Hybrids of amphibian chytrid show high virulence in native hosts

Hybridization of parasites can generate new genotypes with high virulence. The fungal amphibian parasite Batrachochytrium dendrobatidis (Bd) hybridizes in Brazil’s Atlantic Forest, a biodiversity hotspot where amphibian declines have been linked to Bd, but the virulence of hybrid genotypes in native hosts has never been tested. We compared the virulence (measured as host mortality and infection burden) of hybrid Bd genotypes to the parental lineages, the putatively hypovirulent lineage Bd-Brazil and the hypervirulent Global Pandemic Lineage (Bd-GPL), in a panel of native Brazilian hosts. In Brachycephalus ephippium, the hybrid exceeded the virulence (host mortality) of both parents, suggesting that novelty arising from hybridization of Bd is a conservation concern. In Ischnocnema parva, host mortality in the hybrid treatment was intermediate between the parent treatments, suggesting that this species is more vulnerable to the aggressive phenotypes associated with Bd-GPL. Dendropsophus minutus showed low overall mortality, but infection burdens were higher in frogs treated with hybrid and Bd-GPL genotypes than with Bd-Brazil genotypes. Our experiment suggests that Bd hybrids have the potential to increase disease risk in native hosts. Continued surveillance is needed to track potential spread of hybrid genotypes and detect future genomic shifts in this dynamic disease system.


Results
Brachycephalus ephippium and I. parva (host species with direct development). The ranked mortality by treatment (from highest to lowest) for B. ephippium was: hybrid, Bd-GPL, and Bd-Brazil. (χ 2 = 33.938; d.f. = 3; p < 0.0001; Fig. 1a). Mortality in B. ephippium exposed to Bd-Brazil was similar to the control (p = 0.100; Fig. 1a). Mortality was first observed in frogs exposed to Bd-Brazil on day 35 and 2/9 frogs (22%) died by the end of the experiment on day 40 (Fig. 1a). In contrast, frogs exposed to Bd-GPL (p = 0.002) and the hybrid (p < 0.0001) had higher mortality rates than in the control, and the mortality rate was higher in the hybrid treatment than in the Bd-GPL treatment (p = 0.0008). Mortality was first observed in frogs exposed to the hybrid on day 19 and 9/9 frogs (100%) died by day 34 (Fig. 1a). Mortality was first observed in frogs exposed to Bd-GPL on day 26 and 6/9 frogs (67%) died by the end of the experiment (Fig. 1a).
The ranked mortality by treatment for I. parva was: Bd-GPL, hybrid, and Bd-Brazil (χ 2 = 36.723; d.f. = 3; p < 0.0001; Fig. 1b). Mortality in I. parva exposed to Bd-Brazil was similar to the control (p = 0.071; Fig. 1b). Mortality was first observed in frogs treated with Bd-Brazil on day 52 and 2/5 frogs (40%) died by the end of the experiment on day 58 (Fig. 1b). In contrast, frogs exposed to Bd-GPL (p < 0.0001) and the hybrid (p = 0.0003) had higher mortality rates than in the control, and the mortality rate was higher in the Bd-GPL treatment than in the hybrid treatment (p < 0.0001). Mortality was first observed in frogs exposed to Bd-GPL on day 18 and 6/6 frogs (100%) died by day 27 (Fig. 1b). Mortality was first observed in frogs exposed to the hybrid on day 31 and 6/7 frogs (86%) died by the end of the experiment (Fig. 1b). All direct-developing individuals that were not experimentally inoculated survived during both experiments.

Dendropsophus minutus (host species with aquatic larval development).
We did not detect differences in mortality of D. minutus among infection treatments or between infection and control treatments (χ 2 = 8.297; d.f. = 9, p = 0.504). Mortality was first observed in frogs exposed to Bd-Brazil on day 19  We detected differences in infection loads on day 60 within and among treatments (p < 0.001; Fig. 3). Average infection loads on frogs exposed to hybrids and Bd-GPL were highly variable within treatments (hybrids: 7,356 g.e.; 8,679 g.e.; 248,663 g.e.; Bd-GPL: 9,893 g.e.; 49,316 g.e.; 141,879 g.e.; Fig. 3). Average infection loads within the Bd-Brazil treatment were less variable and were generally lower than in frogs treated with hybrids and Bd-GPL (Bd-Brazil: 3,437 g.e.; 17,614 g.e.; 26,169 g.e.; Fig. 3). Thirteen of 20 control frogs carried natural Bd infections. Average infection loads on control frogs were lower than those on treatment frogs, likely reflecting what they were carrying in the field (Fig. 3). Passage rate was not a significant predictor of Bd infection loads, independent of Bd lineage (F = 0.228, p = 0.634).

Discussion
Amphibian declines and extirpations throughout the Atlantic Forest have been attributed to Bd, and the zone of hybridization between Bd-Brazil and Bd-GPL is within the region with the clearest signature of disease-related mortality, underscoring the need to test the virulence of hybrid genotypes in native hosts 30,42 . In our direct-developing host species, mortality rates of frogs exposed to a hybrid genotype were higher (B. ephippium) This finding indicates that hybrid virulence is context specific; in certain scenarios these genotypes may produce disease outcomes that far exceed parental ranges, while in others these genotypes may elicit intermediate disease outcomes. Since it is well-established that Bd has a dynamic genome and is now nearly globally distributed, this finding highlights the importance of global surveillance to detect future genomic shifts in this disease system that could lead to new outbreaks of chytridiomycosis, especially because a non-Brazilian host (Lithobates sylvaticus) also exhibited increased mortality in response to a Bd hybrid relative to the parental genotypes in a preliminary trial 51 . In addition, in other chytrid fungi, sexual reproduction produces a thick-walled resting stage that is capable of tolerating desiccation and high levels of heat and salinity 52 . Resting spores can also be produced by asexual reproduction, as in the chytrid Rhizophydium brooksianum 53 , which is in the same taxonomic order (Rhizophydiales) as Bd. No definitive evidence of a resting stage in Bd has been observed to date 54,55 , but this would have important implications for the geographic and temporal scales at which transmission of Bd could occur.
The relative virulence of hybrid genotypes and Bd-GPL appeared to depend on host factors, highlighting the context dependency of disease outcomes that is a hallmark of this disease system 56,57 . Compared to I. parva,  B. ephippium died with relatively low Bd burdens, suggesting a defense tradeoff in which B. ephippium invested more resources in resistance defenses (minimizing parasite burden) at the expense of tolerance defenses (minimizing harm caused by parasites, such as mortality effects in this study) 58 . Theoretically, resistance strategies are expected to reduce pathogen fitness, eliciting a strong antagonistic host-pathogen co-evolution 59,60 . The effectiveness of an immune defense strategy that is heavily influenced by host-pathogen co-evolution should be positively correlated with the length of time that the host has been exposed to the parasite. In accordance with this expectation, B. ephippium exhibited the longest incubation periods and highest survival rate (variables indicating the most developed resistance defenses) when exposed to Bd-Brazil, the lineage with which this species has probably co-existed in the wild for the longest time period. It follows that incubation periods and survival were intermediate when B. ephippium was exposed to Bd-GPL, the lineage with which this species has probably co-existed in the wild for a time period intermediate between Bd-Brazil and hybrid genotypes. Lastly, this species exhibited the shortest incubation periods and lowest survival rate, as well as the highest infection burdens at the time of death (variables indicating the least developed resistance defenses), when exposed to hybrid genotypes, the group with which this species has probably co-existed in the wild for the shortest time period, or not at all.
These findings indicate that B. ephippium may be particularly vulnerable to infections by hybrid genotypes in nature, primarily due to the relative novelty of these genotypes. The vulnerability of B. ephippium to novel pathogens may be linked to its association with patchy, high-altitude habitats, which may have limited its exposure to pathogens throughout its evolutionary history or lowered its immunogenetic diversity [61][62][63] . In the case of Bd, anthropogenic habitat alteration may tip the host-pathogen balance further in favor of the pathogen, as deforestation may promote the evolutionary isolation of B. ephippium while at least some relatively novel genotypes of Bd appear highly competent at dispersing across even the most fragmented landscapes in the Atlantic Forest, such as our B. ephippium collection site at Jundiái 42 . Previous studies indicate that F1 hybrids often show hybrid vigor because of outbreeding enhancement, whereas F2 hybrids might express hybrid breakdown as a result of recombination 26 . Considering that all living Bd hybrid isolates are first-generation crosses (TYJ pers. comm.) 64 , we are unable to test whether our results for B. ephippium represent an instance of hybrid vigor that could weaken through subsequent crosses 16 . However, an F2 backcrossed to Bd-Brazil was observed in the field and its survival provides some indication that parasitemia of Bd hybrids may not be limited to the F1 generation 64 .
In contrast to B. ephippium, I. parva died with relatively high Bd burdens, suggesting the reverse defense tradeoff in which I. parva invested more resources in tolerance defenses at the expense of resistance defenses. Tolerance strategies are not expected to negatively affect the success of pathogen populations, so we would expect relatively weak co-evolutionary pressures between parasites and hosts that invest primarily in this type of defense strategy 59,60 . This may help to explain why the responses of I. parva to our panel of Bd treatments were not correlated with the relative length of time to which this species was probably exposed to each lineage in the wild. Rather, it is possible that this species was more responsive to the expression of hybrid phenotypes that were intermediate between the hypervirulent Bd-GPL and the hypovirulent Bd-Brazil, resulting in intermediate incubation periods and mortality in frogs exposed to the hybrid compared to Bd-GPL and Bd-Brazil. Our findings suggest that I. parva is particularly vulnerable to pathogens with aggressive phenotypes, such as Bd-GPL, regardless of the extent to which this species has shared an evolutionary history with co-occurring pathogens. Thus, an important avenue for future study is to determine the genetic or physiological factors that make Bd-GPL particularly damaging to hosts and the ecological backdrop against which these genotypes emerged. Immunological comparisons of disease progression among Bd genotypes and host species are necessary to verify the mechanisms we have proposed to explain the host-specific patterns in our data. Nevertheless, our results offer convincing evidence that both Bd-GPL and hybrid genotypes are virulent in the Atlantic Forest.
Compared to hybrid genotypes and Bd-GPL, frogs treated with Bd-Brazil had the lowest ranked mortality in both direct-developing host species and the lowest infection burdens in D. minutus, consistent with the hypothesis that Bd-Brazil has shared a long co-evolutionary history with endemic Brazilian frogs and is hypovirulent in endemic hosts 28,38,42,50 . A recent study also found that the Bd-Brazil genotype CLFT 001 (isolated from the Atlantic Forest) exhibited lower in vitro growth performance than the Bd-GPL genotype CJB5-2 and the Bd-Brazil genotype UM 142 (unknown geographic origin) 65 , another indication that the relative threat of endemic Atlantic Forest Bd genotypes is low. In contrast, the only other study that tested the relative virulence of Bd-Brazil and Bd-GPL reported that 50% of hosts died when exposed to Bd-Brazil and that the virulence of Bd-Brazil (one genotype) fell within the range of virulence shown by Bd-GPL (three genotypes) 66 . This conflicting study used the Bd-Brazil genotype UM 142, different Bd-GPL genotypes (from the eastern U.S. and Panama), and a North American host species. Our conflicting results may thus reflect host-independent variability in virulence within Bd lineages or suggest that Bd-Brazil has higher virulence toward non-native hosts. Evidence suggests that humans are facilitating the spread of Bd in South America 67 and globally 38 . For instance, the Bd-Brazil genotype UM 142 was isolated from a bullfrog (Lithobates catesbeianus) collected from a U.S. amphibian market. We reiterate the concern raised by Becker et al. 66 that lineages endemic to one region may lead to declines of naïve host populations in other regions.
Terrestrial, direct-developing frog species have typically been considered less vulnerable to chytridiomycosis than species with more aquatic life histories that may experience high levels of exposure to aquatic Bd zoospores [68][69][70] . However, both of our terrestrial study species acquired heavy Bd infections and experienced mortality from chytridiomycosis under laboratory conditions mimicking the microhabitat of direct-developing frogs in nature, consistent with laboratory data for other Brazilian direct-developing species 63 . In the wild, Brazilian direct-developers had high Bd infection loads 71,72 , but low infection prevalence 63 , the latter of which could be underestimated if frogs die quickly from infections or if sick frogs remain stationary in hidden refugia 63,73 . Even if low prevalence of Bd is currently facilitating population persistence of direct-developing host species in Brazil, this host-pathogen balance is precarious in an era of global change 61 . For example, in Puerto Rico, Bd dynamics in the direct-developing species Eleutherodactylus coqui shifted from enzootic to epizootic when extreme drought SCiENtifiC REPORTS | (2018) 8:9600 | DOI:10.1038/s41598-018-27828-w conditions associated with global climate change forced frogs to congregate in humid refugia, increasing transmission and reinfection rates 73,74 . Moreover, reduced levels of recruitment stemming from increased Bd-related mortality of juvenile E. coqui has led to recent, low-level population declines 75,76 , similar to other examples of negative population effects from Bd in the absence of drastic epizootic events [77][78][79] . Future research should investigate the potential effects of Bd on the population persistence of Brazil's diverse direct-developing amphibian fauna, especially considering that the narrow geographic ranges of many Brazilian direct-developers leave them vulnerable to other natural and anthropogenic stressors and that Bd caused population declines of a direct-developing Arthroleptis in Cameroon 35 and likely the extinction of several direct-developing species in the Atlantic Forest (see Supplementary Table S2).
In contrast to our direct-developing species, mortality rates of D. minutus were low across treatments, suggesting that this species is relatively tolerant to Bd regardless of variation in the genetic attributes of the fungus. This finding is consistent with previous studies of D. minutus in the laboratory 63,80 and in the wild 81 . Patterns of infection loads in D. minutus by lineage matched mortality rates in our direct-developing species, with the lowest infection loads in frogs exposed to Bd-Brazil and higher infection loads in frogs exposed to Bd-GPL and hybrid genotypes, corroborating the evidence from our direct-developing species that Bd-Brazil is less virulent in native hosts. Infection loads were also less variable among genotypes within Bd-Brazil than within Bd-GPL and hybrid genotypes, which could reflect consistency in host immune responses stemming from long-term exposure to Bd-Brazil.
Our study shows that hybridization can be associated with high levels of virulence in Bd. Hybrid Bd genotypes emerged relatively recently in Brazil, at some point after the introduction of Bd-GPL to Brazil in the last few centuries 41 . The known distribution of hybrid genotypes is small (an isolated mountain range in the Atlantic Forest), which could suggest that it is functioning primarily as a parasite with intermediate rather than extreme fitness relative to parental populations and could be outcompeted by quick-dispersing Bd-GPL. However, it is unknown whether the current geographic distribution of hybrid genotypes is a product of their short evolutionary history (i.e., they might currently be spreading), their specific microhabitat requirements, or gaps in field sampling. Nevertheless, their coexistence with Bd-GPL suggests that they could be adept competitors in Brazilian landscapes. Applying newly developed, non-invasive techniques that can discriminate among Bd genotypes on skin swabs would help to paint a more complete picture of the spatial distribution of Bd lineages in Brazil as well as coinfection dynamics 82 . The same techniques could also be used with contemporary and retrospective sampling of preserved specimens to increase our understanding of which genotypes and lineages are associated with amphibian mortality in Brazil, track the spread of Bd-GPL through time, and determine whether the geographic distributions of hybrid genotypes and Bd-Brazil have changed over time 82 . Another useful avenue for future study is to determine how coexistence and hybridization of Bd genotypes in Brazil could influence host population recoveries through adaptive responses, which have been documented in other regions in the decades following mass declines 83,84 .
Our results do not point definitively to a single genetic culprit of Bd-related amphibian declines in Brazil. Bd-GPL is likely to have played a large role given its high level of virulence in some hosts and widespread geographic distribution 42 , but the hybrid zone overlaps the region with significant evidence of disease-related mortality 30 , and our results indicate that some host species are especially vulnerable to new genotypes, suggesting a possible role of hybrids in declines. Taking all these factors into account, a plausible scenario for disease-linked amphibian declines in Brazil is that the impacts of the introduction of Bd-GPL were exacerbated by the emergence of hybrids, possibly by overloading host immune systems with an even more genetically and spatiotemporally diverse assemblage of pathogen strains. As much as evolutionary novelty can aid species adaptation in an era of rapid environmental change, our findings underscore that this plasticity can also be advantageous for parasites, with serious consequences for the persistence of host populations 13,16 .

Materials and Methods
Study species. We selected three experimental host species with varying life histories and levels of tolerance (i.e., ability to minimize harm caused by parasites, such as mortality) to Bd. Dendropsophus minutus (Hylidae: Dendropsophinae) is a habitat generalist tree frog with indirect development and a close association with water bodies throughout its life history. The geographic range of this species covers most of tropical South America 85,86 , and it exhibits relatively high survival rates when challenged with Bd-GPL under laboratory conditions 63,80 . Ischnocnema parva and Brachycephalus ephippium (Brachycephalidae) are direct-developing leaf litter frogs that occur in southeastern Brazil 85 and exhibit relatively low survival rates when challenged with Bd-GPL under laboratory conditions 63 . All three species are listed as Least Concern by the International Union for the Conservation of Nature 87 . In a vulnerability assessment of amphibians of the Brazilian Atlantic Forest, all three species were considered to have large geographic ranges and high local abundances but D. minutus and I. parva were classified as having wide habitat specificities, whereas B. ephippium was classified as having a narrow habitat specificity due to its association with high-altitude habitats 47 .
We collected adults of our study species in the municipality of Jundiaí, near Serra do Japi (B. ephippium and D. minutus) and in the municipality of São Luiz do Paraitinga, adjacent to Parque Estadual da Serra do Mar, Núcleo Santa Virgínia (I. parva), São Paulo state, Brazil. Both collection sites are located to the north of the Bd hybrid zone identified by Jenkinson et al. 41 . Some D. minutus tested positive for Bd at the time of collection but we elected not to treat infections to avoid any negative treatment-associated side effects and instead controlled for natural infections in our statistical analyses 88 . We only used I. parva and B. ephippium that tested negative for Bd in the field. For the experiments, frogs were randomly assigned to treatments and housed individually in plastic containers with sterile moist sphagnum (all species) and a sterile leaf for cover (B. ephippium and I. parva). Frogs were fed pinhead crickets ad libitum during the experiments.  42 . Tadpoles were screened in the field with a 10X hand lens for signs of chytrid infection by assessing the level of oral tissue dekeratinization 89 . Animals with signs of Bd infection were euthanized and oral tissues excised. Infected tissues were prepared for pathogen isolation on 1% tryptone agar with 0.2 mg. mL −1 penicillin-G and 0.4 mg.mL −1 streptomycin sulfate. Isolates of Bd were maintained on 1% tryptone agar at 21-23 °C until sufficient growth had occurred for DNA extraction. Isolates were genotyped to determine Bd lineage/group (Bd-Brazil, Bd-GPL, or hybrid) following the procedures described in Jenkinson et al. 41 . Cultures were maintained at 4 °C at Universidade Estadual de Campinas, UNICAMP, Brazil, and passaged every 4 mo.
Challenge experiment. We cultured three hybrid, three Bd-Brazil, and four Bd-GPL genotypes (see Supplementary Table S1) in Petri dishes containing 1% tryptone agar at 19 °C for 7 d. The three hybrid genotypes represent all living hybrid isolates, and analyses of whole genome sequences indicate that all are F1 hybrids (TYJ pers. comm.) 64 . The Bd-Brazil and Bd-GPL genotypes were selected haphazardly. To inoculate frogs with Bd (day zero), we filled each Petri dish with 5 ml of distilled water for 30 minutes and scraped the substrate with a sterile scalpel to facilitate zoospore release. We then transferred the liquid contents of each dish to a sterile beaker, sampled 1 ml of the solution to quantify the zoospore concentration with a hemocytometer, and diluted the solution with distilled water to obtain the desired zoospore concentration for experimental inoculations.
We inoculated B. ephippium and I. parva with hybrid genotype CLFT 160 (B. ephippium: n = 9; I. parva: n = 7), Bd-Brazil genotype CLFT 150 (B. ephippium: n = 9; I. parva: n = 5), and Bd-GPL genotype CLFT 156 (B. ephippium: n = 9; I. parva: n = 6). Due to technical difficulties with preparing Bd-GPL for this component of the experiment, we were unable to use one of the three Bd-GPL genotypes that were tested with D. minutus. We inoculated each frog individually in a Petri dish containing 3.375 × 10 6 zoospores in 1.5 ml of distilled water for 45 minutes. We exposed additional individuals to the same volume of distilled water as controls (B. ephippium: n = 8; I. parva: n = 6).
Survival was monitored daily, dead animals were noted, and dying animals were euthanized with an overdose of the anesthetic MS-222 if they showed lack of righting response, which is a typical sign of advanced stages of chytridiomycosis 90 . Dead animals were swabbed immediately following the protocol described by Hyatt et al. 91 . The experiment concluded on day 60 (D. minutus), 58 (I. parva), or 40 (B. ephippium; length of experiment truncated because this species can become stressed after long periods in captivity), at which point all remaining animals were swabbed and euthanized.
We extracted DNA from skin swabs using 50 ml PrepMan Ultra and screened samples for Bd presence and load using Taqman qPCR assays 92 . For D. minutus, we used Bd genotype-specific standard curves (for each genotype used in the experiment) ranging from 0.1 to 1000 zoospore genome equivalents (g.e.). For B. ephippium and I. parva, we built standard curves (0.1-1000 g.e.) using CLFT 159, a Bd-GPL genotype isolated from a Hylodes frog collected in the Atlantic Forest. We were unable to standardize infection load data for B. ephippium and I. parva due to unforeseen culturing difficulties with one of our genotypes, but we feel confident in using non-standardized infection loads in our analyses for these species because standardization did not influence the overall patterns in our data for D. minutus (see Supplementary Fig. S1).
All experimental protocols were approved by Instituto Chico Mendes de Conservação da Biodiversidade -Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis/Brazil (Permits 29964-11, 27745-13, and 57098-1) and the local Animal Care and Use Committee (Comissão de Ética no Uso de Animal -CEUA/ UNESP permit #29/2016). Our experiment was carried out in accordance with all ethics guidelines and regulations.
Statistical analyses. We used proportional hazards survival analyses 93 to compare mortality rates among frogs exposed to Bd-Brazil, Bd-GPL, and hybrids, independently for each species. For B. ephippium and I. parva, we compared mortality rates among one genotype from each of the three treatments. For D. minutus, we compared mortality rates among nine Bd genotypes within the three treatments, including genotype as a fixed effect.
We used a Generalized Linear Mixed Model (GLMM) to compare infection loads on day 60 among D. minutus exposed to Bd-Brazil, Bd-GPL, and hybrids. In this model, we included the following explanatory variables: genotype, lineage/group, and the interaction between genotype and lineage/group as fixed effects, and infection load at the time of capture in the wild as a random effect. We also performed a GLMM to test for effects of passage rate on Bd loads of D. minutus with Bd lineage/group as a random effect.
For B. ephippium and I. parva, we performed independent General Linear Models (standard least squares) to compare infection loads at the time of mortality between frogs exposed to Bd-GPL and hybrids. For these models, we included log 10 -transformed infection loads as the response variable and Bd lineage/group (Bd-GPL or hybrid), host species (B. ephippium or I. parva), and the interaction between lineage/group and host species as explanatory variables.
Data availability. The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.