Several studies in the lab use computational molecular evolution analyses to scan ant genomes and search for the molecular basis of adaptations related to the social life of ants and other social insects, including: social communication based on chemical signals synthesis (e.g. hydrocarbon anabolism) and perceptions (e.g. olfactory receptors); variation of social behavior and social structure (monogyne vs. polygyne); and the genes underlying variation in social information transfer between different foraging workers in the same colony.
Genetic basis for chemical communication in Cataglyphis desert ants
This project aims to identify genes responsible for chemical signaling in the Israeli ant Cataglyphis niger, using a combined approach of population genomic sequencing and chemical analysis. We started using this species as a model in collaboration with the lab of Abraham Hefetzin Tel Aviv University, who pioneered the study of this system using chemical ecology approaches.
Fig. 2: Tal and Daphna processing Cataglyphis ants collected from Bezet Beach.
Before we can analyze the population genomics of this species, we first need a reference genome sequence. This was the MSc project of Tal: sequencing and assembling the reference genome for Cataglyphis niger. He also compared alternative approaches to de novo genome sequencing and gene annotation, in the special case of haplodiploid species and showed that it's advantageous to use a single haploid male for both DNA and RNA extraction.
The reference genome will form the foundations for the population genomic study. First, we used 117 brothers from a single nest for QTL mapping study. Shani used these samples for both genomic sequencing and chemical analysis of cuticular hydrocarbons (the pheromones used by ants for nestmate recognition). She constructed the first RADseq library ever done in Israel (Restriction-associated DNA sequencing), and the results came out top quality:
These raw sequence reads then went through a lot of processing and bioinformatics, done by Pnina and Besan. Besan constructed a linkage map for the 26 chromosomes of C. niger, with the help of Abraham Korol and using his software MultiPoint. She then conducted QTL mapping of the chemical traits of these ants (quantities of cuticular hydrocarbons). She identified many such QTLs, at least one for every trait, and almost every chromosome has some hint of QTLs.
GWAS: We also collected a much more extensive population sample, from 50 colonies in Betzet beach. Shani constructed whole-genome libraries of 6 workers from each colony (300 genomes altogether). Combined with chemical analysis of the same samples, these results will allow us to test for an association between genetic variation in specific genes and variation in the cuticular hydrocarbons i.e. a genome-wide association study (GWAS).
Social polymorhpism: Previous studies identified three clades within this species complex: C. niger, C. savignyi, and C. drusus. The three were distinct monophyletic clades according to their mitochondrial haplotypes, and they also display distinct social colony structures: C. niger is polygyne, whereas C. savignyi and C. drusus are monogyne. In a joint paper with the Hefetz lab (Reiner-Brodetzki et al. 2019) we used RAD sequencing and population structure analysis of thousands of SNP loci to show that these three mitochondrial clades are not monophyletic in terms of their nuclear DNA. In fact, there is no detectable genetic divergence among them, and they form one un-differentiated population cluster in the STRUCTURE results:
We also conducted a maximum likelihood test for gene flow (using 3s) and rejected the null model of no gene flow between these clades. That is, there is significant evidence for gene flow between all these populations and between colonies of different social structure. Finally, we showed that the spermatheca of polygyne queens with the C. niger mitotype contains mostly sperm with the C. savignyi mitotype. That is, polygyne queens mate mostly with males from monogyne colonies. Therefore, C. niger is one species that harbors a social polymorphism. Naturally, our next goal is to reveal the molecular basis for this polymorphism. Is it genetic? epigenetic? Is it a supergene as in the case of other ants such as Solenopsis invicta and Formica selysi? Stay tuned!
Evolution of social structure in Solenopsis fire ants
Another study system in our lab is the fire ant Solenopsis invicta. This species is a particularly useful model because the genetic basis for a Mendelian trait affecting social behavior is known to be a non-recombining “social chromosome” that is similar to a Y sex chromosome (Wang et al. 2013). This chromosome determine the behavior of ants in this species leading to dramatic differences in their social structure: one social form allow only one queen per nest (monogyne) while the other form accept many additional queens to the same nest (polygyne). The recent evolution of this system involved many micro- and macro-mutations that alter social behavior and organization. We use inter-species comparative genomics and intra-species population genomics to study the evolution of this social chromosome and other genetic elements that underlie the various biological functions responsible for social behavior, especially chemical communication.
The population genetics of this species is also interesting as a case study of a high-impact invasive species. Fire ants were inadvertently introduced from north Argentina to Alabama, USA in the 1930's. They quickly grew to very large and dense populations, spread across south-eastern USA and later were introduced from the US to various islands, Australia and China. This species causes great damages to both ecology and agriculture. Annual losses of $750 million were estimated in the United States alone.
In a collaboration with the lab of DeWayne Shoemaker (USDA) we use these well studied populations to investigate how a social insect becomes adapted to invasiveness. By sequencing whole genomes of many individuals from population samples we identify hundreds of thousands of polymorphic sites across the genome and compare their allele frequencies between native and introduced populations. In her PhD studies, Pnina applies a range of statistical inference methods to reconstruct the recent history of these populations and identify genomic regions that experienced positive selection. These genes may be responsible for recent local adaptation of different populations in the native and the introduced range.
Fig. 3: Invasion history of the fire ant Solenopsis invicta.
Universal adaptations of ants - olfactory receptors
The first whole genome sequencing of seven ant species allowed us to search for genes and gene families underlying adaptations of ants in general. Whole genomes scans for positive selection using dN/dS tests (site and branch-site tests in PAML) identified genes implicated in functional categories related to morphology, metabolism, and behavior. Focused analyses of olfactory receptors from two ant species (about 300 per species) identified subfamilies of receptors that underwent rampant ant-specific gene duplications and positive selection. These analyses generated candidate genes for further investigation of adaptive evolution of communication in ant societies.
Fig 1: Gene tree of odorant receptors from two ant species (green and blue) and one wasp (black). Branches with positive selection are marked in red (Roux et al. 2014).
In her MSc thesis, Rana expanded this analysis to many more ant species, including Solenopsis fire ants. Amir conducted a focused study of a subfamily of receptors found on the fire ant social chromosome (see below). These analyses revealed specific receptors that underwent dramatic evolution in the fire ant lineage. These candidate genes are now tested in functional assays for their potential role in the regulation of social behavior and social organization.
Evolution of sex determination in ants and bees
All 200,000 Hymenoptera species (including ants, bees, wasps and sawflies) have haplodiploid sex determination. They do not have sex chromosomes. Instead these insects may be either haploids that develop into males, or diploids that develop into females. While this general principle is common to all species in this large insect order, the underlying molecular genetic mechanisms are diverse and dynamic throughout evolution. One type of mechanism is complementary sex determination (CSD). CSD was first discovered in the honey bee Apis mellifera. Also the genes responsible for this mechanism were first discovered in the honey bee: feminizer and csd. These two genes are homologs of the transformer gene found in other insects. They were thought to be the result of a gene duplication event in the honey bees.
In one of the first studies of sex determination genes in ant genomes we discovered that almost every ant genome sequenced has two copies of this gene, like the honey bee. Surprisingly, each ant lineage appears to have its own independent duplication of the ancestral gene transformer. We put forward the hypothesis that this pattern is the result of a single ancestral duplication followed by concerted evolution of the two copies (Privman et al. 2013).
Fig. 4: Gene tree of transformer homologs in ants and bees. Stars mark apparent gene duplications. Arrows indicate evidence for concerted evolution (gene conversion events).
More recently, we are collaborating with the lab of John Wang to characterize the sex determination locus of the fire ant Solenopsis invicta and its population genetics.