Evolution of the nitric oxide synthase family in vertebrates and novel insights in gill development.

Nitric oxide (NO) is an ancestral key signalling molecule essential for life and has enormous versatility in biological systems, including cardiovascular homeostasis, neurotransmission and immunity. Although our knowledge of NO synthases (Nos), the enzymes that synthesize NO in vivo, is substantial, the origin of a large and diversified repertoire of nos gene orthologues in fishes with respect to tetrapods remains a puzzle. The recent identification of nos3 in the ray-finned fish spotted gar, which was considered lost in this lineage, changed this perspective. This finding prompted us to explore nos gene evolution, surveying vertebrate species representing key evolutionary nodes. This study provides noteworthy findings: first, nos2 experienced several lineage-specific gene duplications and losses. Second, nos3 was found to be lost independently in two different teleost lineages, Elopomorpha and Clupeocephala. Third, the expression of at least one nos paralogue in the gills of developing shark, bichir, sturgeon, and gar, but not in lamprey, suggests that nos expression in this organ may have arisen in the last common ancestor of gnathostomes. These results provide a framework for continuing research on nos genes' roles, highlighting subfunctionalization and reciprocal loss of function that occurred in different lineages during vertebrate genome duplications.

The bowfin genome illuminates the developmental evolution of ray-finned fishes.

The bowfin (Amia calva) is a ray-finned fish that possesses a unique suite of ancestral and derived phenotypes, which are key to understanding vertebrate evolution. The phylogenetic position of bowfin as a representative of neopterygian fishes, its archetypical body plan and its unduplicated and slowly evolving genome make bowfin a central species for the genomic exploration of ray-finned fishes. Here we present a chromosome-level genome assembly for bowfin that enables gene-order analyses, settling long-debated neopterygian phylogenetic relationships. We examine chromatin accessibility and gene expression through bowfin development to investigate the evolution of immune, scale, respiratory and fin skeletal systems and identify hundreds of gene-regulatory loci conserved across vertebrates. These resources connect developmental evolution among bony fishes, further highlighting the bowfin's importance for illuminating vertebrate biology and diversity in the genomic era.

Fatty acid-binding protein genes of the ancient, air-breathing, ray-finned fish, spotted gar (Lepisosteus oculatus).

With the advent of high-throughput DNA sequencing technology, the genomic sequence of many disparate species has led to the relatively new discipline of genomics, the study of genome structure, function and evolution. Much work has been focused on the role of whole genome duplications (WGD) in the architecture of extant vertebrate genomes, particularly those of teleost fishes which underwent a WGD early in the teleost radiation >230 million years ago (mya). Our past work has focused on the fate of duplicated copies of a multigene family coding for the intracellular lipid-binding protein (iLBP) genes in the teleost fishes. To define the evolutionary processes that determined the fate of duplicated genes and generated the structure of extant fish genomes, however, requires comparative genomic analysis with a fish lineage that diverged before the teleost WGD, such as the spotted gar (Lepisosteus oculatus), an ancient, air-breathing, ray-finned fish. Here, we describe the genomic organization, chromosomal location and tissue-specific expression of a subfamily of the iLBP genes that code for fatty acid-binding proteins (Fabps) in spotted gar. Based on this work, we have defined the minimum suite of fabp genes prior to their duplication in the teleost lineages ~230-400 mya. Spotted gar, therefore, serves as an appropriate outgroup, or ancestral/ancient fish, that did not undergo the teleost-specific WGD. As such, analyses of the spatio-temporal regulation of spotted gar genes provides a foundation to determine whether the duplicated fabp genes have been retained in teleost genomes owing to either sub- or neofunctionalization.

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.

Genome evolution and meiotic maps by massively parallel DNA sequencing: spotted gar, an outgroup for the teleost genome duplication.

Genomic resources for hundreds of species of evolutionary, agricultural, economic, and medical importance are unavailable due to the expense of well-assembled genome sequences and difficulties with multigenerational studies. Teleost fish provide many models for human disease but possess anciently duplicated genomes that sometimes obfuscate connectivity. Genomic information representing a fish lineage that diverged before the teleost genome duplication (TGD) would provide an outgroup for exploring the mechanisms of evolution after whole-genome duplication. We exploited massively parallel DNA sequencing to develop meiotic maps with thrift and speed by genotyping F(1) offspring of a single female and a single male spotted gar (Lepisosteus oculatus) collected directly from nature utilizing only polymorphisms existing in these two wild individuals. Using Stacks, software that automates the calling of genotypes from polymorphisms assayed by Illumina sequencing, we constructed a map containing 8406 markers. RNA-seq on two map-cross larvae provided a reference transcriptome that identified nearly 1000 mapped protein-coding markers and allowed genome-wide analysis of conserved synteny. Results showed that the gar lineage diverged from teleosts before the TGD and its genome is organized more similarly to that of humans than teleosts. Thus, spotted gar provides a critical link between medical models in teleost fish, to which gar is biologically similar, and humans, to which gar is genomically similar. Application of our F(1) dense mapping strategy to species with no prior genome information promises to facilitate comparative genomics and provide a scaffold for ordering the numerous contigs arising from next generation genome sequencing.

Chloride inhibition of nitrite uptake for non-teleost Actinopterygiian fishes.

Fish that transport environmental chloride with a gill uptake mechanism (gill epithelial Cl(-)/HCO(3)(-)cotransport exchange system), also transport nitrite into plasma through the same mechanism. Because of the relationship between nitrite uptake and the gill chloride uptake mechanism, nitrite uptake can provide insight regarding the method of chloride uptake for fish. This study was designed to determine if non-teleost fishes concentrate nitrite in their plasma, and to determine if chloride inhibits nitrite uptake in non-teleost fish. To determine if bowfin Amia calva, spotted gar Lepisosteus oculatus, alligator gar Atractosteus spatula, and paddlefish Polyodon spathula concentrate environmental nitrite in their plasma, individuals were exposed to concentrations of 0, 1, 10, or 100 mg/L nitrite-N. After exposure, all species had plasma nitrite-N concentrations greater than environmental levels. To determine if chloride inhibits nitrite uptake for spotted gar, alligator gar, and paddlefish, fish were exposed to 1 mg/L nitrite-N and 20 mg/L chloride as calcium chloride, or to 1 mg/L nitrite-N only. Chloride effectively prevented nitrite from being concentrated in the plasma of all species. It appears that non-teleost fish concentrate nitrite in their plasma via their chloride uptake mechanism and that this is an ancestral characteristic for teleost.