Papers

In Press
Rosser, N. *, Seixas, F. *, Queste, L. M., Cama, B., Mori-Pezo, R., Kryvokhyzha, D., Nelson, M., et al. (In Press). Hybrid speciation driven by multilocus introgression of ecological traits. Nature , xxx, 000.Abstract
Hybridisation allows adaptations to be shared among lineages and may trigger the evolution of new species. However, convincing examples of homoploid hybrid speciation remain rare because it is challenging to demonstrate that hybridisation was critical in generating reproductive isolation. Here we combine population genomic analysis with quantitative trait locus mapping of species-specific traits to dissect a new case of hybrid speciation in Heliconius butterflies. We show that H. elevatus is a hybrid species that is sympatric with both parents and has persisted as an independently evolving lineage for at least 180,000 years. This is despite pervasive and ongoing gene flow with one parent, H. pardalinus, which homogenises 99% of their genomes. The remaining 1% introgressed from the other parent, H. melpomene, and is scattered widely across the H. elevatus genome in islands of divergence from H. pardalinus. These islands harbour multiple traits under disruptive selection, including colour pattern, wing shape, hostplant preference, sex pheromones and mate choice. Collectively, these traits place H. elevatus on its own adaptive peak and permit coexistence with both parents. Our results show that speciation was driven by introgression of ecological traits and that speciation with gene flow is possible with a multilocus genetic architecture.
2024
Peñalba, J. V., Runemark, A., Meier, J. I., Singh, P., Wogan, G. O. U., Sánchez-Guillén, R., Mallet, J., et al. (2024). The Role of Hybridization in Species Formation and Persistence. Cold Spring Harbor Perspectives in Biology , a041445. Publisher's VersionAbstract
Hybridization, or interbreeding between different taxa, was traditionally considered to be rare and to have a largely detrimental impact on biodiversity, sometimes leading to the breakdown of reproductive isolation and even to the reversal of speciation. However, modern genomic and analytical methods have shown that hybridization is common in some of the most diverse clades across the tree of life, sometimes leading to rapid increase of phenotypic variability, to introgression of adaptive alleles, to the formation of hybrid species, and even to entire species radiations. In this review, we identify consensus among diverse research programs to show how the field has progressed. Hybridization is a multifaceted evolutionary process that can strongly influence species formation and facilitate adaptation and persistence of species in a rapidly changing world. Progress on testing this hypothesis will require cooperation among different subdisciplines.
penalba_et_al_2024_hybridizationreview.pdf
10.1101/cshperspect.a041445
Page, E., Queste, L. M., Rosser, N., Salazar, P. A., Nadeau, N. J., Mallet, J., Srygley, R. B., et al. (2024). Pervasive mimicry in flight behavior among aposematic butterflies. Proceedings of the National Academy of Sciences, USA , 121 (11), e2300886121. Publisher's VersionAbstract
Flight was a key innovation in the adaptive radiation of insects. However, it is a complex
trait influenced by a large number of interacting biotic and abiotic factors, making it
difficult to unravel the evolutionary drivers. We investigate flight patterns in neotropical
heliconiine butterflies, well known for mimicry of their aposematic wing color patterns.
We quantify the flight patterns (wing beat frequency and wing angles) of 351 individuals
representing 29 heliconiine and 9 ithomiine species belonging to ten color pattern
mimicry groupings. For wing beat frequency and up wing angles, we show that heliconiine
species group by color pattern mimicry affiliation. Convergence of down wing
angles to mimicry groupings is less pronounced, indicating that distinct components of
flight are under different selection pressures and constraints. The flight characteristics
of the Tiger mimicry group are particularly divergent due to convergence with distantly
related ithomiine species. Predator-driven selection for mimicry also explained variation
in flight among subspecies, indicating that this convergence can occur over relatively
short evolutionary timescales. Our results suggest that the flight convergence is driven
by aposematic signaling rather than shared habitat between comimics. We demonstrate
that behavioral mimicry can occur between lineages that have separated over evolutionary
timescales ranging from <0.5 to 70 My.
page_et_al._2024_flight_mimicry_pnas.pdf
Mallet, J. (2024). Colour patterns in Lepidoptera evolved mainly as signals to predators. Heliconius are indeed Müllerian mimics. Reply to Mouy (2022). heliconius.org. Publisher's VersionAbstract
A recent paper in the journal Evolutionary Ecology by Henri Mouy, an independent researcher and theoretician who gives his address as “City of Westminster, London, UK,” argues that aposematism (warning colour) and Müllerian mimicry are problematic hypotheses. Instead, he claims that the bright colours and apparent colour pattern mimicry act primarily as an intraspecific or interspecific communication device to enable aggregation. In this critique, I show that the long-standing understanding that aposematic and mimetic colour patterns communicate directly with predators is much more likely than Mouy’s aggregation hypothesis.
2023
Thawornwattana, Y., Huang, J., Flouri, T., Mallet, J., & Yang, Z. (2023). Inferring the direction of introgression using genomic sequence data. Molecular Biology and Evolution , 40 (8), msad178. Publisher's VersionAbstract
Genomic data are informative about the history of species divergence and interspecific gene flow, including the direction, timing, and strength of gene flow. However, gene flow in opposite directions generates similar patterns in multilocus sequence data, such as reduced sequence divergence between the hybridizing species. As a result, inference of the direction of gene flow is challenging. Here we investigate the information about the direction of gene flow present in genomic sequence data using likelihood-based methods under the multispecies-coalescentwith-introgression model. We analyze the case of two species, and use simulation to examine cases with three or four species. We find that it is easier to infer gene flow from a small population to a large one than in the opposite direction, and easier to infer inflow (gene flow from outgroup species to an ingroup species) than outflow (gene flow from an ingroup species to an outgroup species). It is also easier to infer gene flow if there is a longer time of separate evolution between the initial divergence and subsequent introgression. When introgression is assumed to occur in the wrong direction, the time of introgression tends to be correctly estimated and the Bayesian test of gene flow is often significant, while estimates of introgression probability can be even greater than the true probability. We analyze genomic sequences from Heliconius butterflies to demonstrate that typical genomic datasets are informative about the direction of interspecific gene flow, as well as its timing and strength.
OPEN ACCESS
Thawornwattana, Y., Seixas, F. A., Yang, Z., & Mallet, J. (2023). Major patterns in the introgression history of Heliconius butterflies. eLife , 12, RP90656. Publisher's VersionAbstract
Gene flow between species is an important evolutionary process that can facilitate adaptation and lead to species diversification. It also makes reconstruction of species relationships difficult. Here, we use the full-likelihood multispecies coalescent (MSC) approach to estimate species phylogeny and major introgression events in Heliconius butterflies from whole-genome sequence data. We obtain a robust estimate of species branching order among major clades in the genus, including the "melpomene-silvaniform" group, which shows extensive historical and on-going gene flow. We obtain chromosome-level estimates of key parameters in the species phylogeny, including species divergence times, present-day and ancestral population sizes as well as the direction, timing, and intensity of gene flow. Our analysis leads to a phylogeny with introgression events that differ from those obtained in previous studies. We find that H. aoede most likely represents the earliest- branching lineage of the genus and that "silvaniform" species are paraphyletic within the melpomene-silvaniform group. Our phylogeny provides new, parsimonious histories for the origins of key traits in Heliconius, including pollen feeding and an inversion involved in wing pattern mimicry. Our results demonstrate the power and feasibility of the full-likelihood MSC approach for estimating species phylogeny and key population parameters despite extensive gene flow. The methods used here should be useful for analysis of other difficult species groups with high rates of introgression.
OPEN ACCESS
Xiong, T., Tarikere, S., Rosser, N., Li, X., Yago, M., & Mallet, J. (2023). A polygenic explanation for Haldane's Rule in butterflies. Proceedings of the National Academy of Sciences, USA , 120 (44), e2300959120. Publisher's VersionAbstract
The fitness of animal hybrids follows two empirical rules: hybrids of the heterogametic sex aremore unfit (Haldane’s Rule), and the sex chromosome is disproportionately involved in incompatibility (the large-X/Z effect). Whether these rules result from genetic mechanisms shared across taxa remains unknown, and existing explanations rarely consider female heterogametic taxa such as butterflies. Here, we investigate hybrid incompatibilities in Papilio and Heliconius butterflies, and show that defects coincide with unbalanced introgression between the Z chromosome and its genetic background. This polygenic mechanism predicts both rules because introgressed ancestry on the Z chromosome is more skewed in females, and is more variable than on all autosomes. Therefore, the explanation for both rules in butterflies shares little similarity with prevailing theories relying on dominance.
xiong_et_al_2023_polygenic_haldanes_rule.pdf
2022
Huang, J., Thawornwattana, Y., Flouri, T., Mallet, J., & Yang, Z. (2022). Inference of gene flow between species under misspecified models. Molecular Biology and Evolution , 39 (12), 1-19. Publisher's VersionAbstract
Genomic sequence data provide a rich source of information about the history of species divergence and interspecific
hybridization or introgression. Despite recent advances in genomics and statistical methods, it remains challenging to infer
gene flow, and as a result, one may have to estimate introgression rates and times under misspecified models. Here we
use mathematical analysis and computer simulation to examine estimation bias and issues of interpretation when the
model of gene flow is misspecified in analysis of genomic datasets, for example, if introgression is assigned to the wrong
lineages. In the case of two species, we establish a correspondence between the migration rate in the continuous migration
model and the introgression probability in the introgression model. When gene flow occurs continuously through time
but in the analysis is assumed to occur at a fixed time point, common evolutionary parameters such as species divergence
times are surprisingly well estimated. However, the time of introgression tends to be estimated towards the recent end of
the period of continuous gene flow. When introgression events are assigned incorrectly to the parental or daughter
lineages, introgression times tend to collapse onto species divergence times, with introgression probabilities underestimated.
Overall, our analyses suggest that the simple introgression model is useful for extracting information concerning
between-specific gene flow and divergence even when the model may be misspecified. However, for reliable inference of
gene flow it is important to include multiple samples per species, in particular, from hybridizing species.
OPEN ACCESS
Xiong, T., & Mallet, J. (2022). On the impermanence of species: the collapse of genetic incompatibilities in hybridizing populations. Evolution , 76 (11), 2498-2512. Publisher's VersionAbstract
Species pairs often become genetically incompatible during divergence, which is an important source of reproductive isolation. An idealized picture is often painted where incompatibility alleles accumulate and fix between diverging species. However, recent studies have shown both that incompatibilities can collapse with ongoing hybridization, and that incompatibility loci can be polymorphic within species. This paper suggests some general rules for the behavior of incompatibilities under hybridization. In particular, we argue that redundancy of genetic pathways can strongly affect the dynamics of intrinsic incompatibilities. Since
fitness in genetically redundant systems is unaffected by introducing a few foreign alleles, higher redundancy decreases the stability of incompatibilities during hybridization, but also increases tolerance of incompatibility polymorphism within species. We use simulations and theories to show that this principle leads to two types of collapse: in redundant systems, exemplified by classical Dobzhansky–Muller incompatibilities, collapse is continuous and approaches a quasi-neutral polymorphism between broadly sympatric species, often as a result of isolation-by-distance. In nonredundant systems, exemplified by co-evolution among genetic elements, incompatibilities are often stable, but can collapse abruptly with spatial traveling waves. As both types are common, the proposed principle may be useful in understanding the abundance of genetic incompatibilities in natural populations.
xiong_mallet_2022_impermanence_of_species.pdf
Mallet, J., & Mullen, S. P. (2022). Reproductive isolation is a heuristic, not a measure: a commentary on Westram et al., 2022. Journal of Evolutionary Biology , 35 (9), 1175-1182. Publisher's VersionAbstract
Reproductive isolation is the heuristic basis of the biological species concept, but what is it? Westram et al. (this issue) propose that it is a measurable quantity, "barrier strength," that prevents gene flow among populations. However, their attempt to make the concept of reproductive isolation more scientific is unlikely to satisfy the diverse opinions of all evolutionary biologists. There are many different opinions about the nature of species, even under the biological species concept. Complete reproductive isolation, where gene flow is effectively zero, is regarded by some biologists as an important end point of speciation. Others, including Westram et al., argue for a more nuanced approach, and they also suggest that reproductive isolation may differ in different parts of the genome due to variation in genetic linkage to divergently selected loci. In contrast to both these approaches, we favour as a key criterion of speciation the stable coexistence of divergent populations in sympatry. Obviously, such populations must be reproductively isolated in some sense, but neither the fraction of the genome that is exchanged, nor measures of overall barrier strength acting on neutral variation will yield very precise predictions as to species status. Although an overall measure of reproductive isolation is virtually unattainable for these reasons, its early generation components, such as assortative mating, divergent selection, or hybrid inviability and sterility are readily measurable and remain informative. For example, we can make the prediction that to remain divergent in sympatry, almost all sexual species will require strong assortative mating, as well as some sort of ecological or intrinsic selection against hybrids and introgressed variants.
mallet_mullen_2022_on_westram_ri_2.pdf
OPEN ACCESS
Rosser, N., Seixas, F., & Mallet, J. (2022). Sympatric speciation by allochrony? Molecular Ecology , 31, 3975-3978. https://doi.org/10.1111/mec.16599Abstract
Sympatric speciation was once thought most improbable, but careful
study of some systems, particularly the apple maggot (Rhagoletis
pomonella
) and related Rhagoletis species, has led to its reinstatement as
a likely mode of speciation in some cases. Different species and host
races in this clade of flies often have highly specialized host preference,
and along with frequent evolutionary shifts to different fruit species
between sister taxa, there is a likely effect of the timing of adult
emergence that follows host fruiting phenology. This is known as
"allochronic" isolation (from the Greek, meaning "different timing").
This overview covers recent discoveries by Inskeep et al. (Molecular Ecology, 2021) showing
how allochrony is a major factor in preventing gene flow between a pair
of sister species of Rhagoletis on different host fruits. Although the
authors do not claim to prove sympatric speciation, it does seem very
likely, and the work clearly underscores how readily host shifts via
allochrony can aid sympatric speciation.
rosser_seixas_mallet_2022_allochrony_commentary.pdf
Mallet, J. (2022). The making of a moth man (book review of: Grant, Bruce S. 2021. Observing Evolution). Evolution , 76 (6), 1362-1365. Publisher's Version mallet_2022_the_making_of_a_moth_man.pdf
Xiong, T., Li, X., Yago, M., & Mallet, J. (2022). Admixture of evolutionary rates across a butterfly hybrid zone. eLife , 11, e78135. Publisher's VersionAbstract
Hybridization is a major evolutionary force that can erode genetic differentiation between species, whereas reproductive isolation maintains such differentiation. In studying a hybrid zone between the swallowtail butterflies Papilio syfanius and Papilio maackii (Lepidoptera: Papilionidae), we made the unexpected discovery that genomic substitution rates are unequal between the parental species. This phenomenon creates a novel process in hybridization, where genomic regions most affected by gene flow evolve at similar rates between species, while genomic regions with strong reproductive isolation evolve at species-specific rates. Thus, hybridization mixes evolutionary rates in a way similar to its effect on genetic ancestry. Using coalescent theory, we show that the rate-mixing process provides distinct information about levels of gene flow across different parts of genomes, and the degree of rate-mixing can be predicted quantitatively from relative sequence divergence (FSTFST) between the hybridizing species at equilibrium. Overall, we demonstrate that reproductive isolation maintains not only genomic differentiation, but also the rate at which differentiation accumulates. Thus, asymmetric rates of evolution provide an additional signature of loci involved in reproductive isolation
Mallet, J., Seixas, F., & Thawornwattana, Y. (2022). 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.
mallet_et_al._2022_enc_biodiv_species_concepts.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 , 71 (5), 1159-1177. 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
2021
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 , 28, 2912-2930. Publisher's Version
Mallet, J. (2021). Subspecies, Semispecies, Superspecies. In Encyclopedia of Biodiversity . Elsevier. mallet_2021_subspecies_superspecies_semispecies_encyc_of_biodiv.pdf
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.
edelman_mallet_2021_ann_rev_genet_introgression.pdf
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.

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