Supplemental file summarizing the occurrence of bacterial ASVs in controls and crane fecal samples from A bird's-eye view of phylosymbiosis: weak signatures of phylosymbiosis among all 15 species of cranes

In numerous animal clades, the evolutionary history of host species drives patterns of gut microbial community structure, resulting in more divergent microbiota with increasing phylogenetic distance between hosts. This phenomenon, termed phylosymbiosis, has been observed in diverse evolutionary lineages, but has been difficult to detect in birds. Previous tests of phylosymbiosis among birds have been conducted using wild individuals, and thus interspecific differences in diet and environment may have masked a phylogenetic signal. Therefore, we tested for phylosymbiosis among all 15 species of cranes (family Gruidae) housed in the same captive environment and maintained on identical diets. 16S rRNA sequencing revealed that crane species harbour distinct gut microbiota. Overall, we detected marginally significant patterns of phylosymbiosis, the strength of which was increased when including the estimates of absolute microbial abundance (rather than relative abundance) derived from microbial densities determined by flow cytometry. Using this approach, we detected the statistically significant signatures of phylosymbiosis only after removing male cranes from our analysis, suggesting that using mixed-sex animal cohorts may prevent the detection of phylosymbiosis. Though weak compared with mammals (and especially insects), these results provide evidence of phylosymbiosis in birds. We discuss the potential differences between birds and mammals, such as transmission routes and host filtering, that may underlie the differences in the strength of phylosymbiosis.

在众多动物支系（clades）中，宿主物种的演化历史塑造了肠道微生物群落结构的模式：宿主间系统发育距离越远，其肠道微生物组（microbiota）的差异程度越高。这一被称为系统发育共生（phylosymbiosis）的现象已在多种演化支系中被观测到，但在鸟类中却难以被检测到。此前针对鸟类系统发育共生的检测均基于野生个体开展，因此物种间的饮食与环境差异可能掩盖了系统发育信号（phylogenetic signal）。为此，我们针对同处人工饲养环境、饲喂相同日粮的15种鹤科（Gruidae）鸟类开展了系统发育共生检测。16S rRNA测序（16S rRNA sequencing）结果显示，不同鹤类物种拥有独特的肠道微生物组。总体而言，我们检测到了边缘显著的系统发育共生模式；当纳入通过流式细胞术（flow cytometry）测定的微生物密度所推导的微生物绝对丰度（而非相对丰度）指标时，该模式的显著性有所提升。采用该分析方法后，我们仅在剔除雄性鹤类样本后才检测到了具有统计学显著性的系统发育共生特征，这表明使用混合性别动物队列（cohort）可能会阻碍系统发育共生的检测。尽管与哺乳动物（尤其是昆虫）相比，该信号强度较弱，但本研究结果为鸟类存在系统发育共生现象提供了实证支持。我们还讨论了鸟类与哺乳动物之间可能存在的潜在差异（如微生物传播途径与宿主过滤机制），这些差异或为两者系统发育共生强度差异的核心成因。