Ancient fish lineages illuminate toll-like receptor diversification in early vertebrate evolution.

Since its initial discovery over 50 years ago, understanding the evolution of the vertebrate RAG- mediated adaptive immune response has been a major area of research focus for comparative geneticists. However, how the evolutionary novelty of an adaptive immune response impacted the diversity of receptors associated with the innate immune response has received considerably less attention until recently. Here, we investigate the diversification of vertebrate toll-like receptors (TLRs), one of the most ancient and well conserved innate immune receptor families found across the Tree of Life, integrating genomic data that represent all major vertebrate lineages with new transcriptomic data from Polypteriformes, the earliest diverging ray-finned fish lineage. Our analyses reveal TLR sequences that reflect the 6 major TLR subfamilies, TLR1, TLR3, TLR4, TLR5, TLR7, and TLR11, and also currently unnamed, yet phylogenetically distinct TLR clades. We additionally recover evidence for a pulse of gene gain coincident with the rise of the RAG-mediated adaptive immune response in jawed vertebrates, followed by a period of rapid gene loss during the Cretaceous. These gene losses are primarily concentrated in marine teleost fish and synchronous with the mid Cretaceous anoxic event, a period of rapid extinction for marine species. Finally, we reveal a mismatch between phylogenetic placement and gene nomenclature for up to 50% of TLRs found in clades such as ray-finned fishes, cyclostomes, amphibians, and elasmobranchs. Collectively, these results provide an unparalleled perspective of TLR diversity and offer a ready framework for testing gene annotations in non-model species.

A chromosome-level genome assembly of longnose gar, Lepisosteus osseus.

Holosteans (gars and bowfins) represent the sister lineage to teleost fishes, the latter being a clade that comprises over half of all living vertebrates and includes important models for comparative genomics and human health. A major distinction between the evolutionary history of teleosts and holosteans is that all teleosts experienced a genome duplication event in their early evolutionary history. As the teleost genome duplication occurred after teleosts diverged from holosteans, holosteans have been heralded as a means to bridge teleost models to other vertebrate genomes. However, only three species of holosteans have been genome-sequenced to date, and sequencing of more species is needed to fill sequence sampling gaps and provide a broader comparative basis for understanding holostean genome evolution. Here we report the first high quality reference genome assembly and annotation of the longnose gar (Lepisosteus osseus). Our final assembly consists of 22,709 scaffolds with a total length of 945 bp with contig N50 of 116.61 kb. Using BRAKER2, we annotated a total of 30,068 genes. Analysis of the repetitive regions of the genome reveals the genome to contain 29.12% transposable elements, and the longnose gar to be the only other known vertebrate outside of the spotted gar and bowfin to contain CR1, L2, Rex1, and Babar. These results highlight the potential utility of holostean genomes for understanding the evolution of vertebrate repetitive elements, and provide a critical reference for comparative genomic studies utilizing ray-finned fish models.

A highly diverse set of novel immunoglobulin-like transcript (NILT) genes in zebrafish indicates a wide range of functions with complex relationships to mammalian receptors.

Multiple novel immunoglobulin-like transcripts (NILTs) have been identified from salmon, trout, and carp. NILTs typically encode activating or inhibitory transmembrane receptors with extracellular immunoglobulin (Ig) domains. Although predicted to provide immune recognition in ray-finned fish, we currently lack a definitive framework of NILT diversity, thereby limiting our predictions for their evolutionary origin and function. In order to better understand the diversity of NILTs and their possible roles in immune function, we identified five NILT loci in the Atlantic salmon (Salmo salar) genome, defined 86 NILT Ig domains within a 3-Mbp region of zebrafish (Danio rerio) chromosome 1, and described 41 NILT Ig domains as part of an alternative haplotype for this same genomic region. We then identified transcripts encoded by 43 different NILT genes which reflect an unprecedented diversity of Ig domain sequences and combinations for a family of non-recombining receptors within a single species. Zebrafish NILTs include a sole putative activating receptor but extensive inhibitory and secreted forms as well as membrane-bound forms with no known signaling motifs. These results reveal a higher level of genetic complexity, interindividual variation, and sequence diversity for NILTs than previously described, suggesting that this gene family likely plays multiple roles in host immunity.

Transcriptome annotation reveals minimal immunogenetic diversity among Wyoming toads, Anaxyrus baxteri.

Briefly considered extinct in the wild, the future of the Wyoming toad (Anaxyrus baxteri) continues to rely on captive breeding to supplement the wild population. Given its small natural geographic range and history of rapid population decline at least partly due to fungal disease, investigation of the diversity of key receptor families involved in the host immune response represents an important conservation need. Population decline may have reduced immunogenetic diversity sufficiently to increase the vulnerability of the species to infectious diseases. Here we use comparative transcriptomics to examine the diversity of toll-like receptors and major histocompatibility complex (MHC) sequences across three individual Wyoming toads. We find reduced diversity at MHC genes compared to bufonid species with a similar history of bottleneck events. Our data provide a foundation for future studies that seek to evaluate the genetic diversity of Wyoming toads, identify biomarkers for infectious disease outcomes, and guide breeding strategies to increase genomic variability and wild release successes.

Single-minded 2 is required for left-right asymmetric stomach morphogenesis.

The morphogenesis of left-right (LR) asymmetry is a crucial phase of organogenesis. In the digestive tract, the development of anatomical asymmetry is first evident in the leftward curvature of the stomach. To elucidate the molecular events that shape this archetypal laterality, we performed transcriptome analyses of the left versus right sides of the developing stomach in frog embryos. Besides the known LR gene pitx2, the only gene found to be expressed asymmetrically throughout all stages of curvature was single-minded 2 (sim2), a Down Syndrome-related transcription factor and homolog of a Drosophila gene (sim) required for LR asymmetric looping of the fly gut. We demonstrate that sim2 functions downstream of LR patterning cues to regulate key cellular properties and behaviors in the left stomach epithelium that drive asymmetric curvature. Our results reveal unexpected convergent cooption of single-minded genes during the evolution of LR asymmetric morphogenesis, and have implications for dose-dependent roles of laterality factors in non-laterality-related birth defects.

Holosteans contextualize the role of the teleost genome duplication in promoting the rise of evolutionary novelties in the ray-finned fish innate immune system.

Over 99% of ray-finned fishes (Actinopterygii) are teleosts, a clade that comprises half of all living vertebrate species that have diversified across virtually all fresh and saltwater ecosystems. This ecological breadth raises the question of how the immunogenetic diversity required to persist under heterogeneous pathogen pressures evolved. The teleost genome duplication (TGD) has been hypothesized as the evolutionary event that provided the substrate for rapid genomic evolution and innovation. However, studies of putative teleost-specific innate immune receptors have been largely limited to comparisons either among teleosts or between teleosts and distantly related vertebrate clades such as tetrapods. Here we describe and characterize the receptor diversity of two clustered innate immune gene families in the teleost sister lineage: Holostei (bowfin and gars). Using genomic and transcriptomic data, we provide a detailed investigation of the phylogenetic history and conserved synteny of gene clusters encoding diverse immunoglobulin domain-containing proteins (DICPs) and novel immune-type receptors (NITRs). These data demonstrate an ancient linkage of DICPs to the major histocompatibility complex (MHC) and reveal an evolutionary origin of NITR variable-joining (VJ) exons that predate the TGD by at least 50 million years. Further characterizing the receptor diversity of Holostean DICPs and NITRs illuminates a sequence diversity that rivals the diversity of these innate immune receptor families in many teleosts. Taken together, our findings provide important historical context for the evolution of these gene families that challenge prevailing expectations concerning the consequences of the TGD during actinopterygiian evolution.

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.

Transcriptome Ortholog Alignment Sequence Tools (TOAST) for phylogenomic dataset assembly.

BACKGROUND: Advances in next-generation sequencing technologies have reduced the cost of whole transcriptome analyses, allowing characterization of non-model species at unprecedented levels. The rapid pace of transcriptomic sequencing has driven the public accumulation of a wealth of data for phylogenomic analyses, however lack of tools aimed towards phylogeneticists to efficiently identify orthologous sequences currently hinders effective harnessing of this resource. RESULTS: We introduce TOAST, an open source R software package that can utilize the ortholog searches based on the software Benchmarking Universal Single-Copy Orthologs (BUSCO) to assemble multiple sequence alignments of orthologous loci from transcriptomes for any group of organisms. By streamlining search, query, and alignment, TOAST automates the generation of locus and concatenated alignments, and also presents a series of outputs from which users can not only explore missing data patterns across their alignments, but also reassemble alignments based on user-defined acceptable missing data levels for a given research question. CONCLUSIONS: TOAST provides a comprehensive set of tools for assembly of sequence alignments of orthologs for comparative transcriptomic and phylogenomic studies. This software empowers easy assembly of public and novel sequences for any target database of candidate orthologs, and fills a critically needed niche for tools that enable quantification and testing of the impact of missing data. As open-source software, TOAST is fully customizable for integration into existing or novel custom informatic pipelines for phylogenomic inference. Software, a detailed manual, and example data files are available through github carolinafishes.github.io.

Spotted Gar and the Evolution of Innate Immune Receptors.

The resolution of the gar genome affords an opportunity to examine the diversification and functional specialization of immune effector molecules at a distant and potentially informative point in phylogenetic development. Although innate immunity is effected by a particularly large number of different families of molecules, the focus here is to provide detailed characterization of several families of innate receptors that are encoded in large multigene families, for which orthologous forms can be identified in other species of bony fish but not in other vertebrate groups as well as those for which orthologs are present in other vertebrate species. The results indicate that although teleost fish and the gar, as a holostean reference species, share gene families thought previously to be restricted to the teleost fish, the manner in which the members of the multigene families of innate immune receptors have undergone diversification is different in these two major phylogenetic radiations. It appears that both the total genome duplication and different patterns of genetic selection have influenced the derivation and stabilization of innate immune genes in a substantial manner during the course of vertebrate evolution.

Alternative haplotypes of antigen processing genes in zebrafish diverged early in vertebrate evolution.

Antigen processing and presentation genes found within the MHC are among the most highly polymorphic genes of vertebrate genomes, providing populations with diverse immune responses to a wide array of pathogens. Here, we describe transcriptome, exome, and whole-genome sequencing of clonal zebrafish, uncovering the most extensive diversity within the antigen processing and presentation genes of any species yet examined. Our CG2 clonal zebrafish assembly provides genomic context within a remarkably divergent haplotype of the core MHC region on chromosome 19 for six expressed genes not found in the zebrafish reference genome: mhc1uga, proteasome-ß 9b (psmb9b), psmb8f, and previously unknown genes psmb13b, tap2d, and tap2e We identify ancient lineages for Psmb13 within a proteasome branch previously thought to be monomorphic and provide evidence of substantial lineage diversity within each of three major trifurcations of catalytic-type proteasome subunits in vertebrates: Psmb5/Psmb8/Psmb11, Psmb6/Psmb9/Psmb12, and Psmb7/Psmb10/Psmb13. Strikingly, nearby tap2 and MHC class I genes also retain ancient sequence lineages, indicating that alternative lineages may have been preserved throughout the entire MHC pathway since early diversification of the adaptive immune system ∼500 Mya. Furthermore, polymorphisms within the three MHC pathway steps (antigen cleavage, transport, and presentation) are each predicted to alter peptide specificity. Lastly, comparative analysis shows that antigen processing gene diversity is far more extensive than previously realized (with ancient coelacanth psmb8 lineages, shark psmb13, and tap2t and psmb10 outside the teleost MHC), implying distinct immune functions and conserved roles in shaping MHC pathway evolution throughout vertebrates.

The confounding complexity of innate immune receptors within and between teleost species.

Teleost genomes encode multiple multigene families of immunoglobulin domain-containing innate immune receptors (IIIRs) with unknown function and no clear mammalian orthologs. However, the genomic organization of IIIR gene clusters and the structure and signaling motifs of the proteins they encode are similar to those of mammalian innate immune receptor families such as the killer cell immunoglobulin-like receptors (KIRs), leukocyte immunoglobulin-like receptors (LILRs), Fc receptors, triggering receptors expressed on myeloid cells (TREMs) and CD300s. Teleost IIIRs include novel immune-type receptors (NITRs); diverse immunoglobulin domain containing proteins (DICPs); polymeric immunoglobulin receptor-like proteins (PIGRLs); novel immunoglobulin-like transcripts (NILTs) and leukocyte immune-type receptors (LITRs). The accumulation of genomic sequence data has revealed that IIIR gene clusters in zebrafish display haplotypic and gene content variation. This intraspecific genetic variation, as well as significant interspecific variation, frequently confounds the identification of definitive orthologous IIIR sequences between teleost species. Nevertheless, by defining which teleost lineages encode (and do not encode) different IIIR families, predictions can be made about the presence (or absence) of specific IIIR families in each teleost lineage. It is anticipated that further investigations into available genomic resources and the sequencing of a variety of multiple teleost genomes will identify additional IIIR families and permit the modeling of the evolutionary origins of IIIRs.

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 identification of additional zebrafish DICP genes reveals haplotype variation and linkage to MHC class I genes.

Bony fish encode multiple multi-gene families of membrane receptors that are comprised of immunoglobulin (Ig) domains and are predicted to function in innate immunity. One of these families, the diverse immunoglobulin (Ig) domain-containing protein (DICP) genes, maps to three chromosomal loci in zebrafish. Most DICPs possess one or two Ig ectodomains and include membrane-bound and secreted forms. Membrane-bound DICPs include putative inhibitory and activating receptors. Recombinant DICP Ig domains bind lipids with varying specificity, a characteristic shared with mammalian CD300 and TREM family members. Numerous DICP transcripts amplified from different lines of zebrafish did not match the zebrafish reference genome sequence suggesting polymorphic and haplotypic variation. The expression of DICPs in three different lines of zebrafish has been characterized employing PCR-based strategies. Certain DICPs exhibit restricted expression in adult tissues whereas others are expressed ubiquitously. Transcripts of a subset of DICPs can be detected during embryonic development suggesting roles in embryonic immunity or other developmental processes. Transcripts representing 11 previously uncharacterized DICP sequences were identified. The assignment of two of these sequences to an unplaced genomic scaffold resulted in the identification of an alternative DICP haplotype that is linked to a MHC class I Z lineage haplotype on zebrafish chromosome 3. The linkage of DICP and MHC class I genes also is observable in the genomes of the related grass carp (Ctenopharyngodon idellus) and common carp (Cyprinus carpio) suggesting that this is a shared character with the last common Cyprinidae ancestor.

Multigene families of immunoglobulin domain-containing innate immune receptors in zebrafish: deciphering the differences.

Five large multigene families encoding innate-type immune receptors that are comprised of immunoglobulin domains have been identified in bony fish, of which four do not possess definable mammalian orthologs. The members of some of the multigene families exhibit unusually extensive patterns of divergence and the individual family members demonstrate marked variation in interspecific comparisons. As a group, the gene families reveal striking differences in domain type and content, mechanisms of intracellular signaling, basic structural features, haplotype and allelic variation and ligand binding. The potential functional roles of these innate immune receptors, their relationships to immune genes in higher vertebrate species and the basis for their adaptive evolution are of broad interest. Ongoing investigations are expected to provide new insight into alternative mechanisms of immunity.