The harsh environment and thin air of mountain landscapes and highland plateaus is a fertile testing ground for broad evolutionary questions about how animals scrape by in hostile environments. Evolution comes with a lot of baggage and different species may hit upon unique solutions to common problems simply because they had different ancestral starting points. For small, active songbirds, a perennial problem at high altitude is supplying enough oxygen to fuel metabolism.

Haemoglobin is critical to oxygen transport. It is responsible for shuttling oxygen collected in the lungs to the rest of the body and is implicated in adaptation to low oxygen (hypoxia). Many animals that are well adapted to hypoxia have haemoglobins with high oxygen binding affinities. An international team of researchers led by Jay Storz from the University of Nebraska, and Fumin Lei, from the Chinese Academy of Sciences and the University of Chinese Academy of Sciences, compared haemoglobin function in high- and low-altitude birds to find out whether changes in haemoglobin function had similar molecular bases in different evolutionary lineages.

The team collected muscle and blood samples from 17 species of tit from high-altitude sites on the Qinghai-Tibet plateau, in the mountains of southwestern China, and from lowlands throughout eastern China. They extracted haemoglobin from the blood samples and subjected it to a battery of tests to characterize its oxygen binding properties. As haemoglobin function is sensitive to subtle differences in the chain of amino acids that comprise the protein, the team also sequenced the haemoglobin genes for each species from portions of muscle. Next, they constructed a haemoglobin family tree to suss out the evolutionary relationships between the haemoglobins. Finally, the team manufactured their own haemoglobin with custom sequence mutations to match specific changes in the amino acid sequence with effects on oxygen binding affinity.

The haemoglobin of the species that live at high altitude had a higher affinity for oxygen than that of their lowland relatives, meaning that their blood was better at grabbing onto oxygen, a useful feature for life in hypoxia. The high-altitude haemoglobins also displayed a slew of amino acid substitutions absent from those of the low-altitude species. Different branches of the family tree generally had unique sets of substitutions, suggesting that there are multiple, independent molecular pathways by which the affinity of haemoglobin for oxygen can be increased. However, the team identified one case where a pair of species found the same solution for living the high life. Both the grey-crested tit, Lophophanes dichrous, and the ground tit, Parus humilis, swapped a threonine residue for an alanine residue at position 34 in one of the two chains of amino acids that comprise haemoglobin, but this substitution was absent in their lowland cousins. Intrigued, the researchers tested whether this modification had any consequences for oxygen transport by reconstructing the haemoglobin of the last common ancestor shared by the grey-crested tit and ground tit and comparing its functional properties with those of the tits’ haemoglobins. Both of the living species had haemoglobin with higher affinity for oxygen than that of their ancestor, but the differences were attributable to other amino acid alterations elsewhere in the amino acid chains. While the substitution at position 34 alone improved the affinity of haemoglobin for oxygen, it wasn't enough to make a fully fledged, modern highland haemoglobin: additional substitutions were essential for high-altitude adaptation.

High-altitude birds take quite a few liberties with the biochemical properties of their haemoglobin, considering it's so integral to oxygen transport and survival. Even in the rare cases where elements are shared, different families still rely on subtle twists to get it just right. In solving the riddle of life in thin air, birds of a feather do not flock together.

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