Towards complete and error-free genome assemblies of all vertebrate species.

High-quality and complete reference genome assemblies are fundamental for the application of genomics to biology, disease, and biodiversity conservation. However, such assemblies are available for only a few non-microbial species1-4. To address this issue, the international Genome 10K (G10K) consortium5,6 has worked over a five-year period to evaluate and develop cost-effective methods for assembling highly accurate and nearly complete reference genomes. Here we present lessons learned from generating assemblies for 16 species that represent six major vertebrate lineages. We confirm that long-read sequencing technologies are essential for maximizing genome quality, and that unresolved complex repeats and haplotype heterozygosity are major sources of assembly error when not handled correctly. Our assemblies correct substantial errors, add missing sequence in some of the best historical reference genomes, and reveal biological discoveries. These include the identification of many false gene duplications, increases in gene sizes, chromosome rearrangements that are specific to lineages, a repeated independent chromosome breakpoint in bat genomes, and a canonical GC-rich pattern in protein-coding genes and their regulatory regions. Adopting these lessons, we have embarked on the Vertebrate Genomes Project (VGP), an international effort to generate high-quality, complete reference genomes for all of the roughly 70,000 extant vertebrate species and to help to enable a new era of discovery across the life sciences.

Developmental constraint shaped genome evolution and erythrocyte loss in Antarctic fishes following paleoclimate change.

In the frigid, oxygen-rich Southern Ocean (SO), Antarctic icefishes (Channichthyidae; Notothenioidei) evolved the ability to survive without producing erythrocytes and hemoglobin, the oxygen-transport system of virtually all vertebrates. Here, we integrate paleoclimate records with an extensive phylogenomic dataset of notothenioid fishes to understand the evolution of trait loss associated with climate change. In contrast to buoyancy adaptations in this clade, we find relaxed selection on the genetic regions controlling erythropoiesis evolved only after sustained cooling in the SO. This pattern is seen not only within icefishes but also occurred independently in other high-latitude notothenioids. We show that one species of the red-blooded dragonfish clade evolved a spherocytic anemia that phenocopies human patients with this disease via orthologous mutations. The genomic imprint of SO climate change is biased toward erythrocyte-associated conserved noncoding elements (CNEs) rather than to coding regions, which are largely preserved through pleiotropy. The drift in CNEs is specifically enriched near genes that are preferentially expressed late in erythropoiesis. Furthermore, we find that the hematopoietic marrow of icefish species retained proerythroblasts, which indicates that early erythroid development remains intact. Our results provide a framework for understanding the interactions between development and the genome in shaping the response of species to climate change.

Bone microstructure and bone mineral density are not systemically different in Antarctic icefishes and related Antarctic notothenioids.

Ancestors of the Antarctic icefishes (family Channichthyidae) were benthic and had no swim bladder, making it energetically expensive to rise from the ocean floor. To exploit the water column, benthopelagic icefishes were hypothesized to have evolved a skeleton with "reduced bone," which gross anatomical data supported. Here, we tested the hypothesis that changes to icefish bones also occurred below the level of gross anatomy. Histology and micro-CT imaging of representative craniofacial bones (i.e., ceratohyal, frontal, dentary, and articular) of extant Antarctic fish species specifically evaluated two features that might cause the appearance of "reduced bone": bone microstructure (e.g., bone volume fraction and structure linear density) and bone mineral density (BMD, or mass of mineral per volume of bone). Measures of bone microstructure were not consistently different in bones from the icefishes Chaenocephalus aceratus and Champsocephalus gunnari, compared to the related benthic notothenioids Notothenia coriiceps and Gobionotothen gibberifrons. Some quantitative measures, such as bone volume fraction and structure linear density, were significantly increased in some icefish bones compared to homologous bones of non-icefish. However, such differences were rare, and no microstructural measures were consistently different in icefishes across all bones and species analyzed. Furthermore, BMD was similar among homologous bones of icefish and non-icefish Antarctic notothenioids. In summary, "reduced bone" in icefishes was not due to systemic changes in bone microstructure or BMD, raising the prospect that "reduced bone" in icefish occurs only at the gross anatomic level (i.e., smaller or fewer bones). Given that icefishes exhibit delayed skeletal development compared to non-icefish Antarctic fishes, combining these phenotypic data with genomic data might clarify genetic changes driving skeletal heterochrony.

Multiple independent chromosomal fusions accompanied the radiation of the Antarctic teleost genus Trematomus (Notothenioidei:Nototheniidae).

BACKGROUND: Chromosomal rearrangements are thought to be an important driving force underlying lineage diversification, but their link to speciation continues to be debated. Antarctic teleost fish of the family Nototheniidae (Notothenioidei) diversified in a changing environmental context, which led to ecological, morphological, and genetic differentiation among populations. In addition, extensive chromosomal repatterning accompanied species divergence in several clades. The most striking karyotypic changes involved the recent species radiation (about 10 My) of the genus Trematomus, with chromosomal pair numbers ranging between 29 and 12. These dramatic reductions in chromosome number resulted mostly from large-scale chromosome fusions. Multiple centric and/or tandem fusions have been hypothesized in at least seven of the twelve recognized Trematomus species. To reconstruct their evolutionary history, we employed comparative cytogenomics (BAC-FISH and chromosome painting) to reveal patterns of interspecific chromosomal orthologies across several notothenioid clades. RESULTS: We defined orthologous chromosomal segments of reference, termed Structural Units (SUs). SUs were identified in a total of 18 notothenioid species. We demonstrated for the first time that SUs were strongly conserved across every specimen examined, with chromosomal syntenies highlighting a paucity of intrachromosomal macro-rearrangements. Multiple independent fusions of these SUs were inferred in the Trematomus species, in contrast to the shared SU fusions in species of the sister lineage Notothenia. CONCLUSIONS: The SU segments were defined units of chromosomal rearrangement in the entire family Nototheiidae, which diverged from the other notothenioid families 20 My ago. Some of the identified chromosomal syntenies within the SUs were even conserved in their closest relatives, the family Eleginopsidae. Comparing the timing of acquisition of the fusions in the closely related genera Notothenia and Trematomus of the nototheniid species family, we conclude that they exhibit distinct chromosomal evolutionary histories, which may be relevant to different speciation scenarios.

Embryogenesis and early skeletogenesis in the antarctic bullhead notothen, Notothenia coriiceps.

BACKGROUND: Environmental temperature influences rates of embryonic development, but a detailed staging series for vertebrate embryos developing in the subzero cold of Antarctic waters is not yet available from fertilization to hatching. Given projected warming of the Southern Ocean, it is imperative to establish a baseline to evaluate potential effects of changing climate on fish developmental dynamics. RESULTS: We studied the Bullhead notothen (Notothenia coriiceps), a notothenioid fish inhabiting waters between -1.9 and +2 °C. In vitro fertilization produced embryos that progressed through cleavage, epiboly, gastrulation, segmentation, organogenesis, and hatching. We compared morphogenesis spatially and temporally to Zebrafish and medaka. Experimental animals hatched after about 6 months to early larval stages. To help understand skeletogenesis, we analyzed late embryos for expression of sox9 and runx2, which regulate chondrogenesis, osteogenesis, and eye development. Results revealed that, despite their prolonged developmental time course, N. coriiceps embryos developed similarly to those of other teleosts with large yolk cells. CONCLUSIONS: Our studies set the stage for future molecular analyses of development in these extremophile fish. Results provide a foundation for understanding the impact of ocean warming on embryonic development and larval recruitment of notothenioid fish, which are key factors in the marine trophic system. Developmental Dynamics 245:1066-1080, 2016. © 2016 Wiley Periodicals, Inc.

Evolution in an extreme environment: developmental biases and phenotypic integration in the adaptive radiation of antarctic notothenioids.

BACKGROUND: Over the past 40 million years water temperatures have dramatically dropped in the Southern Ocean, which has led to the local extinction of most nearshore fish lineages. The evolution of antifreeze glycoproteins in notothenioids, however, enabled these ancestrally benthic fishes to survive and adapt as temperatures reached the freezing point of seawater (-1.86 °C). Antarctic notothenioids now represent the primary teleost lineage in the Southern Ocean and are of fundamental importance to the local ecosystem. The radiation of notothenioids has been fostered by the evolution of "secondary pelagicism", the invasion of pelagic habitats, as the group diversified to fill newly available foraging niches in the water column. While elaborate craniofacial modifications have accompanied this adaptive radiation, little is known about how these morphological changes have contributed to the evolutionary success of notothenioids. RESULTS: We used a 3D-morphometrics approach to investigate patterns of morphological variation in the craniofacial skeleton among notothenioids, and show that variation in head shape is best explained by divergent selection with respect to foraging niche. We document further an accelerated rate of morphological evolution in the icefish family Channichthyidae, and show that their rapid diversification was accompanied by the evolution of relatively high levels of morphological integration. Whereas most studies suggest that extensive integration should constrain phenotypic evolution, icefish stand out as a rare example of increased integration possibly facilitating evolutionary potential. Finally, we show that the unique feeding apparatus in notothenioids in general, and icefish in particular, can be traced to shifts in early developmental patterning mechanisms and ongoing growth of the pharyngeal skeleton. CONCLUSION: Our work suggests that ecological opportunity is a major factor driving craniofacial variation in this group. Further, the observation that closely related lineages can differ dramatically in integration suggests that this trait can evolve quickly. We propose that the evolution of high levels of phenotypic integration in icefishes may be considered a key innovation that facilitated their morphological evolution and subsequent ecological expansion.

Evolution and function of the globin intergenic regulatory regions of the antarctic dragonfishes (Notothenioidei: Bathydraconidae).

As the Southern Ocean cooled to -1.8 °C over the past 40 My, the teleostean clade Notothenioidei diversified and, under reduced selection pressure for an oxygen-transporting apparatus, became less reliant on hemoglobin and red blood cells. At the extreme of this trend, the crown group of Antarctic icefishes (Channichthyidae) lost both components of oxygen transport. Under the decreased selection scenario, we hypothesized that the Antarctic dragonfishes (Bathydraconidae, the red-blooded sister clade to the icefishes) evolved lower blood hemoglobin concentrations because their globin gene complexes (α- and ß-globin gene pairs linked by a regulatory intergene) transcribe globin mRNAs less effectively than those of basal notothenioids (e.g., the Nototheniidae [notothens]). To test our hypothesis, we 1) sequenced the α/ß-intergenes of the adult globin complexes of three notothen and eight dragonfish species and 2) measured globin transcript levels in representative species from each group. The typical nototheniid intergene was ∼3-4 kb in length. The bathydraconid intergenes resolved into three subclasses (long [3.8 kb], intermediate [3.0 kb], and short [1.5-2.3 kb]) that corresponded to the three subclades proposed for the taxon. Although they varied in length due to indels, the three notothen and eight dragonfish intergenes contained a conserved ∼90-nt element that we have previously shown to be required for globin gene transcription. Using the quantitative polymerase chain reaction, we found that globin mRNA levels in red cells from one notothen species and from one species of each dragonfish subclade were equivalent statistically. Thus, our results indicate that the bathydraconids have evolved adult globin loci whose regulatory intergenes tend to be shorter than those of the more basal nototheniids yet are equivalent in transcriptional efficacy. Their low blood hemoglobin concentrations are most likely due to reduction in hematocrit.

Evolutionary mutant models for human disease.

Although induced mutations in traditional laboratory animals have been valuable as models for human diseases, they have some important limitations. Here, we propose a complementary approach to discover genes and mechanisms that might contribute to human disorders: the analysis of evolutionary mutant models in which adaptive phenotypes mimic maladaptive human diseases. If the type and mode of action of mutations favored by natural selection in wild populations are similar to those that contribute to human diseases, then studies in evolutionary mutant models have the potential to identify novel genetic factors and gene-by-environment interactions that affect human health and underlie human disease.

Adaptations and Diversity of Antarctic Fishes: A Genomic Perspective.

Antarctic notothenioid fishes are the classic example of vertebrate adaptive radiation in a marine environment. Notothenioids diversified from a single common ancestor ∼22 Mya to between 120 and 140 species today, and they represent ∼90% of fish biomass on the continental shelf of Antarctica. As they diversified in the cold Southern Ocean, notothenioids evolved numerous traits, including osteopenia, anemia, cardiomegaly, dyslipidemia, and aglomerular kidneys, that are beneficial or tolerated in their environment but are pathological in humans. Thus, notothenioids are models for understanding adaptive radiations, physiological and biochemical adaptations to extreme environments, and genetic mechanisms of human disease. Since 2014, 16 notothenioid genomes have been published, which enable a first-pass holistic analysis of the notothenioid radiation and the genetic underpinnings of novel notothenioid traits. Here, we review the notothenioid radiation from a genomic perspective and integrate our insights with recent observations from other fish radiations.

A parasite outbreak in notothenioid fish in an Antarctic fjord.

Climate changes can promote disease outbreaks, but their nature and potential impacts in remote areas have received little attention. In a hot spot of biodiversity on the West Antarctic Peninsula, which faces among the fastest changing climates on Earth, we captured specimens of two notothenioid fish species affected by large skin tumors at an incidence never before observed in the Southern Ocean. Molecular and histopathological analyses revealed that X-cell parasitic alveolates, members of a genus we call Notoxcellia, are the etiological agent of these tumors. Parasite-specific molecular probes showed that xenomas remained within the skin but largely outgrew host cells in the dermis. We further observed that tumors induced neovascularization in underlying tissue and detrimentally affected host growth and condition. Although many knowledge gaps persist about X-cell disease, including its mode of transmission and life cycle, these findings reveal potentially active biotic threats to vulnerable Antarctic ecosystems.

Molecular pedomorphism underlies craniofacial skeletal evolution in Antarctic notothenioid fishes.

BACKGROUND: Pedomorphism is the retention of ancestrally juvenile traits by adults in a descendant taxon. Despite its importance for evolutionary change, there are few examples of a molecular basis for this phenomenon. Notothenioids represent one of the best described species flocks among marine fishes, but their diversity is currently threatened by the rapidly changing Antarctic climate. Notothenioid evolutionary history is characterized by parallel radiations from a benthic ancestor to pelagic predators, which was accompanied by the appearance of several pedomorphic traits, including the reduction of skeletal mineralization that resulted in increased buoyancy. RESULTS: We compared craniofacial skeletal development in two pelagic notothenioids, Chaenocephalus aceratus and Pleuragramma antarcticum, to that in a benthic species, Notothenia coriiceps, and two outgroups, the threespine stickleback and the zebrafish. Relative to these other species, pelagic notothenioids exhibited a delay in pharyngeal bone development, which was associated with discrete heterochronic shifts in skeletal gene expression that were consistent with persistence of the chondrogenic program and a delay in the osteogenic program during larval development. Morphological analysis also revealed a bias toward the development of anterior and ventral elements of the notothenioid pharyngeal skeleton relative to dorsal and posterior elements. CONCLUSIONS: Our data support the hypothesis that early shifts in the relative timing of craniofacial skeletal gene expression may have had a significant impact on the adaptive radiation of Antarctic notothenioids into pelagic habitats.

Genome enablement of the notothenioidei: genome size estimates from 11 species and BAC libraries from 2 representative taxa.

The perciform suborder Notothenoidei provides a compelling opportunity to study the adaptive radiation of a marine species flock in the cold Southern Ocean surrounding Antarctica. To enable genome-level studies of these psychrophilic fishes, we estimated the sizes of the genomes of 11 Antarctic species and generated high-quality BAC libraries for 2, the notothen Notothenia coriiceps and the icefish Chaenocephalus aceratus. Our results indicate that evolution of phylogenetically derived notothenioid families, [e.g., the icefishes (Channichthyidae)], was accompanied by genome expansion. Species (n=6) of the basal family Nototheniidae had C values that ranged between 0.98 and 1.20 pg, whereas those of the icefishes, the notothenioid crown group, were 1.66-1.83 pg (n=4 species). The BAC libraries VMRC-19 (N. coriiceps) and VMRC-21 (C. aceratus) comprised 12X and 10X coverage of the respective genomes and had average insert sizes of 138 and 168 kb. Greater than 60% of paired BAC ends sampled from each library ( approximately 0.1% of each genome) contained repetitive sequences, and the repetitive element landscapes of the 2 genomes (13.4% of the N. coriiceps genome and 14.5% for C. aceratus) were similar. The representation and depth of coverage of the libraries were verified by identification of multiple Hox gene contigs: six discrete Hox clusters were found in N. coriiceps and at least five Hox clusters were found in C. aceratus. Given the unusual anatomical and physiological adaptations of the notothenioids, the availability of these BAC libraries sets the stage for expanded analysis of the psychrophilic mode of life.

Adaptations to environmental change: Globin superfamily evolution in Antarctic fishes.

The ancient origins and functional versatility of globins make them ideal subjects for studying physiological adaptation to environmental change. Our goals in this review are to describe the evolution of the vertebrate globin gene superfamily and to explore the structure/function relationships of hemoglobin, myoglobin, neuroglobin and cytoglobin in teleost fishes. We focus on the globins of Antarctic notothenioids, emphasizing their adaptive features as inferred from comparisons with human proteins. We dedicate this review to Guido di Prisco, our co-author, colleague, friend, and husband of C.V. Ever thoughtful, creative, and enthusiastic, Guido spearheaded study of the structure, function, and evolution of the hemoglobins of polar fishes - this review is testimony to his wide-ranging contributions. Throughout his career, Guido inspired younger scientists to embrace polar biological research, and he challenged researchers of all ages to explore evolutionary adaptation in the context of global climate change. Beyond his scientific contributions, we will miss his warmth, his culture, and his great intellect. Guido has left an outstanding legacy, one that will continue to inspire us and our research.

Author Correction: Historical contingency shapes adaptive radiation in Antarctic fishes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

From Africa to Antarctica: Exploring the Metabolism of Fish Heart Mitochondria Across a Wide Thermal Range.

The thermal sensitivity of ectotherms is largely dictated by the impact of temperature on cellular bioenergetics, particularly on mitochondrial functions. As the thermal sensitivity of bioenergetic pathways depends on the structural and kinetic properties of its component enzymes, optimization of their collective function to different thermal niches is expected to have occurred through selection. In the present study, we sought to characterize mitochondrial phenotypic adjustments to thermal niches in eight ray-finned fish species occupying a wide range of thermal habitats by comparing the activities of key mitochondrial enzymes in their hearts. We measured the activity of four enzymes that control substrate entrance into the tricarboxylic acid (TCA) cycle: pyruvate kinase (PK), pyruvate dehydrogenase complex (PDHc), carnitine palmitoyltransferase (CPT), and hydroxyacyl-CoA dehydrogenase (HOAD). We also assayed enzymes of the electron transport system (ETS): complexes I, II, I + III, and IV. Enzymes were assayed at five temperatures (5, 10, 15, 20, and 25°C). Our results showed that the activity of CPT, a gatekeeper of the fatty acid pathway, was higher in the cold-water fish than in the warmer-adapted fish relative to the ETS (complexes I and III) when measured close to the species optimal temperatures. The activity of HOAD showed a similar pattern relative to CI + III and thermal environment. By contrast, PDHc and PK did not show the similar patterns with respect to CI + III and temperature. Cold-adapted species had high CIV activities compared to those of upstream complexes (I, II, I + III) whereas the converse was true for warm-adapted species. Our findings reveal a significant variability of heart mitochondrial organization among species that can be linked to temperature adaptation. Cold-adapted fish do not appear to compensate for PDHc activity but likely adjust fatty acids oxidation through higher activities of CPT and HOAD relative to complexes I + III.

Intergeneric hybrids inform reproductive isolating barriers in the Antarctic icefish radiation.

Interspecific hybridization or barriers to hybridization may have contributed to the diversification of Antarctic icefishes (Channichthyidae), but data supporting these hypotheses is scarce. To understand the potential for hybridization and to investigate reproductive isolating mechanisms among icefish species, we performed in vitro fertilization experiments using eggs from a female blackfin icefish Chaenocephalus aceratus and sperm from a male of another genera, the ocellated icefish Chionodraco rastrospinosus. Sequencing of genomic and mitochondrial DNA confirmed the intergeneric hybrid nature of resulting embryos which successfully developed and hatched as active larvae at about four and a half months during the Antarctic winter. This result demonstrates the compatibility of gametes of these two species and the viability of resulting zygotes and larvae. Due to logistic constraints and the slow developmental rate of icefishes, we could not test for long-term hybrid viability, fertility, fitness, or hybrid breakdown. Analysis of our fishing records and available literature, however, suggests that the strongest barriers to hybridization among parapatric icefish species are likely to be behavioral and characterized by assortative mating and species-specific courtship and nesting behaviors. This conclusion suggests that, in long-lived fish species with late sexual maturity and high energetic investment in reproduction like icefishes, pre-mating barriers are energetically more efficient than post-mating barriers to prevent hybridization.

Retinal oxygen supply shaped the functional evolution of the vertebrate eye.

The retina has a very high energy demand but lacks an internal blood supply in most vertebrates. Here we explore the hypothesis that oxygen diffusion limited the evolution of retinal morphology by reconstructing the evolution of retinal thickness and the various mechanisms for retinal oxygen supply, including capillarization and acid-induced haemoglobin oxygen unloading. We show that a common ancestor of bony fishes likely had a thin retina without additional retinal oxygen supply mechanisms and that three different types of retinal capillaries were gained and lost independently multiple times during the radiation of vertebrates, and that these were invariably associated with parallel changes in retinal thickness. Since retinal thickness confers multiple advantages to vision, we propose that insufficient retinal oxygen supply constrained the functional evolution of the eye in early vertebrates, and that recurrent origins of additional retinal oxygen supply mechanisms facilitated the phenotypic evolution of improved functional eye morphology.

Antarctic blackfin icefish genome reveals adaptations to extreme environments.

Icefishes (suborder Notothenioidei; family Channichthyidae) are the only vertebrates that lack functional haemoglobin genes and red blood cells. Here, we report a high-quality genome assembly and linkage map for the Antarctic blackfin icefish Chaenocephalus aceratus, highlighting evolved genomic features for its unique physiology. Phylogenomic analysis revealed that Antarctic fish of the teleost suborder Notothenioidei, including icefishes, diverged from the stickleback lineage about 77 million years ago and subsequently evolved cold-adapted phenotypes as the Southern Ocean cooled to sub-zero temperatures. Our results show that genes involved in protection from ice damage, including genes encoding antifreeze glycoprotein and zona pellucida proteins, are highly expanded in the icefish genome. Furthermore, genes that encode enzymes that help to control cellular redox state, including members of the sod3 and nqo1 gene families, are expanded, probably as evolutionary adaptations to the relatively high concentration of oxygen dissolved in cold Antarctic waters. In contrast, some crucial regulators of circadian homeostasis (cry and per genes) are absent from the icefish genome, suggesting compromised control of biological rhythms in the polar light environment. The availability of the icefish genome sequence will accelerate our understanding of adaptation to extreme Antarctic environments.

Divergent Hemogen genes of teleosts and mammals share conserved roles in erythropoiesis: analysis using transgenic and mutant zebrafish.

Hemogen is a vertebrate transcription factor that performs important functions in erythropoiesis and testicular development and may contribute to neoplasia. Here we identify zebrafish Hemogen and show that it is considerably smaller (∼22 kDa) than its human ortholog (∼55 kDa), a striking difference that is explained by an underlying modular structure. We demonstrate that Hemogens are largely composed of 21-25 amino acid repeats, some of which may function as transactivation domains (TADs). Hemogen expression in embryonic and adult zebrafish is detected in hematopoietic, renal, neural and gonadal tissues. Using Tol2- and CRISPR/Cas9-generated transgenic zebrafish, we show that Hemogen expression is controlled by two Gata1-dependent regulatory sequences that act alone and together to control spatial and temporal expression during development. Partial depletion of Hemogen in embryos by morpholino knockdown reduces the number of erythrocytes in circulation. CRISPR/Cas9-generated zebrafish lines containing either a frameshift mutation or an in-frame deletion in a putative, C-terminal TAD display anemia and embryonic tail defects. This work expands our understanding of Hemogen and provides mutant zebrafish lines for future study of the mechanism of this important transcription factor.