Results
After filtering, the final ASV table encompassed 3923 unique ASVs, included in a total of 39 bacteria phyla. The most abundant phyla among the species studied were Firmicutes, Bacteroidota, Actinobacteroidota, Proteobacteroidota and Campylobacterota.
Gut bacterial diversity, measured through alpha-diversity indices, was significantly different between localities considering the number of Observed ASVs and PD indices (F-statistics > 39.74; p < 0.02), with species from Lisbon showing consistently higher alpha-diversity indices than the ones from Moledo, (Fig. 2). No differences between localities were found with the Shannon index (F-statistics = 8.33; p = 0.07). Moreover, neither species or sex had a significant effect on microbial alpha-diversity (F < 1.95; p > 0.11), but P. siculus had higher diversity than the native P. virescens . Microbial structure, measured through beta-diversity indices, was significantly different between localities (R2 > 0.03; p < 0.04) and species (R2 > 0.08; p < 0.03) (Fig. 3). In general, pairwise differences in beta-diversity between species were found in species collected from Lisbon (Table 1), while no differences were found between sexes. In samples collected in Moledo, no differences were found in beta-diversity between species or sexes.
Although no differences were found in the proportion of the most abundant phyla between species or sexes, among the most abundant genera some differences were observed (Fig. 4). In the case of species in Moledo, sex influenced the proportion of the genusCorynebacterium (F-statistics = 4.46, p = 0.02) (Table 2). Differences in the proportion of the most abundant taxa between P. siculus and P. virescens were found for an unidentified genus belonging to the orderCorynebacterium(F-statistics = 6.66, p = 0.003) and forOdoribacter  (F-statistics = 10.10, p = 0.0002) (Table 2).
Pearson correlation test only showed significantly positive correlations between SVL and bacterial alpha-diversity (for Shannon index) for males of the invasive species P. siculus (Fig. 5).
Results from FEAST software indicated that the level of bacterial transmission between sympatric species in both populations (Parque das Nações and Moledo) was high. Nevertheless, while bacterial transmission was balanced in both directions between the syntopic P. lusitanicus and P. bocagei (estimated transmission from P. bocagei towards P. lusitanicus was ~ 71% on average, and from P. lusitanicus towards P. bocagei was ~ 69% on average), between the two sympatric species in Lisbon there was a more biased transmission, with P. virescensseemingly having a higher contribution towards P. siculus gut microbiota (transmission estimates from P. virescens towardsP. siculus was ~ 72% on average, and fromP. siculus towards P. virescens it was about 55% on average).