Wolbachia screening of field collected samples
From 253 screened wasps, 41.1% (104 individuals) displayed Wolbachia infection. We found no evidence for multiple
infections as no chromatograms contained multiple peaks. Individual wsp and MLST phylogenies confirm the monophyly of identified
strains (Table S1; Fig. S1); the wsp tree contained five major
clades while the MLST tree contained six. wsp clade assignment
mostly matched with MLST markers, with the exception of the MLST clades
associated with F. trichocerasa subsp. pleioclada and F. microdyctia , which were contained within the same wspclade (Table S1; Fig. S1). We therefore followed wsp clade
designation for all wasps apart from wsp clade six which was
split into two (wspC6_1 and wspC6_2) giving six identified Wolbachia clades.
While 77% of all Ceratosolen armipes (the pollinator of lowlandF. itoana ) were infected with Wolbachia, only one (out of
34) of the mid-elevation C. sp (ex. mid-elevationFicus umbrae ) was infected. Similarly, ca. 63% of C.
“kaironkensis” (ex. highland F. microdyctia ) were
infected. These wasps and their fig hosts form monophyletic clades with
species replacing each other in parapatry (Fig. 3), mid-elevation F. umbrae (largely Wolbachia free) and highland F.
microdyctia being sister species and lowland F. itoana the
outgroup (Souto-Vilaros et al., 2018). Alternative populations of the
four Ceratosolen pollinator species associated with the single
species F. arfakensis showed disjunct infection statuses, with
26% infected overall but with different infection frequencies and
strains at different elevations (e.g., wspC3 in the lowlands and wsp C2 in the highlands). For F. trichocerasa (a single
species comprising two distinct subspecies) the proportion of infected
wasp pollinators differed between host fig subspecies with lowland
subsp. trichocerasa and highland subsp. pleioclada having
84% and 54% infection frequency, respectively. Strain identity was
also largely distinct to a given subspecies. In the case of F.
wassa (a genetically homogenous entity across the gradient hosting a
two major pollinator clades), only 10% of pollinator wasps (all
individuals from highland populations) were infected. Overall, sister
species/populations of wasps usually had different Wolbachia infection status or strain type (Fig. 3 & Fig. S1). These sister
species of wasps were not infected by monophyletic MLST or wsp (except wspC6 ) clades of Wolbachia.
wsp strains appear restricted to lowlands or highlands. For
instance, wsp clades 1, 6_1 and 6_2 are present in wasps from
elevations above 2,200m while the rest occur in the lowlands (below
1,200m). An exception is for wasps originating from the mid-elevation
site (here considered as 1,700m) “Degenumbu” where both lowland and
highland strains occur. For instance, wsp clade 1 (a highland
strain) occurs in F. wassa wasps from this location; similarly, wsp clade 2, a lowland strain occurs in F. arfakensis wasps from 1,700m. Overall, bar a few exceptions, strain type segregates
by (sub)species while infection status seems to be influenced by
elevation.
Simulation of Wolbachia distribution among host species
under the ‘contact contingency’ hypothesis
Our wolPredictor simulation was able to predict positive strain
associations at up to 88.46% (92/104 individuals at species clustering
levels of 10-13; SI runs ‘pleio ’ 4, 5, 12 & 18) accuracy against
the empirically observed infection statuses across our fig wasp
phylogeny (Fig. 4). Predictive accuracy of greater than 80% was found
in 16 of 20 runs at species clustering levels of 10-19. Investigation at
these species delimitation assessments show high congruence with species
diversity patterns in Souto-Vilarós et al. (2019), notably with wasps
from F. arfakensis , F. pleioclada and F. wassa split into two or three putative species featuring alternate Wolbachia strain statuses. The highest accurate overall strain
prediction, 65.61%, (30 positive and 136 negative predictions)
regularly occurred at species clustering levels of 5-7 – with the wolPurger function removing around 30 positive predictions and
adding >100 negative predictions. In general, improved
negative strain accuracy often trades-off with losses in correct
positive strain predictions. High non-infection prediction accuracy
occurs at lower species clustering levels where large singleton clades
within communities are ascribed negative Wolbachia associations.
One-sample T-tests of these best-scoring results against 1000 randomly
generated predictions (mean = 14.16% accuracy for positive strains
only; mean = 37.86% accuracy including negative strains) indicates that
our model simulation predicts Wolbachia infection status with
significantly higher accuracy (t = -698.98, d.f. = 999, p <
2.2e-16 for positive strains only; t = -335.61, d.f. = 999, p
< 2.2e-16 including negative strains). As a further control,
we also ran 100 wolPredictor simulations (see SI files:pleio_shuff* ) with assayed wsp clades randomly shuffled
– the best predictive power for positive strains fell to around 46.15%
(mean = 40.19%) significantly less than our best prediction for
positive strains (t = -33542, d.f. = 99, p < 2.2e-16).