The genetics of monarch butterfly migration and warning colouration

The monarch butterfly (Danaus plexippus) has a large distribution worldwide. It occurs in North, Central, and South America, Caribbean, and it has recently dispersed to other locations, such as Oceania and Africa. Two traits of this butterfly are incredibly intriguing: their annual migration in North America, and their warning coloration.

Among the populations spread out around the globe, only the population of North America has a migrant behavior. Monarchs migrate thousands of kilometers from northern United States and southern Canada to overwinter in Mexico. In Spring, they begin mating and flying back to the North. This long annual migration process happens throughout the life time of more than one generation. Regarding their warning coloration, what is intriguing about it is the occurrence of an intense polymorphism in this trait when compared to other butterflies of the same genus. Monarchs and other Danaus butterflies have by default bright orange wings. This bold coloration warns predators about their toxicity. However, in a monarch population from Hawaii, some butterflies have white instead of orange wings.

In this paper, authors investigate, through comparative population genomics, the genetic base for such migration and color polymorphisms of the monarch butterfly.

The migration of the monarchs

By comparing around 32 million SNPs (single nucleotide polymorphisms) of 89 butterflies of both the migrant North American population and non-migrant populations around the world, authors first determined what is the evolutionary origin of the monarchs and its dispersal history. Second, they screened for regions of the genome that are possibly associated with the migration behavior of monarchs. Lastly, they explored one of the screened genes to investigate if it has, in fact, functions related to migration.

The evolutionary history of the monarchs: The authors suggested that, contrary to a priori expectations, the basal lineage of the monarch is the North American lineage and that it has dispersed worldwide in subsequent three independent dispersal events. This is supported by a consensus neighbor-joining tree based on 1000 bootstraps (fig. 1d). In my opinion, authors could have chosen to display such tree as a rooted tree. This would make clearer the idea that the North American population is the basal lineage. By using a PCA (principal component analysis) plot of the genetic data among populations, they also showed that the different populations (North America, Pacific crossing, Central America and Atlantic crossing) differ genetically, i.e. sample locations are clearly grouped per populations (fig. 1e). The genetic structure and individual ancestry analysis of populations was tested across varying cluster numbers and also provides support that the North American population is the monarch basal lineage (fig. 1f).

Screening of genome regions associated with migratory behavior: The authors identified a genomic region highly divergent between migratory and non-migratory populations and likely to be under natural selection. This is supported by figure 2a, in which they compared genome-wide population divergence using an adaptation of a statistical model called Population-Branch Statistic (PBS). More information about PBS can be found here. By constraining the search to DNA windows with a negative Tajima’s D (that indicates genomic regions evolving under a non-random process) and that are highly conserved in the migratory population, they found a genomic region of 21 Kb highly divergent between migratory and not migratory populations and likely to be under selection (fig. 2a). The authors found multiple signatures of selection in this genome region using diverse population genetic statistics (fig. 2b), which gives further support to their screen. By investigating which annotated genes are present in this 21 Kb DNA region, the authors found three genes: F-box protein, an uncharacterized transmembrane protein, and collagen type IV subunit alpha-1 (fig. 2c). Out of these three screened genes, the authors discussed only the possible function of the collagen IV to the migratory behavior of the monarchs. Surprisingly, they do not further discuss the other two genes found by their screening, although these genes were also found to be highly divergent and under selection.

Exploring the function of collagen IV subunit alpha 1: The gene collagen IV is believed to be linked to the migratory behavior given its importance for muscle morphogenesis and function. Furthermore, this gene showed signals of divergent selection between migratory and non-migratory populations, with high fixation index (Fst) and sequence divergence (Dxy) (fig. 3a). Haplotype divergence between migrant and non-migrant population was also remarkably elevated for the collagen IV, despite the fact that this is a comparison of haplotypes of the same species (fig. 3b). Interestingly, the ancient version of the collagen IV haplotype seems to be the non-migratory one (fig. 3c). This suggest that, if this haplotype is indeed strongly related to migration, the species was originated in North America from a non-migrant ancestor, evolved the current migration behavior, and when it further dispersed worldwide, it has reverted to the ancient non-migratory behavior. In order to test for the function of the collagen IV gene, the authors measured the expression of this gene in adult monarch from migrant and non-migrant populations. Contrary to logic expectations, this gene was down regulated in migrant populations (fig. 3e). This led the authors to hypothesize that it would be acting on aspects of flight efficiency, i.e. that flying would be less demanding for migrant than non-migrant populations. To further investigate this hypothesis, they tested the metabolic rates of both migrants and non-migrant monarchs. As predicted, they found the flight metabolic rates of the migrant butterflies to be significantly lower than the non-migrant ones, but the resting metabolic rates to be similar among individuals of the two migratory behaviors (fig. 3f).

The take home-message of this part of the paper is that after a genome-wide screening they found one gene, the collagen IV subunit 1, which is highly divergent between migratory and non-migratory populations. Also, this gene seems to be under divergent selection and to affect flight function, and therefore, might be part of the genetic base behind the migration polymorphism of the monarch butterfly.

The color polymorphism of the monarchs

In order to investigate the genetic basis of the color polymorphism, the authors sequenced the genome of 12 additional monarch butterflies from a population in Hawaii where the white type occurs. The white type is believed to be caused by a deleterious mutation in a color production pathway (the ommochrome biosynsthesis). The authors, therefore, scanned the genomes of wild and white type monarch butterflies looking for association patterns between SNPs and wing coloration. In figure 4b, they highlighted a genomic region in which there was a high association between SNPs and wing color. This region contains a myosin gene (that is believed to be associated with pigment transportation) under strong purifying selection (fig. 4c), suggesting that the genetic basis of the white type is associated to pigment transportation rather than pigment production. As a drawback of this analysis I would mention that some SNPs with high probability of association with the color polymorphism (particularly two of them, just on the left side of the top ones highlighted by the authors in fig. 4b) were ignored by the authors without any explanation/discussion.

Wrapping up…

In this paper the authors revealed part of the genetic basis of two intriguing traits of monarch butterflies. Also interestingly they re-wrote the evolutionary history of this species and suggested that, contrary to expectations, the species has most likely originated in North America from a non-migrant ancestor. Later they became migratory in North America, and when they dispersed worldwide, they reverted to the original non-migratory behavior. The North American population of monarch butterflies is endangered, and as it is the basal lineage, it contains higher genetic diversity than the dispersed lineages. The data of this paper show that a disappearance of the monarchs in North America would represent a significant reduction in the genetic diversity of this species and a complete loss of the migratory behavior. This paper is, therefore, a good example of how powerful genomic analyses are and how useful they can be to answer evolutionary, ecological and even conservation biology questions!

Zhan, S., Zhang, W., Niitepõld, K., Hsu, J., Haeger, J., Zalucki, M., Altizer, S., de Roode, J., Reppert, S., & Kronforst, M. (2014). The genetics of monarch butterfly migration and warning colouration Nature, 514 (7522), 317-321 DOI: 10.1038/nature13812

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