Introduction
Supergenes are regions of the genome that contain multiple genes held in
strong linkage disequilibrium due to severely restricted recombination
(Schwander, Libbrecht, & Keller, 2014). They are often formed by
chromosomal rearrangements, such as inversions, which inhibit crossing
over during meiosis, thereby facilitating the preservation of favorable
allelic combinations across numerous loci (Faria, Johannesson, Butlin,
& Westram, 2019; Schwander et al., 2014). Chromosomal rearrangements
are fundamental to sex chromosome evolution (Hoffmann & Rieseberg,
2008; White, Kitano, & Peichel, 2015) and are now recognized as
taxonomically widespread, evolutionarily significant mediators of
complex trait variation (Faria et al., 2019; Schwander et al., 2014;
Wellenreuther & Bernatchez, 2018). For example, supergenes have been
discovered that determine variation in social behavior, sexual
dimorphism, and dispersal strategies in animals as diverse as insects
(Purcell, Brelsford, Wurm, Perrin, & Chapuisat, 2014; Wang et al.,
2013), birds (Küpper et al., 2016; Lamichhaney et al., 2015; Tuttle et
al., 2016), and fishes (Kirubakaran et al., 2016; Pearse et al., 2019).
Despite their acknowledged role in facilitating the evolution of complex
trait polymorphisms, supergenes are not without evolutionary costs.
Diminished recombination and resulting linkage disequilibrium within
supergenes are expected to impede the ability of selection to act on
individual mutations, with the result that spread of beneficial
mutations and elimination of deleterious ones are constrained by the
genetic background on which they arise (Hill-Robertson interference)
(Hill & Robertson, 1966; Wang et al., 2013). The subsequent reduction
in the efficacy of natural selection typically culminates in some level
of degeneration of the non-recombining region, usually manifested as
accumulation of fixed deleterious mutations, loss of genetic diversity,
suppression of gene expression, gene deletion, and/or proliferation of
transposable elements (Bachtrog et al., 2011; Stolle et al., 2019;
Tuttle et al., 2016). Thus, the complex makeup of supergenes as mosaics
of tightly linked adaptive, neutral, and even deleterious genetic
variants makes dissection and elucidation of their genotype to phenotype
map especially challenging.
The fire ant Solenopsis invicta displays naturally occurring
variation in fundamental, ecologically important traits comprising two
social syndromes (monogyny and polygyny) that collectively are under the
control of a supergene (Wang et al., 2013). The eponymous difference in
the syndromes is the number of reproductive (egg-laying) queens in a
colony, with colonies of the monogyne form containing only a single
reproductive queen and colonies of the polygyne form containing few to
many queens (Gotzek and Ross 2007; Tschinkel 2006). The difference in
queen number corresponds with differences in numerous individual-level
attributes including: the extent of energy reserves, types of dispersal
behaviors, and reproductive ontogenies of young queens (C. DeHeer,
Goodisman, & Ross, 1999; C. J. DeHeer, 2002); the fecundity of mature
reproductive queens (DeHeer 2002; Tschinkel 2006); and the form of
worker discrimination directed toward queens attempting to join a colony
(Keller & Ross, 1998). The S. invicta supergene regulating
social form arose with the appearance of multiple adjacent chromosomal
inversions that span more than 13Mb of DNA on the “social chromosome”
and include over 400 mapped protein-coding genes (Y. Huang, Dang, Chang,
Wang, & Wang, 2018; Wang et al., 2013; Yan et al., 2020). Recombination
is dramatically reduced between the inverted region of the social
chromosome (denoted as the Sb haplotype) and its homologs that
show synteny with the presumed ancestral haplotype (denoted SB )
(Stolle et al., 2019; Wang et al., 2013; Yan et al., 2020). Moreover,Sb/Sb homokaryotypes (homozygotes) in the invasive U.S. range
effectively do not survive to reproduce (Hallar, Krieger, & Ross,
2007); thus, like the mammalian Y-chromosome, recombination is strongly
reduced between variants of the Sb haplotype as well as between
the Sb and SB haplotypes. The entirety of the fire ant
supergene can be characterized as an enormous, complex genomic module
that collectively regulates colony social form and allied traits while
propagating via simple Mendelian inheritance (Keller & Ross, 1998; Wang
et al., 2013).
Despite a recent surge of interest in the importance of supergenes in
regulating complex trait variation (Schwander et al., 2014;
Wellenreuther & Bernatchez, 2018), progress in disentangling the
functional mechanisms by which they exert their phenotypic effects has
been slow. As suggested above, supergenes may harbor relatively few
genes with direct functional effects on adaptive traits that reside in a
sea of elements with neutral or even deleterious fitness consequences,
and identification of such loci is complicated by the strong linkage
disequilibrium observed across the supergene region. Moreover,
supergene-associated traits may be regulated by a combination of protein
structural variants and variant DNA regulatory elements, necessitating
careful investigation and evaluation of the role of each type, as well
as possible joint effects (e.g., allele-specific expression of
protein-coding genes). In S. invicta , recent studies have defined
the specific inversion breakpoints in the Sb haplotype (Y. Huang
et al., 2018; Yan et al., 2020) and have begun to characterize protein
sequence divergence between the Sb and SB haplotypes in
both the invasive U.S. range (Pracana, Priyam, Levantis, Nichols, &
Wurm, 2017) and native South American range (Yan et al., 2020). However,
fundamental questions remain as to how differences between the supergene
haplotypes, ranging in scope from single nucleotide polymorphisms, to
copy number variants, to major structural rearrangements, influence gene
regulation and induce alternate complex social phenotypes (Fontana et
al., 2019; Gotzek & Ross, 2007; Y. C. Huang & Wang, 2014).
To explore in greater detail the genotype to phenotype relationship
between the Sb supergene haplotype and the fire ant polygyne
syndrome, we generated and analyzed RNA-seq data from brains and ovaries
of individual, sexually mature, similarly-aged unmated queens (gynes) of
both social forms of S. invicta . Organ-specific sampling was
utilized to minimize the effects of variable tissue composition, with
brains and ovaries targeted because they are likely the primary organs
mediating the behavioral and reproductive differences between the social
forms. Samples of gynes with all three social chromosome genotypes
(SB/SB, SB/Sb, and Sb/Sb ) were included in our experiment
(Figure 1), which is noteworthy by virtue of inclusion of the rare gynes
from polygyne nests bearing either homozygous genotype. We focus on gene
expression effects of the two most conspicuous factors relevant to fire
ant social organization: an individual’s social chromosome (supergene)
genotype and its natal colony social form (developmental environment).