INTRODUCTION
A healthy gut microbiome is thought to be both diverse and relatively stable (García-García et al. 2019; Rinninella et al. 2019) but may be heavily affected by a variety of extrinsic and intrinsic factors, including host genetics, habitat, and diet (Hird et al.2015; Rothschild et al. 2018). The composition and stability of a healthy microbiome may change as animals undergo recurrent physiological stressors, such as migration or changing climates across seasons (Sommeret al. 2016; Carey and Assadi-Porter 2017). Increased understanding of both variation and stability of gut microbial ecologies related to recurrent physiological stressors can further elucidate host adaptation to repetitive stress. Here, we ask what changes and what remains consistent within the gut microbiome of a migratory bird species across multiple time points and locations within the annual cycle.
Species experiencing seasonal variation in habitat, diet, or physiological stressors often exhibit correlated changes in their microbiome (Maurice et al. 2015; Sommer et al. 2016; Renet al. 2017; Smits et al. 2017; Drovetski et al.2019). Migratory animals may undergo seasonal fluctuations in metabolic needs that, in combination with changing habitats and diets, result in variable microbiota composition across their annual cycles, but the extent to which this occurs remains unclear (Jenni and Jenni-Eiermann 1998; Grond et al. 2018).
Gut microbiota of some migratory bird species have been characterized at discrete portions of the annual cycle, revealing substantial bacterial diversity (Lewis et al. 2016; Risely et al. 2017; Wuet al. 2018; Cao et al. 2020). Different environments, such as breeding grounds, wintering grounds, and stopover sites during migration, have been shown to impact the overall composition of gut microbiota, likely through exposure from local microbial communities or food sources (Lewis et al. 2017; Wu et al. 2018). Additionally, physiological adaptations of migratory birds, such as intestinal atrophication during active migration, may further affect gut microbiota (Grond et al. 2018). Given the variability of gut microbiota and strong environmental effect, it may be difficult to directly correlate variation in gut microbiota to ongoing biological processes, specific host traits, or environmental factors without temporal sampling across different time points of the annual cycle. (Hird et al. 2014; Capunitan et al. 2020; Song et al. 2020). Here we recaptured individuals multiple times on their tropical wintering and temperate breeding grounds to better understand local and temporal variability in gut microbiota thus reducing sources of variability known to be associated with sampling different individuals and different populations (Flores et al. 2014; Hirdet al. 2014; Baxter et al. 2015).
Until now, no migratory songbird has been sampled at multiple time points and locations across their annual cycles. Migratory birds have complicated annual cycles that involve twice-annual movements often spanning thousands of kilometers between stationary breeding and wintering periods. Once captured, researchers typically have no way to relocate or recapture the same individuals outside of the original capture site, especially for species with expansive wintering and breeding ranges and with populations that may number in the millions. This inhibits sampling from the same population, let alone the same individual, at multiple points in the annual cycle. As a result, one must attempt to measure and control for confounding factors, such as between population differences, and account for high inter-individual variability (Flores et al. 2014; Hird et al. 2014; Baxteret al. 2015). Thus, our inability to study the same individuals across the annual cycle has impeded identification and understanding of variation within birds associated with seasonal movement.
The Kirtland’s Warbler (Setophaga kirtlandii) provides an unusual opportunity for studying changes across the annual cycle in a migratory species. Their small population size as well as restricted breeding and wintering ranges (Cooper et al. 2019) make it feasible to relocate individuals across seasons (Cooper et al. 2018; Cooper and Marra 2020). Following substantial population declines, only 167 singing males were recorded in 1974 and again in 1987, based on breeding surveys (Kepler et al. 1996). Through extensive conservation management efforts, the population has increased to approximately 2,300 singing males of which 97% breed across a relatively small area in Michigan’s Lower Peninsula. This species winters primarily in the scrub forests of The Bahamas (Cooper et al. 2019), more than 2,000 km from the breeding grounds. For this study, we radio-tagged individuals on the wintering grounds and then relocated and recaptured the same birds on the breeding grounds in Michigan through the use of automated telemetry towers. We used 16S rRNA next generation sequencing technologies to catalogue the bacterial communities of individuals. Our goals were to: (1) characterize the bacterial diversity of Kirtland’s Warblers at three unique periods of the annual cycle at the population and individual level; (2) evaluate host sex, age, period of annual cycle, and location effect on abundance and diversity of gut microbiota; and (3) determine if a core bacterial profile for Kirtland’s Warblers exists and if so, establish a species-specific pattern.