Genomics of Secondarily Temperate Adaptation in the Only Non-Antarctic Icefish.

White-blooded Antarctic icefishes, a family within the adaptive radiation of Antarctic notothenioid fishes, are an example of extreme biological specialization to both the chronic cold of the Southern Ocean and life without hemoglobin. As a result, icefishes display derived physiology that limits them to the cold and highly oxygenated Antarctic waters. Against these constraints, remarkably one species, the pike icefish Champsocephalus esox, successfully colonized temperate South American waters. To study the genetic mechanisms underlying secondarily temperate adaptation in icefishes, we generated chromosome-level genome assemblies of both C. esox and its Antarctic sister species, Champsocephalus gunnari. The C. esox genome is similar in structure and organization to that of its Antarctic congener; however, we observe evidence of chromosomal rearrangements coinciding with regions of elevated genetic divergence in pike icefish populations. We also find several key biological pathways under selection, including genes related to mitochondria and vision, highlighting candidates behind temperate adaptation in C. esox. Substantial antifreeze glycoprotein (AFGP) pseudogenization has occurred in the pike icefish, likely due to relaxed selection following ancestral escape from Antarctica. The canonical AFGP locus organization is conserved in C. esox and C. gunnari, but both show a translocation of two AFGP copies to a separate locus, previously unobserved in cryonotothenioids. Altogether, the study of this secondarily temperate species provides an insight into the mechanisms underlying adaptation to ecologically disparate environments in this otherwise highly specialized group.

Molecular mechanism and history of non-sense to sense evolution of antifreeze glycoprotein gene in northern gadids.

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 nongenic 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.

Evolution of chaperome gene expression and regulatory elements in the antarctic notothenioid fishes.

Confined within the cold-stable Southern Ocean, Antarctic notothenioid fishes have undergone an evolutionary loss of the inducible heat shock response (HSR), while facing perpetual low-temperature challenges to cellular proteostasis. This study examines how evolution in chronic cold has affected the shared cellular apparatus that mediates proteostasis under normal and heat stressed states. To deduce Antarctic-specific changes, we compared native expression levels across the full suite of chaperome genes and assessed the structural integrity of two crucial HSR regulators - Heat Shock Factor 1 (HSF1) that activates HSR, and heat shock elements (HSEs), the binding sites for HSF1 - between Antarctic fishes and the basal temperate notothenioid Eleginops maclovinus. Native expression levels of Antarctic fish chaperomes showed very modest changes overall, contrary to the common view of constitutive upregulation in the cold. Only a few cytosolic HSP70 genes showed greater transcription, with only the ancestrally-inducible HSPA6 strongly upregulated across all Antarctic species. Additionally, the constant cold has apparently not relaxed the selective pressures on maintaining HSF1 and HSEs in Antarctic fish. Instead, we found HSF1 experienced intensified selective pressure, with conserved sequence changes in Antarctic species suggesting optimization for non-heat-stress functional roles. HSEs of the HSP70 gene family have largely remained conserved in canonical sequence motifs and copy numbers as in E. maclovinus, showing limited impact of relaxed selective pressure. This study shows that evolution in chronic cold has led to both subtle and distinctive changes in the cellular apparatus for proteostasis and HSR, with functional consequences amenable to experimental evaluation.

Effects of warming rates on physiological and molecular components of response to CTMax heat stress in the Antarctic fish Harpagifer antarcticus.

Maximum and minimum Critical thermal limits (CTMax and CTMin) have been studied extensively to assess thermal tolerance in ectotherms by means of ramping assays. Notothenioid fish have been proposed as particularly sensitive to temperature increases related to global climate change. However, there are large gaps in our understanding of the thermal responses of these extreme cold-adapted fish in assays with heating rates. We evaluated the effects of two commonly used heating rates (0.3 and 1 °C/min) on the cellular stress responses in the intertidal Antarctic fish Harpagifer antarcticus immediately after CTMax was reached, and at 2 and 4 h of recovery time in ambient water. We compared CTMax values, the relative transcript expression of genes relvant to heat shock response (Hsc70, Hsp70, Grp78), hypoxia (Hif1-α, LDHa, GR), ubiquitination (Ube2), and apoptosis (SMAC/DIABLO), and five plasma parameters - glucose, lactate, total protein, osmolality and cortisol. CTMax values between the two heating rates are not significantly different, and both rates elicited a similar stress response at molecular and physiological levels. We found a lack of up-regulated response of heat shock proteins, consistent with other Antarctic notothenioids. The general transcriptional pattern trended to downregulation, which was more evident in the slower 0.3 °C/min rate, and instances of upregulation were mainly related to ubiquitination. The faster 1 °C/min rate, rarely used for Antarctic fish, can be suitable for studying cold-adapted stenothermic fish without overestimating thermal tolerance or inducing damage from longer heat exposure.

A tale of two genes: divergent evolutionary fate of haptoglobin and hemopexin in hemoglobinless Antarctic icefishes.

The evolution of Antarctic notothenioid fishes in the isolated freezing Southern Ocean has led to remarkable trait gains and losses. One of the most extraordinary was the loss of the major oxygen carrier hemoglobin (Hb) in the icefishes (family Channichthyidae). Although the mechanisms of this loss and the resulting compensatory changes have been well studied, the impact of Hb loss on the network of genes that once supported its recycling and disposal has remained unexplored. Here, we report the functional fate and underlying molecular changes of two such key Hb-supporting proteins across the icefish family - haptoglobin (Hp) and hemopexin (Hx), crucial in removing cytotoxic free Hb and heme, respectively. Hp plays a critical role in binding free Hb for intracellular recycling and absent its primary client, icefish Hp transcription is now vanishingly little, and translation into a functional protein is nearly silenced. Hp genotype degeneration has manifested in separate lineages of the icefish phylogeny with three distinct nonsense mutations and a deletion frame shift, as well as mutated polyadenylation signal sequences. Thus, Hb loss appears to have diminished selective constraint on Hp maintenance, resulting in its stochastic, co-evolutionary drift towards extinction. Hx binds free heme for iron recycling in hepatocytes. In contrast to Hp, Hx genotype integrity is preserved in the icefishes and transcription occurs at levels comparable to those in the red-blooded notothenioids. The persistence of Hx likely owes to continued selective pressure for its function from mitochondrial and non-Hb cellular hemoproteins.

Evolution in chronic cold: varied loss of cellular response to heat in Antarctic notothenioid fish.

BACKGROUND: Confined within the freezing Southern Ocean, the Antarctic notothenioids have evolved to become both cold adapted and cold specialized. A marked signature of cold specialization is an apparent loss of the cellular heat shock response (HSR). As the HSR has been examined in very few notothenioid species to-date, it remains unknown whether HSR loss pervades the Antarctic radiation, or whether the broader cellular responses to heat stress has sustained similar loss. Understanding the evolutionary status of these responses in this stenothermal taxon is crucial for evaluating its adaptive potential to ocean warming under climate change. RESULTS: In this study, we used an acute heat stress protocol followed by RNA-Seq analyses to study the evolution of cellular-wide transcriptional responses to heat stress across three select notothenioid lineages - the basal temperate and nearest non-Antarctic sister species Eleginops maclovinus serving as ancestral proxy, the cryopelagic Pagothenia borchgrevinki and the icefish Chionodraco rastrospinosus representing cold-adapted red-blooded and hemoglobinless Antarctic notothenioids respectively. E. maclovinus displayed robust cellular stress responses including the ER Unfolded Protein Response and the cytosolic HSR, cementing the HSR as a plesiomorphy that preceded Antarctic notothenioid radiation. While the transcriptional response to heat stress was minimal in P. borchgrevinki, C. rastrospinosus exhibited robust responses in the broader cellular networks especially in inflammatory responses despite lacking the classic HSR and UPR. CONCLUSION: The disparate patterns observed in these two archetypal Antarctic species indicate the evolutionary status in cellular ability to mitigate acute heat stress varies even among Antarctic lineages, which may affect their adaptive potential in coping with a warming world.

Diverse origins of near-identical antifreeze proteins in unrelated fish lineages provide insights into evolutionary mechanisms of new gene birth and protein sequence convergence.

Determining the origins of novel genes and the genetic mechanisms underlying the emergence of new functions is challenging yet crucial for understanding evolutionary innovations. The convergently evolved fish antifreeze proteins provide excellent opportunities to investigate evolutionary origins and pathways of new genes. Particularly notable is the near-identical type I antifreeze proteins (AFPI) 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, revealing different paths by which a similar protein arose from diverse genomic resources. Comprehensive comparative analyses of de novo sequenced genome of the winter flounder and grubby sculpin, available high-quality genome of the cunner and 14 other relevant species found that the near-identical AFPI originated from a distinct genetic precursor in each lineage. Each independently evolved a coding region for the novel ice-binding protein while retaining sequence identity in the regulatory regions with their respective ancestor. The deduced evolutionary processes and molecular mechanisms are consistent with the Innovation-Amplification-Divergence (IAD) model applicable to AFPI formation in all three lineages, a new Duplication-Degeneration-Divergence (DDD) model we propose for the sculpin lineage, and a DDD model with gene fission for the cunner lineage. This investigation illustrates the multiple ways by which a novel functional gene with sequence convergence at the protein level could evolve across divergent species, advancing our understanding of the mechanistic intricacies in new gene formation.

Model of gene expression in extreme cold - reference transcriptome for the high-Antarctic cryopelagic notothenioid fish Pagothenia borchgrevinki.

BACKGROUND: Among the cold-adapted Antarctic notothenioid fishes, the high-latitude bald notothen Pagothenia borchgrevinki is particularly notable as the sole cryopelagic species, exploiting the coldest and iciest waters of the Southern Ocean. Because P. borchgrevinki is a frequent model for investigating notothenioid cold-adaptation and specialization, it is imperative that "omic" tools be developed for this species. In the absence of a sequenced genome, a well annotated reference transcriptome of the bald notothen will serve as a model of gene expression in the coldest and harshest of all polar marine environments, useful for future comparative studies of cold adaptation and thermal responses in polar teleosts and ectotherms. RESULTS: We sequenced and annotated a reference transcriptome for P. borchgrevinki, with added attention to capturing the transcriptional responses to acute and chronic heat exposures. We sequenced by Roche 454 a normalized cDNA library constructed from pooled mRNA encompassing multiple tissues taken from environmental, warm acclimating, and acute heat stressed specimens. The resulting reads were assembled into 42,620 contigs, 17,951 of which could be annotated. We utilized this annotated portion of the reference transcriptome to map short Illumina reads sequenced from the gill and liver of environmental specimens, and also compared the gene expression profiles of these two tissue transcriptomes with those from the temperate model fish Danio rerio. From this, we identified a conserved group of 58 GO terms, in which terms related to transcription and its regulation, ubiquitin-protein ligase activity, protein ubiquitination, and protein binding among others are more prevalent in the bald notothen, suggesting the pertinent genes play essential roles in cold temperature functioning. CONCLUSION: We sequenced multiple tissue transcriptomes from native and heat-exposed experimental specimens of the high Antarctic, cryopelagic notothenioid P. borchgrevinki to construct a reference transcriptome. In a proof of concept, we utilized the annotated reference transcriptome to profile the gene expression patterns of gill and liver, and identified a suite of over and under-represented GO terms when compared to the tropical water zebrafish suggesting these functions may be important for surviving in freezing waters. The transcriptome resource from this study will aid future investigations of cold adaptation and thermal response of polar ectothermic species.

Evolution of an antifreeze protein by neofunctionalization under escape from adaptive conflict.

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.

Novel mitochondrial genome rearrangements including duplications and extensive heteroplasmy could underlie temperature adaptations in Antarctic notothenioid fishes.

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.

Protein genes in repetitive sequence-antifreeze glycoproteins in Atlantic cod genome.

BACKGROUND: Highly repetitive sequences are the bane of genome sequence assembly, and the short read lengths produced by current next generation sequencing technologies further exacerbates this obstacle. An adopted practice is to exclude repetitive sequences in genome data assembly, as the majority of repeats lack protein-coding genes. However, this could result in the exclusion of important genotypes in newly sequenced non-model species. The absence of the antifreeze glycoproteins (AFGP) gene family in the recently sequenced Atlantic cod genome serves as an example. RESULTS: The Atlantic cod (Gadus morhua) genome was assembled entirely from Roche 454 short reads, demonstrating the feasibility of this approach. However, a well-known major adaptive trait, the AFGP, essential for survival in frigid Arctic marine habitats was absent in the annotated genome. To assess whether this resulted from population difference, we performed Southern blot analysis of genomic DNA from multiple individuals from the North East Arctic cod population that the sequenced cod belonged, and verified that the AFGP genotype is indeed present. We searched the raw assemblies of the Atlantic cod using our G. morhua AFGP gene, and located partial AFGP coding sequences in two sequence scaffolds. We found these two scaffolds constitute a partial genomic AFGP locus through comparative sequence analyses with our newly assembled genomic AFGP locus of the related polar cod, Boreogadus saida. By examining the sequence assembly and annotation methodologies used for the Atlantic cod genome, we deduced the primary cause of the absence of the AFGP gene family from the annotated genome was the removal of all repetitive Roche 454 short reads before sequence assembly, which would exclude most of the highly repetitive AFGP coding sequences. Secondarily, the model teleost genomes used in projection annotation of the Atlantic cod genome have no antifreeze trait, perpetuating the unawareness that the AFGP gene family is missing. CONCLUSIONS: We recovered some of the missing AFGP coding sequences and reconstructed a partial AFGP locus in the Atlantic cod genome, bringing to light that not all repetitive sequences lack protein coding information. Also, reliance on genomes of model organisms as reference for annotating protein-coding gene content of a newly sequenced non-model species could lead to omission of novel genetic traits.

Assembly of the antifreeze glycoprotein/trypsinogen-like protease genomic locus in the Antarctic toothfish Dissostichus mawsoni (Norman).

To investigate the genomic architecture underlying the quintessential adaptive phenotype, antifreeze glycoprotein (AFGP) that enables Antarctic notothenioid survival in the frigid Southern Ocean, we isolated the AFGP genomic locus from a bacterial artificial chromosome library for Dissostichus mawsoni. Through extensive shotgun sequencing of pertinent clones and sequence assembly verifications, we reconstructed the highly repetitive AFGP genomic locus. The locus comprises two haplotypes of different lengths (363.6 kbp and 467.4 kbp) containing tandem AFGP, two TLP (trypsinogen-like protease), and surprisingly three chimeric AFGP/TLP, one of which was previously hypothesized to be a TLP-to-AFGP evolutionary intermediate. The ~100 kbp haplotype length variation results from different AFGP copy number, suggesting substantial dynamism existed in the evolutionary history of the AFGP gene family. This study provided the data for fine resolution sequence analyses that would yield insight into the molecular mechanisms of notothenioid AFGP gene family evolution driven by Southern Ocean glaciation.

Environmental DNA from Marine Waters and Substrates: Protocols for Sampling and eDNA Extraction.

Environmental DNA (eDNA) analysis has emerged in recent years as a powerful tool for the detection, monitoring, and characterization of aquatic metazoan communities, including vulnerable species. The rapid rate of adopting the eDNA approach across diverse habitats and taxonomic groups attests to its value for a wide array of investigative goals, from understanding natural or changing biodiversity to informing on conservation efforts at local and global scales. Regardless of research objectives, eDNA workflows commonly include the following essential steps: environmental sample acquisition, processing and preservation of samples, and eDNA extraction, followed by eDNA sequencing library preparation, high-capacity sequencing and sequence data analysis, or other methods of genetic detection. In this chapter, we supply instructional details for the early steps in the workflow to facilitate researchers considering adopting eDNA analysis to address questions in marine environments. Specifically, we detail sampling, preservation, extraction, and quantification protocols for eDNA originating from marine water, shallow substrates, and deeper sediments. eDNA is prone to degradation and loss, and to contamination through improper handling; these factors crucially influence the outcome and validity of an eDNA study. Thus, we also provide guidance on avoiding these pitfalls. Following extraction, purified eDNA is often sequenced on massively parallel sequencing platforms for comprehensive faunal diversity assessment using a metabarcoding or metagenomic approach, or for the detection and quantification of specific taxa by qPCR methods. These components of the workflow are project-specific and thus not included in this chapter. Instead, we briefly touch on the preparation of eDNA libraries and discuss comparisons between sequencing approaches to aid considerations in project design.

ND6 gene "lost" and found: evolution of mitochondrial gene rearrangement in Antarctic notothenioids.

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 RNA(Glu) (tRNA(Glu)) 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 tRNA(Glu) 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 (ND6(CR)) and tRNA(Glu), with additional copies of tRNA(Thr), tRNA(Pro), 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 ND6(CR) gene in Antarctic notothenioids is transcribed and therefore functional. However, ND6(CR)-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 ND6(CR) under the branch-site model. Collectively, the novel mt ND6(CR) 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.

Structure and dynamics of Antarctic fish neuroglobin assessed by computer simulations.

Neuroglobin (Ngb) is a heme protein, highly conserved along evolution, predominantly found in the nervous system. It is upregulated by hypoxia and ischemia and may have a neuroprotective role under hypoxic stress. Although many other roles have been proposed, the physiological function is still unclear. Antarctic icefishes lack hemoglobin and some species also lack myoglobin, but all have Ngb and thus may help the elucidation of Ngb function. We present the first theoretically derived structure of fish Ngb and describe its behavior using molecular dynamics simulations. Specifically, we sequenced and analyzed Ngbs from a colorless-blooded Antarctic icefish species Chaenocephalus aceratus and a related red-blooded species (Dissostichus mawsoni). Both fish Ngbs are 6-coordinated but have some peculiarities that differentiate them from mammalian counterparts: they have extensions in the N and C termini that can interact with the EF loop, and a gap in the alignment that changes the CD-region structure/dynamics that has been found to play a key role in human neuroglobin. Our results suggest that a single mutation between both fish Ngbs is responsible for significant difference in the behavior of the proteins. The functional role of these characteristics is discussed.

Transcriptomic and genomic evolution under constant cold in Antarctic notothenioid fish.

The antifreeze glycoprotein-fortified Antarctic notothenioid fishes comprise the predominant fish suborder in the isolated frigid Southern Ocean. Their ecological success undoubtedly entailed evolutionary acquisition of a full suite of cold-stable functions besides antifreeze protection. Prior studies of adaptive changes in these teleost fishes generally examined a single genotype or phenotype. We report here the genome-wide investigations of transcriptional and genomic changes associated with Antarctic notothenioid cold adaptation. We sequenced and characterized 33,560 ESTs from four tissues of the Antarctic notothenioid Dissostichus mawsoni and derived 3,114 nonredundant protein gene families and their expression profiles. Through comparative analyses of same-tissue transcriptome profiles of D. mawsoni and temperate/tropical teleost fishes, we identified 177 notothenioid protein families that were expressed many fold over the latter, indicating cold-related up-regulation. These up-regulated gene families operate in protein biosynthesis, protein folding and degradation, lipid metabolism, antioxidation, antiapoptosis, innate immunity, choriongenesis, and others, all of recognizable functional importance in mitigating stresses in freezing temperatures during notothenioid life histories. We further examined the genomic and evolutionary bases for this expressional up-regulation by comparative genomic hybridization of DNA from four pairs of Antarctic and basal non-Antarctic notothenioids to 10,700 D. mawsoni cDNA probes and discovered significant to astounding (3- to >300-fold, P < 0.05) Antarctic-specific duplications of 118 protein-coding genes, many of which correspond to the up-regulated gene families. Results of our integrative tripartite study strongly suggest that evolution under constant cold has resulted in dramatic genomic expansions of specific protein gene families, augmenting gene expression and gene functions contributing to physiological fitness of Antarctic notothenioids in freezing polar conditions.

Propagation of a De Novo Gene under Natural Selection: Antifreeze Glycoprotein Genes and Their Evolutionary History in Codfishes.

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.

Antarctic ecosystems in transition - life between stresses and opportunities.

Important findings from the second decade of the 21st century on the impact of environmental change on biological processes in the Antarctic were synthesised by 26 international experts. Ten key messages emerged that have stakeholder-relevance and/or a high impact for the scientific community. They address (i) altered biogeochemical cycles, (ii) ocean acidification, (iii) climate change hotspots, (iv) unexpected dynamism in seabed-dwelling populations, (v) spatial range shifts, (vi) adaptation and thermal resilience, (vii) sea ice related biological fluctuations, (viii) pollution, (ix) endangered terrestrial endemism and (x) the discovery of unknown habitats. Most Antarctic biotas are exposed to multiple stresses and considered vulnerable to environmental change due to narrow tolerance ranges, rapid change, projected circumpolar impacts, low potential for timely genetic adaptation, and migration barriers. Important ecosystem functions, such as primary production and energy transfer between trophic levels, have already changed, and biodiversity patterns have shifted. A confidence assessment of the degree of 'scientific understanding' revealed an intermediate level for most of the more detailed sub-messages, indicating that process-oriented research has been successful in the past decade. Additional efforts are necessary, however, to achieve the level of robustness in scientific knowledge that is required to inform protection measures of the unique Antarctic terrestrial and marine ecosystems, and their contributions to global biodiversity and ecosystem services.

Metagenomic sequencing of environmental DNA reveals marine faunal assemblages from the West Antarctic Peninsula.

The West Antarctic Peninsula (WAP) is the fastest warming region in Antarctica where climate impact on the cold-adapted marine ecosystem is already visible. To monitor faunal changes in remote vast bodies of Antarctic waters, efficient and informative tools are essential. High-throughput sequencing of environmental DNA (eDNA) has emerged as one such tool for monitoring biodiversity and ecosystems, as it increases detection sensitivity of taxa, and sampling is often simpler and less costly than traditional collection methods. We collected water samples from four WAP shallow (≤300m) shelf regions, recovered the eDNA therein, and performed metagenomic shotgun sequencing and analyses to determine the effectiveness of this method to assess marine benthic faunal diversity; this includes the detection of deep-water predatory king crabs whose potential shoreward expansion to warming shelves has sparked much concern. Using a customized bioinformatics pipeline, we identified abundant signatures of common benthic invertebrate fauna, endemic notothenioid fishes, as well as lithodid king crabs. We also uncovered species richness and diversity comparable to biological inventories compiled by the use of traditional survey methods, supporting the efficacy of the eDNA shotgun sequencing approach. As the rate of eDNA degradation affects faunal detection sensitivity, we also quantified mitochondrial ND2 gene copies in eDNA derived from a WAP icefish and found ND2 copies persisted to at least 20days in the cold WAP water, much longer than values reported for temperate environments. We propose that eDNA metagenomic sequencing complements traditional sampling, and combining both will enable more inclusive biodiversity detection and faunal change monitoring in the vast Southern Ocean.