The Role of Hybridization in Species Formation and Persistence.

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.

On the fast track: hybrids adapt more rapidly than parental populations in a novel environment.

Rates of hybridization are predicted to increase due to climate change and human activity that cause redistribution of species and bring previously isolated populations into contact. At the same time climate change leads to rapid changes in the environment, requiring populations to adapt rapidly in order to survive. A few empirical cases suggest hybridization can facilitate adaptation despite its potential for incompatibilities and deleterious fitness consequences. Here we use simulations and Fisher's Geometric model to evaluate the conditions and time frame of adaptation via hybridization in both diploids and haplodiploids. We find that hybrids adapt faster to new environments compared to parental populations in nearly all simulated scenarios, generating a fitness advantage that can offset intrinsic incompatibilities and last for tens of generations, regardless of whether the population was diploid or haplodiploid. Our results highlight the creative role of hybridization and suggest that hybridization may help contemporary populations adapt to the changing climate. However, adaptation by hybrids may well happen at the cost of reduced biodiversity, if previously isolated lineages collapse into one.

Mechanisms of Intrinsic Postzygotic Isolation: From Traditional Genic and Chromosomal Views to Genomic and Epigenetic Perspectives.

Intrinsic postzygotic isolation typically appears as reduced viability or fertility of interspecific hybrids caused by genetic incompatibilities between diverged parental genomes. Dobzhansky-Muller interactions among individual genes, and chromosomal rearrangements causing problems with chromosome synapsis and recombination in meiosis, have both long been considered as major mechanisms behind intrinsic postzygotic isolation. Recent research has, however, suggested that the genetic basis of intrinsic postzygotic isolation can be more complex and involves, for example, overall divergence of the DNA sequence or epigenetic changes. Here, we review the mechanisms of intrinsic postzygotic isolation from genic, chromosomal, genomic, and epigenetic perspectives across diverse taxa. We provide empirical evidence for these mechanisms, discuss their importance in the speciation process, and highlight questions that remain unanswered.

Genetic modification of the ant Lasius niger using CRISPR-Cas9 technology.

CRISPR-Cas9 has become one of the most prominent gene editing tools available and it has been utilized in various organisms from bacteria to fungi, plants, and animals. In this study, we developed a CRISPR-Cas9 protocol for the black garden ant Lasius niger, a common and easily available study species for lab and field experiments. To create indel mutations using CRISPR-Cas9 in L. niger, we targeted three different locations in a well-studied eye pigmentation gene cinnabar, generating several mutations that disrupt the ommochrome biosynthesis pathway and result in the lack of the pigment and therefore, abnormal eye coloration in adult workers. We also developed a protocol to collect L. niger eggs, inject them with CRISPR-Cas9 construct, and rear the eggs into mature adult workers with the assistance of nursing workers. We demonstrated for the first time in L. niger that CRISPR-Cas9 is an excellent tool to create targeted mutations for this species. Our protocol can be referred to when developing similar studies for other species of ants and eusocial insects.

Rapid and predictable genome evolution across three hybrid ant populations.

Hybridization is frequent in the wild but it is unclear when admixture events lead to predictable outcomes and if so, at what timescale. We show that selection led to correlated sorting of genetic variation rapidly after admixture in 3 hybrid Formica aquilonia × F. polyctena ant populations. Removal of ancestry from the species with the lowest effective population size happened in all populations, consistent with purging of deleterious load. This process was modulated by recombination rate variation and the density of functional sites. Moreover, haplotypes with signatures of positive selection in either species were more likely to fix in hybrids. These mechanisms led to mosaic genomes with comparable ancestry proportions. Our work demonstrates predictable evolution over short timescales after admixture in nature.

Whole-genome analysis of multiple wood ant population pairs supports similar speciation histories, but different degrees of gene flow, across their European ranges.

The application of demographic history modelling and inference to the study of divergence between species has become a cornerstone of speciation genomics. Speciation histories are usually reconstructed by analysing single populations from each species, assuming that the inferred population history represents the actual speciation history. However, this assumption may not be met when species diverge with gene flow, for example, when secondary contact may be confined to specific geographic regions. Here, we tested whether divergence histories inferred from heterospecific populations may vary depending on their geographic locations, using the two wood ant species Formica polyctena and F. aquilonia. We performed whole-genome resequencing of 20 individuals sampled in multiple locations across the European ranges of both species. Then, we reconstructed the histories of distinct heterospecific population pairs using a coalescent-based approach. Our analyses always supported a scenario of divergence with gene flow, suggesting that divergence started in the Pleistocene (c. 500 kya) and occurred with continuous asymmetrical gene flow from F. aquilonia to F. polyctena until a recent time, when migration became negligible (2-19 kya). However, we found support for contemporary gene flow in a sympatric pair from Finland, where the species hybridise, but no signature of recent bidirectional gene flow elsewhere. Overall, our results suggest that divergence histories reconstructed from a few individuals may be applicable at the species level. Nonetheless, the geographical context of populations chosen to represent their species should be taken into account, as it may affect estimates of migration rates between species when gene flow is spatially heterogeneous.

Assembly of a Hybrid Formica aquilonia × F. polyctena Ant Genome From a Haploid Male.

Formica red wood ants are a keystone species of boreal forest ecosystems and an emerging model system in the study of speciation and hybridization. Here, we performed a standard DNA extraction from a single, field-collected Formica aquilonia × Formica polyctena haploid male and assembled its genome using ~60× of PacBio long reads. After polishing and contaminant removal, the final assembly was 272 Mb (4687 contigs, N50 = 1.16 Mb). Our reference genome contains 98.5% of the core Hymenopteran BUSCOs and was pseudo-scaffolded using the assembly of a related species, F. selysi (28 scaffolds, N50 = 8.49 Mb). Around one-third of the genome consists of repeats, and 17 426 gene models were annotated using both protein and RNAseq data (97.4% BUSCO completeness). This resource is of comparable quality to the few other single individual insect genomes assembled to date and paves the way to genomic studies of admixture in natural populations and comparative genomic approaches in Formica wood ants.

Differences in Thermal Tolerance between Parental Species Could Fuel Thermal Adaptation in Hybrid Wood Ants.

AbstractGenetic variability is essential for adaptation and could be acquired via hybridization with a closely related lineage. We use ants to investigate thermal adaptation and the link between temperature and genetic variation arising from hybridization. We test for differences in cold and heat tolerance between Finnish Formica polyctena and Formica aquilonia wood ants and their naturally occurring hybrids. Using workers, we find that the parental individuals differ in both cold and heat tolerances and express thermal limits that reflect their global distributions. Hybrids, however, cannot combine thermal tolerance of parental species as they have the same heat tolerance as F. polyctena but not the same cold tolerance as F. aquilonia. We then focus on a single hybrid population to investigate the relationship between temperature variation and genetic variation across 16 years using reproductive individuals. On the basis of the thermal tolerance results, we expected the frequency of putative F. polyctena alleles to increase in warm years and F. aquilonia alleles to increase in cold years. We find support for this in hybrid males but not in hybrid females. These results contribute to understanding the outcomes of hybridization, which may be sex specific or depend on the environment. Furthermore, genetic variability resulting from hybridization could help hybrid wood ants cope with changing thermal conditions.

Instability of natural selection at candidate barrier loci underlying speciation in wood ants.

Speciation underlies the generation of novel biodiversity. Yet, there is much to learn about how natural selection shapes genomes during speciation. Selection is assumed to act against gene flow at barrier loci, promoting reproductive isolation. However, evidence for gene flow and selection is often indirect and we know very little about the temporal stability of barrier loci. Here we utilize haplodiploidy to identify candidate male barrier loci in hybrids between two wood ant species. As ant males are haploid, they are expected to reveal recessive barrier loci, which can be masked in diploid females if heterozygous. We then test for barrier stability in a sample collected 10 years later and use survival analysis to provide a direct measure of natural selection acting on candidate male barrier loci. We find multiple candidate male barrier loci scattered throughout the genome. Surprisingly, a proportion of them are not stable after 10 years, natural selection apparently switching from acting against to favouring introgression in the later sample. Instability of the barrier effect and natural selection for introgressed alleles could be due to environment-dependent selection, emphasizing the need to consider temporal variation in the strength of natural selection and the stability of the barrier effect at putative barrier loci in future speciation work.

Towards the completion of speciation: the evolution of reproductive isolation beyond the first barriers.

Speciation, that is, the evolution of reproductive barriers eventually leading to complete isolation, is a crucial process generating biodiversity. Recent work has contributed much to our understanding of how reproductive barriers begin to evolve, and how they are maintained in the face of gene flow. However, little is known about the transition from partial to strong reproductive isolation (RI) and the completion of speciation. We argue that the evolution of strong RI is likely to involve different processes, or new interactions among processes, compared with the evolution of the first reproductive barriers. Transition to strong RI may be brought about by changing external conditions, for example, following secondary contact. However, the increasing levels of RI themselves create opportunities for new barriers to evolve and, and interaction or coupling among barriers. These changing processes may depend on genomic architecture and leave detectable signals in the genome. We outline outstanding questions and suggest more theoretical and empirical work, considering both patterns and processes associated with strong RI, is needed to understand how speciation is completed. This article is part of the theme issue 'Towards the completion of speciation: the evolution of reproductive isolation beyond the first barriers'.

Understanding Admixture: Haplodiploidy to the Rescue.

Hybridization has broad evolutionary consequences, from fueling or counteracting speciation to facilitating adaptation to novel environments. Hybridization and subsequent introgression appear widespread along the tree of life. However, our understanding of how distinct evolutionary forces shape admixed genomes and the fate of introgressed genetic variants remains scarce. Most admixture research in animals has focused on diploid organisms. We propose that haplodiploid organisms can help resolve open questions about the genomic consequences of hybridization in natural populations. The ploidy difference between haploid males and diploid females, the availability of genome-wide male haplotypes, and ongoing cases of admixture make haplodiploid organisms promising models to improve our knowledge with regards to the evolution of hybrid genomes.

Genome organization and molecular characterization of the three Formica exsecta viruses-FeV1, FeV2 and FeV4.

We present the genome organization and molecular characterization of the three Formica exsecta viruses, along with ORF predictions, and functional annotation of genes. The Formica exsecta virus-4 (FeV4; GenBank ID: MF287670) is a newly discovered negative-sense single-stranded RNA virus representing the first identified member of order Mononegavirales in ants, whereas the Formica exsecta virus-1 (FeV1; GenBank ID: KF500001), and the Formica exsecta virus-2 (FeV2; GenBank ID: KF500002) are positive single-stranded RNA viruses initially identified (but not characterized) in our earlier study. The new virus FeV4 was found by re-analyzing data from a study published earlier. The Formica exsecta virus-4 genome is 9,866 bp in size, with an overall G + C content of 44.92%, and containing five predicted open reading frames (ORFs). Our bioinformatics analysis indicates that gaps are absent and the ORFs are complete, which based on our comparative genomics analysis suggests that the genomes are complete. Following the characterization, we validate virus infection for FeV1, FeV2 and FeV4 for the first time in field-collected worker ants. Some colonies were infected by multiple viruses, and the viruses were observed to infect all castes, and multiple life stages of workers and queens. Finally, highly similar viruses were expressed in adult workers and queens of six other Formica species: F. fusca, F. pressilabris, F. pratensis, F. aquilonia, F. truncorum and F. cinerea. This research indicates that viruses can be shared between ant species, but further studies on viral transmission are needed to understand viral infection pathways.

Conflict between heterozygote advantage and hybrid incompatibility in haplodiploids (and sex chromosomes).

In many diploid species, the sex chromosomes play a special role in mediating reproductive isolation. In haplodiploids, where females are diploid and males haploid, the whole genome behaves similarly to the X/Z chromosomes of diploids. Therefore, haplodiploid systems can serve as a model for the role of sex chromosomes in speciation and hybridization. A previously described population of Finnish Formica wood ants displays genome-wide signs of ploidally and sexually antagonistic selection resulting from hybridization. Here, hybrid females have increased survivorship but hybrid males are inviable. To understand how the unusual hybrid population may be maintained, we developed a mathematical model with hybrid incompatibility, female heterozygote advantage, recombination and assortative mating. The rugged fitness landscape resulting from the co-occurrence of heterozygote advantage and hybrid incompatibility results in a sexual conflict in haplodiploids, which is caused by the ploidy difference. Thus, whereas heterozygote advantage always promotes long-term polymorphism in diploids, we find various outcomes in haplodiploids in which the population stabilizes either in favour of males, females or via maximizing the number of introgressed individuals. We discuss these outcomes with respect to the potential long-term fate of the Finnish wood ant population and provide approximations for the extension of the model to multiple incompatibilities. Moreover, we highlight the general implications of our results for speciation and hybridization in haplodiploids versus diploids and how the described fitness relationships could contribute to the outstanding role of sex chromosomes as hotspots of sexual antagonism and genes involved in speciation.

Sex allocation conflict between queens and workers in Formica pratensis wood ants predicts seasonal sex ratio variation.

Sex allocation theory predicts parents should adjust their investment in male and female offspring in a way that increases parental fitness. This has been shown in several species and selective contexts. Yet, seasonal sex ratio variation within species and its underlying causes are poorly understood. Here, we study sex allocation variation in the wood ant Formica pratensis. This species displays conflict over colony sex ratio as workers and queens prefer different investment in male and female offspring, owing to haplodiploidy and relatedness asymmetries. It is unique among Formica ants because it produces two separate sexual offspring cohorts per season. We predict sex ratios to be closer to queen optimum in the early cohort but more female-biased and closer to worker optimum in the later one. This is because the power of workers to manipulate colony sex ratio varies seasonally with the availability of diploid eggs. Consistently, more female-biased sex ratios in the later offspring cohort over a three-year sampling period from 93 colonies clearly support our prediction. The resulting seasonal alternation of sex ratios between queen and worker optima is a novel demonstration how understanding constraints of sex ratio adjustment increases our ability to predict sex ratio variation.

Introgression in hybrid ants is favored in females but selected against in males.

Hybridization is not a mere reproductive dead end but has been suggested to play a central role in speciation, for example, by introducing adaptive genetic variation. Our previous study uncovered a unique consequence of hybridization in Formica ants. In a population including two isolated but partially introgressed genetic groups, the females have an apparent hybrid background, whereas the males do not. This situation results in large-scale differences between male and female genomes that are stable throughout generations. Here, we compare genotypes from different developmental stages to investigate how sex-specific introgression and genetic differences between sexes are maintained. We show that strong selection rather than sex-dependent transmission maintains the genetic differences between sexes. All genotype combinations are produced and observed in the eggs of both sexes, but the alleles acquired through hybridization disappear from the haploid males during development from egg to adult as their frequencies drop toward zero. However, the same introgressed alleles increase in frequency and are favored when heterozygous in the females. Genotypes eliminated from males most likely represent incompatibilities arising from hybridization. Our results show an unusual situation of opposite selection, where introgression is favored in diploid females but selected against in haploid males. This finding suggests that introgressed genomic regions harbor both fitness-enhancing and -reducing elements. Our work highlights the complex consequences of hybridization and provides a rare opportunity to observe natural selection in real time in nature.

Not only for egg yolk--functional and evolutionary insights from expression, selection, and structural analyses of Formica ant vitellogenins.

Vitellogenin (Vg), a storage protein, has been extensively studied for its egg-yolk precursor role, and it has been suggested to be fundamentally involved in caste differences in social insects. More than one Vg copy has been reported in several oviparous species, including ants. However, the number and function of different Vgs, their phylogenetic relatedness, and their role in reproductive queens and nonreproductive workers have been studied in few species only. We studied caste-biased expression of Vgs in seven Formica ant species. Only one copy of conventional Vg was identified in Formica species, and three Vg homologs, derived from ancient duplications, which represent yet undiscovered Vg-like genes. We show that each of these Vg-like genes is present in all studied Hymenoptera and some of them in other insects as well. We show that after each major duplication event, at least one of the Vg-like genes has experienced a period of positive selection. This, combined with the observation that the Vg-like genes have acquired or lost specific protein domains suggests sub- or neofunctionalization between Vg and the duplicated genes. In contrast to earlier studies, Vg was not consistently queen biased in its expression, and the caste bias of the three Vg-like genes was highly variable among species. Furthermore, a truncated and Hymenoptera-specific Vg-like gene, Vg-like-C, was consistently worker biased. Multispecies comparisons are essential for Vg expression studies, and for gene expression studies in general, as we show that expression and also, putative functions cannot be generalized even among closely related species.

Insights into the evolution of the CSP gene family through the integration of evolutionary analysis and comparative protein modeling.

Insect chemical communication and chemosensory systems rely on proteins coded by several gene families. Here, we have combined protein modeling with evolutionary analysis in order to study the evolution and structure of chemosensory proteins (CSPs) within arthropods and, more specifically, in ants by using the data available from sequenced genomes. Ants and other social insects are especially interesting model systems for the study of chemosensation, as they communicate in a highly complex social context and much of their communication relies on chemicals. Our ant protein models show how this complexity has shaped CSP evolution; the proteins are highly modifiable by their size, surface charge and binding pocket. Based on these findings, we divide ant CSPs into three groups: typical insect CSPs, an ancient 5-helical CSP and hymenopteran CSPs with a small binding pocket, and suggest that these groups likely serve different functions. The hymenopteran CSPs have duplicated repeatedly in individual ant lineages. In these CSPs, positive selection has driven surface charge changes, an observation which has possible implications for the interaction between CSPs and ligands or odorant receptors. Our phylogenetic analysis shows that within the Arthropoda the only highly conserved gene is the ancient 5-helical CSP, which is likely involved in an essential ubiquitous function rather than chemosensation. During insect evolution, the 6-helical CSPs have diverged and perform chemosensory functions among others. Our results contribute to the general knowledge of the structural differences between proteins underlying chemosensation and highlight those protein properties which have been affected by adaptive evolution.

Segregation distortion causes large-scale differences between male and female genomes in hybrid ants.

Hybridization in isolated populations can lead either to hybrid breakdown and extinction or in some cases to speciation. The basis of hybrid breakdown lies in genetic incompatibilities between diverged genomes. In social Hymenoptera, the consequences of hybridization can differ from those in other animals because of haplodiploidy and sociality. Selection pressures differ between sexes because males are haploid and females are diploid. Furthermore, sociality and group living may allow survival of hybrid genotypes. We show that hybridization in Formica ants has resulted in a stable situation in which the males form two highly divergent gene pools whereas all the females are hybrids. This causes an exceptional situation with large-scale differences between male and female genomes. The genotype differences indicate strong transmission ratio distortion depending on offspring sex, whereby the mother transmits some alleles exclusively to her daughters and other alleles exclusively to her sons. The genetic differences between the sexes and the apparent lack of multilocus hybrid genotypes in males can be explained by recessive incompatibilities which cause the elimination of hybrid males because of their haploid genome. Alternatively, differentiation between sexes could be created by prezygotic segregation into male-forming and female-forming gametes in diploid females. Differentiation between sexes is stable and maintained throughout generations. The present study shows a unique outcome of hybridization and demonstrates that hybridization has the potential of generating evolutionary novelties in animals.