Reviewer #3 (Public Review):

This work seeks to identify a common factor governing priority effects, including mechanism, condition, evolution, and functional consequences. It is suggested that environmental pH is the main factor that explains various aspects of priority effects across levels of biological organization. Building upon this well-studied nectar microbiome system, it is suggested that pH-mediated priority effects give rise to bacterial and yeast dominance as alternative community states. Furthermore, pH determines both the strengths and limits of priority effects through rapid evolution, with functional consequences for the host plant's reproduction. These data contribute to ongoing discussions of deterministic and stochastic drivers of community assembly processes.

Strengths:

Provides multiple lines of field and laboratory evidence to show that pH is the main factor shaping priority effects in the nectar microbiome. Field surveys characterize the distribution of microbial communities with flowers frequently dominated by either bacteria or yeast, suggesting that inhibitory priority effects explain these patterns. Microcosm experiments showed that A. nectaris (bacteria) showed negative inhibitory priority effects against M. reukaffi (yeast). Furthermore, high densities of bacteria were correlated with lower pH potentially due to bacteria-induced reduction in nectar pH. Experimental evolution showed that yeast evolved in low-pH and bacteria-conditioned treatments were less affected by priority effects as compared to ancestral yeast populations. This potentially explains the variation of bacteria-dominated flowers observed in the field, as yeast rapidly evolves resistance to bacterial priority effects. Genome sequencing further reveals that phenotypic changes in low-pH and bacteria-conditioned nectar treatments corresponded to genomic variation. Lastly, a field experiment showed that low nectar pH reduced flower visitation by hummingbirds. pH not only affected microbial priority effects but also has functional consequences for host plants.

Weaknesses:

The conclusions of this paper are generally well-supported by the data, but some aspects of the experiments and analysis need to be clarified and expanded.

The authors imply that in their field surveys flowers were frequently dominated by bacteria or yeast, but rarely together. The authors argue that the distributional patterns of bacteria and yeast are therefore indicative of alternative states. In each of the 12 sites, 96 flowers were sampled for nectar microbes. However, it's unclear to what degree the spatial proximity of flowers within each of the sampled sites biased the observed distribution patterns. Furthermore, seasonal patterns may also influence microbial distribution patterns, especially in the case of co-dominated flowers. Temperature and moisture might influence the dominance patterns of bacteria and yeast.

The authors exposed yeast to nectar treatments varying in pH levels. Using experimental evolution approaches, the authors determined that yeast grown in low pH nectar treatments were more resistant to priority effects by bacteria. The metric used to determine the bacteria's priority effect strength on yeast does not seem to take into account factors that limit growth, such as the environmental carrying capacity. In addition, yeast evolves in normal (pH =6) and low pH (3) nectar treatments, but it's unclear how resistance differs across a range of pH levels (ranging from low to high pH) and affects the cost of yeast resistance to bacteria priority effects. The cost of resistance may influence yeast life-history traits.