Seasonal patterns of infection in ephemeral and permanent ponds
We found that in early spring, just after amphibians in northwest Pennsylvania emerge from hibernation and begin migrating to breeding ponds, Bd prevalence was lower in ephemeral ponds than in permanent ponds. This difference, however, was gone by mid-spring when Bd prevalence peaked at ~ 70% then declined in both pond types (Fig. 2A). Bd load also peaked in spring and was lower in summer, though it rose again in fall in both pond types (Fig. 2B). These seasonal patterns, which are similar to those seen in other sub-tropical and temperate zone amphibian communities (Brannelly et al. 2018; Sonn et al. 2019; Wilber et al. 2022) are perhaps easiest to understand in terms of relationships between temperature and Bd prevalence and load, which we found strong support for (Fig. 2C, D).
Temperature was a significant predictor of Bd infection in the amphibians inhabiting both the ephemeral and permanent ponds in our study area, which is similar to findings of previous studies (Phillott et al. 2013, Sonn et al. 2019). In both pond types we found that Bd load decreased with amphibian body temperature (Fig. 2D). Bd prevalence, on the other hand, was lowest at the extremes of amphibian body temperature and reached a peak in intermediate temperature ranges (Fig. 2C), mirroring the thermal performance limits for Bd growth in culture (Voyles et al. 2017). In the permanent ponds we sampled, Bd infections reached a peak at a lower temperature (~ 11 ºC) than in the ephemeral ponds (~ 15 ºC). Possible explanations for this difference between pond types might include differences in the thermal physiology of distinct Bd lineages (as seen in Voyles et al. 2017; Sheets et al. 2021) or differences in the thermal sensitivity of the defenses of hosts (as seen in Cohen et al. 2017; Moretti et al. 2019) that inhabit these two pond types. Previous studies have attributed higher Bd prevalence in the early spring to the fact that Bd survives well at cool temperatures (Voyles et al. 2012) whereas the immune defenses of amphibians tend to be reduced at cool temperatures (Robak et al. 2019; Rollins-Smith 2020).
The role of amphibian hosts and life stages in pathogen maintenance
Amphibians exhibit a diverse range of breeding (Duellman and Trueb 1986) and overwintering (Neill 1948) strategies that can influence their susceptibility to pathogens as well as their potential to transport pathogens to and between aquatic habitats. The presence of certain species of amphibian hosts in permanent ponds year-round may also contribute to the maintenance of pathogens across the landscape if, for example, tadpoles and/or post-metamorphic amphibians that overwinter in these ponds act as pathogen reservoirs (Wilber et al. 2020). In contrast, because aquatic pathogens, like Bd, are often intolerant of desiccation, their presence in ephemeral ponds may depend upon infected hosts bringing them anew each year. For Bd, we predicted that the most likely source for introduction to newly refilled ephemeral ponds is the amphibians that make use of those ponds for breeding and larval development.
We hypothesized that the higher prevalence of Bd in animals sampled from permanent ponds in early spring, when amphibians first become active, was due to infected overwintering animals shedding zoospores and transmitting the pathogen throughout the winter in permanent ponds (amphibians do not overwinter in ephemeral ponds in our study area). We did not sample overwintering amphibians themselves, so we could not test this directly. However, we did not detect Bd via eDNA sampling in the water during the winter in either pond type (though our sample sizes for this period were very small). eDNA sampling for Bd has a much higher detection threshold than amphibian swab samples (Brannelly et al. 2020), so we cannot rule out the possibility that Bd was in the pond water in winter and we did not detect it. We did, however, detect Bd eDNA during each of the other seasons we sampled. This could be evidence for an infectious zoospore pool being present in ponds during some times of the year, though we cannot rule out the possibility of other sources (e.g., dead cells) for the Bd DNA detected in our water samples.
Some of the frogs in our study area overwinter as larvae in permanent ponds (e.g., American bullfrogs, green frogs) and we hypothesized that infected overwintering larvae could be important contributors to the early spring peak in Bd prevalence and load. However, our findings suggest that this is not the case. We found larvae to have similar Bd loads but lower Bd prevalence than post-metamorphic life stages in both ephemeral and permanent ponds. In permanent ponds the difference in prevalence between life stages was dramatic; prevalence in the post metamorphic animals was close to 40% but only 10% of the larvae we sampled were found to be infected with Bd, suggesting that overwintering larvae are not likely a major source of infectious zoospores in the permanent ponds we sampled. Other studies in the Northeastern United States have shown a wide range of Bd prevalences in tadpoles that overwinter aquatically. For example, Richards-Hrdlicka et al. (2013) found no evidence of infection in American bullfrog tadpoles in Connecticut but Julian et al. (2016) found a wide range of prevalences in green frog tadpoles, which were sampled in May and June after they had overwintered in permanent ponds in Pennsylvania.
Another potential cause for the high prevalence of Bd infection in permanent ponds in early spring could be infected post-metamorphic animals that hibernate in those ponds. The data we collected from the two fenced ephemeral ponds supports this idea. We found that for animals arriving at the fenced ponds in spring, Bd prevalence and load were higher in permanent pond hibernators (American bullfrogs, green frogs, northern leopard frogs) than terrestrial hibernators (all sampled salamander species, wood frog, spring peeper, American toad, and eastern gray treefrog). This, along with the greater Bd prevalence in animals sampled from permanent ponds in early spring (the vast majority of which were permanent pond hibernators), suggests that post-metamorphic anurans hibernating in permanent ponds may be an important contributor to the pool of infectious zoospores in these habitats as well as in ephemeral ponds, which they tend to visit (but not breed in) later in the active season. Despite its potential importance for understanding the risk chytridiomycosis poses to susceptible amphibian hosts, we are unaware of any studies have characterized Bd prevalence or load on individuals during hibernation. However, a survey like ours, which sampled amphibians throughout their active season in Maine, USA, also found that infection prevalence was lower in species that hibernate in terrestrial habitats than that in species that hibernate in aquatic habitats (Longcore et al. 2007).
Sources of Bd in ephemeral ponds
Little is known about how aquatic pathogens like Bd, which cannot persist during extended dry periods (Garmyn et al. 2012), reach habitats that are terrestrial or only episodically aquatic, like ephemeral ponds. In our study system, Bd was consistently detected on animals sampled from ephemeral ponds and we also detected it via eDNA sampling from ephemeral pond water, suggesting that Bd is able to colonize these temporary water bodies consistently once they refill. Spotted salamanders, American toads, and spring peepers, all of which hibernate terrestrially, were the first amphibians to enter our fenced ephemeral ponds with Bd. This suggests that animals entering the pond to breed after overwintering in terrestrial hibernacula are one way that Bd can be brought to ephemeral ponds each spring and that transmission from permanent to ephemeral ponds via infected hosts or environmental sources (e.g., rain or fog, Kolby et al. 2015; Prado et al. 2023) may not be required to sustain Bd in ephemeral pond communities over time. However, the prevalence of Bd infection on ephemeral pond breeders in the early spring was low; only 28 of 137 of the ephemeral pond breeders (8 spotted salamanders, 12 wood frogs, 5 spring peepers, and 2 American toads) entering the fenced ponds in the first month after they became active were infected with Bd. This suggests that stochastic variation could be important to the dynamics of Bd in ephemeral ponds, especially in early spring before visitors from permanent ponds arrive.
Little is known about how hibernation affects the course or outcome of Bd infections in amphibian hosts. The only study we are aware of that has addressed this directly is Rumschlag and Boone (2018), which found that experimental Bd exposure prior to hibernation reduced overwinter survival in the northern leopard frog. It appears that some of the terrestrially hibernating animals in our study emerged infected, which suggests they were able to survive with infection over the long winter, though how infection was acquired and how infection loads may have changed over the winter remain unknown. It may be possible for species hibernating underground to contract Bd infections while hibernating, as Bd has been shown to persist in moist sand for extended periods (12 weeks, Johnson and Speare 2003). Alternatively, given that Bd was detected on many terrestrial hibernators leaving our ephemeral ponds in fall (47/134 infected, including 15 spotted salamanders, 29 wood frogs, 1 spring peeper and 2 American toads), animals may have entered hibernacula infected and maintained those infections over winter. Predicted Bd prevalence and load just after emergence from hibernation were similar to those just prior to hibernation for animals sampled in our ephemeral ponds. This is consistent with the idea that infections are not commonly gained or lost and loads do not increase dramatically during the cold months of hibernation, though we do not have individual-level data to test this explicitly.
While they breed, develop, and hibernate in more permanent water, some of the permanent pond associated amphibians in our study area (e.g., American bullfrogs, green frogs, and northern leopard frogs) are often encountered in and around ephemeral ponds. Due to their time spent in permanent ponds overwinter and in early spring, we wondered whether these hosts might be common vectors bringing Bd to ephemeral ponds as well. We found that Bd prevalence and load on animals entering the fenced ephemeral ponds were greater for amphibian species that hibernate in permanent ponds than for species that hibernate terrestrially. As early as April 5th we began to encounter American bullfrogs, Northern leopard frogs, and green frogs along our drift fences and 60 of the 99 of these animals (60.6%) that we sampled upon entry into the ponds were infected with Bd whereas only 25.4% (66 of 259) of the terrestrially hibernating animals were infected upon their first entry into these ponds. Most of the permanent pond associated animals found entering the fenced ponds with Bd infections were green frogs (50/64 infected). This supports the idea that species associated with permanent ponds play an important role in Bd dynamics of ephemeral ponds in addition to permanent ones. It also suggests that permanent ponds, and the species associated with them, can have a large influence on the distribution of Bd across the landscape.
Genetic diversity of Bd in ephemeral ponds
Despite finding a surprising amount of genetic diversity in Bd across the two fenced ephemeral ponds, there was no appreciable structure to that diversity between ponds or among the amphibian species from which the samples were taken. This lack of genetic structure in Bd was surprising to us because the two ephemeral ponds are separated by ~ 9 km, a distance greater than amphibians in this region are thought to disperse (Smith and Green 2005). The lack of genetic differentiation could be explained by these pond communities being parts of a metapopulation, connected by occasional dispersal (Hamer and McDonnell 2008) or by movement of Bd by more vagile animals (e.g., birds, Burrowes and De la Riva 2017) or through environmental sources like rain or fog (Kolby et al. 2015, Prado et al. 2023). Bd has been detected on the feet of birds, in the gut of crayfish, and on reptiles (Prahl et al. 2020) in previous studies. There was a large amount of overlap in the Bd genetic variants found on animals entering vs. leaving the fenced ponds and also across the spring and summer seasons. The nearly complete lack of genetic structure among Bd from different ponds in our study area stands in stark contrast to the pattern seen among high-elevation lakes in the Sierra Nevada of California (where Bd haplotypes show strong genetic structure and a pattern of isolation by distance among high elevation lakes) and in western Panama (where Bd shows little genetic diversity across geographically distant stream systems) (Rothstein et al. 2021).
In both of our fenced ponds we found a diversity of Bd haplotypes belonging to the globally invasive panzootic lineage (GPL). The GPL comprises two genetic clades: BdGPL-1, which is primarily found in North America and Europe, and BdGPL-2, which is distributed worldwide (Rosenblum et al. 2013, O’Hanlon et al. 2018). GPL-1 is often thought of as a North American lineage and is thought to be ancestral to GPL-2, which is globally distributed and is the lineage responsible for amphibian declines in Central America (James et al. 2015). While GPL-1 and − 2 are both known to be present in the United States (Schloegel et al. 2012), in this study and in a study of a larger sample of ponds from this region (Byrne et al. 2022), we found evidence for a clade with less than 0.7 posterior support that appears to be a mix of Bd GPL1 and GPL2. This finding could be explained by coinfected animals harboring Bd strains belonging to both sublineages. Lab experiments have demonstrated the potential for such coinfection (Jenkinson et al., 2018). Alternatively, it could indicate that recombination has occurred between the two sublineages.
Hybrids between clades of Bd have been reported previously. For example, a hybrid lineage between Bd Brazil and Bd GPL has been described from Brazil and in some host species, infection by this hybrid lineage has been associated with increased mortality (Greenspan et al. 2018). Future work aimed at identifying the cause of the large genetic diversity and ‘mixed’ Bd genotypes is needed to clarify the history of the host-pathogen relationship in this region as well as mechanisms by which these populations have managed to avoid the catastrophic declines that Bd caused in many other amphibian communities.