Surveys for Frog Diversity and Batrachochytrium dendrobatidis in Jamaica AMPHIBIAN DISEASES

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1 vi Carey, C Infectious disease and worldwide declines of amphibian populations, with comments on emerging diseases in coral reef organisms and in humans. Environ. Health Perspect. 108: Chippindale, P. T., A. H. Price, J. J. Wiens, and D. M. Hillis Phylogenetic relationships and systematic revision of Central Texas hemidactyliine plethodontid salamanders Herpetol. Monogr. 14:1 80. Daszak, P., A. Strieby, A. A. Cunningham, J. E. Longcore, C. C. Brown, and D. Porter Experimental evidence that the bullfrog (Rana catesbeiana) is a potential carrier of chytridiomycosis, an emerging fungal disease of amphibians. Herpetol. J. 14: Drew, A., E. J. Allen, and L. J. S. Allen Analysis of climatic and geographic factors affecting the presence of chytridiomycosis in Australia. Dis. Aquat. Org. 68: Gaertner, J. P., M. R. J. Forstner, L. O Donnell, and D. Hahn Detection of Batrachochytrium dendrobatidis in endemic salamander species from Central Texas. EcoHealth 6:20 26., M. A. Gaston, D. 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T., R. A. Knapp, T. Tunstall, and C. J. Briggs Largescale amphibian die-offs driven by the dynamics of an emerging infectious disease. Proc. Natl. Acad. Sci. USA 107: Walker, S. F., M. B. Salas, D. Jenkins, T. W. J. Garner, A.A. Cunningham, A.D. Hyatt, J. Bosch, and M.C. Fisher Environmental detection of Batrachochytrium dendrobatidis in a temperate climate. Dis. Aquat. Org. 77: Herpetological Review, 2012, 43(2), by Society for the Study of Amphibians and Reptiles Surveys for Frog Diversity and Batrachochytrium dendrobatidis in Jamaica Jamaica is home to the world s second most endangered frog assemblage, with 16 of 21 (76%) endemic species recognized as threatened (IUCN 2011). There are 17 endemic Eleutherodactylus (Hedges 1989), five endemic Osteopilus, one of which is unnamed and thus has not been assessed by the IUCN (Moen and Wiens 2008; S. B. Hedges, unpubl. data) and four invasive anurans on the island (Mahon and Aiken 1977). We conducted the first large-scale assessment of the island s amphibians since the 1980s while sampling for the amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd). Prior to our work, Bd was known from all of the other Greater Antillian islands (Burrowes et al. 2004; Diaz et al. 2007; Joglar et al. 2007), several of the Lesser Antillian islands (Alemu et al. 2008; Garcia et al. 2009), IRIS HOLMES* KURT McLAREN BYRON WILSON Department of Life Sciences, University of the West Indies, Mona Campus, Kingston, Jamaica *corresponding author; iah6@cornell.edu and mainland North, Central and South America (Carnaval et al. 2006; Longcore et al. 1999; Fisher et al. 2009), but was not confirmed in Jamaica. Given the documented extinctions and population declines in congeners to Jamaica s frogs from chytridiomycosis in Puerto Rico (Burrowes et al. 2004; Longo et al. 2010) and Central America (Lips et al. 2004; Puschendorf et al. 2006), we were concerned about the conservation implications of epidemic outbreaks of Bd to Jamaica s amphibians. As of September 2010, 6 of Jamaica s 21 described endemic species had not been recorded in over two decades (Hedges and Diaz 2011). The timeframe of their last sighting was similar to that of some chytridiomycosis-related extinctions and extirpations elsewhere in the Caribbean (Burrowes et al. 2004). We conducted this project to investigate the occurrence of Bd per species and location, assess the status of Jamaican frog species, and to provide information to focus future conservation efforts directed at extant endemic species. We sampled for amphibians across Jamaica, spending at least one person-day in the field in the known ranges of every endemic species on the island (Fig. 1; Table 1). We conducted our field work between October 2010 and June We defined a

2 vii Fig. 1. Distribution of sampling locations in Jamaica for the amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd). Proportion of Bd-positive and Bd-negative individuals are indicated per site. Primary land cover types are shown (from Forestry Department, Jamaica: [accessed 3 June 2011]). person-day as the search effort of one person within a 24-h period, generally 2 5 h/day. We sampled closed broadleaf, disturbed broadleaf, short open dry, and swamp forest habitats, as well as mixtures of disturbed broadleaf and small plantations, and suburban habitats and caves. Our field sites ranged from sea level swamp and dry forests to elfin forest at 2250 m on top of Jamaica s highest peak. We used visual/audio encounter surveys beginning at sunset and lasting 2 5 h per site. Frogs were caught by hand using an inverted plastic bag and were housed overnight in the bag. We recorded capture location using a Garmin Global Positioning System Map 60CS in the WGS84 datum. We identified captures to species, and swabbed them ten times on the bottom of each foot, under each thigh, along the stomach, and under the drink patch. We examined frogs for skin sloughing, unusual posture, and other known chytridiomycosis symptoms (Berger et al. 1998; Berger et al. 2005). Frogs that were not collected as voucher specimens were released at their capture location after processing. We thoroughly disinfected all of our gear between each sampling site with a 10% bleach solution, followed by air drying in direct sunlight. We stored the swabs dry at ambient temperature in the field and refrigerated them immediately on return to the laboratory (Hyatt et al. 2007; Skerratt et al. 2008). We extracted the total Bd genome, as well as any DNA Table 1. Amphibian sampling locations in Jamaica (latitude/longitude; WGS 84 datum). Elevation is approximate average search elevation (m). Site number Location Northing Easting Elevation 1 Ecclesdown Millbank Stony River Portland Gap Hollywell Irish Town Grand Ridge Bangor Ridge White River Jackson Bay Cave Belle Aire Cave BBQ Bottom Windsor Troy Quickstep BLRM Rocklands Bird Sanctuary Bluefields Dolphin Head

3 viii Table 2. Batrachochytrium dendrobatidis (Bd) occurrence on frogs in Jamaica. No. Bd positive individuals/total no. individuals sampled per species and location are provided. For site names see Table 1. Genera are: O = Osteopilus; E = Eleutherodactylus; R = Rana; L = Lithobates. 0/0 indicates that the sampling location was in a species range, but the species was not detected. A dash ( ) indicates that the sampling area was outside of the species range, and the species was not found. Bold numbering indicates that the species was found outside its known range. Asterisks indicate invasive species. Location Species Totals (%) O. brunneus 1/3 1/16 0/6 0/0 0/0 1/5 0/0 0/0 0/5 2/5 0/0 1/2 0/12 0/0 6/54 (11.00) O. crucialis 0/0 0/0 0/0 0/0 0/0 (0) O. marianae 0/0 0/0 0/0 0/0 (0) O. wilderi 0/0 0/1 0/0 0/1 0/0 0/0 0/2 (0) O. sp. 0/5 0/5 (0) E. alticola 0/1 0/4 (0) E. andrewsi 1/3 0/0 0/3 1/6 (16.70) E. cavernicola 0/2 0/2 (0) E. cundalli 0/1 0/4 3/15 0/0 0/0 1/9 0/0 0/0 0/0 4/29 (13.80) E. fuscus 2/8 2/8 (25.00) E. glaucoreius 7/17 0/0 1/4 0/0 1/3 3/5 0/1 0/0 0/3 12/33 (36.36) E. gossei 0/8 2/7 1/5 12/63 0/1 2/19 0/9 2/5 0/4 0/0 1/2 0/1 20/124 (16.13) E. grabhami 0/0 0/0 0/0 0/0 (0) E. griphus 0/11 0/14 0/25 (0) E. jamaicensis 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 (0) E. junori 0/0 0/0 (0) E. luteolus 0/9 4/16 0/5 0/4 4/34 (11.76) E. nubicola 0/0 1/3 1/3 2/6 (33.33) E. orcutti 0/0 0/0 0/0 0/0 (0) E. pantoni 4/15 0/6 3/33 6/33 1/27 14/114 (12.28) E. pentasyringos 1/5 0/0 0/6 4/26 (15.38) E. sisyphodemus 0/0 0/0 (0) E. johnstonei* 0/1 0/1 1/13 1/2 0/3 2/2 3/8 1/8 0/1 1/2 10/43 (23.26) E. planirostris* 1/1 1/1 (100%) L. catesbeianus* 0/1 0/1 (0) R. marina* 0/1 0/1 0/12 0/14 (0) Totals (%) 13/51 3/25 2/22 0/4 2/6 16/81 1/7 1/3 3/33 0/2 0/4 7/60 5/14 6/51 13/82 2/22 1/4 0/17 4/41 (%) (25.49) (12.00) (9.09) (0.00) (33.33) (19.75) (14.29) (33.33) (9.09) (0.00) (0.00) (11.67) (35.71) (11.76) (15.85) (9.09) (25.00) (0.00) (9.76)

4 ix present from frog skin cells or other frog skin microbiota, from the swabs using the standard PrepmanUltra protocol (Hyatt et al. 2010). We ran quantitative PCR tests at the Cornell University Life Sciences Core Laboratories Center, Ithaca, New York. We ran samples in singlicate at a 1:10 dilution of the starting stock on an Applied Biosystems ViiA7 machine. All voucher specimens are stored in the Herpetology Laboratory, University of the West Indies, Kingston, Jamaica. Site-specific Bd infection rates ranged from 0 33% (Table 2). All but four sites had at least one Bd-positive frog. The four Bdnegative sites included two caves and a high-elevation cloud forest, each with low sample sizes, and a relatively dry, disturbed coastal broadleaf forest in western Jamaica. The site with the highest Bd prevalence (5/14 frogs infected) was in central Jamaica, at the intersection of small-scale agricultural land and disturbed broadleaf forest. With these exceptions, little geographical pattern is apparent in infection rates. When Bd infection rates per species were pooled across all sites at which each species occurred, most species showed rates between 10 and 20%. Although Bd was not detected on Eleutherodactylus griphus, Osteopilus wilderi, O. sp. (S.B. Hedges, unpubl. data), and E. cavernicola, due to low sample sizes (Skerratt et al. 2008), we cannot reliably draw the conclusion that their infection rates differed from other species sampled. Eleutherodactylus glaucorieus had the highest infection rate at 36% (12/33). All frogs examined were adults, except a very recently metamorphosed O. wilderi sampled in Troy. We were limited to examining leaf litter, caves, and low vegetation, hence canopy-dwelling species (the Osteopilus) were not well represented in our sample. We found no dead or moribund animals, and saw no disease symptoms on the animals we sampled. We were unable to locate seven endemic species during our surveys (Table 2). Two of these species, E. orcutti and E. jamaicensis, have not been observed since the 1980s, and are the island s only two semi-aquatic Eleutherodactylus: E. orcutti is a stream specialist; and E. jamaicensis a bromeliad tank specialist. Because Bd is often transmitted aquatically, semi-aquatic species may be more at risk of Bd infection than terrestrial species (Kilpatrick et al. 2009). We spent considerable search effort on these species, including visiting the type localities of both. We spent 71 person-days (with 2 5 h/day of sampling) searching in the historical range of E. jamaicensis and 27 person-days in the historical range of E. orcutti without seeing an individual or hearing a call of either species, raising concerns about the continued existence of these species. Eleutherodactylus jamaicensis searches were conducted in November 2010 and January, February, March, May, and June of 2011, whereas searches in E. orcutti s range were carried out in January, March, April, May, and June of The other non-sampled Eleutherodactylus are of less concern, as they are highly cryptic terrestrial species. Although the Osteopilus share the semi-aquatic life history strategy with E. jamaicensis, we heard calls from these species while in their ranges. Although we quantified Bd loads in our positive samples, we do not report those data here. Many of the swabs were not refrigerated for up to a week after sampling, due to the remoteness of our sampling sites. We stored the swabs dry in the field at ambient temperature, which was generally between 20 and 30 C. Storage at ambient temperature does not increase the risk of false negatives, but may reduce the detectable Bd zoospore load by up to 67% when temperatures are above 23 C (Skerratt et al. 2008; Van Sluys et al. 2008). Given that our samples were exposed to ambient temperatures for variable amounts of time, between several minutes and seven days, and that our study sites had considerably different ambient temperatures, our measured loads may be inconsistent representations of actual loads. However, we found no loads above 3,000 zoospore equivalents, and the majority of the infected individuals showed less than 100 zoospore equivalents. Even if these measurements represent an average of one third of the original load, they indicate that the frog populations we sampled may not be in immediate danger of chytridiomycosis-related decline (Vredenburg et al. 2010), at least under current environmental conditions and with the current (as yet not genotyped) strain of Bd on the island. Further habitat loss and the introduction of other diseases or more virulent Bd strains are ongoing threats to this highly endangered frog community. Additional sampling is warranted for endemic species which were undetected during this survey. Acknowledgments. We thank D. Luke, C. Sharma, S. Monroe, K. Prospere, S. B. Hedges, K. Lewis, J. Pauel, S. Stewart, M. Grundler, T. Shields, P. Doughty, and L. Johnson for valuable assistance in the field, and P. Tennant, R. Bair, K. Zamudio, A. Longo Barrios, and D. Rodriguez for help with laboratory work. We benefited greatly from the assistance of the Jamaica Caves Organization and the Jamaica Conservation & Development Trust (managers of the Blue and John Crow Mountains National Park). This work was supported by a U.S. Student Fulbright grant, and by awards from the Mohamed Bin Zayed Species Conservation Fund, the MacArthur Foundation, the Fort Worth Zoo and Zoo Miami. Field work was conducted under National Environment and Planning Agency (NEPA) Scientific Research permit 18/27. Literature Cited Alemu, J. B., M. N. E. Cazabon, L. Dempewolf, A. Hailey, R. M. Lehtinen, R. P. Mannette, K. T. Naranjit, and A. C. J. Roach Presence of the chytrid fungus Batrachochytrium dendrobatidis in populations of the critically endangered frog Mannophryne olmonae in Tobago, West Indies. Ecohealth 5: Berger, L., G. Marantelli, L. F. Skerratt, and R. Speare Virulence of the amphibian chytrid fungus Batrachochytrium dendrobatidis varies with the strain. Dis. Aq. Org. 68: , R. Speare, P. Daszak, D. E. Green, A. A. Cunningham, C. L. Goggin, R. Slocombe, M. A. Ragan, A. D. Hyatt, K. R. McDonald, H. B. Hines, K. R. Lips, G. Marantelli, and H. Parkes Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America. Proc. Natl. Acad. Sci. USA 95: Burrowes, P. A., R. L. Joglar, and D. E. 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Speare Survey protocol for detecting chytridiomycosis in all Australian frog populations. Dis. Aq. Org. 80: Van Sluys, M., K. M. Kriger, A. D. Phillott, R. Campbell, L. F. Skerratt, and J. M. Hero Storage of samples at high temperatures reduces the amount of amphibian chytrid fungus Batrachochytrium dendrobatidis DNA detectable by PCR assay. Dis. Aq. Org. 81: Vredenburg, V. T., R. A. Knapp, T. S. Tunstall, and C. J. Briggs Dynamics of an emerging disease drive large-scale amphibian population extinctions. Proc. Natl. Acad. Sci. USA 107: Herpetological Review, 2012, 43(2), by Society for the Study of Amphibians and Reptiles Occurrence of Batrachochytrium dendrobatidis Among Populations of Lithobates clamitans and L. pipiens in Wisconsin, USA Continued efforts to sample for Batrachochytrium dendrobatidis (Bd), the fungus that causes amphibian chytridiomycosis, in the U.S. have begun to provide a more complete understanding of its distribution (Longcore et al. 2007; Muths et al. 2009). In fact, some have even questioned the relevancy for additional non-targeted sampling and have suggested targeting gaps in our understanding of Bd distribution such as in the northern regions of the U.S. (Muths et al. 2009). One such significant northern gap is the state of Wisconsin. The earliest reported detection of Bd in Wisconsin was from specimens preserved in 1969 and 1984 (Ouellet et al. 2005). Since then sampling for Bd has been performed along the Mississippi and St. Croix rivers bordering Wisconsin and Minnesota, detecting its occurrence at numerous localities. Sampling also was performed in southwestern Wisconsin but Bd was not detected there (Sadinski et al. 2010). Although states neighboring Wisconsin have been more thoroughly sampled for Bd JAIMIE L. KLEMISH BROOKE L. JOHNSON SPENCER R. SIDDONS ERIK R. WILD* Department of Biology & Museum of Natural History University of Wisconsin-Stevens Point, Stevens Point, Wisconsin 54481, USA *Corresponding author; ewild@uwsp.edu (Minnesota [Rodriguez et al. 2009; Woodhams et al. 2008], Iowa [Loda and Otis 2009; Steiner and Lehtinen 2008], Illinois [Steiner and Lehtinen 2008], and Michigan [Steiner and Lehtinen 2008; Zellmer et al. 2008; Zippel and Tabaka 2008]), Wisconsin is largely separated from these by geologically and biologically significant borders. To the north and east, Wisconsin is bordered by Lake Superior and Lake Michigan, respectively, and to the west the Mississippi and St. Croix Rivers. Additionally, the state contains one of the highest concentrations of freshwater lakes in the world. Clearly, Wisconsin is not simply a biologically-arbitrary political designation, but rather corresponds to a biologically unique area within which an understanding of the distribution of Bd is important for a more complete documentation of this amphibian pathogen in the U.S. Herein we report the findings of broad-scale sampling for Bd across Wisconsin. We sampled during fall 2009 (5 26 September) and summer 2010 (21 June 9 July) opportunistically at localities within each of the 24 Geographical Management Units (GMU s; Fig. 1) of the Wisconsin Department of Natural Resources. GMUs were used to assure a broad distribution of sampling across the state and because they are based on a mixture of hydrologic basins, county boundaries, and DNR regional boundaries for the purpose of managing water resources: Bd occurrence by GMU is potentially important information for future water resource management decisions. Ten arbitrarily chosen GMU s were sampled during Fall 2009 whereas the remaining 14 (plus two that were sampled