We explored the appropriateness of using cloacal swabs and museum alcohol preserved specimens to investigate the gut microbial communities of wild birds. We first demonstrated that digestive tract regions of wild P. major differ in gut microbial communities, but that these differences are not as prominent as previous studies have documented in other bird species [2, 5, 30, 31]. While compositions differed between cloacal swabs and different gut regions, swabs qualitatively portrayed bacterial community compositions in the entire digestive tract, aligning with a previous study of wild birds [32], as well as reflected gut community compositions from a previous study of wild P. major fecal samples [16]. Microbial communities of alcohol-preserved gut regions did not differ significantly from fresh samples, supporting that museum collections represent a promising resource for analyses of gut microbiomes [37]. Notably, the relative low number of differentially abundant genera in freshly dissected and alcohol preserved specimens only marginally impacted the overall microbial community composition across digestive tract regions.
The possibility of determining the qualitative composition of bird gut microbial communities through cloacal swab sampling support that utilization of this non-invasive method to characterize bird microbiomes is appropriate, despite the quantitative limitations of this approach [32, 33]. Cloacal swabs did not capture a few rare bacterial taxa. This is evident from a significant difference only in OTU richness (Figs. 1a and 1b) but not in diversity indexes that account for both abundance and evenness (Figs. 1c and 1d). The ability of cloacal swabs to capture the majority of common bacterial taxa (Fig. 2) implies that the method would allow for the vast majority of gut symbionts to be identified. Previous work has suggested that fecal sampling better represents gut community compositions than cloacal swabs [2]. Although we did not compare fecal samples with cloacal swabs, the bacterial taxa we acquired from cloacal swabs were similar to taxa characterized in fecal microbiomes of wild P. major in a previous study [16], suggesting that microbiome discrepancies between these sampling methods are minor. Furthermore, the cloacal swab approach also reduces the risk of contamination and removes the problems associated with the typically lower DNA yield from extractions of bird feces [42, 43].
The observed similarity of gut microbial communities in fresh and alcohol preserved specimens opens of for the possibility of utilizing the vast number of museum bird alcohol specimens [37]. Although we did observe high individual variation in microbial communities, which is common in bird gut microbiomes [13, 16, 37, 44], the most common bacterial genera did not differ significantly between preservation methods (Fig. 3). The slight reduction in the relative abundance of members of the phyla Bacteroidetes and Actinobacteria in alcohol preserved specimens could be due to degradation of bacterial DNA. In addition, it may be that ethanol adversely affects DNA extraction, but a previous study suggests that this has a minimal impact on microbial community characterizations [4]. The genera that were differentially abundant between alcohol preserved and fresh specimens were inconsistent across different gut regions, suggesting that they are unlikely to be caused by the alcohol preservation, but rather are due to the naturally high variation between individuals in passerine bird gut microbiomes [16, 37]. Overall, alcohol preserved specimens thus provide a reliable estimate of gut microbial communities, even after two months of storage.
The utilization of museum specimens to investigate wild bird gut microbiomes would allow substantial insights into bird-microbial symbiosis in multiple ways. First, museum collections provide an opportunity to investigate gut microbiomes of a vast number of extant and extinct wild bird species [38–41] to improve our understanding of the ecology and evolution of birds and their gut microbial symbionts. Secondly, museum collections usually also include individuals from multiple geographic regions, allowing insights into host-symbiont biogeography. Finally, collections typically include a long temporal span, enabling investigations into how bird-gut microbial associations change over time. This would allow us to establish natural temporal changes in associations, but also to test for the effects of climate change and habitat alterations caused by anthropogenic effects. Notably, most museum alcohol specimens are older than two months and longer-term effects of preservations on gut microbiome will thus also be needed. New specimen collections should ideally also focus on preserving the entire animal in alcohol (instead of focusing solely on skins and skeletons) and in storage conditions optimal for the possibility of using these collections for microbiome studies [38].