A) Phylogenetic relationships (not to scale) among AFP-producing fish from analysis of complete mitochondrial genomes – or selected nuclear and mitochondrial sequences . Estimated divergence times (Ma, some with 95% highest posterior density limits) – are shown at some nodes. Species names are colored by AF(G)P type as indicated on the right. Representative ribbon structures are shown for types II, III, and I AFPs (PDB 2PY2, 1HG7, 1WFA from top to bottom, red = helix, green = strand, gray = coil). The colored bars at the bottom indicate climate differences marked by the presence (blue) or absence (red) of large ice sheets. Common names of representative AFP-producing fish are indicated but their scientific names are as follows; herring (Clupea harengus), Arctic cod (Boreogadus saida), cunner (Tautogolabrus adspersus), ocean pout (Zoarces americanus), Atlantic snailfish (Liparis atlanticus), dusky snailfish (Liparis gibbus), sea raven (Hemitripterus americanus), longhorn sculpin (Myoxocephalus octodecemspinosus), shorthorn sculpin (Myoxocephalus scorpius), Antarctic toothfish (Dissostichus mawsoni), winter flounder (Pseudopleuronectes americanus) and rainbow smelt (Osmerus mordax). B) Alignment of representative type I skin AFPs from three fishes from three separate orders (winter flounder (M63478.1), longhorn sculpin (AF306348.1) and cunner (JF937681.2). Potential or known ice-binding residues within the 11-aa repeat that show an i, i+4, i+8 spacing pattern are indicated with plus symbols (Ala) and number symbols (Thr) with asterisks denoting residues that are identical in all sequences. Acidic and basic residues are in red and blue font respectively, with Ala highlighted yellow and Thr in white font with black highlighting. Potential helix-stabilizing salt bridges consisting of basic and acidic residues with the more effective i, i+4 separation are double underlined. The cunner isoform is also found in blood .

A) Alignment of Sculpin AFPs. New sequences from shorthorn sculpin cDNAs from liver (Liv), larvae (Lar) or genomic DNA (G) are compared to known shorthorn skin (Skin) and longhorn skin (LHS) sequences. As the deduced peptide sequences are low complexity, they were aligned based on the DNA sequence alignment, which is shown in along with the accession numbers. Thr is highlighted light green and other polar residues are highlighted dark green with white font. Basic residues are highlighted cyan (Lys) or blue (Arg), acidic are highlighted red in black (Asp) or white (Glu) font, hydrophobic residues (except Ala) are highlighted gray and exceptional residues (Pro and Gly) are highlighted yellow. B) Dot matrix comparisons of selected sculpin isoforms. A line indicates a match of at least 9 out of 10 bases. Coding regions are denoted by blue bars.

Symbols and coloring are as in Fig. 2A. A) Alignment of smaller skin and circulatory isoforms from winter flounder liver (WF-Liv, M63478.1) and skin (WF-skin, M63478.1), cunner (JF937681.2), shorthorn sculpin (SHS) SS-8 and Liv5, longhorn sculpin (LHS, AF306348.1) and cunner (JF937681.2). Only WF-Liv possesses a signal peptide (lower case font, difference relative to WF-hyp in blue) and pro-peptide (italics) which is shown on the line above the mature AFP sequence. Amino acids encoded by codons interrupted by an intron in the cunner and flounder liver sequences are indicated with a wavy underline. The intron within the flounder skin gene lies within the 5′ UTR. B) Sequence of the hyperactive type I AFP from winter flounder (WF-hyp, EU188795.1) denoted as in Fig. 2A. This circulating isoform is dimeric and possesses a signal peptide (lower case font) but no pro-sequence. C) Sequence of the two atypical type I AFPs of intermediate length from shorthorn sculpin skin (SHS-skin, AF305502.1) and dusky snailfish (AY455863.1). Thr is seldom found in position i of the 11 aa i, i+4, i+8 pattern of ice-binding residues and this pattern is not necessarily continuous in these longer AFPs. Neither AFP possesses a signal peptide or prosequence.

The height of the color bars represents the fraction of each of the Ala codons in each dataset or sequence. The cDNAs used encoded the following AFPs; WF–hyp, LHS, SHS–skin, Atlantic snailfish AFP and cunner AFP. The number of non-AFP sequences used for each group is as follows: winter flounder, 70; longhorn sculpin, 10; cunner, 10 and four species in the snailfish family (Liparidae), 8. The accession numbers for these sequences are listed in .

A line indicates a match of at least 9 out of 12 bases with red indicating a sense/antisense match. The blue bars denote the coding region (signal peptides excluded). These sequences correspond to those shown in Fig. 3.