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Numerous novel adaptations characterise the radiation of notothenioids, the dominant fish group in the freezing seas of the Southern Ocean. To improve understanding of the evolution of this iconic fish group, here we generate and analyse new genome assemblies for 24 species covering all major subgroups of the radiation, including five long-read assemblies. We present a new estimate for the onset of the radiation at 10.7 million years ago, based on a time-calibrated phylogeny derived from genome-wide sequence data. We identify a two-fold variation in genome size, driven by expansion of multiple transposable element families, and use the long-read data to reconstruct two evolutionarily important, highly repetitive gene family loci. First, we present the most complete reconstruction to date of the antifreeze glycoprotein gene family, whose emergence enabled survival in sub-zero temperatures, showing the expansion of the antifreeze gene locus from the ancestral to the derived state. Second, we trace the loss of haemoglobin genes in icefishes, the only vertebrates lacking functional haemoglobins, through complete reconstruction of the two haemoglobin gene clusters across notothenioid families. Both the haemoglobin and antifreeze genomic loci are characterised by multiple transposon expansions that may have driven the evolutionary history of these genes. The notothenioid radiation is a remarkable group of fish adapted to life in the icy waters of the Southern Ocean. This study investigates the evolutionary history of this group and the basis of their adaption to cold environments through genomic analysis of 24 new genome assemblies.

A fundamental question in evolutionary biology is how genetic novelty arises. De novo gene birth is a recently recognized mechanism, but the evolutionary process and function of putative de novo genes remain largely obscure. With a clear life-saving function, the diverse antifreeze proteins of polar fishes are exemplary adaptive innovations and models for investigating new gene evolution. Here, we report clear evidence and a detailed molecular mechanism for the de novo formation of the northern gadid (codfish) antifreeze glycoprotein (AFGP) gene from a minimal noncoding sequence. We constructed genomic DNA libraries for AFGP-bearing and AFGP-lacking species across the gadid phylogeny and performed fine-scale comparative analyses of the AFGP genomic loci and homologs. We identified the noncoding founder region and a nine-nucleotide (9-nt) element therein that supplied the codons for one Thr-Ala-Ala unit from which the extant repetitive AFGP-coding sequence (cds) arose through tandem duplications. The latent signal peptide (SP)-coding exons were fortuitous noncoding DNA sequence immediately upstream of the 9-nt element, which, when spliced, supplied a typical secretory signal. Through a 1-nt frameshift mutation, these two parts formed a single read-through open reading frame (ORF). It became functionalized when a putative translocation event conferred the essential cis promoter for transcriptional initiation. We experimentally proved that all genic components of the extant gadid AFGP originated from entirely non-genic DNA. The gadid AFGP evolutionary process also represents a rare example of the proto-ORF model of de novo gene birth where a fully formed ORF existed before the regulatory element to activate transcription was acquired.

Mitochondrial genomes are known for their compact size and conserved gene order, however, recent studies employing long-read sequencing technologies have revealed the presence of atypical mitogenomes in some species. In this study, we assembled and annotated the mitogenomes of five Antarctic notothenioids, including four icefishes ( Champsocephalus gunnari , C. esox , Chaenocephalus aceratus , and Pseudochaenichthys georgianus ) and the cold-specialized Trematomus borchgrevinki . Antarctic notothenioids are known to harbor some rearrangements in their mt genomes, however the extensive duplications in icefishes observed in our study have never been reported before. In the icefishes, we observed duplications of the protein coding gene ND6 , two transfer RNAs , and the control region with different copy number variants present within the same individuals and with some ND6 duplications appearing to follow the canonical Duplication-Degeneration-Complementation (DDC) model in C. esox and C. gunnari . In addition, using long-read sequencing and k-mer analysis, we were able to detect extensive heteroplasmy in C. aceratus and C. esox . We also observed a large inversion in the mitogenome of T. borchgrevinki , along with the presence of tandem repeats in its control region. This study is the first in using long-read sequencing to assemble and identify structural variants and heteroplasmy in notothenioid mitogenomes and signifies the importance of long-reads in resolving complex mitochondrial architectures. Identification of such wide-ranging structural variants in the mitogenomes of these fishes could provide insight into the genetic basis of the atypical icefish mitochondrial physiology and more generally may provide insights about their potential role in cold adaptation.

The evolutionary model escape from adaptive conflict (EAC) posits that adaptive conflict between the old and an emerging new function within a single gene could drive the fixation of gene duplication, where each duplicate can freely optimize one of the functions. Although EAC has been suggested as a common process in functional evolution, definitive cases of neofunctionalization under EAC are lacking, and the molecular mechanisms leading to functional innovation are not well-understood. We report here clear experimental evidence for EAC-driven evolution of type III antifreeze protein gene from an old sialic acid synthase ( SAS ) gene in an Antarctic zoarcid fish. We found that an SAS gene, having both sialic acid synthase and rudimentary ice-binding activities, became duplicated. In one duplicate, the N-terminal SAS domain was deleted and replaced with a nascent signal peptide, removing pleiotropic structural conflict between SAS and ice-binding functions and allowing rapid optimization of the C-terminal domain to become a secreted protein capable of noncolligative freezing-point depression. This study reveals how minor functionalities in an old gene can be transformed into a distinct survival protein and provides insights into how gene duplicates facing presumed identical selection and mutation pressures at birth could take divergent evolutionary paths.

The de novo birth of functional genes from non-coding DNA as an important contributor to new gene formation is increasingly supported by evidence from diverse eukaryotic lineages. However, many uncertainties remain, including how the incipient de novo genes would continue to evolve and the molecular mechanisms underlying their evolutionary trajectory. Here we address these questions by investigating evolutionary history of the de novo antifreeze glycoprotein (AFGP) gene and gene family in gadid (codfish) lineages. We examined AFGP phenotype on a phylogenetic framework encompassing a broad sampling of gadids from freezing and non-freezing habitats. In three select species representing different AFGP-bearing clades, we analyzed all AFGP gene family members and the broader scale AFGP genomic regions in detail. Codon usage analyses suggest that motif duplication produced the intragenic AFGP tripeptide coding repeats, and rapid sequence divergence post-duplication stabilized the recombination-prone long repetitive coding region. Genomic loci analyses support AFGP originated once from a single ancestral genomic origin, and shed light on how the de novo gene proliferated into a gene family. Results also show the processes of gene duplication and gene loss are distinctive in separate clades, and both genotype and phenotype are commensurate with differential local selective pressures.

The Antarctic bald notothen, Trematomus borchgrevinki (family Nototheniidae) occupies a high latitude, ice-laden environment and represents an extreme example of cold-specialization among fishes. We present the first, high-quality, chromosome-scale genome of a female T. borchgrevinki individual comprised of 23 putative chromosomes, the largest of which is 65 megabasepairs (Mbp) in length. The total length of the genome 935.13 Mbp, composed of 2,094 scaffolds, with a scaffold N50 of 42.67 Mbp. Annotation yielded 22,192 protein-coding genes while 54.75% of the genome was occupied by repetitive elements; an analysis of repeats demonstrated that an expansion occurred in recent time. Conserved synteny analysis revealed that the genome architecture of T. borchgrevinki is largely maintained with other members of the notothenioid clade, although several significant translocations and inversions are present, including the fusion of orthologous chromosomes 8 and 11 into a single element. This genome will serve as a cold-specialized model for comparisons to other members of the notothenioid adaptive radiation.

Cytogenetics provides a unique platform to study in situ structural, functional, and evolutionary aspects of the genome. As such it holds powerful promise in decoding mechanisms and processes of genome architectural changes and their role in organism’s diversification and evolution. Since the early 80s, such an approach has been applied to the study of the Antarctic notothenioid fishes. In almost three decades, the cytogenetic information has expanded to cover half of the known species inhabiting the high Antarctic waters. Although started 10 years later, cytogenetic studies of species from the Ross sea region have provided valuable contributions to this bulk of knowledge. Here, we synthesize the currently available cytogenetic information on Antarctic notothenioid fishes from the Ross Sea Region, inclusive of both conventional karyotyping and gene mapping. In addition, new karyotypic data on four species ( Lepidonotothen squamifrons , Trematomus scotti , T. loennbergii, and T. lepidorhinus ) are provided. In discussing these data, specific focus is made on the patterns and subtleties of cytogenetic diversity at inter- and intra-specific levels aiming at contributing to the refinement of the knowledge of fish diversity in a region, the Ross Sea area, whose primary ecological value is widely recognized.

Abstract Determining the origins of novel genes and the mechanisms driving the emergence of new functions is challenging yet crucial for understanding evolutionary innovations. Recently evolved fish antifreeze proteins (AFPs) offer a unique opportunity to explore these processes, particularly the near-identical type I AFP (AFPI) found in four phylogenetically divergent fish taxa. This study tested the hypothesis of protein sequence convergence beyond functional convergence in three unrelated AFPI-bearing fish lineages. Through comprehensive comparative analyses of newly sequenced genomes of winter flounder and grubby sculpin, along with available high-quality genomes of cunner and 14 other related species, the study revealed that near-identical AFPI proteins originated from distinct genetic precursors in each lineage. Each lineage independently evolved a de novo coding region for the novel ice-binding protein while repurposing fragments from their respective ancestors into potential regulatory regions, representing partial de novo origination—a process that bridges de novo gene formation and the neofunctionalization of duplicated genes. The study supports existing models of new gene origination and introduces new ones: the innovation–amplification–divergence model, where novel changes precede gene duplication; the newly proposed duplication–degeneration–divergence model, which describes new functions arising from degenerated pseudogenes; and the duplication–degeneration–divergence gene fission model, where each new sibling gene differentially degenerates and renovates distinct functional domains from their parental gene. These findings highlight the diverse evolutionary pathways through which a novel functional gene with convergent sequences at the protein level can evolve across divergent species, advancing our understanding of the mechanistic intricacies in new gene formation.

Evolution of Antarctic notothenioids in the frigid and oxygen-rich Southern Ocean had led to remarkable genomic changes, most notably the gain of novel antifreeze glycoproteins and the loss of oxygen-binding hemoproteins in the icefish family. Recently, the mitochondrial (mt) NADH dehydrogenase subunit 6 (ND6) gene and the adjacent transfer RNAGlu (tRNAGlu) were also reportedly lost. ND6 protein is crucial for the assembly and function of Complex I of the mt electron transport chain that produces adenosine triphosphate (ATP) essential for life; thus, ND6 absence would be irreconcilable with Antarctic notothenioids being thriving species. Here we report our discovery that the ND6 gene and tRNAGlu were not lost but had been translocated to the control region (CR) from their canonical location between ND5 and cytochrome b genes. We characterized the CR and adjacent sequences of 22 notothenioid species representing all eight families of Notothenioidei to elucidate the mechanism and evolutionary history of this mtDNA rearrangement. Species of the three basal non-Antarctic families have the canonical vertebrate mt gene order, whereas species of all five Antarctic families have a rearranged CR bearing the embedded ND6 (ND6CR) and tRNAGlu, with additional copies of tRNAThr, tRNAPro, and noncoding region in various lineages. We hypothesized that an initial duplication of the canonical mt region from ND6 through CR occurred in the common ancestor to the Antarctic clade, and we deduced the succession of loss or modification of the duplicated region leading to the extant patterns of mt DNA reorganization that is consistent with notothenioid evolutionary history. We verified that the ND6CR gene in Antarctic notothenioids is transcribed and therefore functional. However, ND6CR-encoded protein sequences differ substantially from basal non-Antarctic notothenioid ND6, and we detected lineage-specific positive selection on the branch leading to the Antarctic clade of ND6CR under the branch-site model. Collectively, the novel mt ND6CR genotype of the Antarctic radiation represents another major molecular change in Antarctic notothenioid evolution and may reflect an adaptive change conducive to the functioning of the protein (Complex I) machinery of mt respiration in the polar environment, driven by the advent of freezing, oxygen-rich conditions in the Southern Ocean.

The Antarctic icefish Chaenocephalus aceratus lacks the globins common to most vertebrates, hemoglobin and myoglobin, but has retained neuroglobin in the brain. This conserved globin has been cloned, over-expressed and purified. To highlight similarities and differences, the structural features of the neuroglobin of this colourless-blooded fish were compared with those of the well characterised human neuroglobin as well as with the neuroglobin from the retina of the red blooded, hemoglobin and myoglobin-containing, closely related Antarctic notothenioid Dissostichus mawsoni. A detailed structural and functional analysis of the two Antarctic fish neuroglobins was carried out by UV-visible and Resonance Raman spectroscopies, molecular dynamics simulations and laser-flash photolysis. Similar to the human protein, Antarctic fish neuroglobins can reversibly bind oxygen and CO in the Fe(2+) form, and show six-coordination by distal His in the absence of exogenous ligands. A very large and structured internal cavity, with discrete docking sites, was identified in the modelled three-dimensional structures of the Antarctic neuroglobins. Estimate of the free-energy barriers from laser-flash photolysis and Implicit Ligand Sampling showed that the cavities are accessible from the solvent in both proteins.Comparison of structural and functional properties suggests that the two Antarctic fish neuroglobins most likely preserved and possibly improved the function recently proposed for human neuroglobin in ligand multichemistry. Despite subtle differences, the adaptation of Antarctic fish neuroglobins does not seem to parallel the dramatic adaptation of the oxygen carrying globins, hemoglobin and myoglobin, in the same organisms.