Vol 442|3 August 2006|doi:10.

1038/nature04843

LETTERS
The calmodulin pathway and evolution of elongated
beak morphology in Darwin’s finches
Arhat Abzhanov1†, Winston P. Kuo1,2,3†, Christine Hartmann4, B. Rosemary Grant5, Peter R. Grant5
& Clifford J. Tabin1

A classic textbook example of adaptive radiation under natural microarrays were used for a direct comparison of the gene expression
selection is the evolution of 14 closely related species of Darwin’s profiles of several thousand transcripts in stage 26 frontonasal
finches (Fringillidae, Passeriformes), whose primary diversity lies processes (which give rise to the upper beak) of five species of
in the size and shape of their beaks1–6. Thus, ground finches have genus Geospiza: the sharp-beaked finch (Geospiza difficilis), the
deep and wide beaks, cactus finches have long and pointed beaks medium and large ground finches (G. fortis and G. magnirostris),
(low depth and narrower width), and warbler finches have slender and the cactus and large cactus finches (G. scandens and G. conirostris)
and pointed beaks, reflecting differences in their respective diets6. (Fig. 1a; Methods). We first used hierarchical clustering to inspect
Previous work has shown that even small differences in any of the whether the overall expression profiles clustered according to species
three major dimensions (depth, width and length) of the beak have (Methods). The resultant tree illustrates that most of the individual
major consequences for the overall fitness of the birds3–7. Recently
we used a candidate gene approach to explain one pathway
involved in Darwin’s finch beak morphogenesis8. However, this
type of analysis is limited to molecules with a known association
with craniofacial and/or skeletogenic development. Here we use a
less constrained, complementary DNA microarray analysis of the
transcripts expressed in the beak primordia to find previously
unknown genes and pathways whose expression correlates with
specific beak morphologies. We show that calmodulin (CaM), a
molecule involved in mediating Ca21 signalling, is expressed at
higher levels in the long and pointed beaks of cactus finches than
in more robust beak types of other species. We validated this
observation with in situ hybridizations. When this upregulation of
the CaM-dependent pathway is artificially replicated in the chick
frontonasal prominence, it causes an elongation of the upper beak,
recapitulating the beak morphology of the cactus finches. Our
results indicate that local upregulation of the CaM-dependent
pathway is likely to have been a component of the evolution of
Darwin’s finch species with elongated beak morphology and
provide a mechanistic explanation for the independence of beak
evolution along different axes. More generally, our results impli-
cate the CaM-dependent pathway in the developmental regulation
of craniofacial skeletal structures.
To understand the genetic basis of the species-specific beak
morphologies, we previously performed a comparative candidate
gene analysis with developmental genes known to be associated with Figure 1 | Microarray analysis in different finch species. a, Clustering
craniofacial development. We found that a broader and earlier strategy to isolate transcripts whose expression correlated with beak
domain of bone morphogenetic protein 4 (BMP4) expression in morphology. b, The Ward linkage tree showed that most of the individual
the distal neural-crest-derived mesenchyme correlated with the very samples clustered by species. Each individual was sampled two to four times.
deep and wide beak morphology of the ground finches8. This The y-axis is euclidian distance between branches. c, The final clusters of
transcripts, which were upregulated in the comparison between cactus
expression difference was shown to be functionally significant by finches and the sharp-beaked finch and were downregulated or remained
misexpression analysis in chick embryos8. unchanged in the ground finches compared with the sharp-beaked finch.
However, the candidate gene approach did not yield any candi- d, Individual expression profiles clustered by species except for the
dates for pathways that could be involved in evolution of the longer occasional individual profile, probably reflecting a certain overlap in
beak morphology characteristic of the cactus finch species. To morphology or development between the species. Each spot represents the
identify pathways involved in the evolution of long beaks, cDNA length of an individual beak.
1
Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA. 2Decision Systems Group, Brigham and Women’s Hospital, Harvard Medical School,
310 Thorn Building, 75 Francis Street, Boston, Massachusetts 02115, USA. 3Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine,
188 Longwood Avenue, Boston, Massachusetts 02115, USA. 4Institute of Molecular Pathology, Dr Bohr-Gasse 7, A-1030, Vienna, Austria. 5Department of Ecology and
Evolutionary Biology, Princeton University, Princeton, New Jersey 08544, USA. †Present address: Department of Developmental Biology, Harvard School of Dental Medicine,
Boston, Massachusetts 02115, USA.

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expression profiles clustered by species. For example, most individ- the resultant infection was somewhat variable. In more than half of
uals of either G. scandens (4 of 5) or G. conirostris (4 of 4) clustered the cases, the infection was limited to the distal and ventral
together with individuals of the same species (Fig. 1b). The mesenchyme surrounding the developing cartilage and did not
expression profile of one particular individual of G. scandens spread to the skeletal element (Fig. 3d; n ¼ 9 of 16; not shown).
(G.s.5) clustered more closely with those of G. conirostris, probably Because these infected regions closely approximate the domain of
reflecting a certain degree of morphological (and thus developmental) elevated CaM expression in the cactus finches (Fig. 2), we restricted
overlap between these two species (Fig. 1d). G. scandens and our analysis to these embryos. In chicks in which activated CaMKII
G. conirostris collected for this study live on different islands was misexpressed specifically in the distal/ventral mesenchyme, we
(Santa-Cruz and Genovesa, respectively), thus excluding the possi- observed a significant increase in the length of the beaks (length in
bility of species misidentification. We obtained very similar results arbitrary units: 0.78 ^ 0.05 (^s.d.); P , 0.0056, n ¼ 9; more than
when we compared G. fortis and G. magnirostris individuals (largely 10% beak elongation relative to control embryos (Fig. 3a–d)),
distinct expression profiles but some individuals clustering with the whereas the beak width and depth were not affected (Fig. 3, and
other species; not shown). It therefore seems that the analysis is Supplementary Fig. S3).
sensitive to phylogenetic differences between different species of For a better understanding of the phenotype induced by activated
Darwin’s finches. CaMKII in beaks, we studied the expression of markers for skeletal
We then clustered the measurements of signal ratios and intensities cell types, proliferation and cell death (Fig. 3, and Supplementary
for different transcripts to identify genes that were upregulated or Fig. S4). In situ hybridization analysis with probes directed against
downregulated in all individuals of a particular morphology com- skeletogenic genes verified that the cartilage element was indeed
pared with the basal G. difficilis reference (Fig. 1, and Supplementary longer in the distal/ventrally infected embryos (Fig. 3). We found that
Fig. S1; Methods). All of 100 candidates from the resulting final
cluster of cactus finch morphology-specific genes were sequenced to
reveal their identity. These genes were screened to produce candi-
dates that were expressed at moderate or high levels of signal
intensity on the microarray and were expressed at least fivefold
higher in the cactus finches. We found two microarray spots carrying
probes with identical sequences for CaM (Supplementary Fig. S2)
that were both at a much higher level of average signal in the cactus
finch beaks than in the reference sharp-beaked finch (G. difficilis)
(Supplementary Information). Because these clones represented the
most differentially expressed gene that was not an enzyme, a house-
keeping gene or a ribosomal gene and the only representative of a
signalling transduction pathway, we focused on CaM as our primary
candidate associated with the elongated cactus finch beak morphology.
CaM is a Ca2þ-binding protein that can bind to and regulate
many different protein targets and is a key component of a Ca2þ-
dependent signal transduction pathway9. It has not been previously
characterized in either craniofacial or skeletal development.
Our microarray data indicate a possible correlation between
elevated levels of CaM and the more elongated beak morphology
of the cactus finches. To validate this suggestion we performed a
comparative in situ hybridization on embryos of Darwin’s finches
(Fig. 2). We found that CaM was indeed expressed at detectably
higher levels in the distal–ventral mesenchyme of the frontonasal
processes in the cactus finches (n ¼ 3) and large cactus finches
(n ¼ 3) than in similar-sized processes of the ground finches
(Fig. 2). Thus, higher expression of CaM is indeed associated with
the elongated beak morphology of the cactus finches.
To address whether differential levels of CaM-dependent signalling
might be important in the development of distinct beak mor-
phologies in different species of Darwin’s finches, we wished to
elevate the level of CaM-dependent signalling in the developing
chick beak prominence. To accomplish this we used a constitutively
activated form of a downstream effector of CaM, CaM kinase kinase
(CaMKII; M. J. Taschner, S. Schnaiter and C.H., unpublished
observations). In one of the major cellular responses to increased
Ca2þ levels, CaM activates CaMKII. CaMKII, in turn, activates
downstream kinases, enabling them to phosphorylate and hence Figure 2 | Comparative analysis of CaM expression in finches.
activate various targets, such as the transcription factors cAMP- a, b, Geospiza group species displaying distinct beak morphologies form a
response-element-binding protein (CREB), serum response factor monophyletic group. c, The differences in beak morphology are skeletal.
and CREB-binding protein. d, CaM is expressed in a strong distal–ventral domain in the mesenchyme of
Both CaM and CamKII are expressed at low levels in the mesen- the upper beak prominence of the large cactus finch, G. conirostris,
somewhat lower levels in cactus finch, G. scandens, and at significantly lower
chyme of the chicken upper beak process (data not shown). To
levels in the large ground finch and medium ground finch, G. magnirostris
stimulate higher CaM-dependent CAMKII signalling, we used an and G. fortis, respectively. Very low levels of CaM were detected in the
avian retroviral vector carrying the constitutively active form of mesenchyme of G. difficilis, G. fuliginosa and the basal warbler finch
CaMKII (RCAS::CA-CaMKII) to infect the distal mesenchyme of Certhidea olivacea. CaM expression domains are indicated with short arrows
early stage-24 chick frontonasal processes. Although virus injection in d. Scale bar, 1 mm in b. The molecular tree is from ref. 23; images of skulls
was targeted to the distal upper beak prominence with relative ease, are from ref. 6, with permission from the author.
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cell death, as revealed by the TUNEL (TdT-mediated dUTP nick end Consistent with our observations, parallel studies in the laboratory of
labelling) assay, was essentially unchanged relative to the wild-type one of us (C.H.) have also indicated that CaMKII signalling leads to the
condition (not shown). Staining for anti-PCNA (proliferating cell elongation of limb skeletal elements in a non-cell-autonomous manner
nuclear antigen) antibody and labelling with bromodeoxyuridine (M. J. Taschner, S. Schnaiter and C.H, unpublished observations), in a
indicate that proliferation might have been upregulated in the distal similar manner to our observation in the beak.
half of the pre-nasal cartilage element of infected beaks (not shown). There are few examples of the identification and characterization
The expression of cell-cycle regulators cyclin D1 and the activator of developmental pathways responsible for evolutionary morpho-
protein 1 (AP1) family members c-Jun and c-Fos was upregulated in logical change10–15. Previous work on Darwin’s finches has shown that
the distal parts of pre-nasal cartilage and its perichondrium (arrow in selection on adult beak variation results in changes in growth in the
Supplementary Fig. S4J; not shown). As these skeletal tissues were next generation, as a result of genetic correlations between adult and
not infected with the retrovirus, this effect was indirect. The same juvenile expression of the same morphological characters16,17. Here,
genes were downregulated in the mesenchyme, which forms the using a microarray approach, we found evidence that a higher level of
premaxillary dermal bone (arrowheads in Supplementary Fig. S4I). CaM-dependent signalling is both biologically relevant and func-
tionally important for the morphogenesis of the longer beaks of
cactus finches used in a specialized manner to probe cactus flowers
and fruit for nectar.
The avian beak varies between species, and indeed between
individuals of the same species, along at least three different axes:
length, depth and width. In Darwin’s finches it can be seen that there
is both linkage and independence in the variation along these
different axes. For example, in the two species of cactus finches
there is a much weaker genetic correlation between adult beak length
and depth or width than between depth and width themselves. In
contrast, in G. fortis all three correlations are strong18,19. The data
reported here provide at least a partial explanation for this difference
between the finch species. Embryos of the cactus finch species, in
which beak length shows independence from width and length,
strongly express CaM (a factor specifically affecting beak length)
during beak morphogenesis, whereas G. fortis has minimal CaM
expression (Fig. 4).
In considering the independence of beak length from width and
depth, it is particularly intriguing that, here and in our previous
study, we have shown that two different factors (BMP4 and activated
CaMKII), expressed in a similar domain in the beak prominence, result
in changes in growth along different dimensions of the developing beak
(width/depth and length, respectively) without negatively affecting

Figure 3 | Functional analysis of CaM-dependent pathway in beak

development. a, b, Whole head views of embryonic day 10 (E10; HH stage
36) wild-type (a) and RCAS::CA-CaMKII-infected (b) chicken embryos. The Figure 4 | BMP- and CaM-dependent signalling regulates growth along
length of the beak is shown with a red line; the depth of the beak at the base different axes, facilitating the evolution of distinct beak morphologies in
and the depth of the beak at the tip are shown with blue and green lines, Darwin’s finches. a, Developing avian beak is a three-dimensional structure
respectively. c–j, We used RSCH (c, d), Coll IX (e, f), Runx2 (g, h) and that can change along any of the growth axes. b, A beak of the sharp-beaked
PTHrP-Rec (i, j) probes to reveal RCAS infection (RSCH), chondrocytes finch reflects a basal morphology for Geozpiza. The model for BMP4 and
(Coll IX) and early osteoblasts (Runx2). c, e, g, i, Wild type; d, f, h, j, CaM involvement explains development of both elongated and deep/wide
RCAS::CA-CaMKII infected. The star indicates an egg-tooth. Scale bar, beaks of the more derived species. Abbreviations: C, caudal; D, dorsal;
2 mm in a. R, rostral; V, ventral.
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the other axes, explaining the observed independence of these traits. Darwin finch microarray production and usage. A DNA microarray (21,168
Moreover, analysis of BMP4 and CaM expression in chick embryos spots) was printed from a non-normalized poly(A)-primed cDNA library made
infected with RCAS::CA-CaMKII and RCAS::BMP4, respectively, from RNA isolated from multiple (12 individuals) frontonasal processes of
showed that these two molecules do not regulate each other during stage-26 and stage-29 embryos of the medium ground finch, G. fortis. We used
Cy5-labelled probes made from individual frontonasal processes of the four
beak development (data not shown). These two molecules vary derived species of Geospiza for direct comparisons against a common Cy3-labelled
independently of each other in some species as demonstrated by reference sample made from pooled RNA of several (nine individuals) embryos
their reciprocal expression levels in the two medium-sized species of more basal G. difficilis (Fig. 1a, and Supplementary Fig. S1). In most cases we
with thick and with long beak morphologies, whereas they are compared four unrelated individuals from each of the derived cactus finch and
expressed together in the developing beak of the large cactus finch ground finch species (G. scandens, G. conirostris, G. magnirostris and G. fortis)
in good correlation with its robust yet elongated beak morphology against the pooled common reference (Figs 1a and 4, and Supplementary
(Figs 1, 3 and 4)6,8. Theoretically, such modular developmental Fig. S1). RNA from each individual finch beak prominence was independently
regulation need not have been necessary to construct a beak. A amplified and labelled in triplicate with a control dye swap. We used the two
common set of growth-promoting pathways could, in principle, have highest-quality sets of microarray data from each triplicate for clustering.
Raw.gpr files were generated with GenePix 3.0 (Molecular Devices). Normal-
led to outgrowth of the frontonasal process along all three dimen-
ization and statistical analysis of the GPR data files were performed in MatLab
sions. Indeed, a single factor, BMP4, seems to stimulate growth of the (The Math Works). Data were normalized with the Lowess algorithm22. Only
beak along two dimensions, promoting deeper and wider beaks, spots with a signal intensity exceeding the median background þ2 s.d. were
explaining the linkage in their variation. Using a common signal for considered, which left 7,369 spots. The data was log2 transformed.
outgrowth in different axes still permits evolution of form, for Microarray cluster analysis. Clustering analysis and visualization were com-
example through the action of localized antagonists. However, the puted in MatLab. Agglomerative hierarchical clustering was performed by using
developmental programme using a distinct pathway for the long axis the euclidean distance measure: the average linkage and Ward heuristics were
of the beak enhances the ‘evolvability’ along this dimension, by used to connect the gene clusters. For k-means clustering, the k-means algorithm
allowing independent variation along the different axes. Thus, the partitioned the genes into k discrete clusters on the basis of their expression. The
dissociable regulation of growth in different beak axes is an example number k (50) was preselected. The resultant tree illustrates that duplicates of the
amplification/labelling experiments from the same individuals clustered
of the organization of the biology of the organism facilitating the
together (Fig. 1b).
generation of variation on which natural selection can act20.
Measurements of signal ratios and intensities for different transcripts were
It will be of interest to sample other species of Darwin’s finches clustered to identify genes that were upregulated or downregulated in all
with elongated beaks such as the woodpecker finch Cactospiza individuals of a particular species compared with the basal G. difficilis reference.
pallida, as well as other avian species with long beaks, to discover Species-specific clusters were further cross-compared to reveal transcripts that
how generally the CaM-dependent pathway is involved in elongation were consistently upregulated in frontonasal processes of all individuals of the
of the beak skeleton. Upregulation of this pathway at stage 26 is not cactus-finch beak morphology and that remained unchanged or were down-
simply related to low beak depth and width of beaks, because CaM regulated in beak primordia of the ground finches. Conveniently, the cactus
expression was very low in the pointed but slender beaks of the finch has an upper beak depth similar in size to that of the medium ground finch,
warbler finch embryos (Fig. 3). Upregulation of CaMKII activity in whereas the large cactus finch’s beak is more similar in size to that of the large
the distal mesenchyme of the upper beak prominence in chicken ground finch while differing strongly in shape. This allowed us to separate
transcripts exhibiting the size-specific regulation from those with shape-specific
embryos produced a roughly 10% increase in beak length, primarily
regulation.
due to elongation of the pre-nasal cartilage rod, which is in concert Data analysis: hierarchical clustering. Gene expression patterns of nine
with the differences in relative beak length between the cactus finches experimental samples representing five G. scandens and four G. conirostris
and the more basal sharp-beaked finch and other ground finches6. samples were analysed by hierarchical clustering with Ward linkage. The samples
This might indicate that additional pathways and mechanisms have a were divided into two groups on the basis of differences in gene expression.
function in generating the even longer beaks found in other avian Figure 2 shows a cluster dendrogram of the two groups, G. scandens (blue) and
lineages, such as some shorebirds, hummingbirds and Hawaiian G. conirostris (red). Within each branch the siblings were found to cluster tightly
honeycreepers, all highly adaptive. together on a sub-branch.

Received 15 February; accepted 25 April 2006.

METHODS
Collection and treatment of embryonic material from Darwin’s finches. 1. Darwin, C. R. Journal of Researches into the geology and Natural History of the
Under an agreement with the Galápagos National Park, we received quotas for various countries visited during the voyage of H.M.S. Beagle, under the command of
Captain FitzRoy, R.N. 2nd edn (John Murray, London, 1845).
collecting embryos of G. magnirostris, G. fortis, G. fuliginosa and G. scandens
2. Lack, D. Darwin’s Finches (Cambridge Univ. Press, Cambridge, 1947).
from Santa Cruz Island, and G. conirostris, G. difficilis and C. olivacea from 3. Grant, P. R. The Ecology and Evolution of Darwin’s Finches (Princeton Univ. Press,
Genovesa Island. To avoid causing nest defection, only the third egg was collected Princeton, New Jersey, 1999).
shortly after it was laid; it was then incubated at 100 8F (37.8 8C). Embryonic 4. Futuyma, D. J. Evolutionary Biology 3rd edn (Sinauer Associates, Sunderland,
material was fixed in 4% paraformaldehyde in PBS for 2 h at ambient tempera- Massachusetts, 1998).
ture and stored in RNAlater reagent (Ambion) at about 5 8C for two to five 5. Freeman, S. & Herron, J. C. Evolutionary Analysis 3rd edn (Prentice Hall,
weeks. Chick antisense riboprobes (CaM, Coll IX, Runx2 and PTHrP-Rec) were Englewood Cliffs, New Jersey, 2003).
prepared and used on Darwin’s finch embryos as described previously8. We 6. Bowman, R. I. Morphological differentiation and adaptation in the Galapagos
analysed 26 heads of Darwin’s finches: large ground finch (n ¼ 3), medium finches. Univ. Calif. Publ. Zool. 58, 1–-302 (1961).
7. Grant, P. R. & Grant, B. R. Unpredictable evolution in a 30-year study of
ground finch (n ¼ 5), small ground finch (n ¼ 4), large cactus finch (n ¼ 4), Darwin’s finches. Science 296, 707–-711 (2002).
cactus finch (n ¼ 3), sharp-beaked finch (n ¼ 3) and warbler finch (n ¼ 4). 8. Abzhanov, A., Protas, M., Grant, R. B., Grant, P. R. & Tabin, C. J. Bmp4 and
Chicken embryo manipulations and statistical analysis. Fertilized eggs (Spafas) morphological variation of beaks in Darwin’s finches. Science 305, 1462–-1465
were incubated at 100 8F. Embryos were staged as described in ref. 21. The (2004).
RCAS::CA-CamKII construct will be described elsewhere (M. J. Taschner, 9. O’Day, D. H. CaMBOT: profiling and characterizing calmodulin-binding
S. Schnaiter and C.H., unpublished observations). To infect embryos for studies proteins. Cell. Signal. 15, 347–-354 (2003).
in vivo we injected the distal part of the frontonasal process of HH stage-24 10. Burke, A. C., Nelson, C. E., Morgan, B. A. & Tabin, C. Hox genes and the
chick embryos. The infected embryos were collected at embryonic day 10, fixed evolution of vertebrate axial morphology. Development 121, 333–-346 (1996).
11. Averof, M. & Patel, N. H. Crustacean appendage evolution associated with
overnight in 4% paraformaldehyde in PBS, and frozen in OCT (optimal cutting
changes in Hox gene expression. Nature 388, 682–-686 (1997).
temperature) medium for sectioning. Extent of viral infection was assayed by 12. Abzhanov, A. & Kaufman, T. C. Novel regulation of the homeotic gene Scr
hybridization in situ with a virus-specific RSCH probe. The embryonic heads were associated with a crustacean leg-to-maxilliped appendage transformation.
photographed and measured in NIH Image 1.62. These arbitrary measurements Development 126, 1121–-1128 (1999).
were used for the analysis of variance function (ANOVA toolbox; t-test) in 13. Abzhanov, A. & Kaufman, T. C. Crustacean (malacostracan) Hox genes and
Microsoft Excel X to calculate standard deviations and P values for the data. the evolution of the arthropod trunk. Development 127, 2239–-2249 (2000).
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14. Shapiro, M. D. et al. Genetic and developmental basis of evolutionary pelvic Acknowledgements We thank all field assistants and participants of the field
reduction in threespine sticklebacks. Nature 428, 717–-723 (2004). collecting trips—M. Protas, J. Chavez, G. Castaneda, O. Perez, F. Brown,
15. Kimmel, C. B. et al. Evolution and development of facial bone morphology A. Aitkhozhina, M. Gavilanes, M. Paez, K. Petren, J. Podos and S. Kleindorfer—
in threespine sticklebacks. Proc. Natl. Acad. Sci. USA 102, 5791–-5796 for their advice with species identification and other advice; Charles Darwin
(2005). Research Station on Santa Cruz Island and The Galápagos National Park for
16. Grant, P. R. Inheritance of size and shape in a population of Darwin’s finches. permits and logistical support; and M. Kirschner for discussions that led to the
Proc. R. Soc. Lond. B 212, 403–-432 (1983). inception of this project. A.A. was supported by the Cancer Research Fund of a
17. Price, T. D. & Grant, P. R. The evolution of ontogeny in Darwin’s Finches: a Damon Runyon–Walter Winchell Foundation Fellowship. This project was
quantitative genetics approach. Am. Nat. 125, 169–-188 (1985). funded by a program project grant from the NIH to C.J.T.
18. Grant, B. R. & Grant, P. R. Evolutionary Dynamics of a Natural Population. The
Large Cactus Finch of the Galápagos (Univ. Chicago Press, Chicago, Illinois, Author Contributions A.A. performed embryonic material collection, microarray
1989). probe preparation, microarray hybridizations and scanning, sectioning of
19. Grant, P. R. & Grant, B. R. Phenotypic and genetic effects of hybridization in material, in situ hybridizations and CaMKII functional analysis in chicken
Darwin’s finches. Evolution Int. J. Org. Evolution 48, 297–-316 (1994). embryos. W.P.K. conducted all relevant bioinformatics analyses. C.H.
20. Kirschner, M. W. & Gerhart, J. C. The Plausibility of Life: Resolving Darwin’s constructed the RCAS virus carrying the constitutively active version of CaMKII.
Dilemma (Yale Univ. Press, New Haven, Connecticut, 2005). B.R.G. and P.R.G. provided logistics and secured permits for the fieldwork on the
21. Hamburger, V. & Hamilton, H. L. A series of normal stages in the development Galápagos Islands. C.J.T. conceived and supervised the project. A.A., B.R.G.,
of the chick embryo. J. Morphol. 88, 49–-92 (1951). P.R.G. and C.J.T. co-wrote the manuscript. All authors discussed the results and
22. Dudoit, S., Yang, H., Callow, M. J. & Speed, T. P. Statistical methods for commented on the manuscript.
identifying genes with differential expression in replicated cDNA experiments.
Statist. Sin. 12, 111–-139 (2002). Author Information The sequence of Darwin’s finch CaM has been deposited at
23. Petren, K., Grant, B. R. & Grant, P. R. A phylogeny of Darwin’s finches based GenBank under accession number DQ386479, and the microarray data have
on microsatellite DNA length variation. Proc. R. Soc. Lond. B 266, 321–-329 been filed with ArrayExpress under the accession number E-MEXP-702.
(1999). Reprints and permission information is available at npg.nature.com/
reprintsandpermissions. The authors declare no competing financial interests.
Supplementary Information is linked to the online version of the paper at Correspondents and requests for materials should be addressed to C.J.T.
www.nature.com/nature. ([email protected]).

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