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.

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.

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.

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.

Positive and Relaxed Selective Pressures Have Both Strongly Influenced the Evolution of Cryonotothenioid Fishes during Their Radiation in the Freezing Southern Ocean.

Evolution in the chronic cold of the Southern Ocean has had a profound influence on the physiology of cryonotothenioid fishes. However, the suite of genetic changes underlying the physiological gains and losses in these fishes is still poorly surveyed. By identifying the genomic signatures of selection, this study aims to identify the functional classes of genes that have been changed following two major physiological transitions: the onset of freezing temperatures and the loss of hemoproteins. Looking at the changes that followed the onset of freezing temperatures, positive selective pressure was found among a set of broadly acting gene regulatory factors, suggesting a route through which cryonotothenioid gene expression has been retooled for life in the cold. Further, genes related to the cell cycle and cellular adhesion were found under positive selection suggesting that both present key challenges to life in freezing waters. By contrast, genes showing signatures of the relaxation of selective pressure showed a narrower biological impact, acting on genes related to mitochondrial function. Finally, although chronic cold-water temperatures appear correlated with substantial genetic change, the loss of hemoproteins resulted in little observable change in protein-coding genes relative to their red-blooded relatives. Combined, the influence of positive and relaxed selection shows that long-term exposure to cold has led to profound changes in cryonotothenioid genomes that may make it challenging for them to adapt to a rapidly changing climate.

Heat tolerance and its plasticity in Antarctic fishes.

The adaptive radiation of the Antarctic notothenioid ancestral benthic fish stock within the chronic freezing waters of the Southern Ocean gave rise to five highly cold adapted families. Their stenothermy, first observed from several high-latitude McMurdo Sound species, has been of increasing recent interest given the threat of rising polar water temperatures from global climate change. In this study we determined the heat tolerance in a geographically diverse group of 11 Antarctic species as their critical thermal maximum (CTMax). When acclimatized to their natural freezing water temperatures, environmental CTMaxs ranged from 11.95 to 16.17 °C, well below those of fishes endemic to warmer waters. There was a significant regional split, with higher CTMaxs in species from the more northerly and thermally variable Seasonal Pack-ice Zone. When eight of the Antarctic species were warm acclimated to 4 °C all showed a significant increase over their environmental CTMaxs, with several showing plasticity comparable in magnitude to some far more eurythermal fishes. When the accrual of heat tolerance during acclimation was followed in three high-latitude McMurdo Sound species, it was found to develop slowly in two of them, which was correlated with their low metabolic rates.

The genomic basis for colonizing the freezing Southern Ocean revealed by Antarctic toothfish and Patagonian robalo genomes.

BACKGROUND: The Southern Ocean is the coldest ocean on Earth but a hot spot of evolution. The bottom-dwelling Eocene ancestor of Antarctic notothenioid fishes survived polar marine glaciation and underwent adaptive radiation, forming >120 species that fill all water column niches today. Genome-wide changes enabling physiological adaptations and the rapid expansion of the Antarctic notothenioids remain poorly understood. RESULTS: We sequenced and compared 2 notothenioid genomes-the cold-adapted and neutrally buoyant Antarctic toothfish Dissostichus mawsoni and the basal Patagonian robalo Eleginops maclovinus, representing the temperate ancestor. We detected >200 protein gene families that had expanded and thousands of genes that had evolved faster in the toothfish, with diverse cold-relevant functions including stress response, lipid metabolism, protein homeostasis, and freeze resistance. Besides antifreeze glycoprotein, an eggshell protein had functionally diversified to aid in cellular freezing resistance. Genomic and transcriptomic comparisons revealed proliferation of selcys-transfer RNA genes and broad transcriptional upregulation across anti-oxidative selenoproteins, signifying their prominent role in mitigating oxidative stress in the oxygen-rich Southern Ocean. We found expansion of transposable elements, temporally correlated to Antarctic notothenioid diversification. Additionally, the toothfish exhibited remarkable shifts in genetic programs towards enhanced fat cell differentiation and lipid storage, and promotion of chondrogenesis while inhibiting osteogenesis in bone development, collectively contributing to the achievement of neutral buoyancy and pelagicism. CONCLUSIONS: Our study revealed a comprehensive landscape of evolutionary changes essential for Antarctic notothenioid cold adaptation and ecological expansion. The 2 genomes are valuable resources for further exploration of mechanisms underlying the spectacular notothenioid radiation in the coldest marine environment.

RNA-seq analyses of cellular responses to elevated body temperature in the high Antarctic cryopelagic nototheniid fish Pagothenia borchgrevinki.

Through evolution in the isolated, freezing (-1.9°C) Southern Ocean, Antarctic notothenioid fish have become cold-adapted as well as cold-specialized. Notothenioid cold specialization is most evident in their limited tolerance to heat challenge, and an apparent loss of the near universal inducible heat shock (HSP70) response. Beyond these it remains unclear how broadly cold specialization pervades the underlying tissue-wide cellular responses. We report the first analysis of massively parallel RNA sequencing (RNA-seq) to identify gene expression changes in the liver in response to elevated body temperature of a high-latitude Antarctic nototheniid, the highly cold-adapted and cold-specialized cryopelagic bald notothen, Pagothenia borchgrevinki. From a large (14,873) mapped set of qualified, annotated liver transcripts, we identified hundreds of significantly differentially expressed genes following two and four days of 4°C exposure, suggesting substantial transcriptional reorganization in the liver when body temperature was raised 5°C above native water temperature. Most notably, and in sharp contrast to heat stressed non-polar fish species, was a widespread down-regulation of nearly all classes of molecular chaperones including HSP70, as well as polyubiquitins that are associated with proteosomal degradation of damaged proteins. In parallel, genes involved in the cell cycle were down-regulated by day two of 4°C exposure, signifying slowing cellular proliferation; by day four, genes associated with transcriptional and translational machineries were down-regulated, signifying general slowing of protein biosynthesis. The log2 fold differential transcriptional changes are generally of small magnitudes but significant, and in total portray a broad down turn of cellular activities in response to four days of elevated body temperature in the cold-specialized bald notothen.