Journal Club: Butterbutt biology: warblers, migration and mitochondria
SUMMARY: A non-migratory population of songbirds appears to have acquired mitochondria from their close relatives that are migratory, potentially allowing these birds to migrate better
An adult male Butterbutt -- also known as Audubon’s (yellow-rumped) warbler, Setophaga coronata, in-hand.
Image courtesy of David Toews.
A non-migratory population of songbirds appears to have acquired mitochondria from their close relatives that are migratory, potentially allowing these birds to migrate better, according to a newly-published study by a group of researchers based at Canada's University of British Columbia. Mitochondria synthesise the biochemical energy that powers living cells. The team studied a population of neotropical warblers living in the transition zone between the northern (seasonally migratory) form and the southern (resident) form. Using a variety of novel approaches, they compared mitochondrial genetics and function, and migratory behaviour. The researchers found that mitochondria in flight muscles of the migratory birds may be more metabolically efficient, thus capable of powering the energetic demands of migration over longer distances. These findings could have sweeping implications for a variety of research areas, ranging from physiology to conservation biology to evolution.
When I lived in Seattle, springtime meant butterbutts. Oh, they weren't the first migrating birds to appear each spring, but their brilliant lemon-yellow throats and rumps and precise black, grey and white markings were a welcome sight for eyes grown weary of slushy yellow and brown snowpiles.
"Butterbutt" was the amusing and evocative moniker that I first learned for the western population of the widespread North American yellow-rumped warbler (Setophaga [coronata] spp.) species complex. This species complex comprises four distinct types that are either formally recognised as separate species (by the International Ornithological Council) or as distinct subspecies of the yellow-rumped warbler, Setophaga coronata (by the American Ornithologists' Union; figure 1A & B; larger view):
Figure 1A. Distributions of 4 species in yellow-rumped warbler (Setophaga spp.) complex. 3 distinct nuclear groups align with areas shaded blue (myrtle warbler), red/yellow (Audubon's and black-fronted warbler phenotypes), and violet (Goldman's warbler). Hatched areas distinguish myrtle type from black-fronted type mtDNA. B. Sampling localities in SW USA where cryptic mtDNA transition zone was studied. Locations 1–7: western transect; 8–15: eastern transect.
Although I typically saw Audubon's warblers, a small number of stray myrtle warblers also popped up every year, just to keep things interesting. As I soon learned, these are the two seasonally migratory forms of the yellow-rumped warbler -- the myrtle warbler, S. coronata (blue area, figure 1A), breeds in northern boreal forests and in forested areas east of the Rocky Mountains, and winters in eastern North America, Central America, and the Caribbean. Audubon's warbler, S. auduboni (red area, figure 1A), which ranges from the Pacific Northwest all the way to Mexico is more variable: the northern populations breed in forested areas west of the Rocky Mountains and winter in the southwestern United States, Mexico, and Central America whereas the southern populations generally stay put.
There are two sedentary (non-migratory) forms of yellow-rumped warblers: the black-fronted warbler, S. nigrifrons (yellow area, figure 1A), which resides year-round in Mexico; and the Goldman's warbler, S. goldmani (purple area; figure 1A), which consists of a small isolated population of birds that are confined to Guatemala.
Despite obvious differences in plumage and behaviour between these four recognizable taxa, previously published studies that examined diffierences in these birds' chromosomal (nuclear) DNA could only identify three distinct groups; the myrtle warbler group, the Audubon's/black-fronted warbler group, and the Goldman's warbler group (doi:10.1111/j.1365-294X.2011.05055.x). Additionally, those studies revealed that the nuclear genomes within the Audubon's/black-fronted warbler group show a gradual latitudinal gradient that corresponds to specific measurable physical traits (morphometrics) such as wing length (doi:10.1111/j.1365-294X.2011.05055.x).
Further examination of Audubon's warblers' nuclear DNA markers revealed a smooth and gradual transition -- a sort of hybrid zone. But this hybrid zone is special: it results from gene flow from one species into the gene pool of the other by repeated and preferential backcrossing of offspring with one of the two parent species -- a process known as introgression. Technically speaking, Audubon's warblers represent a population that is introgressed with migratory myrtle warbler DNA. The measurable differences in plumage patterns, morphometrics and nuclear DNA in this introgressed population appear to be colour-coded (figure 1A & C; larger view):
Figure 1C. A simplified schematic adapted from Brelsford et al. (2011; doi:10.1111/j.1365-294X.2011.05055.x) to illustrate the discordances between plumage, morphometric, nuclear, and mitochondrial patterns in this system.
Except ... scientists and ornithologists were puzzled by one little mystery: near the border of Utah and Arizona there is an abrupt transition between mitochondrial DNA (mtDNA) lineages: Audubon's warblers either have mtDNA from migratory myrtle warblers or from sedentary black-fronted warblers -- which originally were assumed to be geographically restricted to Mexico. How did black-fronted warbler mtDNA end up so far north?
Furthermore, this mtDNA transition is invisible -- it's only found within birds that are colour-coded as Audubon's warblers. Not only do these birds look like Audubon's warblers, but their morphometrics indicate they are Audubon's warblers -- except for their mitochondria, which either belong to the migratory myrtle warbler lineage or to the non-migratory black-fronted warbler lineage.
"So it's a very useful marker for differentiating species. In this case, finding two species of songbirds sharing the same mtDNA is very surprising, so we set out to find out why."
Considering that these two mitochondrial lineages are carried by two distinct avian taxa that differ in one very important and physiologically demanding life history trait -- seasonal migration -- might this genetic difference affect the functional efficiency of their mitochondria -- and migration?
"We wondered if there could be a functional explanation for it. In other words, could there be something functionally different about this myrtle mitochondria that might have promoted it to introgress across most of the range of the Audubon's warbler?" said Mr Toews in email.
To explore these questions, he and fellow researcher Milica Mandic, a doctoral candidate in comparative physiology at UBC, and their thesis advisors investigated the connections between DNA, physiology and behaviour in this transitional population of yellow-rumped warblers.
"My knowledge of biochemistry and mitochondrial physiology ended mostly with first year biology, so this was a perfect project for a collaboration between the two of us", explained Mr Toews in email.
"[co-author] Milica took the lead on the mitochondrial aspects of the study (e.g. enzyme assays and mitochondrial respiration) and I did the other stuff; neither one of us in isolation could have done this work."
The team used several novel laboratory methods to examine mitochondrial introgression and to measure mitochondrial functional efficiency, and they compared those findings to behavioural and physiological data obtained from several wild populations of Audubon's warblers residing in the transition zone.
As they conducted their studies, the team made two predictions that they tested:
- if there is a link between DNA and behaviour, then the mtDNA transition should correspond to the transition in migratory phenotype
- further, the variation in mitochondrial genotype could support at least some of the variation in the functional efficiency of these mitochondria
Migration: do they or don't they?
In the field in southern Colorado; Haley Kenyon (left), David Toews, Alan Brelsford (right).
Image courtesy of David Toews.
To test whether there is any link between mitochondrial introgression and migratory behavior, the team set up two transects, "eastern" and "western", across the cryptic transition zone and captured 225 individual birds. They took morphometric measurements (i.e., bill, wing, and tail length), photographs, feather samples, and a blood sample from each individual. The birds were given a unique combination of colour bands so they could be tracked in the future and then they were released.
Be free! A colour-banded Audubon's warbler from central Utah. Almost all of the birds we studied for this project were released!
Image courtesy of David Toews.
The team sequenced mtDNA to identify which of the two mitochondrial types that these birds carried. Mapped onto the birds' location, these genetic data were used to estimate both the width of each cryptic transition zone (western [red] and eastern [blue] transects; figure 2A; larger view) and its geographic center.
Now here's where a detailed knowledge of these birds' life history traits played a critical role in the elegant experimental design of this study. First; adult yellow-rumped warblers carry two generations of feathers on their bodies during the spring breeding season. This is because they replace most of their feathers whilst on their breeding ground in autumn and then they replace a subset of their wing feathers during the winter. Not only is this moult pattern predictable, but these feathers are visually distinguishable.
Based on previous research, the team knew that biological tissues and materials -- such as feathers -- contain distinct proportions of elements and their various isotopes that are characteristic of the geographic location where the bird resided whilst growing those particular structures. By experimentally measuring the ratio of an element to its isotopes, one can generate an "isotopic fingerprint" for each geographic location where these birds resided.
Mr Toews and his colleagues realised that by determining the isotopic fingerprints contained within the "breeding feathers" and the "winter feathers" carried by individual birds, they could pinpoint whether that bird had lived in different locations whilst growing each generation of feathers. If the two isotopic fingerprints were different, this would indicate that the bird was migratory.
Second, yellow-rumped warblers are site-faithful -- individuals breed in the same location, and often on the same territory, year after year. So the hydrogen/deuterium (in this case) isotopic fingerprints obtained from the birds' "breeding feathers" could be used to generate a linear regression of capture site latitude versus deuterium values to reconstruct a rough map. This isotopic fingerprint map could then be used to estimate both the latitude of the wintering location and the distance traveled for each individual bird (figure 2B; larger view):
Figure 2. (A) Clines in mtDNA across two transects in a cryptic contact zone in the southwestern United States between myrtle-type and black-fronted type mtDNA. (B) Stable hydrogen isotope data obtained from feather samples across the contact zone. [Filled circles indicate individuals with sedentary (black-fronted) mtDNA and open circles are individuals with migratory (myrtle-type) mtDNA.]
According to their findings, Audubon's warblers captured along both transects ("eastern" transect [blue] and "western" transect [red]) showed the same pattern: birds from the most southerly sites were residents, whereas birds from the more northern sites were migratory. Moreover, the isotopic fingerprint map indicated that individuals in the north traveled seasonally between 4o and 10o latitude.
The isotopic fingerprint map also pointed to a gradual behavioural transition between residents and migrants -- and the center of this transition lies very close to the mtDNA cline centers (arrows in figure 2B) identified for both transects.
Mitochondria: myrtle or black-fronted?
The mtDNA sequences obtained from birds captured along these transects revealed an average 4.1% sequence divergence between myrtle and Audubon's/black-fronted mtDNA. But some genes showed greater sequence variations than did others (figure 3; larger view):
Figure 3. Sequence divergence and the number of fixed amino acid changes between the two mitochondrial clades in the contact zone. [doi:10.1111/evo.12260]
In fact, the genes encoding energy-generating mitochondrial proteins had far greater sequence variations than did genes encoding other types of proteins: the proteins in complex I of the electron transport chain (NADH dehydrogenase) contained far more amino acid (AA) substitutions (16 fixed AA substitutions) between the two mitochondrial types compared to genes encoding proteins in other complexes.
Since the electron transport chain generates the biochemical energy in the form of adenosine triphosphate (ATP) that powers living cells, the team wondered whether these AA differences translated into functional differences in the enzymes themselves.
"In our mtDNA sequencing data we found that complex I had a number of amino acid substitutions, and we were testing whether these amino acid changes might scale up to differences in ... how quickly the enzymes can process a substrate", explained Mr Toews in email.
To examine this question in more detail, the team returned to two locations in the center of the western cryptic transition zone (red arrow, figure 2B) to collect 24 birds. Based on their earlier DNA work, they knew these birds differed only in their mitochondria.
They sampled breast muscle tissue and co-author Ms Mandic assayed the maximal activity for two mitochondrial enzymes, one involved in the electron transport chain (complex I) and the other that is involved in the TCA cycle (citrate synthase) (figure 4; larger view):
Figure 4. Maximal enzyme activity for (A) complex I and (B) citrate synthase from frozen pectoralis major of yellow-rumped warblers in the mtDNA contact zone distinguished as having a myrtle-type (open; n = 8) or black-fronted (filled; n = 15) mitochondria. The horizontal line represents the average maximal enzyme activity for each mitochondrial type. doi:10.1111/evo.12260
"Complex I is most important as it is specifically involved in the energy generating pathway of the mitochondria -- it is the first step in the electron transport chain", explained Mr Toews in email.
Despite the differences in AAs between these enzymes carried by the two mitochondria types, the team didn't find any significant functional differences between them (figure 4). The researchers then tested how efficient the two mitochondrial types are. Basically, there are two ways that physiologists define mitochondrial efficiency: (1) they can either have greater oxygen consumption because the "engine" can run faster (higher state III) or (2) they can have a higher output relative to their background respiration rate (as measured by the acceptor control ratio [ACR]: this is the ratio of the background [state II] respiration rate versus the maximal [state III] respiration rate).
Ms Mandic assayed the mitochondrial types by measuring how quickly and efficiently they consume oxygen and calculated their ACRs (figure 5; larger view):
Figure 5. (A) Respiration rates of permeabilized fibers from pectoralis major of yellow-rumped warblers in the mtDNA contact zone either have myrtle-type (open; n = 9) or black-fronted-type mitochondria (filled; n = 15). (B) rate ratios, show a significant difference (P < 0.05) between myrtle-type and black-fronted-type mitochondria for ACR; the ratio of state III to state II respiration rate.
These data suggest that overall, myrtle-type (northern) mitochondria have a significantly higher output relative to their background respiration rate (ACR), and this greater efficiency is driven primarily by their lower background (state II) respiration rate.
"This hints at the possibility that there may be more 'proton leak' in the southern mitochondria, producing inefficiencies, and that the northern mitochondria is more 'coupled' and efficient", explained Mr Toews in email.
What does all this mean?
"I think the most exciting part of the study is its integrative nature", said Mr Toews.
"We brought together very different types of data; genetic, geographic, isotopic, and biochemical, to try to understand this strange pattern. It was our hope that tackling the problem from numerous angles [would give] people a better idea of what was happening in this system."
Like all good research, this study raises a number of interesting questions. First, if the northern myrtle-type mitochondria is more efficient, why isn't it favoured by selection over the yellow-rumped warblers' entire range?
"We really don't know the answer to this. We speculate in the paper that there may be some tradeoff between the ways the two mitochondria may respire, but we have no data to support this", said Mr Toews in email.
But how does mitochondrial efficiency translate into the day-to-day lives of free-ranging Audubon's warblers?
"Our findings suggest that over generations, the Audubon's warbler may have co-opted the myrtle's mitochondria to better power its own travels", said Mr Toews.
Yellow-rumped warblers are not the only known example of mitochondrial borrowing: last year, Mr Toews and a colleague, Alan Brelsford, identified more than 100 such cases in animals (doi:10.1111/j.1365-294X.2012.05664.x).
"In other systems, such as arctic and brook charr, there may be a thermal component, where one mitochondrial type is better in a given thermal environment", said Mr Toews in email.
Of course, some life history scenarios can generate mitochondrial introgression patterns that don't have a functional basis. Because mitochondria are inherited only from the mother, species where the females typically disperse widely whilst the males remain on or near their natal territory can produce discord between nuclear and mitochondrial DNA.
But this study has greater significance beyond solving one mystery of butterbutt biology.
Since I am an evolutionary biologist, I think the one of this study's most important implications applies to how mtDNA is used to recreate family trees. Basically, evolutionary biologists have typically reconstructed phylogenies of closely-related species based on analysing sequences from portions of one or several mitochondrial genes, often overlooking the fact that mitochondria have a very important function as the power generator of living cells.
"In most cases it works really well to describe relationships among species", Mr Toews pointed out. "But using mtDNA to create phylogenies assumes that there is no natural selection, which a number of studies have now demonstrated is not always true."
"At the end of the day, though, it is becoming so cheap and easy to sequence lots of additional genetic markers from the nuclear genome it is becoming less of an issue."
Toews D.P.L., Mandic M., Richards J.G. & Irwin D.E. (2013). Migration, Mitochondria and the Yellow-rumped Warbler, Evolution. doi:10.1111/evo.12260
David Toews [emails; 25 September, 2 & 3 October 2013]
Toews D.P.L. & Brelsford A. (2012). The biogeography of mitochondrial and nuclear discordance in animals, Molecular Ecology, 21 (16) 3907-3930. doi:10.1111/j.1365-294X.2012.05664.x
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