Mimicry Genetics

The diversity of Heliconius butterflies from Costa Rica.

Neotropical butterflies in the genus Heliconius are unpalatable, aposematic, and they have undergone a recent adaptive radiation in wing color patterns as a consequence of natural selection for Müllerian mimicry. We are working to identify the molecular basis of the genes that control wing pattern diversity in Heliconius and characterize the evolutionary history of divergence and convergence across the clade. This work relies on combination of high-resolution genetic mapping, fine-scale genome-wide association mapping, comparative analyses of gene expression, and genome editing. Much of this work has been done in concert with an international network of collaborative researchers and has resulted in rich genomic resources for Heliconius butterflies and identification of the key genes regulating wing pattern variation across the clade. Our work also explores the evolution and genetics of mimicry in other diverse butterfly clades, including Limenitis, Hypolimnas, Elymnias, and Papilio.

Selected Publications

VanKuren, N. W., D. Massardo, S. Nallu and M. R. Kronforst. 2019. Butterfly mimicry polymorphisms highlight phylogenetic limits of gene reuse in the evolution of diverse adaptations. Molecular Biology and Evolution 36: 2842-2853

Westerman, E., N. VanKuren, D. Massardo, A. Tenger-Trolander, W. Zhang, R. I. Hill, M. Perry, E. Bayala, K. Barr, N. Chamberlain, T. E. Douglas, N. Buerkle, S. E. Palmer and M. R. Kronforst. 2018. Aristaless controls butterfly wing color variation used in mimicry and mate choice. Current Biology 28: 3469-3474.  ScienceDaily  Futurity  Phys.org

Mazo-Vargas, A., C. Concha, L. Livraghi, D. Massardo, R. W. R. Wallbank, L. Zhang, J. D. Papador, D. Martinez-Najera, C. D. Jiggins, M. R. Kronforst, C. J. Breuker, R. D. Reed, N. H. Patel, W. O. McMillan and A. Martin. 2017. Macro-evolutionary shifts of WntA function potentiate butterfly wing pattern diversity. Proc. Natl. Acad. Sci. USA 114: 10701−10706. Nature News & Views  Washington Post  NY Times  Science Magazine  The Atlantic

Kronforst, M. R. and R. Papa. 2015. The functional basis of wing patterning in Heliconius butterflies: the molecules behind mimicry. Genetics 200: 1-19.  2015 Genetics Spotlight   Genes to Genomes

Gallant, J. R, V. E. Imhoff, A. Martin, W. K. Savage, N. L. Chamberlain, B. L. Pote, C. Peterson, G. E. Smith, B. Evans, R. D. Reed, M. R. Kronforst and S. P Mullen. 2014. Ancient homology underlies adaptive mimetic diversity across butterflies.  Nature Communications 5: 4817.  Futurity

Martin A., R. Papa, N. J. Nadeau, R. I. Hill, B. A. Counterman, G. Halder, C. D. Jiggins, M. R. Kronforst, A. D. Long, W. O. McMillan and R. D. Reed. 2012. Diversification of complex butterfly wing patterns by repeated regulatory evolution of a Wnt ligand.  Proc. Natl. Acad. Sci. USA 109: 12632-12637.

Reed, R. D., R. Papa, A. Martin, H. M. Hines, B. A. Counterman, C. Pardo-Diaz, C. D. Jiggins, N. L. Chamberlain, M. R. Kronforst, R. Chen, G. Halder, H. F. Nijhout, and W. O. McMillan. 2011.  optix drives the repeated convergent evolution of butterfly wing pattern mimicry.  Science 333: 1137-1141.  Science Perspective  New York Times  F1000 Opinions

Supergenes

Supergenes

Color pattern variation in the Common Mormon Swallowtail butterfly, Papilio polytes.

We have extended our research on Heliconius to explore the genetic basis of mimicry in a diversity of butterfly species, including one particularly long-lived mystery of evolutionary genetics—the molecular basis of ‘supergene’ mimicry. Using an integrative approach combining genetic and association mapping, transcriptome and genome sequencing, and gene expression analyses, we found that a single gene, doublesex (dsx), controls supergene mimicry in Papilio polytes. This is in contrast to the long-held view that supergenes are likely to be controlled by a tightly linked cluster of loci. Our results ultimately fuse two different hypotheses for the identity of supergenes, showing that a single gene can switch the entire wing pattern among mimicry phenotypes but may require multiple, tightly linked mutations to do so. Our work on supergene mimicry is now expanding along multiple fronts, with particular focus on functional mechanisms in Papilio polytes and comparative genomics across species.

Selected Publications

Palmer, D. H. and M. R. Kronforst. 2020. A shared genetic basis of mimicry across swallowtail butterflies points to ancestral co-option of doublesex. Nature Communications 11: 6.

Palmer, D. H., Y. Q. Tan, S. D. Finkbeiner, A. D. Briscoe, A. Monteiro and M. R. Kronforst. 2018. Experimental field tests of Batesian mimicry in the swallowtail butterfly Papilio polytes. Ecology and Evolution 8: 7657-7666.

Zhang, W., E. Westerman, E. Nitzany, S. Palmer and M. R. Kronforst. 2017. Tracing the origin and evolution of supergene mimicry in butterflies. 2017. Nature Communications 8: 1269.  UChicago ScienceLife  Seeker  Phys.org

Kunte, K., W. Zhang, A. Tenger-Trolander, D. H. Palmer, A. Martin, R. D. Reed, S. P. Mullen and M. R. Kronforst. 2014.  doublesex is a mimicry supergene.  Nature 507: 229-232.  Nature News & Views  Nature News  Science magazine  NY Times  LA Times  University of Chicago  F1000 Opinions

Mating Behavior

Interspecific mating between Heliconius pachinus and Heliconius cydno.

Color pattern mimicry and mating behavior are deeply intertwined in Heliconius butterflies. Previously we showed that Heliconius mate assortatively based on color pattern and that male mate preference is genetically linked to a major Mendelian locus that shifts wing color between white and yellow. Recently we used genome-wide association mapping, gene expression analyses, and genome editing with CRISPR/Cas9 to show that the gene aristaless1 controls the critical white/yellow mimicry locus that butterflies use as a cue during mating. Our work also showed that the derived white allele originated in one species and was subsequently passed to another by hybridization. We continue to pursue the question of how mate preference is generated and what causes preference variation to be linked to the color switch locus. Related to this work, we have invested substantial effort into developing genome editing approaches in Heliconius and other butterflies, studying the impact of female mate preference in Heliconius, and we have also participated in a number of large comparative surveys aimed at tracking the amount and evolutionary consequence of introgression among Heliconius species.

Selected Publications

Westerman, E., N. Antonson, S. Kreutzmann, A. Peterson, S. Pineda, M. R Kronforst and C. F. Olson-Manning. 2019. Behaviour before beauty: signal weighting during mate selection in the butterfly Papilio polytes. Ethology 125: 565-574.

Westerman, E. L., R. Letchinger, A. Tenger-Trolander, D. Massardo, D. Palmer and M. R. Kronforst. 2018. Does male preference play a role in maintaining female limited polymorphism in a Batesian mimetic butterfly? Behavioural Processes 150: 47-58.

Southcott, L. and M. R. Kronforst. 2018. Female mate choice is a reproductive isolating barrier in Heliconius butterflies. Ethology 124:862–869. Dryad Data  bioRxiv Preprint

Chamberlain, N. L., R. I. Hill, D. D. Kapan, L. E. Gilbert and M. R. Kronforst. 2009. Polymorphic butterfly reveals the missing link in ecological speciation.  Science 326: 847-850.  National Science Foundation  Harvard Gazette  UT Austin

Kronforst, M. R., L. G. Young and L. E. Gilbert. 2007. Reinforcement of mate preference among hybridizing Heliconius butterflies.  Journal of Evolutionary Biology 20: 278-285.

Kronforst, M. R., L. G. Young, D. D. Kapan, C. McNeely, R. J. O’Neill and L. E. Gilbert. 2006. Linkage of butterfly mate preference and wing color preference cue at the genomic location of winglessProc. Natl. Acad. Sci. USA 103: 6575-6580.  Faculty1000 Opinions

Migration

A monarch butterfly tethered in our monarch flight simulator.

The monarch butterfly, Danaus plexippus, is famous for its spectacular annual migration across North America, recent worldwide dispersal, and orange warning coloration. Despite decades of study and broad public interest, we know little about the genetic basis of these hallmark traits. Recently, by sequencing and analyzing 101 Danaus genomes from around the globe, we uncovered the history of the monarch’s evolutionary origin and global dispersal, characterized the genes and pathways associated with migratory behavior, and identified the discrete genetic basis of warning coloration. The results substantially changed our understanding of this classic system, showing, for instance, that D. plexippus was ancestrally migratory and dispersed out of North America to occupy its broad distribution. We found striking signatures of selection associated with suites of genes involved in neurogenesis, development, and metabolism but the strongest signatures of selection centered on flight muscle function, resulting in greater flight efficiency among migratory monarchs, and that variation in monarch warning coloration was controlled by a gene not previously implicated in insect pigmentation. We are now working to determine the precise environmental cues that trigger migratory behavior and genetically dissect the migratory syndrome – including behavior, physiology and morphology. This research is of particular importance now because monarch migration is currently experiencing a notable decline. Our work is documenting the evolutionary, genetic, and behavioral distinctiveness of North American monarchs and identifying the functional molecular basis of this amazing natural phenomenon.

Selected Publications

Tenger-Trolander, A. and M. R. Kronforst. 2020. Migration behaviour of commercial monarchs reared outdoors and wild-derived monarchs reared indoors. Proc. R. Soc. B 287: 20201326.  UChicago Medicine News  Phys.org

Talla, V., A. A. Pierce, K. L. Adams, T. J. B. de Man, S. Nallu, F. X. Villablanca, M. R. Kronforst and J. C. de Roode. 2020. Genomic evidence for gene flow between monarchs with divergent migratory phenotypes and flight performance. Molecular Ecology 29: 2567– 2582. ScienceDaily  YouTube

Tenger-Trolander, A., W. Lu, M. Noyes and M. R. Kronforst. 2019. Contemporary loss of migration in monarch butterflies. Proc. Natl. Acad. Sci. USA 116: 14671-14676.  Nature News & Views  Science Magazine  The Atlantic  ScienceDaily  Sciworthy  Anthropocene

Green, D. A. and M. R. Kronforst. 2019. Monarch butterflies use an environmentally sensitive, internal timer to control overwintering dynamics. Molecular Ecology 28: 3642-3655. Dryad Data  ScienceDaily  EarthSky

Zhan, S., W. Zhang, K. Niitepold, J. Hsu, J. F. Haeger, M. P. Zalucki, S. Altizer, J. C. de Roode, S. M. Reppert and M. R. Kronforst. 2014. The genetics of monarch butterfly migration and warning colouration.  Nature 514: 317-321.  Nature News & Views  Science Magazine  NY Times  National Geographic  Washington Post  NBC News

Pierce, A. A., M. P. Zalucki, M. Bangura, M. Udawatta, M. R. Kronforst, S. Altizer, J. F. Haeger and J. C. de Roode. 2014. Serial founder effects and genetic differentiation during worldwide range expansion of monarch butterflies.  Proc. R. Soc. B 281: 20142230.

Herbivory

Herbivory

Herbivory

A monarch caterpillar munching away on a milkweed leaf.

Some of our newest work focuses on the interactions between butterflies and their larval host plants. We have mapped the genetic basis of herbivory in butterflies and plants and compared patterns of gene expression in larvae and their hosts, across the time course of their interaction and across species. We are now in the process of functionally validating the plant and insects genes implicated in herbivory as well as studying their evolution. We are also interested in understanding how future climate change might alter the dynamics of these co-evolved interactions – will plants ‘escape’ herbivores via elevated growth rates or enhanced protection, or will herbivores demolish host plants due to reduced plant defenses or enhanced insect detoxification abilities, or will these systems maintain equilibrium? Furthermore, how are these organismal and ecological changes actually mediated at a molecular level?

Selected Publications

Nallu, S., J. Hill, K. Don, C. Sahagun, W. Zhang, C. Meslin, E. Snell-Rood, N. Clark, N. Morehouse, J. Bergelson, C. Wheat and M. R. Kronforst. 2018. The molecular genetic basis of herbivory between butterflies and their host plants. Nature Ecology & Evolution 2: 1418-1427.  Research Highlight  Behind The Paper

CRISPR/Cas9

CRISPR/Cas9 mosaic knockout of red patterning gene optix in Heliconius melpomene

In the last decade, CRISPR/Cas9 has emerged as an essential tool for genome editing and we have worked hard to apply these methods to butterflies. We use CRISPR/Cas9 genome editing to functionally validate candidate genes and study gene function by generating knock-outs. We are also working to expand the CRISPR methodology toolkit in butterflies and make them an even more tractable experimental system. In addition to CRISPR/Cas9, we utilize a variety of other methods to study functional genomics in butterflies, including comparative transcriptomics, ChIP-Seq, ATAC-Seq and RNAi.

Selected Publications

Westerman, E., N. VanKuren, D. Massardo, A. Tenger-Trolander, W. Zhang, R. I. Hill, M. Perry, E. Bayala, K. Barr, N. Chamberlain, T. E. Douglas, N. Buerkle, S. E. Palmer and M. R. Kronforst. 2018. Aristaless controls butterfly wing color variation used in mimicry and mate choice. Current Biology 28: 3469-3474.  ScienceDaily  Futurity  Phys.org

Mazo-Vargas, A., C. Concha, L. Livraghi, D. Massardo, R. W. R. Wallbank, L. Zhang, J. D. Papador, D. Martinez-Najera, C. D. Jiggins, M. R. Kronforst, C. J. Breuker, R. D. Reed, N. H. Patel, W. O. McMillan and A. Martin. 2017. Macro-evolutionary shifts of WntA function potentiate butterfly wing pattern diversity. Proc. Natl. Acad. Sci. USA 114: 10701−10706. Nature News & Views  Washington Post  NY Times  Science Magazine  The Atlantic

Li, X., D. Fan, W. Zhang, G. Liu, L. Zhang, L. Zhao, X. Fang, L. Chen, Y. Dong, Y. Chen, Y. Ding, R. Zhao, M. Feng, Y. Zhu, Y. Feng, X. Jiang, D. Zhu, H. Xiang, X. Feng, S. Li, J. Wang, G. Zhang, M. R. Kronforst and W. Wang. 2015. Outbred genome sequencing and CRISPR/Cas9 gene editing in butterflies.  Nature Communications 6: 8212.  IGTRCN

Evo Devo

Evo-Devo

Evo Devo

Antibody stain showing aristaless1 (green) expressed during wing scale development in Heliconius cydno.

Evolutionary developmental biology, or evo-devo, is a field of study that focuses on comparative analyses of developmental processes in order to infer how development has evolved. We use the
methods of developmental biology–including in situ hybridization, immunohistochemistry, spatial transcriptomics, and imaging–to compare development across species and populations. Much of this work is focused on understanding how specific genes and mutations ultimately generate distinct phenotypes, from the diverse mimetic color patterns of Heliconius and Papilio butterflies to the divergent mate preference behaviors of white and yellow winged Heliconius cydno.

Selected Publications

Westerman, E., N. VanKuren, D. Massardo, A. Tenger-Trolander, W. Zhang, R. I. Hill, M. Perry, E. Bayala, K. Barr, N. Chamberlain, T. E. Douglas, N. Buerkle, S. E. Palmer and M. R. Kronforst. 2018. Aristaless controls butterfly wing color variation used in mimicry and mate choice. Current Biology 28: 3469-3474.  ScienceDaily  Futurity  Phys.org

Martin A., R. Papa, N. J. Nadeau, R. I. Hill, B. A. Counterman, G. Halder, C. D. Jiggins, M. R. Kronforst, A. D. Long, W. O. McMillan and R. D. Reed. 2012. Diversification of complex butterfly wing patterns by repeated regulatory evolution of a Wnt ligand.  Proc. Natl. Acad. Sci. USA 109: 12632-12637.

kronforst favicon

Genomics

kronforst faviconThe ability to survey and compare genetic variation genome-wide has revolutionized the study of evolutionary genomics. We sequence and compare genomes across species, populations, and individuals to address questions related to phylogenetics, population genetic structure and demographics, the genetic basis of phenotypic variation, and to study the interplay between divergence and gene flow among populations and species. Thankfully, butterflies have small genomes (200-400 Mbp) so we can generate good reference assemblies with relative ease and population resequencing at scale is feasible. On the other hand, poison dart frogs have large genomes (6-9 Gbp) that are highly repetitive, which makes genome assembly and analysis more complicated. Methods for genome sequencing and assembly are constantly improving and these will continue to empower the genomic revolution in evolutionary biology.

Selected Publications

Massardo, D., N. W. VanKuren, S. Nallu, R. R. Ramos, P. Gusmão, K. L. Silva-Brandão, M. M. Brandão, M. B. Lion, A. V. L. Freitas, M. Z. Cardoso and M. R. Kronforst. 2020. The roles of hybridization and habitat fragmentation in the evolution of Brazil’s enigmatic longwing butterflies, Heliconius nattereri and H. hermathena. BMC Biology 18: 84.

Mullen, S. P., N. W. VanKuren, W. Zhang, S. Nallu, E. B. Kristiansen, Q. Wuyun, K. Liu, R. I. Hill, A. D. Briscoe and M. R. Kronforst. 2020. Disentangling population history and character evolution among hybridizing lineages. Molecular Biology and Evolution 37: 1295-1305.

Edelman, N. B., P. B. Frandsen, M. Miyagi, B. Clavijo, J. Davey, R. B. Dikow, G. Garcia-Accinelli, S. M. Van Belleghem, N. Patterson, D. E. Neafsey, R. Challis, S. Kumar, G. R. P. Moreira, C. Salazar, M. Chouteau, B. A. Counterman, R. Papa, M. Blaxter, R. D. Reed, K. K. Dasmahapatra, M. R. Kronforst, M. Joron, C. D. Jiggins, W. Owen McMillan, F. Di Palma, A. J. Blumberg, J. Wakeley, D. Jaffe and J. Mallet. 2019. Genomic architecture and introgression shape a butterfly radiation. Science 366: 594-599.  Science Perspective  Dryad Data  bioRxiv Preprint  ScienceDaily

Zhang, W, K. K. Dasmahapatra, J. Mallet, G. R. P. Moreira and M. R. Kronforst. 2016. Genome-wide introgression among distantly related Heliconius butterfly species.  Genome Biology 17: 25.

Li, X., D. Fan, W. Zhang, G. Liu, L. Zhang, L. Zhao, X. Fang, L. Chen, Y. Dong, Y. Chen, Y. Ding, R. Zhao, M. Feng, Y. Zhu, Y. Feng, X. Jiang, D. Zhu, H. Xiang, X. Feng, S. Li, J. Wang, G. Zhang, M. R. Kronforst and W. Wang. 2015. Outbred genome sequencing and CRISPR/Cas9 gene editing in butterflies.  Nature Communications 6: 8212.  IGTRCN

Heliconius Genome Consortium. 2012. Butterfly genome reveals promiscuous exchange of mimicry adaptations among species.  Nature 487: 94-98.  New York Times  Harvard Gazette