The amphibian chytrid fungus along an altitudinal transect before the first reported declines in Costa Rica

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Short communication

The amphibian chytrid fungus along an altitudinal transect before the first reported declines in Costa Rica Robert Puschendorf *, Federico Bolan˜os, Gerardo Chaves Escuela de Biologı´a, Universidad de Costa Rica, San Jose´, Costa Rica

A R T I C L E I N F O

A B S T R A C T

Article history:

Amphibian populations have declined and disappeared in protected and apparently undis-

Received 23 August 2005

turbed areas around the world, especially in montane areas of the tropics. The amphibian

Received in revised form

chytrid fungus, Batrachochytrium dendrobatidis has been implicated in many of these

28 February 2006

declines. In Costa Rica most declines occurred in the highlands. We examined an amphib-

Accepted 8 March 2006

ian collection made in Braulio Carrillo National Park in 1986 for the amphibian chytrid fun-

Available online 5 May 2006

gus B. dendrobatidis, prior to the well documented amphibian declines in Monteverde, along an altitudinal transect from 100 to 2600 m elev. Skin from the pelvic patch of 202 specimens

Keywords:

corresponding to 30 species was examined histologically to determine whether the disease

Amphibian declines

was present in the highlands of the park before amphibian populations declined. For com-

Chytrid fungus

parison, in 2002 we collected and examined 18 specimens of seven species of Eleuthero-

Costa Rica

dactylus and Craugastor from two other lowland Caribbean sites. The chytrid fungus was

Tropics

present in almost all altitudes in 1986, including lower areas. The pathogen was also found

Reproductive mode

in both species that later declined and in species that did not do so. We detected chytrid

Batrachochytrium dendrobatidis

fungus on amphibians collected at almost all altitudes in 1986, including those sampled at 280 m. B. dendrobatidis was abundant in frogs collected in the 2002 survey, and seems to be endemic in most of Costa Rica. More retrospective museum surveys are needed in order to determine whether it can be found in the area before 1986. Ó 2006 Elsevier Ltd. All rights reserved.

1.

Introduction

The decline and extinction of many amphibian populations in protected areas around the world is an alarming phenomenon with several possible causes (Alford and Richards, 1999; Stuart et al., 2004; Beebee and Griffiths, 2005). In Central America, this phenomenon occurred at elevations above 500 m (Young et al., 2001). Costa Rican declines have been characterized by a general reduction of abundance in almost all amphibian species at mid to high elevations, and by the extirpation of endemics

that used to inhabit the higher parts of the mountains (Pounds et al., 1997; Lips, 1998; Bolan˜os, 2002). In the Neotropics, one of the best indicators of amphibian population decline is the case of the harlequin frogs of the genus Atelopus (Lips et al., 2003a; La Marca et al., 2005). Diurnal and brightly colored these animals are severally threatened, and their sudden disappearances is strongly linked to a general amphibian decline in the area were they occurred (La Marca et al., 2005). What follows is an account of individual Atelopus populations reported in the literature and unpublished reports by other researchers of where these disappeared throughout

* Corresponding author: Present address: Amphibian Disease Ecology Group, School of Tropical Biology, James Cook University, Townsville, Queensland 4811, Australia. Tel.: 617 4781 5719. E-mail address: [email protected] (R. Puschendorf). 0006-3207/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocon.2006.03.010

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Costa Rica’s mountains starting on the northwest and going down to the southeast. The most northern population of any Atelopus species existed at Volca´n Cacao, in the province of Guanacaste (Savage, 2002). Atelopus varius was last seen here in 1990 (Flowers, pers. comm.). Approximately 95 km southeast, harlequin frog populations suffered total extirpation in apparent synchrony with the one of golden toad (Bufo periglenes), in the cloud forest of Monteverde (Pounds and Crump, 1994; Pounds et al., 1997). A few kilometers further south, in the San Ramo´n reserve amphibians suffered strong declines as well, with the apparent loss of many species, including A. varius populations which were last seen in 1986, and were absent in 1990 (Bolan˜os and Ehmcke, 1996; Bolan˜os and Chaves, unpublished). Bufo holdridgei, which was endemic to Cerro Chompipe (Central highlands), and Atelopus senex, also found at this site, may now be extinct (Savage, 2002). In Tapantı´ National Park, Atelopus was last seen in 1986 (Wake, pers. comm.), and in Las Tablas (southwestern highlands) Hyla calypsa and Bufo fastidiosus disappeared at the same time as Atelopus chiriquiensis (Lips, 1998). Significant amphibian declines, including the loss of harlequin frogs have also occurred at Fortuna, in the Panamanian Talamanca region, an area close to the Costa Rican border (Lips, 1999). Twenty-three species of Costa Rican anurans have been affected; 10 of these have not been seen for more than a decade, including A. senex and A. chiriquiensis (Bolan˜os, 2002). Most of Costa Rica’s mountains seem to have been affected by this phenomenon. These striking population declines in Costa Rica have been linked to climate change (Pounds and Crump, 1994; Pounds et al., 1997; Pounds et al., 1999), and to outbreaks of the chytrid fungus Batrachochytrium dendrobatidis (Lips et al., 2003b), a recently discovered species that grows on the epidermis of amphibians (Berger et al., 1998; Pessier et al., 1999; Longcore et al., 1999). It is reported to be non lethal for amphibian larvae (Berger et al., 1999; Longcore et al., 1999). However, Blaustein et al. (2005) found that Bufo boreas tadpoles exposed to this disease experienced increased mortality and behavior that indicate an effected by this pathogen. In culture the fungus grows well at temperatures below 23 °C but dies at temperatures greater than 29 °C (Longcore et al., 1999). This pathogen occurs on at least five continents (Berger et al., 1998; Ron and Merino, 2000; Lips et al., 2004; Bosch et al., 2001; Green and Sherman, 2001; Puschendorf, 2003; Bonaccorso et al., 2004; Weldon et al., 2004) and initial studies of it’s population genetics suggest that it is a clonally reproducing organism with little genetic variation (Morehouse et al., 2003). Woodhams et al. (2003) show that elevating the body temperature of infected animals can clear the disease from their skin. Temperature appears to be an important factor that may limit the effect of chytridiomycosis. Puschendorf (2003) examined skin samples of most of the specimens of A. varius in the Museum of Zoology of the University of Costa Rica, and found an infected specimen that had been collected during a survey in 1986 in Braulio Carillo National park, on the Atlantic slope of the Cordillera Volca´nica Central. Because well-documented declines occurred 75 km west of this site in Monteverde (Pounds and Crump, 1994) two years later, archived frogs collected during the 1986 survey provided an opportunity to check if B. dendrobatidis occurred in this

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137

area prior to disappearance of the golden toad (B. periglenes) in 1989. Declines occurred in this area in the higher portion of the transect, around Cerro Chompipe and Volca´n Barva, although no exact date exists for this, since no systematic studies were conducted here until 1990, when no frogs were found. (Bolan˜os and Chaves, unpublished data). Herpetological research has been carried out in the area of the lower part of the 1986 transect since the late 1940s (Guyer and Donnelly, 2005). In an intense study between 1982 and 1983 nearly all species known historically to this site were present (Clark, 1988); no dramatic changes have occurred since then (Bolan˜os and Chaves, unpublished data), as they have in higher elevations. In 2002, we collected and histologically examined specimens from Reserva Escalera de Mono and Guayaca´n, two lowland sites to see if the chytrid fungus could be found in amphibian populations more recently. Our objective was to determine to what extent and in what species B. dendrobatidis was present in 1986, when amphibian populations were still robust. In an attempt to learn about the evolution of B. dendrobatidis in Costa Rican amphibians, we compare infection status of host species in 1986 with altitude (a surrogate for temperature), reproductive mode and current population status.

2.

Methods

2.1.

Study regions and collection of specimens

2.1.1.

The La Selva–Volca´n Barva expedition of 1986

Braulio Carrillo National Park and La Selva Biological Station encompasses a 46,000 ha reserve (Clark, 1990) on the Atlantic slope of Cordillera Volca´nica Central in Costa Rica. Four different life zones are included in the transect: tropical wet forest, premontane rain forest, lower montane rain forest and mountain rain forest (Holdridge, 1982; Hartshorn and Peralta, 1988; Lieberman et al., 1996). In March and April 1986, a team of biologists including botanists, mammalogists, entomologists, invertebrate zoologists and herpetologists participated in a month long survey from La Selva (30–137 m elev.) to Volca´n Barva (2906 m elev.) during which museum specimens were collected (Timm et al., 1989). No dead or dying animals were encountered during the survey and there was no reason to believe that any of the animals collected were infected. The team of herpetologists worked out of seven camps, situated roughly at 300 m elev., 700 m elev., 1000 m elev., 1500 m elev., 1800 m elev., 2050 m elev. and 2600 m elev. Thirty-five species of anurans were collected. Elevations at these sites were determined using topographic maps. The herpetological specimens were divided between three different museums; we examined all the anuran specimens available from the Museum of Vertebrate Zoology, University of California at Berkeley (MVZ) and the Museo de Zoologı´a, Universidad de Costa Rica (UCR).

2.1.2. The reserva forestal Escalera de Mono (EARTH) and Guayaca´n surveys, 2002 Guayaca´n (10°02 0 N, 83°32 0 W, 650 m elev.) is one of the most herpetologically diverse sites in Costa Rica (Savage, 2002). Reserva Forestal Escalera de Mono at EARTH University

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(10°14 0 N, 83°34 0 W, 40 m elevation) is in the Caribbean lowlands. We collected specimens of Eleutherodactylus and Craugastor by hand at these sites, euthanized with Orajel, and preserved them in the field in formalin. We used these species since they are abundant at both sites throughout the year.

2.2.

Histologic analysis for B. dendrobatidis

We tested 202 specimens, representing 30 species from the La Selva-Barva expedition and 18 specimens of five species from the EARTH and Guayaca´n locations. Six of the eight families of frogs in Costa Rica were represented. Samples were processed in the Laboratory of Pathology at the National Children’s Hospital in San Jose´, Costa Rica. Specimens preserved in formalin were soaked in water for 48 h and transferred to 70% ethanol for final storage. At least 2 mm2 of skin from the pelvic patch was extracted from each individual. Juveniles with a snout vent length of 1.5 cm and smaller, were not used to avoid damaging the specimens. Using routine procedures, the skin samples were run through a Miles Scientific processor (Tissue Tek, VIP 3000), in which they were dehydrated and embedded in paraffin. Four-micron thick sections were stained with hematoxylin and eosin (H&E). Skin sections were examined with a compound microscope for the presence of B. dendrobatidis. Three different morphs of the organism, probably representing different life stages were found in most infected samples: a uninucleated form containing homogenous basophilic cytoplasm; a form with multiple separate nuclei and a form with well defined 2–3 lm round to oval basophilic spores (Pessier et al., 1999). Empty thalli were also common. Ideally ultrastructural (Barr, 1990) and genetic studies should be used to confirm species identification of chytrids. However, as B. dendrobatidis is the only chytrid reported to grow on the amphibian integument, and there are no other known frog parasites with a similar appearance, histological identification was considered adequate. Ultrastructural and genetic studies have shown that B. dendrobatidis occurs in this region (Berger et al., 1998; Lips et al., 2003b; Morehouse et al., 2003).

2.3. Surveyed species population status in Costa Rica and their reproductive mode The status of each species with respect to ‘‘enigmatic’’ population declines was determined using the comments on the individual species accounts of the World Conservation Union Global Amphibian Assessment (GAA) web page (IUCN, Conservation International, and NatureServe, 2004. Global Amphibian Assessment. . Accessed on 26th March, 2005). The comments for these species were mostly derived from the experience of two authors (F.B. and G.C.) who have worked with amphibians in Costa Rica for more than 20 years. The conservation status reported in Table 1, reflects the experience with the species found at Braulio Carrillo National Park, and explains in which cases populations disappeared, are still present or lack data, according too F.B. and G.C. One of the authors (F.B.) was part of the team of herpetologists that worked with the project in 1986, and

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F.B. and G.C. monitored the upper and lower portion of the transect at the beginning of the 1990s, where it was obvious that populations from the upper part of the transect had declined. Reproductive habitat and mode was assigned to each species according to Savage (2002): Lotic (species that breed in fast moving streams or rivers); Lentic (species that breed in swamps, ponds or slow moving bodies of water); plant-breeders; the direct development forest dwellers and direct development stream dweller. The taxonomy of the species follows Savage (2002), Crawford and Smith (2005) and Faivovich et al. (2005). The altitudes were categorized by 300 m intervals. Likelihood ratio G tests were used for the analysis. Although specimens of Eleutherodactylus altae and Craugastor melanostictus were collected during the expedition, we did not include them in the analysis because they are rather uncommon, and it is not clear if they have declined or not.

3.

Results

Of the specimens from the La Selva–Barva area in 1986, 5.94% had B. dendrobatidis present in their integument; 30% of the species had at least one infected individual. All families except Centrolenidae were infected. Of the 13 species, which have since declined, 33%, including A. varius and Rana vibicaria, were infected. But of the 18 species that have not subsequently declined, 42%, including Craugastor fitzingeri and Dendrobates pumilio, were also infected (Table 1). Both Istmohyla pseudopuma and Craugastor podiciferus, two of the few species that can still be found at high altitudes (Pounds et al., 1997), had chytrid in their skin. The distribution of the fungus was spread homogeneously among the different families (G = 3.655, df = 5, P = 0.600). Chytrid fungus infection occurred at almost all altitudes. The highest frequency of individuals infected (17.6%) occurred between 100 and 399 m elev., but high infection rates also occurred at 1000–1299 m elev. and 1900–2200 m elev. (14.3%). The difference among the altitudes was significant (G = 15.54, df = 7, P = 0.030). The main reason was the lack of infected animals between 1300 and 1900 m elev. Species with all reproductive modes were infected, except the direct developing species that inhabit streams. Plant breeders, represented by one species, had a prevalence of 20% infected individuals, followed by lotic breeders (7.1%), direct developing species (5.6%) and lentic breeders (4.3%). The differences in infection rates among the reproductive categories were not significant (G = 2.739, df = 4, P = 0.602). Fungus infections were found in 24% of the Eleutherodactylus specimens in 2002. Rates of infection were: Craugastor talamancae (1/3), Craugastor bransfordii (1/4), Eleutherodactylus diastema (0/2), Craugastor crassidigitus (1/6) and Eleutherodactylus ridens (0/3). All five species are common at present in the Caribbean lowlands.

4.

Discussion

Our results demonstrate that B. dendrobatidis was present in nine frog species in Costa Rica back in 1986, prior to the first documented population decline in Monteverde. In the high-

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Table 1 – Species and number of specimens examined from the La Selva–Barva transect, Braulio Carrillo National Park Species

N

n Infected

Altitudinal range

Bufonidae Atelopus varius Atelopus senex

9 3

1

850–1500 2070–2300

920 –

Declined Declined

Dendrobatidae Dendrobates pumilio

6

1

280–620

620

No decline

280–850 700 1500–1800 1500–1800 2050 960–1500 960–1000 960

– – – – 2050 1000 – –

No decline No decline Declined Declined Declined, but still present Declined Still present Declined

960–1800 1800

– –

Still present Still present

920 280–1000 500–800 960–1020 1500–1800 1460–2050 280–1130 1460–2300 440–660 580–1500 960–990 280–1000 1500–2500 400–1000 350–850

– 280 (2), 1000 800 – – – 280 2300 – – – – – –

Declined Still present Still present Still present Data deficient Declined Still present No decline Still present Still present Data deficient Still present Still present Still present No decline

1800–2400 700–920

2050 (2) –

Declined Declined

Hylidae Smilisca sordida Smilisca phaeota Istmohyla rivularis Istmohyla angustilineata Istmohyla pseudopuma Duellmanohyla uranochroa Duellmanohyla rufioculis Hylomantis lemur Centrolenidae Centrolene prosoblepon Hyalinobatrachium colymbiphyllum

5 1 8 7 14 6 2 1

1 1

7 2

Leptodactylidae Craugastor andi Craugastor fitzingeri Craugastor talamancae Craugastor crassidigitus Craugastor melanostictus Craugastor escoces Craugastor bransfordii Craugastor podiciferus Craugastor megacephalus Eleutherodactlus cruentus Eleutherodactylus altae Eleutherodactylus diastema Eleutherodactylus hylaeformis Eleutherodactylus caryophyllaceus Eleutherodactylus cerasinus

1 6 2 10 4 9 13 19 5 15 2 10 8 8 5

Ranidae Rana vibicaria Rana warschewitschii

11 3

3 1

1 1

2

Elevation infected specimens

Population status

Prevalence of B. dendrobatidis, altitudinal range at which each species was collected along the transect, elevation at which infected specimens were collected, current status of each species in the park, and reproductive mode.

lands, where declines occurred, we found infections not only in species such as R. vibicaria and A. varius which subsequently disappeared from the park, but also in species such as C. podiciferus and I. pseudopuma which have persisted (Pounds et al., 1997). In Monteverde the abundance of I. pseudopuma dropped sharply after the declines, but has shown signs of recovery since (Pounds et al., 1997). Similar patterns have been found in Australia, where chytrid infections occurred in the few persisting populations known of Taudactylus eungellensis, a species that suffered strong declines in the past (Retallick et al., 2004), showing that these remnant populations do coexist with this disease in the present. Factors, such as differences in skin peptides (RollinsSmith et al., 2003), or behavior (Woodhams et al., 2003) could be contributing to create the heterogeneous effect found among species. Although most of the amphibian declines in Costa Rica have occurred at elevations greater than 500 m elev. (Lips,

1998; Lips, 1999; Young et al., 2001), chytrid infections in 1986 were not restricted to the upland areas. The chytrid continues to exist today in the lowlands, in species such as D. pumilio and several species of Eleutherodactylus and Craugastor. Given that the highest proportion of infected animals is in the lower portion of the transect, and finding B. dendrobatidis in the specimens of 2002, shows that all amphibians in the lower parts of Costa Rica should be considered as reservoir for this disease and potentially infected. Specimens extracted for the illegal pet trade should be considered potential vectors for this disease. Also, all species from the lowlands of Costa Rica, brought in for serious programs of captive breeding should be put in quarantine. Chytrid infections in 1986 were not limited to a strictly aquatic habitat. The fact that species such as C. fitzingeri or D. pumilio were infected (and continue to be infected) suggests that this pathogen can survive on the moist forest floor.

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Perhaps it lives there as a saprobe, as suggested by Longcore et al. (1999), and is picked up from the ground. It has been shown to grow on alternative hosts, for which is would be unnecessary for it to need frogs in the environment to persist (Rowley et al., 2006). There were no infected animals collected between 1300 and 1900 m elev. in 1986. Our sample at this elevation is only 73 frogs, so it is possible that this chytrid free category may be an artifact of the sample size. The same applies for the lack of observed infections in species in the direct developing river category, and the Centrolenids. Also, some thought should be given to how consistent the body part chosen to detect the disease is, as in this case with the pelvic patch. We have in fact studied the usefulness of different areas of the body (Puschendorf and Bolan˜os, in press) and have found that in C. fitzingeri, one of the most commonly found species infected along the transect, that the pelvic patch was one of the areas where infections could be most consistently identified. Even when dealing with the best body part for detection, light infections could go undetected with the effort made in this project, and an increase of almost four times the number of ribbons sampled would be needed to reach 95% confidence (Puschendorf and Bolan˜os, in press). It is clear that potentially we underscored detection, and some of the chytrid free categories might be an artifact of this effect. Our results do not support the simple predictions derived from the hypothesis that chytrid infections would occur only in species and sites where subsequent population declines occurred. They do not, however, eliminate this hypothesis. In Costa Rica, the link between die offs and the following disappearance of several frog species attributed to B. dendrobatidis, has been shown to occur and seems to be responsible at least in part for the decline of some amphibian species (Lips, 1998; Lips et al., 2003b). In Costa Rica most of the highland populations declined, and at present the lowland amphibian populations apparently coexist with B. dendrobatidis, which seems to be endemic in large portions of the country (Puschendorf, unpublished). Although it has been suggested that this is a novel pathogen (Daszak et al., 1999; Morehouse et al., 2003; Weldon et al., 2004), some authors suggest that the information available is still not conclusive (Rachowicz et al., 2005; Oullet et al., 2005). Lips et al. (2006), provide strong evidence that in Panama this seems to be an emerging infectious disease, and suggest that it has been invading naive populations as it moves west, coming from Costa Rica. Pounds et al. (2006) hypothesize that micro-scale temperature shifts, both increases and decreases, resulting from large-scale warming and changes in cloud cover frequency may favor the development of chytridiomycosis. Daszak et al. (2003) state that chytridiomycosis shows the clearest link between disease and decline. Too much basic biology and ecology of the pathogen in the wild and its interaction with the environment is still unknown (McCallum, 2005). We strongly support the conclusion of Collins and Storfer (2003) that only with an integrated team of multidisciplinary scientists will we eventually understand why highland anurans are disappearing in the tropics.

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Acknowledgements We thank M.C. Obando and M. Sa´nchez for help with histological preparations and, A. Carranza and the National Childrens Hospital for permission to process samples in their facilities. We also acknowledge W. Eberhard, A. Lizano, B. Kohlmann, L. Berger, K. Lips, A. Pounds, R. Alford, A. Crawford, C. Vaughan, M. Sasa, A. Carnaval, D. Brooks, P. Hanson and B. Young for their helpful suggestions throughout this research and on earlier versions of the manuscript. We thank B. Kubicki for help in the field, and the Museum of Vertebrate Zoology of the University of California at Berkeley and D. Wake for access to their collection. Idea Wild and K. Lips funded this research. It is a contribution of the Museo de Zoologı´a, Universidad de Costa Rica and conducted in association with the Research Analysis Network for Neotropical Amphibians (RANA), which is supported by the National Science Foundation (DEB-0130273).

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