19. G. Fatemifar et al., Hum. Mol. Genet. 22, 3807–3817 (2013).
Fatemifar et al. (2013) showed that HMGA2 is associated with craniofacial features such as the width of the eye region, the width of the lower part of the nose, and the height of the mid-brow prominence.
18. M. N. Weedon et al., Nat. Genet. 40, 575–583 (2008).
Weedon et al. concluded that the HMGA2 gene was one of several that has a role in influencing height in adult humans.
17. X. Zhou, K. F. Benson, H. R. Ashar, K. Chada, Nature 376, 771–774 (1995).
Zhou (1995) found that, in mice, mutant alleles sometimes arise from deleted DNA or from chromosomal inversions.
When these mutations cause the protein Hmgi-c to inactivate and not be expressed in mice, the result is dwarfism. This protein is associated with the HMGA2 gene.
16. K. Pfannkuche, H. Summer, O. Li, J. Hescheler, P. Dröge, Stem Cell Rev. Rep. 5, 224–230 (2009).
Pfannkuche and colleagues (2009) found that HMGA2 is an important factor in embryonic stem cells and seems to magnify other factors, such as the regulation of body height in humans, the repression of certain genes, and several other functions.
15. S. Lamichhaney et al., Nature 518, 371–375 (2015).
The previous tree from Lamichhaney and colleagues in 2015 showed that the initial split between warbler finches and other finches happened 900,000 years ago. Rapid divergence of ground and tree finches occurred 100,000 – 300,000 years ago.
14. A. Abzhanov, M. Protas, B. R. Grant, P. R. Grant, C. J. Tabin, Science 305, 1462–1465 (2004).
Abzhanov and colleagues (2004) analyzed various growth factors that were known to be expressed during craniofacial development of birds. When looking at Darwin's finch species, some factors showed simply showed no correlation whereas other factors showed a correlation with beak size, but not beak shape.
However, researchers did find that the expression of the Bmp4 molecule had strong association with both beak size and shape.
11. P. R. Grant, B. R. Grant, Science 313, 224–226 (2006).
Grant and Grant (2006) reported that the finch species Geospiza fortis diverged in beak size from one of its competitors, G. magnirostris. This divergence happened on an isolated Galapagos island 22 years after G. magnirostris arrived to share a habitat with G. fortis.
Beak size and beak shape are involved in all the major evolutionary shifts in the adaptive radiation of Darwin’s finches (1).
Thanks to the long-term scientific field work of Peter and Rosemary Grant, scientists have developed an understanding of how this speciation has happened.
Jonathan Weiner provides an introduction to this legendary fieldwork in The Beak of the Finch.
For a more scholarly background, check out How and Why Species Multiply by Peter and Rosemary Grant.
Our results provide evidence of two loci with major effects on beak morphology across Darwin’s finches. ALX1, a transcription factor gene, has been associated with beak shape (15), and here we find that HMGA2 is associated with beak size.
Coupled with the team's previous study that showed the ALX1 gene controlled beak shape, this study shows evidence that HMGA2 is associated with beak size.
These findings show how scientific research builds upon itself to continue to not only answer questions, but to continue to ask questions.
Introgressive hybridization
Movement of genes from one species into the gene pool of another species by the repeated crossing of a hybrid with one of its parents.
We genotyped a diagnostic SNP for the HMGA2 locus in medium ground finches on Daphne Major that experienced the severe drought in 2004–2005 (n = 71; 37 survived and 34 died) (11).
To look at the specific finches that experienced the 2004–2005 drought, the researchers genotyped a HMGA2-specific SNP in both survivors and victims (71 total birds).
we genotyped an additional 133 individuals of this species for a haplotype diagnostic SNP (A/G) at nucleotide position 7,003,776 base pairs in scaffold JH739900, ~2.3 kb downstream of HMGA2.
Lamichhaney and colleagues took a closer look at another 133 birds. Specifically, they investigated an SNP at a certain position in the genome.
Within the 17 SNPs, researchers knew that the large finches were homozygous (LL in Figure 2D) for one haplotype group and small finches were homozygous (SS) for another haplotype group. However, what was going on with the medium ground/tree finches?
This particular SNP was shown to be associated with only beak and body size within these medium finches.
we investigated whether the HMGA2 locus is primarily associated with variation in body size, beak size, or both.
HMGA2 had been identified as a candidate gene, and the SNPs within the 525-kb region within HMGA2 had been located. Therefore, the researchers attempted to see which trait this gene was specifically related to.
Segregation is mainly observed in species with intermediate size (medium ground and tree finches).
In contrast to haplotypes present in large and small finches, the medium-sized finches tended to inherit the particular genes from both parents.
This is what we may call a heterozygous condition.
scan comparing large, medium, and small ground finches and tree finches (Table 1) identified seven independent genomic regions with consistent genetic differentiation (ZFST > 5) in each contrast (Fig. 2A and table S2).
Researchers performed pairwise, genome-wide fixation index (see definition in Glossary section) scans across the whole genome. They did this with 15kb windows that were non-overlapping regions.
The following three comparisons were made: 1) large ground/tree versus medium ground/tree; 2) large ground/tree versus small ground/tree; and 3) medium ground/tree versus small ground/tree.
A compilation of SNP calls yielded 44,767,199 variable sites within or between populations. The fixation index score ranged from zero (complete sharing of genetic material) to one (no sharing of genetic material).
Each index value was then transformed into a Z-score, which is simply a measure of how many standard deviations below or above the population mean a raw score is. Further analysis was done if the Z-score of the fixation index was greater than five. See the Supplementary Materials for more information.
We identified 17 SNPs showing high genetic divergence between large and small ground finches and tree finches (FST > 0.8) at nucleotide sites in highly conserved regions across birds and mammals (PhastCons score > 0.8) (Fig. 2C).
The researchers again calculated the fixation index. However, this time it was only for the variable region (~525-kb in size) that contained the HMGA2 gene.
Remember, the fixation index score ranged from zero (complete sharing of genetic material) to one (no sharing of genetic material).
For a good explanation of a SNP, see this video.
missense mutation
Where a single nucleotide base is changed, which results in a different amino acid.
HMGA2 has been associated with variation in height, craniofacial distances, and primary tooth eruption in humans (18, 19)
Fatemifar et al. (2013) showed that HMGA2 is associated with craniofacial features, such as the width of the eye region, the width of the lower part of the nose, and the height of the mid-brow prominence.
Ligon et al. (2005) had previously reported an 8-year-old boy with a shortened HMGA2 gene that exhibited widely-spaced eyes, a large head circumference, and premature dentition.
exhibits severe growth retardation (17)
Zhou (1995) found that, in mice, mutant alleles sometimes arise from deleted DNA or from chromosomal inversions. When these mutations cause the protein Hmgi-c to inactivate and not be expressed in mice, the result is dwarfism. This protein is associated with the HMGA2 gene.
This tree was almost identical to our previous tree (15).
The previous tree from Lamichhaney and colleagues in 2015 showed that the initial split between warbler finches and other finches happened 900,000 years ago. Rapid divergence of ground and tree finches occurred 100,000 — 300,000 years ago.
Hybridization in finches has influenced the evolution of beak shape. Using phylogenetic studies along with genomic data allowed researchers to reveal some of the genetic variation that underlies finch beak diversity.
We combined these data with sequences from 120 birds, including all species of Darwin’s finches and two outgroup species (15)
DNA extraction and whole genome sequencing was performed using samples from 60 birds. In addition, 120 bird samples from a previous study (Lamichhaney 2015) were used.
The sequence reads were analyzed and trimmed using FASTQC software and FASTX software, respectively. To see more specific software used, see the Supplementary Materials
The researchers used the genome assembly of a medium ground finch as a reference genome. The reads from the samples were aligned to this reference.
only one regulatory gene, ALX1, is known and it regulates variation in beak shape (15), which was not associated with survival in 2004–2005.
Lamichhaney et al. (2015) previously scanned the genomes of finch populations that were related but displayed different beak structures. They found that the ALX1 gene was a strong candidate for regulating the variation in beak morphology. ALX1 encodes a protein that is vital in developing structures from embryonic tissue that will form craniofacial structures.
Thus, body size was possibly subject to selection, but beak size was a more important factor affecting the probability of survival independent of body size (11, 12). However, the genetic basis of the selected traits remains unknown.
Grant and Grant (1994) explored hybridization among finch species over 17 years. They concluded that hybrid traits were morphologically intermediate, which indicated the parent genes contributed to the offspring phenotype equally. Hybrids also varied more phenotypically.
Even though both were heritable, beak size seemed to be more important that body size because the beak's relationship to food acquisition.
2004–2005 selection event
The 2004–2005 selection event was a severe drought. For a thorough explanation of the climate of the Galapagos islands, see here.
Beak sizes diverged as a result of a selective disadvantage to medium ground finches with large beaks when food availability declined through competition with large ground finches during a severe drought in 2004–2005 (11).
Grant and Grant (2006) reported that the finch species Geospiza fortis diverged in beak size from one of its competitors, G. magnirostris. This divergence happened on an isolated Galapagos island 22 years after G. magnirostris arrived to share a habitat with G. fortis.
Genotypes associated with large beak size were at a strong selective disadvantage in medium ground finches (selection coefficient s = 0.59).
After the drought, medium ground finches with larger beaks were at a severe disadvantage because they could not compete for food with the large ground finches.
When a genotype is shown to be disadvantaged, what happens to the genes in that gene pool? What happens to those disadvantaged individuals? What happens to the population as a whole?
Furthermore, although some signaling molecules affecting beak dimensions in Darwin’s finches have been identified (14)
Abzhanov and colleagues (2004) analyzed various growth factors that were known to be expressed during craniofacial development of birds. When looking at Darwin's finch species, some factors showed no correlation whereas other factors showed a correlation with beak size, but not beak shape. However, researchers did find that the expression of the Bmp4 molecule had strong association with both beak size and shape.
One of these regions (~525 kb in size) showed the strongest differentiation in all three contrasts. The region included four genes: high mobility AT-hook 2 (HMGA2), methionine sulfoxide reductase B3 (MSRB3), LEM domain-containing protein 3 (LEMD3), and WNT inhibitory factor 1 (WIF1).
This index scan allowed the researchers to narrow down the region that showed the strongest differences between the large, medium, and small ground and tree finches.
The results suggest that the phenotypic effects of these loci are small relative to the effect of the HMGA2 locus.
The investigators were able to rule out the six other loci that may have been associated with beak size, body size, or beak shape.
This SNP showed a highly significant association with beak size, a significant association with body size, and no association with beak shape among medium ground finches (Fig. 2E).
Investigations within the HMGA2 gene showed that it did, in fact, show a high relationship with beak size.
This seems to confirm the fact that HMGA2 played an important role in the evolutionary shift in beak size during the drought.
Previous field studies have documented rare but recurring introgressive hybridization on Daphne Major between medium ground finches and small ground finch immigrants (23)
Introgression, the movement of a gene from one species into the gene pool of another, has been known to speed up a population's response to an evolutionary selective pressure. It does this by increasing genetic diversity.
Grant and Grant documented recurring introgressive hybridization between medium ground finches and small ground finches.
23. P. R. Grant, B. R. Grant, Biol. J. Linn. Soc. London 117, 812–822 (2016).
Introgression, the movement of a gene from one species into the gene pool of another, has been known to speed up a population's response to an evolutionary selective pressure. It does this by increasing genetic diversity.
Grant and Grant documented recurring introgressive hybridization between medium ground finches and small ground finches.
3. W. L. Brown Jr., E. O. Wilson, Syst. Zool. 5, 49–64 (1956).
Brown and Wilson (1956) argued that character displacement was a common part of geographical speciation. They explained that displacement happened most often as a product of genetic and ecological interaction when species first met.
13. P. R. Grant, B. R. Grant, Evolution 48, 297–316 (1994).
Grant and Grant (1994) explored hybridization among finch species over 17 years. They concluded that hybrid traits were morphologically intermediate, which indicated the parent genes contributed to the offspring phenotype equally. Hybrids also varied more phenotypically.
We have shown that the HMGA2 locus played a critical role in this character shift. The selection coefficient at the HMGA2locus (s = 0.59 ± 0.14) is comparable in magnitude to the selection differential on the phenotype and is higher than other examples of strong selection, such as loci associated with coat color in mice (s < 0.42) (25). The main implication of our findings is that a single locus facilitates rapid diversification.
This single HMGA2 locus seems to allow for diversity among finch beak size. However, the exact mechanism for how HMGA2 controls beak size in finches is unknown.
Would the findings of this study provide enough justification for further studies that would attempt to better understand that function of HMGA2?
Thus, we conclude that the relationship between HMGA2 and fitness was mediated entirely by the effect of this locus on beak size or associated craniofacial bones or muscles; developmental research will be necessary to reveal the underlying mechanism for the association. There is no evidence of pleiotropic effects of the gene on other, unmeasured, traits affecting fitness (table S5).
The HMGA2 gene is strongly associated with beak size. Beak size is the main factor that allowed some medium ground and tree finches to survive after a drought.
Thus, the researchers provided a direct link between this particular gene and the fitness of finches.
revealed two major haplotype groups associated with size; 98% of small birds (body weight <16 g) clustered into one group and 82% of the large birds (body weight >17 g) clustered into the other (Fig. 1D). The split between the two haplotypes occurred before the divergence of warbler and nonwarbler finches at the base of the phylogeny (Fig. 1D), about 1 million years ago (Fig. 1C).
Why was it important to not only construct a phylogenetic tree based on the entire genome sequences (Fig. 1C), but also construct one based on the HMGA2 region (Fig. 1D)?
What do these two trees reveal about the differing sizes of finches?
We discovered a genomic region containing the HMGA2 gene that varies systematically among Darwin’s finch species with different beak sizes.
For the first time, researchers have been able to find a gene that controls beak size in Darwin's finches. Why is this finding such an important part of the story of these birds and how they evolved?
As a result of resource competition, they may diverge in traits associated with exploiting these resources (1, 2).
In addition to Darwin's finches, this story about lizards provides another great example.
Jonathan Losos also wrote a popular book, Improbable Destinies: Fate, Chance, and the Future of Evolution, that highlights these anole lizards.
We performed a genome-wide screen for loci affecting overall body size in six species of Darwin’s finches that primarily differ in size and size-related traits: the small, medium, and large ground finches, and the small, medium, and large tree finches (Fig. 1, A and B, and table S1)
The investigators initially had to decide that they were going to use samples from six species of finches. Then, they screened the entire genome of these species to look for genetic variants in different individuals. The objective was to see if any variation at any given location was associated with size and/or size-related traits.
We constructed a maximum-likelihood phylogenetic tree on the basis of this ~525-kb region
The genome-wide fixation index scan found that a region around 525 kb in size showed the most striking differences.
Lamichhaney and colleagues constructed another phylogenetic tree based on the alignment of this region in all of the samples.
We constructed a maximum-likelihood phylogenetic tree on the basis of all 180 genome sequences (Fig. 1C)
The nucleotide alignment of the variable positions from all 180 samples (60 birds plus 120 from previous study) allowed the scientists to generate a phylogeny using software FastTree.
See here to learn more about FastTree and its maximum-likelihood method.
We sequenced 10 birds from each of the six species (total 60 birds) to ~10× coverage per individual, using 2 × 125–base pair paired-end reads. The sequences were aligned to the reference genome from a female medium ground finch (12).
Lamichhaney and colleagues sequenced a total of 60 birds. Sequencing is a technique used to actually read the DNA.
For a history of DNA sequencing and assembly, this resource from HHMI BioInteractive is a great tool.
This video shows specifically how Illumina Sequencing Technology works.
If a finch genome is 1 Gbp (one trillion base pairs), sequencing "to ~10x coverage per individual" would mean that you obtain 10 Gbp of sequencing data.
"Using 2 x 125-base pair paired-end reads" refers to the fact that the fragments sequenced were sequenced from both ends and not just one. Refer to the videos above for more information.
We then genotyped individuals of the Daphne population of medium ground finches that succumbed or survived during the drought of 2004–2005.
Genotyping establishes a genetic code for each individual finch. With birds, this can typically be done using plucked feathers, blood, or eggshell membranes.
The researchers here used blood samples that were collected on FTA paper and then stored at -80°C. FTA paper is treated to bind and protect nucleic acids from degrading.
This type of scanning is used to identify specific gene markers that are highly variable. Researchers wanted to identify a locus that showed beak size variation.
HMGA2 is a chromatin-associated protein
HMGA2 is a gene that plays a role in transcribing HMGA proteins. These proteins are associated with chromatin, the substance that makes up chromosomes and can consist of DNA, RNA, and proteins.
Pfannkuche and colleagues (2009) found that HMGA2 is an important factor in embryonic stem cells and seems to magnify other factors, such as the regulation of body height in humans, the repression of certain genes, and several other functions.
However, it has been difficult to obtain unequivocal evidence for ecological character displacement in nature (8, 9).
Stuart and Losos (2013) found that just over 6% of the cases they reviewed that highlighted character displacement actually showed strong examples. However, one study not only documented ecological character displacement, but also showed it happening in real time. This was the long-term experiment of Darwin's finches by Grant and Grant (2006).
Darwin proposed this as the principle of character divergence [now known as ecological character displacement (3, 4)], a process invoked as an important mechanism in the assembly of complex ecological communities (5, 6).
Darwin states in The Origin of Species that the differences between species is caused by divergence. It is this divergence that acts to minimize the competitive interactions between similar populations.
Brown and Wilson (1956) later argued that this concept (they called it character displacement) was a common part of geographical speciation. They explained that displacement happened most often as a product of genetic and ecological interaction when species first met.
polygenic
Referring to traits that are controlled by two or more genes at different places on different chromosomes.
synteny
When two or more genomic regions are derived from a single ancestor.
directional selection
Type of natural selection where one phenotype is favored over others. This causes the frequency of alleles to shift in the direction of that phenotype.
heterozygotes
An individual that has two different forms of a particular gene on homologous chromosomes.
homozygous
Referring to a particular gene in which all of the alternative forms of that gene are identical on homologous chromosomes.
phenotype
Observable characteristics of an individual.
single-nucleotide polymorphism
Refers to a variation in a single base pair within a DNA sequence.
intrinsic transcriptional activity
An essential copying of DNA into RNA, the first step of gene expression.
genome-wide fixation index
Measure of the genetic differences in and among populations due to genetic structure.
selection differential analysis
Measures the difference between the average genetic value of selected organisms and the average genetic value of all the organisms in the population.
multiple regression
Used to explain the relationship between a dependent variable and one or more independent variables.
heritable
Referring to traits that are transmissible from parent to offspring.
taxa
Groups of one or more populations.
phylogenetic
Referring to the study of the evolutionary history and relationships of various biological species.
speciation
The formation of new species.
haplotypes
Groups of genes inherited from a single parent.
genomic analysis
The process of measuring or comparing features such as DNA sequences, variation of structures, gene functions, or gene expression.
adaptive radiation
Event in which a lineage rapidly diversifies into more species with different adaptations. These events lead to speciation.
morphological
Relating to the structure or form of living organisms.
character displacement
When two competing species overlap in habitat, natural selection may drive one population to use resources that are not used by individuals of the other population. This allows two similar species to coexist.
Ecological
Describing a branch of biology that deals with relationships and interactions among organisms and the environment.
phylogenetic
Referring to the study of the evolutionary history and relationships of various biological species.
morphological
Relating to the structure or form of living organisms.
interspecific competition
Individuals of different species compete for the same resources.
adaptive radiations
Events in which a lineage rapidly diversifies into more species with different adaptations. These events lead to speciation.
speciation
The formation of new species.
heritability
A statistic that describes how much variation of a specific trait can be attributed to genetics. With these finches, it's a measure of how well differences in beak genes account for differences in beak sizes.
natural selection
Process that describes how species adapt to their environment. The organisms that are best adapted tend to survive and reproduce.
ecological
Describing a branch of biology that deals with relationships and interactions among organisms and the environment.
character displacement
When two competing species overlap in habitat, natural selection may drive one population to use resources that are not used by individuals of the other population. This allows two similar species to coexist.