DOI: 10.5061/dryad.rv15dv45s
title: Data from: Chemical defenses shift with the seasonal vertical migration of a Panamanian poison frog

description: Dendrobatid poison frogs sequester lipophilic alkaloids from their
 arthropod prey to use as a form of chemical defense. Some dendrobatid
 frogs seasonally migrate between the leaf litter of the forest floor in
 the dry season to the canopy in the wet season, which may yield
 differences in prey (arthropods) and therefore alkaloid availability over
 space and time. Here, we document a seasonal vertical migration of
 Andinobates fulguritus (the yellow-bellied poison frog) from ground to
 canopy between dry and wet seasons. We observed turnover in alkaloid
 composition between seasons and found that dry season frogs contained a
 lower relative quantity of alkaloids; however, there was no change in
 alkaloid richness between seasons. The 77 alkaloids of 13 structural
 classes identified in this population appear to be derived mostly from
 mites and ants, though the two most common alkaloids were mite derived.
 Our observed shifts in defensive profiles are consistent with
 well-documented turnover in mite and ant communities between seasons and
 vertical strata. As climate change is expected to lengthen and strengthen
 dry seasons in many tropical regions, our results suggest that arboreal
 poison frogs forced to the ground for longer periods of time may see a
 shift in the abundance of alkaloids, possibly decreasing their defensive
 potential. This study provides further predictions for the wide-reaching
 effects of climate change, even as nuanced as charismatic poison frogs
 losing their poisons.
description: 1. Study area In central Panama, we surveyed
 an Isthmian-Atlantic Moist Forest located within the Esteban Alphonso Lee
 Natural Reserve (Lat 9.358555 : Lon 79.7029; 333 – 473 m a.s.l; Figure
 S1), which borders the Chagres and Portobelo National Parks.
 <br> 2. Collection of <em>A.
 fulguritus</em> We surveyed for <em>A.
 fulguritus </em>during the dry (mid-December – March) and wet
 seasons (April – mid-December) (Comita & Engelbrecht 2009, Basham
 & Scheffers 2020). Specifically, in the dry season frogs were
 captured between 4 February 2019 and 9 March 2019, and in the wet season
 between 2 July 2019 and 27 July 2019 (Figure S2). Thus, we attempted to
 standardize the amount of time experienced by individuals in both seasons
 before sampling, with individuals sampled between 2-3 months after the
 beginning of either season (Figure S2). Using survey methods in Scheffers
 <em>et al.</em> (2013) and Basham and Scheffers (2020), we
 conducted vertical, ground-to-canopy surveys for <em>A.
 fulguritus</em>, with each survey centered on a single Espavé tree,
 which is the principal habitat for <em>A. fulguritus</em> in
 this area (EB; unpublished data, Figure 1). A second researcher surveyed
 the ground in a 15 m radius around the tree for 1 hour searching through
 leaf litter, logs and other microhabitats (Heyer <em>et
 al</em>. 1994). During arboreal surveys, we searched for
 <em>A. fulguritus</em> in tree holes, moss, epiphytes and
 other microhabitat structures. For the purpose of this project we selected
 13 adult individuals from each season collected from five sites (Table 1;
 Figure S1), for a total of 26 individual samples. Sites were located in
 contiguous forest at a range of distances from one another (closest sites,
 Tree IDs #114 to #111 = 19 m; furthest sites, Tree IDs #111 to #121 = 701
 m, Figure S1). Samples were chosen to maximize sample size whilst
 representing sites equally across seasons, but due to the limitations of
 sampling and the need to avoid using the same individuals across seasons
 (checked by markings), there was a slight discrepancy in sampling across
 sites (Table 1). However, though tree #114 was represented by a single
 frog in the dry season, the trees #114 and #111 were closely located (19
 m), thus reducing the potential for a significant difference in arthropod
 prey between selected sites. Frogs were collected
 solely from the leaf litter beneath Espavé trees in the dry season and
 from the canopy of Espavé trees in the wet season. Espavé trees are host
 to a wide range of epiphytes, but were dominated by a small number of
 highly abundant moss, orchid, bromeliad, and ludovia spp epiphytes. Thus,
 the variation in arthropod prey availability across sites was reduced
 through the relative homogenization of vegetation structures and resources
 present across all sites. <br> 3. Alkaloid
 Sampling Captured frogs were weighed, measured (mean
 weight = 0.28 g; mean snout-vent length = 14.3 mm), brought to the
 research station, and allowed to rest overnight with ample moisture to
 maintain full hydration. Alkaloids were collected from each frog using a
 transcutaneous amphibian stimulator (TAS; Grant & Land, 2002),
 which applies a weak electric current to the skin, causing the secretion
 of the contents of their granular glands. Following the methods of Bolton
 et al. (2017), the TAS treatment (Frequency:  50 Hz; Pulse width: 2 ms;
 Amplitude: 9 V) was standardized among frogs. The frogs were then wiped
 with discs of absorbent bibulous paper to collect the secretions, which
 were deposited in 1 ml of 100 % methanol in glass vials with Teflon-lined
 caps. Frogs were fully recovered within 5 minutes, and were later released
 at the same point of capture. The TAS is a nonlethal method of collecting
 frog skin alkaloids, and previous studies have found no difference in the
 number and types of alkaloids collected using the TAS method when compared
 to the more traditional, yet lethal whole-skin extraction method (Clark et
 al. 2006, Hantak et al. 2013, Bolton et al. 2017, Schulte et al. 2017).
 However, based on studies of other small dendrobatids (Oophaga pumilio and
 Oophaga granulifera), the quantity of alkaloids extracted using the TAS
 are only proportional to the total quantity present in whole-skin
 extractions of alkaloids (Saporito, unpublished data). Therefore, the
 alkaloid quantities reported here (see Table S1) do not represent the
 total quantity of skin alkaloids present in A. fulguritus, but are instead
 proportional to the total quantity contained in each frog and should be
 compared based on their relative differences. We report quantity as
 micrograms of alkaloid per gram of frog. <br> 4.
 Alkaloid extraction, identification, and quantification
 For each sample, an internal standard of nicotine ((-)-nicotine
 99%, Sigma-Aldrich, Milwaukee, Wisconsin) were added to 0.5 ml of the
 original MeOH extract. This extract was evaporated to dryness with N2, and
 then resuspended in 100 μl of 100 % methanol. Gas chromatography/mass
 spectrometry (GC/MS) was performed for each sample on a Varian Saturn 2100
 T ion trap MS instrument coupled to a Varian 3900 GC with a 30 m x 0.25 mm
 i.d. Varian Factor Four VF-5 ms fused silica column. GC separation of
 alkaloids was achieved using a temperature program from 100 to 280 °C at a
 rate of 10 °C per minute with He as the carrier gas (1 ml/min). Each
 sample was analyzed in triplicate using electron impact MS, and once using
 chemical ionization MS with methanol as the CI reagent.
 All alkaloids from each sample were identified using a
 combination of gas chromatography and mass spectrometry (GC-MS). Following
 the methods detailed in Hovey <em>et al</em>. (2018),
 individual alkaloids were quantified by comparison to the nicotine
 internal standard, and identified by comparing their retention times and
 mass spectral data with those of known alkaloids.
description: This data was collected by the Scheffers Lab at the University of
 Florida and the Saporito Lab at John Carrol University, OH, at significant
 cost of effort and resources. If downloaded for use in
 publications we would appreciate a notification on its usage and would be
 glad to discuss potential research avenues and collaborations.
 Please use this email for contact:
 brett.scheffers@ufl.edu     <br> Metadata  
       <br> Alkaloids: The Alkaloids sheet
 contains all information pertaining to each specific alkloid that was
 recorded from the sample of 26 frogs The information is
 broken down into the retention time, structural class, specific alkaloid
 name, the season from which it was sampled, the quanitity that was
 recorded in micro grams, a tentative label for arthropod origin, and the
 Individual Code for the relevant frog from which it was sampled
     <br> Frogs+Trees: The Frogs+Trees sheet contains
 information pertaining to the characteristics of each sampled frog (mass
 in grams), from when (Season and Date) and where (Height, and tree
 location as longitude and latitude) it was sampled. This data can be
 joined to the Alkaloids sheet using Individual_code as a linking
 variable