Introduction
A myriad of microorganisms can be found living in the gastrointestinal tract of all animals. These microorganisms have a significant impact on host biology and can influence a variety of processes that affect host fitness (Cryan & Dinan, 2012; Thaiss et al. 2016). While certain variations in the composition of gut microbial communities can cause disease (Martin et al. 2014; Boursier et al. 2016), gut microbiome may also increase resistance to pathogens, besides being important for xenobiotics metabolism, nutrient uptake and energy acquisition [e.g. Vavre & Kremer, 2014; Rowland et al. 2018). Moreover, gut microbiota may also contribute towards host adaptation to environment changes by enabling a response to new challenges, such as exploitation of novel food sources (Delsuc et al. 2014; Hammer & Bowers, 2015). Ultimately, gut microbiome can have a major impact on host development, behaviour and fitness, with cascading effects to the dynamics of ecosystems (Thaiss et al. 2016). In turn, it can also be modulated by several host traits, such as host evolutionary history, sex and size, as well as the external environment, such as habitat or prey availability (e.g., Muegge et al. 2011; Xavier et al. 2019). In addition, social interactions between hosts can also influence the gut microbiome in many animal species, although these mechanisms remain less studied (see review by Archie & Tung, 2015).
Gut microbiome dynamics has been studied in many mammals (e.g., Thaiss et al. 2016), birds (e.g., Hird et al. 2015), fishes (e.g., Xavier et al. 2020) and amphibians (e.g., Bletz et al. 2016). Comparatively fewer studies have been performed in reptiles, and only a handful of these addressed lizards. Nevertheless, studies showed that maternal transmission of gut microbiota to offspring can occur in squamate reptiles (Kohl et al. 2017). Additionally, microbiota can be acquired by reptiles through horizontal transmission from the environment or through interaction with other organisms (e.g. predatory encounters, Colston, 2017). Host systematics and ecology were also seen to be important drivers of gut microbiota diversity in reptiles. For example, feeding habits influence the gut microbiota of the Chinese crocodile lizard, Shinisaurus crocodilurus Ahl 1930, with potential effects on host health due to the influence of diet on the abundances of pathogenic or opportunistic gut bacteria (Jiang at al. 2017). Diet and habitat of the Australian water dragon,Intellagama lesueurii (Gray, 1831), also have an effect on its gut microbiome, with lizards living in urban areas presenting higher bacterial diversity than populations living in natural habitats (Littleford-Colquhoun et al. 2019). Moreover, host systematics and habitat also influence the gut microbiota of venomous snakes (Smith et al. 2021).
Here, we analyze and compare the diversity, composition and structure of gut bacterial communities of five related lacertid species captured in Portugal. Individuals of Podarcis bocagei (Lopez-Seoane, 1885) and Podarcis lusitanicus Geniez, Sá-Sousa, Guillaume, Cluchier and Crochet, 2014, were sampled in Moledo (North of Portugal) where they live in syntopy. Invasive Podarcis siculus (Rafinesque-Schmaltz, 1810) and native Podarcis virescens Geniez, Sá-Sousa, Guillaume, Cluchier and Crochet, 2014, were sampled from Parque das Nações (Lisbon) where they live in sympatry. Finally, a population of the invasiveTeira dugesii (Milne-Edwards, 1829) was sampled in the Alcantara Docks in Lisbon. All five species exhibit sexual dimorphism, with males usually being larger than females, and they are mostly insectivorous (Geniez et al. 2014; Carretero et al. 2015), although P. siculus and Teira dugesii may also occasionally consume some fruits or flowers (Mačát et al. 2015).Podarcis species are considered model organisms to study ecotoxicology, immune/histochemical reactions, among other processes [e.g. Bicho et al. 2013; Luís et al. 2019); however, microbiome studies are still largely lacking, with only three studies available to data. Two studies investigated two species endemic to the Balearic Islands (Spain), Podarcis lilfordi (Günther, 1874) andPodarcis pityusensis (Bosca, 1883), with results indicating that islet, time since separation from mainland and seasonality are significant factors contributing to their gut microbiome (Baldo et al. 2018, Alemany et al. 2022). Another recent study compared the gut microbiota of two Italian populations of P. siculus (mainland vs island) demonstrating that there were considerable differences between the two (Buglione et al., 2022).
Our main objective was to determine whether locality, which also corresponded to two different habitats (rural vs urbanized) and host factors such as species, size and sex modulate the gut bacterial diversity of these five lizards. To achieve this, we used cloacal swabs to obtain a proxy for gut bacterial communities which were characterized by sequencing the V4 region of the 16S rRNA gene. Swabs were preferred to fecal samples as these more accurately reflect microbial communities residing in lower gut and cloacal tissues (Bunker et al. 2021).