Results
After filtering, the final ASV table encompassed 3923 unique ASVs, included in a total of 39 bacteria phyla. The most abundant phyla were Firmicutes, Bacteroidota, Actinobacteroidota, Proteobacteroidota and Campylobacterota (Fig. 2)
Gut bacterial alpha-diversity was significantly different between Moledo populations (P. bocagei and P. lusitanicus ) and Lisbon populations (P. siculus , P. virescens and T. dugesii ) (Table 1). Lisbon populations, those from an urbanized habitat, showed consistently higher alpha-diversity indices, withP. siculus having higher diversity than the native P. virescens. (Fig. 3). Microbial beta-diversity was also significantly different between localities when considering the Bray-Curtis (R2 = 0.186, p < 0.001) and the Unifrac unweighted (R2 = 0.180, p < 0.001) indices (Fig. 4 and Table 1).
Although no differences were found in the proportion of the most abundant phyla between P. bocagei and P. lusitanicus , significant differences were found in the proportion of the genusCorynebacterium (F-statistics = 6.823, p = 0.013) (Fig. 5). Differences in the proportion of the most abundant taxa between P. siculus and P. virescens were found at genus levels for an unidentified genus belonging to the order Gastranaerophilales (F-statistics = 6.324, p = 0.003), Corynebacterium (F-statistics = 6.887, p = 0.002), Kocuria (F-statistics = 4.639, p = 0.0138),Staphylococcus (F-statistics = 6.767, p = 0.002), andOdoribacter  (F-statistics = 11.609, p = 6.398e-05) (Fig. 6). Additionally, for P. siculus and P. virescens, both species and sex significantly affected the abundance ofAkkermansia (sex: F-statistics = 5.191, p = 0.026; species: F-statistics = 3.467, p = 0.038) (Fig 6 and 7) and the interaction between species and sex (species*sex) significantly affected the proportion of Romboutsia (F-statistics = 3.475, p = 0.038) andPseudomonas (F-statistics = 3.412, p = 0.040) (Fig. 7).
Pearson correlation test only showed significantly positive correlations between SVL and bacterial alpha-diversity (for Shannon indice) for males of the in the invasive species P. siculus (Fig. 8).
Results from FEAST software indicate that the level of bacterial transmission between sympatric species in both populations (Parque das Nações and Moledo) was high. Nevertheless, while between the syntopicP. lusitanicus and P. bocagei bacterial transmission was balanced in both directions (P. bocagei P. lusitanicus~ 71% on average, and P. lusitanicus P. bocagei ~ 69% on average), the other two sympatric species showed a more biased transmission, with P. virescenshaving a higher contribution towards P. siculus (P. virescens P. siculus of 72% on average, and P. siculusP. virescens of about 55% on average).