Introduction

Wildlife extinctions are not typically attributed to infectious diseases, yet there are a few examples showing pathogens as the direct cause of local extinctions [1–3]. Some of them have been linked to chytridiomycosis, an emerging infectious disease [4,5]. In amphibians, chytridiomycosis results from a skin infection caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd). In the tropics, the most severe declines have been documented in comparatively cooler and humid areas above 500 m [6], most likely because lower temperatures (17–25°C) are optimal for Bd growth and water facilitates the propagation and dispersal of the aquatic, flagellated Bd zoospores [7]. Although the rapid spread of Bd into apparently Bd-free areas throughout the world is well-documented [8–12], the process by which Bd spreads is still not clearly understood [13].

Bd has rapidly spread throughout the highlands of Central America. In the late 1980s, the disappearance of golden toads (Incilius periglenes) and declines of other anuran populations in the protected cloud forest of Monteverde, Costa Rica (Fig 1), were the first alerts to what later became a predictable pattern of declines spreading towards Panamá [14,15]. Shortly thereafter, between 1993 and 1997 additional cases of amphibian population declines or extinctions were reported in the highlands close to the border with Panamá at Las Tablas, Costa Rica, and Fortuna, Panamá (Fig 1)[16,17]. The sites experiencing population declines were located at altitudes above 500 m, and the fastest declining species were stream dwellers with aquatic tadpoles [6]. Bd was spreading west to east in a wave like pattern [8]. High elevation sites in western and central Panamá were then intensively studied prior to and during the arrival of Bd, these studies documented amphibian population declines after the arrival of Bd in this region (Fig 1)[16–19].

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Fig 1. Sites where Bd has been detected in the past (filled circles), and sites where we sampled Túngara frog (Physalaemus [Engystomops] pustulosus) populations (open triangles).

Year in parenthesis corresponds to year of decline or the year that Bd was first detected.

https://doi.org/10.1371/journal.pone.0155745.g001

Only two studies have investigated Bd infection spread in eastern Panamá. Rebollar et al. [20] sampled populations along the lowlands of east Panamá, and Woodhams et al. [19] sampled sites in central Panamá. These studies did not detect a clear pattern of wave-like spread from west to east as observed in the highlands of Panamá. It is also possible that another wave of Bd from South America crossed into Panamá [8]. Currently, the dynamics of Bd spread in the lowlands of Panamá, where environmental conditions are not ideal for Bd, are still unclear and require additional investigation.

Evaluating the spread of a pathogen in a single, abundant, and well-characterized host can help to predict spread dynamics while controlling for host phylogenetic diversity. The Túngara frog (Physalaemus [Engystomops] pustulosus) is a common species occupying lowland habitats ranging from Mexico to Colombia, Venezuela, and a small portion of the Guyana Shield. Its range parallels many highland regions where Bd and amphibian declines have been extensively documented [8–13]. The adults and tadpoles of this species use both permanent and ephemeral bodies of water in habitats ranging from urban areas to pristine forests. Thus, the Túngara frog is an ideal species in which to characterize the spread of Bd through populations of a lowland host. Here, we (i) report the Bd infection of túngara frogs at two sites west of the Panamá Canal where Bd is enzootic, and (ii) present data documenting the spread of Bd in the lowlands of central and east Panamá from 2009 to 2014.

Methods

We sampled Túngara frog populations in Panamá from 2009 to 2014, each year during the rainy season (June to November), which falls within their reproductive season. In most cases, populations were sampled during two reproductive seasons. Lowland sites in east and central Panamá ranged from 12 to 67 m in altitude (Fig 1). We also sampled highland populations at El Valle (elevation 600 m; Fig 1) in central Panamá, and at Chiriquí near the town of Cuesta de Piedra (elevation 460–967 m; Fig 1) in western Panamá. This altitude is the highest known Túngara population in Panamá. Chiriquí was also our western most site and was located 30 km east of sites where other amphibian species experienced declines in 1993 [17].

In central Panamá, we sampled two lowland sites east of the Panamá Canal, Gamboa and Summit, ranging from 46 to 98 m in elevation (Fig 1). These two lowland sites are located on the eastern side of the Panamá Canal but are separated by the Chagres River. Gamboa is a small town north of the river that is surrounded by rainforest. Here we included Pipeline Road, which intersects a protected rainforest in the Soberanía National Park. Summit is located south of the river and includes portions of the Soberanía National Park. We sampled along the main road, on trails and dirt roads within the National Park.

In east Panamá, we sampled four sites. Metetí and Yaviza are located along and at the very end of the Inter-American Highway, respectively (Fig 1). Here, we sampled disturbed, deforested habitats, in puddles on dirt roads and cattle ranches. Further east, along the Tuira River, we sampled at El Real (Fig 1), a small town surrounded by less disturbed habitat and in Darién National Park. In Darién National Park, we sampled around the Rancho Frío field station (Fig 1) at an average of 50 m of elevation. This site is predominantly primary rainforest with a tall canopy, thick lianas and an understory dominated by palms.

Results

In total, we sampled 1695 P. pustulosus adults across Panamá from 2009 to 2014 (Fig 1). Sample size, prevalence, and 95% binomial confidence intervals are shown in Table 1. We considered sites with sample sizes greater than 60 individuals and no positive samples as Bd-negative [25]. The site Chiriquí, in western Panamá, was positive for Bd in 2010 and 2013. In central Panamá, El Valle was positive for Bd in 2009. In 2010, Gamboa samples were negative for Bd, but Summit, which is only 8 km to the south, was positive. Bd reached Gamboa in 2011, and in following years we detected Bd positive samples in both Summit and Gamboa. By 2014, prevalence had reached 26% in Gamboa (Table 1). Populations in Metetí were Bd naive in 2011 but positive in 2013. Farther east, Yaviza was naive for Bd in 2013, but by 2014 prevalence reached approximately 6%. By 2014, Bd was also present in P. pustulosus populations from El Real and Rancho Frío, Darién National Park (Table 1).

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Table 1. Bd prevalence and infection intensity per site and year in Túngara frog (Physalaemus [Engystomops] pustulosus) populations sampled in this study (n = number of individuals sampled; Positive = number of individuals detected positive for Bd; 95% CI = 95% binomial distribution confidence intervals; Average intensity = average of number of zoospore equivalents in infected frogs per population).

Sites are arranged west to east by longitude.

https://doi.org/10.1371/journal.pone.0155745.t001

Discussion

Data for the spread of Bd in the lowlands of Middle America are scarce. Here we estimated the rate of spread of Bd in Túngara frogs based on the first detection of Bd, and we assumed that Bd spread in a wave-like fashion [8]. In the lowlands of Panamá, Bd spread at a similar rate among Túngara frogs (54 km/year) when compared to other amphibian species tested in this area (30–174 km/year) [19]. There was one exception. The rate of spread from Summit across the Chagres River to Gamboa was slower than the average (8 km/year).

In western Panamá, we sampled in Chiriquí, at elevations where amphibian population declines have been severe in the past [16]. Since Bd is now enzootic in this area [19] and at El Valle in central Panamá, it is not surprising that the Túngara frog populations from Chiriquí and El Valle were positive in 2010, and 2009, respectively. We also found the highest average loads of Bd in Chiriquí (3570 z.e., Table 1). We could not estimate the rate of Bd dispersal among Túngara frog populations between Chiriquí and El Valle as they were already infected at the time we sampled. In 2007, Woodhams et al. [19] reported Bd positive individuals (30% prevalence) among three species at Soberanía National Park, including the Summit area where we sampled in 2010, and they suggested that Bd was already enzootic during their study. In the same year, Gamboa, was still naive and we most likely sampled before Bd arrived. In 2011, we detected Bd at Gamboa for the first time, one year earlier than previously reported for this area [20].

We cannot discern whether the source of infection of the Gamboa populations was from Summit Túngara frogs or from other species of frogs in Gamboa. Regardless, there is a relatively large time lag between when Bd was detected in Túngara frogs from Gamboa compared to when Bd was first detected in Túngara frogs from Summit. The mechanism by which Bd spreads is unknown, but it has been suggested that Bd could survive and could be carried in mud, and thus easily dispersed by humans [26]. In Túngara frogs, Bd did not spread as fast as expected from Summit to Gamboa, which are only 8 km apart and connected by a well-traveled road and bridge over the Chagres River.

Certain geographic features, like rivers, could impede the spread of Bd. The Chagres River, which is about 100 m wide and has water all year long, separates Gamboa and Summit. Genetic studies demonstrate that the Chagres River is a geographical barrier for gene flow between Túngara frog populations [27], thus it is possible that limited migration between these populations could slow the spread of Bd whether Túngara frogs contracted Bd from conspecifics or heterospecifics. Bd does seem to have spread rapidly in the lowlands towards eastern Panamá. An alternative explanation for the spread of Bd from east to west throughout all of Panamá is that populations in Darién, and elsewhere in far eastern Panamá, were infected by a wave coming from the south [8]. These two scenarios should be tested through a phylogenetic analysis of Bd throughout the Túngara frog's range in Central and South America. As such data are currently unavailable, it is difficult to determine the recent origin of Bd in eastern Panamá; therefore, our estimates of rate of spread should be viewed with this potential caveat in mind.

Woodhams et al. [19] conservatively estimated that Bd spread east and would have reached Tortí in September of 2012, but [28] reported two positives out of 93 samples of other species of frogs at this site in 2010. There are no other published reports on the presence of Bd among amphibian species from this specific area. In 2011, we sampled in Metetí, approximately 70 km southeast of Tortí, and found this site to be Bd naive. Rebollar et al. [20] recorded Bd positive samples from Nuevo Vigía in 2012, just 26 km east of Metetí, where we detected low Bd prevalence in 2013, thus supporting a wave-like Bd spread from west to east in Túngara frogs.

Species that carry Bd asymptomatically and share the habitat with more vulnerable species can potentially function as Bd spreaders. Túngara frog population declines have not been reported and were not evident during our study. If the prevalence and dispersal of Bd is density dependent, then the spread of Bd along the lowlands might be enhanced by abundant and apparently resistant species serving as reservoirs. Túngara frogs are known to disperse between breeding sites at distances up to 200 m [29]. They also share the habitat with a wide variety of species; thus, they could contribute to the rapid dispersal of Bd if they are effective carriers. Moreover, if most species in the lowlands are less susceptible to Bd or Bd is less virulent [7], then the high diversity and abundance of hosts could further facilitate the dispersal of Bd.

Chytridiomycosis is linked to some of the most severe population declines and extinctions of wildlife yet recorded. While substantial efforts have been aimed at understanding the spread and pathogenicity in the more vulnerable highland frog species, we know relatively little about the dynamics of Bd in tropical lowland regions of the world. Lowland species, however, could be reservoirs and dispersal agents between areas where amphibian species are more vulnerable to Bd infection. As highlighted by our results, even though lowland regions are typically characterized by less favorable climatic conditions for Bd [7], by harboring asymptomatic Bd infections, lowland amphibian populations could potentially play an important role in the spread of Bd across tropical regions.

Acknowledgments

For help in the field we thank Emma and Lucy Ryan, Tony Alexander, Alex Jordan, Meghan Still, Ty Hoskin, Mahudy Díaz, Samuel Sucre, and for logistical support we thank SENAFRONT, ANAM, and STRI. We thank Teofil Nakov and Robert Puschendorf for comments on the manuscript. We also thank Michael R.J. Forstner at Texas State University for instrumentation support, and Mahudy Díaz at STRI for laboratory assistance.