AY AF Xenotoca eiseni NC NC NC NC Xenotoca melanosoma AY AF Xenotoca variatus AY AF Zoogoneticus quitzeoensis AY EU X Zoogoneticus tequila AY AF Gymnotiformes Gymnotiformes AB Apteronotus albifrons AB Apteronotus albifrons AB Apteronotus albifrons AB Apteronotus albifrons Lampriformes Lophiiformes Lutjanidae Macrouridae Moronidae Mugiliformes Myctophiformes Ophidiiformes Osmeriformes Pleuronectiformes Lampriformes Lophius Lutjanus Macrouridae Morone Mugil Myctophiformes Ophidiiformes Osmeriformes Pleuronectiformes NC Stylephorus chordatus AP Lophius americanus NC Lutjanus sebae AP Bathygadus antrodes NC Morone saxatilis NC Mugil cephalus AP Neoscopelus microchir NC Sirembo imberbis AP Alepocephalus agassizii GQ Cynoglossus abbreviatus NC Stylephorus chordatus AP Lophius americanus NC Lutjanus sebae AP Bathygadus antrodes NC Morone saxatilis NC Mugil cephalus AP Neoscopelus microchir NC Sirembo imberbis AP Alepocephalus agassizii GQ Cynoglossus abbreviatus NC Stylephorus chordatus AP Lophius americanus NC Lutjanus sebae AP Bathygadus antrodes NC Morone saxatilis NC Mugil cephalus AP Neoscopelus microchir NC Sirembo imberbis AP Alepocephalus agassizii GQ Cynoglossus abbreviatus Alfaro cultratus X EF Alfaro hubberi Aplocheilichthys normani X X Aplocheilichthys spilauchen X X NC Stylephorus chordatus AP Lophius americanus NC Lutjanus sebae AP Bathygadus antrodes NC Morone saxatilis NC Mugil cephalus AP Neoscopelus microchir NC Sirembo imberbis AP Alepocephalus agassizii GQ Cynoglossus abbreviatus 15

28 Belonesox belizanus EU X HM cascajalensis FJ hartwegi FJ EF EF holdridgei BHU68308 parismina FJ rhabdophora EF EF EF roseni BRU68300 terrabensis EF EF EF Carlhubbsia kidderi FJ Carlhubbsia stuarti EF EF EF Cnesterodon decemmaculatus EF EF EF Cnesterodon hypselurus GU GU Cnesterodon septentrionalis X Dactylophallus denticulatus FN FJ Dactylophallus ramsdeni FJ Dactylophallus sp. DDEN10 FJ Fluviphylax pygmaeus EF EF EF Fluviphylax simplex X X affinis AP AP AP AP atrora EF EF EF caymanensis GCU18115 eurystoma GEU18206 geiseri GGU18207 heterochir GHU18208 hispaniolae GHU18209 holbrooki GU X X hubbsi GHU18211 EF hurtadoi GHU18212 luma X X manni GMU18214 marshi GMU18215 melapleura GMU18216 nicaraguensis GNU18217 oligosticta GOU18218 panuco GPU18219 punctata FN GPU18220 puncticulata FN GPU18221 rhizophorae FN GRU18223 sexradiata EU GSU18224 sp. LLSTC4571 HM HM

29 vittata EF EF EF wrayi EF EF EF yucatana EU GYU18226 zarskei GU Girardinus creolus FN EF EF EF Girardinus metallicus FN FJ EF EF Girardinus microdactylus FN FJ Girardinus rivasi FJ Girardinus sp. GMIC19 FJ Glaridichthys falcatus FN FJ Glaridichthys uninotatus FJ Heterandria bimaculata EU EF EF EF Heterandria formosa AF EF EF Heterandria jonesi EF EF EF Heterophallus milleri EF EF EF Heterophallus rachovii HM HM Limia caymanensis X AF Limia dominicensis EF EF EF Limia garnieri X Limia grossidens X Pseudolimia heterandria HQ HQ Limia melanogaster EF EF EF Limia melanonotata X AF Limia nigrofasciata X AF Limia pauciradiata X AF Limia perugiae X AF Limia rivasi X Limia sulfurophila X Limia tridens EF EF EF Limia versicolor X AF Limia vittata FN X AF Limia zonata X AF Micropoecilia bifurca GU GU Micropoecilia branneri GU GU Acanthophacelus obscura GQ Micropoecilia parae (French Guiana) GU GU Micropoecilia parae (Suriname) GU GU

30 Micropoecilia picta GU GU Micropoecilia picta (Trinidad and Tobago) GU GU Acanthophacelus reticulata GU GU Micropoecilia sarrafae X X Acanthophacelus wingei GU GU Acanthophacelus wingei (Venezuela) GU GU Neoheterandria cana X X Neoheterandria elegans X X Neoheterandria tridentiger EF EF EF Pamphorichthys araguaiensis GU GU Pamphorichthys hasemani HQ HQ Pamphorichthys hollandi HM HQ HQ Pamphorichthys minor GU GU Pamphorichthys scalpridens HQ HQ Phallichthys amates X EF X Phallichthys pittieri FJ Phallichthys quadripunctatus X X Phallichthys tico EF EF EF Phalloceros caudimaculatus EF EF Phalloptychus januarius EF Mollienesia butleri X X Mollienesia catemaconis AF Mollienesia caucana EF EF EF Mollienesia chica X X Mollienesia gilli X AF Mollienesia gracilis X X Mollienesia latipinna X AF Mollienesia latipunctata EF EF EF Mollienesia mexicana limanturi X X Mollienesia mexicana mexicana X X Mollienesia orri X AF Mollienesia petenensis Campeche X X Mollienesia petenensis MP523 X X 18

31 Mollienesia salvatoris X X Mollienesia sphenops X AF Mollienesia sulphuraria AF Mollienesia velifera X X vivipara HQ HQ vivipara (Trinidad and Tobago) HQ HQ baenschi AF AF balsas X X catemaco EU AF AF elongata AF AF fasciata AF EF AF gracilis AF AF hnilickai AF EF AF infans AF AF latidens AF AF lucida AF AF monacha AF AF occidentalis AF AF paucimaculata AF AF pleurospilus EU presidionis AF AF prolifica AF AF retropinna AF X scarlii AF AF sonoriensis DQ DQ turneri AF AF turrubarensis AF AF viriosa AF AF Priapella chamulae X X Priapella compressa X EF X Priapella intermedia AY EF EF EF Priapella olmecae EF EF X Priapichthys annectens EF EF EF Priapichthys darienensis X X Priapichthys panamensis X Priapichthys puetzi DQ Pseudopoecilia festae EF EF EF Quintana atrizona FN EF EF EF Scolichthys greenwayi EF EF EF

32 Scolichthys iota X X Tomeurus gracilis EF EF Xenodexia ctenolepis EF EF EF Xenophallus umbratilis FJ EF alvarezi EU X X andersi XAU06495 birchmanni X X clemenciae X continens X X cortezi X X couchianus X X evelynae EF EF EF gordoni X X hellerii FJ X FJ X maculatus AP AP AP AP malinche XMU06516 mayae AF meyeri X X milleri XMU06518 mixei montezumae X X monticolus X X multilineatus X X nezahualcoyotl X X nigrensis X X pygmaeus X X signum X X variatus X X xiphidium X EF X Polymixiidae Polymixia NC Polymixia japonica NC Polymixia japonica NC Polymixia japonica NC Polymixia japonica Pristigasteridae Chirocentrus dorab NC NC NC NC NC Pellona NC Pellona NC Pellona NC Pellona Pristigasteridae Pellona flavipinnis flavipinnis flavipinnis flavipinnis Rivulidae Rivulus hartii X AF Salmoniformes Oncorhynchus mykiss NC NC NC NC Scorpaeniformes Scorpaeniformes EU Sebastes ruberrimus EU Sebastes ruberrimus EU Sebastes ruberrimus Siluriformes Ictalurus punctatus NC NC NC NC AP AP AP AP Stomiiformes Stomiiformes Chauliodus sloani Chauliodus sloani Chauliodus sloani Chauliodus sloani Synbranchiformes Monopterus albus NC NC NC NC Tetraodontidae Takifugu rubripes NC NC NC NC

33 Tetraodontidae Tetraodon nigroviridis NC NC NC NC Valenciidae Valencia hispanica Zeidae Zeus faber NC NC NC NC Zoarcoidea Lycodes NC Lycodes toyamensis NC Lycodes toyamensis NC Lycodes toyamensis NC Lycodes toyamensis Nuclear Genes Family Species CCND1 D2 D29 D8 ENC1 Glyt Anablepidae Anableps anableps X X Anablepidae Anableps dowei X X Anablepidae Jenynsia lineata X X Anablepidae Jenynsia multidentata X X Anablepidae Oxyzygonectes dovii X X Aphredoderidae Aphredoderus sayanus EU EU Aplocheilidae Fenerbahce formosus X X Aplocheilidae Aphyoplatys duboisi X X Aphyosemion Aplocheilidae bitaeniatum X Aplocheilidae Aplocheilus lineatus X X Aplocheilidae Epiplatys annulatus X X Aplocheilidae Fundulopanchax X Fundulopanchax gardneri X Fundulopanchax gardneri Argentiniformes Argentina EU Argentina sialis EU Argentina sialis Atheriniformes Atheriniformes Aulopiformes Synodus EU Synodus foetens EU Synodus foetens Batrachoidiformes Porichthys EU Porichthys plectrodon Beloniformes Oryzias latipes EF EF Beryciformes Beryciformes EU Myripristis violacea EU Myripristis violacea Characiformes Characiformes Cichlidae Cichlasomatinae EU Herichthys cyanoguttatus EU Herichthys cyanoguttatus Cichlidae Oreochromis Clupeidae EF Oreochromis niloticus EF Oreochromis niloticus Dorosoma cepedianum EU Cypriniformes Danio rerio EF EF Cypriniformes Notemigonus crysoleucas EU EU Cypriniformes Semotilus atromaculatus EF EF Cyprinodontidae Cubanichthys cubensis X X Cyprinodontidae Cubanichthys pengelleyi X X Cyprinodontidae Cyprinodon variegatus X X Cyprinodontidae Floridichthys carpio X X Cyprinodontidae Jordanella floridae X X 21

34 Cyprinodontidae Orestias X Orestius agassizzi X Orestias agassizii Esociformes Esox lucius EU EU Fundulidae Fundulus cingulatus X X Fundulidae Fundulus lineolatus X X Fundulidae Lucania goodei X X Fundulidae Lucania parva X X Gadidae Gadus morhua EU EU Gasterosteus Gasterosteiformes aculeatus Ensembl 63 AB Gonorynchiformes Chanos chanos EU Allodontichthys hubbsi Allodontichthys polylepis Allodontichthys tamazulae Allodontichthys zonistius Alloophorus robustus Allotoca catarinae Allotoca diazi Allotoca dugesii Allotoca goslinei Allotoca maculata Allotoca meeki Allotoca regalis Allotoca sp. MNCN3676 Allotoca zacapuensis Ameca splendens X X Ataeniobius toweri X X Chapalichthys encaustus Chapalichthys pardalis X X Characodon audax Characodon lateralis Crenichthys baileyi Crenichthys nevadae X X Empetrichthys latos Girardinichthys multiradiatus Girardinichthys viviparus X X Goodea atripinnis Goodea gracilis X X Hubbsina turneri Ilyodon amecae Ilyodon furcidens X X Ilyodon whitei 22

35 Ilyodon xantusi Neotoca bilineata Profundulus guatemalensis Profundulus labialis X X Profundulus punctatus Skiffia bilineatus Skiffia francesae Skiffia lermae Skiffia multipunctata X X Xenoophorus captivus X X Xenotaenia resolanae Xenotoca eiseni X X Xenotoca melanosoma Xenotoca variatus Zoogoneticus quitzeoensis X X Zoogoneticus tequila Gymnotiformes Gymnotiformes AB Gymnotus carapo Lampriformes Lampriformes EU Metavelifer multiradiatus EU Regalecus glesne Lophiiformes Lophius EU Lophius gastrophysus Lutjanidae Lutjanus Macrouridae Macrouridae Moronidae Morone Mugiliformes Mugil EF Lutjanus mahogoni EU Coryphaenoides rupestris EF Morone chrysops EU Mugil curema EF Lutjanus mahogoni EU Coryphaenoides rupestris EF Morone chrysops EU Mugil curema Myctophiformes Myctophiformes EF Brotula EF Brotula Ophidiiformes Ophidiiformes multibarbata multibarbata Osmeriformes Osmeriformes Pleuronectiformes Pleuronectiformes EU Pleuronectes platessa Alfaro cultratus X X Alfaro hubberi Aplocheilichthys normani X X Aplocheilichthys spilauchen X X Belonesox belizanus X X cascajalensis hartwegi holdridgei parismina rhabdophora X X 23

36 roseni terrabensis Carlhubbsia kidderi Carlhubbsia stuarti X X Cnesterodon decemmaculatus GU GU Cnesterodon hypselurus GU GU Cnesterodon septentrionalis X X Dactylophallus denticulatus Dactylophallus ramsdeni Dactylophallus sp. DDEN10 Fluviphylax pygmaeus Fluviphylax simplex X X affinis EU EU atrora caymanensis eurystoma geiseri heterochir hispaniolae holbrooki X X hubbsi hurtadoi luma X X manni marshi melapleura nicaraguensis oligosticta panuco punctata puncticulata rhizophorae sexradiata sp. LLSTC4571 vittata wrayi yucatana zarskei Girardinus creolus Girardinus metallicus X X 24

37 Girardinus microdactylus Girardinus rivasi Girardinus sp. GMIC19 Glaridichthys falcatus Glaridichthys uninotatus Heterandria bimaculata Heterandria formosa X X Heterandria jonesi Heterophallus milleri Heterophallus rachovii Limia caymanensis X X Limia dominicensis GU GU Limia garnieri X X Limia grossidens X X Pseudolimia heterandria HQ HQ Limia melanogaster GU GU Limia melanonotata X X Limia nigrofasciata X X Limia pauciradiata X X Limia perugiae X X Limia rivasi X X Limia sulfurophila X X Limia tridens X X Limia versicolor X X Limia vittata X X Limia zonata X X Micropoecilia bifurca GU GU Micropoecilia branneri GU GU Acanthophacelus obscura Micropoecilia parae (French Guiana) GU GU Micropoecilia parae (Suriname) GU GU Micropoecilia picta GU GU Micropoecilia picta (Trinidad and Tobago) GU GU Acanthophacelus reticulata GU GU

38 Micropoecilia sarrafae X X Acanthophacelus wingei GU GU Acanthophacelus wingei (Venezuela) GU GU Neoheterandria cana X X Neoheterandria elegans X X Neoheterandria tridentiger X X Pamphorichthys araguaiensis GU GU Pamphorichthys hasemani HQ HQ Pamphorichthys hollandi HQ HQ Pamphorichthys minor GU GU Pamphorichthys scalpridens HQ HQ Phallichthys amates X X Phallichthys pittieri Phallichthys quadripunctatus X X Phallichthys tico X X Phalloceros caudimaculatus X X Phalloptychus januarius X X Mollienesia butleri X X Mollienesia catemaconis Mollienesia caucana GU GU Mollienesia chica X X Mollienesia gilli X X Mollienesia gracilis X X Mollienesia latipinna X X Mollienesia latipunctata GU GU Mollienesia mexicana limanturi X X Mollienesia mexicana mexicana X X Mollienesia orri X X Mollienesia petenensis Campeche X X Mollienesia petenensis MP523 X X Mollienesia salvatoris X X Mollienesia sphenops X X Mollienesia sulphuraria Mollienesia velifera X X 26

39 vivipara HQ HQ vivipara (Trinidad and Tobago) HQ HQ baenschi X X balsas X X catemaco X X elongata X X fasciata X X gracilis X X hnilickai infans X X latidens X X lucida X X monacha X X occidentalis X X paucimaculata X X pleurospilus presidionis X X prolifica X X retropinna X X scarlii X X sonoriensis turneri X X turrubarensis X X viriosa X X KJ KJ Priapella chamulae Priapella compressa DQ DQ DQ KJ KJ Priapella intermedia AY Priapella olmecae X X Priapichthys annectens X X Priapichthys darienensis X X Priapichthys panamensis X X Priapichthys puetzi Pseudopoecilia festae X X Quintana atrizona Scolichthys greenwayi Scolichthys iota X X Tomeurus gracilis X X Xenodexia ctenolepis X X Xenophallus umbratilis X X 27

40 alvarezi AY DQ DQ DQ KJ KJ andersi AY DQ DQ DQ KJ KJ birchmanni DQ DQ DQ KJ KJ clemenciae AY DQ DQ DQ KJ KJ continens DQ DQ DQ KJ KJ cortezi DQ DQ DQ KJ KJ couchianus AY DQ DQ DQ evelynae DQ DQ DQ KJ KJ gordoni DQ DQ DQ KJ KJ hellerii AY DQ DQ DQ maculatus AY DQ DQ malinche DQ DQ DQ mayae meyeri DQ DQ DQ KJ KJ milleri DQ DQ DQ mixei AY KJ KJ montezumae AY DQ DQ DQ KJ KJ monticolus AY KJ KJ multilineatus DQ DQ DQ KJ KJ nezahualcoyotl DQ DQ DQ KJ KJ nigrensis DQ DQ DQ KJ KJ pygmaeus DQ DQ DQ KJ KJ signum AY DQ DQ DQ KJ KJ variatus DQ DQ DQ KJ KJ xiphidium DQ DQ DQ Polymixiidae Polymixia EU Polymixia japonica EU Polymixia japonica Pristigasteridae Chirocentrus dorab EU EU EU EU Pristigasteridae Pellona Pellona flavipinnis Pellona flavipinnis Rivulidae Rivulus hartii X X Salmoniformes Oncorhynchus mykiss EF EF Scorpaeniformes Scorpaeniformes EU Sebastes ruberrimus EU Sebastes ruberrimus Siluriformes Ictalurus punctatus EF EF EU EU Stomiiformes Stomiiformes Stomias boa Stomias boa Synbranchiformes Monopterus albus EU EU Tetraodontidae Takifugu rubripes Ensembl 63 NM Tetraodon Tetraodontidae nigroviridis Ensembl 63 AJ Valenciidae Valencia hispanica X X Zeidae Zeus faber EU EU

41 Zoarcoidea Lycodes EF Lycodes terraenovae EF Lycodes terraenovae Family Species myh6 RH Rag1 SH3PX3 x-src Anablepidae Anableps anableps X X X X X Anablepidae Anableps dowei X X X X X Anablepidae Jenynsia lineata X X X X X Jenynsia Anablepidae multidentata X X X X X Anablepidae Oxyzygonectes dovii X X X X X Aphredoderidae Aphredoderus sayanus EU DQ FJ EU Aplocheilidae Fenerbahce formosus X X X X X Aplocheilidae Aphyoplatys duboisi X X X X X Aphyosemion Aplocheilidae bitaeniatum X X X Aplocheilidae Aplocheilus lineatus X X X X Aplocheilidae Epiplatys annulatus X X X X Aplocheilidae Argentiniformes Atheriniformes Aulopiformes Batrachoidiforme s Fundulopanchax Argentina Atheriniformes Synodus Porichthys X Fundulopanchax gardneri EU Argentina sialis EU Labidesthes sicculus X Fundulopanchax gardneri EU Argentina silus FJ Melanotaenia X Fundulopanchax gardneri FJ896426; Argentina sialis X Fundulopanchax gardneri EU Argentina sialis australis AY AY DQ Synodus EU Synodus foetens intermedius Synodus foetens EU Synodus foetens EU Porichthys plectrodon X Fundulopan chax gardneri Beloniformes Oryzias latipes EF Ensembl 63 EF EF Ensembl 63 Beryciformes Beryciformes EU Myripristis violacea MBU57538 Myripristis berndti EU Sargocentron cornutum EU Myripristis violacea Characiformes Cichlidae Cichlidae Clupeidae Characiformes EU Pygocentrus nattereri EU Cichlasoma Herichthys cyanoguttatus EF Oreochromis niloticus AMU12328; Astyanax mexicanus EU Chalceus macrolepidotus HQ Herichthys cyanoguttatus DQ Oreochromis tanganicae EU Herichthys cyanoguttatus EF Oreochromis niloticus Cichlasomatinae GU AY Oreochromis Oreochromis niloticus Dorosoma cepedianum EU DQ Cypriniformes Danio rerio EF NM NM EF Cypriniformes Notemigonus crysoleucas EU FJ EF Cypriniformes Semotilus atromaculatus EF EU GU JF Cyprinodontidae Cubanichthys cubensis X X X X Cyprinodontidae Cubanichthys pengelleyi X X X U02347 Cyprinodontidae Cyprinodon variegatus X X X X Cyprinodontidae Floridichthys carpio X X X X Cyprinodontidae Jordanella floridae X X X X Cyprinodontidae Orestias X Orestias agassizii X Orestias agassizii X Orestias agassizii X Orestias agassizii 29

42 Esociformes Esox lucius EU AY AY EU Fundulidae Fundulus cingulatus X X X X X Fundulidae Fundulus lineolatus X X X X X Fundulidae Lucania goodei X X X X X Fundulidae Lucania parva X X X X Gadidae Gadus morhua EU AF FJ EU Gasterosteiforme s Gasterosteus aculeatus AB EU Ensembl 63 AB Ensembl 63 Gonorynchiforme s Chanos chanos EU AY Allodontichthys hubbsi Allodontichthys polylepis Allodontichthys tamazulae Allodontichthys zonistius Alloophorus robustus Allotoca catarinae Allotoca diazi Allotoca dugesii Allotoca goslinei Allotoca maculata Allotoca meeki Allotoca regalis Allotoca sp. MNCN3676 Allotoca zacapuensis Ameca splendens X X X X X Ataeniobius toweri X X X X X Chapalichthys encaustus Chapalichthys pardalis X X X X X Characodon audax Characodon lateralis Crenichthys baileyi FJ Crenichthys nevadae X X X X X Empetrichthys latos Girardinichthys multiradiatus Girardinichthys viviparus X X X X X Goodea atripinnis Goodea gracilis X X X X X Hubbsina turneri Ilyodon amecae Ilyodon furcidens X X X X X Ilyodon whitei Ilyodon xantusi 30

43 Neotoca bilineata Profundulus guatemalensis Profundulus labialis X X X X Profundulus punctatus Skiffia bilineatus Skiffia francesae Skiffia lermae Skiffia multipunctata X X X X X Xenoophorus captivus X X X X X Xenotaenia resolanae Xenotoca eiseni X X X X X Xenotoca melanosoma Xenotoca variatus Zoogoneticus quitzeoensis X X X X X Zoogoneticus tequila Gymnotiformes Gymnotiformes FJ Apteronotus albifrons FJ Apteronotus albifrons EU Apteronotus albifrons Lampriformes Lampriformes EU Regalecus glesne AY Regalecus glesne EF Regalecus glesne EU Regalecus glesne Lophiiformes Lutjanidae Macrouridae Moronidae Mugiliformes Myctophiformes Ophidiiformes Osmeriformes Pleuronectiforme s Lophius Lutjanus Macrouridae Morone Mugil Myctophiformes Ophidiiformes Osmeriformes Pleuronectiformes EU Lophius gastrophysus EF Lutjanus mahogoni EU Coryphaenoides rupestris EF Morone chrysops EU Mugil curema EU Neoscopelus macrolepidotus EF Brotula multibarbata EU Thaleichthys pacificus EU Pleuronectes platessa EF Lophius budegassa EF Lutjanus analis AY Coryphaenoides rupestris EU Morone saxatilis EF Mugil cephalus EU Stenobrachius leucopsarus EF Brotula barbata EU Alepocephalus antipodianus EU Pleuronectes platessa EF Lophius budegassa EU Lutjanus mahogoni FJ Coryphaenoides rupestris GU Morone chrysops AY Mugil curema EU Neoscopelus macrolepidotus AY Petrotyx sanguineus AY Thaleichthys pacificus AF Pseudopleuronect es americanus EU Lophius gastrophysus EF Lutjanus mahogoni EU Coryphaenoides rupestris EF Morone chrysops EU Mugil curema EU Neoscopelus macrolepidotus EF Brotula multibarbata EU Thaleichthys pacificus EU Pleuronectes platessa Alfaro cultratus X X EF X X Alfaro hubberi AHU06589 Aplocheilichthys normani X X X X X Aplocheilichthys spilauchen X X X X X Belonesox belizanus X X EF X X cascajalensis hartwegi EF holdridgei parismina X X EF X X 31

44 rhabdophora roseni terrabensis EF Carlhubbsia kidderi FJ Carlhubbsia stuarti X X EF X X Cnesterodon decemmaculatus GU GU EF GU GU Cnesterodon hypselurus GU GU GU GU GU Cnesterodon septentrionalis X X X X X Dactylophallus denticulatus FJ Dactylophallus ramsdeni Dactylophallus sp. DDEN10 FJ185 Fluviphylax pygmaeus EF U02350 Fluviphylax simplex X X X X X affinis EU EF EU atrora EF caymanensis eurystoma geiseri heterochir hispaniolae holbrooki X X X X X hubbsi hurtadoi luma X X X X X manni marshi melapleura nicaraguensis oligosticta panuco punctata puncticulata rhizophorae sexradiata sp. LLSTC4571 vittata EF wrayi EF yucatana zarskei Girardinus creolus EF

45 Girardinus metallicus X X EF X X Girardinus microdactylus FJ Girardinus rivasi FJ Girardinus sp. GMIC19 FJ Glaridichthys falcatus FJ Glaridichthys uninotatus FJ Heterandria bimaculata EF Heterandria formosa X X EF X X Heterandria jonesi EF Heterophallus milleri EF Heterophallus rachovii Limia caymanensis X X X X X Limia dominicensis GU GU EF GU GU Limia garnieri X X X X X Limia grossidens X X X X X Pseudolimia heterandria HQ HQ HQ HQ HQ Limia melanogaster GU GU EF GU GU Limia melanonotata X X X X X Limia nigrofasciata X X X X X Limia pauciradiata X X X X X Limia perugiae X X X X X Limia rivasi X X X X X Limia sulfurophila X X X X X Limia tridens X X X X X Limia versicolor X X X X X Limia vittata X X X X X Limia zonata X X X X X Micropoecilia bifurca GU GU GU GU GU Micropoecilia branneri GU GU GU GU GU Acanthophacelus obscura Micropoecilia parae (French Guiana) GU GU GU GU GU Micropoecilia parae (Suriname) GU GU GU GU GU Micropoecilia picta GU GU GU GU GU Micropoecilia picta (Trinidad and Tobago) GU GU GU GU GU Acanthophacelus reticulata GU GU EF GU GU Micropoecilia X 33

46 sarrafae X X X X Acanthophacelus wingei GU GU GU GU GU Acanthophacelus wingei (Venezuela) GU GU GU GU GU Neoheterandria cana X X X X X Neoheterandria elegans X X EF X X Neoheterandria tridentiger X X EF X X Pamphorichthys araguaiensis GU GU GU GU GU Pamphorichthys hasemani HQ HQ HQ HQ HQ Pamphorichthys hollandi HQ HQ HQ HQ HQ Pamphorichthys minor GU GU GU GU GU Pamphorichthys scalpridens HQ HQ HQ HQ HQ Phallichthys amates X X X X X Phallichthys pittieri Phallichthys quadripunctatus X X X X X Phallichthys tico X X EF X X Phalloceros caudimaculatus X X EF X X Phalloptychus januarius X X EF X X Mollienesia butleri X X X X X Mollienesia catemaconis Mollienesia caucana GU GU EF GU U02355 Mollienesia chica X X X X X Mollienesia gilli X X X X X Mollienesia gracilis X X X X X Mollienesia latipinna X X X X X Mollienesia latipunctata GU GU EF GU GU Mollienesia mexicana limanturi X X X X X Mollienesia mexicana mexicana X X X X X Mollienesia orri X X X X X Mollienesia petenensis Campeche X X X X X Mollienesia petenensis MP523 X X X X X Mollienesia salvatoris X X X X X Mollienesia sphenops X X X X X Mollienesia sulphuraria Mollienesia velifera X X X X X vivipara HQ HQ HQ HQ HQ

47 vivipara (Trinidad and Tobago) HQ HQ HQ HQ HQ baenschi X X X X X balsas X X X X X catemaco X X X X X elongata X X X X X fasciata X X EF X X gracilis X X X X X hnilickai EF infans X X X X X latidens X X X X X lucida X X X X X monacha X X X X X occidentalis X X X X X paucimaculata X X X X X pleurospilus presidionis X X X X X prolifica X X X X X retropinna X X X X X scarlii X X X X X sonoriensis turneri X X X X X turrubarensis X X X X X viriosa X X X X X KJ KJ KJ KJ KJ Priapella chamulae KJ KJ KJ KJ KJ Priapella compressa Priapella intermedia EF X Priapella olmecae X X EF X X Priapichthys annectens X X EF X X Priapichthys darienensis X X X X X Priapichthys panamensis X X X X X Priapichthys puetzi Pseudopoecilia festae X X EF X X Quintana atrizona EF Scolichthys greenwayi EF Scolichthys iota X X X X X Tomeurus gracilis X X EF X X Xenodexia ctenolepis X X EF X Xenophallus umbratilis X X X X X alvarezi KJ KJ KJ KJ KJ

48 andersi DQ KJ KJ KJ KJ KJ birchmanni KJ KJ KJ KJ KJ clemenciae KJ KJ KJ KJ KJ continens KJ KJ KJ KJ KJ cortezi KJ KJ KJ KJ KJ couchianus evelynae DQ KJ KJ KJ KJ gordoni KJ KJ KJ KJ KJ hellerii KJ maculatus DQ malinche DQ XMU06614 mayae meyeri KJ KJ KJ KJ KJ milleri DQ XMU06616 mixei KJ KJ KJ KJ KJ montezumae KJ KJ KJ KJ KJ monticolus KJ KJ KJ KJ KJ multilineatus KJ KJ KJ KJ KJ nezahualcoyotl KJ KJ KJ KJ KJ nigrensis KJ KJ KJ KJ KJ pygmaeus KJ KJ KJ KJ KJ signum KJ KJ KJ KJ KJ variatus KJ KJ KJ KJ KJ xiphidium Polymixiidae Polymixia EU Polymixia japonica AY Polymixia nobilis AY Polymixia japonica EU Polymixia japonica Pristigasteridae Chirocentrus dorab EU DQ EU Pellona AY Pellona Pristigasteridae Pellona flavipinnis flavipinnis Rivulidae Rivulus hartii X X X U02357 Salmoniformes Oncorhynchus mykiss EF NM NM EF Scorpaeniformes Scorpaeniformes EU Sebastes ruberrimus EF Sebastes rubrivinctus EU Pterois lunulata EU Sebastes ruberrimus Siluriformes Ictalurus punctatus EF AF DQ EF Stomiiformes Stomiiformes EU Stomias boa GQ Stomias atriventer EU Stomias boa Synbranchiforme s Monopterus albus EU AY EU Tetraodontidae Takifugu rubripes Ensembl 63 Ensembl 63 AY Ensembl 63 Tetraodon Tetraodontidae nigroviridis Ensembl 63 Ensembl 63 Ensembl 63 Ensembl 63 Valenciidae Valencia hispanica X X X X Zeidae Zeus faber EU EU FJ EU Zoarcoidea Lycodes EF Lycodes terraenovae EU Lycodes brevipes EF Lycodes terraenovae 36

49 Family Species Ptr RAB27 RYR3 T36 beta_actin Anablepidae Anableps anableps Anablepidae Anableps dowei Anablepidae Jenynsia lineata Anablepidae Jenynsia multidentata Anablepidae Oxyzygonectes dovii Aphredoderidae Aphredoderus sayanus EU EU Aplocheilidae Fenerbahce formosus Aplocheilidae Aphyoplatys duboisi Aplocheilidae Aphyosemion bitaeniatum Aplocheilidae Aplocheilus lineatus Aplocheilidae Epiplatys annulatus Aplocheilidae Fundulopanchax Argentiniformes Argentina EU Argentina sialis EU Argentina sialis Atheriniformes Atheriniformes EU Labidesthes sicculus EU Labidesthes sicculus Aulopiformes Synodus EU Synodus foetens EU Synodus foetens Batrachoidiformes Porichthys EU Porichthys plectrodon Beloniformes Oryzias latipes EF EF Beryciformes Beryciformes EU Myripristis violacea EU Myripristis violacea Characiformes Characiformes EU Pygocentrus nattereri EU Pygocentrus nattereri Cichlidae Cichlasomatinae HQ Cichlasoma Herichthys cyanoguttatus Cichlidae Clupeidae EF Oreochromis niloticus EF Oreochromis niloticus Oreochromis Dorosoma cepedianum EU Cypriniformes Danio rerio EF EF Cypriniformes Notemigonus crysoleucas EU EU Cypriniformes Semotilus atromaculatus EF EF Cyprinodontidae Cubanichthys cubensis Cyprinodontidae Cubanichthys pengelleyi Cyprinodontidae Cyprinodon variegatus Cyprinodontidae Floridichthys carpio Cyprinodontidae Jordanella floridae Cyprinodontidae Orestias Esociformes Esox lucius EU EU Fundulus Fundulidae cingulatus Fundulidae Fundulus lineolatus Fundulidae Lucania goodei 37

50 Fundulidae Lucania parva Gadidae Gadus morhua EU Gasterosteus Gasterosteiformes aculeatus EF EF Gonorynchiformes Chanos chanos EU Allodontichthys hubbsi Allodontichthys polylepis Allodontichthys tamazulae Allodontichthys zonistius Alloophorus robustus Allotoca catarinae Allotoca diazi Allotoca dugesii Allotoca goslinei Allotoca maculata Allotoca meeki Allotoca regalis Allotoca sp. MNCN3676 Allotoca zacapuensis Ameca splendens Ataeniobius toweri Chapalichthys encaustus Chapalichthys pardalis Characodon audax Characodon lateralis Crenichthys baileyi Crenichthys nevadae Empetrichthys latos Girardinichthys multiradiatus Girardinichthys viviparus Goodea atripinnis Goodea gracilis Hubbsina turneri Ilyodon amecae Ilyodon furcidens Ilyodon whitei Ilyodon xantusi Neotoca bilineata Profundulus guatemalensis Profundulus labialis Profundulus punctatus 38

51 Skiffia bilineatus Skiffia francesae Skiffia lermae Skiffia multipunctata Xenoophorus captivus Xenotaenia resolanae Xenotoca eiseni Xenotoca melanosoma Xenotoca variatus Zoogoneticus quitzeoensis Zoogoneticus tequila Gymnotiformes Gymnotiformes EU Apteronotus albifrons Lampriformes Lampriformes EU Regalecus glesne Lophiiformes Lophius EU Lophius gastrophysus Lutjanidae Lutjanus EF Lutjanus mahogoni EF Lutjanus mahogoni Macrouridae Macrouridae EU Coryphaenoides rupestris Moronidae Morone EF Morone chrysops EF Morone chrysops Mugiliformes Mugil EU Mugil curema EU Mugil curema Myctophiformes Myctophiformes EU Neoscopelus macrolepidotus Ophidiiformes Ophidiiformes EF Brotula multibarbata EF Brotula multibarbata Osmeriformes Osmeriformes EU Thaleichthys pacificus Pleuronectiformes Pleuronectiformes HM Pleuronectes platessa EU Pleuronectes platessa Alfaro cultratus Alfaro hubberi Aplocheilichthys normani Aplocheilichthys spilauchen Belonesox belizanus cascajalensis hartwegi FJ holdridgei parismina rhabdophora roseni terrabensis Carlhubbsia kidderi Carlhubbsia stuarti 39

52 Cnesterodon decemmaculatus Cnesterodon hypselurus Cnesterodon septentrionalis Dactylophallus denticulatus FJ Dactylophallus ramsdeni FJ Dactylophallus sp. DDEN10 FJ Fluviphylax pygmaeus Fluviphylax simplex affinis EU atrora caymanensis eurystoma geiseri heterochir hispaniolae holbrooki hubbsi hurtadoi luma manni marshi melapleura nicaraguensis oligosticta panuco punctata puncticulata rhizophorae sexradiata sp. LLSTC4571 vittata wrayi yucatana zarskei Girardinus creolus FJ Girardinus metallicus FJ Girardinus microdactylus FJ Girardinus rivasi FJ Girardinus sp. GMIC19 FJ Glaridichthys falcatus FJ

53 Glaridichthys uninotatus FJ Heterandria bimaculata Heterandria formosa Heterandria jonesi Heterophallus milleri Heterophallus rachovii Limia caymanensis Limia dominicensis Limia garnieri Limia grossidens Pseudolimia heterandria Limia melanogaster Limia melanonotata Limia nigrofasciata Limia pauciradiata Limia perugiae Limia rivasi Limia sulfurophila Limia tridens Limia versicolor Limia vittata Limia zonata Micropoecilia bifurca Micropoecilia branneri Acanthophacelus obscura Micropoecilia parae (French Guiana) Micropoecilia parae (Suriname) Micropoecilia picta Micropoecilia picta (Trinidad and Tobago) Acanthophacelus reticulata Micropoecilia sarrafae Acanthophacelus wingei Acanthophacelus wingei (Venezuela) 41

54 Neoheterandria cana Neoheterandria elegans Neoheterandria tridentiger Pamphorichthys araguaiensis Pamphorichthys hasemani Pamphorichthys hollandi Pamphorichthys minor Pamphorichthys scalpridens Phallichthys amates Phallichthys pittieri FJ Phallichthys quadripunctatus Phallichthys tico Phalloceros caudimaculatus Phalloptychus januarius Mollienesia butleri Mollienesia catemaconis Mollienesia caucana Mollienesia chica Mollienesia gilli Mollienesia gracilis Mollienesia latipinna Mollienesia latipunctata Mollienesia mexicana limanturi Mollienesia mexicana mexicana Mollienesia orri Mollienesia petenensis Campeche Mollienesia petenensis MP523 Mollienesia salvatoris Mollienesia sphenops Mollienesia sulphuraria 42

55 Mollienesia velifera vivipara vivipara (Trinidad and Tobago) baenschi balsas catemaco elongata fasciata gracilis hnilickai infans latidens lucida monacha occidentalis paucimaculata pleurospilus presidionis prolifica retropinna scarlii sonoriensis turneri turrubarensis viriosa Priapella chamulae Priapella compressa DQ Priapella intermedia AY Priapella olmecae Priapichthys annectens Priapichthys darienensis Priapichthys panamensis Priapichthys puetzi Pseudopoecilia festae Quintana atrizona FJ Scolichthys greenwayi Scolichthys iota Tomeurus gracilis Xenodexia ctenolepis 43

56 Xenophallus umbratilis alvarezi AY DQ andersi AY DQ birchmanni DQ clemenciae AY DQ continens DQ cortezi DQ couchianus AY DQ evelynae DQ gordoni DQ hellerii AY DQ maculatus AY DQ malinche DQ mayae meyeri DQ milleri DQ mixei AY montezumae AY DQ monticolus AY multilineatus DQ nezahualcoyotl DQ nigrensis DQ pygmaeus DQ signum AY DQ variatus DQ xiphidium DQ Polymixiidae Polymixia EU Polymixia japonica EU Polymixia japonica Pristigasteridae Chirocentrus dorab EU EU Pellona EU Pristigasteridae Pellona flavipinnis Pellona flavipinnis Rivulidae Rivulus hartii Salmoniformes Oncorhynchus mykiss EF EF Scorpaeniformes Scorpaeniformes EU Sebastes ruberrimus EU Sebastes ruberrimus Siluriformes Ictalurus punctatus EF EF EU Stomiiformes Stomiiformes Stomias boa Synbranchiformes Monopterus albus EU EU Tetraodontidae Takifugu rubripes Ensembl 63 Ensembl 63 Tetraodon Tetraodontidae nigroviridis Ensembl 63 Ensembl 63 Valenciidae Valencia hispanica 44

57 Zeidae Zeus faber EU EU Zoarcoidea Lycodes EF Lycodes terraenovae EF Lycodes terraenovae Family Species sreb tbr1 zic1 Plagl2Z Anableps Anablepidae anableps Anablepidae Anableps dowei Anablepidae Jenynsia lineata Anablepidae Jenynsia multidentata Anablepidae Oxyzygonectes dovii Aphredoderidae Aphredoderus sayanus EU EU EU Aplocheilidae Fenerbahce formosus Aplocheilidae Aphyoplatys duboisi Aplocheilidae Aphyosemion bitaeniatum Aplocheilidae Aplocheilus lineatus Aplocheilidae Epiplatys annulatus Aplocheilidae Fundulopanchax Argentiniformes Argentina EU Argentina sialis EU Argentina sialis EU Argentina sialis EU Argentina sialis Atheriniformes Atheriniformes EU Labidesthes sicculus EU Labidesthes sicculus EU Labidesthes sicculus Aulopiformes Synodus Batrachoidiformes Porichthys EU Synodus foetens EU Synodus foetens EU Porichthys plectrodon EU Synodus foetens EU Porichthys plectrodon EU Synodus intermedius Beloniformes Oryzias latipes EF EF EF EF EU EU EU Myripristis Myripristis Myripristis Beryciformes Beryciformes violacea violacea violacea Characiformes Cichlidae Cichlidae Clupeidae EU Myripristis violacea EU AB Pygocentrus Citharinus Characiformes nattereri congicus HQ EU EU Cichlasoma Cichlasoma Cichlasoma Herichthys Herichthys Herichthys Cichlasomatinae cyanoguttatus cyanoguttatus cyanoguttatus EF EF EF Oreochromis Oreochromis Oreochromis Oreochromis niloticus niloticus niloticus Dorosoma cepedianum EU EU EU Pygocentrus nattereri EU Cichlasoma Herichthys cyanoguttatus EF Oreochromis niloticus Cypriniformes Danio rerio EF EF EF EF Cypriniformes Notemigonus crysoleucas EU EU EU EU Cypriniformes Semotilus atromaculatus EF JF EF EF Cyprinodontidae Cubanichthys cubensis Cyprinodontidae Cubanichthys pengelleyi Cyprinodontidae Cyprinodon variegatus Cyprinodontidae Floridichthys carpio Cyprinodontidae Jordanella floridae Cyprinodontidae Orestias 45

58 Esociformes Esox lucius EU EU EU Fundulidae Fundulus cingulatus Fundulidae Fundulus lineolatus Fundulidae Lucania goodei Fundulidae Lucania parva Gadidae Gadus morhua EU EU EU002 Gasterosteus Gasterosteiformes aculeatus AB AB AB EF Gonorynchiformes Chanos chanos EU EU EU EU Allodontichthys hubbsi Allodontichthys polylepis Allodontichthys tamazulae Allodontichthys zonistius Alloophorus robustus Allotoca catarinae Allotoca diazi Allotoca dugesii Allotoca goslinei Allotoca maculata Allotoca meeki Allotoca regalis Allotoca sp. MNCN3676 Allotoca zacapuensis Ameca splendens Ataeniobius toweri Chapalichthys encaustus Chapalichthys pardalis Characodon audax Characodon lateralis Crenichthys baileyi Crenichthys nevadae Empetrichthys latos Girardinichthys multiradiatus Girardinichthys viviparus Goodea atripinnis Goodea gracilis Hubbsina turneri Ilyodon amecae Ilyodon furcidens Ilyodon whitei Ilyodon xantusi 46

59 Neotoca bilineata Profundulus guatemalensis Profundulus labialis Profundulus punctatus Skiffia bilineatus Skiffia francesae Skiffia lermae Skiffia multipunctata Xenoophorus captivus Xenotaenia resolanae Xenotoca eiseni Xenotoca melanosoma Xenotoca variatus Zoogoneticus quitzeoensis Zoogoneticus tequila Gymnotiformes Gymnotiformes EU Apteronotus albifrons EU Apteronotus albifrons EU Apteronotus albifrons EU Apteronotus albifrons Lampriformes Lampriformes EU Regalecus glesne EU Regalecus glesne EU Regalecus glesne Lophiiformes Lutjanidae Lophius Lutjanus EU Lophius gastrophysus EF Lutjanus mahogoni EU i EF Lutjanus mahogoni EU Lophius gastrophysus EF Lutjanus mahogoni EU Lophius gastrophysus EU Lutjanus mahogoni Macrouridae Macrouridae Moronidae Morone EF Morone chrysops EF Morone chrysops EF Morone chrysops EF Morone chrysops Mugiliformes Mugil EU Mugil curema EU Mugil curema EU Mugil curema EU Mugil curema Myctophiformes Myctophiformes EU Neoscopelus macrolepidotus EU Neoscopelus macrolepidotus EU Neoscopelus macrolepidotus Ophidiiformes Ophidiiformes EF Brotula multibarbata EF Brotula multibarbata EF Brotula multibarbata EF Brotula multibarbata Osmeriformes Pleuronectiformes Osmeriformes Pleuronectiformes EU Thaleichthys pacificus EU Pleuronectes platessa EU Thaleichthys pacificus EU Pleuronectes platessa EU Thaleichthys pacificus EU Pleuronectes platessa Alfaro cultratus Alfaro hubberi Aplocheilichthys normani Aplocheilichthys spilauchen Belonesox belizanus cascajalensis hartwegi holdridgei parismina rhabdophora EU Thaleichthys pacificus EU Pleuronectes platessa 47

60 roseni terrabensis Carlhubbsia kidderi Carlhubbsia stuarti Cnesterodon decemmaculatus Cnesterodon hypselurus Cnesterodon septentrionalis Dactylophallus denticulatus Dactylophallus ramsdeni Dactylophallus sp. DDEN10 Fluviphylax pygmaeus Fluviphylax simplex affinis EU EU EU EU atrora caymanensis eurystoma geiseri heterochir hispaniolae holbrooki hubbsi hurtadoi luma manni marshi melapleura nicaraguensis oligosticta panuco punctata puncticulata rhizophorae sexradiata sp. LLSTC4571 vittata wrayi yucatana zarskei Girardinus creolus 48

61 Girardinus metallicus Girardinus microdactylus Girardinus rivasi Girardinus sp. GMIC19 Glaridichthys falcatus Glaridichthys uninotatus Heterandria bimaculata Heterandria formosa Heterandria jonesi Heterophallus milleri Heterophallus rachovii Limia caymanensis Limia dominicensis Limia garnieri Limia grossidens Pseudolimia heterandria Limia melanogaster Limia melanonotata Limia nigrofasciata Limia pauciradiata Limia perugiae Limia rivasi Limia sulfurophila Limia tridens Limia versicolor Limia vittata Limia zonata Micropoecilia bifurca Micropoecilia branneri Acanthophacelus obscura Micropoecilia parae (French Guiana) Micropoecilia parae (Suriname) Micropoecilia picta Micropoecilia picta (Trinidad and Tobago) 49

62 Acanthophacelus reticulata Micropoecilia sarrafae Acanthophacelus wingei Acanthophacelus wingei (Venezuela) Neoheterandria cana Neoheterandria elegans Neoheterandria tridentiger Pamphorichthys araguaiensis Pamphorichthys hasemani Pamphorichthys hollandi Pamphorichthys minor Pamphorichthys scalpridens Phallichthys amates Phallichthys pittieri Phallichthys quadripunctatus Phallichthys tico Phalloceros caudimaculatus Phalloptychus januarius Mollienesia butleri Mollienesia catemaconis Mollienesia caucana Mollienesia chica Mollienesia gilli Mollienesia gracilis Mollienesia latipinna Mollienesia latipunctata Mollienesia mexicana limanturi Mollienesia mexicana mexicana Mollienesia orri 50

63 Mollienesia petenensis Campeche Mollienesia petenensis MP523 Mollienesia salvatoris Mollienesia sphenops Mollienesia sulphuraria Mollienesia velifera vivipara vivipara (Trinidad and Tobago) baenschi balsas catemaco elongata fasciata gracilis hnilickai infans latidens lucida monacha occidentalis paucimaculata pleurospilus presidionis prolifica retropinna scarlii sonoriensis turneri turrubarensis viriosa Priapella chamulae Priapella compressa Priapella intermedia Priapella olmecae 51

64 Priapichthys annectens Priapichthys darienensis Priapichthys panamensis Priapichthys puetzi Pseudopoecilia festae Quintana atrizona Scolichthys greenwayi Scolichthys iota Tomeurus gracilis Xenodexia ctenolepis Xenophallus umbratilis alvarezi andersi birchmanni clemenciae continens cortezi couchianus evelynae gordoni hellerii maculatus malinche mayae meyeri milleri mixei montezumae monticolus multilineatus nezahualcoyotl nigrensis pygmaeus signum variatus xiphidium Polymixiidae Polymixia HM Polymixia nobilis EU Polymixia japonica EU Polymixia japonica Pristigasteridae Chirocentrus dorab EU EU EU

65 Pristigasteridae Pellona EU Pellona castelnaeana EU Pellona flavipinnis EU Pellona flavipinnis Rivulidae Rivulus hartii Salmoniformes Oncorhynchus mykiss EF EF EF EF Scorpaeniformes Scorpaeniformes EU Sebastes ruberrimus EU Sebastes ruberrimus EU Sebastes ruberrimus Siluriformes Ictalurus punctatus EF EF EF EF Stomiiformes Stomiiformes EU Stomias boa EU Stomias boa EU Stomias boa Synbranchiformes Monopterus albus EU EU EU EU Tetraodontidae Takifugu rubripes Ensembl 63 Ensembl 63 Ensembl 63 Ensembl 63 Tetraodontidae Tetraodon nigroviridis Ensembl 63 Ensembl 63 Ensembl 63 Ensembl 63 Valenciidae Valencia hispanica Zeidae Zeus faber EU EU EU Zoarcoidea Lycodes EF Lycodes terraenovae EF Lycodes terraenovae EF Lycodes terraenovae EF Lycodes terraenovae 53

66 Supplementary Table 4 Gene segments and primers. GENE PRIMER ME PRIMER SEQUENCE (5'-3') Cytb cytb-if CAATGAATYTGRGGNGGYTTYTC Cytb-IF2 Cytb-IR2 CytbMicro-IF2 CytbPamp-IF2 HA (16249) LA (15058) LimIF-cytb LimIR-cytb MicroIF-cytb MicroIR-cytb GTHGGNTAYGTCCTCCCMTGAGGAC GGARAAKCCHCCTCARATTCAYTG CAGTAGACAACGCCACCYTAAC GGGGCTACCGTAATTACCAACC CAACGATCTCCGGTTTACAAGAC GTGACTTGAAAAACCACCGTTG CAGTTGACAACGCCACCCTAAC GGGTTAGGGTGGCGTTGTCAAC CAATGAATCTGAGGGGGATTTTC GCGTTGTCTACTGAAAATCC ND2 C-Trp_ND2_REV CTGAGGGCTTTGAAGGCCC C-Trp_Pam_ND2_REV ND2-IF1 ND2-IF2 ND2-IR1 ND2-IR2 ND2-IRPamp ND2ASN ND2GLN ND2Pamp-IR1 ND2Pamp-IR2 GTCTAAGGAATTATCCTAAG CTCCGAAAAATCYTAGCRTAYTC CTMCGAAAARTYCTYGCATACTC CGGAGYTGGACTTGGTTAAAHCC CGKAGTTGKACTTGGTTAAAVCC GTGGGAAGGTTATTGTTAAATAG CGCGTTTAGCTGTTAACTAA CTACCTGAAGAGATCAAAC GAGGGGCAAGTTTTTGTCAGG GGGAGTTAATGTGTGTGGTGCG ENC1 CutA-IF GACCTAGAAAAGAGGCACTG CutA-IR cutainf cutainm CutAinR LF1 LF2 LR1 LR2 GCTTCATCTACCAGTTCCTTGC ATTGGATTAACTATGACCTAGAA GCCAAAGAGATAATCCC TTAATCCAATTAAGGGCAGC GACATGCTGGAGTTTCAGGA ATGCTGGAGTTTCAGGACAT ACTTGTTRGCMACTGGGTCAAA AGCMACTGGGTCAAACTGCTC Glyt BrachrhapIR-Gylt GGATCCATACCTCGTACTTAAC MF1 MF2 MF2-IF MR1 MR2 MR2-IR TomgracIR-Gylt GGACTGTCMAAGATGACCACMT ACATGGTACCAGTATGGCTTTGT CCAGGCATGGACCTTCACTAC CCCAAGAGGTTCTTGTTRAAGAT GTAAGGCATATASGTGTTCTCTCC CTGCTCATCTTTTTCTAAGTG CTGCTCGTCTTTGTCTAAGTG SH3PX3 FundcingIF-HMG20A CATCCAAAGGATCAGCCGCCGC PF1 PF2 PF2-IF PR1 PR2 GTATGGTSGGCAGGAACYTGAA GACGTTCCCATGATGGCWAAAAT CATCCACAGTATCAGTCGCCTC CAAACAKCTCYCCGATGTTCTC CATCTCYCCGATGTTCTCGTA 54

67 PR2-IR CTGTGATCTCTGTGCCTCACCTC LucGoodIF-HMG20A CATCCAGAGTATCAGCCGCCGC MYH6 AF1 CATMTTYTCCATCTCAGATAATGC AF2 AR1 AR2 Pak6-IF Pak6-IR GGAGAATCARTCKGTGCTCATCA ATTCTCACCACCATCCAGTTGAA CTCACCACCATCCAGTTGAACAT GAGACGTAYCTGCTGGARAAGTC GGCTTYTGRTTGGACAGGATYTG Rag1 H2932 RAG1 GAGAARCGRACAGCCTTYTC H3405 RAG1 H4054 RAG1 L2492 RAG1 L2891 RAG1 L3371 RAG1 Pamp-IFRag1 Pamp-IFRag1 PampRag1-IF2 PampRag1-IF2 Rag1PampIF Rag1GoodIF GCNGAGACTCCTTTGACTCTGTC GTGTAGAGCCARTGRTGYTT CCWGCTGTITGYYTGGCCATIMG AAGGAGTGYTGYGATGGCATGGG GARCGYTAYGAAATATGGAG GCCACCTGTCCAGCCAAGGAGTG GCCACCTGTCCAGCCAAGGAGTG GGAACGTTATGAAATTTGGAGAAC GGAACGTTATGAAATTTGGAGAAC GCCAGTGATGAGGATGAATG GCCGAGGTAGTTTGTGAACTG Rh Rh1039R TGCTTGTTCATGCAGATGTAGA Rh193F Rh545F Rh667R CNTATGAATAYCCTCAGTACTACC GCAAGCCCATCAGCAACTTCCG AYGAGCACUGCAUGCCCU X-src FundgairdIF-xsrc GCTCGGAGATCTCTGTGCACGTAGTTC Girardin-xsrcF IFAnableps-xsrx IFFluv-xsrc IFGamhol-xsrc IFJenynsia-xsrx IFLuc-xsrc IFLimia-xsrx IFLimia-xsrx IFPoec-xsrc IFPoeciliop-xsrx IFXiphoph-xsrc IR2Cyp-xsrc IRAnableps-xsrc IRGamhol1-xsrc IRJennynsia-xsrc IRLuc-xsrc Luc-xsrcR X-src C X-src D GCGAGCCAACCAAAATCAGCCAC CCCTAAATGTTCCCACCTTTCATC CGCCTCCCTCAGCTGGTGGAC CGGAGCATTGCACTATACTGTC CCCTAAATGTTCCCTCTTTTCGTCAG GCCGGAGCATTGAACTATATTG CAAGCTAAAGGTAAGAAAATATTC CAAGCTAAAGGTAAGAAAATATTC GCGTACAGCTGAACCAGCTTC GCTAATGGTAAGAAAATATTC CGCAGCAAATCTTTAAACAAC GGTCAGAATAAAACCTTAAATC CTCAAGTTAGTGGTAGAAAATAC GCTTGAGATGCTATACCTTTTTG CTCAAGTTGGTTGTAAAAAG CAGCCTCATTACTGAATTTCTC GGRACCATGTCCCCCGAGGCBTTC CTCAATCAGGCGAGCCAACCAAAATC ACGGCACCACACAGGTGGCGATCAA Primer abbreviations: Micro=Micropoecilia Pamp=Pamphorichthys Lim=Limia 55

68 Brachrhap= Tomgrac=Tomerus Fundgaird=Fundulopanchax Girardin= Girardinichthys Luc=Lucania Fluv=Fluviphylax Gamhol= Poec= Xiphoph= Cyp=Cyprinodon Poeciliop= 56

69 Supplementary Table 5 Newick ML phylogram (-ln ) for 22 partitions and 293 terminals. (((((((((((((((((((((((((((((((((((((((((((_hurtadoi: ,_vittata: ): ,(gamb usia_panuco: ,_marshi: ): ): ,_atr ora: ): ,(_rhizophorae: ,_punctata: ): ): ,_zarskei: ): E- 4,((((_oligosticta: ,_caymanensis: E- 6): ,_puncticulata: ): ,_yucatana: ): ,((_manni: E- 6,_hubbsi: ): ,_nicaraguensis: ): ): ): ,((_melapleura: ,_wrayi: ): ,_hispaniolae: ): ): ,(((_affinis: ,_sp_LLSTC4571: ): ,_holbrooki: ): ,(_geiseri: ,_heterochir: ): ): ): ,(_eurystoma: ,_sexradiata: ): ): ,_luma: ): ,(Heterophallus_milleri: ,H eterophallus_rachovii: ): ): ,belonesox_belizanus: ): ,(((((((((((_couchianus: ,_gordoni: ): E- 4,_meyeri: ): ,_xiphidium: ): ,Xi phophorus_variatus: ): e- 4,_evelynae: ): ,_milleri: ): ,_maculatus: ): E- 4,_andersi: ): ,(((_signum: ,_mayae: ): ,(_alvarezi: ,_hellerii: ): ): ,((_mixei: ,_clemenciae: ): ,_monticolus: ): ): ): ,((((((_nigrensis: ,_multilineatus: E- 4): ,_birchmanni: E-4): E- 4,_continens: ): ,((_cortezi: ,_montez umae: ): e-4,_malinche: ): e- 4): ,_nezahualcoyotl: ): ,_pygmaeus: ): ): ,(Heterandria_bimaculata: ,Heterandria_jonesi: ): ): ): ,((Priapella_compressa: ,Priapella_chamulae: ): ,(Priapella_olmecae: ,Priapella_intermedia: ): ): ): ,((Carlhubbsia_stuarti: ,Carlhubbsia_kidderi: E- 6): ,(Scolichthys_greenwayi: ,Scolichthys_iota: ): ): ): ,((((((((((_hnilickai: ,_catemaco: ): ,_gracilis: ): ,_pleurospilus: ): ,(_presidionis: ,_turneri: ): ): ,(_scarlii: ,_turrubarensis: ): ): ,((_latidens: ,_fasciata: ): , _baenschi: ): ): ,((((((_occidentalis: ,poeciliop sis_sonoriensis: ): ,_lucida: ): , _prolifica: ): ,_infans: ): ,(_mon acha: ,_viriosa: ): ): ,_balsas: ): ): ,((_retropinna: ,_elongata: ): ,_paucimaculata: ): ): ,((Neoheterandria_cana: ,Neoheterandria_tridentiger: ): ,Neoheterandria_elegan s: ): ): ,((((((((((_terrabensis: , _roseni: ): ,_rhabdophora: ): ,brachyrhaph is_holdridgei: ): ,_hartwegi: ): ,(((phallicht hys_amates: ,phallichthys_pittieri: ): ,phallichthys_quadripunctatus: ): ,Phallichthys_tico: ): ): ,(( _parismina: ,_cascajalensis: e- 4): ,(Priapichthys_puetzi: ,Priapichthys_annectens: ): ): ): ,(Alfaro_hubberi: ,Alfaro_cultratus: ): ): ,Xenophallus_umbratilis: ): ,(Priapichthys_panamensis: ,Heterandria_formosa: ): ): ,(Priapichthys_darienensis: ,Pseudopoecilia_festae: ): ): ): ): ,(((((((Girardinus_sp_GMIC19: ,Girardinus_rivasi: ): ,Girardinus_ microdactylus: ): ,girardinus_metallicus: ): ,girardinus_ creolus: ): ,(glaridichthys_falcatus: ,glaridichthys_uninotatus: ): ): ,((Dactylophallus_sp_DDEN10: ,Dactylophallus_ramsdeni: ): E- 4,Dactylophallus_denticulatus: ): ): ,Quintana_atrizona: ): ): ,(((((((((((((((_Limia_rivasi: E- 4,_Limia_tridens: E-4): E-4,_Limia_melanonotata: E- 6): E-4,_Limia_perugiae: E- 4): ,_Limia_sulfurophila: ): ,((_Limia_garnieri: ,_Limia_grossidens: E-4): E-4,_Limia_nigrofasciata: E- 4): ): ,(_Limia_dominicensis: ,_Limia_pauciradiata: ): ): ,(_Limia_vittata: ,_Limia_caymanensis: ): ): ,(_Limia_versicolor: ,_Limia_zonata: ): ): ,_Limia_melanogaster: ): , _Pseudolimia_heterandria: ): ,(((_Pamphorichthys_hollandi: ,Poe cilia_pamphorichthys_araguaiensis: ): ,_pamphorichthys_hasemani: ) : ,(_Pamphorichthys_minor: ,_Pamphorichthys_scalpridens: ): ): ): ,(((((((((_ Mollienesia_mexicana_limanturi: ,_Mollienesia_gracilis: ): E- 57

70 4,_Mollienesia_sulphuraria: ): ,((_Mollienesia_sphenops: ,Po ecilia_mollienesia_catemaconis: ): ,_mollienesia_mexicana_mexicana: ): ): ,(_Mollienesia_gilli: ,_Mollienesia_salvatoris: ): E- 4): ,_Mollienesia_orri: ): ,_Mollienesia_butleri_MR04: ): ,_Mollienesia_chica: ): ,(((_Mollienesia_petenensi s_mp523: e-4,_mollienesia_petenensis_campeche: e- 4): ,_Mollienesia_latipunctata: ): ,(_Mollienesia_velifera_MP73 7: ,_Mollienesia_latipinna: ): ): ): ,_Mollienesia_caucana: ): ): ,( vivipara: , vivipara_trinidad_and_tobago: ): ): ,(( (((_Micropoecilia_sarrafae: ,_Micropoecilia_branneri: ): , _Micropoecilia_bifurca: ): ,(_Micropoecilia_parae_French_Guiana: ,Poe cilia_micropoecilia_parae_suriname: ): ): ,(_micropoecilia_picta_trinida d_and_tobago: ,_micropoecilia_picta: ): ): ,((p oecilia_acanthophacelus_reticulata: e-4,_acanthophacelus_obscura: e- 4): ,(_Acanthophacelus_wingei_Venezuela: ,_Acanthophacelus_wingei: E- 4): ): ): ): ,((Cnesterodon_septentrionalis: ,Cnesterodon_decemmaculatus: ): ,Cnesterodon_hypselurus: ): ): ): ,Phalloceros_caudimaculatus: ): ,Ph alloptychus_januarius: ): ,tomeurus_gracilis: ): ,xenodexi a_ctenolepis: ): ,(((jenynsia_multidentata: ,jenynsia_lineata: ): ,(Anableps_dovii: ,Anableps_anableps: ): ): ,Oxyzygonectes_dovii: ): ): ,(Fluviphylax_simplex: ,Fluviphylax_pygmaeus: ): ): ,(((Aplocheilichthys_normani: ,Aplocheilichthys_spilauchen: ): ,Valencia_hispanica: ): ,Orestias: ): ): ,(((((((((((((((((Allotoca_diazi: ,Allotoca_ meeki: ): e- 4,Allotoca_catarinae: ): ,Allotoca_zacapuensis: ): ,(Al lotoca_dugesii: ,allotoca_goslinei: ): ): ,allotoca_sp_m NCN3676: ): ,Allotoca_maculata: ): ,Allotoca_regalis: ): ,Hubbsina_turneri: ): E- 4,((((Skiffia_francesae: ,Skiffia_multipunctata: E- 4): ,Skiffia_lermae: ): ,Skiffia_bilineatus: ): ,((Girardinichthys_multiradiatus: ,Girardinichthys_viviparus: ): ,Neo toca_bilineata: ): ): ): ,ataeniobius_toweri: ): ,(((((((Chapalichthys_pardalis: ,Chapalichthys_encaustus: ): ,Alloophorus_robustus: ): E- 4,Xenotoca_variatus: ): ,Ameca_splendens: ): ,((Zoogo neticus_quitzeoensis: ,zoogoneticus_tequila: ): ,xenoophorus_captiva: ): ): ,(Xenotoca_melanosoma: ,Xenotoca_eiseni: ): ): ,(Goodea_gracilis: ,Goodea_atripinnis: E- 4): ): ): ,((((Ilyodon_whitei: E- 4,Ilyodon_xantusi: E- 4): ,(Ilyodon_furcidens: ,Ilyodon_amecae: E-4): E- 4): ,Xenotaenia_resolanae: ): ,(((Allodontichthys_hubbsi: ,Allodontichthys_tamazulae: ): ,Allodontichthys_polylepis: ): ,Allodontichthys_zonistius: ): ): ): ,(Characo don_lateralis: ,characodon_audax: ): ): ,((crenichthys_bai leyi: ,crenichthys_nevadae: ): ,empetrichthys_latos: ): ): ,(Profundulus_punctatus: ,Profundulus_guatemalensis: ): ): ,Profundulus_labialis: ): ,(((Lucania_goodei: ,Lucania_parvae: ): ,(Fundulus_lineolatus: ,Fundulus_cingulatus: ): ): ,((Jordanella_floridae: ,Cyprinodon_variegatus: ): ,Floridichthys_carpio: ): ): ): ): ,(Cubanichthys_pengelleyi: ,Cubanichthys_cubensis: ): ): ,(((((Aphyosemion_bitaeniatum: ,Fundulopanchax: ): , Adamas_formosus: ): ,(Aphyoplatys_duboisi: ,Epiplatys_annulatus: ): ): ,Aplocheilus_lineatus: ): ,Rivulus_hartii: ): ): ,(Oryzias_latipes: ,Atheriniformes: ): ): ,(Oreochromis: ,Cichlasomatinae: ): ): ,Mugil: ): ,(Pleuronectiformes: ,Monopterus_albus: ): ): ,((((Tetraodon_nigroviridis: ,Takifugu_rubripes: ): ,Lophius: ): ,(Lutjanus: ,Morone: ): ): ,((Lycodes: ,Gasterosteus_aculeatus: ): ,Scorpaeniformes: ): ): ): ,(Ophidiiformes: ,Porichthys: ): ): ,Beryciformes: ): ,Lampriformes: ): ,(Polymixia: ,Zeus_faber: ): ): ,Myctophiformes: ): ,Synodus: ): ,((Gadus_morhua: ,Macrouridae: ): ,Aphredoderus_sayanus: ): ): ,(Osmeriformes: ,Stomiiformes: ): ): ,((Oncorhynchus_mykiss: ,Esox_lucius: ): ,Argentina: ): ): ,(((((Semotilus_atromaculatus: ,Notemigonus_c rysoleucas: ): ,danio_rerio: ): ,((characiformes: ,Ictalurus_punctatus: ): ,Gymnotiformes: ): ): ,Chanos_chanos: ): ,((Pellona: ,Dorosoma_cepedianum: ): ,Chirocentrus_dorab: ): ): ); 58 doi: /nature

71 Supplementary Table 6 Regression models that explain variation in life history traits within the family as a function of the natural log-transformed matrotrophy index. Model comparisons Regression parameters Fit parameters Best vs Alternative! 2 for P for Model Coef SE * df / df adj F P d ln ML AIC Model " i LRT df LRT Sexual selection index OLS Y-Int / x OU vs OLS Slope / OU vs PGLS PGLS Y-Int / Slope / OU Y-Int / Slope / Relative gonopodium length OLS Y-Int / x OU vs OLS Slope / OU vs PGLS PGLS Y-Int / Slope / OU Y-Int / Slope / Size dimorphism index body weight OLS Y-Int / OLS vs PGLS > # 0 # < 0.05 # Slope / OLS vs OU PGLS Y-Int / Slope / OU Y-Int / x Slope / Sexual size dimorphism body weight OLS Y-Int / x OLS vs PGLS > # 0 # < 0.05 # Slope / OLS vs OU PGLS Y-Int / Slope / OU Y-Int / x x Slope / Log 10 male body weight OLS Y-Int / OU vs OLS Slope / OU vs PGLS PGLS Y-Int / Slope / OU Y-Int / Slope / Log 10 female body weight OLS Y-Int / OU vs OLS Slope / OU vs PGLS PGLS Y-Int / Slope / OU Y-Int / Slope / Size dimorphism index standard length 59

72 OLS Y-Int / x OLS vs PGLS > # 0 # < 0.05 # Slope / OLS vs OU PGLS Y-Int / Slope / OU Y-Int / x Slope / Sexual size dimorphism standard length OLS Y-Int / x OLS vs PGLS > # 0 # < 0.05 # Slope / OLS vs OU PGLS Y-Int / Slope / OU Y-Int / x Slope / Log 10 male standard length OLS Y-Int / x OU vs OLS Slope / OU vs PGLS PGLS Y-Int / Slope / OU Y-Int / x Slope / Log 10 female standard length OLS Y-Int / x OU vs OLS Slope / OU vs PGLS PGLS Y-Int / Slope / OU Y-Int / x Slope / Best-fit models are indicated in bold. * Degrees of freedom were corrected to allow for soft polytomies 59,60. Based on! I and LRTs, the OU model is statistically significantly better than the two alternative models. Based on! I and LRTs, there is statistically no significant difference in the fit between the OLS and OU models (both are statistically significantly better than PGLS). Notes on model comparisons (for more details see Lavin et al., 2008): When comparing models, nested or not, the one with the lowest AIC is considered to be the best. The difference in AIC between models (! i = AIC i - AIC min, where AIC i is the AIC value for alternative model i and AIC min is the AIC value of the best model) provides an heuristic indicator of the support for the alternative model:! i < 2 suggests that there is substantial support for alternative model i; 4 <! i < 7 indicates that the alternative model has considerably less support and;! i > 10 signifies that the alternative model is very unlikely 61. When one model is a nested subset of the other, likelihood ratio tests (LRTs) can be used to compare them. Twice the difference in ln likelihoods between models (D = -2 [maximum likelihood for best model maximum likelihood for alternative model]) is assumed to be distributed asymptotically as a " 2 distribution with degrees of freedom equal to the difference in the number of parameters in the two models. # LRTs can also be used to compare PGLS and OLS models, which have the same number of parameters. In such comparisons with 0 df, a difference in likelihoods > (which is the ninety-fifth percentile of the distribution of " 2 with 1 df) is often taken to indicate a significant difference (P > 0.05) in the fit of the two models

73 Argentina Osmeriformes Pellona Chanos chanos Oncorhynchus mykiss Stomiiformes Polymixia Dorosoma cepedianum Esox lucius Beryciformes Fig. S1. ML phylogram (-ln likelihood = ) obtained with RAxML for the 22 partitions. Bootstrap support percentages are provided in Fig. S2. Chirocentrus dorab Characiformes Myctophiformes Semotilus atromaculatus Notemigonus crysoleucas Gadus morhua Macrouridae Lycodes Danio rerio Gymnotiformes Aphredoderus sayanus Lutjanus Zeus faber Lampriformes Morone Synodus Ictalurus punctatus Scorpaeniformes Mugil Oreochromis Gasterosteus aculeatus Lophius Cichlasomatinae Monopterus albus Atheriniformes Pleuronectiformes Profundulus labialis 5.0 Ophidiiformes Tetraodon nigroviridis Takifugu rubripes Epiplatys annulatus Characodon audax Allodontichthys polylepis Ilyodon amecae Girardinichthys multiradiatus Girardinichthys viviparus Allotoca maculata Allotoca dugesii Anableps anableps Aphyosemion bitaeniatum Allodontichthys tamazulae Chapalichthys pardalis Cyprinodon variegatus Profundulus guatemalensis Crenichthys nevadae Ilyodon whitei Ilyodon furcidens Goodea gracilis Xenoophorus captiva Alloophorus robustus Skiffia francesae Jenynsia multidentata Fundulopanchax Cubanichthys pengelleyi Crenichthys baileyi Jordanella floridae Allodontichthys zonistius Xenotaenia resolanae Goodea atripinnis Ameca splendens Chapalichthys encaustus Skiffia multipunctata Hubbsina turneri Allotoca sp. MNCN3676 Allotoca diazi Valencia hispanica Oxyzygonectes dovii Anableps dovii Aphyoplatys duboisi Ilyodon xantusi Floridichthys carpio Xenotoca melanosoma Xenotoca eiseni Jenynsia lineata Oryzias latipes Rivulus hartii Aplocheilus lineatus Lucania goodei Allotoca meeki Aplocheilichthys normani Aplocheilichthys spilauchen Phalloptychus januarius Phalloceros caudimaculatus Cnesterodon septentrionalis Cnesterodon decemmaculatus Acanthophacelus wingei Venezuela Acanthophacelus wingei Micropoecilia picta Trinidad and Tobago vivipara Trinidad and Tobago Mollienesia velifera Mollienesia latipinna Mollienesia latipunctata Mollienesia petenensis MP523 Mollienesia chica Mollienesia butleri Mollienesia orri Mollienesia salvatoris Mollienesia mexicana limanturi Mollienesia gracilis Limia versicolor Limia rivasi Glaridichthys falcatus Priapichthys puetzi retropinna balsas Carlhubbsia stuarti Pamphorichthys hasemani Pamphorichthys araguaiensis Limia melanogaster Limia dominicensis Limia grossidens Limia sulfurophila Limia melanonotata Girardinus rivasi Priapichthys darienensis Priapichthys panamensis Xenophallus umbratilis Alfaro cultratus Priapella intermedia pygmaeus continens turrubarensis monticolus signum milleri Porichthys Adamas formosus Cubanichthys cubensis Lucania parvae Fundulus cingulatus Profundulus punctatus Empetrichthys latos Characodon lateralis Zoogoneticus quitzeoensis Zoogoneticus tequila Ataeniobius toweri Fundulus lineolatus Allodontichthys hubbsi Xenotoca variatus Neotoca bilineata Skiffia bilineatus Skiffia lermae Allotoca regalis Allotoca goslinei Allotoca zacapuensis Allotoca catarinae Orestias Fluviphylax simplex Fluviphylax pygmaeus Xenodexia ctenolepis Cnesterodon hypselurus Acanthophacelus reticulata Acanthophacelus obscura Micropoecilia picta Micropoecilia parae French Guiana Micropoecilia parae Suriname Micropoecilia bifurca Micropoecilia branneri vivipara Mollienesia petenensis Campeche Mollienesia gilli Mollienesia sulphuraria Pamphorichthys minor Pamphorichthys scalpridens Pseudolimia heterandria Limia zonata Limia vittata Limia pauciradiata Limia garnieri Limia perugiae Limia tridens Dactylophallus sp. DDEN10 Dactylophallus ramsdeni Glaridichthys uninotatus Girardinus creolus Girardinus metallicus Girardinus microdactylus Girardinus sp. GMIC19 Pseudopoecilia festae parismina cascajalensis Phallichthys quadripunctatus Phallichthys amates hartwegi holdridgei rhabdophora terrabensis roseni Neoheterandria cana Neoheterandria tridentiger paucimaculata elongata Carlhubbsia kidderi monacha viriosa Priapella compressa prolifica baenschi hnilickai Scolichthys greenwayi Scolichthys iota Priapella chamulae Heterandria bimaculata nezahualcoyotl montezumae clemenciae mayae alvarezi hellerii andersi variatus latidens fasciata scarlii presidionis turneri Heterandria jonesi malinche cortezi birchmanni couchianus gordoni lucida gracilis nigrensis infans pleurospilus multilineatus evelynae xiphidium meyeri catemaco Belonesox belizanus Heterophallus milleri Heterophallus rachovii luma eurystoma sexradiata geiseri heterochir holbrooki affinis sp. LLSTC4571 hispaniolae melapleura nicaraguensis manni yucatana puncticulata oligosticta caymanensis zarskei rhizophorae punctata atrora hurtadoi vittata panuco marshi Tomeurus gracilis Mollienesia mexicana mexicana Mollienesia sphenops Mollienesia catemaconis Pamphorichthys hollandi Limia caymanensis Limia nigrofasciata Alfaro hubberi Micropoecilia sarrafae Mollienesia caucana Quintana atrizona Dactylophallus denticulatus Heterandria formosa Priapichthys annectens Phallichthys tico Phallichthys pittieri Neoheterandria elegans Priapella olmecae mixei maculatus occidentalis sonoriensis wrayi hubbsi

74 27 Chirocentrus dorab Pellona Dorosoma cepedianum Chanos chanos 98 Danio rerio Semotilus atromaculatus Notemigonus crysoleucas Gymnotiformes Characiformes Ictalurus punctatus Argentina Oncorhynchus mykiss Esox lucius Osmeriformes Stomiiformes Aphredoderus sayanus Gadus morhua Macrouridae Synodus Myctophiformes Polymixia Zeus faber 84 Lampriformes Beryciformes Ophidiiformes Porichthys Scorpaeniformes Lycodes Gasterosteus aculeatus Lutjanus Morone Lophius Tetraodon nigroviridis Takifugu rubripes 77 Pleuronectiformes Monopterus albus 75 Mugil Oreochromis Cichlasomatinae Oryzias latipes Atheriniformes Rivulus hartii Aplocheilus lineatus Aphyoplatys duboisi Epiplatys annulatus Adamas formosus Aphyosemion bitaeniatum Fundulopanchax Cubanichthys pengelleyi Cubanichthys cubensis Floridichthys carpio Jordanella floridae Cyprinodon variegatus Lucania goodei Lucania parvae Fundulus lineolatus Fundulus cingulatus Profundulus labialis Profundulus punctatus Profundulus guatemalensis Empetrichthys latos Crenichthys baileyi Crenichthys nevadae Characodon lateralis Characodon audax Allodontichthys zonistius Allodontichthys polylepis Allodontichthys hubbsi Allodontichthys tamazulae Xenotaenia resolanae Ilyodon whitei Ilyodon xantusi Ilyodon furcidens Ilyodon amecae Goodea gracilis Goodea atripinnis Xenotoca melanosoma Xenotoca eiseni Xenoophorus captiva Zoogoneticus quitzeoensis Zoogoneticus tequila Ameca splendens Xenotoca variatus Alloophorus robustus Chapalichthys pardalis Chapalichthys encaustus Ataeniobius toweri Neotoca bilineata Girardinichthys multiradiatus Girardinichthys viviparus Skiffia bilineatus Skiffia lermae Skiffia francesae Skiffia multipunctata Hubbsina turneri 9 Allotoca regalis 75 Allotoca maculata Allotoca sp. MNCN Allotoca dugesii Allotoca goslinei Allotoca zacapuensis Allotoca catarinae Allotoca diazi Allotoca meeki Orestias 27 Valencia hispanica Aplocheilichthys normani Aplocheilichthys spilauchen 99 Fluviphylax simplex Fluviphylax pygmaeus Oxyzygonectes dovii Jenynsia multidentata Jenynsia lineata Anableps dovii Anableps anableps Xenodexia ctenolepis Tomeurus gracilis Phalloptychus januarius Phalloceros caudimaculatus Cnesterodon hypselurus Cnesterodon septentrionalis Cnesterodon decemmaculatus 99 Acanthophacelus reticulata Acanthophacelus obscura Acanthophacelus wingei Venezuela Acanthophacelus wingei Micropoecilia picta Trinidad and Tobago Micropoecilia picta 97 Micropoecilia parae French Guiana Micropoecilia parae Suriname Micropoecilia bifurca Micropoecilia sarrafae Micropoecilia branneri vivipara vivipara Trinidad and Tobago Mollienesia caucana Mollienesia velifera Mollienesia latipinna Mollienesia latipunctata Mollienesia petenensis MP523 Mollienesia petenensis Campeche Mollienesia chica 91 Mollienesia butleri Mollienesia orri Mollienesia gilli Mollienesia salvatoris Mollienesia sulphuraria Mollienesia mexicana limanturi Mollienesia gracilis Mollienesia mexicana mexicana Mollienesia sphenops Mollienesia catemaconis Pamphorichthys minor Pamphorichthys scalpridens Pamphorichthys hasemani Pamphorichthys hollandi Pamphorichthys araguaiensis Pseudolimia heterandria Limia melanogaster Limia versicolor Limia zonata Limia vittata Limia caymanensis 92 Limia dominicensis Limia pauciradiata Limia nigrofasciata Limia garnieri Limia grossidens Limia sulfurophila Limia perugiae Limia melanonotata Limia rivasi Limia tridens Quintana atrizona Dactylophallus denticulatus Dactylophallus sp. DDEN10 Dactylophallus ramsdeni Glaridichthys falcatus Glaridichthys uninotatus Girardinus creolus 76 Girardinus metallicus Girardinus microdactylus Girardinus sp. GMIC19 Girardinus rivasi Priapichthys darienensis Pseudopoecilia festae Priapichthys panamensis Heterandria formosa Xenophallus umbratilis Alfaro hubberi Alfaro cultratus parismina cascajalensis Priapichthys puetzi Priapichthys annectens Phallichthys tico Phallichthys quadripunctatus Phallichthys amates Phallichthys pittieri hartwegi 55 holdridgei rhabdophora terrabensis roseni Neoheterandria elegans Neoheterandria cana Neoheterandria tridentiger 87 paucimaculata retropinna elongata balsas monacha viriosa infans prolifica lucida occidentalis sonoriensis baenschi latidens fasciata 63 scarlii turrubarensis presidionis turneri pleurospilus gracilis hnilickai catemaco 71 Carlhubbsia stuarti Carlhubbsia kidderi Scolichthys greenwayi Scolichthys iota Priapella compressa Priapella chamulae Priapella olmecae Priapella intermedia 61 Heterandria bimaculata Heterandria jonesi pygmaeus nezahualcoyotl malinche cortezi montezumae continens birchmanni nigrensis multilineatus monticolus mixei clemenciae signum mayae alvarezi hellerii andersi 83 maculatus 58 milleri 87 evelynae 78 variatus 55 xiphidium meyeri couchianus gordoni Belonesox belizanus 97 Heterophallus milleri Heterophallus rachovii luma eurystoma sexradiata geiseri heterochir holbrooki affinis sp. LLSTC hispaniolae melapleura wrayi Fig. S2. Maximum likelihood bootstrap support percentages mapped onto the ML tree nicaraguensis manni hubbsi yucatana puncticulata oligosticta caymanensis zarskei 11 rhizophorae punctata 18 atrora hurtadoi vittata panuco marshi