Nature of a Greenbeard Gene in the Fire Ant

Genes generally spread in populations by increasing the survival and/or reproductive success of the carrier organism. However, some genetic elements gain a transmission advantage without increasing the host organism’s fitness. Such pieces of DNA –called « selfish » genetic elements (SGEs)– induce intragenomic conflicts, which have far-reaching consequences on the evolution of genomes and organisms. Greenbeard genes are a special type of SGEs, which achieve a transmission bias by increasing the fitness of other individuals also carrying the greenbeard gene. Such phenotype, known as a "Greenbeard effect", can only occur if a gene or a group of tightly linked genes produce 1: a conspicuous phenotype, 2: the ability to recognize this phenotype, allowing the bearer individual to discriminate carriers from non-carriers and 3: a nepotistic behavior in favor of carriers. Because of these requirements, Greenbeard effects have been presumed to be rare in nature. The first identified Greenbeard genetic element was identified in the fire ant Solenopsis invicta. In this species, the Greenbeard effect is produced by a single Mendelian genomic element: the Social b (Sb) supergene. Sb-bearer workers kill non-bearer queens (SB/SB) to replace them by carrying-queens (SB/Sb). The objective of the project GreenAnt is to take advantage of the fire ant Greenbeard system to identify and characterize the genetic elements involved in this SGE. The aim of my project is to determine how workers discriminate Greenbeard-carrier queens from non-carriers.

The objectives of GreenAnt action is to gain greater insight into the molecular genetic basis underlying the greenbeard effect produced by the social supergene evolving in S. invicta. We conducted multiple complementary analyses such as gene content variation, molecular evolutionary signature and gene expression, to identify the genetic bases of the Greenbeard effect in S. invicta.
The first objective is to investigate the Supergene molecular evolution. Using an extensive dataset of sequenced haploid males from multiple socially polymorphic species carrying either the SB or the Sb haplotype, we managed to understand more in depth the recent evolutionary history of the supergene. We show that the three inversions constituting the Solenopsis social supergene emerged sequentially during the speciation process between S. invicta and S. richteri. The two first inversions arose in the ancestral population of the two species and the third one in S. richteri. Once completed, the supergene then introgressed S. invicta first, and from there the three other species. Surprisingly, the supergene introgression occurred despite hybridization being very uncommon among some of the species. In a subsequent study, we analysed the evolutionary fate of the Sb haplotype. We uncovered moderate traces of degeneracy, essentially because the Sb haplotype is rarely found at the homozygous state. Surprisingly, we also uncovered traces of recombination between the SB and the Sb haplotypes. Using both sources of information, we conducted an investigation of candidate genes associated with the phenotypes produced by the Sb haplotype. We identified 48 candidate genes.
The second objective is to conduct a gene expression analyses in organs in which the Greenbeard effect is mediated. The project is more specifically orientated toward the ability of Greenbeard-carrier workers to discriminate carrier from non-carrier queens. Recognition of Sb-queens could be mediated by the product of one or several gene(s) expressed in the antennae or in the brain. We compared patterns of gene expression in antennae and brains between workers of different genotypes, using workers brain RNA-seq data produced in the lab and antennae RNA-seq data produced by Dang et al. 2019. Such analysis allowed us to reduce even further the number of candidate genes.
Lastly, the third objective is to functionally identify the gene allowing Sb-bearer workers to discriminate egg-laying queens. We managed to adapt the CRISPR-Cas9 technique on S. invicta to transform workers. However, while transformation is successful, and allowed us to knockout one candidate gene, we met unexpected challenges which are currently addressed in the lab. Overall, this project has been successful in identifying high quality candidate genes that would be involved in the Greenbeard effect. Only a handful of genes are reuniting the conditions to be involved in the ability of Greenbeard-carrier workers to discriminate carrier from non-carrier queens.

Understanding how complex traits requiring multiple novel mutations arise and are maintained in populations is a long-standing question in evolutionary biology. The GreenAnt project allowed us to gain insight into the evolutionary history of a social supergene associated with a Greenbeard effect. The action contributed to the acquisition of data that will enable the testing of predictions and proposing a list high-quality candidate genes involved in the Greenbeard effect, which allows the social supergene to maintain and spread in Solenopsis invicta and closely related species. No manuscript has yet been submitted for publication. However, a first manuscript is ready and currently going through final proof-reading by co-authors before submission. We plan to submit this manuscript to an influential multidisciplinary journal such as PNAS. Two other manuscripts are in the process of writing, and should be ready for submission in the second half of 2022. The first one is a review about social supergenes identified in Ant, the second about the identification of candidate genes for the ability of Sb-bearer workers to discriminate egg-laying queens. Our results are likely to receive great interest from the evolutionary biology community interested in the evolution of supergenes and evolutionary conflicts. So far, Dr. Helleu has presented preliminary results of this action at two internal seminars of the University of Lausanne. Because of the covid crisis most international conferences were cancelled. However, Dr Helleu plans to present the final results of the action at a forthcoming international meeting organized by the European Society for Evolutionary Biology (ESEB 2021, in Prague, Czech Republic).

Periodic Reporting for period 1 - GreenAnt (Nature of a Greenbeard Gene in the Fire Ant)

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