Rapid hybrid speciation in Darwin’s finches
We might think of all hybrids as unnatural or, at the very least, sterile like mules. Hybrids pose problems for conservation; should we prioritize "pure" species over the hybridized? Why do we classify one organism as a new species but another as only a hybrid? As we discover that hybridization occurs more often than we once thought, we are learning that hybridization can be important to population health as a source of genetic variation. Here, we investigate a hybridization event that began in 1981 on the island of Daphne Major in the Galápagos. It all starts with a migrant from another island nicknamed Big Bird.
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Homoploid hybrid speciation in animals has been inferred frequently from patterns of variation, but few examples have withstood critical scrutiny. Here we report a directly documented example from its origin to reproductive isolation. An immigrant Darwin’s finch to Daphne Major in the Galápagos archipelago initiated a new genetic lineage by breeding with a resident finch (Geospiza fortis). Genome sequencing of the immigrant identified it as a G. conirostris male that originated on Española >100km from Daphne. From the second generation onwards the lineage bred endogamously, and despite intense inbreeding, was ecologically successful and showed transgressive segregation of bill morphology. This example shows that reproductive isolation, which typically develops over hundreds of generations, can be established in only three.
Interbreeding of two species may result in the formation of a new species, reproductively isolated from the parental species (1). Hybrid speciation without chromosomal doubling, that is homoploid hybrid speciation, is rare (1–4). Possible examples have been reported in plants (4), butterflies (5), flies (6), fish (7), mammals (8) and birds (9). However, only one of these involving Heliconius butterflies (5), and three additional examples involving Helianthus sunflowers (3, 10), meet stringent criteria that have been proposed for recognizing that hybridization was the cause of speciation (2). Here we report the results of a combined ecological and genomic study of Darwin’s finches that documents hybrid speciation in the wild from its inception to the development of reproductive isolation.
An immature male finch immigrated to the small Galápagos Island of Daphne Major (0.34 km2) in 1981 (11–13). It resembled the medium ground finch, Geospiza fortis, but was 70% larger and sang a unique song. Assignment tests with microsatellite markers from finches on neighboring islands indicated it was likely a G. fortis x G. scandens hybrid originating on the adjacent large island of Santa Cruz 8km from Daphne (11). We followed the survival and breeding of this individual and its descendants for six generations over the next 31 years.
The immigrant (generation 0) bred with a G. fortis female and one of its F1offspring bred with another G. fortis female, but all other matings occurred within this lineage, endogamously, and therefore from generation 2 onwards the lineage behaved as an independent species relative to other birds on the island (Fig. 1). Generations 4-6 were derived from a single brother-sister mating in generation 3. Despite close inbreeding, members of the lineage experienced high fitness as judged by their reproductive output and high survival (12). At maximum (2010) eight breeding pairs and 36 individuals were present on the island, and on our most recent visit (2012) there were eight breeding pairs and 23 individuals of generations 3 to 6. From observations and experiments with ground finches (12, 13), bill morphology is likely to be a key factor in the success of these birds. The ability of finches to efficiently exploit the large woody fruits of Tribulus cistoides in dry seasons, and particularly during droughts and limited food supply, is a function of bill size, especially bill depth (12). Also, finch species imprint on features of their parents early in life, and later when choosing a mate they discriminate between members of their own and other species on the basis of bill size and shape, as well as body size and song (13).
We combined morphological measurements and whole genome sequencing of the great majority of individuals in the new lineage to establish the genetic basis of the founder population and characteristics associated with its success. We (a) assigned the founder male to species and source population, (b) confirmed pedigree assignments from observations and sequence data (14), (c) quantified patterns of gene transmission between generations, (d) assessed genetic diversity, and (e) searched for genetic clues to the success of the lineage. Since members of the new lineage are conspicuously large, we refer to it as the Big Bird lineage (12).
A phylogenetic tree analysis showed that the founder male (5110) was not a G. fortis x G. scandens hybrid as previously hypothesized (12), but a G. conirostris (Fig. 2A). This species (large cactus finch) occurs on Española and its satellite Gardner (Fig. 2B) and nowhere else in the Galápagos archipelago; a population on Genovesa, formerly classified as G. conirostris, was recently reclassified as G. propinqua (15). Immigration from Española is remarkable and unexpected because it is located more than 100 km from Daphne Major, and a large island (Santa Cruz) lies between them (Fig. 2B). Rare long-distance movements of finches in the archipelago have been detected before, but until recently it was assumed these birds were vagrants that did not stay to breed (16–18).
The founder had an inbreeding coefficient (F) of 0.19 and appeared to be a typical member of the source population of G. conirostris from Española (F = -0.04-0.31) in terms of average genome-wide homozygosity, and ADMIXTURE (19) analysis classified it as a normal G. conirostris (Fig. 2C). The inbreeding coefficient was negative in the F1 generation (Fig. 2D) as a result of the interspecies hybridization (12, 13). A gradual increase in homozygosity was then observed over the next five generations (Fig. 2D), as expected from the small number of breeding pairs (1-8) causing genetic drift. Genome-wide average nucleotide diversity (π) showed a similar pattern, with a decline from 0.17 in generation 1 to ~0.13 in generations 4-6; values for G. fortis and G. conirostris were 0.15 and 0.16, respectively (fig. S1). Furthermore, the extensive linkage disequilibrium across the genome is consistent with a recent hybridization event (fig. S2). The Big Bird lineage also exhibited low quantitative variation. The population, all generations combined, varied less in bill length as measured by the coefficient of variation (3.82 ± 0.42, mean ± sem, n = 42) than G. fortis (7.55 ± 0.69, n = 60, P < 0.005) and G. conirostris(6.35 ± 0.56, n = 64, P <0.01), and varied less in bill depth than G. conirostris(5.02 ± 0.55 vs. 7.71 ± 0.68, P < 0.05). The low values probably represent low additive genetic variation because the traits are highly heritable in Geospizaspecies (13, 20).
The ecological success and reproductive isolation of the Big Bird lineage were most likely due to large bill and body size, and a distinctive song (12). We undertook a more detailed morphological analysis of the new lineage, together with both of the parental species G. conirostris and G. fortis [(14), table S1]. In body size, the members of the Big Bird lineage are intermediate on average between the means of the parental species (Fig. 3A), but closer to the G. fortis mean (table S2) as expected from their predominantly G. fortisgenetic composition and polygenic inheritance. However, in contrast they are more similar to G. conirostris in bill size (Fig. 3A, fig. S3, and tables S1 and S2), despite the minority representation of G. conirostris genes in generation 3 and onwards. This represents a substantial allometric shift in the Big Bird lineage, possibly caused by natural selection. Selection is plausible because shifts in the elevations of static allometries have been produced relatively easily in a few generations of artificial selection in laboratory populations of animals (21). The pattern of change also has the characteristics of transgressive segregation (10, 22). This is the production of progeny with extreme phenotypes beyond the range of parents that is likely caused by epistasis, which has been detected in other hybridizing finch species on Daphne (23), and/or by combining complementary alleles at different loci from different populations in F2 and further generations (22, 24, 25).
In order to investigate the genetic basis for the relatively large bills of the Big Birds we examined the genotypes at HMGA2 and ALX1, two closely linked loci (7 Mb apart) previously shown to be associated with variation in bill morphology in Darwin’s finches (15, 26). At the HMGA2 locus, the allele frequency of the L allele associated with large bill size was 60.8% in generations 4-6 (fig. S4 and table S5). From generation 3 onwards all Big Birds were homozygous for ALX1 B alleles associated with blunt bills. A closer examination revealed that two variants of the B allele, denoted B1 and B2, were segregating among the Big Birds (fig. S5 and table S5); the two alleles differ by 9 nucleotide substitutions within the 240 kb region showing a strong association with bill shape (14). The B1 allele originated from the founder male that was genotyped as P/B1, where P refers to pointed, whereas the B2allele originated from G. fortis (table S5). Interestingly, B2/B2 homozygotes had significantly shorter bills than the other two genotypes: F2,32 = 10.5, P = 0.0003; Tukey post hoc tests, B2/B2 < B1/B1 (P = 0.005) and B2/B2 < B1/B2 (P= 0.0002). Although these associations should be confirmed with larger sample sizes, they are consistent with the hypothesis that the ALX1 locus in Darwin’s finches involves an allelic series with different effects on bill morphology (15).
ALX1 and HMGA2 have large effects on bill dimensions, which are polygenic traits that are affected by other gene variants and these may have changed in frequency due to a combination of natural selection and random drift. A trend of increasing bill size across generations (F1,42 = 6.0, P = 0.018, adj r2 = 0.10) is more indicative of selection than of drift. In 2009, the only year with sufficient samples for an analysis of mortality, 19 adult survivors to the following year had a larger mean bill size than five adults that died (F1,22 = 8.30, P = 0.009). The most important component of bill size is bill depth, and an increase in this dimension (Fig. 3B) is noteworthy for two reasons. First, the increase was independent of body size (Fig. 3C). The genetic correlation between bill size and body size that potentially constrains independent evolution of bill size is not known. However in G. fortis the genetic correlation between bill depth and body mass is strongly positive (0.67 ± 0.10 sem) (23). Second, bill length did not change in the population (fig. S6), hence bills became not only larger but progressively blunter on average across generations (fig. S6). A possible scenario is that transgressive segregation produced genotype combinations that have been favored by natural selection causing the shift in beak morphology. The net result was morphologically based ecological segregation from the three sympatric competitor species G. fortis, G. scandens and G. magnirostris (Fig. 3D).
The final stage in speciation is the development of reproductive isolation from the parent population. In Darwin’s finches a premating barrier to interbreeding is established by a difference in song and morphology (12, 13). The test of reproductive isolation requires sympatry with the parental population(s) or a surrogate experiment, for example with finch models and/or playback of tape-recorded song (27). The new population on Daphne is reproductively isolated from one of the parental populations, G. fortis, but whether it is reproductively isolated from the other, G. conirostris on Española, is unknown because experiments have not been done there. Nevertheless, it is likely that the founder population has already become reproductively isolated from G. conirostris as bill size has changed in relation to body size (Fig. 3A). Together these traits are used as cues in the choice of mates arising from cultural, non-genetic, imprinting (12, 13). Of particular relevance, experiments on Daphne with G. scandens showed that altering bill size in relation to body size of finch models significantly reduced responses from males (28). Additionally, males of the founder population sing a different song from G. conirostris on Española and Gardner, probably as a result of imperfect copying of a Daphne finch by the founder after it had first learned its father’s song on Española (or Gardner) (13). Song and morphology are cues used in mate choice, and typically result in the avoidance of interspecific mating.
“...to understand the mechanism of speciation, the focus should be on cases of incipient speciation rather than on completed ones” (29). We have taken advantage of witnessing a rare colonization event to directly document the fate of a population founded by a single immigrant and his G. fortis mate. The newly founded population of Darwin’s finches is an incipient hybrid species, reproductively isolated and ecologically segregated from coexisting finch species (Fig. 3D). The key features of success of the new lineage are reproductive isolation based on learned song and morphology, transgressive segregation producing novel phenotypes, and the availability of underexploited food resources. Homoploid hybrid speciation is believed to be a generally slow process extending over hundreds of generations (29), but as the present example shows it can be established in only three generations. Thus in small islands or island-like settings it may be easier to achieve than is currently believed (1, 30–32).
Homoploid hybrid speciation of the Big Bird lineage exemplifies the potential evolutionary importance of rare and chance events. Expansion of the population from two individuals to three dozen was conditioned on the founder being a male with a distinctive song (14), and facilitated by the chance occurrence of strong selection against large bill size in a competitor species, G. fortis, in 2004-05 (12, 26). The selection event, in turn, was mediated by G. magnirostris, a species that established a breeding population in 1983. Joint occurrence of rare and extreme events such as these may be especially potent in ecology and evolution (33, 34).
Materials and Methods
Figs. S1 to S7
Tables S1 to S5
REFERENCES AND NOTES
1. R. J. Abbott, N. H. Barton, J. M. Good, Mol. Ecol. 25, 2325-2332 (2016).
2. M. Schumer, G. G. Rosenthal, P. Andolfatto, Evolution 68, 1553-1560 (2014).
3. B. L. Gross, L. H. Rieseberg, J. Hered. 96, 241-252 (2005).
4. S. B. Yakimowski, L. H. Rieseberg, Am. J. Bot. 101, 1247-1258 (2014).
5. J. Mavarez, C. A. Salazar, E. Bermingham, C. Salcedo, C. D. Jiggins, M. Linares, Nature 441, 868-871 (2006).
6. D. Schwarz, B. M. Matta, N. L. Shakir-Botteri, B. A. McPheron, Nature 436, 546-549 (2005).
7. J. H. Kang, M. Schartl, R. B. Walter, A. Meyer, BMC Evol. Biol. 13, 25 (2013).
8. P. A. Larsen, M. R. Marchan-Rivadeneira, R. J. Baker, Proc. Natl. Acad. Sci. U.S.A. 107, 11447-11452 (2010).
9. J. S. Hermansen, S. A. Saether, T. O. Elgvin, T. Borge, E. Hjelle, G. -P. Saetre, Mol. Ecol. 20, 3812-3822 (2011).
10. L. H. Rieseberg, C. Van Fossen, A. M. Desrochers, Nature 375, 313-316 (1995).
11. P. R. Grant, B. R. Grant, Proc. Natl. Acad. Sci. U.S.A. 106, 20131-20148 (2009).
12. B. R. Grant, P. R. Grant, 40 Years of Evolution: Darwin's Finches on Daphne Major Island (Princeton Univ. Press, 2014).
13. P. R. Grant, B. R. Grant, How and Why Species Multiply (Princeton Univ. Press, 2008).
14. See supplementary materials.
15. S. Lamichhaney, J. Berglund, M. S. Almen, K. Maqbool, M. Grabherr, A. Martinez-Barrio, M. Promerova, C. -J. Rubin, C. Wang, N. Zamani, B. R. Grant, P. R. Grant, M. T. Webster, L. Andersson, Nature 518, 371-375 (2015).
16. P. R. Grant, B. R. Grant, Philos. Trans. R. Soc. Lond. B Biol Sci. 365, 1065-1076 (2010).
17. D. L. Lack, Darwin's Finches (Cambridge Univ. Press, 1947).
18. H. S. Swarth, Occas. Pap. Calif. Acad. Sci. 18, 1-299 (1931).
19. D. H. Alexander, J. Novembre, K. Lange, Genome Res. 19, 1655-1664 (2009).
20. L. F. Keller, P. R. Grant, B. R. Grant, K. Petren, Heredity 87, 325-336 (2001).
21. G. H. Bolstad, J. A. Cassara, E. Marquez, T. F. Hansen, K. van der Linde, D. Houle, C. Pelabon, Proc. Natl. Acad. Sci. U.S.A. 112, 13284-13289 (2015).
22. L. H. Rieseberg, M. A. Archer, R. K Wayne, Heredity 83, 363-372 (1999).
23. P. R. Grant, B. R. Grant, Evolution 48, 297-316 (1994).
24. K. J. Parsons, Y. H. Son, R. Craig Albertson, Evol. Biol. 38, 306-315 (2011).
25. R. Stelkens, O. Seehausen, Evolution 63, 884-897 (2009).
26. S. Lamichhaney, F. Han, J. Berglund, C. Wang, M. S. Almen, M. T. Webster, B. R. Grant, P. R. Grant, L. Andersson, Science 352, 470-474 (2016).
27. B. R. Grant, P. R. Grant, Biol. J. Linn. Soc. Lond. 76, 545-556 (2002).
28. L. M. Ratcliffe, P. R. Grant, Anim. Behav. 31, 1139-1153 (1983).
29. A. W. Nolte, D. Tautz, Trends Genet. 26, 54-58 (2010).
30. J. Mavarez, M. Linares, Mol. Ecol. 17, 4181-4185 (2008).
31. Marie Curie SPECIATION Network, Trends Ecol. Evol. 27, 27-39 (2012).
32. R. J. Abbott, M. J. Hegarty, S. J. Sixcock, A. C. Brennan, Taxon 59, 1375-1386 (2010).
33. R. Paine, M. Tegner, E. Johnson, Ecosystems (N. Y.) 1, 535-545 (1998).
34. P. R. Grant, B. R. Grant, R. B. Huey, M. T. J. Johnson, A. H. Knoll, J. Schmitt, Philos. Trans. R. Soc. Lond. B Biol. Sci. 372, 20160146 (2017).
35. B. Li, H. Li, P. Parker, J. Wang, GigaScience 10.5524/100040 (2012).
36. S. Purcell, B. Neale, K. Todd-Brown, L. Thomas, M. A. R. Ferreira, D. Bender, J. Maller, P. Sklar, P. I. W. de Bakker, M. J. Daly, P. C. Sham, AM. J. Hum. Genet. 81, 559-575 (2007).
37. J. Huisman, Mol. Ecol. Resour. 17, 1775-0998 (2017).
38. M. N. Price, P. S. Dehal, A. P. Arkin, PLOS ONE 5, e9490 (2010).
39. P. Danecek, A. Auton, G. Abecasis, C. A. Albers, E. Banks, M. A. DePristo, R. E. Handsaker, G. Lunter, G. T. Marth, S. T. Sherry, G. McVean, R. Durbin, Bioinformatics 27, 2156-2158 (2011).
40. P. R. Grant, Proc. R. Soc. London 212, 403-432 (1981).
NSF (USA) funded the collection of material under permits from the Galápagos and Costa Rica National Parks Services and the Charles Darwin Research Station, and in accordance with protocols of Princeton University’s Animal Welfare Committee. The project was supported by the Knut and Alice Wallenberg foundation and the Swedish Research Council. Sequencing was performed by the SNP&SEQ Technology Platform, supported by Uppsala University and Hospital, SciLifeLab and Swedish Research Council. Computer resources were supplied by UPPMAX. We would also like to thank two anonymous reviewers for valuable comments on the paper. The Illumina reads have been submitted to the short reads archive (http://www.ncbi.nlm.nih.gov/sra) with the accession number PRJNA392917. Raw tree files for constructing Fig. 2A and figs. S4, S5, and S7 have been submitted to the TreeBASE database with submission ID S21803 (http://purl.org/phylo/treebase/phylows/study/TB2:S21803?format=html). PRG and BRG collected the material. PRG, BRG and LA conceived the study. LA and MTW led the bioinformatic analysis of data. SL and FH performed the bioinformatic analysis. PRG, BRG, SL, and LA wrote the paper with input from the other authors. All authors approved the manuscript before submission.