Ingo Braasch

The African coelacanth genome provides insights into tetrapod evolution.

The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.

The Ghost of Selection Past: Rates of Evolution and Functional Divergence of Anciently Duplicated Genes

Abstract. The duplication of genes and even complete genomes may be a prerequisite for major evolutionary transitions and the origin of evolutionary novelties. However, the evolutionary mechanisms of gene evolution and the origin of novel gene functions after gene duplication have been a subject of many debates. Recently, we compiled 26 groups of orthologous genes, which included one gene from human, mouse, and chicken, one or two genes from the tetraploid Xenopus and two genes from zebrafish. Comparative analysis and mapping data showed that these pairs of zebrafish genes were probably produced during a fish-specific genome duplication that occurred between 300 and 450 Mya, before the teleost radiation (Taylor et al. 2001). As discussed here, many of these retained duplicated genes code for DNA binding proteins. Different models have been developed to explain the retention of duplicated genes and in particular the subfunctionalization model of Force et al. (1999) could explain why so many developmental control genes have been retained. Other models are harder to reconcile with this particular set of duplicated genes. Most genes seem to have been subjected to strong purifying selection, keeping properties such as charge and polarity the same in both duplicates, although some evidence was found for positive Darwinian selection, in particular for Hox genes. However, since only the cumulative pattern of nucleotide substitutions can be studied, clear indications of positive Darwinian selection or neutrality may be hard to find for such anciently duplicated genes. Nevertheless, an increase in evolutionary rate in about half of the duplicated genes seems to suggest that either positive Darwinian selection has occurred or that functional constraints have been relaxed at one point in time during functional divergence.

The spotted gar genome illuminates vertebrate evolution and facilitates human-teleost comparisons

To connect human biology to fish biomedical models, we sequenced the genome of spotted gar (Lepisosteus oculatus), whose lineage diverged from teleosts before teleost genome duplication (TGD). The slowly evolving gar genome has conserved in content and size many entire chromosomes from bony vertebrate ancestors. Gar bridges teleosts to tetrapods by illuminating the evolution of immunity, mineralization and development (mediated, for example, by Hox, ParaHox and microRNA genes). Numerous conserved noncoding elements (CNEs; often cis regulatory) undetectable in direct human-teleost comparisons become apparent using gar: functional studies uncovered conserved roles for such cryptic CNEs, facilitating annotation of sequences identified in human genome-wide association studies. Transcriptomic analyses showed that the sums of expression domains and expression levels for duplicated teleost genes often approximate the patterns and levels of expression for gar genes, consistent with subfunctionalization. The gar genome provides a resource for understanding evolution after genome duplication, the origin of vertebrate genomes and the function of human regulatory sequences.

The genome of the platyfish, Xiphophorus maculatus , provides insights into evolutionary adaptation and several complex traits

Several attributes intuitively considered to be typical mammalian features, such as complex behavior, live birth and malignant disease such as cancer, also appeared several times independently in lower vertebrates. The genetic mechanisms underlying the evolution of these elaborate traits are poorly understood. The platyfish, X. maculatus, offers a unique model to better understand the molecular biology of such traits. We report here the sequencing of the platyfish genome. Integrating genome assembly with extensive genetic maps identified an unexpected evolutionary stability of chromosomes in fish, in contrast to in mammals. Genes associated with viviparity show signatures of positive selection, identifying new putative functional domains and rare cases of parallel evolution. We also find that genes implicated in cognition show an unexpectedly high rate of duplicate gene retention after the teleost genome duplication event, suggesting a hypothesis for the evolution of the behavioral complexity in fish, which exceeds that found in amphibians and reptiles.

Multiple Sex-Associated Regions and a Putative Sex Chromosome in Zebrafish Revealed by RAD Mapping and Population Genomics

Within vertebrates, major sex determining genes can differ among taxa and even within species. In zebrafish (Danio rerio), neither heteromorphic sex chromosomes nor single sex determination genes of large effect, like Sry in mammals, have yet been identified. Furthermore, environmental factors can influence zebrafish sex determination. Although progress has been made in understanding zebrafish gonad differentiation (e.g. the influence of germ cells on gonad fate), the primary genetic basis of zebrafish sex determination remains poorly understood. To identify genetic loci associated with sex, we analyzed F2 offspring of reciprocal crosses between Oregon *AB and Nadia (NA) wild-type zebrafish stocks. Genome-wide linkage analysis, using more than 5,000 sequence-based polymorphic restriction site associated (RAD-tag) markers and population genomic analysis of more than 30,000 single nucleotide polymorphisms in our *ABxNA crosses revealed a sex-associated locus on the end of the long arm of chr-4 for both cross families, and an additional locus in the middle of chr-3 in one cross family. Additional sequencing showed that two SNPs in dmrt1 previously suggested to be functional candidates for sex determination in a cross of ABxIndia wild-type zebrafish, are not associated with sex in our AB fish. Our data show that sex determination in zebrafish is polygenic and that different genes may influence sex determination in different strains or that different genes become more important under different environmental conditions. The association of the end of chr-4 with sex is remarkable because, unique in the karyotype, this chromosome arm shares features with known sex chromosomes: it is highly heterochromatic, repetitive, late replicating, and has reduced recombination. Our results reveal that chr-4 has functional and structural properties expected of a sex chromosome.

Evolution of pigment synthesis pathways by gene and genome duplication in fish.

BackgroundColoration and color patterning belong to the most diverse phenotypic traits in animals. Particularly, teleost fishes possess more pigment cell types than any other group of vertebrates. As the result of an ancient fish-specific genome duplication (FSGD), teleost genomes might contain more copies of genes involved in pigment cell development than tetrapods. No systematic genomic inventory allowing to test this hypothesis has been drawn up so far for pigmentation genes in fish, and almost nothing is known about the evolution of these genes in different fish lineages.ResultsUsing a comparative genomic approach including phylogenetic reconstructions and synteny analyses, we have studied two major pigment synthesis pathways in teleost fish, the melanin and the pteridine pathways, with respect to different types of gene duplication. Genes encoding three of the four enzymes involved in the synthesis of melanin from tyrosine have been retained as duplicates after the FSGD. In the pteridine pathway, two cases of duplicated genes originating from the FSGD as well as several lineage-specific gene duplications were observed. In both pathways, genes encoding the rate-limiting enzymes, tyrosinase and GTP-cyclohydrolase I (GchI), have additional paralogs in teleosts compared to tetrapods, which have been generated by different modes of duplication. We have also observed a previously unrecognized diversity of gchI genes in vertebrates. In addition, we have found evidence for divergent resolution of duplicated pigmentation genes, i.e., differential gene loss in divergent teleost lineages, particularly in the tyrosinase gene family.ConclusionMainly due to the FSGD, teleost fishes apparently have a greater repertoire of pigment synthesis genes than any other vertebrate group. Our results support an important role of the FSGD and other types of duplication in the evolution of pigmentation in fish.

Many genes in fish have species-specific asymmetric rates of molecular evolution

BackgroundGene and genome duplication events increase the amount of genetic material that might then contribute to an increase in the genomic and phenotypic complexity of organisms during evolution. Thus, it has been argued that there is a relationship between gene copy number and morphological complexity and/or species diversity. This hypothesis implies that duplicated genes have subdivided or evolved novel functions compared to their pre-duplication proto-orthologs. Such a functional divergence might be caused by an increase in evolutionary rates in one ortholog, by changes in expression, regulatory evolution, insertion of repetitive elements, or due to positive Darwinian selection in one copy. We studied a set of 2466 genes that were present in Danio rerio, Takifugu rubripes, Tetraodon nigroviridis and Oryzias latipes to test (i) for forces of positive Darwinian selection; (ii) how frequently duplicated genes are retained, and (iii) whether novel gene functions might have evolved.Results25% (610) of all investigated genes show significantly smaller or higher genetic distances in the genomes of particular fish species compared to their human ortholog than their orthologs in other fish according to relative rate tests. We identified 49 new paralogous pairs of duplicated genes in fish, in which one of the paralogs is under positive Darwinian selection and shows a significantly higher rate of molecular evolution in one of the four fish species, whereas the other copy apparently did not undergo adaptive changes since it retained the original rate of evolution. Among the genes under positive Darwinian selection, we found a surprisingly high number of ATP binding proteins and transcription factors.ConclusionThe significant rate difference suggests that the function of these rate-changed genes might be essential for the respective fish species. We demonstrate that the measurement of positive selection is a powerful tool to identify divergence rates of duplicated genes and that this method has the capacity to identify potentially interesting candidates for adaptive gene evolution.

The Endothelin System: Evolution of Vertebrate-Specific Ligand–Receptor Interactions by Three Rounds of Genome Duplication

Morphological innovations like the acquisition of the neural crest as well as gene family expansions by genome duplication are considered as major leaps in the evolution of the vertebrate lineage. Using comparative genomic analyses, we have reconstructed the evolutionary history of the endothelin system, a signaling pathway consisting of endothelin ligands and their G protein-coupled receptors. The endothelin system plays a key role in cardiovascular regulation as well as in the development of diverse neural crest derivatives like pigment cells and craniofacial bone structures, which are hot spots of diversity in vertebrates. However, little is known about the origin and evolution of the endothelin system in the vertebrate lineage. We show that the endothelin core system, that is, endothelin ligands (Edn) and their receptors (Ednr), is a vertebrate-specific innovation. The components of the endothelin core system in modern vertebrate genomes date back to single genes that have been duplicated during whole-genome duplication events. After two rounds of genome duplication during early vertebrate evolution, the endothelin system of an ancestral gnathostome consisted of four ligand and four receptor genes. The previously unknown fourth endothelin ligand Edn4 has been kept in teleost fish but lost in tetrapods. Bony vertebrates generally possess three receptor genes, EdnrA, EdnrB1, and EdnrB2. EdnrB2 has been lost secondarily in the mammalian lineage from a chromosome that gave rise to the sex chromosomes in therians (marsupials and placentals). The endothelin system of fishes was further expanded by a fish-specific genome duplication and duplicated edn2, edn3, ednrA, and ednrB1 genes have been retained in teleost fishes. Functional divergence analyses suppose that following each round of genome duplication, coevolution of ligands and their binding regions in the receptors has occurred, adjusting the endothelin signaling system to the increase of possible ligand-receptor interactions. Furthermore, duplications of genes involved in the endothelin system are associated with functional specialization for the development of particular neural crest derivatives. Our results support an important role for newly emerging ligands and receptors as components of signaling pathways and their expansion through genome duplications in the evolution of the vertebrate neural crest.

Deep conservation of wrist and digit enhancers in fish

Significance The fossil record shows that the wrist and digits have an aquatic origin, becoming recognizable in a group of (mostly extinct) fish that contained robust fins. Do the fins of living fishes have the equivalent of these structures? Because comparisons of fin and limb morphology have been inconclusive, we sought to investigate this question using developmental and molecular data. By utilizing a nonmodel fish (the spotted gar), we find that the regulatory networks that control “wrist and digit”-building genes (Hox) are deeply conserved between fish and tetrapods. The genomic architecture described here defines Hox gene activity in fins and limbs as equivalent, in turn suggesting equivalence between the distal bones of fish fins and the wrist and/or digits of tetrapods. There is no obvious morphological counterpart of the autopod (wrist/ankle and digits) in living fishes. Comparative molecular data may provide insight into understanding both the homology of elements and the evolutionary developmental mechanisms behind the fin to limb transition. In mouse limbs the autopod is built by a “late” phase of Hoxd and Hoxa gene expression, orchestrated by a set of enhancers located at the 5′ end of each cluster. Despite a detailed mechanistic understanding of mouse limb development, interpretation of Hox expression patterns and their regulation in fish has spawned multiple hypotheses as to the origin and function of “autopod” enhancers throughout evolution. Using phylogenetic footprinting, epigenetic profiling, and transgenic reporters, we have identified and functionally characterized hoxD and hoxA enhancers in the genomes of zebrafish and the spotted gar, Lepisosteus oculatus, a fish lacking the whole genome duplication of teleosts. Gar and zebrafish “autopod” enhancers drive expression in the distal portion of developing zebrafish pectoral fins, and respond to the same functional cues as their murine orthologs. Moreover, gar enhancers drive reporter gene expression in both the wrist and digits of mouse embryos in patterns that are nearly indistinguishable from their murine counterparts. These functional genomic data support the hypothesis that the distal radials of bony fish are homologous to the wrist and/or digits of tetrapods.

Pigmentation Pathway Evolution after Whole-Genome Duplication in Fish

Whole-genome duplications (WGDs) have occurred repeatedly in the vertebrate lineage, but their evolutionary significance for phenotypic evolution remains elusive. Here, we have investigated the impact of the fish-specific genome duplication (FSGD) on the evolution of pigmentation pathways in teleost fishes. Pigmentation and color patterning are among the most diverse traits in teleosts, and their pigmentary system is the most complex of all vertebrate groups. Using a comparative genomic approach including phylogenetic and synteny analyses, the evolution of 128 vertebrate pigmentation genes in five teleost genomes following the FSGD has been reconstructed. We show that pigmentation genes have been preferentially retained in duplicate after the FSGD, so that teleosts have 30% more pigmentation genes compared with tetrapods. This is significantly higher than genome-wide estimates of FSGD gene duplicate retention in teleosts. Large parts of the melanocyte regulatory network have been retained in two copies after the FSGD. Duplicated pigmentation genes follow general evolutionary patterns such as the preservation of protein complex stoichiometries and the overrepresentation of developmental genes among retained duplicates. These results suggest that the FSGD has made an important contribution to the evolution of teleost-specific features of pigmentation, which include novel pigment cell types or the division of existing pigment cell types into distinct subtypes. Furthermore, we have observed species-specific differences in duplicate retention and evolution that might contribute to pigmentary diversity among teleosts. Our study therefore strongly supports the hypothesis that WGDs have promoted the increase of complexity and diversity during vertebrate phenotypic evolution.