Our understanding of phylogenetic relationships among bony fishes has been transformed by analysis of a small number of genes, but uncertainty remains around critical nodes. Genome-scale inferences so far have sampled a limited number of taxa and genes. Here we leveraged 144 genomes and 159 transcriptomes to investigate fish evolution with an unparalleled scale of data: >0.5 Mb from 1,105 orthologous exon sequences from 303 species, representing 66 out of 72 ray-finned fish orders. We apply phylogenetic tests designed to trace the effect of whole-genome duplication events on gene trees and find paralogy-free loci using a bioinformatics approach. Genome-wide data support the structure of the fish phylogeny, and hypothesis-testing procedures appropriate for phylogenomic datasets using explicit gene genealogy interrogation settle some long-standing uncertainties, such as the branching order at the base of the teleosts and among early euteleosts, and the sister lineage to the acanthomorph and percomorph radiations. Comprehensive fossil calibrations date the origin of all major fish lineages before the end of the Cretaceous.

The existence of males and females, which are often strikingly different in morphology, reproductive strategies and behavior, is one of the most widespread phenomena in biology. However, the genetic mechanisms that generate this ubiquitous pattern are surprisingly diverse and do not follow a phylogenetic pattern. Sex-determination mechanisms can differ between even closely related species and arise frequently and independently. Fish provide a paradigmatic example, as their sex-determination mechanisms range from environmental to different modes of genetic determination. The evolutionary meaning of this remarkable plasticity is unknown. For genetic sex determination, where the trigger for female or male development comes from the genetic constitution of the individual, the evolution of sex-determination mechanisms is connected to a very peculiar genomic process, namely the formation of sex chromosomes [1][2][3][4] .To improve understanding of the function and evolution of sex chromosomes, their genetic organization must be deciphered.However, owing to their degenerate nature and high repetitive DNA content, sex chromosomes pose almost insurmountable problems in deciphering their gene content and organization. So far, only the human 5 , chimpanzee 6 and rhesus macaque Y chromosomes 7 and the male-specific region on the Y chromosome of one fish, the medaka 8 , have been sequenced. These analyses have nevertheless provided important insights into the evolution of Y chromosomes, their genomic organization and their degeneration processes, as well as predictions as to their likely evolutionary fate 9-12 .Much less genomic information exists on W chromosomes because, as with Y chromosomes, they are predominantly highly repetitive in nature. The prevailing theory of the evolution of sex chromosomes predicts that degeneration of the heterogametic sex chromosome is a stepwise process that occurs over an extended period of time. We therefore reasoned that an evolutionarily young W chromosome Whole-genome sequence of a flatfish provides insights into ZW sex chromosome evolution and adaptation to a benthic lifestyle Genetic sex determination by W and Z chromosomes has developed independently in different groups of organisms. To better understand the evolution of sex chromosomes and the plasticity of sex-determination mechanisms, we sequenced the whole genomes of a male (ZZ) and a female (ZW) half-smooth tongue sole (Cynoglossus semilaevis). In addition to insights into adaptation to a benthic lifestyle, we find that the sex chromosomes of these fish are derived from the same ancestral vertebrate protochromosome as the avian W and Z chromosomes. Notably, the same gene on the Z chromosome, dmrt1, which is the male-determining gene in birds, showed convergent evolution of features that are compatible with a similar function in tongue sole. Comparison of the relatively young tongue sole sex chromosomes with those of mammals and birds identified events that occurred during the early phase of sex-chromosome evolution. Pertinent to...

Environmental sex determination (ESD) occurs in divergent, phylogenetically unrelated taxa, and in some species, cooccurs with genetic sex determination (GSD) mechanisms. Although epigenetic regulation in response to environmental effects has long been proposed to be associated with ESD, a systemic analysis on epigenetic regulation of ESD is still lacking. Using half-smooth tongue sole (Cynoglossus semilaevis) as a model-a marine fish that has both ZW chromosomal GSD and temperature-dependent ESD-we investigated the role of DNA methylation in transition from GSD to ESD. Comparative analysis of the gonadal DNA methylomes of pseudomale, female, and normal male fish revealed that genes in the sex determination pathways are the major targets of substantial methylation modification during sexual reversal. Methylation modification in pseudomales is globally inherited in their ZW offspring, which can naturally develop into pseudomales without temperature incubation. Transcriptome analysis revealed that dosage compensation occurs in a restricted, methylated cytosine enriched Z chromosomal region in pseudomale testes, achieving equal expression level in normal male testes. In contrast, female-specific W chromosomal genes are suppressed in pseudomales by methylation regulation. We conclude that epigenetic regulation plays multiple crucial roles in sexual reversal of tongue sole fish. We also offer the first clues on the mechanisms behind gene dosage balancing in an organism that undergoes sexual reversal. Finally, we suggest a causal link between the bias sex chromosome assortment in the offspring of a pseudomale family and the transgenerational epigenetic inheritance of sexual reversal in tongue sole fish.

9 l e t t e r sFlatfish have the most extreme asymmetric body morphology of vertebrates. During metamorphosis, one eye migrates to the contralateral side of the skull, and this migration is accompanied by extensive craniofacial transformations and simultaneous development of lopsided body pigmentation 1-5 . The evolution of this developmental and physiological innovation remains enigmatic. Comparative genomics of two flatfish and transcriptomic analyses during metamorphosis point to a role for thyroid hormone and retinoic acid signaling, as well as phototransduction pathways. We demonstrate that retinoic acid is critical in establishing asymmetric pigmentation and, via cross-talk with thyroid hormones, in modulating eye migration. The unexpected expression of the visual opsins from the phototransduction pathway in the skin translates illumination differences and generates retinoic acid gradients that underlie the generation of asymmetry. Identifying the genetic underpinning of this unique developmental process answers long-standing questions about the evolutionary origin of asymmetry, but it also provides insight into the mechanisms that control body shape in vertebrates.