Atlantic cod is composed of multiple migratory and stationary populations widely distributed in the North Atlantic Ocean. The Northeast Arctic cod (NEAC) population in the Barents Sea undertakes annual spawning migrations to the northern Norwegian coast. Although spawning occurs sympatrically with the stationary Norwegian coastal cod (NCC), phenotypic and genetic differences between NEAC and NCC are maintained. In this study, we resolve the enigma by revealing the mechanisms underlying these differences. Extended linkage disequilibrium (LD) and population divergence were demonstrated in a 17.4-Mb region on linkage group 1 (LG1) based on genotypes of 494 SNPs from 192 parents of farmed families of NEAC, NCC or NEACxNCC crosses. Linkage analyses revealed two adjacent inversions within this region that repress meiotic recombination in NEACxNCC crosses. We identified a NEAC-specific haplotype consisting of 186 SNPs that was fixed in NEAC sampled from the Barents Sea, but segregating under Hardy-Weinberg equilibrium in eight NCC stocks. Comparative genomic analyses determine the NEAC configuration of the inversions to be the derived state and date it to ~1.6-2.0 Mya. The haplotype block harbours 763 genes, including candidates regulating swim bladder pressure, haem synthesis and skeletal muscle organization conferring adaptation to long-distance migrations and vertical movements down to large depths. Our results suggest that the migratory ecotype experiences strong directional selection for the two adjacent inversions on LG1. Despite interbreeding between NEAC and NCC, the inversions are maintaining genetic differentiation, and we hypothesize the co-occurrence of multiple adaptive alleles forming a 'supergene' in the NEAC population.

A major challenge in evolutionary biology is to identify the genes underlying adaptation. The oxygentransporting haemoglobins directly link external conditions with metabolic needs and therefore represent a unique system for studying environmental effects on molecular evolution. We have discovered two haemoglobin polymorphisms in Atlantic cod populations inhabiting varying temperature and oxygen regimes in the North Atlantic. Three-dimensional modelling of the tetrameric haemoglobin structure demonstrated that the two amino acid replacements Met55b 1 Val and Lys62b 1 Ala are located at crucial positions of the a 1 b 1 subunit interface and haem pocket, respectively. The replacements are proposed to affect the oxygen-binding properties by modifying the haemoglobin quaternary structure and electrostatic feature. Intriguingly, the same molecular mechanism for facilitating oxygen binding is found in avian species adapted to high altitudes, illustrating convergent evolution in water-and air-breathing vertebrates to reduction in environmental oxygen availability. Cod populations inhabiting the cold Arctic waters and the low-oxygen Baltic Sea seem well adapted to these conditions by possessing the high oxygen affinity Val55-Ala62 haplotype, while the temperature-insensitive Met55-Lys62 haplotype predominates in the southern populations. The distinct distributions of the functionally different haemoglobin variants indicate that the present biogeography of this ecologically and economically important species might be seriously affected by global warming.

Haemoglobins from mammals of sub-Arctic and Arctic species, as well as fetal human Hb, are all characterized by a significantly lower Delta H of oxygenation compared with the majority of mammalian haemoglobins from temperate species (exceptions are represented by some cold-resistant species, such as cow, horse and pig). This has been interpreted as an adaptive mechanism of great importance from a physiological point of view. To date, the molecular basis of this thermodynamic characteristic is still not known. In the present study, we show that binding of extra chloride (with respect to adult human Hb) ions to Hb would significantly contribute to lowering the overall heat of oxygenation, thus providing a molecular basis for the low effect of temperature on the oxygenation-deoxygenation cycle. To this aim, the oxygen binding properties of bovine Hb, bear (Ursus arctos) Hb and horse Hb, which are representative of this series of haemoglobins, have been studied with special regard to the effect of heterotropic ligands, such as organic phosphates (namely 2,3-diphosphoglycerate) and chloride. Functional results are consistent with a mechanism for ligand binding that involves an additional binding site for chloride ion. Analysis of computational chemistry results, obtained by the GRID program, further confirm the hypothesis that the reason for the lower Delta H of oxygenation is mainly due to an increase in the number of the oxygen-linked chloride-binding sites.

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