Evolutionary genomics of socially polymorphic populations of Pogonomyrmex californicus.

BACKGROUND: Social insects vary considerably in their social organization both between and within species. In the California harvester ant, Pogonomyrmex californicus (Buckley 1867), colonies are commonly founded and headed by a single queen (haplometrosis, primary monogyny). However, in some populations in California (USA), unrelated queens cooperate not only during founding (pleometrosis) but also throughout the life of the colony (primary polygyny). The genetic architecture and evolutionary dynamics of this complex social niche polymorphism (haplometrosis vs pleometrosis) have remained unknown. RESULTS: We provide a first analysis of its genomic basis and evolutionary history using population genomics comparing individuals from a haplometrotic population to those from a pleometrotic population. We discovered a recently evolved (< 200 k years), 8-Mb non-recombining region segregating with the observed social niche polymorphism. This region shares several characteristics with supergenes underlying social polymorphisms in other socially polymorphic ant species. However, we also find remarkable differences from previously described social supergenes. Particularly, four additional genomic regions not in linkage with the supergene show signatures of a selective sweep in the pleometrotic population. Within these regions, we find for example genes crucial for epigenetic regulation via histone modification (chameau) and DNA methylation (Dnmt1). CONCLUSIONS: Altogether, our results suggest that social morph in this species is a polygenic trait involving a potential young supergene. Further studies targeting haplo- and pleometrotic individuals from a single population are however required to conclusively resolve whether these genetic differences underlie the alternative social phenotypes or have emerged through genetic drift.

Social isolation shortens lifespan through oxidative stress in ants.

Social isolation negatively affects health, induces detrimental behaviors, and shortens lifespan in social species. Little is known about the mechanisms underpinning these effects because model species are typically short-lived and non-social. Using colonies of the carpenter ant Camponotus fellah, we show that social isolation induces hyperactivity, alters space-use, and reduces lifespan via changes in the expression of genes with key roles in oxidation-reduction and an associated accumulation of reactive oxygen species. These physiological effects are localized to the fat body and oenocytes, which perform liver-like functions in insects. We use pharmacological manipulations to demonstrate that the oxidation-reduction pathway causally underpins the detrimental effects of social isolation on behavior and lifespan. These findings have important implications for our understanding of how social isolation affects behavior and lifespan in general.

Optimizing ddRAD sequencing for population genomic studies with ddgRADer.

Double-digest Restriction-site Associated DNA sequencing (ddRADseq) is widely used to generate genomic data for non-model organisms in evolutionary and ecological studies. Along with affordable paired-end sequencing, this method makes population genomic analyses more accessible. However, multiple factors should be considered when designing a ddRADseq experiment, which can be challenging for new users. The generated data often suffer from substantial read overlaps and adaptor contamination, severely reducing sequencing efficiency and affecting data quality. Here, we analyse diverse datasets from the literature and carry out controlled experiments to understand the effects of enzyme choice and size selection on sequencing efficiency. The empirical data reveal that size selection is imprecise and has limited efficacy. In certain scenarios, a substantial proportion of short fragments pass below the lower size-selection cut-off resulting in low sequencing efficiency. However, enzyme choice can considerably mitigate inadvertent inclusion of these shorter fragments. A simple model based on these experiments is implemented to predict the number of genomic fragments generated after digestion and size selection, number of SNPs genotyped, number of samples that can be multiplexed and the expected sequencing efficiency. We developed ddgRADer - http://ddgrader.haifa.ac.il/ - a user-friendly webtool and incorporated these calculations to aid in ddRADseq experimental design while optimizing sequencing efficiency. This tool can also be used for single enzyme protocols such as Genotyping-by-Sequencing. Given user-defined study goals, ddgRADer recommends enzyme pairs and allows users to compare and choose enzymes and size-selection criteria. ddgRADer improves the accessibility and ease of designing ddRADseq experiments and increases the probability of success of the first population genomic study conducted in labs with no prior experience in genomics.

Ancient DNA from a lost Negev Highlands desert grape reveals a Late Antiquity wine lineage.

Recent excavations of Late Antiquity settlements in the Negev Highlands of southern Israel uncovered a society that established commercial-scale viticulture in an arid environment [D. Fuks et al., Proc. Natl. Acad. Sci. U.S.A. 117, 19780-19791 (2020)]. We applied target-enriched genome-wide sequencing and radiocarbon dating to examine grapevine pips that were excavated at three of these sites. Our analyses revealed centuries long and continuous grape cultivation in the Southern Levant. The genetically diverse pips also provided clues to ancient cultivation strategies aimed at improving agricultural productivity and ensuring food security. Applying genomic prediction analysis, a pip dated to the eighth century CE was determined to likely be from a white grape, to date the oldest to be identified. In a kinship analysis, another pip was found to be descendant from a modern Greek cultivar and was thus linked with several popular historic wines that were once traded across the Byzantine Empire. These findings shed light on historical Byzantine trading networks and on the genetic contribution of Levantine varieties to the classic Aegean landscape.

Putting hornets on the genomic map.

Hornets are the largest of the social wasps, and are important regulators of insect populations in their native ranges. Hornets are also very successful as invasive species, with often devastating economic, ecological and societal effects. Understanding why these wasps are such successful invaders is critical to managing future introductions and minimising impact on native biodiversity. Critical to the management toolkit is a comprehensive genomic resource for these insects. Here we provide the annotated genomes for two hornets, Vespa crabro and Vespa velutina. We compare their genomes with those of other social Hymenoptera, including the northern giant hornet Vespa mandarinia. The three hornet genomes show evidence of selection pressure on genes associated with reproduction, which might facilitate the transition into invasive ranges. Vespa crabro has experienced positive selection on the highest number of genes, including those putatively associated with molecular binding and olfactory systems. Caste-specific brain transcriptomic analysis also revealed 133 differentially expressed genes, some of which are associated with olfactory functions. This report provides a spring-board for advancing our understanding of the evolution and ecology of hornets, and opens up opportunities for using molecular methods in the future management of both native and invasive populations of these over-looked insects.

The social supergene dates back to the speciation time of two Solenopsis fire ant species.

Colony social organization of multiple Solenopsis fire ant species is determined by a supergene with two haplotypes SB and Sb, which are similar to X/Y sex chromosomes. The ancestral monogyne (single-queen) social form has been associated with homozygous SB/SB queens, while queens in colonies with the derived polygyne (multi-queen) social structure are heterozygous SB/Sb. By comparing 14 Solenopsis invicta genomes and the outgroup S. fugax, we dated the formation of the supergene to 1.1 (0.7-1.6) million years ago, much older than previous estimates, and close to the estimated time of speciation of the two socially polymorphic species S. invicta and S. richteri. We also used 12 S. invicta and S. richteri genomes to compare the evolutionary distances between these species and the distances between the social haplotypes, and found them to be similar. A phylogenetic analysis suggested that the monophyletic Sb clade is more closely related to S. richteri SB haplotypes than to S. invicta SB haplotypes. We conclude that the formation of the supergene occurred concomitantly with the process of speciation of the Solenopsis socially-polymorphic clade, and hypothesize that the Sb variant first arouse in one incipiently-speciating population and then introgressed into the other populations or species.

Comparative study of population genomic approaches for mapping colony-level traits.

Social insect colonies exhibit colony-level phenotypes such as social immunity and task coordination, which are produced by the individual phenotypes. Mapping the genetic basis of such phenotypes requires associating the colony-level phenotype with the genotypes in the colony. In this paper, we examine alternative approaches to DNA extraction, library construction, and sequencing for genome wide association studies (GWAS) of colony-level traits using a population sample of Cataglyphis niger ants. We evaluate the accuracy of allele frequency estimation from sequencing a pool of individuals (pool-seq) from each colony using either whole-genome sequencing or reduced representation genomic sequencing. Based on empirical measurement of the experimental noise in sequenced DNA pools, we show that reduced representation pool-seq is drastically less accurate than whole-genome pool-seq. Surprisingly, normalized pooling of samples did not result in greater accuracy than un-normalized pooling. Subsequently, we evaluate the power of the alternative approaches for detecting quantitative trait loci (QTL) of colony-level traits by using simulations that account for an environmental effect on the phenotype. Our results can inform experimental designs and enable optimizing the power of GWAS depending on budget, availability of samples and research goals. We conclude that for a given budget, sequencing un-normalized pools of individuals from each colony provides optimal QTL detection power.

Publisher Correction: The Interplay between Incipient Species and Social Polymorphism in the Desert Ant Cataglyphis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Has gene expression neofunctionalization in the fire ant antennae contributed to queen discrimination behavior?

Queen discrimination behavior in the fire ant Solenopsis invicta maintains its two types of societies: colonies with one (monogyne) or many (polygyne) queens, yet the underlying genetic mechanism is poorly understood. This behavior is controlled by two supergene alleles, SB and Sb, with ~600 genes. Polygyne workers, having either the SB/SB or SB/Sb genotype, accept additional SB/Sb queens into their colonies but kill SB/SB queens. In contrast, monogyne workers, all SB/SB, reject all additional queens regardless of genotype. Because the SB and Sb alleles have suppressed recombination, determining which genes within the supergene mediate this differential worker behavior is difficult. We hypothesized that the alternate worker genotypes sense queens differently because of the evolution of differential expression of key genes in their main sensory organ, the antennae. To identify such genes, we sequenced RNA from four replicates of pooled antennae from three classes of workers: monogyne SB/SB, polygyne SB/SB, and polygyne SB/Sb. We identified 81 differentially expressed protein-coding genes with 13 encoding potential chemical metabolism or perception proteins. We focused on the two odorant perception genes: an odorant receptor SiOR463 and an odorant-binding protein SiOBP12. We found that SiOR463 has been lost in the Sb genome. In contrast, SiOBP12 has an Sb-specific duplication, SiOBP12b', which is expressed in the SB/Sb worker antennae, while both paralogs are expressed in the body. Comparisons with another fire ant species revealed that SiOBP12b' antennal expression is specific to S. invicta and suggests that queen discrimination may have evolved, in part, through expression neofunctionalization.

The Interplay between Incipient Species and Social Polymorphism in the Desert Ant Cataglyphis.

In social insects, due to considerable polyphenism as well as high level of hybridization, the delimitation of species can be challenging. The genus Cataglyphis presents a high level of diversification, making it an excellent model with which to study evolutionary paths. Israel appears to be a "hot spot" for recent speciation in this genus. Although previous studies have described multiple species of Cataglyphis in Israel, a recent genetic study has questioned the existence of some of these historically described species. The present study focuses on an apparent species complex, the C. niger species complex which includes C. niger, C. savigyi, and C. drusus that are distinguishable by their mitochondrial DNA (and therefore named mitotypes) but not by their nuclear DNA. Using a multi-method approach (genetics, chemistry and behavior), we show that these mitotypes also differ in their social structures and are readily distinguishable by their cuticular hydrocarbons profiles. While most populations of the different mitotypes are allopatric, at our study site they are sympatric, but nonetheless maintain the observed differences between them. This raises the evolutionary question: Are these incipient species that have diverged with gene flow, or is this a case of social and chemical polymorphism that is maintained within a single species? Unveiling the interplay between social polyphenism and species segregation is at the core of evolutionary biology.

Speciation and hybridization in invasive fire ants.

BACKGROUND: A major focus of evolutionary biology is the formation of reproductive barriers leading to divergence and ultimately, speciation. Often, it is not clear whether the separation of populations is complete or if there still is ongoing gene flow in the form of rare cases of admixture, known as isolation with migration. Here, we studied the speciation of two fire ant species, Solenopsis invicta and Solenopsis richteri, both native to South America, both inadvertently introduced to North America in the early twentieth century. While the two species are known to admix in the introduced range, in the native range no hybrids were found. RESULTS: We conducted a population genomic survey of native and introduced populations of the two species using reduced representation genomic sequencing of 337 samples. Using maximum likelihood analysis over native range samples, we found no evidence of any gene flow between the species since they diverged. We estimated their time of divergence to 190,000 (100,000-350,000) generations ago. Modelling the demographic history of native and introduced S. invicta populations, we evaluated their divergence times and historic and contemporary population sizes, including the original founder population in North America, which was estimated at 26 (10-93) unrelated singly-mated queens. CONCLUSIONS: We provide evidence for complete genetic isolation maintained between two invasive species in their natïve range, based, for the first time, on large scale genomic data analysis. The results lay the foundations for further studies into different stages in the formation of genetic barriers in dynamic, invasive populations.

A comparative analysis of methods for de novo assembly of hymenopteran genomes using either haploid or diploid samples.

Diverse invertebrate taxa including all 200,000 species of Hymenoptera (ants, bees, wasps and sawflies) have a haplodiploid sex determination system, where females are diploid and males are haploid. Thus, hymenopteran genome projects can make use of DNA from a single haploid male sample, which is assumed advantageous for genome assembly. For the purpose of gene annotation, transcriptome sequencing is usually conducted using RNA from a pool of individuals. We conducted a comparative analysis of genome and transcriptome assembly and annotation methods, using genetic sources of different ploidy: (1) DNA from a haploid male or a diploid female (2) RNA from the same haploid male or a pool of individuals. We predicted that the use of a haploid male as opposed to a diploid female will simplify the genome assembly and gene annotation thanks to the lack of heterozygosity. Using DNA and RNA from the same haploid individual is expected to provide better confidence in transcript-to-genome alignment, and improve the annotation of gene structure in terms of the exon/intron boundaries. The haploid genome assemblies proved to be more contiguous, with both contig and scaffold N50 size at least threefold greater than their diploid counterparts. Completeness evaluation showed mixed results. The SOAPdenovo2 diploid assembly was missing more genes than the haploid assembly. The SPAdes diploid assembly had more complete genes, but a higher level of duplicates, and a greatly overestimated genome size. When aligning the two transcriptomes against the male genome, the male transcriptome gave 2-3% more complete transcripts than the pool transcriptome for genes with comparable expression levels in both transcriptomes. However, this advantage disappears in the final results of the gene annotation pipeline that incorporates evidence from homologous proteins. The RNA pool is still required to obtain the full transcriptome with genes that are expressed in other life stages and castes. In conclusion, the use of a haploid source material for a de novo genome project provides a substantial advantage to the quality of the genome draft and the use of RNA from the same haploid individual for transcriptome to genome alignment provides a minor advantage for genes that are expressed in the adult male.

Molecular evolution of juvenile hormone esterase-like proteins in a socially exchanged fluid.

Socially exchanged fluids are a direct means by which an organism can influence conspecifics. It was recently shown that when workers of the carpenter ant Camponotus floridanus feed larval offspring via trophallaxis, they transfer Juvenile Hormone III (JH), a key developmental regulator, as well as paralogs of JH esterase (JHE), an enzyme that catalyzes the hydrolysis of JH. Here we combine proteomic, phylogenetic and selection analyses to investigate the evolution of this esterase subfamily. We show that Camponotus JHE-like proteins have undergone multiple duplications, experienced positive selection, and changed tissue localization to become abundantly and selectively present in trophallactic fluid. The Camponotus trophallactic esterases have maintained their catalytic triads and contain a number of positively-selected amino acid changes distributed throughout the protein, which possibly reflect an adaptation to the highly acidic trophallactic fluid of formicine ants. To determine whether these esterases might regulate larval development, we fed workers with a JHE-specific pharmacological inhibitor to introduce it into the trophallactic network. This inhibitor increased the likelihood of pupation of the larvae reared by these workers, similar to the influence of food supplementation with JH. Together, these findings suggest that JHE-like proteins have evolved a new role in the inter-individual regulation of larval development in the Camponotus genus.

Evolution of Olfactory Functions on the Fire Ant Social Chromosome.

Understanding the molecular evolutionary basis of social behavior is a major challenge in evolutionary biology. Social insects evolved a complex language of chemical signals to coordinate thousands of individuals. In the fire ant Solenopsis invicta, chemical signals are involved in the determination of a polymorphic social organization. Single-queen (monogyne) or multiqueen (polygyne) social structure is determined by the "social chromosome," a nonrecombining region containing ∼504 genes with two distinct haplotypes, SB and Sb. Monogyne queens are always SBB, while polygyne queens are always SBb. Workers discriminate monogyne from polygyne queens based on olfactory cues. Here, we took an evolutionary genomics approach to search for candidate genes in the social chromosome that could be responsible for this discrimination. We compared the SB and Sb haplotypes and analyzed the evolutionary rates since their divergence. Notably, we identified a cluster of 23 odorant receptors in the nonrecombining region of the social chromosome that stands out in terms of nonsynonymous changes in both haplotypes. The cluster includes twelve genes formed by recent Solenopsis-specific duplications. We found evidence for positive selection on several tree branches and significant differences between the SB and Sb haplotypes of these genes. The most dramatic difference is the complete deletion of two of these genes in Sb. These results suggest that the evolution of polygyne social organization involved adaptations in olfactory genes and opens the way for functional studies of the molecular mechanisms underlying social behavior.

Neofunctionalization in Ligand Binding Sites of Ant Olfactory Receptors.

Chemical communication is fundamental for the operation of insect societies. Their diverse vocabulary of chemical signals requires a correspondingly diverse set of chemosensory receptors. Insect olfactory receptors (ORs) are the largest family of chemosensory receptors. The OR family is characterized by frequent expansions of subfamilies, in which duplicated ORs may adapt to detect new signals through positive selection on their amino acid sequence. Ants are an extreme example with ∼400 ORs per genome-the highest number in insects. Presumably, this reflects an increased complexity of chemical communication. Here, we examined gene duplications and positive selection on ant ORs. We reconstructed the hymenopteran OR gene tree, including five ant species, and inferred positive selection along every branch using the branch-site test, a total of 3326 tests. We find more positive selection in branches following species-specific duplications. We identified amino acid sites targeted by positive selection, and mapped them onto a structural model of insect ORs. Seventeen sites were under positive selection in six or more branches, forming two clusters on the extracellular side of the receptor, on either side of a cleft in the structure. This region was previously implicated in ligand activation, suggesting that the concentration of positively selected sites in this region is related to adaptive evolution of ligand binding sites or allosteric transmission of ligand activation. These results provide insights into the specific OR subfamilies and individual residues that facilitated adaptive evolution of olfactory functions, potentially explaining the elaboration of chemical signaling in ant societies.

Positive selection on sociobiological traits in invasive fire ants.

The fire ant Solenopsis invicta and its close relatives are highly invasive. Enhanced social cooperation may facilitate invasiveness in these and other invasive ant species. We investigated whether invasiveness in Solenopsis fire ants was accompanied by positive selection on sociobiological traits by applying a phylogenomics approach to infer ancient selection, and a population genomics approach to infer recent and ongoing selection in both native and introduced S. invicta populations. A combination of whole-genome sequencing of 40 haploid males and reduced-representation genomic sequencing of 112 diploid workers identified 1,758,116 and 169,682 polymorphic markers, respectively. The resulting high-resolution maps of genomic polymorphism provide high inference power to test for positive selection. Our analyses provide evidence of positive selection on putative ion channel genes, which are implicated in neurological functions, and on vitellogenin, which is a key regulator of development and caste determination. Furthermore, molecular functions implicated in pheromonal signalling have experienced recent positive selection. Genes with signatures of positive selection were significantly more often those overexpressed in workers compared with queens and males, suggesting that worker traits are under stronger selection than queen and male traits. These results provide insights into selection pressures and ongoing adaptation in an invasive social insect and support the hypothesis that sociobiological traits are under more positive selection than nonsocial traits in such invasive species.

Oral transfer of chemical cues, growth proteins and hormones in social insects.

Social insects frequently engage in oral fluid exchange - trophallaxis - between adults, and between adults and larvae. Although trophallaxis is widely considered a food-sharing mechanism, we hypothesized that endogenous components of this fluid might underlie a novel means of chemical communication between colony members. Through protein and small-molecule mass spectrometry and RNA sequencing, we found that trophallactic fluid in the ant Camponotus floridanus contains a set of specific digestion- and non-digestion related proteins, as well as hydrocarbons, microRNAs, and a key developmental regulator, juvenile hormone. When C. floridanus workers' food was supplemented with this hormone, the larvae they reared via trophallaxis were twice as likely to complete metamorphosis and became larger workers. Comparison of trophallactic fluid proteins across social insect species revealed that many are regulators of growth, development and behavioral maturation. These results suggest that trophallaxis plays previously unsuspected roles in communication and enables communal control of colony phenotypes.

Higher expression of somatic repair genes in long-lived ant queens than workers.

Understanding why organisms senesce is a fundamental question in biology. One common explanation is that senescence results from an increase in macromolecular damage with age. The tremendous variation in lifespan between genetically identical queen and worker ants, ranging over an order of magnitude, provides a unique system to study how investment into processes of somatic maintenance and macromolecular repair influence lifespan. Here we use RNAseq to compare patterns of expression of genes involved in DNA and protein repair of age-matched queens and workers. There was no difference between queens and workers in 1-day-old individuals, but the level of expression of these genes increased with age and this up-regulation was greater in queens than in workers, resulting in significantly queen-biased expression in 2-month-old individuals in both legs and brains. Overall, these differences are consistent with the hypothesis that higher longevity is associated with increased investment into somatic repair.

Patterns of positive selection in seven ant genomes.

The evolution of ants is marked by remarkable adaptations that allowed the development of very complex social systems. To identify how ant-specific adaptations are associated with patterns of molecular evolution, we searched for signs of positive selection on amino-acid changes in proteins. We identified 24 functional categories of genes which were enriched for positively selected genes in the ant lineage. We also reanalyzed genome-wide data sets in bees and flies with the same methodology to check whether positive selection was specific to ants or also present in other insects. Notably, genes implicated in immunity were enriched for positively selected genes in the three lineages, ruling out the hypothesis that the evolution of hygienic behaviors in social insects caused a major relaxation of selective pressure on immune genes. Our scan also indicated that genes implicated in neurogenesis and olfaction started to undergo increased positive selection before the evolution of sociality in Hymenoptera. Finally, the comparison between these three lineages allowed us to pinpoint molecular evolution patterns that were specific to the ant lineage. In particular, there was ant-specific recurrent positive selection on genes with mitochondrial functions, suggesting that mitochondrial activity was improved during the evolution of this lineage. This might have been an important step toward the evolution of extreme lifespan that is a hallmark of ants.