Discussion
Our experimental design leverages samples from two organs and the full breadth of social chromosome genotypes and social forms to provide a high-resolution snapshot of the transcriptomic effects of these factors in fire ants, with the aim of exploring the direct and indirect genetic effects of the supergene. Our focal samples, young queens (gynes) embarking on nuptial flights, arguably are the most relevant caste/life stage on which to conduct such a study. These potential future reproductives were collected while engaged in a pivotal activity that immediately precedes nest founding (monogyne form) or joining (polygyne form), thus marking the initiation of a queen’s reproductive life. Fire ant workers are obligately sterile, though essential to colony fitness, while males are short-lived and appear to play no meaningful role in colony social life. Inclusion of SB/SB gynes from both monogyne and polygyne colonies facilitates direct comparison of the gene regulatory consequences of the contrasting developmental environments of the two colony types, while sampling two organs of particular relevance to the alternate social syndromes (brains mediating queen dispersal/nest-founding behaviors and ovaries enabling reproduction) from individual ants, with appropriate biological replication, allows us to begin linking individual facets of the complex phenotype of polygyny to putative genetic mechanisms. Expression profiling of two organs also provides the opportunity to screen for loci with putative constitutive regulatory effects, that is, genes differentially expressed in multiple cellular contexts and thus possibly mediating body-wide effects ofSb . Finally, inclusion of the rare homozygous Sb/Sbgenotype allows exploration of the potential genetic causes of the documented, presumably endogenous effective lethality of this genotype.
The genome-wide perspective. Considering the striking number of individual- and colony-level differences between the monogyne and polygyne forms of S. invicta ―e.g., in worker behavior towards queens, queen cuticular chemical profiles and reproductive trajectories, worker gene expression patterns, and many other biological features (Eliyahu et al., 2011; Keller & Ross, 1998; Trible & Ross, 2016; Wang et al., 2008)―we predicted that differences in the social environment in which gynes are reared would be reflected in gene expression differences well beyond those that can be explained by social chromosome genotype alone. Although we observed no significantly differentially expressed genes (DEGs) directly attributable to colony social form in our multifactorial-model analysis (FDR < 0.01), polygyne-rearedSB/Sb gynes did exhibit many more DEGs in ovaries when compared to monogyne-reared SB/SB gynes (205 DEGs) than when compared to polygyne-reared SB/SB gynes (43 DEGs; Figure 3). This result mirrors the previously observed small, but statistically significant, higher weight of monogyne-reared, as compared to polygyne-reared,SB/SB gynes (all SB/SB gynes, however, are far heavier than polygyne-reared SB/Sb or Sb/Sb gynes) (C. DeHeer et al., 1999). Gyne weight in fire ants reflects amount of storage reserves accumulated during sexual maturation and profoundly affects prospects for successful independent founding (e.g., the ability to produce workers solitarily), versus colony joining, as an individual reproductive strategy (C. J. DeHeer, 2002). Our gene expression results point to an interaction between the effects of natal colony type and supergene-mediated gene regulation that is superimposed on the small effect of natal colony type alone.
We predicted that gene expression differences associated with social chromosome genotype would preferentially involve genes located within the supergene region (Nipitwattanaphon et al., 2013; Wang et al., 2013). The reason is that extensive sequence divergence exists between theSb and SB haplotypes of the supergene in U.S. populations of S. invicta (estimated at 1.4 SNPs per Kb; (Pracana, Priyam, et al., 2017; Wang et al., 2013)), contrasted with the rest of the genome, which recombines freely and is subject to constant high levels of admixture between the social forms (Ross, Krieger, Keller, & Shoemaker, 2007; Ross, Shoemaker, Krieger, DeHeer, & Keller, 1999; Shoemaker, Deheer, Krieger, & Ross, 2006), mainly via polygyne-derivedSB/Sb gynes mating with monogyne-derived SB (haploid) males (Ross & Shoemaker, 2006). Consequently, linkage disequilibrium outside of the supergene region is minimal in both social forms (Ross & Shoemaker, 2018; Yan et al., 2020). Our data expand upon the previous finding that genes differentially expressed in association with social chromosome genotype are disproportionately localized within the supergene region (Nipitwattanaphon et al., 2013; Wang et al., 2013), illustrating the importance of supergene cis -regulatory evolution. Nonetheless, many of the genes differentially expressed in association with social chromosome genotype are located elsewhere in the genome, illustrating that trans -acting effects, or other epistatic interactions involving the supergene, are also widespread and potentially of major significance in the evolution of the supergene and the alternate social forms in fire ants. This is not surprising given the prevalence of trans-acting effects documented in supergene systems mediating sexual dimorphism (Parsch & Ellegren, 2013; Wijchers & Festenstein, 2011) and, strikingly, even in the relatively small (~100kb) supergene regulating mimicry in Papilio polytes butterflies (Kunte et al., 2014).
An important, related point is that the majority of supergene-driven DEGs in our multifactorial-model comparisons show higher expression in individuals carrying a copy of the Sb supergene haplotype, both within and outside of the supergene region (Figures 2, S2). Preliminary indications of preferentially elevated gene expression in the presence of the Sb haplotype have been reported (Wang et al., 2013), but the broad scope of this phenomenon was not established previously. Thus, the observed “degenerative expansion” (Stolle et al., 2019) of the Y-like Sb supergene haplotype is, somewhat paradoxically, accompanied by an increase in transcriptional activity for manySb alleles at loci within the supergene. This Sbupregulation could be driven by a combination of cis- regulatory evolution, increased chromatin accessibility, and/or recentSb- specific gene duplication events that appear to have occurred in conjunction with the expansion of the Sb haplotype (Fontana et al., 2019; Stolle et al., 2019).
Although such gene duplication within the inversion-derived Sbhaplotype likely explains some of the widespread Sb -mediated upregulation observed in our analyses, we think it is unlikely to be the sole driver of this phenomenon for three reasons. First, while Fontana et al. (2019) show a general increase in transcript levels from genes with Sb- specific paralogs, they found no significant correlation between gene expression level and copy number, implying that regulatory element evolution may play a larger role than copy number in observed expression differences between SB and Sb haplotypes inS. invicta . Second, we detected several genes in our analysis that exhibited differential expression without significant allele-specific expression, indicating that their increased transcription is facilitated by both the Sb and SBhaplotypes rather than being a simple consequence of possession of extra copies of the Sb -linked variants. Finally, while a number of our candidate genes have undergone gene duplication events, the increase in gene expression we observe in Sb -carrying individuals is significantly higher than one would expect from a simple gene duplication event (or even several), with log2-fold changes greater than 5 in many duplicated genes (Table 1). Ultimately, the Sb -upregulated DEGs we report reflect an increase in the number of identical or highly similar transcripts in Sb- carrying individuals that collectively influence downstream pathways and phenotypes, no matter the specific causal genetic architecture.
Our comparisons of supergene effects on gene expression in the brains and ovaries revealed some key differences in the profiles of the two organs. In particular, ovary DEGs are less concentrated in the supergene than brain DEGs (Figure 2D). This indicates that regulatory effects of the supergene may be more indirect in the ovaries than the brain, involving more diffuse sets of genetic pathways regulated largely bytrans -acting effects. Moreover, many more DEGs are detected in ovaries than the brain in comparisons of the two homozygous classes (SB/SB versus Sb/Sb ; Figure 2), suggesting that the transcriptional consequences of supergene homozygosity are particularly pronounced in the ovarian tissues of Sb/Sb gynes. Because these individuals do not survive to reproduce in U.S. populations, it was not possible to gauge the effects of these expression differences with respect to fecundity; however, maturing Sb/Sb gynes have been shown to suffer curtailed development in terms of other physiological markers associated with onset of oogenesis compared to gynes of the other two genotypes (Hallar et al., 2007).
The supergene perspective. Given that minimal levels of recombination are observed within the Sb haplotype (Ross & Shoemaker, 2018; Wang et al., 2013; Yan et al., 2020), its constituent genes are effectively inherited as a single Mendelian element and are in near-complete linkage disequilibrium. Nonetheless, expression profiles and associated selective pressures are expected to vary among supergene genes in relation to their specific functions. Indeed, we observed spatially heterogeneous gene expression patterns across the supergene region in S. invicta gynes, with genes expressed more highly inSb than SB , as well as genes showing the opposite pattern, clustered together more commonly than expected by chance.
We highlighted two regions of the supergene with these distinctive gene and allele-specific expression patterns (Figure 5,S5). The first, here denoted region 1, exhibits minimal differential expression in theSB/SB-SB/Sb comparison, while showing extensiveSB/SB -biased expression in the SB/SB-Sb/Sb comparison. Concurrently, this region shows disproportionate SB -biased allele expression in heterozygous SB/Sb individuals. These results point to diminished expression of the alleles within the Sb haplotype, compensated by means of overexpression of the SB versions of those genes. We hypothesize that genes subject to this SB dosage compensation in region 1 (and elsewhere) contribute to the observed inviability of Sb/Sb queens (Hallar et al., 2007). Dosage compensation is a commonly observed phenomenon in sex chromosome systems (Disteche, 2012; White et al., 2015) and has been documented recently in the supergene of the white-throated sparrow (Sun, Huh, Zinzow-kramer, Maney, & Yi, 2018). A second region of the supergene, here denoted region 2, contains genes exhibiting strong Sb -biased gene expression and Sb allele-specific expression (Figure 5), indicating upregulation driven largely by expression of theSb -linked alleles. We hypothesize that genes in region 2 (and other genes conforming to this regulatory pattern) act as functional drivers of the polygyne syndrome and thus are important candidates of major effect on this social polymorphism.
Gene-specific, functional observations. We leveraged our sampling scheme to search for genes exhibiting differential expression patterns that parallel specific phenotypic differences observed between gynes of each social chromosome genotype. Specifically, we classified DEGs into four categories: brain-specific, ovary-specific,Sb/Sb -specific, and constitutive (Table 1). Among eight detected brain-specific DEGs, Ribonuclease H-like is notable for having a human ortholog linked to splicing and neurological disfunction (Bin et al., 2016). Additionally, among the twelve detected ovary-specific DEGs,sphingosine-1-phosphate lyase is notable for having aDrosophila ortholog involved in sphingolipid metabolism that is linked to reproductive deficiencies (Phan et al., 2007). This gene also stands out as the only characterized gene in the ovary-specific category with lower expression in heterozygotes than SB/SB homozygotes.
Among eight detected Sb/Sb- specific DEGs (those with differences in both organs but solely in the Sb/Sb -SB/SB comparison),pre-mRNA-splicing factor Slu7-like and peptide deformylase, mitochondrial-like are notable for their putative impacts on the production of properly spliced and processed proteins. Finally,
among the thirteen detected constitutive DEGs (those with differences in all comparisons of Sb- supergene presence/absence) we identified notable genes with orthologs involved in metabolism (NADH dehydrogenase ), odorant perception (pheromone binding protein GP-9-like , also referred to as an odorant binding protein) (Leal, 2013), and brain development and sperm motility (growth arrest-specific protein-8 ) (zur Lage, Newton, & Jarman, 2019).Gas-8 may be implicated in differences in traits as diverse as gyne dispersal behavior (C. DeHeer et al., 1999) and male reproductive capacity (Lawson, Vander Meer, & Shoemaker, 2012). Interestingly, one of the constitutive supergene-effect loci we identify appears to be located outside of the supergene region: nose-resistant to fluoxetine protein 6-like is affiliated with defects in lipid transport to the ovaries and with lifespan in Caenorhabditis elegans and humans (Brejning et al., 2014; Choy & Thomas, 1999). This gene is a candidate for direct, trans -regulation by the Sb supergene and may contribute to the diminished early fecundity of polygyneSB/Sb queens relative to SB/SB queens (C. J. DeHeer, 2002; Keller & Ross, 1993). However, given recent evidence that duplicate genes may arise by translocation into the Sb supergene from other genomic locations (Fontana et al., 2019), improved Sb assemblies and annotation will be necessary to further explore this hypothesis.
Pheromone binding protein GP-9-like belongs to one of the best studied gene families in S. invicta : the insect odorant binding protein (OBP) family (Keller & Ross, 1998, 1999; Pracana, Levantis, et al., 2017). These genes are thought to produce proteins that function as molecular carriers, transporting odorant molecules to their receptors in some canonical study systems, though OBPs likely also function outside of the chemosensory system as general chaperone molecules (Leal, 2013). In S. invicta , OBPs are of particular interest, as it has long been known that the fire ant social polymorphism is marked by fixed amino acid differences at an OBP, General protein-9 (encoded by the geneGp-9 ) (Ross, 1997). We found that the most strongly differentially expressed OBP-encoding gene in our data was not the widely studied Gp-9 , but a paralog, OBP12 , that exists as duplicated tandem genes on the Sb haplotype and as a single-copy gene on the SB haplotype (Fontana et al., 2019; Pracana, Levantis, et al., 2017). This Sb -specific duplication ofOBP12 corresponds with a unique increase in gene expression andSb allele-specific expression among S. invicta OBP genes (reads from both Sb paralogs evidently mapped to the singleOBP12 encoded in the SB reference genome in our analyses; Figure S4). Our data are largely consistent with the recent findings by Dang et al. (2019) regarding increased expression of OBP12 in antennae of Sb -carrying workers, though we also observed increased OBP12 expression in both the brains and ovaries ofSb -carrying gynes, which may mitigate against the notion of antennal-specific neofunctionalization (Dang, Cohanim, Fontana, Privman, & Wang, 2019). Additionally, the >12-fold increase in expression we observed for OBP12 in Sb -carrying individuals compared to SB/SB individuals is much higher than the simple doubling one might expect if the duplicate was expressed similarly to its progenitor (Table 1, Figure S4). This points to effects of gene regulatory evolution. Our findings are consistent with the hypothesis that duplication of OBP12 in the Sb haplotype and subsequent Sb- upregulation of the derivative OBP12 may facilitate some differences in sensory functions and response behaviors between monogyne and polygyne fire ants (Keller & Ross, 1998; Trible & Ross, 2016).
Conclusion. We find that the gene regulation underlying a major alternate form of social organization in fire ants is more complex than the simple inheritance patterns or broad structural differences in the implicated supergene may superficially suggest. As hypothesized (Y. C. Huang & Wang, 2014), gene expression evolution appears to play a large role in the evolution of the complex phenotype of polygyny in S. invicta . We show that possession of theSb inversion-mediated haplotype by a queen leads to changes in gene expression both within and beyond the boundaries of the constitutive supergene inversions, that such supergene-mediated effects interact with those of the developmental environment, and that genes differentially expressed in queens bearing different supergene genotypes are heterogeneously distributed along the supergene. Further work exploring the interplay of protein sequence, gene expression, and regulatory element evolution is necessary to better understand the genetic causes of a fundamental shift in fire ant colony social organization with widespread behavioral, ecological, and economic consequences (Tschinkel 2006).
Acknowledgements
We thank Dietrich Gotzek for the idea to sample gynes embarking on mating flights, Bob Schmitz and Nick Rohr for library preparation, Yannick Wurm and Rodrigo Pracana for providing linkage mapping and OBP annotation information, and the Georgia Advanced Computing Resource Center, a partnership between the University of Georgia’s Office of the Vice President for Research and Office of the Vice President for Information Technology. This work was supported by U.S. NSF grants to B.H. and K.R. (1755130) and K.R. (1354479) and U.S. Federal Hatch funds to B.H. and K.R.