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 .