Results
The output of our bioinformatics
pipeline successfully recovered 94.03% of all Wolbachiaamplicons and yielded read count numbers between 4658 and 36548 per
sample (summed over all seven Wolbachia genes; for details see
File S1 and S2). This high coverage enabled us to identify a diversity
of Wolbachia strains within and across hosts. Among 75Wolbachia -positive samples (59 scale insect samples and 16
associates), 68% were infected with a single Wolbachia strain
and 32% were infected with more than one Wolbachia strain (20
double and 4 triple infected). Among nine scale insect species with
samples from more than one population, intra-species infection
polymorphism was observed in Cryptes baccatus, Eriococcus
confusus, Icerya purchasi, Parasaissetia nigra and Sphaerococcus
ferrugineus (File S2). Among 29
scale insect species that were screened in this study, 6 species were
always found co-infected (File S2). For example, two screened samples ofAkermes scrobiculatus were coinfected with strains belonging to
Supergroup A (w Ake1) and Supergroup B (w Ake2) and two
samples of Cystococcus echiniformis were coinfected with strains
belonging to Supergroup A (w Sph1) and Supergroup F
(w Cys1).
A total of 63 strains were identified and clustered into 31 strain
groups belonging to three Wolbachia -supergroups (Figures 1, S2
and File S4). Most of the strains belong to Supergroups A (38) and B
(21), but we also identified three strains groups from Supergroup F:w Cys1 and w Sph5 (respectively infecting Cystococcus
echiniformis and Sphaerococcus ferrugineus ), and w Sph3
(infecting two specimens of S . ferrugineus ). Based on the
MLST database, these are the first Supergroup F strains reported in
Australia. Although w Cys1 is placed within Supergroup F, it forms
a unique clade compared to other reported Supergroup F strains (Figure
S2).
The most diverse and abundant Wolbachia strain group in our
dataset is w Sph1, which includes 12 closely related strains and
was detected in 23 samples belonging to eight scale insect, four wasp
and one ant species (Figure 2, S3 and File S2, S4). Similar strains
which are grouped within w Sph1 were reported before in several
Australian ant species (MLST ST = 54, 19, 478 and 112) (Russell 2012).
Based on the MLST database, it appears that this strain group has a
cosmopolitan distribution (Oceania, North America, Europe, Asia and
South Africa) and has already been reported in various insect groups
(e.g., ST 19 in Coleoptera, Hymenoptera, Lepidoptera and Orthoptera). By
contrast, some of the scale insect species are infected with uniqueWolbachia strains that were not observed in other scale insects
or reported in any other insects (by searching both MLST and GenBank on
October 1st2021), including infection of Apiomorpha variabilis withw Aphi1 and co-infection of Coccus hesperidium withw Coc1 and w Coc2.
ML trees based on a 947 bp alignment of scale insect genes (including
COI, 28S and 18S), and based on 3065 bp aligned Wolbachia genes
(including MLST, wsp, and 16S) are shown in Figure 2. In addition, an
interactive figure of Wolbachia sharing among all host species,
including associates, is provided in File S5. Evidence of numerous host
shifting events can be seen in these figures. Both the Parafit test
(ParaFitGlobal = 0.0008, P -value = 0.27) and the PACo test
(m2XY = 49.3897, P -value =
0.2056) were non-significant.
Therefore,
there is no evidence of
phylogenetic congruence between Wolbachia and their scale insect
hosts, in the sense that non-independent evolution of the two groups is
not supported by these tests.
Our Wolbachia sharing models revealed that incorporating
phylogenetic distance substantially improved model fit (change in DIC =
-5.88), and had a significant effect in the model (P<0.0001).
The effect was highly non-linear, with high sharing probabilities at
high relatedness that quickly dropped to near zero at greater
phylogenetic distances (Figure 3). In contrast, incorporating geographic
home range overlap slightly improved model fit (change in DIC = -2.76),
but had no significant effect in the model (P=0.199). Inspecting the
shape of the effect was not revealing. Furthermore, there was no
significant effect of geographic distance between sampling locations
(change in DIC > -2). Therefore, we do not interpret this
effect as representing strong support for geographic effects onWolbachia sharing.
In searching for Wolbachia strains in pairs consisting of a scale
insect and its direct associate, we found the same Wolbachiastrain groups in one out of five ants, one out of two flies, and three
out of seven wasps. In the
ant-Eriococcus sp1 pair that is co-infected with w Sph4 andw Apio2 strain groups, both the ant and the scale insect are
infected with the same w Sph4 strain (w Sph4.4), but
different w Apio2 strains (w Apio2.4 in the ant andw Apio2.5 in the scale insect, which differ by 4bp in gatB gene).
The three wasp-scale insect pairs with similar Wolbachia strain
groups have a single infection (wasp- Ascelis schraderi pair withw Sph1.11, wasp- Apiomorpha floralis pair with w Api
2.1 and wasp-Lachnodius eucalypti pair with w Lac). The
fly-Icerya seychellarum pair share identical strains ofw Cal2.2. We did not find the same strain group in the single
tested moth-scale insect and two beetle-scale insect pairs. As in scale
insects overall, the most common strain group shared between scale
insects and associates is the w Sph1 strain group (File S2).