How do hummingbirds thrive in the Andes?
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
extraordinary 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 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
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
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 binding affinities
always possessed either the two-site
genotypes β13Ser-β83Ser or β13Gly-
β83Ser. They also found that
hummingbirds whose Hb showed
enhanced O binding affinities were
high-elevation species.
In contrast, those species with the
lowest Hb-O 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."
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