Introduction
As high-throughput sequencing becomes more accessible, microbiome studies are expanding beyond humans and mammalian model systems to non-model organisms. It has become clear that the community of microbes within the gut of all animals plays an important role not only in host physiology but also their ecology and evolution (Archie & Theis, 2011; McFall-Ngai et al., 2013; Moran et al., 2019; Reese & Dunn, 2018). To better understand the wide-ranging implications of host-microbe co-evolution and the variety of functions the gut microbiome can provide for the host, it is critical to study a broad diversity of host species with a broad diversity of environmental pressures and adaptations. Conducting such research on wild populations is particularly important because captivity has been shown to change the composition of gut microbiomes (Hird, 2017; Keenan et al., 2013; Kohl et al., 2017).
The function and the composition of the gut microbiome can be highly specialized along different regions of the gastrointestinal (GI) tract, which vary in acidity, specific macromolecules secreted by gut epithelia, oxygen content, and other conditions that affect which microbes can thrive (Beasley et al., 2015; Reese & Dunn, 2018). Regional localization of the gut microbiome is well established in both model and non-model systems (Beasley et al., 2015; Colston et al., 2015; Kohl et al., 2017; Shterzer et al., 2020; Videvall et al., 2018; Yasuda et al., 2015). Among oviparous vertebrates, the terminus of the GI tract is shared with the reproductive tract, as the cloaca is the site of defecation, copulatory intromission, and oviposition. Thus, the cloacal microbiome of birds and reptiles likely experiences unique selective pressures relative to the rectal microbiome of mammals. For instance, the cloacal microbiome may be affected by breeding season, sex, degree of promiscuity, and other aspects of host reproduction and behavior (Escallón et al., 2019; Lee, 2015; White et al., 2011).
In the striped plateau lizard, Sceloporus virgatus , free-ranging females lay their eggs in soil burrows at the onset of the summer monsoon rains and then leave, providing no further parental care (Rose, 1981). Timing oviposition during the monsoon season appears to be unique to S. virgatus among temperate lizard species, and has been hypothesized to have a large selective impact on the species (Vinegar, 1975). We propose a strong selective impact on the species’ microbiome as well, resulting in a cloacal microbiome that protects host’s eggs from fungal pathogens that thrive during the wet monsoon period (Bunker et al., in review). Indeed, microbes are transferred from the cloacae of S. virgatus females to eggshells during egg laying, reducing fungal attachment to eggs and improving hatch success (Bunker et al., in review). Given this specialized ecological function, we further hypothesized that the gut microbiome of S. virgatus females will express a high degree of localization along the GI and reproductive tracts. While such localization has been found in several other reptile species (Colston et al., 2015; Costello et al., 2010; Keenan et al., 2013; Kohl et al., 2017), we predicted a pattern in contrast to that found in these other reptiles; specifically, we predicted that the cloaca will show reduced microbial diversity and a unique community structure relative to upper regions. To test this, we compared the microbiome of the cloaca, lower and upper intestinal regions, and the oviduct. Because previous work on the S. virgatus microbiome has been based on cloacal swab samples (Bunker et al., in review; Martin et al., 2010), we additionally assessed whether the cloacal microbiome determined from cloacal swabs was representative of cloacal tissue or other regions of the GI and reproductive tracts. We predicted that the less invasive swab samples would serve as a good proxy for the more invasive tissue sampling of the cloaca, but would be less suitable as a proxy of upper GI and reproductive tract tissues.
It is common among studies of reptiles and birds for cloacal swabs and/or fecal samples to be used as a proxy for the gut microbiome as a whole (Colston et al., 2015; Escallón et al., 2019; Hong et al., 2011; Jiang et al., 2017; Kohl et al., 2017; Kreisinger et al., 2015). These methods are attractive as they are non-destructive, minimally invasive, and allow for repeat sampling of the same individuals and communities over time (Berlow et al., 2020; Videvall et al., 2018). However, a microbial community recovered from swabs or feces may represent only a portion of the gut due to the regional localization. While only a few studies have directly compared the two non-invasive sampling approaches, unique community structures have been recovered by cloacal swab and fecal sampling in zebra finches and ostriches (Berlow et al., 2020; Videvall et al., 2018). Here, we add to this important work by also directly comparing microbial communities collected from cloacal swab and fecal samples of S. virgatus individuals. If the communities are similar, feces may inoculate the cloaca with microbes as they pass through it. However, we predicted that the communities will be distinct in S. virgatus , indicating selection for the particular cohort of microbes that occupy the cloaca, due in part to the natural history ofS. virgatus (as detailed above). If indeed the cloacal and fecal microbial communities are distinct, it is possible that cloacal swab samples can be contaminated by small particles of fecal material that remain in the cloaca following defecation and then attach to the swab. Unintended fecal contamination of swab samples may or may not be visible on the swab but could potentially help explain the high variation recovered from such samples in other studies (Videvall et al., 2018; Williams & Athrey, 2020; Bunker and Weiss, unpublished data). To test these ideas, we compared microbial communities detected from cloacal swabs collected before and after defecation. If contamination was occurring, we predicted that communities recovered from cloacal swabs immediately after defecation would have higher diversity and a different community structure than swabs taken prior to defecation.