Field surveys and sampling
We carried out eight field surveys between November 2013 and September 2015 at numerous amphibian habitats over ca. 177 km2 in the Hula Valley. Daytime surveys for L. nigriventer involved searching terrestrial habitats as well as various water bodies where we used dip nets to search for adults, tadpoles and egg clutches. Night surveys only involved visual inspection of water bodies and their banks. To minimise disturbance, sites were usually not inspected more than twice a week. Equipment and shoes were either completely dried or disinfected with Virkon S or 10% bleach solution between locations (i.e. those not connected by waterways) following Johnson et al. (2003). Detailed coordinates of confirmed locations are not published to avoid disturbance and collecting, but have been communicated to the Israel Nature and Parks Authority.
Metamorphosed individuals were captured with gloved hands, photographed in dorsal and ventral views and morphometric measurements were taken. We collected tissue and buccal swabs for assessing genetic variation, and took skin and cloacal swabs for exploring the microbial communities of L. nigriventer and other local amphibians. Skin swabs were also used for Bd screening (Hyatt et al., 2007). Before swabbing, frogs were rinsed with sterilised distilled water to remove transient bacteria (Culp et al., 2007; Lauer et al., 2007; Rebollar et al., 2014). Water volume was adjusted based on SVL (50–150 ml). The skin of each individual was swabbed dorsally and ventrally using two separate swabs with each side receiving 10 strokes.
Tissue samples and buccal swabs were directly stored in 95–99% ethanol, while skin swabs were immediately placed on ice and transferred to freezer storage (–20 °C) within 8 hours. All metamorphosed individuals were released back to their collection site after examination. None were sacrificed but dead individuals (e.g. due to predation, road kills) were preserved in ethanol. Several tadpoles died shortly after capture and were fixed in 70% ethanol and later preserved in 70% ethanol or 5% formalin.
External transmitters (SOPR-2038; Wildlife Materials International, Inc.) were fitted to seven large (> 70 mm) individuals with an elastic waistband. Specimens were kept in a terrarium for up to three days to ensure that the waistband was not causing undue harm. Released individuals were tracked with a TRX-16 receiver and 3-element folding antenna (Wildlife Materials International, Inc.). Tracking was completed twice a day for up to 18 days, but individuals were only visually inspected every second or third day in order to minimise disturbance (Fig. 2 O).