Discussion
Understanding the historical eco-evolutionary processes that determine
the structure of biodiversity are primary goals in ecology and
evolutionary biology. Therefore, the current understanding that a large
proportion of arthropod diversity (Zug & Hammerstein 2015) harbours a
non-systematically distributed agent of speciation constitutes a major
academic challenge with respect to identifying predictable processes
that generate biodiversity. Here we introduce the ‘contact contingency’
predictive model for Wolbachia strain distributions based on
phylogenetic relationships, ecological contact, and host abilities to
determine their own infection status, that shows remarkable accuracy on
an empirical fig wasp dataset sampled from elevational transects in
Papua New Guinea. We also present the ‘oviposition trade-off’ model to
account for the influence of post-zygotic fitness losses imposed by Wolbachia which could potentially invalidate our proposal. We
show that post-zygotic fecundity losses may be tolerated under
multiple-mating scenarios when considering the ecological
characteristics of the fig syconia microcosm. We hope our work
stimulates further debate around these phenomena in order to either
accept and refine our proposed mechanism, or to prompt a dismissal
accompanied by a more parsimonious theoretical framework that accounts
for distributions of Wolbachia endosymbionts at the host
taxonomic scales we are querying. Our models may be particularly suited
to the unusual ecology of fig wasps, but their underlying dynamics may
contribute to differing degrees among other ecological systems (Box 1).
An inspection of Wolbachia strain distributions among our fig
wasp phylogenies reveals suggests that systemic processes may be in
operation. Wasp populations at different elevations display different
infection patterns. For example, 77% of Ceratosolen armipes are
infected but only one out of 34 individuals of its clade that pollinates Ficus umbrae carries Wolbachia . Further, there are Wolbachia strains that are restricted to lowland and highland
elevations, even among the same host (e.g., F. arfakensis ). As
such, parapatric populations of pollinating wasps often have contrasting
infection status that may represent RI, mediated by either uni- or
bi-directional CI. Our data repudiate vertical transmission
co-divergence hypotheses. However, we consider it important to note
that this also repudiates a horizontal exchange hypothesis, as Wolbachia strains invariably do not occupy all or multiple wasp
clades infecting the same host species/complex, despite potential for
ecological contact. These results are consistent with the idea that Wolbachia only infects certain insect groups under particular
host-adaptive circumstances.
Systemically generated patterns in host Wolbachia statuses could
arise if infection is largely controlled by endosymbiont abilities to
manipulate their own infection status as is often posited (Werrenet al. 2008); for example, because particular clades of host
lineages’ immune responses are unable to repel the infection. However,
our observed patterns do not obviously support manipulation by the
endosymbiont given the correlation with abiotic drivers such as
altitude, and no known a priori reason to assume that host
immunity is principally determined by abiotic circumstances. Relative
fitness costs in host mating potential, fecundity, or inappropriate sex
ratio distortion associated with Wolbachia invasion may also
generate the conclusion that Wolbachia is the chief architect of
its own success. However, it is known that Wolbachia offers host
fitness benefits that may have ecological contingencies (Zug &
Hammerstein 2015; Correa & Ballard 2016). We may consider a host insect
species that exhibits a broad phenotypic range, say for ovipositor
length that has distinct optima at different altitudinal ranges
according to host plant morphological divergence. Any mechanism that
prevented reproductive events between extreme phenotypes (yielding
intermediate morphs) at inappropriate altitudes would be favoured
providing the benefits (increased local fitness) outweighed any
deleterious costs.
Our wolPredictor simulation of the ‘contact contingency’
hypothesis attempts to model such a scenario that is founded on: (i)
host abilities to modulate/purge Wolbachia when evolutionarily
apposite; and (ii) Wolbachia causing a circumstantial host
fitness advantage. Our hypothesis is that recently evolved
sister-species diversifying within the same host community are at a
selective advantage when they harbour alternate strains of Wolbachia. Divergent strains facilitate the initial stages of
speciation; this is because we expect that closely related, co-diverging Wolbachia strains would not confer RI between hosts. Given that Wolbachia infection does appear to contain some fitness cost
component (Zug & Hammerstein 2015), we predict that these patterns
should not be evident among diverging lineages that are not in regular
ecological contact, thus leaving Wolbachia subject to host
immunosuppression due to the circumstantial absence of any RI-enforcing
benefit. Finally, as Wolbachia typically drops out of host
lineages after approximately 7 million years (Bailly-Bechet et
al. 2017), and because alternative mechanisms of RI that require
cytogenetic or morphological modification may take longer to evolve
(Bordenstein et al. 2001), we predict that closely-related
species that have not recently diverged should also be purged of Wolbachia infection. This would reflect a hypothesis that
observed lineage dropout (Bailly-Bechet et al. 2017) results from
temporal changes in the adaptive benefits of Wolbachia , which may
subsequently become redundant (the adaptive decay hypothesis).
Simulation results among our fig wasp data are particularly impressive
(ca. 88% accuracy for positive strains) when the species delimitation
algorithm matches the empirical understanding of ecological diversity
patterns (Souto-Vilarós et al. 2019). In particular, when
multiple congeners within fig host communities are algorithmically
predicted among F. arfakensis , F. trichocerasa subsp. pleioclada and F. wassa , wolPredictor ascribes
multiple strains within these communities that reflect the data.
Prediction accuracy when also considering negative (uninfected) Wolbachia strains is less precise. This may be due to variation
in the empirical data where negative and positive strains co-occur
within clades or the tendency for non-infected individuals to appear
among lineages that comprise multiple/incipient species within a single
host fig (e.g., F. wassa - infection rate = ca. 10%), where
wolPredictor ascribes positive Wolbachia associations
(such patterns may actually represent unidirectional RI whilst we model
the bidirectional mechanism).
To model the ‘adaptive decay’ hypothesis, our wolPurger function
operates to remove Wolbachia from lineages after extended
evolutionary timescales. However, as it uniformly removes Wolbachia across all samples exceeding the distance threshold it
constitutes a crude method that may not always be appropriately applied
across lineages. Inconsistencies in strain associations could result
from other drivers we have not accounted for, and alternative mechanisms
of RI (hypothetically rendering Wolbachia redundant) may appear
at different rates across lineages possibly due to serendipitous genomic
architecture or unconsidered ecological contingencies. For example,
among fig wasps, syconia access is partially controlled by relative
syconia-wasp size (Bronstein 1987), which mechanically prevents
hybridisation opportunity among some species.
Furthermore, under a simple model of panmixis and infinite population
size, CI is predicted to sweep to fixation, contrary to the population
level polymorphism observed in our data. Theoretical expectations on how
CI spreads through populations are largely determined by population
structure (Engelstädter & Telschow 2009). However, this depends on
perfect transmission and infection rates may still eventually decay even
if fixation is achieved. Fig wasps are both haplodiploid and inbred.
Haplodiploidy can facilitate the survival of infected haploid males
(Breeuwer & Werren 1990), which like inbreeding can result in both a
higher invasion threshold and a reduced stable equilibrium frequency
(Engelstädter & Hurst 2006). These considerations deserve further
attention, but they may, at least in part, explain infection frequencies
below 100% as observed in our study system.
Overall, our wolPredictor simulation of the ‘contact
contingency’ hypothesis is a rule-based algorithm that manages to
capture much of the embedded structure in a dataset that presents a
superficially stochastic appearance. Thus, it suggests that some
environmentally contingent symbiotic benefits (Correa & Ballard 2016)
may systematically sum to yield predictable Wolbachia distributions. Our methods cannot test for precise mechanisms
determining Wolbachia distributions across our study systems, and
the algorithm underpinning wolPredictor may inadvertently
represent some other set of real-world contingencies.
Moreover, we acknowledge that our model may be considered problematic
since Wolbachia -mediated CI is a post-zygotic mechanism that
elicits an immediate fitness cost in host fecundity. However, it is
feasible that the unique life-histories and ecological conditions of fig
wasps means they may be tolerant of CI: oviposition sites are at
especially high premium (Dunn et al. 2015), fig wasps are known
to produce surplus eggs (Dunn et al. 2011), and co-evolved
species are renowned for precise tolerances in interacting traits that
may render hybridisation particularly costly. This critique prompts our
‘oviposition trade-off’ hypothesis and second simulation model. Here we
investigated the impact of CI in multiple-mating scenarios when
considering the oviposition limiting constraints of fig syconia. We show
that inclusive fitness of multiple mated females can be higher despite
fecundity losses providing that egg load reduction (or selective
ovipositioning) facilitates oviposition into higher-quality fig ovules
that are less vulnerable to parasitoid attack (Dunn et al. 2008).
Notably, the parameter values (i.e., conspecific mating levels and
relative fitnesses) that yielded CI favouring outcomes are realistically
achievable among natural fig wasp populations. The results imply that
bi-directional CI may adaptively evolve in fig wasps without
accompanying mechanisms.
The interaction of CI-inducing Wolbachia on multiple-mating in
fig wasps has not been studied but we found a single study in Drosphila demonstrating Wolbachia associated reductions in
sperm competition abilities (Champion de Crespigny & Wedell 2006).
Given the unusual reproductive manipulations of haplodiploid Hymenoptera
such as selective fertilisation, adjustment of sex-ratios, and control
over oviposition order according to ploidy, and given the dearth of
research of CI under multiple-mating conditions, it is entirely possible
that such dynamics are at play among fig wasps at least. Thus, future
work examining whether incompatible matings result in differential
pre-oviposition embryo mortality, and whether selective oviposition of
conspecific versus heterospecific eggs occurs would be of great value.
It may be that RI-inducing Wolbachia constitutes a mutualist
symbiont preventing the inefficient use of highly valuable oviposition
sites with intermediate hybrid form, lower fitness offspring.
We further note that our models diverge from some conventionally held
opinions regarding Wolbachia and host manipulation/purging (e.g.,
Werren et al. 2008) capabilities and a general view thatWolbachia is in conflict with its hosts in many respects (e.g.,
Charlat et al. 2007). While it has been shown that Wolbachia may employ microRNAs to alter host gene expression
(Hussain et al. 2011), it has not been investigated whether any
Red Queen-type arms race dynamics facilitate host resistance – as our
model implies. We argue that despite potential pitfalls it is difficult
to propose an alternative systemic model or explain observed structural
patterns as random, within our or other published datasets. For example,
an overview of malvantheran fig wasps shows that communities featuring
singleton congeners invariably display negative Wolbachia associations while the reverse is true for multi-congener communities
(Haine & Cook 2005). Finally, we also contend that the most
parsimonious interpretation of reported empirical patterns (i.e.,
sister-species hosting paraphyletic infections) leads to a view that the
interests of Wolbachia may be aligned with their hosts under
conditions of ecological speciation. Wolbachia is known to impart
some host benefits (Zug & Hammerstein 2015) and we are yet to fully
understand the nuances, trade-offs and ecological contingencies that
determine whether it is rendered circumstantially advantageous.
Our proposed models are particularly suited to testing in fig wasps due
to the high degree of easily-collatable host-specificity relationships
exhibited when wasps co-occur within the fig microcosm. Such processes
may also be subtly at play among other taxa where ecological contact is
not easily assessed – such data have not been previously incorporated
into a predictive phylogenetic model. Variables such as ecological
contact may act along a continuum among species rather than the easily
assessable binary states observed in fig wasps. Indeed, a suite of
variables that comes into play in fig wasp communities, e.g., degrees of
inbreeding or variation in oviposition site quality, may differentially
contribute to different systems and may interact with other factors
regarding Wolbachia cost-benefit trade-offs that determine
infection status. Thus, fig wasps may offer an ideal window into
understanding not only the determinant ecological contingencies at play
but also offer insight into the nature of the types of variables that
may be significant or even overridden by other factors (for example, we
note that our ‘contact contingency’ hypothesis is pertinent to all fig
wasps whereas the ‘oviposition trade-off’ hypothesis is relevant to
pollinators and non-pollinators that enter the fig). Even with these
system-specific limitations in mind, the vital issue then may be that
for most of global diversity we simply do not have the kind of detailed
ecological information that can reliably inform us about the processes
underpinning community assembly. Our theoretical reasoning may therefore
be extremely generalizable and we urge that model communities
representing different ecosystem states are identified and investigated
(Box 1) under a proposed methodological framework (Box 2).
Our models imply a genomic mechanism among insects making Wolbachia tolerance/purging a highly labile and evolutionarily
unstable trait that would concomitantly render RI events a relatively
trivial occurrence. Other potentially similar mechanisms have been
documented in other taxa (see also Box 1). In the hymenopteran Nasonia, Wolbachia -induced CI has been shown to precede
other incompatibility mechanisms (Bordenstein et al. 2001). In Drosophila , readily occurring genomic inversions (Noor & Bennett
2009) often serve to maintain RI among closely-related
species/subspecies in sympatry (although CI is also found in
fruit-flies; Merçot & Charlat 2004), while such phenomena are less
prominent in allopatry (Noor et al. 2001). And among certain
gastropods, a single gene mutation coding for shell chirality can cause
RI between sister-species (Hoso et al. 2010). Under the islands of speciation paradigm (Noor & Bennett 2009), regions of
genomic divergence incrementally build until differences between
lineages yield distinct evolutionary trajectories. A conundrum exists in
explaining how this might generate entirely separate lineages under
ecologically driven divergence in sympatry and in the face of gene flow.
Future work may reveal that it is highly labile, binary decision-making
reproductive switches such as CI or chromosomal inversions that resolve
this puzzle by providing tipping point mechanisms that promote
evolutionary schisms when net selective pressures favour speciation.
The stark disparity between recent advances in genomic data accrual
relative to the laborious efforts required to record
phenotypic/ecological data is well acknowledged – our work highlights
the need to ameliorate this. Biodiversity cannot be simplistically
evaluated as a metric indicating number of species nor considered solely
as the outcome of interactions between closely-related species within
the same trophic level. There is a growing consensus that we need to
consider interactions both within and between all trophic levels whilst
also identifying what constitutes significant versus trivial dynamics
(i.e., intensity of interaction; Segar et al. 2020), or, more
generally, ecological contingency. Thus, we face a massive challenge to
document community dynamics of not only obviously tractable
relationships (e.g., competition between focal species) but also of both
mutualistic (e.g., pollination, seed dispersal, nutrient sequestration,
mycorrhizal) and antagonistic factors (e.g., parasitic, disease) that
may sometimes be bacterial, fungal or viral in origin. Failure to
account for such agents may mean we never fully disentangle the myriad
determinants of ecosystem dynamics nor quantify the relative
contributions of stochastic (viz . neutral; Hubbel 2001)
processes.
CONCLUSIONS Our results indicate that Wolbachia distributions are predictably
structured among an arthropod dataset based on a predictive model
invoking adaptive responses in host fig wasps. A parsimonious
interpretation of these findings suggests that ecologically contingent
co-evolutionary benefits of Wolbachia induced CI, particularly
with respect to opportunity for lineage diversification, may
systematically sum to yield predictable distributions despite initial
appearances that the endosymbiont is stochastically distributed at some
taxonomic resolutions. In particular, our data suggests that future work
assessing biodiversity patterns among arthropods should incorporate Wolbachia infection data (alongside other microorganisms) as an
added modelling dimension in order to account for a potentially
confounding variable. Our aim is to stimulate debate and subsequent
research in unravelling a rather puzzling phenomenon within arthropod
biodiversity.
Acknowledgments : We thank villagers from all collecting sites for
both providing local assistants and offering us accommodation during our
stay along the transect. We also would like to think all staff of the
New Guinea Binatang Research Centre in Papua New Guinea and the Papua
New Guinea Department of Environment and Conservation for help granting
export permits. We thank Sylvain Charlat for constructive criticism and
for the improvement of this manuscript. We acknowledge funding from the
Grant Agency of the Czech Republic (grant number 15-24571S). STS
acknowledges departmental support from Harper Adams University.