ARTICLE doi:10.1038/nature14181 Evolution of Darwin’s finches and their beaks revealed by genome sequencing Sangeet Lamichhaney1*, Jonas Berglund1*, Markus Sa¨llman Alme´n1, Khurram Maqbool2, Manfred Grabherr1, Alvaro Martinez-Barrio1, Marta Promerova´1, Carl-Johan Rubin1, Chao Wang1, Neda Zamani1,3, B. Rosemary Grant4, Peter R. Grant4, Matthew T. Webster1 & Leif Andersson1,2,5 Darwin’s finches, inhabiting the Gala´pagos archipelago and Cocos Island, constitute an iconic model for studies of speci- ation and adaptive evolution. Here we report the results of whole-genome re-sequencing of 120 individuals representing all of the Darwin’s finch species and two close relatives. Phylogenetic analysis reveals important discrepancies with the phenotype-based taxonomy. We find extensive evidence for interspecific gene flow throughout the radiation. Hybrid- ization has given rise to species of mixed ancestry. A 240 kilobase haplotype encompassing the ALX1 gene that encodes a transcription factor affecting craniofacial development is strongly associated with beak shape diversity across Darwin’s finch species as well as within the medium ground finch (Geospiza fortis), a species that has undergone rapid evolution of beak shape in response to environmental changes. The ALX1 haplotype has contributed to diversification of beak shapes among the Darwin’s finches and, thereby, to an expanded utilization of food resources. Adaptive radiations are particularly informative for understanding the diversification throughout phylogeny, and report the discovery of a locus ecological and genetic basis of biodiversity1,2. Those causes are best iden- with a major effect on beak shape. tified in young radiations, as they represent the early stages of diver- sification when phenotypic transitions between species are small and Considerable nucleotide diversity interpretable and extinctions are likely to be minimal3. Darwin’s finches We generated approximately103 sequence coverage per individual bird are a classic example of such a young adaptive radiation3,4. They have using 2 3 100 base-pair (bp) paired-end reads (Extended Data Fig. 1). diversified in beak sizes and shapes, feeding habits and diets in adapt- Reads were aligned to the genome assembly of a female medium ground ing to different food resources4,5 (Extended Data Table 1). The radiation finch (G. fortis)14. We identified Z- and W-linked scaffolds on the basis is entirely intact, unlike most other radiations, none of the species hav- of significant differences in read depth between males (ZZ) and females ing become extinct as a result of human activities4. (ZW) (Supplementary Table 1) and generated a G. fortis mtDNA se- Fourteen of the currently recognized species evolved from a common quence through a combined bioinformatics and experimental approach. ancestor in the Gala´pagos archipelago (Fig. 1a) in the past 1.5 million Stringent variant calling revealed approximately 45 million variable years according to mitochondrial DNA (mtDNA) dating6; a fifteenth sites within or between populations. We found a considerable amount species inhabits Cocos Island. The radiation proceeded rapidly as a of genetic diversity within each population, in the range 0.3 3 1023 to result of strong isolation from the South American continent, genera- 2.2 3 1023 (Extended Data Table 2), similar to that reported in other tion of new islands by volcanic activity, climatic oscillations caused by bird populations15 including island populations of the zebra finch16. the El Nin˜o phenomenon, and sea level changes associated with glacial We used these estimates of diversity to estimate effective population sizes and interglacial cycles over the past million years that led to repeated of Darwin’s finch species within a range of 6,000–60,000 (Supplemen- alternations of island formation and coalescence7,8. tary Text). Extensive sharing of genetic variation among populations Traditional taxonomy of Darwin’s finches is based on morphology3, was evident, particularly among ground and tree finches, with almost no and has been largely supported by observations of breeding birds4,5 and fixed differences between species in each group (Extended Data Fig. 2). genetic analysis6,9. However, the branching order of several recently diverged taxa is unresolved6 and genetic analysis of phylogeny has been Genome-based phylogeny limited to mtDNA and a few microsatellite loci. Some candidate genes According to the classical taxonomy of Darwin’s finches, supported by for beak development are differentially expressed in species with dif- morphological and mitochondrial (cytochrome b) data, warbler finches ferent beak morphologies10–12, but the loci controlling genetic variation were the first to branch off, and ground and tree finches constitute the in beak diversity among Darwin’s finches remain to be discovered. most recent major split3,6,9. Our maximum-likelihood phylogenetic tree Here we report results from whole genome re-sequencing of 120 based on autosomal genome sequences is generally consistent with cur- individuals representing all Darwin’s finch species and two closely rent taxonomy, but shows several interesting deviations (Fig. 1b). First, related tanagers, Tiaris bicolor and Loxigilla noctis13. For some species Geospiza difficilis occurring on six different islands forms a polyphyl- we collected samples from multiple islands (Fig. 1a). We comprehen- etic group separated into three distinct groups: (1) populations occu- sively analyse patterns of intra- and interspecific genome diversity and pying the highlands of Pinta, Santiago and Fernandina, (2) populations phylogenetic relationships among species. We find widespread evid- occupying the low islands of Wolf and Darwin in the northwest3,6,9 and ence of interspecific gene flow that may have enhanced evolutionary (3) the population on Genovesa in the northeast. This is consistent with 1Department of Medical Biochemistry and Microbiology, Uppsala University, SE-751 23 Uppsala, Sweden. 2Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, SE- 75007 Uppsala, Sweden. 3Department of Plant Physiology, Umea˚ University, SE-901 87 Umea˚, Sweden. 4Department of Ecology and Evolutionary Biology, Princeton University, Princeton, New Jersey 08544, USA. 5Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, Texas 77843-4458, USA. *These authors contributed equally to this work. 19 FEBRUARY 2015 | VOL 518 | NATURE | 371 ©2015 Macmillan Publishers Limited. All rights reserved RESEARCH ARTICLE C. olivacea_S Warbler finches a b Vegetarian finch * C. fusca_E Cocos finch Darwin Sharp-beaked ground finches G. difficilis_D * Tree finches Cocos All other ground finches Wolf P. inornata_C C. fusca_L Outgroups G. difficilis_W * P. crassirostris_Z 1º P. inornata_C Galápagos G. difficilis_P * C. psittacula_P South America * G. difficilis_F * G. difficilis_S Pinta G. conirostris_G * G. magnirostrs_G G. difficilis_P G. difficilis_G Genovesa C. pauper_F G. fuliginosa_S * * C. parvulus_Z G. difficilis_S Marchena * C. psittacula_P C. olivacea_S C. heliobates_I G. fortis_M * 0º * C. pallidus_Z Santiago G. scandens_M * G. difficilis_D G. fuliginosa_Z * G. difficilis_F C. parvulus_Z G. difficilis_W C. pallidus_Z G. fuliginosa_Z Fernandina Daphne P. crassirostris_Z * * G. fuliginosa_S * G. fortis_M Santa * G. difficilis_G Cruz C. fusca_L G. scandens_M Isabela * * C. heliobates_I G. conirostris_G 1º San Cristóbal * C. pauper_F G. magnirostris_G C. fusca_E * G. conirostris_E G. conirostris_E 0 100 km Floreana 92º 91º 90º Española * T. bicolor_B L. noctis_B Figure 1 | Sample locations and phylogeny of Darwin’s finches. local support on the basis of the Shimodaira–Hasegawa test are marked by a, Geographical origin of samples; the letter after the species name is the asterisks. The colour code for groups of species applies to both panels. Taxa that abbreviation used for geographical origin. The map is modified from ref. 30. showed deviations from classical taxonomy are underscored. b, Maximum-likelihood trees based on all autosomal sites; all nodes having full an earlier version of the taxonomy, in which these three groups were clas- sified as distinct species on the basis of morphological differences17,18. Second, Geospiza conirostris on Espan˜ola showed the highest gen- a C.olivacea_S b etic similarity to another species, Geospiza magnirostris, whereas G. 1 G. magnirostris_G 827 ± 157 C. fusca_E conirostris on Genovesa clustered with Geospiza scandens (Fig.1b).Here, 291 2 G. difficilis_W phenotypic similarity parallels genetic similarity; G. conirostris on C. fusca_L ± Genovesa have a pointed beak similar to G. scandens, whereas those 901 123 3 G. difficilis_P on Espan˜ola have a blunt beak more similar to the beaks of G. magnir- P. crassirostris_Z ostris (Extended Data Fig. 3). P. inornata_C 4 T. bicolor_B 546 ± 74 D = 0.098 231 ± 32 G. difficilis_F 39% A network constructed from autosomal genome sequences indicates G. difficilis_S P = 5 X 10–113 418,099 ± 231 34% conflicting signals in the internal branches of ground and tree finches 529 81 G. difficilis_P 361,033 that may reflect incomplete lineage sorting and/or gene flow (Extended C. pauper_F 27% 205 ± 50 174 C. psittacula_P Data Fig. 3). The exact branching order of the most recently evolved 469 ± 101 C. parvulus_Z 296,072 ± C. pallidus_Z 204 83 171 1 ground and tree finches should be interpreted with caution as it may C. heliobates_I B B A G. difficilis_D 2 change with additional sampling. Since our data