No signal of geographic home range on Wolbachia strain sharing
As a first step in the host shift process, Wolbachia need to physically reach the recipient host species, which requires direct or indirect species interactions. Therefore, it is expected that one should observe host shifting among species with an overlap in geographic distribution. However, our model indicates that the geographic home range of scale insect species has no significant contribution toWolbachia sharing (Figure 3). This finding may firstly relate to the age of infection. Estimation of the Wolbachia infection age and consequently the intervals of host-shift events is controversial, with vastly different estimates across different case studies being reported (from a few thousand (Turelli et al. 2018; Cooper et al. 2019) to nine million years (Bailly-Bechet et al. 2017), see also Sanaei et al. (2021a)). If the changes in the host geographic distribution occur faster than Wolbachia host shift events, the current geographic distribution may not be able to explain host-shift events (and thus, we would need to reconstruct the historical home range). In addition, the geographic distribution of a given species is not necessarily representative of the realised niche of that species, including ecological connectivity (Pulliam 2000; Kearney 2006; Peterson & Soberón 2012). Therefore, two species may have the same geographic distribution but have no direct or even indirect physical interactions (e.g., via sharing foods or other resources). In that case, host ecological niche may be a better tool to explain Wolbachia host-shifting. However, ecological niches are technically harder to measure, especially when trying to account for ecological interactions.

Role of scale insect associate species in Wolbachia host shifts

Another possible reason why host geographic distance effect has such a weak effect on Wolbachiasharing is host shifting via ecological vectors. Such vectors can carry on the infection, either temporarily or permanently, and transmit it to a recipient species at a distant geographic location from the donor species. From prey-predator (Johanowicz & Hoy 1996; Le Clec’h et al. 2013) to host-parasitoid (Vavre et al. 1999; Kageyama et al. 2010; Tzuri et al. 2020) and trophallaxis interactions (Ramalho & Moreau 2020), there are several direct and indirect ecological pathways which can be routes of Wolbachia transfer. Intimacy of direct physical interaction between ants and scale insects may provide a route of microbial exchange, as seen in other hemipteran groups (Pringle & Moreau 2017; Ivens et al. 2018). Moreover, Gruwell et al., (2007) found plausible routes of Cardinium horizontal transmission between armoured scale insects and their associate parasitoids.Wolbachia may also utilize these ecological routes to spread within scale insect communities.
In a previous study, a positive correlation between Wolbachiainfection in scale insects and their associates indicated that ants may play a role in host-shifting (Sanaei et al. 2021b). Here, we found that only one out of five case of infected ant-scale insect pairs shares the same Wolbachia strain. While we do not have enough statistical power to test which route of transfer is the most common and in which directions these transfers take place, our data support the hypothesis that the associates tested in the current study play a role in host shifting. Although positive correlations were not previously observed between infection of scale insects and their associates (Sanaei et al. 2021b), here we observed sharing similar Wolbachia strains between pairs of scale insect and not only ants but also wasps, and flies (File S3). In addition, infection by the super-spreader stain “w Sph1” of several species of scale insects, ants, and wasps is another source of evidence for a substantial contribution of associate species in Wolbachia host-shifting in scale insects.
This study provided the first insight into Wolbachia strain diversity in scale insects, revealed a high portion of co-infected samples and detected w Sph1 as one the most common strain ofWolbachia in scale insects. We also found that the host phylogenetic distance effect plays a critical role in host shifting in scale insects. In future studies, the methodology suggested by this study could be applied to a larger data set to detect the factors influencing host-shifting on the global perspective.