Introduction
An estimated 80% of animal species have complex life cycles (CLCs)
wherein metamorphosis separates two or more discrete, post-embryonic
life stages (Wilbur, 1980). Although disagreements persist over
diagnostic criteria, the general consensus is that metamorphosis
involves an irreversible transformation in morphology that is typically
accompanied by a pronounced change in ecology (Bishop et al., 2006).
This change results in specialized stages optimized for distinct
ecological niches (Benesh, Chubb, & Parker, 2013; Bishop et al., 2006;
Ebenman, 1992; Istock, 1967; Moran, 1994). One explanation for the
prevalence of CLCs is that independent adaptations at the different
phases allow for optimal growth at some stages and optimal reproductive
success at other stages (Bryant, 1969; Moran, 1994; Truman & Riddiford,
2019). Central to this explanation is the idea that pleiotropy creates
genetic correlations across ontogeny that constrain evolution when
traits beneficial for one stage are detrimental to another (Haldane,
1932).
The adaptive decoupling hypothesis (ADH) proposes that metamorphosis
evolved as a mechanism for optimizing genetic correlations between life
stages, thereby facilitating the independent evolution of traits when
opposing selection pressures are experienced during different life
stages (Ebenman, 1992; Haldane, 1932; Istock, 1967; Moran, 1994;
Wigglesworth, 1954; Wilbur, 1980). A key prediction of the ADH is that
traits that experience antagonistic selection across development will be
genetically decoupled between distinct life stages and that more
ecologically distinct life stages should have greater decoupling. To
date, tests of this prediction have been mixed, with some studies
supporting the ADH (Anderson, Scott, & Dukas, 2016; Blouin, 1992;
Bonett & Blair, 2017; Goedert & Calsbeek, 2019; Hilbish, Winn, &
Rawson, 1993; Jacobs et al., 2006; Loeschcke & Krebs, 1996; Medina,
Vega-Trejo, Wallenius, Symonds, & Stuart-Fox, 2020; Parichy, 1998;
Phillips, 1998; Saenko, Jerónimo, & Beldade, 2012; Sherratt,
Vidal-García, Anstis, & Keogh, 2017; Wollenberg Valero et al., 2017),
others refuting (Chippindale et al., 1998; Crean, Monro, & Marshall,
2011; Fellous & Lazzaro, 2011; Watkins, 2001; Wilson & Krause, 2012),
and some with equivocal results (Aguirre, Blows, & Marshall, 2014;
Helle, Johansson, Lederer, & Lind, 2010). However, many of these
studies are limited by using only a few morphological traits and not
taking stage-specific selection pressures into account when evaluating
predictions of the ADH. Importantly, if metamorphosis is an adaptation
for optimizing genetic independence, then the magnitude of trait
decoupling should depend on the strength of antagonistic selection.
Testing this prediction of the ADH will require quantifying decoupling
for large and diverse collections of traits that vary in the extent to
which they experience antagonistic selection.
Whole-transcriptome gene-expression data obtained from multiple life
stages provide an ideal collection of traits for evaluating the extent
to which patterns of genetic decoupling fit predictions of the ADH
(Collet & Fellous, 2019). First, because all stages of the life cycle
must be encoded by a single genome, dramatic phenotypic changes that
accompany metamorphosis must be mediated by changes in gene expression.
Second, transcriptomes provide a large number of quantitative traits,
all measured in comparable units of gene expression, that can be readily
compared across life stages. Third, the genes included in a
transcriptome cover a wide range of biological functions that should
vary somewhat predictably in the extent to which they experience
antagonistic selection across the life cycle. Following the logic of the
ADH, this variation in selection should generate predictable variation
in gene-expression decoupling. For example, genes involved in basic
cellular functions (i.e., housekeeping genes) should be more genetically
coupled than genes that mediate ecological interactions that change
across the life cycle.
Transcriptome-wide patterns of decoupling should also vary with the
magnitude of the ecological change accompanying metamorphosis. The most
extreme metamorphic transformations occur in holometabolous insects,
whose defining characteristic is a distinct pupal stage that separates
two completely different body plans (Gilbert, Tata, & Atkinson, 1996;
Heming, 2003; Kristensen, 1999). Importantly, this profound
transformation enables one stage to be optimized for feeding and growth
(the larval stage) and a second stage for dispersal and reproduction
(the adult stage). The genetic independence of larval and adult traits
proposed by the ADH may explain, in part, why holometabolous insects are
one of the most evolutionarily successful and diverse lineages on the
planet (Ebenman, 1992; Haldane, 1932; Istock, 1967; Moran, 1994;
Rainford, Hofreiter, Nicholson, & Mayhew, 2014; Truman & Riddiford,
2019; Wigglesworth, 1954; Wilbur, 1980). In some holometabolous
lineages, pronounced ecological and morphological transformations also
occur between successive larval instars. This phenomenon, which has been
dubbed hypermetamorphosis (Belles, 2011), provides a valuable
opportunity to test the prediction that transcriptome-wide levels of
genetic decoupling between life stages will increase with the
dissimilarity of the fitness landscapes to which they are adapting.
Looking beyond metamorphosis, the rationale underlying the ADH applies
more generally to any scenario in which a single genome expresses
multiple distinct phenotypes that are subject to opposing selection
pressures (Collet & Fellous, 2019). Arguably the best-studied scenario
of genetic decoupling evolving in response to antagonistic pleiotropy
involves alternative phenotypes of a single life stage: adult males and
adult females. Just as stage-limited gene expression can reduce genetic
correlations across life stages of organisms with CLCs, sex-biased gene
expression can enable the independent evolution of male and female
traits, leading to the evolution of sexual dimorphism (Assis, Zhou, &
Bachtrog, 2012; Ellegren & Parsch, 2007; Parsch & Ellegren, 2013;
Perry, Harrison, & Mank, 2014; Proschel, Zhang, & Parsch, 2006). An
important distinction between CLCs and sexual dimorphism is that only in
CLCs must all alternative phenotypes (i.e., life stages) have non-zero
fitness for a novel pleiotropic allele to spread in a population (Collet
& Fellous, 2019). More generally, for the same net fitness difference
between alternative phenotypes, selection against an allele with
opposing fitness effects in different life stages may be stronger than
selection against an allele with opposing fitness effects in different
sexes. For this reason, trait decoupling should be more pronounced for
ecologically distinct life stages than for different sexes.
To date, only a handful of studies have evaluated the prediction that
gene-expression traits will be decoupled across metamorphic boundaries
as it pertains to the adaptive decoupling hypothesis (Fellous &
Lazzaro, 2011; Jacobs et al., 2006; Saenko et al., 2012; Wollenberg
Valero et al., 2017). Furthermore, to our knowledge, no study has
evaluated whether gene-expression decoupling varies predictably with the
type of life-cycle transition or gene function, and few studies have
directly compared patterns of sex-biased and stage-biased gene
expression (but see (Ometto, Shoemaker, Ross, & Keller, 2011; Perry et
al., 2014)). To these ends, we take advantage of a hypermetamorphic and
sexually dimorphic species of insect with a well-characterized ecology
and annotated genome, the redheaded pine sawfly (Neodiprion
lecontei , order: Hymenoptera; family: Diprionidae) (Figure 1).
In addition to the complete metamorphic event that occurs during the
pupal stage, there are two metamorphic transitions that occur within the
larval stage of the redheaded pine sawfly that result in pronounced
changes in coloration and behavior (Atwood & Peck, 1943; Coppel &
Benjamin, 1965; Linnen, O’Quin, Shackleford, Sears, & Lindstedt, 2018)
and references therein) (Figure 1A). The first metamorphic transition is
a shift from a “cryptic” to an “aposematic” feeding larval morph and
is less dramatic than the other transitions (hereafter, “minor
metamorphosis”). The cryptic morph is lightly pigmented, ingests only
the exterior of pine needles while avoiding the toxic resinous core, and
retreats to the base of the needle when predators are near. By contrast,
the aposematic morph is heavily pigmented, ingests the entire needle,
and sequesters the toxic pine resins for use in dramatic anti-predator
defensive displays. A more striking transition occurs when the
aposematic morph molts into a “dispersing” final instar (hereafter,
“major metamorphosis”). The dispersing morph is solitary, non-feeding,
less intensely pigmented, and migrates to the litter or soil to spin a
cocoon. Complete metamorphosis occurs within the cocoon. The non-feeding
adult stage is dedicated entirely to reproduction. Sexually dimorphic
adults are highly specialized for sex-specific tasks. Males are
excellent fliers and use bipectinate antennae to detect female
pheromones from considerable distances. In contrast, females remain near
the cocoon eclosion site and use serrate antennae to search for suitable
oviposition sites in Pinus needles (Anderbrant, 1993). Like most
hymenopterans, N. lecontei females lay a combination of
fertilized and unfertilized eggs that will develop into diploid females
and haploid males, respectively.
The hypermetamorphic life cycle of the redheaded pine sawfly, the wealth
of natural history data for this species, and the logic of the ADH
enables us to make a priori predictions about how levels of
genetic decoupling (inferred here from the magnitude of differential
gene expression) will vary among genes categories, developmental stages,
and sexes. We predict that: 1) The extent of gene-expression decoupling
should increase with the ecological dissimilarity of the life-stages
(Figure 1B). 2) Across the transcriptome, the most pronounced
gene-expression decoupling will be observed for genes that mediate
ecological changes across development. 3) Because traits expressed in
different individuals (sexes) may experience weaker selection for
decoupling than traits expressed in multiple life stages of a single
individual, we predict trait decoupling between the sexes will be less
extreme than that observed between metamorphic events (Figure 1B). To
test these predictions, we generated expression data for 9,304 genes via
whole-transcriptome sequencing of males of each N. lecontei life
stage and adults of both sexes.