While fungi present at individual sampling locations varied among years, the effect of sampling year on the overall fungal community was modest and smaller than the effects of space or substrate type. Patterns of fungal specificity among substrates observed in a single year persisted over the duration of the study (Fig. 2), and fungi that were widely distributed in one year tended to also be common in other years (Fig. S2). Year explained less than one percent of community variation (Fig. 1), so while the effect was statistically significant, its biological significance is dubious. These findings suggests that the epiphytic fungal community was largely stable at the broad, site-level scale. The same set of taxa are present from year to year at similar levels of abundance and within the same substrates, though their exact locations within a site may shift. This is consistent with findings in soil systems that inter-annual changes tend to be small compared to intra-annual seasonal ones [26, 31].
In contrast to community composition, alpha diversity did change among years. There was an increase in per sample OTU richness from 2015 to 2016, but it remained stable from 2016 to 2017 (Fig. S3). The reason for this increase is unknown. It could have been related to an environmental change or a change in our sample preservation methods. Longer term monitoring is needed to better understand the dynamics and drivers of fungal biodiversity in the epiphytic environment.
If fungal communities varied randomly from year to year, we would expect temporal dissimilarities to match those seen at larger spatial distances, in which there is no detectable spatial structure and distance decay curves have reached an asymptote (generally > 1 meter in this system, ). Instead, inter-annual dissimilarities matched those seen at much shorter distances, indicating that communities within individual sampling locations were more similar to each other over time than expected due to chance, and that there is continuity of fungi over the course of multiple years. When Jaccard dissimilarities were partitioned into turnover and nestedness components, turnover predominated (Fig. 5). This indicates there was not just a simple gain or loss of OTUs and fungal diversity with time, but that both were happening simultaneously, resulting in turnover of community members.
Inter-annual turnover within sample locations, as measured by Bray-Curtis and Jaccard dissimilarity varied among substrates (Figs. 4, 5). When compared to spatial turnover in the same substrate, however, the spatial distances needed to see the same amount of Bray-Curtis dissimilarity were remarkably similar among substrates, ranging from 5.7 to 8.3 centimeters (Table S1). For example, while live bryophytes had the greatest OTU turnover with time, they also had the highest levels of change across space. Live bryophytes may have the highest inter-annual dissimilarity and OTU turnover because living bryophyte tissues are actively growing, creating new substrate for fungi to colonize, unlike the underlying dead bryophytes or tree bark. The growing bryophytes may also be producing anti-fungal compounds [47, 48] which could limit the growth rate or longevity of fungi. Live bryophytes, when present, were also the top-most substrate layer, leaving them most exposed to incoming fungal spores and disturbance, both of which could increase fungal community turnover. Dead bryophytes and host tree bark are less exposed and, we expect, more physically and chemically stable, allowing for greater fungal community stability.
Individual fungal OTUs were more likely to persist to the next year if they were at high relative abundance in the first year (Fig. 6, Table S2). The least abundant OTUs making up 0.01% or less of a sample seldom persisted to the next year. In contrast, the most abundant OTUs usually did persist. This suggests that fungi able to establish themselves and accumulate biomass can maintain their presence for at least a year. Greater persistence in higher abundance fungi also implies stability in ecological functions. Fungi that are more abundant and have greater biomass presumably have more functional significance than low abundance ones represented by only a few reads and that might only be inactive spores. The larger ecological impacts of these more abundant fungi, possibly including decomposition and nutrient cycling, inhibiting plant growth via disease, or promoting plant growth through mycorrhizal symbiosis, can persist and remain stable over multiple years. This temporal stability of high abundance fungi coupled with the high spatial turnover seen in this system  could create small but persistent patches with greater or lesser suitability for germination and growth of epiphytes, for example.
These dominant fungi likely drive the patterns of temporal stability in this system. The spatial distance needed to see the amounts of Jaccard dissimilarity observed among years was typically much higher than for Bray-Curtis. The Jaccard metric only considers presence-absence and is blind to relative abundance. All fungi, regardless of relative abundance are given the same weight, and the many low abundance, but non-persistent, taxa create high dissimilarity values. With Bray-Curtis, the more abundant taxa, which tend to be more persistent, drive down the dissimilarity values.
The dominance of certain fungi could be due to their early arrival at that location compared to other fungi [49, 50]. Early colonizing microbes, however, do not always persist long term, as has been shown in decaying logs  and biofilms . The host tree branches used in this study were also several years old, as we avoided sampling branch tips or twigs, making it unlikely that we were observing early stages of succession. Wide variation in relative abundances could also be due to environmental factors, with particular fungi being better suited to different environments, as seen with fungi assorting among different substrate types (Fig. 1). High and low relative abundances may also be a function of the traits and life histories of different fungi, with some being small, short-lived ruderal species while others are larger with greater longevity. It is unclear how much of the gain and loss of OTUs is due to life history strategies, particularly short-lived r-strategists which might live less than one year even in favorable circumstances, versus failure of fungi to establish in the first place, regardless of their life history. Sampling at shorter time intervals would allow for greater insights into the dynamics of these diverse and ever-changing communities.
The number of dominant fungi was low, with the overwhelming majority of OTU observations being less than one percent of a sample. Most fungi were also rare, with the majority of OTUs being observed in three or fewer samples over the course of the study. This, combined with high spatial turnover, implies that most fungi in the epiphytic environment are physically small and short lived and likely rely on stochastic spore dispersal to spread within and between branches, rather than mycelial growth. Despite this high temporal and spatial turnover, with many fungi appearing and disappearing erratically, there is an underlying stability, both at the site and sample scale, of common and high abundance fungi that are able to persist and, presumably, have sustained functional impact on their environment.