Introduction
Recognising the conditions that favour speciation is critical if we are
to understand the extent and structure of biodiversity.
Moreover, species interactions,
both between and within trophic levels, can be significant contributors
to diversification processes and are sculpted by evolutionary forces,
which in combination with abiotic drivers deliver an ecosystem or
community’s (dynamic) state (Harmon et al. 2019). Thus, an in
depth understanding of adaptive processes alongside their ecological
contingencies (e.g., interaction strengths and polarities; Segaret al. 2020) is a fundamental component of the diagnostic tool
kit essential for achieving standard objectives in ecology.
Apropos of this, increasing emphasis on the arthropod microbiome as a
modifier of ecological interaction strength (e.g., Hansen & Moran 2014)
underlines the need to consider endosymbionts as part of the extended
phenotype. The rise and fall of microbial partners is an
eco-evolutionary process, driving and being driven by ecological
interactions between organisms and their environment. One such
endosymbiotic bacterium, Wolbachia , infects up to 40% of
arthropod species and often plays a key role in speciation (Werrenet al. 2008; Zug & Hammerstein 2015). Wolbachia commonly
induces cytoplasmic incompatibility (CI) via sexual sterility between
infected males and females that are either uninfected
(unidirectional CI) or carry an alternative strain
(bidirectional CI) (Beckmann et al. 2017, 2019; LePageet al. 2017). CI may therefore promote reproductive isolation
(RI) between populations or incipient host species and increase the
speed or likelihood of speciation by restricting geneflow (Bordensteinet al. 2001; Zimmer 2001; Telschow et al. 2007), a
critical factor in sympatric and ecological speciation otherwise caused
by correlations between divergent traits, mate choice and/or habitat
choice (Gavrilets 2004).
In some non-arthropod taxa, Wolbachia is an essential mutualist
and accordingly shows strict co-divergence with hosts (Casiraghiet al. 2001; Balvín et al. 2018). Among arthropods, Wolbachia lineages are mostly facultative and evolutionarily
unstable symbionts generally exhibiting host co-phylogenetic
incongruence (Shoemaker et al. 2002; Yang et al. 2012;
Jäckel et al. 2013; Zug & Hammerstein 2015), although exceptions
are known where essential mutualism appears likely (e.g., Dedeineet al. 2001; Raychoudhury et al. 2009; Hamm et al.2014). At broader taxonomic scales (e.g., families, orders), a
non-random distribution of Wolbachia has been noted (Engelstädter
& Hurst 2006; Weinert et al. 2015), viewed as the consequence of
accelerated host switching among closely related species from highly
speciose clades (Engelstädter & Hurst 2006). However, at reduced scales Wolbachia often appears idiosyncratically distributed (Shoemakeret al. 2002; Smith et al. 2012; Yang et al. 2012;
Jäckel et al. 2013; Zug & Hammerstein 2015), as closely related
hosts often harbour paraphyletic strains. These strains are paraphyletic
within the context of the complete Wolbachia phylogeny, such that
grouping strains from two closely related hosts renders them
paraphyletic. Horizontal exchange also occurs between unrelated species
(Shoemaker et al. 2002; Zug & Hammerstein 2015; Bailly-Bechetet al. 2017). Counterintuitively, this may not be readily
predicted from close ecological contact (Haine & Cook 2005; Jäckelet al. 2013; Gerth et al. 2013) but incidences where it
has been recorded (Sintupachee et al. 2006; McFrederick & Rehan
2016; Miraldo & Duplouy 2019) suggest that outcomes may be context
dependant. Many studies conclude that infection status depends on the
ability of Wolbachia to manipulate its arthropod hosts (Werrenet al. 2008; Zug & Hammerstein 2015), which may add to the sense
that it is non-systemically distributed. Testable models linking
eco-evolutionary processes to distribution patterns and ecological
context remain critically absent.
As Wolbachia mediated CI results in post-zygotic mortality,
initial fitness losses due to reduced fecundity are costly, meaning that
selection may be expected to operate on hosts to purge Wolbachia .
However, Wolbachia is posited to facilitate reproductive
isolation between incipient species in combination with reduced hybrid
fitness, even when only unidirectional pre-zygotic isolation operates
(Shoemaker et al. 1999). The maladaptation of intermediate forms
is central to models of sympatric/ecological speciation which may be
likely under bi-directional CI as documented in closely-related,
co-occurring Nasonia wasps (Bordenstein & Werren 2007). Thus, it
is possible that Wolbachia represents a tolerable cost
(contingent on ecological circumstances), rendering host fitness
advantage (i.e., via hybrid avoidance) the prime determinant of
infection status rather than the bacterium’s manipulative capability.
Predictive phylogenetic models of Wolbachia distribution have not
previously incorporated the intensity of ecological contact between
insect lineages that (a), provides a direct opportunity for horizontal
exchange of microbes or genetic material, and (b), provides a
contingency axis of whether RI is required. When speciation occurs in
allopatry, specific mechanisms of RI may not necessarily evolve as the
nascent species are not in contact (Coyne & Orr 2004). This may also be
true if newly formed species specialise on different resources in
sympatry (Nosil 2012). However, a mechanism of pre- or post-mating RI is
required if ecological contact occurs, when the species use the same
resources and overlap in space and time (e.g., Via & Hawthorne 2002).
Wolbachia typically drops out of host lineages after
approximately 7 million years (±5.2-9.6) (Bailly-Bechet et al.2017), contributing to the lack of correlated host-symbiont divergence
and adding weight to the idea that purging may occur. Compared with Wolbachia , alternative mechanisms of RI that require cytogenetic
or morphological modification may take longer periods of time to evolve
(Bordenstein et al. 2001), and thus may not be responsive enough
to changing ecological circumstances that favour diversification,
particularly in a sympatric setting. These lines of evidence suggest
that observed lineage dropout (Bailly-Bechet et al. 2017) may
result from temporal changes in the relative adaptive benefits of Wolbachia (as alternative mechanisms of RI evolve), that may
subsequently become redundant and eradicated if hosts can mediate their
own infection statuses (e.g., via physiological immune responses) –
hereafter termed the adaptive decay hypothesis.
Fig wasps (Chalcidoidea), where Wolbachia prevalence is ca. 60%,
appear to be a prime candidate for CI manipulation because many closely
related and often cryptic species (both pollinating and non-pollinating)
share an enclosed reproductive space (i.e., fig inflorescences), where
they regularly come into contact giving potential for hybridisation
(Molbo et al. 2003; Darwell et al. 2014; Yu et al.2019). Moreover, inbreeding is also common favouring female biased
sex-ratios, strain fidelity through vertical transmission, and reduced
allospecific (i.e., ex community) encounter rates – all
increasing barriers to gene flow (Branca et al. 2009). Due to the
confined nature of fig syconia (i.e., fig inflorescences), co-occurring
incipient species must rapidly employ RI barriers (Nosil 2012) to avoid
any hybridisation costs. Fig wasp studies often show paraphyletic Wolbachia infections across sister-species (Shoemaker et
al. 2002; Haine & Cook 2005; Yang et al. 2012), while species
occupying fig communities that do not contain congeners invariably
display negative Wolbachia statuses (Haine & Cook 2005).
Importantly, while these factors may obviously and measurably dominate
fig wasp community structure, their influence may be apparent in all
ecological systems to differing degrees.
Hybridisation between highly adapted lineages of wasps, with narrow
abiotic niches and extreme matching for host fig interacting traits,
presents a rather extreme cost. We develop a model which selects for
ecologically contingent host tolerance of otherwise costly Wolbachia in this system, thus imposing pre-zygotic selection and
reduced gene flow between lineages. We propose that sister
populations/incipient species of wasps, associated with diverging fig
hosts, should be infected with paraphyletic Wolbachia strains
when in close ecological contact. Thus, Wolbachia should
facilitate adaptive divergence. Subsequently, we model purging ofWolbachia after alternative mechanisms of RI are established
across evolutionary time (see Fig. 1). This contact contingency hypothesis leads to a predictive system that would elicit an apparently
stochastic distribution, with respect to the host phylogeny, similar to
those frequently observed.
While the unusual ecological conditions of fig wasps, including the
potential for complementary pre-zygotic (e.g., behavioural) barriers,
may be sufficient to permit tolerance of post-zygotic fecundity
reduction, we also develop a second model to singularly account for
post-zygotic dynamics. We consider the heightened value of oviposition
sites which are finite for pollinating fig wasps as they are unable to
leave fig syconia after entry (Cook & Segar 2010). In monoecious fig
species syconium oviposition sites are more valuable towards the centre
where parasitoid wasp ovipositors typically do not penetrate (e.g., Dunnet al. 2008). As intermediate hybrid forms exhibit marked fitness
reductions within co-evolved systems, the costs of reduced fecundity may
prove tolerable if hybrid eggs are not wasted on premium oviposition
sites. This could feasibly occur in two ways among fig wasps: (i) via
preferential oviposition of favoured non-hybrid embryos (Hymenoptera are
at least known to manipulate the oviposition order of haploid versus
diploid eggs as well as adjust sex ratios; Raja et al. 2008); or
(ii) via differential mortality affecting unviable hybrids before
oviposition (an undocumented but plausible phenomenon). This is
contingent on multiple mating events occurring within syconia (e.g.,
Murray 1990; Greeff et al. 2003), so that fig wasp foundresses
carry egg loads of high versus low fitness embryos.
We model this oviposition trade-off hypothesis by simulating
pre-oviposition egg mortality causing reduced egg load, meaning zero
fitness is attributed to lost hybrid embryos. However, as fig wasps are
known to prioritise oviposition into favourable sites, remaining
non-hybrid eggs receive greater average fitness as they are
probabilistically oviposited towards the syconium centre as opposed to
their average position when mixed together with viable hybrids of
reduced fitness (Fig. 2). We examine these trade-offs under different
frequencies of conspecific-heterospecific mating opportunity scenarios.
As part of an ongoing study investigating patterns of co-speciation
between several monophyletic fig (Ficus , Moraceae) species
complexes and their pollinating wasps, we tested our primary ‘contact
contingency’ hypothesis, which explains how Wolbachia infection
may be an adaptive responses to diversification pressures in host wasps.
This is accomplished by presentation of empirical data of pollinating
wasps screened for Wolbachia , and then using Python
programming to simulate our proposed mechanism incorporating ecological
contact and phylogenetic relationships. We then test our secondary
‘oviposition trade-off’ hypotheses which accounts for Wolbachiapost-zygotic fecundity costs by modelling disparities in oviposition
site quality again using Python programming. Our field site,
located along a steep elevational gradient in Papua New Guinea, features
a steep clinal turnover of Ficus species (Segar et al.2017) with species complexes comprising lowland/highland sister species
or morphologically homogenous species with wide elevational ranges
(Souto-Vilarós et al. 2018, 2019).