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 a major predictor of microbial diversity and structure. Plausibly, differences in habitat may lead to differences in the composition and diversity of the gut microbiome of animals (e.g. Amato et al. 2013; Xavier et al. 2021). The two habitats in which the lizards from this study were captured are very different, with lizards from Lisbon living in a highly urbanized and artificial habitat, with greater environmental disturbance, compared to lizards from Moledo, which live in a semi-natural habitat. Specifically, we detected a consistently higher microbial diversity in the species from Lisbon which could be possibly explained by the higher variety of diet items consumed. Podarcis siculus diet is viewed as extremely opportunistic, and can include human food waste (e.g. cheese and pasta), fruits, other lizards and small carrion (Mo & Mo, 2021; Mačát et al. 2015; Capula & Aloise, 2011; personal observations).
Although the species we sampled in urban areas can also reside in rural habitats, and vice-versa, our sampling design did not allow comparisons of gut bacterial communities between conspecifics residing in these two habitats. Nevertheless, urbanization is frequently seen to restructure the gut microbiome of animals (e.g. Stothart et al. 2019), with increases in microbiome alpha diversity reported for some reptiles (avian and non-avian) and mammals (Dillard et al. 2022). For example, higher gut bacterial diversities were previously reported in a study from populations of the Australian water dragon residing urban environments when compared to those inhabiting natural environments, presumably driven by differences in the diet (Littleford-Colquhoun et al. 2019). Additionally, authors hypothesized that environmental microbiota, which may be horizontally transferred to lizards, could also be more diverse in urban habitat than in semi-natural ones (Littleford-Colquhoun et al. 2019). A similar pattern was observed in urban crested anole lizards, white-crowned sparrows as well as coyotes (Dillard et al., 2022; Phillips et al. 2018; Sugden et al. 2020). Interestingly, Dillard et al. (2022) found increased similarities between the gut microbiota of these three animals and human populations in urbanized habitats. Different hypothesis have been put forward to explain this trend, including that it could be caused by increased heterogeneity of urban land cover (Phillips et al. 2018), higher consumption of human food waste (Sugden et al. 2020) and acquisition of human microbiota in urban habitats (Dillard et al. 2022). We hypothesize that the higher microbiome diversity in lizards from the urban environment could also be related with the aforementioned factors, but further studies including conspecific lizards from urban and natural habitats are needed to determine the generality of this pattern.
Gut microbial diversity (alpha-diversity) did not significantly differ between lizard species and there were no differences in bacterial community structure (beta-diversity) between the two syntopic species,P. bocagei and P. lusitanicus , sampled at Moledo. Additionally, our analysis of potential bacterial transmission between these two syntopic lizards indicates a high and balanced bi-directional transmission of bacteria between the two species (ca. 70%), indicating a high similarity between their gut microbiota (Shenhav et al. 2019). This is not surprising as the two species have high dietary overlap and similarity in their habitat occupancy. Moreover, it is likely they consume the same or very similar prey items (Kaliontzopoulou et al. 2011), and also encounter each other frequently. On the contrary, there were significant differences between the structure of gut bacterial communities (beta-diversity) of the different species of lizards sampled in Lisbon, with lower and unbalanced estimates for bacterial transmission between the two sympatric species, P. siculus andP. virescens . The invasive P. siculus was estimated to receive a higher proportion of bacteria from the native P. virescens than vice versa (ca. 72% vs 55% on average). These differences 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. Although the populations of P. siculus andP. virescens are found living in sympatry, occupying roughly the same area, they are rarely in syntopy, although sightings of these two species within 50 m of each other have been recorded (Ribeiro & Sá-Sousa, 2018; personal observations).
The proportion of some of the most abundant bacterial genera found in our study also differed between P. virescens and P. siculus, but not between P. lusitanicus and P. bocagei . 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 (Moeller et al. 2013; Moeller et al. 2014), including reptiles (Smith et al. 2021). It is also interesting to note that the gut microbiota of individuals of P. siculus sequenced herein and those from Italy by Buglione et al. (2022) share the most abundant bacterial phyla but differ at the level of the most represented bacterial genera.
Comparatively, host sex had a negligible effect on gut microbiota of the studied lizards, albeit it significantly affected the abundance of Corynebacterium in lizards from Moledo. These results agree with those from a previous study on two Podarcis species from the Balearic islands, in which sex had no influence on the gut microbial structure (Alemany et al. 2022). However, an interesting result was that size of males of P. siculus was positively related with bacterial diversity. This lizard can reach larger sizes than the other studied species (Carretero & Silva-Rocha, 2015, Damas-Moreira et al. 2019). Furthermore,Podarcis siculus can be bolder and more aggressive than nativePodarcis species (Downes & Bauwens, 2002), and also more exploratory and better at exploiting food resources when compared to the native P. virescens at our study location (Damas-Moreira et al. 2019, 2020). These behaviors can be associated with the displacement ofP. virescens from gardens now inhabited by P. siculus(Ribeiro & Sá-Sousa, 2018) and can also be leading to a wider ecological and trophic niche, and consequently to the correlation found as well as to the slightly higher average microbiome diversity observed for P. siculus .
The present study contributes to the existing knowledge on the effects of the environment 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 diet and urbanization on the microbiome of Podarcis and the use of sympatric lizards as models to test the effects of behavior on lizard microbial composition.