Marine Biology (2018) 165:181 https://doi.org/10.1007/s00227-018-3440-x

ORIGINAL PAPER

Gene expression of antifreeze protein in relation to historical distributions of fsh species

A. Yamazaki1,2 · Y. Nishimiya3 · S. Tsuda3 · K. Togashi1 · H. Munehara4

Received: 13 March 2018 / Accepted: 22 October 2018 / Published online: 1 November 2018 © Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract Gene expression of antifreeze proteins (AFPs) facilitates various species of fsh to survive in near-freezing seawater. The marine genus Myoxocephalus (Teleostei; ) is widely distributed from Arctic to Subarctic and is known to express type I AFP. Here, we clarify gene structures and the antifreeze activity of three Myoxocephalus species and discuss their cold adaptation and their geographic movements across a global-level time scale. These fshes were collected during winter in 2014–2015 at Hokkaido and Alaska. A total of 14–16 amino acid sequences were determined as type I AFP, some of which exhibit the same amino acid sequence among species. The Subarctic species contains many amino acid sequences, and the antifreeze activities are lowest. In contrast, the Arctic species contains a few active proteins. Mitochondrial DNA analysis suggests that the Atlantic and Arctic species migrated to those oceans 7.9 and 3.0 million years ago (Mya), respec- tively. Since the AFP sequences of Myoxocephalus are similar to each other regardless of their diference in the timing of migration, the ancestral species of this genus may have developed the AFP gene(s) approximately 7.9 Mya, before speciation within the genus. It is speculated that these survived the cold environment in the Arctic Ocean during the Pliocene– Pleistocene by acquisition of the type I AFP in their body fuids.

Introduction

Many marine species inhabiting icy seawater produce anti- freeze proteins (AFPs) or antifreeze glycoproteins (AFGPs) to prevent their body fuids from freezing (DeVries 1984; Responsible Editor: C. Eizaguirre. Cheng 1988; Davies 2014). Following the frst discovery Reviewed by Undisclosed experts. of AFGPs in Antarctic notothenioid fshes (DeVries and Wohlschlag 1969), type I–III AFPs have been successively Electronic supplementary material The online version of this found in winter founder, sea raven and , respectively article (https​://doi.org/10.1007/s0022​7-018-3440-x) contains (Duman and DeVries 1976; Slaughter et al. 1981; Hew et al. supplementary material, which is available to authorized users. 1984; Li et al. 1985). These proteins are thought to inhibit * A. Yamazaki ice growth by irreversible adsorption to the surface of nas- [email protected] cent ice crystals generated at the moment of water freezing (Raymond and DeVries 1977). These proteins are thought to 1 Graduate School of Environmental Sciences, Hokkaido University, N10W5, Sapporo 060‑0810, Japan facilitate these fshes to live in icy niche habitats. Generally, the AFP expression level and its antifreeze activity are high 2 Nanae Fresh‑Water Station, Field Science Center for Northern Biosphere, Hokkaido University, 2‑9‑1 in winter and low in summer. Sakuracho, Nanae town Kameda‑gun, Hokkaido 041‑1105, The remarkable structural diversity of fsh-derived AF(G) Japan Ps has been regarded as an example of convergent evolution 3 Bioproduction Research Institute, National Institute to acquire the same ice-binding function (e.g., Chen et al. of Advanced Industrial Science and Technology, 2‑17‑2‑1 1997; Graham et al. 2013). The repetitive AFGP tripeptide Tsukisamu‑Higashi, Toyohira‑ku, Sapporo 062‑8517, Japan (Thr-Ala/Pro-Ala) coding sequences are drastically diferent 4 Usujiri Fisheries Station, Field Science Center between Antarctic notothenioids and Arctic cod, suggest- for Northern Biosphere, Hokkaido University, 152 Usujiri, ing that they arose from duplications of two distinct, short Hakodate 041‑1613, Japan

Vol.:(0123456789)1 3 181 Page 2 of 11 Marine Biology (2018) 165:181 ancestral sequences with a diferent permutation of three exposure to subzero temperatures below their freezing point codons for the same tripeptide (Chen et al. 1997). Type I by residing in deeper waters, where they can avoid or reduce AFPs containing a monomeric α-helical structure with a the probability of ice contact. The TH values of three Myox- high alanine (Ala) content are encoded by at least three unre- ocephalus species are seasonally modulated, with maximum lated genes identifed from founders, snailfshes, sculpins values in winter. Low et al. (1998; 2001) determined AFP and cunner (Graham et al. 2013). sequences from M. scorpius and M. octodecemspinosus and The sculpin family () consists of demersal fshes demonstrated that transcription of these AFP genes increase of 282 described species categorized into 70 genera (Nel- in March and rapidly decrease until May. Tsuda and Miura son et al. 2016). Most of the species are mainly distributed (2005) showed that type I AFP is produced by some fshes in the North Pacifc Ocean, while nine genera (Artediellus, living in coastal areas of the Northwest Pacifc Ocean where Enophrys, Gymnocanthus, Hemilepidotus, Icelus, Myoxo- the sea temperature is usually above 0 °C, suggesting that cephalus, Megalocottus, Porocottus, Triglops) encompassing not only Arctic but also mid-latitude species contain AFP in a total of 31 species inhabit the Arctic Ocean (Mecklenburg their body fuids. The sea surface temperature at the coastal et al. 2011). Cottidae is the dominant family in the Arctic region of the Sea of Okhotsk during last glacial period was Ocean, followed by Zoarcidae, which comprises 34 spe- below 0 °C in winter season (Harada et al. 2006). Drifting cies (Mecklenburg et al. 2011). Cottidae arose in the North ice seasonally covers the Sea of Okhotsk and the sea tem- Pacifc Ocean (Briggs et al. 2013) and expanded its distri- perature is also below 0 °C, therefore, the species inhabiting bution from the Northeast Pacifc Ocean to the Northwest around Hokkaido would be expected to have AFP. However, Pacifc, Arctic, and Atlantic Oceans (Briggs et al. 2013). the activity levels and genetic structures of these AFPs have Many marine species are thought to have migrated to the not been clarifed and are presumably diferent between Arc- Arctic Ocean from the North Pacifc Ocean via the Bering tic and non-Arctic species. Strait approximately 12–6 Mya, 5–3 Mya, or more recently Here, we focus on the cold-adapted fshes Myoxocepha- after the Strait had opened (Harrison and Crespi 1999; lus, which are widely distributed from Arctic to mid-latitude Briggs 2003, 2007a, b). Further studies showed that the seas. We examined the AFP gene structures and expression migration of several Cottidae species also occurred approxi- mechanisms of the genus Myoxocephalus, especially the mately 5–3 Mya (Yokoyama and Goto 2005; Yamazaki et al. Arctic–Subarctic species M. polyacanthocephalus and Sub- 2013). arctic species M. stelleri and M. brandtii, which are distrib- Cottidae-derived AFP was initially identifed from the uted in Alaska and Hokkaido. Based on the analyses, cold warty sculpin, Myoxocephalus verrucosus (Raymond et al. adaptation of Myoxocephalus and its historical migration to 1975), which is distributed across the high latitudes of the diferent temperature environments will be discussed on a northern hemisphere (Mecklenburg et al. 2002; Froese and global-level time scale. Pauly 2015). The Myoxocephalus genus comprises 15–16 species that occur widely from the North Pacifc and Arc- tic Oceans to the North Atlantic Ocean (Mecklenburg et al. Materials and methods 2002; Amaoka et al. 2011; Mecklenburg et al. 2011; Froese and Pauly 2015). Four species—M. scorpius, M. aenaeus, Sample collection M. quadricornis and M. q. thompsonii—inhabit the Arctic Ocean, while two other species—M. polyacanthocephalus Fresh livers from a frog sculpin M. stelleri and a great scul- and M. jaok—migrate to the Arctic Ocean only in summer pin M. polyacanthocephalus were collected from Notsuke, (Mecklenburg et al. 2007; Mecklenburg et al. 2011). Iso- northern Hokkaido (43.568155°N, 145.225606°W) in Feb- forms of type I AFP were also identifed from the shorthorn ruary 2002 by ice under waiting seine fshing (Table 1). sculpin, Myoxocephalus scorpius, in Newfoundland, Canada The fresh fn clips from eight specimens of the great scul- (Hew et al. 1980), the longhorn sculpin, M. octodecemspi- pin were collected from Dutch Harbor (53.872228°N, nosus, and the grubby sculpin, M. aenaeus (Reisman et al. 166.561668°W), Unalaska Island, Alaska, in March 2014 1987; Chakrabartty et al. 1988; Low et al. 2001). Duman by scuba diving. Fresh livers and fn clips from a total of and DeVries (1974) determined the thermal hysteresis (TH), 11 specimens of the frog sculpin M. stelleri and 11 speci- which is defned as the diference between melting and freez- mens of the snow sculpin M. brandtii were collected from ing points of an AFP solution and is used as an indicator of Mori (42.111372°N, 140.597437°E), southwest Hokkaido, the activity of AFPs, for several Myoxocephalus species, once a month from November 2014 to April 2015 by fshing. and demonstrated that their TH values vary with sampling These tissues were collected from sculpins anesthetized with location and distribution depth. The presence of ice at deep m-aminobenzoate methanesulfonate and stocked overnight area is unlikely because of the efect of depth pressure on at − 4 °C in RNAlater® Stabilization Solution (life technol- the freezing point of water. Many northern species survive ogies™, USA), and then stocked at − 80 °C. Muscle tissue

1 3 Marine Biology (2018) 165:181 Page 3 of 11 181

Table 1 Sample information examined in every month for M. brandtii and M. stelleri and M. polyacanthocephalus Species Location Date N ST TH only in March for .

M. polyacan- AK 19 Mar, 2014 8 3.6 0.80 ± 0.09 RNA extractions thocephalus NT 27 Feb, 2002 1 – – M. brandtii MR 31 Jan, 2015 1 5.6 0.13 ± 0.01 Total RNA was extracted from fn clips and livers collected MR 28 Feb, 2015 1 3.5 0.12 ± 0.01 at Dutch Harbor and Mori using the QuickGene RNA tis- MR 26 Mar, 2015 3 2.8 0.42 ± 0.07 sue kit S II (QuickGene 800, Kurabo, Japan) and DNase MR 16 Apr, 2015 4 7 0.30 ± 0.05 I Amplifcation Grade (Invitrogen™, USA) following the M. stelleri NT 27 Feb, 2002 1 – – manufacturer’s instructions. For the specimen collected at MR 28 Nov, 2014 1 11.2 0.15 ± 0.03 Notsuke, the total RNA was extracted from livers using the MR 31 Jan, 2015 1 5.6 0.16 ± 0.02 RNeasy Mini Kit (QIAGEN, Germany) following the manu- MR 28 Feb, 2015 1 3.5 0.15 ± 0.02 facturer’s instructions. MR 26 Mar, 2015 2 2.8 0.37 ± 0.05 MR 16 Apr, 2015 5 7 0.20 ± 0.02 cDNA library construction and screening

N, sample number; ST, sea temperature; TH, thermal hysteresis; AK, Alaska; NT, Notsuke; MR, Mori For the specimens collected at Dutch Harbor, the poly-A RNA (mRNA) was purifed from total RNA using Oligo- tex™ –dT30 < Super > mRNA Purification Kit (Takara, was immediately preserved at − 25 °C for TH measurement. Japan) following the manufacturer’s instructions. A com- These treatments were according to the Hokkaido University plementary DNA (cDNA) library of the great sculpin was Regulations of Experimentation. constructed from purifed mRNA using cDNA Library Con- struction Kit (Takara, Japan) according the manufacturer’s Measurements of antifreeze activities and thermal instructions. The vectors were transformed to Escherichia hysteresis coli by heat shock method, and incubated on LB plates for 16 h at 37 °C. A skin cDNA library was hybridized to a DeVries (1982) showed that the dialyzed blood without nylon membrane in 2.5 mL of hybrid bufer and 5 µL of AFP has no TH activity. Several articles showed that type probe (5 mM) at 55 °C overnight. A fragment of the 5′ I–III AFP are successfully purifed from the fsh muscle untranslated region (UTR, 5′-CAAACA​ AAA​ AGC​ ATC​ TTG​ ​ homogenates, and described that the fsh muscle is a rich AGACGC​ TCC​ TGT​ TGT​ GAA​ TCA​ GTT​ TAA​ TCA​ ATT​ TAA​ ​ source of AFP (Yamashita et al. 2003; Nishimiya et al. ATG​TGT​GGT​TAA​AAA​CCC​GCT​GCT​TA-3′) was used as 2005, 2008; Mahatabuddin et al. 2017). It has also been the probe, which was labeled with 5-digoxigenin (Fasmac showed that the ice-shaping ability of AFP is not nullifed Ltd., Japan). The fnal wash was performed in 1 × SSC and by the contaminants present in the body fuid, such as blood 0.1% SDS at 55 °C for 10 min. Colony PCR was performed serum. We hence used the minced muscle homogenates as a to detect colonies with a positive signal. source material to examine the TH activity. After weighing the muscle tissues, they were homogenized by Beadbeater mRNA purifcation, cDNA syntheses and sequencing (Biospec, USA) with an equal amount of distilled water and centrifuged at 15,000 rpm for 15 min to obtain the super- For the specimens collected at Mori, the frst-strand cDNA natant solutions for measurement of TH activity. The solu- was synthesized from the RNA using SuperScript VILO tions were lyophilized for storage, measured to calculate the (Invitrogen™, USA) according to the manufacturer’s dry weight and later dissolved in water to prepare 200 mg/ instructions. mL solutions for TH measurement. The measurement was Nested PCR was conducted to select candidate AFP genes performed with a temperature-controlled stage mounted on from each RT-PCR amplicon. The fve primers (5′1, 5′2, a Leica DMLB100 photomicroscope (Leica Microsystems, Deg1, 3′1 and 3′2) designed by Graham et al. (2013) and Wetzlar, Germany) with a Linkam THMS 600 temperature a newly designed primer LS-ST-P2 5′-CGA​GCT​AAA​CAA​ controller (Linkam Scientifc Instruments Ltd, Tadworth, AAG​TGA​GAATG-3′ were used in three combinations (5′1 Surrey, UK) according to the procedures described by and 3′1, 5′2 and 3′1, and LS-ST-P2 and 3′1) in the frst poly- Takamichi et al. (2007). Images of ice crystals were captured merase chain reaction (PCR), and then the same four prim- using a color video 3CCD camera (Sony, Tokyo, Japan), and ers except 5′1 and 3′1 primers in all possible combinations the temperature status was viewed on a display and simulta- were used as the second PCR with the frst PCR products neously saved as a video fle. The dependence of TH activity serving as the DNA templates. The concentration of each on the temperature of the natural marine environment was primer was 0.4 µM. KOD-Plus-Ver. 2 including KOD DNA

1 3 181 Page 4 of 11 Marine Biology (2018) 165:181 polymerase was used for both PCRs according to the manu- incubated on LB/amp/IPTG/X-gal plates for 12 h at 37 °C. facturer’s instructions (Toyobo, Japan). PCR products from a The plasmids were purifed using Quantum prep plasmid single RT-PCR amplicon were pooled and sub-cloned using mini prep kit (BIO-RAD, USA). The cloned DNA-encod- the TArget Clone™-Plus-system (Toyobo, Japan) accord- ing AFPs were sequenced using the BigDye Terminator ing to the manufacturer’s instructions. Transformations into V3.1 Cycle Sequencing Kit and ABI 3100 genetic analyzer E. coli (ECOS™ Competent E. coli JM109, Nippongene, (Applied Biosystems). Japan) were conducted by the heat shock method, and bac- teria were then incubated on LB/amp/IPTG/X-gal plates Bioinformatics analyses containing ampicillin for 12 h at 37 °C. After blue/white selection, all the white colonies were restreaked and ampli- Sequence manipulation and alignments were conducted fed by colony PCR. Each reaction was conducted in a total using MEGA6 (Tamura et al. 2013). Codon usage was also volume of 50 µL, which consisted of 0.2 µM of each primer calculated within MEGA6. (universal T7 and T3 promoter primers), 1x EmeraldAmp PCR Master Mix (Takara Bio Inc., Japan), and the picked Phylogenetic analyses and divergence time DNA sample. The colony was amplifed by PCR, with pre- estimation denaturing at 95 °C for 1 min, 30 cycles of denaturing at 94 °C for 30 s, annealing at 60 °C for 30 s and extending at To identify the phylogenetic relationships among the genus 72 °C for 3 min, with a fnal extension at 72 °C for 10 min. Myoxocephalus and estimate the time when the Arctic and PCR products were purifed using NucleoSpin® Gel and Atlantic species migrated to these oceans, 11 Myoxocepha- PCR Clean-up according to the manufacturer’s instructions lus species were used in these analyses (Arctic: M. scorpius, (Macherey-Nagel, Germany). Sequencing was conducted M. quadricornis quadricornis, M. q. thompsonii, Atlantic: with two directions using same universal primers (T7 and T3 M. aenaeus, Pacifc: M. octodecemspinosus, M. niger, M. promoters) using Applied Biosystems 3730 × 1 DNA ana- polyacanthocephalus, M. brandtii, M. stelleri, M. ochoten- lyzer (Life Technologies, USA) by Macrogen Japan (Kyoto, sis, M. jaok). One Hexagrammos lagocephalus, six Gymno- Japan). The sequences of M. scorpius and M. octodecemspi- canthus species (G. tricuspis, G. pistilliger, G. intermedius, nosus were cited from GenBank data (Table S1). G. herzensteini, G. galeatus, G. detrisus) and Scorpaenich- For the specimens collected at Notsuke, mRNA was thys marmoratus were used as outgroups in the phylogenetic purifed from total RNA using the same kit. The frst-strand analysis. The six Gymnocanthus species were also analyzed cDNA was synthesized from the mRNA using SuperScript for divergence time estimation. These samples were col- II (Invitrogen™, USA) according to the manufacturer’s lected in Hokkaido and Alaska, and some related sequences instructions. PCR was performed using the templates of were cited from GenBank (Table S1). Ex-Taq DNA polymerase (Takara, Japan), and three newly Mitochondrial DNA was extracted from the fin clip designed primers (forward: LS-ST-P1 5′-CGA​GCT​AAA​ of all samples using a Quick Gene DNA extraction kit CAA​AAG​TGA​GAA​TGG​-3′, or LS-ST-P2, reverse: stypeI- (Kurabo, Japan) following the manufacturer’s protocol. The AFPb1 5′-GAG​CGT​CTC​AAG​ATG​CTT​TTT​GTT​TN-3′). cytochrome b (cytb) and cytochrome c oxidase subunit I Another reverse primer PolyT-EcoRI-1 5′-GAG​AGA​GAG​ (COI) region were amplifed by PCR with the FishF1 (5′- AGA​GAG​AGA​GAA​TTC​TTT​TTT​TTT​TTT​TTT​TTT​-3′) TCAACC​ AAC​ CAC​ AAA​ GAC​ ATT​ GGC​ AC-3​ ′) and FishR1 was used with the forward primer LS-ST-P2, when the (5′-TAG​ACT​TCT​GGG​TGG​CCA​AAG​AAT​CA-3′) primers fragment did not amplify by the combinations with three designed by Ward et al. (2005) and the Cyt-L (5′-ATGGCA​ ​ primers. The second PCR was performed with LS-ST-P2 AGCCTA​ CGA​ AAA​ A-3​ ′) and Cyt-R (5′-TCCTAA​ GGC​ CTT​ ​ and stypeIAFPb1 after the frst amplifcation with LS-ST- GTT​TTC​TA-3′) primers designed by Kimura et al. (2007), P2 and PolyT-EcoRI-1. Each reaction was conducted in a with pre-denaturing at 94 °C for 5 min, 35 cycles of denatur- total volume of 50 µL, which consisted of 0.5 µM of each ing at 94 °C for 30 s, annealing at 58 °C for COI or 55 °C primer, 1 × Ex Taq bufer, 2.5 mM of each dNTP mixture, for cytb for 30 s, and extending at 72 °C for 30 s, and a fnal and 0.5 µL of cDNA template. The PCR conditions are as extension at 72 °C for 7 min. Sequencing was conducted by follows: denaturing at 94 °C for 3 min, 30 cycles of pre- Macrogen Japan (Kanagawa, Japan). extending at 94 °C for 1 min, annealing at 52 °C for 1 min Sequences were aligned using MEGA6 with default set- and 72 °C for 1 min, and fnal extending at 72 °C for 1 min. tings and adjusted by eye. Gaps were identifed and deleted The PCR products obtained were purifed and ligated into using MAFFT ver. 7 (Katoh and Standley 2013) with the pGEM-T Easy Vector Systems (Promega, USA) according E-INS-i option and trimAl ver. 1.2 (Capella-Gutierrez et al. to the manufacturer’s instructions. Transformations into E. 2009; Katoh and Standley 2013) with no gap option. Kaku- coli (E. coli DH5α Competent Cells, Takara, Japan) were san4 (Tanabe 2011) was used to determine the appropriate conducted by the heat shock method, and bacteria were then model for each gene. The maximum likelihood method was

1 3 Marine Biology (2018) 165:181 Page 5 of 11 181 also explored. The phylogenetic analysis is to infer species M. polyacanthocephalus relationships of the sculpins, not the relationship between 20.0 M. brandtii COI and cytb, which are the two partitions of data in the 1. 0 M. stelleri dataset by the ML method using RAxML ver. 7.2.8 (Stama- Sea temperature takis 2006). The robustness of nodes was estimated from 15.0 1000 bootstrap replicates (Felsenstein 1985). 0. 8

Estimations of divergence times followed the method of Sea temperature (°C) Yamazaki et al. (2013). The overall substitution rate (rgene .6 gamma) and rate-drift parameter (sigma2 gamma) were set 10. 0 at G (1, 330) and G (1, 10) using a strict molecular clock assumption with a 5.5–4.8 Mya constraint to the divergence between G. tricuspis, G. intermedius and G. pistilliger, and a 0. 40 Thermal Hysteresis (°C)

0.7–1.9 Mya constraint to the divergence between M. quad- 5. 0 ricornis quadricornis and M. q. thompsonii (Kontula and Väinölä 2003; Yamazaki et al. 2013). 0. 2

Defnition of distributional zones 0. 0 0. 0

The distributional zones were divided into three zones: Nov Dec JanFeb MarApr Arctic, Subarctic (between the Arctic zone and Subarctic boundary), and temperate zones (between the Subarctic and Fig. 1 Monthly thermal hysteresis values evaluated for 200 mg/mL Subtropical boundaries). The Arctic zone was defned by solutions from these species and the average sea temperature. Error bars represent SDs. The crystal images that showed the highest activ- Mecklenburg et al. (2011). ity were shown for each species. The average sea temperature shown with gray line was observed at Usujiri Fisheries Station Results 3′-untranslated regions (UTR) show high identity, with more Antifreeze activities and thermal hysteresis than 80% identity within SI and more than 90% identity within MI and LI between overlapping regions. The 3′-UTR Myoxocephalus brandtii and M. stelleri showed low TH val- has 36.3–87.7% identity among isoforms and 24.1% identity ues in January and February, which became the highest in among all sequences. March and then decreased by April (Fig. 1). M. polyacan- A total of 142 fragments corresponding to AFPs were thocephalus collected in Alaska showed higher TH values sequenced and these encoded 14 distinct AFPs in M. pol- compared with the other Subarctic species. yacanthocephalus, 16 in M. brandtii and 14 in M. stelleri (Table 2). These 44 sequences represent 31 new AFP var- Genetic structure of AFP iants as some of these sequences were present in two or more species or were previously known from M. scorpius. The 142 AFP clones that were sequenced encoded a total of This brings the total number of known sequences from this 44 distinct AFP sequences. These three Myoxocephalus spe- genus to 41, with only nine of these being found in two or cies show a preference for a particular Ala codon within the more species (Fig. 2). The number of sequences that were AFP sequences. Between 48.3 and 49.4% of the Ala residues found exclusively in M. polyacanthocephalus, M. brandtii, are encoded by the GCC codon. Three groups according or M. stelleri are 7, 10 and 9, respectively. The amino acid to a diference in the peptide length were determined; (1) sequence of (1) one protein is common to among all Myoxo- 35- or 36-residue short isoform (SI), (2) 40-residue medium cephalus species except M. octodecemspinosus, (2) three isoform (MI), and (3) 55-residues or longer isoform (LI). proteins are shared between M. scorpius and M. polyacan- The deduced AFP sequences from SI and MI are similar to thocephalus, (3) two proteins are shared among M. polya- the 42-aa longhorn sculpin skin sequence (LHS, Low et al. canthocephalus, M. brandtii and M. stelleri, (4) one protein 2001) and the sequences from LI are similar to the 92-aa is shared by M. polyacanthocephalus and M. brandtii, and shorthorn sculpin skin sequence (SHS, Low et al. 1998, Fig. (5) two proteins are shared by M. brandtii and M. stelleri S1). In the coding region, there is 56.0% identity among SI, (Fig. 2). 69.4% identity among MI, and 87.5% identity among LI. For a protein shared by M. scorpius and M. polya- All the three isoforms show 24.0% identity (SI-MI: 40.0% canthocephalus, we detected amino acid replacements identity, SI-LI: 40% identity, MI-LI: 58.3% identity). The (mutations) of A23K (i.e., A23 to K23) in SI, and T13L

1 3 181 Page 6 of 11 Marine Biology (2018) 165:181

Table 2 Characteristics of AFP amino acid sequences for each species Msc Mo Mp Mb Mst

Nucleotide length (bp) – – 211–877 233–693 226–543 Amino acid (aa) length (residues) 33–42 42 35–40 25–66 35–66 N of total aa sequences 9 1 14 16 14 N of shared aa sequences 4 0 7 6 5 N of species-specifc aa sequences 5 1 7 10 9 Ratio of species-specifc aa sequences 0.12 (0.56) 0.02 (1.00) 0.17 (0.50) 0.24 (0.63) 0.22 (0.64) Ratio of species-specifc aa sequences (SI) 0.15 (0.44) 0.02 (1.00) 0.19 (0.36) 0.07 (0.13) 0.31 (0.57) Ratio of species-specifc aa sequences (MI) 0.10 (0.11) – 0.20 (0.14) 0.50 (0.31) 0 (0) Ratio of species-specifc aa sequences (LI) – – – 0.60 (0.19) 0.20 (0.07)

Sequences of M. scorpius and M. octodecemspinosus were cited from GenBank. The ratio of the species is represented within parentheses Msc, M. scorpius; Mo, M. octodecemspinosus; Mp, M. polyacanthocephalus; Mb, M. brandtii; Mst, M. stelleri in MI (Fig. 2). Two species-specifc proteins of M. stel- Discussion leri had mutations in the ice-binding residues of A15T and A19G in SI. The number of salt bridges consisting of Antifreeze activity and AFP genetic structure basic (Lys or Arg) and acidic (Asp or Glu) residues, which are known as the helix stabilizer, in i and i + 4 was 1–2 in Generally, the antifreeze activity increases when the sea tem- SI and MI for each species. The ice-binding residue and perature starts falling, and declines when the sea temperature the salt bridge could not be accurately determined for LI. starts rising. The two species M. brandtii and M. stelleri All sequence data were deposited in GenBank (Accession showed no antifreeze activity from November to February, numbers: MH745429–MH745570). high activity at March and declined at April. The monthly average sea temperature around southwest Hokkaido in 2014–2015 was lowest at February (2.7 °C) and gradually Phylogenetic relationships and divergence time rose after March (3.4 °C); however, the sea temperature at estimation sampling was lowest in March (Table 1). Furthermore, there is no risk for freezing around southwest Hokkaido. Although In all, 1220 bp from two loci were sequenced from 24 we need further experiments, the high antifreeze activity a species. The lengths of the partial cytb and partial COI month later after the lowest sea temperature might suggest sequences were 612 bp, including 215 variable sites and the decline of antifreeze activity for the two species. 178 parsimony informative sites, and 608 bp, including The deduced AFP sequences in this study were similar 180 variable sites and 158 parsimony informative sites, to SHS and LHS with high similarity (more than 80%). respectively. The eleven Myoxocephalus species formed Our results show that sculpins have at least three isoforms a monophyletic group, with a high bootstrap value (96%), including a lot of substitutions, suggesting that several gene and consisted of two lineages supported by bootstrap val- duplications occurred in the sculpin-derived type I AFP ues of 100%: lineage A comprised the Atlantic species, gene family. The sculpin-derived AFP genes include various while lineage B comprised the Pacifc and the Arctic spe- sequences (length and substitutions) than other types (cod- cies (Fig. S2). Lineage A diverged into the M. q. com- ing region: 25-66-aa, 24.0% identity among all isoforms, plex, M. aenaeus and M. octodecemspinosus (95%). In non-coding region: 24.1% identity among all isoforms). lineage B, M. scorpius diverged at frst, and subsequently For example, the winter founder, which express 37–40-aa M. brandtii, M. ochotensis, M. stelleri, M. polyacantho- residue type I AFP and 195-aa residue hyperactive type I cephalus, M. jaok diverged in that order (≥ 62%). The rela- AFP, shows 72.1–82.2% identity between 3′-UTR of skin tionships between M. aenaeus and M. octodecemspinosus, and liver isoforms (Gong et al. 1996). The fsh type III AFP and M. brandtii and M. ochotensis difer from previously including QAE and SP isoforms shows > 75% identity within reported studies (Kontula and Väinölä 2003; Podlesnykh QAE isoform, > 90% identity within SP isoform and < 55% and Moreva 2014). The other branches are consistent with identity among isoforms (Hew et al. 1988), and 91% iden- these studies. tity in the non-coding region (Desjardins et al. 2012). The The divergence time of the two lineages was estimated spruce budworm AFP including two isoforms is consisted as 7.9 Mya, and the speciation occurred after 2.9 Mya of 49–50 and 79–81-aa residues (Tyshenko et al. 2005). (Fig. 3). Diverged sequences in Myoxocephalus species indicate that

1 3 Marine Biology (2018) 165:181 Page 7 of 11 181

Short isoform 1 10 20 30 Msc Mo Mp Mb Mst MVA PARAAAKTAAD AKAAAAK TAAD AAKAAAK TAAK MDAPARAAAKTAAD AKAAAAK TAAD AAKAAAK TAAK MDAPARAAAKTAAD AKAAAAKTAAGAAKAAAKTAAK ---PARAAAKTAAD ALAAANK TAAD AAAAAARTAAK ---PARAAAETAAD ALAAANK TAAD AAAAAARTAAK MDAPARAAAKTAAD ALAAANK TKAD AAAAAAK TAAK MDAPARAAAKTAAD ALAAANK TAAD AAAAAAK TAAK MDAPARAAAKTAAD TLAAANK TAAD AAAAAAK TAAK MDAPARAATKTAAD ALAAANK TAAD AAAAAAK TAAK MDAPARAAAKTAAD ALAAANK TAAD AAAAADK TAAK MDAPARAAAKTAAGALGAGNKTAAD AAAAAAK TGAK KDAPARAAAKTAAD ALGAANKTAAGAAAAAAKTGVK MDAPARAAAKTAAD ALGAANK TAAD AAAAAAK TAAK MYAPARAAAKTAAD ALAAATK TAAD AAAAAAK TAAK MDAPARAAAKTAAD ALAAATK TAAD AAAAAAK TAAK MDAPARAAAKTAAD AKAAAAK TAAD AAKAAAK SAK MNAPARAAAKTAAD ALAAAKK TAAD AAKAAAK SAK MDAPARAAAKTAAD ALAAAKK TAAD AAKAAAK SAK MDAPARAAAKTAAD ALAAAKK TAAD AAAAAAK SAK MDAPARAAAKTAAD ALAAANK TAAD ATAAAAK SAK MDAPARAAAKTAAD ALAAAKK TAAD AAAAA-K SAK MDAPARAAAATAAAAKAAAEKTAAD AAAAAKSAK MNAPARAAAKTAAD ALAAAKK TAAD AAAAAAAA ---PARAAAKTAAD ALAAAKK TAAD AAAAAAAK MDAPARAAAKTAAD ALAAANK TAAK MDAPARAAAATAAAA-----KTAAK

Middle isoform 1 10 20 30 40 Msc Mo Mp Mb Mst MDAPAR--AAAATAAAAKAAAEKTAVDALAAAEATKAAAARA MDAPAR--AAAATAAAAKAAAEKTAAD ALAAAEATAAAAARA MDGPAR--AAAATAAAAKAAAEKTAAD ALAAAEATKAAAARA MDAPAR--AAAATAAAAKAAAEKTAAD AAAAAAATKAAAARA MDAPAR--AAAKTAAD AKAAAEAVAAKAKADAEKTAADAARA MDAPAR--AAAATAATAAAAAEKTAAD ALAAAEATKAAAARA MDAPAR--AAAKTAAD ALAAAEK TAAD ALAAAEATKAAAARA MDAPAK--AAAKTAAD AKAAAAK TAAD ALAAANKTAAAAKAAAK MDAPARAAAAAKTAAD AKAAAAK TAADALAAAEATKAAAARA MDGETPAQKAARLAAAAAALAAKTAAD AAAKAAAIAAAAASA MDGETPAGKAARLAAAAALAA-KTAAD AAAKAAAIAAAAA

Long isoform 1102030405060 Msc Mo Mp Mb Mst MDAPARAAAFAAATAAEAAALTASTAATAAATTAANAAAAAAATAITAAAAAAAAAANAAAAAAAV MDAPARAAAAAAATAAEAAALTASTAATAAATTAANAAAAAAATAITAAAAAAAAAANAAAAV MDAPARAAAATAATAAEAAALTASTAATAAATTAANAAAAAAATAITAAAAAAAAAANAAAAV MDAPARAAAAAAATAAEAAALTASTAATAAATTAANAAAAAAAAAANAAAAAAAV

Fig. 2 Amino acid sequences of all type I antifreeze protein isoforms Gly) are highlighted yellow. The closed circle represents the deter- determined for four species of Myoxocephalus. The sequences from mined sequence for each species. The putative ice-binding residues M. scorpius and M. octodecemspinosus were cited from KF381183– are shown with # (Thr) and + (others), and the residues constructing 90, AF306348, SS3 and SS8. Thr residues are colored green. Polar a salt bridge are underlined. Hyphen (-) represents gaps. Asterisk (*) residues (except Thr) are highlighted light green. Basic residues are indicates the conserved site. Species abbreviations are as follows; highlighted blue (Lys) or purple (Arg), acidic residues are highlighted Msc, M. scorpius; Mo, M. octodecemspinosus; Mp, M. polyacantho- red (Asp and Glu), hydrophobic residues (except Ala) are highlighted cephalus; Mb, M. brandtii; Mst, M. stelleri gray (Met, Leu, Ile, Phe and Val) and exceptional residues (Pro and type I AFP genes in Myoxocephalus are the multigene family crystals to inhibit their growth (i.e., to provide antifreeze including a lot of copies. activity) (Wierzbicki et al. 1996; Wierzbicki et al. 2008; Type I AFP contains threonyl (Thr) residues at regular Davies 2014). The potential ice-binding site of M. scorpius intervals along its α-helical structure, and this side of the comprises Thr11, Ala15, Ala19, Thr22, Ala26, and Ala30 molecule (i.e., the ice-binding site) binds to embryonic ice (Graham et al. 2013), and a missense mutation that afects

1 3 181 Page 8 of 11 Marine Biology (2018) 165:181

■ G. herzensteini

■ G. detrisus 100 97 ■ G. galeatus

- ■ G. tricuspis

- ■ G. pistilliger (a) 100 ■ G. intermedius Kontula & Väinölä (2003) ■ M. q. quadricornis M. q. quadricornis

100 ■ M. q. thompsonii M. q. thompsonii A 93 ■ M. octodecemspinosus* M. octodecemspinosus

77 ■ M. aenaeus* M. aenaeus ★ 100 ■ M. niger (b) ★ B 100 ■ M. scorpius Podlenykh & Moreva (2014)

100 ■ M. ochotensis* M. brandtii

■ M. brandtii* M. ochotensis 91 ■ M. stelleri M. jaok - ■ M. polyacanthocephalus M. stelleri ■ Arctic 76 ■ Arctic–Subarctic 89 ■ M. jaok ■ Subarctic 1.0

12.5 10.0 7.5 5.0 2.5 0.0 Mya

Fig. 3 Estimated divergence time for Gymnocanthus and Myoxo- represents the distributional zone; the Arctic zone is colored blue, cephalus species based on two mitochondrial genes. The lateral bar the Subarctic zone is dark purple, and the temperate zone is red. The indicates the divergence time (million years ago). The 95% CIs are asterisks on the species name represent the species that branch points shown as gray bars on each node. The branches where Arctic spe- difer from previous studies. a The molecular phylogenetic relation- cies diverged are shown with stars. Lineage names are denoted with ship was inferred by Kontula and Väinölä (2003). Dotted line indi- capital letters (A and B). The gray squares indicate the time when the cates the branches where difer from this study. b The molecular phy- Bering Strait opened. The colored square neighboring species name logenetic relationship was inferred by Podlenykh and Moreva (2014)

Thr or Ala in the ice-binding site decreases antifreeze activ- mutations accumulated in M. stelleri, the selection pressure ity (Baardsnes et al. 1999; Baardsnes et al. 2001; Wierz- to each sequence in M. stelleri might have decreased gradu- bicki et al. 2008). In particular, replacement of Thr with Ser ally, because this species does not require a high level of causes a signifcant decrease (Haymet et al. 1998; Zhang and freeze resistance. Laursen 1998), while replacement with Val or Leu does not signifcantly change the activity (Chao et al. 1997; Haymet Acquisition of AFP and colonization of the Arctic et al. 1998). In SS3, which was isolated from M. scorpius, a Ocean mutation at Ala12, Ala17 and Ala23 caused a 20% decrease in antifreeze activity (Bang et al. 2013). The initial episodic formation of sea ice in marginal shelf Amino acid substitutions that do not afect antifreeze areas began 47.5 Mya, and seasonal sea ice formation activity were identifed in M. scorpius and M. polyacan- started 47 Mya in ofshore areas of the central Arctic thocephalus (T13L in MI), and in M. brandtii (T22V in MI, (Stickley et al. 2009). There would be selection pressure Fig. 2). In contrast, a missense mutation that decreased anti- for acquisition of cold resistance during this period in the freeze activity was observed in M. scorpius and M. polya- Arctic. After that, a strong ice age started 2.9–2.4 Mya, canthocephalus (A23K in SI). Several missense mutations following the closing of the Bering Strait 3.0 Mya (Raymo were observed in M. stelleri (A15T, A17G and A19G in 1994). The Bering Strait opened during the warm periods SI). M. stelleri also exhibited substitutions on a side of of 12.0–6.0 Mya and 5.4–3.5 Mya (Harrison and Crespi the protein that includes the salt bridges that stabilize the 1999; Kafanov 1999; Marincovich and Gladenkov 1999; α-helical structure (Marqusee and Baldwin 1987; Graham Briggs 2007a). Considering that AFP genes derived et al. 2013). The mutations are sometimes accumulated by a from three Myoxocephalus species are homologs with M. relaxed constraint (Akashi 1994; Begun et al. 2000; Schmid scorpius and M. octodecemspinosus, it would seem that and Aquadro 2001). In addition, the multigene family can the AFP genes in Myoxocephalus would have originated reasonably stake a claim as the largest family in muticel- from an ancestral gene before the divergence occurred lular organisms (Benton 2015). Considering that a lot of within the genus, approximately 7.9 Mya. The genus

1 3 Marine Biology (2018) 165:181 Page 9 of 11 181

Myoxocephalus likely originated approximately 10 Mya, of Fish and Game. The fshes sampled for the study were acquired by and then the allopatric species diversifcation occurred fshing and scuba diving. around the archipelago in the North Pacifc Ocean from 5.0 Mya (Fig. 3). Our estimates of the divergence times Myoxocephalus within species indicate that expansions to References the Arctic Ocean occurred separately in lineages A and B; the frst expansion would have occurred in lineage A Akashi H (1994) Synonymous codon usage in Drosophila mela- during 10–6 Mya, and the second expansion would have nogaster: natural selection and translational accuracy. Genetics occurred in M. scorpius during 6–1 Mya. When the Bering 136:927–935 Strait opened, the lineage A species had migrated to the Amaoka K, Nakaya K, Yabe M (2011) The pictorial book of all of the Arctic Ocean 10–6 Mya, and M. scorpius had migrated fshes of Hokkaido. The Hokkaido Shimbun Press, Sapporo Baardsnes J, Kondejewski LH, Hodges RS, Chao H, Kay C, Davies PL 5.4–3.5 Mya. Indeed, some Cottidae species, such as G. (1999) New ice-binding face for type I antifreeze protein. FEBS tricuspis and Cottus (freshwater species), migrated to the J 463:87–91 Arctic region approximately 4–3 Mya (Yokoyama and Baardsnes J, Jelokhani-Niaraki M, Kondejewski LH, Kuiper MJ, Kay Goto 2005; Yamazaki et al. 2013). After the acquisition of CM, Hodges RS, Davies PL (2001) Antifreeze protein from short- horn sculpin: identifcation of the ice-binding surface. Protein Sci AFP (at least 7.9 Mya), some Myoxocephalus species had 10:2566–2576. https​://doi.org/10.1101/ps.26501​ migrated to the Arctic and Atlantic Ocean during warm Bang JK, Lee JH, Murugan RN, Lee SG, Do H, Koh HY, Shim HE, periods. Therefore, during the cold period of 2.9–2.4 Mya, Kim HC, Kim HJ (2013) Antifreeze peptides and glycopeptides, they could survive this ice age and the subsequent ice age. and their derivatives: potential uses in biotechnology. Mar Drugs 11:2013–2041. https​://doi.org/10.3390/md110​62013​ These species survived the ice ages, and M. polyacan- Begun DJ, Whitley P, Todd BL, Waldrip-Dail HM, Clark AG (2000) thocephalus diverged after 2.5 Mya and maintains high Molecular population genetics of male accessory gland proteins antifreeze activity at present. Furthermore, M. brandtii and in Drosophila. Genetics 156:1879–1888 M. stelleri, which diverged at approximately the same time Benton R (2015) Multigene family evolution: perspectives from insect chemoreceptors. Trends Ecol Evol 30:590–600. https​://doi. as M. polyacanthocephalus, exhibit reduced antifreeze org/10.1016/j.tree.2015.07.009 activity. In conclusion, our results demonstrate that the Briggs JC (2003) Marine centres of origin as evolutionary engines. J ancestral species of Myoxocephalus acquired AFP before Biogeogr 30:1–18 this genus diverged and that the Arctic species maintain Briggs JC (2007a) Marine biogeography and ecology: invasions and introductions. J Biogeogr 34:193–198. https​://doi.org/10.111 high antifreeze activity, while the Subarctic species show 1/j.1365-2699.2006.01632​.x reduced antifreeze activity. The genetic structures also dif- Briggs JC (2007b) Marine longitudinal biodiversity: causes and con- fer between the Arctic and Subarctic species, and M. stel- servation. Divers Distrib 13:544–555. https​://doi.org/10.111 leri appears to have accumulated several missense muta- 1/j.1472-4642.2007.00362​.x Briggs JC, Bowen BW, McClain C (2013) Marine shelf habitat: bio- tions in its AFP gene. geography and evolution. J Biogeogr 40:1023–1035. https​://doi. org/10.1111/jbi.12082​ Acknowledgements We appreciate associate Prof. H. Kondo and Dr. Capella-Gutierrez S, Silla-Martinez JM, Gabaldon T (2009) trimAl: a Y. Hanada for their useful advice on antifreeze protein analyses. We tool for automated alignment trimming in large-scale phylogenetic also thank Prof. Y. Koya, assistant Prof. S. Awata, and Mr. N. Sato, analyses. Bioinformatics 25:1972–1973. https​://doi.org/10.1093/ the owner of the dive shop “GruntSculpin”, the Alaska Department of bioin​forma​tics/btp34​8 Fish and Game, the Mac Enterprise, and graduate students of Usujiri Chakrabartty A, Hew CL, Shears MA, Fletcher GL (1988) Primary Fisheries Station for sampling assistance. Lastly, we appreciate two structures of the alanine-rich antifreeze polypeptides from grubby anonymous reviewers for their useful advice. sculpin, Myoxocephalus aenaeus. Can J Zool 66:403–408 Chao H, Houston ME, Hodges RS, Kay CM, Sykes BD, Loewen MC, Funding This study was funded by Hokkaido University Grant for Davies PL, Sönnichsen FD (1997) A diminished role for hydro- Research Activities Abroad, Research Fellow of Japan Society for the gen bonds in antifreeze protein binding to ice. Biochemistry Promotion of Science (JSPS Research Fellow), and a Grant-in-Aid for 36:14652–14660 Scientifc Research from JSPS (Grant-in-Aid 26-1530, 15K13760, Chen L, DeVries AL, Cheng CH (1997) Convergent evolution of anti- 25304011, 26292098). freeze glycoproteins in Antarctic notothenioid fsh and Arctic cod. Proc Natl Acad Sci USA 94:3817–3822 Cheng CH (1988) Evolution of the diverse antifreeze proteins. Curr Compliance with ethical standards Opin Genet Dev 8:715–720 Davies PL (2014) Ice-binding proteins: a remarkable diversity of struc- Conflict of interest The authors declare that they have no confict of tures for stopping and starting ice growth. Trends Biochem Sci interest. 39:548–555. https​://doi.org/10.1016/j.tibs.2014.09.005 Desjardins M, Graham LA, Davies PL, Fletcher GL (2012) Antifreeze Ethical approval All applicable international, national, and institu- protein gene amplifcation facilitated niche exploitation and spe- tional guidelines for the use of were followed. All procedures ciation in wolfsh. FEBS J 279:2215–2230. https​://doi.org/10.11 performed in studies involving animals were in accordance with the 11/j.1742-4658.2012.08605​.x ethical standards of the institution or practice at which the studies were DeVries AL (1982) Biological antifreeze agents in coldwater fshes. conducted. Collecting permits were provided by the Alaska Department Comp Biochem Physiol A Mol Integr Physiol 73:627–640

1 3 181 Page 10 of 11 Marine Biology (2018) 165:181

DeVries AL (1984) Role of glycopeptides and peptides in inhibition data. Mol Ecol 12:3179–3184. https​://doi.org/10.1046/j.1365- of crystallization of water in polar fshes. Philos Trans R Soc 294X.2003.01963​.x Lond Ser B Biol Sci 304:575–588 Li X-M, Trinh K-Y, Hew CL (1985) Structure of an antifreeze polypep- DeVries AL, Wohlschlag DE (1969) Freezing Resistance in tide and its precursor from the ocean pout, Macrozoarces ameri- Some Antarctic Fishes. Science 163:1073–1075. https​://doi. canus. J Biol Chem 260:12904–12909 org/10.1126/scien​ce.163.3871.1073 Low WK, Miao M, Ewart KV, Yang DSC, Fletcher GL, Hew CL Duman JG, DeVries AL (1974) The role of macromolecular anti- (1998) Skin-type antifreeze protein from the shorthorn sculpin, freezes in cold water fshes. Comp Biochem Physiol A Mol Myoxocephalus scorpius: expression and characterization of a Mr Integr Physiol 52:193–199 9,700 recombinant protein. J Biol Chem 273:23098–23103. https​ Duman JG, DeVries AL (1976) Isolation, characterization, and phys- ://doi.org/10.1074/jbc.273.36.23098​ ical properties of protein antifreezes from the winter founder, Low WK, Lin Q, Stathakis C, Miao M, Fletcher GL, Hew CL (2001) Pseudopleuronectes americanus. Comp Biochem Physiol A Isolation and characterization of skin-type, type I antifreeze poly- Mol Integr Physiol 54:375–380. https​://doi.org/10.1016/0305- peptides from the longhorn sculpin, Myoxocephalus octodecems- 0491(76)90260​-1 pinosus. J Biol Chem 276:11582–11589. https://doi.org/10.1074/​ Felsenstein J (1985) Confdence limits on phylogenies: an approach jbc.M0092​93200​ using the bootstrap. Evolution 39:783–791. https​://doi. Mahatabuddin S, Hanada Y, Nishimiya Y, Miura A, Kondo H, Davies org/10.2307/24086​78 PL, Tsuda S (2017) Concentration-dependent oligomerization of Froese R, Pauly D (2015) FishBase. World Wide Web electronic an alpha-helical antifreeze polypeptide makes it hyperactive. Sci publication. http://www.fshb​ase.org/searc​h.php. Accessed 13 Rep 7:42501. https​://doi.org/10.1038/srep4​2501 Jun 2015 Marincovich LJ, Gladenkov AY (1999) Evidence for an early opening Gong Z, Ewart KV, Hu Z, Flethcer GL, Hew CL (1996) Skin antifreeze of the Bering Strait. Nature 397:149–151 protein genes of the winter founder, Pleuronectes americanus, Marqusee S, Baldwin RL (1987) Helix stabilization by Glu-… encode distinct and active polypeptides without the secretory sig- Lys + salt bridges in short peptides of de novo design. Proc Natl nal and prosequences. J Biol Chem 271:4106–4112. https​://doi. Acad Sci 84:8898–8902 org/10.1074/jbc.271.8.4106 Mecklenburg CW, Mecklenburg TA, Thorsteinson LK (2002) Fishes Graham LA, Hobbs RS, Fletcher GL, Davies PL (2013) Helical anti- of Alaska. American Fisheries Society, Maryland freeze proteins have independently evolved in fshes on four Mecklenburg CW, Stein DL, Sheiko BA, Chernova NV, Mecklenburg occasions. PLoS One 8:e81285. https​://doi.org/10.1371/journ​ TA, Holladay BA (2007) Russian-American long-term census of al.pone.00812​85 the Arctic: Benthic fshes Trawled in the Chukchi Sea and Ber- Harada N, Ahagon N, Sakamoto T, Uchida M, Ikehara M, Shibata Y ing Strait, August 2004. Northwest Nat 88:168–187. https​://doi. (2006) Rapid fuctuation of alkenone temperature in the south- org/10.1898/1051-1733(2007)88%5b168​:rlcot​a%5d2.0.co;2 western Okhotsk Sea during the past 120 ky. Glob Planet Change Mecklenburg CW, Møller PR, Steinke D (2011) Biodiversity of arctic 53:29–46. https​://doi.org/10.1016/j.glopl​acha.2006.01.010 marine fshes: and zoogeography. Mar Biodivs 41:109– Harrison MK, Crespi BJ (1999) Phylogenetics of cancer crabs (Crus- 140. https​://doi.org/10.1007/s1252​6-010-0070-z tacea: Decapoda: Brachyura). Mol Phylogen Evol 12:186–199. Nelson JS, Grande TC, Wilson MV (2016) Fishes of the World. Wiley, https​://doi.org/10.1006/mpev.1998.0608 Hoboken Haymet A, Ward LG, Harding MM, Knight CA (1998) Valine sub- Nishimiya Y, Sato R, Takamichi M, Miura A, Tsuda S (2005) Co- stituted winter founder ‘antifreeze’: preservation of ice growth operative efect of the isoforms of type III antifreeze protein hysteresis. FEBS Lett 430:301–306 expressed in Notched-fn eelpout, elongatus Kner. FEBS Hew CL, Fletcher GL, Ananthanarayanan VS (1980) Antifreeze pro- J 272:482–492. https://doi.org/10.1111/j.1742-4658.2004.04490​ .x​ teins from the shorthorn sculpin, Myoxocephalus scorpius: isola- Nishimiya Y, Mie Y, Hirano Y, Kondo H, Miura A, Tsuda S (2008) tion and characterization. Can J Biochem 58:377–383. https://doi.​ Mass preparation and technological development of an antifreeze org/10.1139/o80-049 protein. Synthesiology 1:7–14 Hew CL, Slaughter D, Joshi SB, Fletcher GL, Ananthanarayanan VS Podlesnykh AV, Moreva IN (2014) Variability and relationships of the (1984) Antifreeze polypeptides from the Newfoundland ocean Far Eastern species of sculpins Myoxocephalus and Megalocottus pout, Macrozoarces americanus: presence of multiple and com- (Cottidae) based on mtDNA markers and karyological data. Russ positionally diverse components. J Comp Physiol B 155:81–88. J Genet 50:949–956. https://doi.org/10.1134/s1022​ 79541​ 40901​ 17​ https​://doi.org/10.1007/BF006​88795​ Raymo M (1994) The initiation of Northern Hemisphere glaciation. Hew CL, Wang NC, Joshi S, Fletcher GL, Scott GK, Hayes PH, Annu Rev Earth Pl Sci 22:353–383 Buettner B, Davies PL (1988) Multiple genes provide the basis Raymond JA, DeVries AL (1977) Adsorption inhibition as a mecha- for antifreeze protein diversity and dosage in the ocean pout, Mac- nism of freezing resistance in polar fshes. Proc Natl Acad Sci rozoarces americanus. J Biol Chem 263:12049–12055 USA 74:2589–2593 Kafanov A (1999) Neogene Macoma (Bivalvia, Tellinidae) migra- Raymond JA, Lin Y, DeVries AL (1975) Glycoprotein and protein tion from the Pacifc to the Atlantic through the Bering Strait: antifreezes in two Alaskan fshes. J Exp Zool A Comp Exp Biol taxonomic and biogeographic remarks. Boll-Soc Paleontol Ital 193:125–130. https​://doi.org/10.1002/jez.14019​30112​ 38:77–86 Reisman HM, Fletcher GL, Kao MH, Shears MA (1987) Antifreeze Katoh K, Standley DM (2013) MAFFT multiple sequence alignment proteins in the grubby sculpin, Myoxocephalus aenaeus and the software version 7: improvements in performance and usability. tomcod, Microgadus tomcod: comparisons of seasonal cycles. Mol Biol Evol 30:772–780. https://doi.org/10.1093/molbe​ v/mst01​ ​ Environ Biol Fishes 18:295–301 0 Schmid KJ, Aquadro CF (2001) The evolutionary analysis of Kimura MR, Yanagimoto T, Munehara H (2007) Maternal identifca- “Orphans” from the Drosophila genome identifes rapidly diverg- tion of hybrid eggs in Hexagrammos spp. by means of multiplex ing and incorrectly annotated genes. Genetics 159:589–598 amplifed product length polymorphism of mitochondrial DNA. Slaughter D, Fletcher GL, Ananthanarayanan VS, Hew CL (1981) Anti- Aquatic Biol 1:187–194. https​://doi.org/10.3354/ab000​19 freeze proteins from the sea raven, americanus. Kontula T, Väinölä R (2003) Relationships of palearctic and nearctic Further evidence for diversity among fsh polypeptide antifreezes. ‘glacial relict’Myoxocephalus sculpins from mitochondrial DNA J Biol Chem 256:2022–2026

1 3 Marine Biology (2018) 165:181 Page 11 of 11 181

Stamatakis A (2006) RAxML-VI-HPC: maximum likelihood-based Wierzbicki A, Taylor MS, Knight CA, Madura JD, Harrington JP, phylogenetic analyses with thousands of taxa and mixed models. Sikes CS (1996) Analysis of shorthorn sculpin antifreeze protein Bioinformatics 22:2688–2690 stereospecifc binding to (2–1 0) faces of ice. Biophys J 71:8–18 Stickley CE, St John K, Koç N, Jordan RW, Passchier S, Pearce RB, Wierzbicki A, Knight CA, Salter EA, Henderson CN, Madura JD Kearns LE (2009) Evidence for middle Eocene Arctic sea ice from (2008) Role of nonpolar amino acid functional groups in the sur- diatoms and ice-rafted debris. Nature 460:376–379 face orientation-dependent adsorption of natural and synthetic Takamichi M, Nishimiya Y, Miura A, Tsuda S (2007) Effect of antifreeze peptides on ice. Cryst Growth Des 8:3420–3429 annealing time of an ice crystal on the activity of type III anti- Yamashita Y, Miura R, Takemoto Y, Tsuda S, Kawahara H, Obata H freeze protein. FEBS J 274:6469–6476. https​://doi.org/10.111 (2003) Type II antifreeze protein from mid-latitude freshwater 1/j.1742-4658.2007.06164​.x fsh, Japanese smelt (Hypomesus nipponensis). Biosci Biotechnol Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Biochem 67:461–466 molecular evolutionary genetics analysis version 6.0. Mol Biol Yamazaki A, Markevich A, Munehara H (2013) Molecular phylogeny Evol 30:2725–2729. https​://doi.org/10.1093/molbe​v/mst19​7 and zoogeography of marine sculpins in the genus Gymnocanthus Tanabe AS (2011) Kakusan4 and Aminosan: two programs for com- (Teleostei; Cottidae) based on mitochondrial DNA sequences. Mar paring nonpartitioned, proportional and separate models for com- Biol 160:2581–2589. https://doi.org/10.1007/s0022​ 7-013-2250-4​ bined molecular phylogenetic analyses of multilocus sequence Yokoyama R, Goto A (2005) Evolutionary history of freshwater data. Mol Ecol Resour 11:914–921. https​://doi.org/10.111 sculpins, genus Cottus (Teleostei; Cottidae) and related taxa, as 1/j.1755-0998.2011.03021​.x inferred from mitochondrial DNA phylogeny. Mol Phylogen Evol Tsuda S, Miura A (2005) Antifreeze proteins originating in fshes, 36:654–668. https​://doi.org/10.1016/j.ympev​.2005.06.004 Japan. US Patent 2005/0019856 A1, application no. 10/496,104 Zhang W, Laursen RA (1998) Structure-function relationships in a type Tyshenko MG, Doucet D, Walker VK (2005) Analysis of antifreeze I antifreeze polypeptide the role of threonine methyl and hydroxyl proteins within spruce budworm sister species. Insect Mol Biol groups in antifreeze activity. J Biol Chem 273:34806–34812 14:319–326. https​://doi.org/10.1111/j.1365-2583.2005.00562​.x Ward RD, Zemlak TS, Innes BH, Last PR, Hebert PD (2005) DNA barcoding Australia’s fsh species. Philos Trans R Soc Lond B Biol Sci 360:1847–1857. https​://doi.org/10.1098/rstb.2005.1716

1 3