Discussion
In this study, we characterized the gut bacterial microbiota of five lizard species from Portugal (the native Podarcis virescens ,P. bocagei and P. lusitanicus, and the introduced P. siculus and Teira dugesii ) using a metabarcoding approach. Our results showed that locality was the main predictor of microbial diversity, significantly influencing microbiota composition and structure. Moreover, host sex and size also had an effect, albeit more discrete, in gut bacterial communities. All lizards shared the same most abundant bacteria phyla with results being in accordance with what has been found in other studies in lizards [e.g. 43, 44].
The two habitats in which lizards were captured are very different, with lizards from Lisbon living in an urbanized and artificial habitat, with greater environmental disturbance, compared to lizards from Moledo, which live in a semi-natural habitat. Plausibly, differences in habitat may lead to differences in the composition and diversity of the gut microbiome [45]. We detected higher microbiota diversity in the more urbanized environment which could be explained by the higher variety of diet items, which may also include human food waste. Additionally, environmental microbiota, which could be horizontally transferred to lizards, may also be more diverse in urban habitat than in semi-natural ones [17]. We hypothesize that habitat disturbance, the co-existence with humans and urban animals (such as cats, dogs and rats), may influence dietary behaviour and contribute to a higher bacterial load in the environment, which can then be acquired by lizards. These results agree with those on the Australian water dragon, where the gut microbiome of lizards in urban areas was more diverse compared to those residing in semi-natural habitats [17].
The proportion of some of the most abundant bacterial phyla and genera found in our study differed between lizard species at each locality. Additionally, we found that the species and sex also had an effect in the abundance of some of the gut microbiota components in P.virescens and P. siculus . The influence of host taxonomy in gut microbiota, which is a proxy not only for host genetics but also its general ecology, has been reported in many animals [46, 47] including reptiles [18]. The influence of sex in the abundance of major bacterial groups, has also been reported in striped Plateau lizards (Sceloporus virgatus Smith, 1938), with sex-specific cloaca microbiomes being related to different hormone levels [48]. In our study, the influence of sex was small when compared to the effect of habitat and host taxonomy. Nonetheless, the influence it exerts could also be linked to differences in the ecology of the two sexes. Importantly, the difference in size between female and male lizards may lead to slight differences in feeding behavior, and consequently on the gut microbial diversity.
Interestingly, we found a positive correlation between lizard lenght and alpha bacterial diversity in males from P. siculus . This lizard is larger than the other studied species and is also a very successful invasive species [27, 28]. Indeed, males of P. siculus can be more aggressive than males of the native Podarcis species [49], and also more exploratory, bolder, and better at exploiting food resources when compared to the native P. virescens in our study location [28, 29]. These behaviours can be associated with the displacement of P. virescens from gardens now inhabited byP. siculus [50] and can also be leading to a wider ecological and trophic niche, and consequentially to a higher microbiome diversity in P. siculus .
Finally, our analysis of potential bacterial transmission between the lizards living in sympatry, indicates a balanced transmission between species at Moledo and unbalanced transmission between species in Lisbon, with the invasive P. siculus estimated to receive a higher proportion of bacteria from the native P. virescens . These differences in estimates of bacterial transmission may reflect different non-exclusive aspects of the ecology of the hosts, such as the similarity in dietary niches and habitat occupancy (i.e., horizontal bacterial transmission from the environment). Differences in the microbiome of P. siculus and P. virescens could be related to an increased habitat occupancy and successful adaptation to the environment by the invasive species, which facilitated the acquisition of a higher quantity of local microbiota upon its arrival. These results could also be reflecting an increased ability to exploit a variety of food resources, or most likely a combination of both. The populations ofP. siculus and P. virescens live in sympatry, occupying roughly the same area, but rarely in syntopy, although sightings of these two species within 50 m of each other have been recorded [50, pers. obs.]. On the other hand, both Podarcis species from Moledo are considered syntopic and may have greater overlap and similarity in their habitat occupancy. Moreover, is very likely they consume the same or very similar prey items [24], and also encounter each other more frequently. All these factors may explain the much more balanced transmission we found between these two species.
The present study contributes to the existing knowledge on the effects of environmental and host factors on the dynamics of the gut microbiome of lizards. Our results also set the stage for future research exploring the influence of other factors on the microbiome, particularly diet, as well as the use of sympatric Podarcis lizards as models to test the effects of behaviour on lizard microbial composition.