New research finds that hummingbird species living at high altitudes have evolved hemoglobin with enhanced oxygen-binding properties so they can thrive in oxygen-poor environments. This enhanced oxygen-binding property is derived from the same mutations that arose independently in these birds' hemoglobin genes.

When people climb mountains, they can experience health problems at elevations above 1,500 metres (4,900 feet) due to "thin air" -- basically, there are fewer molecules of oxygen present at higher elevations, so humans struggle to meet their oxygen needs. Yet, even whilst humans gasp for breath, they've noticed that birds, such as migratory geese flying above their heads, remain unaffected. Equally as remarkable as these avian sojourners, but less well-studied, are birds that actually live out their entire lives at high elevations. Since avian metabolisms are much faster than those of mammals, birds have correspondingly greater oxygen demands, so how do they survive at high altitudes, where oxygen is so limited?

I recently ran across a beautiful study that explores this paradox in hummingbirds. In this paper, a group of scientists reveal how a (seemingly) tiny genetic substitution changes the structure of the blood protein, hemoglobin, thereby enhancing its oxygen-binding properties. In turn, these genetic substitutions (or "mutations", if you prefer) have opened up new possibilities for hummingbirds by allowing some species to colonise high elevation home ranges in South America's Andes Mountains. Moreover, the team reports that multiple hummingbird species independently evolved these same changes in their hemoglobin structure, a process known as parallel adaptation.

Hummingbirds comprise a New World family of (mostly) tropical birds. Due to their tiny body size and colourful iridescent plumage, they are sometimes referred to as flying jewels, which has inspired evocative common names such as sunangels, mountain-gems, hillstars and sunbeams. These diminutive birds fuel their intense lifestyles with nectar and insects since, even amongst birds, they have exceptionally high metabolic rates.

"On a gram per gram basis, hovering hummingbirds use energy at about 10 times the rate of a highly trained human athlete at their peak performance", writes ornithologist Christopher Witt in email.

Professor Witt is an associate professor of biology and the curator of birds at the Museum of Southwestern Biology at the University of New Mexico. In addition to investigating the distribution of tropical New World bird species throughout time and space, he is interested in deciphering the physiological framework that allows some hummingbird species to live comfortably at high altitudes despite their extraordinary energy requirements.

Since hummingbirds have such remarkable energy demands, the last place that one might expect to encounter them is high in the mountains. Yet this is exactly where some species live. To investigate this paradox, Professor Witt teamed up with evolutionary geneticist Jay Storz, associate professor of biological sciences at the University of Nebraska-Lincoln. One of the research areas that particularly interests Professor Storz is deciphering the nature of evolutionary change, and he's fascinated by the repeatability and predictability of molecular changes in the oxygen-binding blood protein, hemoglobin, as it evolves to meet the physiological demands of life in oxygen-poor environments.

Professors Storz and Witt recently published a paper that investigates the overlap between evolutionary changes in hemoglobin's oxygen-binding capacity in hummingbirds and how improving this particular property has allowed these birds to colonise high-elevation habitats. To do this work, Professors Storz and Witt assembled a team of researchers and together, they designed a series of experiments to examine the multiple facets of this phenomenon: genetics, protein structure and protein function (physiology) -- all of which affect and are shaped by these species' evolutionary, geographical and ecological circumstances.

Is there a connection between oxygen-binding efficiency of hemoglobin and elevation?

The research team first determined whether there really is a relationship between oxygen-binding affinity of hemoglobin and the elevation where these hummingbird species live. They began by identifying three Andean hummingbird lineages -- the Coquettes (blue shading in figure 1A), the Brilliants (orange shading), and the Emeralds (yellow shading) -- that encompass closely related species that either are lowland or moderate elevation species or are high elevation specialists that routinely occur above 4,200 metres. The team then measured and mapped their hemoglobin (Hb) oxygen-binding affinities and the elevation where these species occur onto their phylogeny (a DNA-based family tree; see Figure 1A; larger view):

This analysis revealed that hemoglobin from birds that typically live at high elevations does correspond to enhanced oxygen-binding affinities.

A more rigorous analysis using a different method also found a strong statistically significant relationship between the oxygen-binding affinity of hemoglobin and the average elevation where these birds live (Figure 1B; larger view):

But did this enhanced oxygen-binding affinity result from changes in same locations within the birds' hemoglobin genes? And were these genetic substitutions identical between different species?

Are specific genetic substitutions in hemoglobin repeated?

Hemoglobins (Hbs) are large iron-containing metalloproteins comprised of four subunits; two α-globin protein chains and two β-globin chains. Each Hb can bind up to four O 2 molecules and four iron atoms. Contained inside red blood cells of nearly all vertebrates, the primary "job" of Hb is to transport oxygen to the tissues to fuel metabolism and to remove CO 2 and other gaseous waste products. Although these genes and their resulting proteins are large, only a very few amino acids actually bind oxygen, so the team proposed that the substitutions that affect Hb's binding properties should also be limited.

To identify the Hb mutations and to determine whether these substitutions are repeated in different species, the team aligned the sequences for β-globin genes for the three hummingbird lineages against the sequence estimated for the ancestral hummingbird (Anc 1). They found that species living at the highest elevations (more than 3,000 metres; grey shading; figure 2) substituted the same amino acid (serine) at just two positions (β13 and β83) in their β-globin sequences (red boxes; figure 2; larger view):

Of the 21 amino acids that make up eukaryotic proteins, serine is one of the smallest and it is polar (different regions of the molecule carry either a partial positive or a partial negative charge) -- thus, substituting serine could have profound biochemical and structural effects upon Hb function, so the researchers investigated these parameters too.

They measured the Hb-O 2 binding affinities for three species within each of the three hummingbird lineages, for a total of nine species. They found that those species with the highest Hb-O 2 binding affinities always possessed either the two-site genotypes β13Ser-β83Ser or β13Gly-β83Ser. They also found that hummingbirds whose Hb showed enhanced O 2 binding affinities were high-elevation species.

In contrast, those species with the lowest Hb-O 2 binding affinities always possessed the two-site genotype β13Gly-β83Gly. All of these hummingbirds were either predominantly or exclusively lowland species.

When the team examined the structure of the two-site genotype β13Ser-β83Ser hemoglobin and compared that to the structure for two-site genotype β13Gly-β83Gly hemoglobin, they found distinct structural alterations that likewise affected the oxygen-binding affinities. (Structural data not shown here, but you are welcome to peek at it if you're truly curious.)

They characterised the functional effects of these substitutions by creating recombinant globin proteins using genetic engineering techniques and studying them. The team also reconstructed the inferred ancestral hemoglobin gene sequences and characterised them, too.

Are hemoglobin substitutions predictable?

Globin genes are numerous and large, so there are plenty of places where an adaptive mutation could occur at the genetic level without being identical to similarly advantageous mutations seen in other species.

To answer the question whether these same genotypic substitutions occur in a predictable way amongst Andean hummingbirds, the team sequenced the β-globin gene for 63 hummingbird species. Then they mapped the β13 and β83 substitutions onto a phylogeny that had been published in 2007 by a different research group (doi:10.1080/10635150701656360; Figure 4; larger view):

They also mapped other information onto the above phylogeny. First, the small pie diagrams at each node indicate the probability that each genotype occurred at the same rate through several reversible stepwise transitions, as indicated in the small inset diagram in the upper left corner.

The end of each tree branch corresponds to one species and is colour-coded according to the upper limit of that species' elevational range (as decoded by the colour key inset on the left). The basal portion of the tree branches corresponds to shared ancestral species and is color-coded to reflect estimates of ancestral elevation ranges. The branch lengths are proportional to the passage of time (except where indicated by "//").

Species' scientific names appear at the end of the appropriate branch and the names of lineages are indicated on the right. Bolded species names correspond to those that were included in the experimental analysis of Hb function.

Analysis of this figure found that mapping the β13-β83 genotype and native elevation onto this phylogeny described a relationship that was highly significant.

"The amino acid mutations at sites 13 and 83 in the beta-chain subunit are the only changes that occurred in parallel and they are very strongly associated with the among-species variation in hemoglobin-O2 affinity", writes Professor Storz in email.

Parsimony analysis revealed the Gly-to-Ser substitution occurred independently at least 17 times (at least 4 times at β13 and at least 13 times at β83). How did the researchers know these mutations were independent?

"If a given mutation is shared between a pair of distantly related species -- and the same mutation is not found in close relatives of either species -- then we can infer that the mutation occurred twice independently, and was not simply inherited from a common ancestor", explains Professor Storz in email.

Further, corresponding ecological preferences were also identified: estimates for each hummingbird species' native elevation indicated they shifted their range upwards and downwards in conjunction with repeated substitutions and back-substitutions at β13 and β83.

"Hummingbirds are remarkable for their degree of elevational specialization -- the average Andean species has an elevational distribution that spans [roughly] 1100 metres", explains Professor Witt in email.

"These elevational distributions are also conserved at deep levels in the phylogeny -- i.e. elevation doesn't shift willy-nilly during evolution."

What does all this mean?

This research demonstrates the evolutionary relationship between genes and biochemistry to physiology and ecology.

"We've shown that highland species have mutations that closely related lowland species don't have", summarises Professor Storz in email.

In this study, Professors Storz and Witt identify a specific evolutionary adaptation in Andean hummingbirds -- enhanced oxygen-binding affinity in hemoglobin -- establish its connection to specific genetic mutations, describe transitions in biochemical structure and function of hemoglobin, and then show how those changes affect the elevations where the species live.

"The appearance or disappearance of these mutations in the phylogeny is almost perfectly associated with shifts in elevation in the direction that we would predict", writes Professor Witt in email.

"This the most spectacular known example of parallel evolution -- same gene, same nucleotide position on that gene, same nucleotide substitution, same amino acid replacement, same environmental context, demonstrated functional effect, 17 times, and in lineages that have been separate for millions of years", writes Professor Witt in email.

"It's exciting to have discovered a key genetic mechanism by which physiological specialization occurs."

This study goes one step further by testing a particularly interesting hypothesis that -- at the molecular level -- adaptive evolution may be more predictable than previously imagined.

"This pattern of repeated change suggests that natural selection has hit upon the same solutions time and time again", writes Professor Storz.

"Our findings demonstrate that these two positions on the beta-chain are important for adapting to altitude, but that doesn't mean that they're the only hemoglobin sites that are important, nor that hemoglobin is the only gene involved in high-altitude adaptation", adds Professor Witt.

"What's remarkable is that they're so important as to have happened repeatedly and predictably in conjunction with evolutionary shifts in elevation."

Besides being an elegant piece of work, this research highlights some important conservation concerns. One example is the escalator to extinction hypothesis, which suggests that global warming is forcing some species to move to higher elevations in search of cooler temperatures -- until there is nowhere left to go (i. e.; doi:10.1111/j.1523-1739.2007.00852.x).

Already, field observations have found that predicted upwards movement is underway, but this study suggests this may not last long because the mutations necessary to allow species to colonise and thrive in high-elevation habitats may not occur quickly enough. As a consequence, low altitude species may die out before adapting to higher elevations, which may protect high-altitude species from direct competition for limited resources.

"If these birds are specialized on oxygen pressure in the mountains, rather than low temperatures, this could prevent the 'escalator to extinction' effect", writes Professor Witt in email. "In which low-elevation bird species displace higher elevation ones as temperatures warm up."

Sources:

Projecto-Garcia J., Natarajan C., Moriyama H., Weber R.E., Fago A., Cheviron Z.A., Dudley R., McGuire J.A., Witt C.C. & Storz J.F. Repeated elevational transitions in hemoglobin function during the evolution of Andean hummingbirds, Proceedings of the National Academy of Sciences, doi:10.1073/pnas.1315456110 [OA]

Jay Storz: emails [4, 5 & 6 December 2013]

Christopher Witt emails [6 December 2013]

McGuire J., Witt C., Altshuler D. & Remsen J.V. (2007). Phylogenetic Systematics and Biogeography of Hummingbirds: Bayesian and Maximum Likelihood Analyses of Partitioned Data and Selection of an Appropriate Partitioning Strategy, Systematic Biology, 56 (5) 837-856. doi:10.1080/10635150701656360 [OA]

Sekercioglu C.H., Schneider S.H., Fay J.P. & Loarie S.R. (2008). Climate Change, Elevational Range Shifts, and Bird Extinctions, Conservation Biology, 22 (1) 140-150. doi:10.1111/j.1523-1739.2007.00852.x [OA]

Another physiological challenge to high-altitude living, especially for small-bodied creatures that rapidly lose body heat, is surviving cold temperatures. But this problem has also been solved by hummingbirds: How do hummingbirds survive cold nights?

Many thanks to the authors of this study and to my wonderful twitter followers who kindly emailed me the PNAS PDF, mere nanoseconds after I requested it: @soozaphone, @CMBuddle and @Jackg00de.

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GrrlScientist can also be found here: Maniraptora. She's very active on twitter @GrrlScientist and sometimes lurks on social media: facebook, G+, LinkedIn, and Pinterest.