Xiong, T., Li, X., Yago, M., & Mallet, J. (Forthcoming). Genetic barriers to gene flow separate divergent substitution rates across a butterfly hybrid zone. BioRxiv , 2021, .2009.2027.461223. Publisher's VersionAbstract
Substitution rate defines the fundamental timescale of molecular evolution which often varies in a species-specific manner. However, it is unknown under what conditions lineage-specific rates can be preserved between natural populations with frequent hybridization. Here, we show in a hybrid zone between two butterflies Papilio syfanius and Papilio maackii that genome-wide barriers to gene flow can effectively separate different rates of molecular evolution in linked regions. The increased substitution rate in the lowland lineage can be largely explained by temperature-induced changes to the spontaneous mutation rate. A novel method based on entropy is developed to test for the existence of barrier loci using a minimal number of samples from the hybrid zone, a robust framework when system complexity far exceeds sample information. Overall, our results suggest that during the process of speciation, the separation of substitution rates can occur locally in the genome in parallel to the separation of gene pools.
Mallet, J. (Forthcoming). Subspecies, Semispecies, Superspecies. In Encyclopedia of Biodiversity . Elsevier. mallet_2021_subspecies_superspecies_semispecies_encyc_of_biodiv.pdf
Mallet, J., Seixas, F., & Thawornwattana, Y. (Forthcoming). Species, concepts of. In Encyclopedia of Biodiversity . Elsevier.Abstract
What are species? We take a historical approach to demonstrate how our understanding of the nature of species has changed since Darwin's time until the current postgenomic era. If species are fundamental units of nature (like cells, or organisms, say), as some scientists believe, then their underlying basis has yet to be revealed. Instead, it seems more likely that the species rank in taxonomy is a man-made distinction; a heuristic cut-off that can perhaps be agreed upon bybiologists to enable communication on issues of biodiversity, human health, agriculture, and conservation.
Xiong, T., & Mallet, J. (Forthcoming). The collapse of genetic incompatibilities in a hybridizing population. BioRxiv , 2021.01.08.425971v1. Publisher's VersionAbstract
Genetic incompatibility has long been considered to be a hallmark of speciation due to its role in reproductive isolation. Previous analyses of the stability of epistatic incompatibility show that it is subject to collapse upon hybridization. In the present work, we derive explicitly the distribution of the lifespan of two-locus incompatibilities, and show that genetic drift, along with recombination, is critical in determining the time scale of collapse. The first class of incompatibilities, where derived alleles separated in parental populations act antagonistically in hybrids, survive longer in smaller populations when incompatible alleles are (co)dominant and tightly linked, but collapse more quickly when they are recessive. The second class of incompatibilities, where fitness is reduced by disrupting co-evolved elements in gene regulation systems, collapse on a time scale proportional to the exponential of effective recombination rate. Overall, our result suggests that the effects of genetic drift and recombination on incompatibility’s lifespan depend strongly on the underlying mechanisms of incompatibilities. As the time scale of collapse is usually shorter than the time scale of establishing a new incompatibility, the observed level of genetic incompatibilities in a particular hybridizing population may be shaped more by the collapse than by their initial accumulation. Therefore, a joint theory of accumulation-erosion of incompatibilities is in need to fully understand the genetic process under speciation with hybridization
Mallet, J. (2022). The making of a moth man (book review of: Grant, Bruce S. 2021. Observing Evolution). Evolution. Publisher's Version mallet_2022_the_making_of_a_moth_man.pdf
Rosser, N., Edelman, N. B., Queste, L. M., Nelson, M., Seixas, F., Dasmahapatra, K. K., & Mallet, J. (2022). Complex basis of hybrid female sterility and Haldane’s rule in Heliconius butterflies: Z-linkage and epistasis. Molecular Ecology , 31, 959-977. Publisher's VersionAbstract
Hybrids between diverging populations are often sterile or inviable. Hybrid unfitness usually evolves first in the heterogametic sex – a pattern known as Haldane’s rule. The genetics of Haldane’s Rule have been extensively studied in species where the male is the heterogametic (XX/XY) sex, but its basis in taxa where the female is heterogametic (ZW/ZZ), such as Lepidoptera and birds, is largely unknown. Here, we analyse a new case of female hybrid sterility between geographic subspecies of Heliconius pardalinus. The two subspecies mate freely in captivity, but female F1 hybrids in both directions of cross are sterile. Sterility is due to arrested development of oocytes after they become differentiated from nurse cells, but before yolk deposition. We backcrossed fertile male F1 hybrids to parental females, and mapped quantitative trait loci (QTLs) for female sterility. We also identified genes differentially expressed in the ovary, and as a function of oocyte development. The Z chromosome has a major effect, similar to the “large X effect” in Drosophila, with strong epistatic interactions between loci at either end of the Z chromosome, and between the Z chromosome and autosomal loci on chromosomes 8 and 20. Among loci differentially expressed between females with arrested vs. non-arrested ovary development, we identified six candidate genes known also from Drosophila melanogaster and Parage aegeria oogenesis. This study is the first to characterize hybrid sterility using genome mapping in the Lepidoptera. We demonstrate that sterility is produced by multiple complex epistastic interactions often involving the sex chromosome, as predicted by the dominance theory of Haldane’s Rule.
Thawornwattana, Y., Seixas, F. A., Yang, Z., & Mallet, J. (2022). Full-likelihood genomic analysis clarifies a complex history of species divergence and introgression: the example of the erato-sara group of Heliconius butterflies. Systematic Biology , xxx (00), 000-000. Publisher's VersionAbstract
Introgression plays a key role in adaptive evolution and species diversification in many groups of species including Heliconius butterflies. However, frequent hybridization and gene flow between species makes estimation of the species phylogeny challenging, especially for rapidly speciating species within adaptive radiations. Here, we analyze the genomic sequences from six members of the erato-sara clade of Heliconius butterflies to infer the species phylogeny and cross-species introgression events. To avoid heterozygote phasing errors in haploid sequences commonly produced by genome assembly methods, we processed and compiled unphased diploid sequence alignments, with the analytical methods averaging over uncertainties in heterozygote phase resolution. We use likelihood-based methods under the multispecies coalescent (MSC) model with and without gene flow to accommodate random fluctuations in genealogical history across the genome due to deep coalescence. There is robust evidence of introgression across the genome, both among distantly related species deep in the phylogeny and between sister species in shallow parts of the tree. We obtain estimates of population parameters such as introgression times and probabilities, species divergence times, and population sizes for modern and ancestral species. We confirm ancestral gene flow between the sara clade and an ancestral population of likely hybrid origin of H. telesiphe, a likely hybrid origin of H. hecalesia, and gene flow between the sister species H. erato and H. himera. Our approach also demonstrates how introgression among ancestral species can explain the history of two chromosomal inversions deep in the phylogeny of the group. For the first time, we not only test for the presence of cross-species gene flow, but also estimate its direction, timing and magnitude, extracting rich historical information of species divergence and gene flow from genomic data
Doré, M., Willmott, K., Leroy, N., Chazot, N., Mallet, J., Freitas, A. V. L., Hall, J. P. W., et al. (2021). Anthropogenic pressures coincide with Neotropical biodiversity hotspots in a flagship butterfly group. Diversity and Distributions , xx (000). Publisher's Version
Edelman, N. B., & Mallet, J. (2021). Prevalence and adaptive impact of introgression. Annual Review of Genetics , 55, 265-283. Publisher's VersionAbstract
Alleles that introgress between species can influence the evolutionary and ecological fate of species exposed to novel environments. Hybrid offspring of different species are often unfit, and yet it has long been argued that introgression can be a potent force in evolution, especially in plants. Over the last two decades, genomic data have increasingly provided evidence that introgression is a critically important source of genetic variation and that this additional variation can be useful in adaptive evolution of both animals and plants. Here, we review factors that influence the probability that foreign genetic variants provide long-term benefits (so-called adaptive introgression) and discuss their potential benefits. We find that introgression plays an important role in adaptive evolution, particularly when a species is far from its fitness optimum, such as when they expand their range or are subject to changing environments.
Seixas, F. A., Edelman, N. B., & Mallet, J. (2021). Synteny-based genome assembly for 16 species of Heliconius butterflies, and an assessment of structural variation across the genus. Genome Biology and Evolution , 13 (7), evab069. Publisher's VersionAbstract
Heliconius butterflies (Lepidoptera: Nymphalidae) are a group of 48 neotropical species widely studied in evolutionary research. Despite the wealth of genomic data generated in past years, chromosomal level genome assemblies currently exist for only two species, Heliconius melpomene and H. erato, each a representative of one of the two major clades of the genus. Here, we use these reference genomes to improve the contiguity of previously published draft genome assemblies of 16 Heliconius species. Using a reference-assisted scaffolding approach, we place and order the scaffolds of these genomes onto chromosomes, resulting in 95.7-99.9% of their genomes anchored to chromosomes. Genome sizes are somewhat variable among species (270-422 Mb) and in one small group of species (H. hecaleH. elevatus and H. pardalinus) expansions in genome size are driven mainly by repetitive sequences that map to four small regions in the H. melpomene reference genome. Genes from these repeat regions show an increase in exon copy number, an absence of internal stop codons, evidence of constraint on non-synonymous changes, and increased expression, all of which suggest that at least some of the extra copies are functional. Finally, we conducted a systematic search for inversions and identified five moderately large inversions fixed between the two major Heliconius clades. We infer that one of these inversions was transferred by introgression between the lineages leading to the erato/sara and burneyi/doris clades. These reference-guided assemblies represent a major improvement in Heliconius genomic resources that enable further genetic and evolutionary discoveries in this genus.
Rosser, N., Shirai, L. T., Dasmahapatra, K. K., Mallet, J., & Freitas, A. V. L. (2021). The Amazon river is a suture zone for a polyphyletic group of co-mimetic heliconiine butterflies. Ecography , 43 (2), 177-187. Publisher's Version
Mallet, J. (2020). Alternative views of biological species: reproductively isolated units or genotypic clusters? National Science Review , 8 (7), 1401-1407. Publisher's VersionAbstract

Wang et al. have recently outlined their thoughts on two ideas about species: the biological species concept (BSC), and the genic view of speciation. Although both are based on reproductive isolation, the genic view, they argue, is likely preferred due to the possibility of allowing considerable gene flow among species, which is arguably what recent genomic data shows. These data, however, mostly failed to distinguish between the BSC and the genic view according to Wang et al., because it could not be ruled out that the observed introgression occurred early in speciation, when both models allow gene flow. I propose that the lack of resolution in the authors' debate is chiefly due to the difficulty of deciding when speciation is "complete" under both views tested. I agree with Wang et al. that the study of reproductive isolation is worthwhile in order to understand speciation, but I prefer to use a simpler, third criterion for speciation: the acquisition of genetic differences that allow persistence of distinguishable populations in spite of geographic overlap and the potential for continued gene flow. Under this multilocus "genotypic cluster" view, gene flow may take place at any time after species are recognized, and we do not have to decide whether gene flow is early or late in the speciation process. I detail recent genomic evidence from Anopheles mosquitoes and Heliconius butterflies showing that such "leaky" species seem to be able to coexist in spite of massive levels of introgression, often among non-sister species that show hybrid sterility in one sex.

Gauthier, J., de Silva, D. L., Gompert, Z., Whibley, A., Houssin, C., Le Poul, Y., McClure, M., et al. (2020). Contrasting genomic and phenotypic outcomes of hybridization between pairs of mimetic butterfly taxa across a suture zone. Molecular Ecology , 29 (7), 1328-1343. Publisher's Version
Qiao, L., Yan, Z. -wen, Xiong, G., Hao, Y. -jin, Wang, R. -xin, Hu, H., Song, J. -bo, et al. (2020). Excess melanin precursors rescue defective cuticular traits in stony mutant silkworms probably by upregulating four genes encoding RR1-type larval cuticular proteins. Insect Biochemistry and Molecular Biology , 119, 103315. Publisher's VersionAbstract
Melanin and cuticular proteins are vital cuticle components in insects. Cuticular defects caused by mutations in cuticular protein-encoding genes can obstruct melanin deposition. The effects of changes in melanin on the expression of cuticular protein-encoding genes, the cuticular and morphological traits, and the origins of these effects are unknown. We found that the cuticular physical characteristics and the expression patterns of larval cuticular protein-encoding genes markedly differed between the melanic and non-melanic integument regions. By using four p multiple-allele color pattern mutants with increasing degrees of melanism (+p, pM, pS, and pB), we found that the degree of melanism and the expression of four RR1-type larval cuticular protein-encoding genes (BmCPR2, BmLcp18, BmLcp22, and BmLcp30) were positively correlated. By modulating the content of melanin precursors and the expression of cuticular protein-encoding genes in cells in tissues and in vivo, we showed that this positive correlation was due to the induction of melanin precursors. More importantly, the melanism trait introduced into the BmCPR2 deletion strain Dazao-stony induced up-regulation of three other similar chitin-binding characteristic larval cuticular protein-encoding genes, thus rescuing the cuticular, morphological and adaptability defects of the Dazao-stony strain. This rescue ability increased with increasing melanism levels. This is the first study reporting the induction of cuticular protein-encoding genes by melanin and the biological importance of this induction in affecting the physiological characteristics of the cuticle.
Gillespie, R. G., Bennett, G. M., De Meester, L., Feder, J. L., Fleischer, R. C., Harmon, L. J., Hendry, A. P., et al. (2020). Comparing adaptive radiations across space, time, and taxa. Journal of Heredity , 111 (1), 1-20. Publisher's VersionAbstract
Adaptive radiation plays a fundamental role in our understanding of the evolutionary process. However, the concept has provoked strong and differing opinions concerning its definition and nature among researchers studying a wide diversity of systems. Here, we take a broad view of what constitutes an adaptive radiation, and seek to find commonalities among disparate examples, ranging from plants to invertebrate and vertebrate animals, and remote islands to lakes and continents, to better understand processes shared across adaptive radiations. We surveyed many groups to evaluate factors considered important in a large variety of species radiations. In each of these studies, ecological opportunity of some form is identified as a prerequisite for adaptive radiation. However, evolvability, which can be enhanced by hybridization between distantly related species, may play a role in seeding entire radiations. Within radiations, the processes that lead to speciation depend largely on (1) whether the primary drivers of ecological shifts are (a) external to the membership of the radiation itself (mostly divergent or disruptive ecological selection) or (b) due to competition within the radiation membership (interactions among members) subsequent to reproductive isolation in similar environments, and (2) the extent and timing of admixture. These differences translate into different patterns of species accumulation and subsequent patterns of diversity across an adaptive radiation. Adaptive radiations occur in an extraordinary diversity of different ways, and continue to provide rich data for a better understanding of the diversification of life
Edelman, N. B., Frandsen, P. B., Miyagi, M., Clavijo, B., Davey, J., Dikow, R. B., García-Accinelli, G., et al. (2019). Genomic architecture and introgression shape a butterfly radiation. Science , 366 (November 1), 594-599. Publisher's VersionAbstract

Following gene flow in butterfly genomes

The role of hybridization in evolution and species radiations has long been debated. In Heliconius butterflies, introgression was a major factor in their radiation, and the genetic variation it imparted into species is variable across the genome. Edelman et al. developed a new sequencing strategy and produced 20 Heliconius genomes (see the Perspective by Rieseberg). They also developed a means by which to identify genetic variation that originates from incomplete lineage sorting versus hybridization. Applying this model to their newly developed genomes, they investigated the evolutionary history of the genus and, in particular, the impact of introgression.

Science, this issue p. 594; see also p. 570.

We used 20 de novo genome assemblies to probe the speciation history and architecture of gene flow in rapidly radiating Heliconius butterflies. Our tests to distinguish incomplete lineage sorting from introgression indicate that gene flow has obscured several ancient phylogenetic relationships in this group over large swathes of the genome. Introgressed loci are underrepresented in low-recombination and gene-rich regions, consistent with the purging of foreign alleles more tightly linked to incompatibility loci. Here, we identify a hitherto unknown inversion that traps a color pattern switch locus. We infer that this inversion was transferred between lineages by introgression and is convergent with a similar rearrangement in another part of the genus. These multiple de novo genome sequences enable improved understanding of the importance of introgression and selective processes in adaptive radiation.

Mallet, J. (2019). Reply to Andrew Brower’s critique of the evidence for hybridization among Heliconius butterfly species in the wild. Zootaxa , 4679, 577-595. Publisher's VersionAbstract
Andrew Brower recently published a long article in this journal that seeks to dismantle evidence for hybridization between species of Heliconius butterflies. The main evidence that Brower criticizes here is given in two papers published by my colleagues and myself in 2007. In this reply, I briefly defend our evidence, and at greater length provide additional background information to help establish the credibility of the evidence even more firmly than previously
Rosser, N., Queste, L. M., Cama, B., Edelman, N. B., Mann, F., Mori Pezo, R., Morris, J., et al. (2019). Geographic contrasts between pre‐ and postzygotic barriers are consistent with reinforcement in Heliconius butterflies. Evolution , 73 (9), 1821-1838. Publisher's VersionAbstract
Identifying the traits causing reproductive isolation and the order in which they evolve is fundamental to understanding speciation. Here, we quantify prezygotic and intrinsic postzygotic isolation among allopatric, parapatric, and sympatric populations of the butterflies Heliconius elevatus and Heliconius pardalinus. Sympatric populations from the Amazon (H. elevatus and H. p. butleri) exhibit strong prezygotic isolation and rarely mate in captivity; however, hybrids are fertile. Allopatric populations from the Amazon (H. p. butleri) and Andes (H. p. sergestus) mate freely when brought together in captivity, but the female F1 hybrids are sterile. Parapatric populations (H. elevatus and H. p. sergestus) exhibit both assortative mating and sterility of female F1s. Assortative mating in sympatric populations is consistent with reinforcement in the face of gene flow, where the driving force, selection against hybrids, is due to disruption of mimicry and other ecological traits rather than hybrid sterility. In contrast, the lack of assortative mating and hybrid sterility observed in allopatric populations suggests that geographic isolation enables the evolution of intrinsic postzygotic reproductive isolation. Our results show how the types of reproductive barriers that evolve between species may depend on geography
Chazot, N., Willmott, K. R., Lamas, G., Freitas, A. V. L., Piron-Prunier, F., Arias, C. F., Mallet, J., et al. (2019). Renewed diversification following Miocene landscape turnover in a Neotropical butterfly radiation. Global Ecology and Biogeography , 28 (8), 1118–1132. Publisher's VersionAbstract

The landscape of the Neotropical region has undergone dynamic evolution throughout the Miocene, with the extensive Pebas wetland occupying western Amazonia between 23 and c. 10 Ma and the continuous uplift of the Andes mountains. The complex interaction between the Andes and Amazonia probably influenced the trajectory of Neotropical biodiversity, but evidence from time-calibrated phylogenies of groups that diversified during this period is lacking. We investigate the role of these landscape transformations in the dynamics of diversification in the Neotropical region using a 26-Myr-old endemic butterfly radiation. Location Neotropics. Time period Oligocene to present. Major taxa studied Ithomiini butterflies.

We generated one of the most comprehensive time-calibrated molecular phylogenies of a large clade of Neotropical insects, the butterfly tribe Ithomiini, comprising 340 species (87% of extant species) and spanning 26 Myr of diversification. We applied a large array of birth–death models and historical biogeography estimations to assess the dynamics of diversification and biotic interchanges, especially at the Amazonia–Andes interface.

Our results suggest that the Amazonian Pebas wetland system played a major role in the timing and geography of diversification of Ithomiini, by constraining dispersal and diversification in the Amazon basin until c. 10 Ma. During the Pebas wetland period, Ithomiini diversification mostly took place in the Andes, where terrestrial habitats were not affected. An explosion of interchanges with Amazonia and with the Northern Andes accompanied the demise of the Pebas system (11–8 Ma) and was followed by local diversification in those areas, which led to a substantial renewal of diversification.

Main conclusions
Many studies on Neotropical diversity have focused only on the Andes, whereas we show that it is the waxing and waning of the Pebas mega-wetland, interacting with Andean uplift, that determined the timing and patterns of regional interchanges and diversification in Ithomiini.

Ray, D. A., Grimshaw, J. R., Halsey, M. K., Korstian, J. M., Osmanski, A. B., Sullivan, K. A., Wolf, K. A., et al. (2019). Simultaneous TE analysis of 19 heliconiine butterflies yields novel insights into rapid TE-based genome diversification and multiple SINE births and deaths. Genome Biology and Evolution , 11 (8), 2162-2177. Publisher's VersionAbstract
Transposable elements (TEs) play major roles in the evolution of genome structure and function. However, because of their repetitive nature, they are difficult to annotate and discovering the specific roles they may play in a lineage can be a daunting task. Heliconiine butterflies are models for the study of multiple evolutionary processes including phenotype evolution and hybridization. We attempted to determine how TEs may play a role in the diversification of genomes within this clade by performing a detailed examination of TE content and accumulation in 19 species whose genomes were recently sequenced. We found that TE content has diverged substantially and rapidly in the time since several subclades shared a common ancestor with each lineage harboring a unique TE repertoire. Several novel SINE lineages have been established that are restricted to a subset of species. Furthermore, the previously described SINE, Metulj, appears to have gone extinct in two subclades while expanding to significant numbers in others. This diversity in TE content and activity has the potential to impact how heliconiine butterflies continue to evolve and diverge