How the microbiome interacts with hosts across evolutionary time is poorly understood. To address this question, datasets comprised of many host species are required to conduct comparative analyses. Here, we have analyzed 142 intestinal microbiome samples from 92 birds belonging to 74 species from Equatorial Guinea, using the 16S rRNA gene. Using four definitions for microbial taxonomic units (97%OTU, 99%OTU, 99%OTU with singletons removed, ASV), we conducted alpha and beta diversity analyses and used phylogenetic comparative methods to assess the evolution of the microbiome as a trait of bird species. We find that raw abundances and diversity varied between the datasets but relative patterns were largely consistent across datasets. Host taxonomy, diet and locality were significantly associated with microbiomes, at generally similar levels using three distance metrics. Phylogenetic comparative methods were used to assess the relationship between the microbiome as a trait of a host species and the underlying bird phylogeny. We find that a neutral Brownian motion model does not explain variation in microbiomes. Instead, a White Noise model that indicates the trait contains little to no phylogenetic signal, is most likely across many definitions of "microbiome trait". While there was some support for the Ornstein-Uhlenbeck model (that invokes selection), the level of support was similar to our White Noise simulation, further supporting the White Noise model as the best explanation for the evolution of the microbiome as a trait of avian hosts. Our study highlights the qualitatively minor impact that different analytical choices can have on results and that biological interpretations can be robust to method choice.

Table 1. Sample information for the 92 individuals in this study. Fig.2 refers to the samples placement on Fig.2 in the manuscript. SampleID is the unique name given the sample, which is a combination of the first three letters of the genus, the first three letters of the species and the last three digits of the LSUMNS Bnumber given upon deposition to the museum (the full Bnumber for each specimen is 90XXX). The number of sample replicates (Sample Reps) and PCR replicates (PCR Reps) indicates which individuals had more than one sample taken. Other data are bird order, family, genus, species, sampling locality (see Fig. 1), sampling province, diet and weight (g).