Microbiota taxonomic composition
There were 2,526 unique ASVs present in the samples. Twenty-four phyla were identified and samples were dominated by Firmicutes (52.6%), Proteobactiera (35.1%), and Actinobacteriota (8.5%; Fig. 2). All other phyla had mean relative abundances of <1%. Across the 58 samples, there were 551 genera identified and twelve genera had relative abundances >1%. The most commonly observed genera wereCandidatus Arthromitus (22.3%), Escherichia-Shigella(13.8%), Enterococcus (13.5%), Ligilactobacillus(9.1%), Klebsiella (7.8%), Cronobacter (4.8%),Erysipelatoclostridium (2.3%), Rothia (2.2%),Clostridium sensu stricto 1 (1.9%), Campylobacter(1.6%), Corynebacterium (1.4%), and Kocuria (1.1%).
At the phylum level, location significantly affected the relative abundance of Firmicutes and Proteobacteria (Table 1). Specifically, nestlings from urban sites had higher relative abundances of Firmicutes (χ2 = 13.74, Padj = 0.008) and lower abundances of Proteobacteria (χ2 = 8.35,Padj = 0.01) compared to nestlings from non-urban field sites (Figure 2). At the genus level, location significantly affected the abundance of Candidatus Arthromitus,Klebsiella , Erysipelatoclostridium , and Rothia(Table 1, Figure S3). Candidatus Arthromitus was highly abundant in urban nestlings, but rarely observed in fecal samples collected from non-urban nestlings (Figure 3, χ2 = 10.94,Padj = 0.01). Conversely, Klebsiella ( χ2=7.13, Padj = 0.03) andErysipelatoclostridium2 = 6.62,Pad j = 0.03) were observed at higher abundances in fecal samples from non-urban nestlings (Figure 3). The genusRothia was more common in nestlings from urban nests and was rarely observed in non-urban nestlings (χ2 = 7.29,Pad j = 0.03). This trend was difficult to visualize due to two individuals that had very high relative abundance values (Figure S4: one non-urban nestling: 41.89% Rothia , and one urban nestling: 63.43% Rothia ). Additional outliers were detected and removed using the boxplot.stats function in R (non-urban: 1.25%, urban nestlings: 8.36%, 2.77%, 2.15%, 0.73%). Location (urban, non-urban) still significantly affected the relative abundance of Rothia with the two greatest outliers removed (Figure S4: χ2 = 10.11, Pad j = 0.01) and with the five additional outliers identified by the boxplot.stats function removed (shown in Fig 3, χ2 = 12.57,Pad j = 0.005).
Parasite treatment did not significantly influence the relative abundance of the three most abundant phyla or the abundance of any of the twelve most abundant genera (Table 1). When analyzing only nestling samples from sham-fumigated nests, there was still a significant effect of location on the abundance of Firmicutes and Proteobacteria (Figure S5). However, there was no effect of urbanization on the abundance of the top twelve genera within the sham-fumigated nests (Table 1).