Study sites

The study was conducted at four streamside sites within the tropical rainforests of northern Queensland, Australia: Python Creek (“Tully Gorge”, 145°35'E 17°46'S, 200 m asl) and an unnamed creek (“Lower Tully”, 145°41'E 17°48'S; 70 m asl) in Tully Falls Forest Reserve, an unnamed creek in Kirrama State Forest (“Kirrama”, 145°52 E 18°11 S; 200 m asl) and Frenchman Creek, in Wooroonooran National Park (“Babinda”, 145°55' E 17°20' S 20–100 m asl). A marked transect at each stream served as a reference for frog locations.

Study species

Frogs of three species were tracked: the Stony Creek Frog L. lesueuri (31 females, 37 males), which has not experienced population declines12, the Green Eyed Treefrog L. serrata (10 females, 13 males), which declined during epidemics of chytridiomycosis in the late 1980s and early 1990s and subsequently recovered12 and the Waterfall Frog L. nannotis (29 females, 8 males), which was extirpated from many sites during chytridiomycosis epidemics12. The taxonomy of L. lesueuri was revised in 200413. Two newly recognized species, L. jungguy and L. wilcoxii, occur in sympatry in the study region, hybridise and cannot reliably be distinguished morphologically13. Our study populations could have included either species, mixtures of both and hybrid individuals. Population declines have not been observed in either species. We therefore continue to refer to the study species as L. lesueuri, while recognizing that our samples contain unknown proportions of the two morphologically indistinguishable species. All three species are large to medium sized hylids (males 5.4–12.5 g, females 6.5–41.3 g).

Tracking

At initial capture, frogs were weighed and swabbed on the ventral surface for diagnostic PCR using a sterile rayon swab (Medical Wire & Equipment Co. (Bath) Ltd., Wiltshire, UK). Swab samples were analysed using diagnostic PCR or quantitative PCR14.

Tracking devices (radio transmitters, or diode tags for harmonic direction finding, depending on size of the frog16) were then fitted in situ and frogs were released at their point of capture after less than five minutes of handling. Tracking followed protocols we have previously published15.

Surveys lasted 16 days and were conducted in the winter/dry season (July-September) and the summer/wet season (February-April) at two sites for each species. Litoria genimaculata and L. nannotis were tracked simultaneously at the same streams during 2004 and L. lesueuri were tracked during 2005. During surveys, the location of each frog was determined once during the day (0900–1800 h) and once at night (1900–0400 h). Whenever possible, the temperatures of located frogs and their substrates were recorded.

When we could visually locate individual frogs and were able to reach within 0.5 m of them, temperature of the dorsal body surface was measured using a Raytek ST80 Pro-Plus Non-contact Thermometer (RAYST80, emissivity set to 0.95). This technique obtains body temperature readings within 0.5°C of cloacal temperatures17. When the infrared thermometer could not be used, if the frog was fitted with a temperature-sensitive transmitter, we recorded the pulse interval of the telemetry signal by timing 90 pulses with a stopwatch. This was converted to temperature using individual calibration curves for each transmitter.

On average, each frog was located 17 times (minimum 5, maximum 28). We excluded data from the night following tag attachment from analyses due to the potential short-term behavioural effects of handling18. Effects of tags on behavior are unlikely to persist after the first night of tag attachment16. The weights of tracked individuals did not change significantly over the study period (Wilcoxon Signed Ranks Test; Z = −1.361, p = 0.173, n = 70). Research was carried out under Scientific Purposes Permits issued by the Queensland Parks and Wildlife Service (WISP01715204 and WITK01715604) and approved by the James Cook University Animal Care and Ethics Committee (A863).

Data analyses

On swabs analysed using qPCR, we measured between 6 and 10,527 zoospore genome equivalents per swab. As ~20% of our samples were analysed via PCR only, we included only infection prevalence in our statistical analyses. Because tracking did not occur at the same sites for all three species, we could not include site as a factor in orthogonal models. We initially examined our data for possible site-specific effects using Fisher's exact tests to determine whether infection prevalence differed significantly among sites within seasons for each species. It did not (Fisher's exact tests; all p > 0.05). We thus omitted site as a factor in all further analyses. We next examined whether individual thermal history, measured as the proportion of body temperatures of each individual that were greater than 25°C, was affected by species, gender and season, using an ANOVA in SPSS version 19. This analysis showed that thermal histories were affected by all those factors. Finally, we examined how Bd infection status (0 = infected, 1 = uninfected) was affected by species, gender, season and the thermal histories of individuals (proportion of body temperatures above 25°C), using a set of models created using the generalized linear models procedure in SPSS version 19 with a binary logistic link function (Table 2). We used Akaike's information criterion, corrected for finite sample size (AICc) to evaluate model fit, using the criteria of Burnham and Anderson19. Our best fitting models were averaged to obtain the final model19.