A Tale of Two Genes: Divergent Evolutionary Fate of Haptoglobin and Hemopexin in Hemoglobinless Antarctic Icefishes Kevin T

RESEARCH ARTICLE A tale of two genes: divergent evolutionary fate of haptoglobin and hemopexin in hemoglobinless Antarctic icefishes Kevin T. Bilyk1,2,*,‡, Xuan Zhuang2,3,*, Katherine R. Murphy2,4 and C.-H. Christina Cheng2

ABSTRACT freezing (DeVries and Cheng, 2005). In contrast to such an adaptive The evolution of Antarctic notothenioid fishes in the isolated freezing fitness gain, the evolutionary transformations also included a no less Southern Ocean has led to remarkable trait gains and losses. One of impactful trait loss: the disappearance of the universal vertebrate – the most extraordinary was the loss of the major oxygen carrier oxygen transport protein hemoglobin in one of the families the ’ hemoglobin (Hb) in the icefishes (family Channichthyidae). Although icefishes (Channichthyidae) (Sidell and O Brien, 2006). the mechanisms of this loss and the resulting compensatory changes Icefishes are the only known vertebrates to be devoid of have been well studied, the impact of Hb loss on the network of genes erythrocytes and hemoglobin (Hb) as adults. Instead, oxygen is that once supported its recycling and disposal has remained transported as dissolved gas in the circulating plasma, at about only unexplored. Here, we report the functional fate and underlying 10% of the concentration in the red-blooded notothenioids (Sidell ’ molecular changes of two such key Hb-supporting proteins across and O Brien, 2006). All 16 species of the icefish family lack Hb β the icefish family – haptoglobin (Hp) and hemopexin (Hx), crucial in owing to the ancestral deletion of the -globin genes in the Hb αβ removing cytotoxic free Hb and heme, respectively. Hp plays a critical coding -globin gene cluster (Cocca, et al., 1995), which occurred role in binding free Hb for intracellular recycling and absent its primary an estimated 8 to 9 million years ago (Near et al., 2006). In addition, client, icefish Hp transcription is now vanishingly little, and translation six icefish species have further lost the expression of myoglobin into a functional protein is nearly silenced. Hp genotype degeneration (Mb), the key hemoprotein in oxidative muscles, in the heart. In has manifested in separate lineages of the icefish phylogeny with three contrast to the single origin of Hb loss, Mb loss was erratic, resulting distinct nonsense mutations and a deletion frame shift, as well as from distinct mutational events in four separate occasions (Sidell ’ mutated polyadenylation signal sequences. Thus, Hb loss appears to and O Brien, 2006). have diminished selective constraint on Hp maintenance, resulting in Past research on the icefishes has greatly advanced our its stochastic, co-evolutionary drift towards extinction. Hx binds free understanding of the mechanisms behind Hb and Mb loss, and heme for iron recycling in hepatocytes. In contrast to Hp, Hx genotype the compensatory cellular, anatomical and physiological changes ’ integrity is preserved in the icefishes and transcription occurs at levels that followed (Sidell and O Brien, 2006). However, little is known comparable to those in the red-blooded notothenioids. The persistence about the evolutionary impact of the hemoprotein losses on the of Hx likely owes to continued selective pressure for its function from network of protein partners that once supported their function. In the mitochondrial and non-Hb cellular hemoproteins. homeostatic balance of erythropoiesis [new red blood cell (RBC) formation] and eryptosis (destruction of senescent RBCs), KEY WORDS: Notothenioid, Channichthyidae, Relaxed selection partnering proteins are involved in the essential processes of recovery and disposal of free Hb and heme iron, which are otherwise INTRODUCTION cytotoxic because of their strong oxidizing properties (Chiabrando The isolated, frigid Southern Ocean has become well recognized as a et al., 2011). As Hb and the RBCs that carry it headed towards vast natural laboratory where extreme polar conditions have wrought extinction in the icefish ancestor, co-evolutionary changes very extraordinary evolutionary transformations. Today, its fish fauna is likely would have propagated across the supporting proteins. The predominated by the five families of Antarctic notothenioid fishes fate of these supporting and partnering proteins has largely that evolved in situ, forming an adaptive radiation and a rare marine remained an unexplored part of the unique evolutionary history of species flock (Eastman, 2005). The success of these fishes in freezing the icefishes. Permanent absence of the hemoproteins would be waters owes to the evolution of a novel life-saving trait, the antifreeze expected to result in relaxation of selection pressure on the glycoprotein (Chen et al., 1997), which inhibits the growth of ice maintenance of their auxiliary systems (Lahti et al., 2009) and, as crystals in blood and body fluids, thereby preventing organismal such, Hb loss (with Mb loss in addition) could be likened to the proverbial tip of the iceberg, with a much larger base of changes in partnering or supporting protein-coding genes. 1Department of Biology, Western Kentucky University, 1906 College Heights Blvd, Heme, with its iron center, is a strong oxidant, thus Hb will cause #11080, Bowling Green, KY 42101-1080, USA. 2Department of Animal Biology, oxidative damage to surrounding molecules and tissues should it University of Illinois at Urbana-Champaign, 515 Morrill Hall, 505 S. Goodwin Avenue, Urbana, IL 61801, USA. 3Department of Ecology & Evolution, University of become free from its cellular carrier. The recovery and disposal of Chicago, 1101 E. 57th Street, Chicago, IL 60637, USA. 4Laboratories of Analytical free plasma Hb and heme from RBC lysis owing to cellular Biology, National Museum of Natural History, Smithsonian Institution, senescence, infections or wounds depend on the direct action of two P.O. Box 37012, MRC 183, Washington, DC 20013-7012, USA. *These authors contributed equally to this work key plasma proteins, haptoglobin (Hp) and hemopexin (Hx) (Wicher and Fries, 2006). These bind with high affinity to their ‡ Author for correspondence ([email protected]) ligands, Hb and heme, respectively (Wicher and Fries, 2010). K.T.B., 0000-0002-2262-2703 Therefore, they are excellent models for investigating the impact of relaxed selection from the Hb-less condition on the evolutionary

Received 11 July 2018; Accepted 6 February 2019 fate of Hb-supporting proteins in Antarctic icefishes. Hp is well Journal of Experimental Biology

1 RESEARCH ARTICLE Journal of Experimental Biology (2019) 222, jeb188573. doi:10.1242/jeb.188573 known as a scavenger of free Hb in vertebrates. In mice and humans, A260/A280 ratios (Epoch Take3 Microplate Spectrophotometer, Hp has also been found to play various roles in the immune or BioTek Instruments). RNA integrity was verified by visualizing inflammatory response. It modulates or regulates the balance of 1 μg of each sample run on a 2 mol l−1 formaldehyde/1% agarose immune cells in mice (Arredouani et al., 2003; Huntoon et al., 2008) denaturing gel. and stimulates angiogenesis in vasculitis (inflammation of blood vessels) in humans (Cid et al., 1993). Whether Hp in teleost fishes Amplification of Hp and Hx cDNA plays similar immune roles besides binding free Hb is currently RT-PCR amplification for Hp and Hx cDNA was performed as an unknown. Hx binds to free heme, which has sources beyond the Hb initial means to evaluate the transcriptional status of the two genes of RBCs. In the absence of RBC as the major source of free heme in and the integrity of the protein coding sequence. Approximately icefishes, free heme could still be produced from other heme- 10 μg of each RNA sample was first treated with 2 units of DNase I containing proteins including mitochondrial cytochromes and (New England Biolabs) at 37°C for 10 min to degrade potential microsomal cytochrome P450s, and other cellular hemoproteins contaminating genomic DNA, then purified using E.Z.N.A. HiBind including Mb in icefish species that express it. We hypothesize that Mini Columns (Omega Bio-Tek). The DNA-free total RNA was the initial loss of Hb in the icefish lineage would commence a primed with oligodT and reverse transcribed to generate the relaxation of selective maintenance of Hp and Hx functions, the first-strand cDNA using SuperScript II reverse transcriptase extent of which would reflect the differences between the reservoirs (Invitrogen). To amplify Hp and Hx cDNA, gene-specific primers of client proteins of these two scavengers. Here, we investigate the (Table S1) were designed using the genomic and transcriptomic evolutionary fate of these two primary Hb and heme scavenger resources available at the time, namely, the reference transcriptomes proteins in the Antarctic icefishes to gain new insight into the nature prepared for P. borchgrevinki (Bilyk and Cheng, 2013), D. mawsoni and extent of the impact of Hb loss on partnering molecules. (Chen et al., 2008) and the icefish C. aceratus (Shin et al., 2012). For Hx, a pair of UTR primers, Hx_UTR_F and Hx_UTR_R MATERIALS AND METHODS successfully amplified Hx cDNA from both red-blooded Fish tissues and blood sampling notothenioids and all 15 icefishes. For Hp, the UTR primers Most tissues for DNA and RNA extraction were taken from our Hp_UTR_F and Hp_UTR_R successfully amplified full-length Hp existing inventory of preserved or frozen Antarctic fish samples. cDNA from four icefishes but not the others (Table S1). A priori,it Fresh blood plasma for isolation of Hb-binding proteins was obtained was unknown whether the null result was due to absence of Hp for seven icefish species that we were able to catch in the West transcription or primer mismatch. Pairing the Hp_UTR_R primer with Antarctic Peninsula (WAP) waters during our field season in 2014. a forward primer Hp_ATG_F that anneals to the start of the protein These included Chaenocephalus aceratus, Chaenodraco wilsoni, coding sequence (the first 27 nt inclusive of ATG start) produced an Champsocephalus gunnari, Chionobathyscus dewitti, Chionodraco amplicon covering nearly full-length CDS (coding domain rastrospinosus, Cryodraco antarcticus and Pseudochaenichthys sequences) for three additional icefish species. This suggested that georgianus. Blood plasma was also collected from two red-blooded divergence in the 5′ UTR sequence in various icefishes may have notothenioids, Notothenia coriiceps and Akarotaxis nudiceps,to contributed to the null amplification. We thus attempted 5′RACE to serve as positive controls for native Hp detection. Live fish were obtain species specific 5′ UTR sequences to design the UTR forward anesthetized with tricaine methanesulfonate (MS-222) (Western primer but without success, suggesting absence or extremely low level Chemical) at 1 g per 15 liters seawater, bled from the caudal vein of transcription not amenable to amplification. Thus in total, we using heparin-coated syringes, after which the fish were returned to obtained full-length protein CDS from four icefish species, and nearly the seawater holding tank to recover. The blood plasma was isolated full-length CDS for three other icefishes. Amplified Hp and Hx cDNA after pelleting blood cells by centrifugation at 7000 rpm (4650 g)at were sequenced with BigDye Terminator v.3 Cycle Sequencing 4°C for 20 min. Tissues were also dissected from several individuals chemistry (Applied BioSystems) by direct sequencing or following of these species and either frozen in liquid nitrogen or preserved in cloning into the pGemTeasy vector (Promega). 90% molecular grade ethanol at −20°C until use. All fish handling complied with the University of Illinois at Urbana-Champaign Molecular evolution of Hp and Hx in the icefishes IACUC approved protocol 12123. Hp and Hx protein coding sequences were investigated for evidence of changed selective pressure upon Hb loss in icefishes. RNA extraction We used RELAX (Wertheim et al., 2014), a general hypothesis test Plasma Hp and Hx are synthesized in the liver (Chiabrando et al., using a codon-based phylogenetic framework to assess whether 2011), thus we extracted total RNA from liver samples of 15 of the selective constraint became relaxed or intensified gene-wide in test 16 known icefish species, missing only Channichthys rhinoceratus, species compared with reference species. The complete coding for which we lacked liver samples. RNA was also isolated from four sequences of Hp, Hx and mitochondrial ND2 genes for a subset of red-blooded notothenioids, Dissostichus mawsoni, Pagothenia teleost fishes were obtained from Ensembl to serve as the reference borchgrevinki and Notothenia coriiceps (family Nototheniidae), set. Additional notothenioid Hp and Hx sequences came from the as well as Gymnodraco acuticeps (family Bathydraconidae), to Parachaenichthys charcoti genome (Ahn et al., 2017) and the serve as positive controls, for both RT-PCR amplification of Hp and Eleginops maclovinus reference transcriptome (Bilyk et al., 2018). Hx cDNA as well as northern blot analysis. Approximately 50 mg of We then performed six comparisons to detect molecular tissue was homogenized in 1 ml TRIzol (Invitrogen) using 0.5 mm evolutionary signals: (i) all icefish branches against all red- zirconium oxide beads and a Bullet Blender StormR (Next blooded Antarctic notothenioid branches, (ii) all icefish branches Advance), and RNA extraction from the homogenate followed the against reference teleosts, (iii) all red-blooded Antarctic manufacturer’s (TRIzol) instructions. The final RNA pellets were notothenioid branches against reference teleosts, (iv) the single dissolved in 50–100 μl of 0.5× TE (5 mmol l−1 Tris–HCl, ancestral branch to icefish against all other icefish branches, (v) the 0.5 mmol l−1 EDTA, pH 8.0). RNA concentrations were single ancestral branch to icefish against all red-blooded estimated using absorbance at 260 nm (A260) and RNA purity by notothenioid branches, and (vi) the single ancestral branch to Journal of Experimental Biology

2 RESEARCH ARTICLE Journal of Experimental Biology (2019) 222, jeb188573. doi:10.1242/jeb.188573 icefish against reference teleosts. Table S2 lists the species and D. mawsoni and E. maclovinus BAC libraries had been constructed in accession numbers of sequences used in this analysis. Only the four our lab for use in various studies, whereas the C. aceratus library was icefish species with full-length Hp coding sequences were included available through Children’s Hospital Oakland Research Institute in this analysis. To reconstruct phylogenetic relationships of these (CHORI) BAC Resources Center. The macroarray filters of the BAC species needed for the RELAX analyses, we used the ND2 libraries were screened for the Hp loci using P32dATP-labeled probes sequence rather than Hp or Hx, as the latter do not possess enough generated from the C. aceratus or D. mawsoni Hp cDNA using the informative sites to resolve the relatedness of icefishes. We aligned same hybridization process described above but washed less the complete ND2 gene sequences with codon constraint using stringently, to approximately 50°C. Putative positive BAC clones MUSCLE (Edgar, 2004), and evaluated for models of nucleotide were isolated from the D. mawsoni and E. maclovinus libraries, and substitution using jModelTest (Posada, 2008). The best fit model those of C. aceratus were purchased from CHORI. These were further GTR+I+G was implemented in Bayesian phylogenetic analyses verified by Southern blot probing of the NotI digested recombinant using MrBayes v.3.2.6 (Huelsenbeck and Ronquist, 2001). We ran BAC plasmid DNA. The positive BAC clones were found to form a the Markov chain Monte Carlo simulation for 4 million generations single contig group on fingerprinted contig (FPC) analysis (Soderlund with four chains and sampled every 100 generations. We then used et al., 2000), thus there is a single Hp locus represented by any of the Tracer 1.4.1 (Drummond and Rambaut, 2007) to examine the trace BAC clones within the group. The plasmid DNA of one selected files to ensure the chains reached convergence, and discarded the positive clone for each species was electroporated into the first 25% of trees as burn-in. The consensus tree (Fig. S2) was used TransForMax EPI300 Escherichia coli strain (Epicentre), which as the phylogenetic framework for RELAX analysis. Translated was induced to produce the plasmid at high copy number to generate protein sequences for Hp and Hx were aligned using MUSCLE, sufficient DNA for sequencing. Sequencing libraries were and the sequences were manually trimmed to remove gap regions constructed using the Roche GS Titanium Nextera DNA Sample and premature stops. The codon aligned nucleotide sequences were Prep Kit and sequenced on a Roche/454 GS FLX+ (Roche/454 Life then analyzed using RELAX from the HyPhy software package Sciences) at the Roy J. Carver Biotechnology Center at the University (Pond et al., 2005) implemented on the Datamonkey Adaptive of Illinois at Urbana-Champaign. The sequence reads were assembled Evolution Server (http://datamonkey.org/; Weaver et al., 2018). for each species using Newbler v2.7. The sequence contig containing the Hp gene was analyzed for the presence and integrity of proximal Northern blot analysis promoter elements within approximately 1 kbp upstream of the start We utilized northern blot to provide a tissue-wide view of the codon using JASPAR (Khan et al., 2017). transcriptional status of Hp and Hx in liver among the icefishes, and as a means to validate loss of transcription where RT-PCR Isolation and sequencing the large terminal Hp exon from amplification produced no amplicon. Northern blot analysis genomic DNA was carried out on the 15 icefishes with available total liver RNA, The sequenced Hp loci were used to design a 3′ flanking region primer and four representative red-blooded notothenioids as positive (Hp_3Flank_R), which was then paired with a forward primer controls. Eight micrograms of total RNA was resolved on a 1.1% (Hp_E4_F) to amplify much of the large terminal exon (exon 4) and agarose formaldehyde gel at 110 V for 65 min, and imaged. The gel part of the downstream 3′ flanking sequence of the Hp gene from was gently shaken at room-temperature sterile-distilled water for genomic DNA of all 16 icefish species and two red-blooded control 30 min to remove excess formaldehyde, then vacuum-blotted species, D. mawsoni and G. acuticeps. This provided a means toverify (Amersham VacuGene, GE Health Sciences) onto Hybond N the continued presence of the gene within the genomes of the icefish membrane (Amersham, GE Health Sciences) and UV crosslinked species where we could not amplify cDNA products. Genomic DNA (Stratalinker, Agilent Technologies). The membrane was was isolated from liver or muscle using standard Tris/EDTA/SDS prehybridized at 55°C in PerfectHyb (Sigma-Aldrich) for several tissue lysis, phenol/chloroform extraction and final ethanol hours, then hybridized at the same temperature overnight to P32- precipitation. PCR amplified products were treated with SAP/ExoI dATP (Perkin Elmer) labeled probes of Hp or Hx. Probes were (shrimp alkaline phosphatase/Exonuclease I) and then sequenced synthesized using random heptamer priming of templates consisting with BigDye Dye Terminator v.3.1 chemistry (Applied BioSystems) of equal amounts of Hp or Hx cDNA derived from the red-blooded by direct sequencing or after cloning into pGemTeasy vector notothenioid D. mawsoni and the icefish C. aceratus. The (Promega). hybridized blot was washed with increasing stringency, to 0.1× SSC (15 mmol l−1 sodium chloride, 1.5 mmol l−1 sodium citrate)/ Isolation of Hb-binding plasma proteins by affinity 0.5% SDS (sodium dodecyl sulfate) at 55°C for Hp and 57°C for chromatography Hx. The washed blot was autoradiographed on a phosphor storage Fresh native plasma collected for seven icefish species were used for screen (Kodak) overnight and scanned on a STORMR 860 immune detection of physiological levels of circulating Hp protein. phosphoimager (Molecular Dynamics) to obtain the image. Anticipating low physiological Hp levels, we also enriched Between the two gene probes, the blot was stripped of hybridized the Hb-binding protein fraction by affinity chromatography. Hb of probes by immersion in 0.1% SDS that was heated to boiling. the plentiful WAP species N. coriiceps wasusedasbaittoisolate Hb-binding proteins. Spun N. coriiceps erythrocytes were Isolation and sequencing of the Hp genomic loci washed twice with notothenioid phosphate buffered saline (PBS; −1 −1 To characterize the control region of the Hp gene, we sequenced select 12 mmol l NaH2PO4,86mmoll Na2HPO4, with osmolality Hp-positive large-insert DNA genomic clones isolated from BAC adjusted with NaCl to 550 mOsm, the physiological level of native (bacterial artificial chromosome vector pCC1BAC, Epicentre) notothenioid plasma), then lysed in five volumes of an ice cold, libraries of the icefish C. aceratus (Detrich et al., 2010), the hypotonic solution of 10 mmol l−1 Tris (pH 8), and centrifuged at red-blooded Antarctic notothenioid D. mawsoni (Nicodemus- 16,000 g for 10 min at 4°C to pellet cellular debris. The supernatant Johnson et al., 2011) and the basal temperate notothenioid was chromatographed by gravity flow on a 1.5×20 cm column of a −1 Eleginops maclovinus for evolutionarily relevant comparison. The Sephadex G-75 (GE Healthcare) in 10 mmol l Tris (pH 8), and the Journal of Experimental Biology

3 RESEARCH ARTICLE Journal of Experimental Biology (2019) 222, jeb188573. doi:10.1242/jeb.188573 visibly red Hb-containing elution fraction was collected. This purified essential for Hx function. A mammalian (rabbit) Hx sequence Hb was coupled to CNBr-activated Sepharose™ 4B (GE Healthcare) (Morgan et al., 1993) was used as reference to identify the following the manufacturer’s instructions, and then packed in a Polyp- distinctive sequence features and functional residues of Hx in Prep column (BioRad). To capture Hb-binding proteins inclusive of notothenioid sequences. Using the X-ray crystal structure of this Hp, 1.5–2 ml plasma from each species was diluted 3-fold with Hx (Protein Data Bank accession number: PDB|1QHU|A chain, notothenioid PBS, and run through the column three times by gravity doi:10.2210/pdb1que/pdb) (Paoli, et al., 1999) as template, Hx flow. After thoroughly washing with 30 column volumes of protein structural models of two icefishes, C. wilsoni and notothenioid PBS, the bound proteins were eluted with 5 ml of P. macropterus, and the red-blooded N. coriiceps were obtained 8moll−1 urea. The eluatewasthen dialyzed overnight in 10 mmol l−1 by homology modeling using the SWISS-MODEL server Tris (pH 8) at 4°C and the dialyzed proteins were lyophilized. (Waterhouse, et al., 2018) to assess global and local folds.

Antibody synthesis and western blot analyses RESULTS To detect the presence of Hp in native plasma, and in the affinity Hp and Hx cDNA sequences column enriched Hb-binding protein fraction, we used an anti-Hp We successfully amplified full-length or nearly full-length Hp cDNA polyclonal antibody in western blot analysis. The anti-Hp antibody was by RT-PCR for seven of the 15 available icefish species and four red- custom produced (by GenScript) in rabbit against several in silico blooded control species. The sequences have been deposited in determined antigenic epitopes from conserved regions of notothenioid GenBank under accession numbers MH548902–MH548912. Full- Hp sequences we obtained for icefishes. Synthetic peptides of these length cDNAs were obtained for four icefish species: Pagetopsis epitopes were made with a terminal cysteine added for conjugation to macropterus, C. aceratus, C. wilsoni and C. hamatus.TheC. wilsoni keyhole limpet hemocyanin. Antibodies generated with the epitope and C. hamatus Hp cDNAs encode an intact protein of 315 amino DKVTPIPLPERGQDC provided the strongest binding and sensitivity acids, whereas C. aceratus and P. macropterus cDNAs contained in subsequent tests. To first test the specificity and sensitivity of the independent nonsense mutations leading to premature termination at anti-Hp antibodies, we expressed and purified recombinant Hp of the amino acid positions 24 and 133, respectively (Fig. 1). Potential 5′ icefish Chionodraco hamatus, which was also used as an icefish Hp UTR sequence divergence and/or extremely low or absence of positive control in western immunoblotting, as well as for determining Hp transcription precluded amplification of full-length cDNA appropriate antibody dilution factors. The C. hamatus Hp cDNA was in the other icefish species. The use of an inner primer that cloned into the pET26b+ expression vector upstream of the HisTag and anneals to the first nine codons (27 nt) of the signal peptide to pair transformed into Rosetta2(DE3)pLysS host cell (Novagen, Millipore- with the 3′ UTR primer succeeded in amplifying partial (nearly full Sigma). A verified recombinant cell clone was grown at 16°C in length) CDS for three additional icefish species, Neopagetopsis lysogeny broth containing 50 µg ml−1 kanamycin and 34 µg ml−1 ionah, Dacodraco hunteri and Chionodraco myersi (Fig. 1). These chloramphenicol to an OD600 of 0.6, at which point protein expression nearly full-length cDNAs serve as evidence that the Hp gene in was induced by adding IPTG (isopropyl β-D-1-thiogalactopyranoside), these three species remained transcriptionally active. Interestingly, followed by further growth of the culture for 4 h. Cells were harvested N. ionah Hp contained a 12 nt deletion (residues 169–172) by centrifugation and lysed in BugBusterR protein extraction reagent without resulting in a reading frame shift, while D. hunteri Hp (Novagen, Millipore-Sigma). Hp was extracted from inclusion sustained the same position 24 nonsense mutation as C. aceratus,and bodies and then purified using His-bind column chromatography additionally a downstream frame-shift mutation at positions 52–54 (Millipore-Sigma) following the manufacturer’s instructions. The (Fig. 1, Fig. S1). purified Hp was eluted in buffer containing 1 mol l−1 imidazole and In contrast to Hp, we successfully RT-PCR amplified Hx cDNA 6moll−1 urea, and the concentration was measured using a Bradford from all available (15 of 16) icefish species and the four red-blooded protein assay kit (BioRad). controls, encoding a protein of 426 to 443 amino acids depending Western blot analyses of Hp were carried out on samples of native on species (Fig. 2). These Hx sequences have been deposited plasma samples and the lyophilized, affinity column enriched in GenBank under accession numbers MH546081–MH546099. Hb-binding protein fraction of the two representative red-blooded Readily amplifiable icefish Hx cDNAs indicate sustained species N. coriiceps and A. nudiceps, and the seven icefish transcription of this gene throughout the icefish family. The Hx species with available fresh plasma, and the icefish C. hamatus protein coding sequences show no apparent molecular defect (Fig. 2), recombinant Hp as positive control. Protein samples were resolved on indicating continued integrity of the Hx gene in the icefishes. precast 8–12% gradient SDS-PAGE gels (Invitrogen), and electrophoretically transferred to Immun-Blot PVDF membrane Molecular evolution of Hp and Hx in the icefishes (Bio-Rad). The membrane was then shaken gently in 5% gelatin With the phylogenetic framework for the target notothenioids (Sigma) for 1 h at room temperature to block non-specific sites, then species and other teleost fishes (Fig. S2), the RELAX test identified incubated for 2 h in PBST (PBS with 1% Tween 20) with a 1:1000 a significant relaxation in selective pressure on the Hp sequence of dilution of the primary anti-Hp antibody (stock concentration both the Hb-less icefishes and the red-blooded notothenioids 0.864 mg ml−1), followed by washing three times with PBST. The relative to the reference teleost fishes. In both comparisons, the washed blot was incubated for 1 h with the secondary goat anti-rabbit estimated intensity of selection parameter, K, was found to be below antibody diluted 1:20,000 in PBST from a 0.8 mg ml−1 stock. After 1, indicating that selection strength has been reduced along the test washing with PBST, the membrane was treated with enhanced branches (Table 1). However, direct comparison between the chemiluminescence (ECL) western blotting substrate (Pierce), and icefishes and the red-blooded Antarctic notothenioids failed to find the signal was imaged with a LI-COR C-DiGit Imaging System. a significant change in selective pressure, as did all of the comparisons against the ancestral icefish branch. In contrast, Hx Functional integrity of Hx inferred from structural models showed signs of intensification in selective pressure (K>1) in both We examined the icefish Hx sequence features and modeled their the icefishes and the red-blooded Antarctic notothenioids when protein structures to assess the integrity of structural components compared against the other teleosts (Table 2). Journal of Experimental Biology

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Predicted signal peptide sequence Pro-sequence

D. rerio D. mawsoni N. coriiceps P. borchgrevinki G. acuticeps P. macropterus N. ionah (partial) D. hunteri frame 1 * D. hunteri frame 3 C. aceratus * C. wilsoni C. myersi (partial) C. hamatus C. aceratus and D. hunteri premature stop codons D. hunteri frameshift

D. rerio D. mawsoni N. coriiceps P. borchgrevinki G. acuticeps P. macropterus * N. ionah (partial) D. hunteri frame 3 C. aceratus C. wilsoni C. myersi (partial) C. hamatus P. macropterus premature stop codon

D. rerio D. mawsoni N. coriiceps P. borchgrevinki G. acuticeps P. macropterus N. ionah (partial) D. hunteri frame 3 C. aceratus C. wilsoni C. myersi (partial) C. hamatus

D. rerio D. mawsoni N. coriiceps P. borchgrevinki G. acuticeps P macropterus N. ionah (partial) D. hunteri frame 3 C. aceratus C. wilsoni C. myersi (partial) C. hamatus

Fig. 1. Haptoglobin (Hp) amino acid alignment. Hp alignment for the icefish species where the Hp could be isolated in whole or in part, along with several control red-blooded species. The predicted signal peptide and the prosequence are indicated. The D. hunteri sequence is split over two lines to keep the front and rear segments in frame despite a frameshift mutation. The light blue bar above the alignment denotes the region sequenced for all 16 icefish species from genomic DNA as displayed in Fig. 4B. Species list: Danio rerio, Dissostichus mawsoni, Notothenia coriiceps, Pagothenia borchgrevinki, Gymnodraco acuticeps, Pagetopsis macropterus, Neopagetopsis ionah, Dacodraco hunteri, Chaenocephalus aceratus, Chaenodraco wilsoni, Chionodraco myersi and Chionodraco hamatus.

Northern blot assessment of Hp and Hx transcription with those of the red-blooded notothenioids. Contrasting with Hp, To ascertain whether technical issues (primer mismatch, non- Hx transcription appears qualitatively robust in the icefishes, albeit optimal RT and PCR conditions, etc.) solely contributed to the levels based on hybridization intensity are highly variable, unsuccessful amplification of Hp cDNA in some of the icefish but the variability was similarly observed in the red-blooded species, we performed a northern blot to broadly assess liver species (Fig. 3C). Hp transcription (Fig. 3). The results showed that among the 15 examined icefish species, Hp mRNA was weakly detected in Hp genomic loci and Hp genes of icefishes five of the icefishes: P. macropterus, C. aceratus, C. wilsoni, Sequencing results for the Hp genomic loci in E. maclovinus, C. hamatus and C. rastrospinosus (Fig. 3B), three of which had D. mawsoni and the icefish C. aceratus are deposited in the NCBI produced full-length Hp cDNA on RT-PCR amplification. SRA (accession numbers SRR5878014–SRR5878018), and the Hp

The transcript abundances, however, are miniscule compared gene structure is diagrammed for all three species in Fig. 4A. The Journal of Experimental Biology

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D. mawsoni N. coriiceps P. borchgrevinki G. acuticeps C. esox C. gunnari P. macropterus P. maculatus N. ionah P. georgianus D. hunteri C. aceratus C. dewitti C. antarticus C. atkinsoni C. wilsoni C. myersi C. hamatus C. rastrospinosus First blade disulfide bridge site

Linker D. mawsoni N. coriiceps P. borchgrevinki G. acuticeps C. esox C. gunnari P. macropterus P. maculatus N. ionah P. georgianus D. hunteri C. aceratus C. dewitti C. antarticus C. atkinsoni C. wilsoni C. myersi C. hamatus C. rastrospinosus Fourth blade disulfide bridge site First blade disulfide bridge site

Fig. 2. See next page for legend. Journal of Experimental Biology

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Fig. 2. Hemopexin (Hx) amino acid alignment. Hx alignment for the Pseudochaenichthys georgianus and Cryodraco antarcticus at 15 icefish species for which RNA was available along with several control amino acid position 174 in the full-length protein (Fig. 1). In red-blooded species. Red and blue bars above the alignment denote the addition, we discovered a mutated polyadenylation signal sequence, sequence repeats comprising the two characteristic four-bladed β-propeller Hx domains. The linker sequence connecting the two domains is also labeled AATAGA instead of the canonical AATAAA, in five of the above the alignment. The pair of cysteine residues forming the disulfide species (Fig. 4C). bridges linking the first and fourth blade of each propeller domain are labeled Using the Hp genomic loci of E. maclovinus, D. mawsoni and the below the alignment and highlighted in bright yellow. Additional disulfide icefish C. aceratus, we searched for control element motifs within bridge forming cysteine pairs are shown in Fig. S4. Species list: Dissostichus the proximal 1 kbp sequence ahead of translational start site and mawsoni, Notothenia coriiceps, Pagothenia borchgrevinki, Gymnodraco located a logically placed TATA box at approximately 80 bp acuticeps, Champsocephalus esox, Champsocephalus gunnari, Pagetopsis macropterus, Pagetopsis maculatus, Neopagetopsis ionah, upstream from the ATG start in all three species. Additionally, Pseudochaenichthys georgianus, Dacodraco hunteri, Chaenocephalus putative cis-promoter sites for transcriptional factors including aceratus, Chionobathyscus dewitti, Cryodraco antarcticus, Cryodraco HNF1, HNF4, CEBPB/D and DBP found to be present in the two atkinsoni, Chaenodraco wilsoni, Chionodraco myersi, Chionodraco hamatus red-blooded species are conserved in the icefish (Fig. 5). and Chionodraco rastrospinosus. Translational and functional status of icefish Hp complete gene was assembled into a single contig for E. maclovinus Expecting native Hp levels would be low in icefishes, a recombinant and D. mawsoni, but was split between two contigs in the icefish C. hamatus Hp protein was generated to confirm the specificity and C. aceratus. However, this split did not preclude obtaining sensitivity of the Hp antibody, and the titration western blot showed the complete protein coding sequence, or intron/exon boundaries. that the antibody can detect Hp down to as low as 0.2 ng µl−1 The Hp CDS from spliced exon sequences of the gene from the (Fig. S3A). Subsequent western blot analysis on native blood C. aceratus locus assembly corroborates the occurrence of the plasma (Fig. 6A,B) revealed a single strong immunopositive band in premature stop codon at amino acid position 24 found in the cDNA the control red-blooded notothenioids N. coriiceps and A. nudiceps sequence (Fig. 1). We attempted to amplify the Hp gene from at an estimated size of 37 kDa, close to the average molecular mass genomic DNA of the icefish species for which we could not obtain of approximately 35 kDa calculated from translated Hp sequences. cDNA, but this was unsuccessful. The 5.7 kbp Hp genomic In contrast, western blot of native blood plasma from the icefishes sequence of D. mawsoni is quite long, primarily owing to the large failed to detect Hp at their natural plasma concentrations. intron 2 (Fig. 4A), but we were able to PCR amplify this sequence The failure to detect Hp in icefish blood plasma could reflect from genomic DNA by long-distance PCR (results not shown). The an absence of expression or natural expression at levels below failure to amplify the icefish Hp genomic sequences suggests that the 0.2 ng µl−1 sensitivity of our anti-Hp antibodies. Hb from intron 2 might have further expanded, perhaps at the repeat-rich red-blooded N. coriiceps was used as bait in an affinity column region that had led to the split of the gene in our assembly of the for testing the Hb binding ability of Hp in the blood plasma of C. aceratus Hp genomic locus from BAC clone DNA. We therefore the Antarctic icefishes if present. This affinity column also serves utilized the 3′ flanking sequence of the Hp locus of C. aceratus to to concentrate the icefish plasma Hp from physiological levels, develop a reverse primer and paired it with a forward primer at the where expressed, providing a highly sensitive assay for its start of exon 4, and successfully amplified a large part of the presence. Using a Hb-binding protein fraction enriched by Hp gene for all 16 icefish species in the family. Readily amplifiable affinity chromatography, the presence of Hp was again confirmed Hp genomic sequences indicated that the Hp gene (or at least the in the red-blooded notothenioids(Fig.6C,D),andalsoinoneof bulk of the gene amplified) persists in the genomes all 16 icefishes. the seven icefishes, C. wilsoni, showing a putative 40 kDa Hp The partial sequences are shown in Fig. 4B, and have been protein (Fig. 6B,C, Fig. S3B). The enrichment by Hb affinity deposited in GenBank under accession numbers MH546100– column was approximately 300-fold, which by definition means MH546112. These partial Hp sequences revealed another distinct the C. wilsoni Hp is capable of binding Hb, but is expressed at premature stop (position 39 of the partial sequence; Fig. 4B) in extremely low levels.

Table 1. Results of the RELAX test for changes in selective pressure on haptoglobin (Hp) Test branch Background branches Model Parameters log likelihood AICc K LRT statistic P-value Icefishes Red-blooded Alternative 65 −7664.3 15,459.9 0.99 0 1 Antarctic notothenioids Null 64 −7664.3 15,457.9 All other teleosts Alternative 65 −7664.4 15,460.2 0.67* 4.25 0.039 Null 64 −7666.6 15,462.4 Red-blooded All other teleosts Alternative 65 −7666.2 15,463.8 0.63‡ 11.96 0.001 Antarctic notothenioids Null 64 −7672.2 15,473.7 Ancestral icefish branch All other icefish branches Alternative 65 −7671.7 15,474.8 1.86 0.89 0.345 Null 64 −7672.2 15,473.7 Red-blooded Alternative 65 −7666.2 15,463.7 1.23 0.32 0.547 Antarctic notothenioids Null 64 −7666.3 15,461.9 All other teleosts Alternative 65 −7664.2 15,459.8 0.77 0.91 0.34 Null 64 −7664.7 15,458.7 AICc is the Akaike information criterion corrected for small sample size. K is the selection intensity parameter; for a significant result, K>1 indicates an intensification of selective pressure, and K<1 indicates that selective strength has been relaxed. LRT is the likelihood ratio test result. The estimates of the three rate classes, ω1, ω2 and ω3, are provided below where a significant change in selective pressure was identified, with the proportion of sites given in parentheses. *Background branches: ω1=0.02(68.37%), ω2=0.84(29.56%), ω3=288.98(2.07%). Foreground branches: ω1=0.08(68.37%), ω2=0.89(29.56%), ω3=43.69(2.07%). ‡ Background branches: ω1=0.03(69.55%), ω2=0.86(28.14%), ω3=305.23(2.31%). Foreground branches: ω1=0.11(69.55%), ω2=0.91(28.14%), ω3=35.98(2.31%). Journal of Experimental Biology

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Table 2. Results of the RELAX test for changes in selective pressure on hemopexin (Hx) Test branch Background branches Model Parameters log likelihood AICc K LRT statistic P-value Icefishes Red-blooded Alternative 65 −9925.3 20,044.7 0 −15.07 1 Antarctic notothenioids Null 64 −9956.8 20,027.6 All other teleosts Alternative 65 −9939.4 20,009.8 8.05* 85.5 0 Null 64 −9982.2 20,093.3 Red-blooded All other teleosts Alternative 65 −9944.8 20,020.7 4.07‡ 24.36 0 Antarctic notothenioids Null 64 −9957.0 20,043.0 Ancestral icefish branch All other icefish branches Alternative 65 −9958.3 20,047.6 0.94 0.04 0.835 Null 64 −9958.3 20,045.7 Red-blooded Alternative 65 −9972.4 20,075.8 2.49 −3.79 1 Antarctic notothenioids Null 64 −9970.5 20,070.0 All other teleosts Alternative 65 −9947.4 20,025.8 8.33 2.82 0.093 Null 64 −9948.8 20,026.6 K is the selection intensity parameter; for a significant result, K>1 indicates an intensification of selective pressure, and K<1 indicates that selective strength has been relaxed. LRT is the likelihood ratio test result. *Background branches: ω1=0.01(44.69%), ω2=0.14(40.87%), ω3=1.60(14.43%). Foreground branches: ω1=0.00(44.69%), ω2=0.00 (40.87%), ω3=44.62(14.43%). ‡Background branches: ω1=0.01(51.85%), ω2=0.17(34.37%), ω3=1.57(13.78%). Foreground branches: ω1=0.00(51.85%), ω2=0.00(34.37%), ω3=6.28(13.78%).

In silico inference of functional integrity of notothenioid Hx Hb and heme binding function. This study provides the first We examined the canonical sequence features and functional examination of this evolutionary domino in the Antarctic icefish residues, as well as protein structure models of translated Hx protein family, and the outcome from the release of selective pressure due to sequences of the notothenioid fishes to assess functionality. The the absence of RBCs and Hb. The contrasting fates we observed for well-studied mammalian Hx consists of two distinctive homologous the two candidate genes offers insight into the impact of Hb loss that Hx domains connected at 90 deg to each other by a short linker, with may fall on the network of its supporting genes. each domain comprising four sequence repeats that form a four- bladed β-propeller structure. Three pairs of cysteine residues in each Divergent evolutionary fate of Hp and Hx domain form three disulfide bridges, and a pair of histidine residues, The Hb-null state of the entire icefish clade means that the selective one in the linker and the other in repeat 1 of the C-terminal domain, pressure to maintain functional Hp as an Hb-scavenger would have coordinate the heme ligand and stabilize it in the binding pocket been removed at the origin of the icefishes, with the expectation that between the two domains (Paoli et al., 1999; Piccard et al., 2007; the trait would continue to degenerate and potentially become Shrimal and Gilmore, 2013). These sequence features and extinct. However, Hp exhibits a peculiar pattern of persistence and functional residues are conserved in both the red-blooded and loss across vertebrate taxa. Chicken (bird) and western clawed frog white-blooded notothenioids as indicated in the sequence alignment (amphibian) lacked the Hp gene, with a different scavenging protein (Fig. 2) with additional details for three representative species (PIT54) replacing Hp as the free Hb scavenger in chicken (Wicher selected for Hx structural modeling (Fig. S4). Using the mammalian and Fries, 2006). Hp is present and expressed at high levels in (rabbit) Hx X-ray crystal structure (1HQU chain A) (Paoli et al., cartilaginous fishes, but its Hb binding affinity is low or nil, 1999) as template, homology modeling of the translated Hx suggesting that Hp may serve other, as yet unknown, non-Hb sequences of a representative red-blooded notothenioid scavenging function in these fishes (Redmond et al., 2018). In N. coriiceps and the two icefishes C. wilsoni and P. macropterus teleost fishes, the Hp gene is present in various species, and native showed high degree of conservation of global structure and the two serum of the Japanese pufferfish binds Hb (Wicher and Fries, 2006), four-bladed propeller domains (Fig. 7). The GMQE (global model but trout Hb does not, again suggesting it persists in trout for a non- quality estimation; range 0 to 1) scores of all three notothenioid Hb binding role(s) (Redmond et al., 2018). With only two teleost models are 0.71, indicating high reliability of the models. QMEAN species in which Hb-binding has been assessed and producing (qualitative model energy analysis) scores are −1.45, −1.37 and contradictory results, it remains unclear whether Hp in general has −1.70 for N. coriiceps, C. wilsoni and P. macropterus, respectively, other non-Hb binding functions in teleosts in general, or whether it indicating good agreement between the modeled structures and is species specific, and what the function(s) may be. In this study, experimental structures as they are much higher than the −4.0 cut- we showed that the Hp gene is present in the basal notothenioid E. off score for low-quality models (Benkert et al., 2011). These results maclovinus and across the derived Antarctic species, both red- and are consistent with the notothenioid Hx cDNAs being translatable white-blooded. Further, functional results indicate an Hb binding by into a functional protein. a plasma protein (very likely Hp) in the red-blooded Antarctic notothenioids N. coriiceps and A. nudiceps (Fig. 6B) and the icefish DISCUSSION C. wilsoni (Fig. 6D). These establish that the Hp genotype and The evolution of Hp and Hx in vertebrates as scavengers of Hb-binding function is a shared plesiomorphic trait in the cytotoxic free Hb and heme was intimately tied to the appearance of Notothenioidei suborder. If the only or predominant role for Hp is Hb, emerging in fishes as Hb became present at high corpuscular Hb binding in the notothenioids, then the loss of Hb at the origin of concentrations (Wicher and Fries, 2010). The ancestor of the the icefishes would have removed its major, if not the sole, client Antarctic icefishes suffered genetic loss of this important oxygen protein, relaxing selective constraint for its maintenance. transport protein, yet survived and diversified into 16 species (Kock, The hypothesized loss in selective pressure on Hp maintenance 2005). This trait loss begs the question of how it in turn affected the was corroborated by the near silencing of Hp transcription and fate of partnering protein genes that once supported erythropoiesis translation across the icefishes we examined. With the less sensitive and eryoptosis, particularly the Hp and Hx proteins with dedicated but more global detection by northern blot, Hp transcription in Journal of Experimental Biology

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A amplifying Hp cDNAs from seven of the 15 available icefishes, Red-blooded Icefish indicating that Hp transcription exists at low levels in these seven species (Fig. 1). The inability to amplify Hp cDNA by RT-PCR or us detect mRNA by northern blot for the other eight species

ionah hunteri rastrospinosus

D. mawsoniP. borchgrevinkiN. coriicepsG. acuticepsC. esox C. gunnariP. macropterusP. maculat N. P. georgianusD. C. aceratusC. dewittiC. antarcticusC. atkinsoniC. wilsoni C. myersi C. hamatusC. indicatesthat either the transcription level is below the detection limit of these two methods, or that these species may have lost Hp transcription entirely. The mutated polyadenylation signal sequence in five species – C. rhinoceratus, C. aceratus, C. dewitti, C. atkinsoni and C. rastrospinosus (Fig. 4C) – likely contributes to mRNA instability, obviating ready detection. Investigations of the translational status of the Hp gene provided an even greater contrast. Only one icefish species, C. wilsoni, showed a protein of expected size for Hp on western blot (Fig. 6C,D) after its Hb-binding protein fraction in the plasma had been greatly enriched (∼300-fold) for Hp using Hb-affinity column chromatography. Thus, physiological Hp protein levels must be extremely low even if expressed, and below the threshold of detection (0.2 ng μl−1) of our antibodies (Fig. S3A). No Hp could be immuno-detected in the other six icefish species with available B fresh plasma, whether in the native plasma (Fig. 6A,B) or affinity Red-blooded Icefish column enriched Hb-binding protein fraction from a large volume of

us plasma (Fig. 6C,D). This shows a paucity of functional protein product at physiological conditions either from translation levels too

ionah hunteri rastrospinosus D. mawsoniP. borchgrevinkiN. coriicepsG. acuticepsC. esox C. gunnariP. macropterusP. maculat N. P. georgianusD. C. aceratusC. dewittiC. antarcticusC. atkinsoniC. wilsoni C. myersi C. hamatusC. low to measure, or inability of any synthesized Hp to bind Hb, or no Hp protein synthesis at all. Although terminal evidence of relaxed selective pressure, in the form of loss-of-function mutations, occurred only in the icefishes, tests for shifts in selective pressure detected signs of relaxation in both the icefish and the red-blooded Antarctic notothenioids (Table 1). Additionally, direct comparison against the red-blooded Antarctic notothenioids failed to find a significant signature of further relaxation specific to the icefishes. One possible reason for this shared relaxation in selective pressure is a potential lower incidence of free plasma Hb, even in the red-blooded species, owing C to much lower hematocrits and mean corpuscular Hb concentrations Red-blooded Icefish in Antarctic notothenioids compared with temperate fishes (Wells et al., 1980), and with the expected slower turnover of RBCs given us their extreme low body temperature. But it is only among the icefish

ionah hunteri rastrospinosus species in which the need for Hb binding had been fully removed D. mawsoniP. borchgrevinkiN. coriicepsG. acuticepsC. esox C. gunnariP. macropterusP. maculat N. P. georgianusD. C. aceratusC. dewittiC. antarcticusC. atkinsoniC. wilsoni C. myersi C. hamatusC. that nonsense mutations were capable of reaching fixation. Ultimately, if there is only relatively weak further relaxation in selective pressure with the origin of the icefishes than this may not be readily detectable using existing approaches. In marked contrast to Hp, northern blot showed that Hx is clearly transcribed across all 15 of the tested icefishes, some at prominent levels (Fig. 3C). The Hx mRNA levels are variable among icefish species, but variations are also observed in the red-blooded notothenioids. Whether they resulted from differing levels of physiological demands for Hx function among individual fishes at the time of sampling is unclear. Regardless of mRNA abundance on northern blot, full-length Hx cDNA was readily amplified from liver RNA for all icefish species (Fig. 2), indicating that this heme scavenging gene is actively transcribed regardless of presence or Fig. 3. Northern blot analysis of Hp and Hx transcription in red- and white- absence of Hb. Although Hb would be the largest reservoir of heme, blooded notothenioid fishes. (A) Liver RNA samples run on a 1.1% in its absence in icefishes, demands for Hx function could arise from denaturing gel for northern blot analysis showing RNA integrity. (B) Northern a variety of other cellular client hemoproteins. These include blot hybridized with Hp cDNA probe. (C) Northern blot hybridized with Hx cytochromes of the respiratory chain in mitochondria, which occur at cDNA probe. greatly increased density in icefish cells than in the red-blooded species to facilitate cellular O2 transport, heart Mb in icefish species icefishes has greatly declined in comparison with the red-blooded that synthesize it (O’Brien and Mueller, 2010), microsomal and relatives and appears non-existent in several species (Fig. 3B). With mitochondrial cytochrome P450s, as well as neuroglobin (Cheng, repeated optimization, the more sensitive RT-PCR succeeded in et al., 2009) and cytoglobin (Cuypers, et al., 2017). This level of Journal of Experimental Biology

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E. maclovinus 378 bp 2401 bp 773 bp

Exon 1 Exon 2 Exon 3 Exon 4 5 bp 89 bp 284 bp 561 bp

D. mawsoni 522 bp 3804 bp 839 bp

Exon 1 Exon 2 Exon 3 Exon 4 5 bp 86 bp 284 bp 561 bp

C. aceratus gDNA amplification site 522 bp 829 bp

Exon 1 Exon 2 Exon 3 Exon 4 5 bp 86 bp 284 bp 570 bp

B 10 20 30 40 50 60 70 80 90 100

D. mawsoni G. acuticeps C. esox C. gunnari P. macropterus P. maculatus P. georgianus N. ionah D. hunteri C. rhinoceratus C. aceratus C. dewitti C. antarcticus C. atkinsoni C. wilsoni C. myersi C. hamatus C. rastrospinosus P. georgianus and C. antarcticus premature stop codons 110 120 130 140 150 160 170 D. mawsoni G. acuticeps C. esox C. gunnari P. macropterus P. maculatus P. georgianus N. ionah D. hunteri C. rhinoceratus C. aceratus C. dewitti C. antarcticus C. atkinsoni C. wilsoni C. myersi C. hamatus C. rastrospinosus

C 10 20 30 40 50 60 70 80 90 D. mawsoni G. acuticeps C. esox C. gunnari P. macropterus P. maculatus P. georgianus N. ionah D. hunteri C. rhinoceratus C. aceratus C. dewitti C. antarcticus C. atkinsoni C. wilsoni C. myersi C. hamatus C. rastrospinosus Stop codon Polyadenylation signal

Fig. 4. See next page for legend. Journal of Experimental Biology

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Fig. 4. Hp genomic structure of three notothenioids and partial Hp gene an intensification in both the icefishes and red-blooded Antarctic structure of 16 icefishes. (A) Complete Hp gene structure assembled from notothenioids relative to the reference teleost species (Table 2). Heme 454 sequencing of Hp loci of three selected notothenioid species, occurs in four (a, b, c and d1) common types in vertebrate E. maclovinus, D. mawsoni and C. aceratus. The 3′ Hp gene region to PCR amplify from genomic DNA for sequencing is demarcated on the Hp gene of hemoproteins, with heme b being the most abundant as the C. aceratus. (B) Amino acid sequence alignment of the CDS in the 3′ Hp gene prosthetic group of Hb and Mb (Chapman et al., 1997). The detected region for all 16 icefishes in the family. A third distinct premature stop codon intensification in selective pressure among the Antarctic notothenioids shared by P. georgianus and C. antarcticus was identified. (C) Alignment of 3′ may again reflect the absence or reduced incidence of free heme from flanking sequence of the 16 icefishes, showing mutated polyadenylation signal Hb in the white-blooded and red-blooded species, respectively, and the sequence (AATAGA) in 5 of the 16 icefishes. resulting optimization of the Hx protein in the face of what is likely a marked change in the prevalence of heme types. client demand appears sufficient to maintain steady levels of Hx Alternatively, this could represent a shift in the gene primarily transcription. The Hx cDNA sequences of all the icefish species do responsible for heme binding in the Antarctic notothenioids. Most not contain any deleterious mutations and are thus likely translated teleost species have two WAP65 (Warm Acclimation Protein 65) into functional Hx proteins (Fig. 2). Our sequence and structural genes that are similar to the mammalian Hx. Although differences in analyses support the functionality of the encoded Hx protein. The their physiological function remain unclear, both retain heme strong conservation of the distinctive dual Hx domains with the binding motifs (Machado et al., 2014; Diaz-Rosales et al., 2014). sequence repeats and functional residues (Fig. 2, Fig. S4) and of the Only one of these could be found in the available transcriptomes and global and domain structures with known Hx (Fig. 7) across genomes of the Antarctic notothenioids (WAP65-2), suggesting that red-blooded notothenioids and the icefishes serve to support that the other has been lost. The detected signature of intensified proteins encoded by their Hx cDNA would be functional. The selective pressure could thus reflect this gene now being co-opted difference in expression of the Hp and Hx genes therefore lies in the into the additional roles formerly carried out by WAP65-1. fact that only the Hp function experiences truly relaxed selection, thus drastic trait reduction was only observed for Hp. Independent losses of Hp in the icefish lineage In contrast to Hp, and in keeping with Hx genic integrity and Underlying Hp expression loss is a more complex and interesting transcription, estimates of the strength of selective pressure showed pattern of molecular lesions that are independent of phylogenetic

–780 –710 –700 E. maclovinus D. mawsoni C. aceratus HNF4A

–700 –690 –680 –670 –660 –650 –640 –630 –620 –610 E. maclovinus D. mawsoni C. aceratus HNF4A –600 –590 –580 –570 –560 –550 –540 –530 –520 –510 E. maclovinus D. mawsoni C. aceratus

–500 –490 –480 –470 –460 –450 –440 –430 –420 –410 E. maclovinus D. mawsoni C. aceratus CEBPB/D CEBPB

–400 –390 –380 –370 –360 –350 –340 –330 –320 –310 E. maclovinus D. mawsoni C. aceratus CEBPB/D DBP/CEBPB

–300 –290 –280 –270 –260 –250 –240 –230 –220 –210 E. maclovinus D. mawsoni C. aceratus

–200 –190 –180 –170 –160 –150 –140 –130 –120 –110 E. maclovinus D. mawsoni C. aceratus

–100 –90 –80 –70 –60 –50 –40 –30 –20 –10 0 E. maclovinus D. mawsoni C. aceratus TATA Box HNF1A Start codon

Fig. 5. Overview of transcriptional factor binding sites upstream of the Hp gene in E. maclovinus, D. mawsoni and C. aceratus. The sequence was screened 1 kb upstream of the start codon to identify cis-acting regulatory elements. The core promotor TATA box and other putative transcriptional factor binding sites are color highlighted in the foreground. Journal of Experimental Biology

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A C Hb-binding proteins

nnari

lasma C. hamatus p P. georgianus C. aceratus C. dewitti C. antarcticus C. wilsoni C. rastrospinosus Standard 1 expressed HpN. coriiceps N. coriiceps C. gu Standard 2

nnari

N. coriiceps A. nudiceps C. gu P. georgianus C. aceratus C. dewitti C. antarcticus C. wilsoni C. rastrospinosus

250 kDa 150 kDa 250 kDa 100 kDa 75 kDa 150 kDa 116 kDa 100 kDa 50 kDa 97 kDa

75 kDa 37 kDa 66 kDa

50 kDa 45 kDa

25 kDa 37 kDa 31 kDa

20 kDa 25 kDa 20 kDa 21 kDa 15 kDa 14 kDa 15 kDa

Hb-binding proteins BD nnari nnari lasma p C. hamatus N. coriiceps N. coriiceps P. georgianus C. dewitti C. wilsoni N. coriiceps Standard 1 C. gu C. aceratus C. rastrospinosusStandard 2 A. nudiceps C. gu P. georgianus C. aceratus C. dewitti C. antarcticus C. wilsoni expressed Hp C. antarcticus 250 kDa C. rastrospinosus 150 kDa 250 kDa 100 kDa 75 kDa 150 kDa 50 kDa 116 kDa 100 kDa 37 kDa 97 kDa 75 kDa 66 kDa 25 kDa 50 kDa 45 kDa 20 kDa 37 kDa

31 kDa 15 kDa 25 kDa 20 kDa 21 kDa

15 kDa 14 kDa

Fig. 6. Western blot analysis for Hp. (A) SDS-PAGE of crude blood plasma proteins, stained with Coomassie Brilliant Blue. Each lane contains 0.5 µl plasma. (B) Western blot hybridization of crude blood plasma proteins with anti-Hp antibody at the concentration of 0.864 µg ml−1. (C) SDS-PAGE of Hb binding proteins from affinity column, stained with Coomassie Brilliant Blue. Lanes 5 to 11 each contains the amount of Hb-binding proteins from icefish plasma. As positive controls, lane 4 contains Hb-binding proteins from 5 µl plasma of the red-blooded fish N. coriiceps, and lane 3 contains 0.075 µl plasma of N. coriiceps. (D) Western blot hybridization of eluted Hb-binding proteins with anti-Hp antibody at the concentration of 0.864 µg ml−1. relatedness, as summarized in Fig. 8 from cDNA (Fig. 1) and Interestingly, despite CDS mutation(s), the Hp gene of genomic sequences (Fig. 3). Three distinct nonsense mutations P. macropterus, P. georgianus, D. hunteri and C. aceratus (at amino acid positions 24, 133 and 174) occurred in five species – continued to be transcribed (Fig. 1), perhaps owing to the P. macropterus, P. georgianus, D. hunteri, C. aceratus and stochastic nature of the degeneration process. The combination of C. antarcticus – occupying separate branches in the icefish intact and pseudogenized Hp genes among the icefishes suggest phylogeny (Fig. 8). The D. hunteri Hp gene additionally sustained we are observing differing progression of non-functionalization a 7-nt deletion (at positions 52–54), causing a reading frame shift and among these species. The Hp gene, and perhaps others like it, truncation of the CDS shortly downstream. Any of these mutations were not lost with the initial disappearance of Hb, but rather their will lead to a truncated protein if translated, compromising or sequences are drifting towards functional extinction in a sporadic obliterating the Hp function. Thus, non-functionalization of the manner, and independently among the icefish lineages. Hp is Hp trait upon Hb loss has involved multiple independent molecular synthesized as a preproprotein, with an N-terminal signal peptide mechanisms akin to the independent loss of Mb in various that targets it to the secretory pathway and extracellular export, icefish lineages. and proteolytic cleavage of the proprotein that generates the Journal of Experimental Biology

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Fig. 7. Structural models of notothenioid ABHx by homology modeling using X-ray C C crystal structure of mammalian Hx as N N template. (A) Rabbit Hx structure (Protein Data Bank accession number: PDB|1QHU|A chain, doi:10.2210/pdb1que/pdb) showing the two homologous domains at 90 deg to each other, and the heme ligand bound at the center and constrained by the linker. The orange box indicates the four-blade propeller structure of the C-terminal domain. The models of Hx from the red-blooded notothenioid N. coriiceps Hx (B), and the icefishes C. wilsoni (C) and P. macropterus (D) show high degree of conservation of global structure and the four-blade propeller Hx domains. Models CDwere generated using SWISS MODEL. C C NN

biologically active mature protein (Wicher and Fries, 2006). An start of the mature protein (Fig. 1) (Wicher and Fries, 2006). interesting observation of note is that the nonsense mutation at Thus, the entire mature protein CDS remains free of deleterious amino acid position 24 in C. aceratus Hp is located in the pro mutations, and in principle, the Hp gene of C. aceratus could sequence of the preproprotein. The predicted proprotein cleavage evolve to encode a cytoplasmic protein should there be selection sequence is ‘RSRR’ at sites 38–41, with the Met on 42 being the pressure for its continuance.

Icefish phylogeny Gene Transcript Protein Non-functionalization mechanism Channichthyidae Champsocephalus esox + – NA

Champsocephalus gunnari + – –

Pagetopsis macropterus ++ NA Premature stop codon at site133

Pagetopsis maculatus + – NA

Pseudochaenichthys georgianus + –– Premature stop codon at site174

Neopagetopsis ionah ++ NA

Dacodraco hunteri ++ NA Frameshift owing to a 7 nt deletion

Channichthys rhinoceratus + –NA Mutated polyadenylation site

Chaenocephalus aceratus ++ – Premature stop codon at SP

Chionobathyscus dewitti + –– Mutated polyadenylation site

Cryodraco antarcticus + –– Premature stop codon at site 174

Cryodraco atkinsoni + –NA Mutated polyadenylation site

Chaenodraco wilsoni ++ +

Chionodraco myersi ++ NA

Chionodraco hamatus ++ NA 1 Chionodraco rastrospinosus ++ – Mutated polyadenylation site

Fig. 8. Summary of icefish Hp status at gene, transcript and protein levels. All icefish species have the Hp gene, while the Hp transcript is only present in some of these icefish species. Only one examined species retains Hp protein expression, and it shows a greatly reduced expression level compared with red-blooded relatives. A ‘+’ sign demarcates that the gene, transcript or protein was amplified or detected; a ‘–’ sign demarcates that they were tested and either not amplified or not detected. NA denotes species for which appropriate samples were not available. The phylogenetic tree is adapted from

Near et al. (2003). Journal of Experimental Biology

13 RESEARCH ARTICLE Journal of Experimental Biology (2019) 222, jeb188573. doi:10.1242/jeb.188573

Evolution of Hp control elements red-blooded N. coriiceps; however, the integrity of Bty in icefishes Apart from changes in the protein coding region, functional and the basis of its paltry expression remained unknown. diminution of Hp could also have resulted from changes in the More recent work on the evolution of the erythropoiesis network regulatory elements that control gene expression. Obtaining the Hp investigated evolution of transcriptional activity in hematopoietic genomic sequences from large DNA insert BAC clones for the basal tissues in the icefish C. hamatus (Xu et al., 2015). This study red-blooded E. maclovinus as an ancestral proxy – the red-blooded identified substantial remodeling of the hematopoietic programs in Antarctic D. mawsoni and the icefish C. aceratus – allowed us to icefish with large-scale reduced expression of hematopoiesis-related investigate the evolution of the control region, and the impact of genes compared with a red-blooded notothenioid, including many relaxed selective pressure on cis regulatory elements controlling Hp transcriptional factors essential for erythropoiesis. However, in expression. The core promotor, the TATA box, remains intact in the investigating two icefish species, Desvignes et al. (2016) found icefish and identical to that of the two red-blooded species (Fig. 5). broad conservation of miRNAs that regulate erythropoiesis in Several transcriptional factors (TFs) are known to have essential white-blooded Antarctic icefish, despite their lacking RBCs, and roles in affecting liver-specific gene expression and in controlling suggested possible additional roles outside of erythropoiesis. the activity of Hp as part of the acute phase response (APR) in In these studies, the focus on single species or small numbers of inflammation. These include HNF1, HNF3, HNF4, HNF6, C/EBP species limits understanding the evolution of these systems in and DBP (Pelletier et al., 1998; Schrem et al., 2002; Odom et al., the diversification of the icefishes, perhaps missing broader 2004). Among these, the induction of Hp during the APR is itself evolutionary trends. In addition, no study thus far has considered regulated through C/EBP beta and delta in hepatocytes (Baumann the fate of those genes associated with the aftermath of RBC and Gauldie, 1994). senescence, the recycling of otherwise strongly oxidizing and thus The prolonged loss of selective pressure for Hp in icefishes would cytotoxic free Hb and heme, which should have been concomitantly expectedly provide the opportunity for sequence degradation in the impacted by the loss of selective pressures from the disappearance of cis regulatory elements that these TFs interact with, leading to the RBCs and Hb. Therefore, the present study is the first to address this drastic reduction of Hp expression that we observed. Surprisingly, hitherto unexplored evolutionary perspective, and assess the HNF1A, HNF4A, CEBP-B, CEBP-D and DBP remained conserved functional outcome and the underlying molecular mechanism of in C. aceratus (Fig. 5). Persistence of these elements, however, does two primary supporting genes across the entire icefish family. not necessarily mean that the transcription of the Hp gene is actively engaged by these TFs, as transcription does have multiple levels of Conclusions control. It only shows that the reduction in selective pressure thus far The icefishes represent a rich, ongoing ‘evolutionary experiment’. has not impacted these putative regulatory elements. It remains The loss of Hb, the major and abundant oxygen transport protein, unclear whether the icefish Hp gene would be transcriptionally simultaneously relaxed selective pressure on the maintenance of responsive during activation of the APR. Regardless, only Hp genes suites of supporting genes, and generated novel selective pressures without deleterious mutations in the protein CDS, such as that of that compel emergence or reprogramming of other suites of genes C. wilsoni, which was also able to synthesize the protein, would be to sustain the unique Hb-less physiology and life history. The fate functionally relevant. of the two relevant genes Hp and Hx contributes to the With the partial Hp genomic sequences spanning the 3′ flanking understanding of gene fate under relaxed selection in the Hb-less region, we identified an atypical polyadenylation signal sequence state. Although Hx has lost its primary client protein, remaining specific to the icefish. The A-to-G mutation in the fifth position of client proteins appear to exert sufficient selective pressure to the polyadenylation signal sequence in the icefishes (Fig. 4) has ensure Hx functional persistence. In contrast, it is unknown been found to result in the most drastic reduction in the accuracy and whether teleost Hp has non-Hb client proteins. The Hp gene in efficiency of the cleavage of the precursor mRNA and polyA tailing several icefishes has sustained deleterious mutations, and the in a systematic examination of the positional effect of point protein is undetectable for all except one tested species, which mutations of AATAAA on these processes (Sheets et al., 1990). The strongly suggest that should it play any accessory role it is either altered polyadenylation reduces the pool of polyA+ mRNA, and minimally essential or readily replaceable. Thus, Hb is very likely ultimately reduces the amount of final protein product (Lutz and the dominant, if not the sole client, and its loss would have all but Moreira, 2011). The 3′ portion of the Hp genomic sequences we fully relaxed selective pressure on the maintenance of Hp. This obtained for all 16 icefishes in the family showed that five species would allow the gene to drift down various mutational paths, now share this mutated polyadenylation signal sequence (Fig. 4C). ultimately leading to the widespread expression loss and non- Their appearance in phylogenetically distinct lineages throughout functionalization observed across the icefish family, exemplifying the clade further corroborate relaxation on the necessity to regularly a clear case of co-evolutionary loss caught in action. This work synthesize transcripts, resulting in stochastic loss. shows that a more complete understanding of the impacts of Hb loss requires investigations into the dual sides of this impact, both Fate of genes associated with the production versus the creation of new selective pressures for compensatory destruction of RBCs adaptations and the relaxation of selective pressures on former Many genes play partnering roles in supporting Hb and Mb supporting genes. Further, the complex pattern of loss seen among proteostasis. Studies of icefishes in this regard thus far have the icefishes suggests that the impacts of relaxed selective pressure logically focused on genes in the erythropoiesis pathways, in part to remain ongoing, underscoring the importance of family-wide identify mechanisms behind the erythrocyte non-production in assessments of evolutionary impacts to achieve comprehensive these fish. An early study examined one candidate gene, Bty (blood understanding. thirsty) (Detrich and Yergeau, 2004). In the zebrafish test system, Acknowledgements Bty protein expression was found to be required for erythrocyte We would like to thank the staff and contractors at Palmer Stations and aboard production and Hb synthesis (Yergeau et al., 2005). This was the RV LM Gould for their assistance in carrying out this project. We would further expressed at much lower levels in the icefish C. aceratus than in the like to acknowledge the work of two undergraduate students, Elizabeth Kalmanek Journal of Experimental Biology

14 RESEARCH ARTICLE Journal of Experimental Biology (2019) 222, jeb188573. doi:10.1242/jeb.188573 and Nicholas Bart, which contributed to preliminary amplifications of Hp and Detrich, H. W., , III and Yergeau, D. A. (2004). Comparative genomics in Hx sequences. Finally, the authors would like to thank Dr Daniel Macqueen for erythropoietic gene discovery: synergisms between the Antarctic icefishes and thoughtful comments and critiques that helped refine this work. the zebrafish. Methods Cell Biol. 77, 477-505. Detrich, H. W., , III, Stuart, A., Schoenborn, M., Parker, S. K., Methé,B.A.and Competing interests Amemiya, C. T. (2010). Genome enablement of the notothenioidei: genome size The authors declare no competing or financial interests. estimates from 11 species and BAC libraries from 2 representative taxa. J. Exp. Zool. B Mol. Dev. Evol. 314, 369-363. DeVries, A. L. and Cheng, C.-H. C. (2005). Antifreeze proteins and organismal Author contributions freezing avoidance in polar fishes. Fish Physiol. 22, 155-201. Conceptualization: K.T.B., C.-H.C.C.; Methodology: K.T.B., X.Z., K.R.M., Diaz-Rosales, P., Pereiro, P., Figueras, A., Novoa, B. and Dios, S. (2014). The C.-H.C.C.; Investigation: K.T.B., X.Z., K.R.M., C.-H.C.C.; Writing - original draft: warm temperature acclimation protein (Wap65) has an important role in the K.T.B., X.Z.; Writing - review & editing: K.T.B., X.Z., C.-H.C.C.; Funding acquisition: inflammatory response of turbot (Scophthalmus maximus). Fish Shellfish K.T.B., C.-H.C.C. Immunol. 41, 80-92. Drummond, A. J. and Rambaut, A. (2007). BEAST: Bayesian evolutionary Funding analysis by sampling trees. BMC Evol. Biol. 7, 214. This work was funded by the US National Science Foundation Division of Eastman, J. T. (2005). The nature of the diversity of Antarctic fishes. Polar Biol. 28, Polar Programs grant ANT-1341701 to K.T.B. and C.-H.C.C. 93-107. Edgar, R. C. (2004). MUSCLE: multiple sequence alignment with high accuracy and Data availability high throughput. Nucleic Acids Res. 32, 1792-1797. Huelsenbeck, J. P. and Ronquist, F. (2001). MRBAYES: Bayesian inference of All sequence data have been deposited in NCBI’s GenBank under accession phylogenetic trees. Bioinformatics 17, 754-755. numbers MH548902–MH548912 for Hp cDNA sequences, MH546100–MH546112 Huntoon, K. M., Wang, Y., Eppolito, C. A., Barbour, K. W., Berger, F. G., for Hp 3′ flanking sequences and MH546081–MH546099 for Hx cDNA sequences. Shrikant, P. A. and Baumann, H. (2008). The acute phase protein haptoglobin Reads for the Hp loci of the three targeted species were deposited in the SRA regulates host immunity. J. Leukoc. Biol. 84, 170-181. – under accessions SRR5878014 SRR5878018. The sequence alignments used Khan, A., Fornes, O., Stigliani, A., Gheorghe, M., Castro-Mondragon, J. 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