High DNA Sequence Variability at the α1 Na/K-ATPase Locus of Artemia franciscana (Brine Shrimp): Polymorphism in a Gene for Salt-Resistance in a Salt-Resistant Organism

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Abstract
We previously reported that the Na/K-ATPase α1 subunit coding gene showed signs of being a very polymorphic locus in Artemia franciscana. This species is adapted to highly saline waters, and the Na/K-ATPase α1 isoform presumably plays a key role in this adaptation. Therefore, we were interested in further study of the α1 Na/K-ATPase polymorphisms to examine whether they might be due to an adaptation to salt resistance driven by natural selection. Using coding sequences from 10 genomic clones and 3 cDNAs, we observed that most substitutions are in synonymous positions (88.8%). The 12 nonsynonymous substitutions code for conservative amino acid replacements with an apparent scattered distribution across functional domains of the protein. Interspecific comparison between these sequences and two genomic clones from Artemia parthenogenetica containing 1,122 bp of the α1 Na/K-ATPase locus coding sequence showed independence of the synonymous/nonsynonymous ratio in the comparison within A. franciscana and between A. franciscana and A. parthenogenetica, which fits the neutral model of evolution. Since there were no previous studies on DNA polymorphism for other A. franciscana genes, we also studied variability at the Actin 302 locus for comparison. Both loci were amplified by reverse transcription–polymerase chain reaction, and 20 sequences were obtained for each. This study shows that the amplified region of the α1 Na/K-ATPase gene is 3.5 times as polymorphic as the Actin 302 gene and 2.9 times as heterozygotic. Interestingly, under a model of neutral evolution, the data observed would be expected with a probability of approximately 0.05, suggesting an excess of intraspecific variation of α1 Na/K-ATPase with respect to Actin 302. Restriction fragment length polymorphism studies show similar patterns of polymorphism along the ∼41-kb span of the α1 Na/K-ATPase locus. Most of the nucleotide differences are linked in a few haplotypes, although recombination events are also inferred from the data. We propose a possible explanation for the high polymorphic levels at the α1 Na/K-ATPase locus which invokes positive selection acting tightly to the locus in transiently isolated or semi-isolated subpopulations.